U.S. patent application number 16/306754 was filed with the patent office on 2021-07-22 for use and composition of buffer formulation with multiple ph values and protein digestion enhancer.
The applicant listed for this patent is ECO-GEO BIO-TECHNOLOGY COMPANY LIMITED. Invention is credited to Fu-An Chen, Ta-Lu Shen.
Application Number | 20210219569 16/306754 |
Document ID | / |
Family ID | 1000005535590 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210219569 |
Kind Code |
A1 |
Shen; Ta-Lu ; et
al. |
July 22, 2021 |
USE AND COMPOSITION OF BUFFER FORMULATION WITH MULTIPLE PH VALUES
AND PROTEIN DIGESTION ENHANCER
Abstract
A composition for enhancing protein digestion is disclosed,
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.
Inventors: |
Shen; Ta-Lu; (Taipei City,
TW) ; Chen; Fu-An; (Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ECO-GEO BIO-TECHNOLOGY COMPANY LIMITED |
Taipei City |
|
TW |
|
|
Family ID: |
1000005535590 |
Appl. No.: |
16/306754 |
Filed: |
March 16, 2018 |
PCT Filed: |
March 16, 2018 |
PCT NO: |
PCT/CN2018/079343 |
371 Date: |
December 3, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23K 10/28 20160501;
A23C 9/1322 20130101 |
International
Class: |
A23K 10/28 20160101
A23K010/28; A23C 9/13 20060101 A23C009/13 |
Claims
1-3. (canceled)
4. A composition for enhancing protein digestion, comprising: at
least one acid component being one selected from a group consisting
of an organic acid, a phosphoric acid and a combination thereof; at
least one base component being one selected from a group consisting
of an organic base, a phosphate and a combination thereof, wherein
the at least one acid component and the at least one base component
conjugate with each other to form a buffer formulation; and a
protein digestion enhancer being one selected from a group
consisting of an ascorbic acid, a salt of the ascorbic acid and a
combination thereof.
5. A composition as claimed in claim 4, which is added into a
protein nutritional supplement, wherein the protein nutritional
supplement is one of a formulated milk powder and an animal
feed.
6. A composition as claimed in claim 4, wherein the acid component
is one selected from a group consisting of a formic acid, an acetic
acid, a propionic acid, a butyric acid, a malic acid, a fumaric
acid, a lactic acid, a citric acid and the phosphoric acid, and
either of the organic base and the phosphate is one of an alkali
metal salt and an alkaline earth metal salt.
7. A composition as claimed in claim 6, wherein the organic acid is
the citric acid, and the organic base is one of a sodium citrate
and a potassium citrate.
8. A composition as claimed in claim 4, wherein the buffer
formulation is evenly mixed with the protein digestion
enhancer.
9. A composition as claimed in claim 4, wherein the buffer
formulation has multiple pHs.
10. A composition as claimed in claim 4, wherein the ascorbic acid
has a concentration range from 60 ppm to 1000 ppm.
11. A composition as claimed in claim 4, wherein the salt of the
ascorbic acid is one selected from a group consisting of a sodium
salt, a potassium salt, a calcium salt, a magnesium salt and a
combination thereof.
12. A composition as claimed in claim 4, wherein the composition
enhances digestion capability of a pepsin and a trypsin in an
environment with a pH of 2-7.5.
13. A composition as claimed in claim 12, wherein the composition
enhances digestion capability of the pepsin and the trypsin in the
environment with the pH of 5-6.
14. A composition as claimed in claim 4, wherein the composition is
manufactured as one being selected from a group consisting of a
powder, a particle, a tablet, a micron-particle, a liquid and a
capsule.
15. A composition as claimed in claim 4, wherein the ascorbic acid
is an L-ascorbic acid.
16. A method for increasing at least one of digestion and
absorption efficacies, the method comprising steps of: providing a
composition 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 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; and administering the composition to one of a human being
and an animal.
17. A method as claimed in claim 16, wherein the buffer formulation
has multiple pHs.
18. A method as claimed in claim 16, wherein the buffer formulation
steadily sustains an activity of the ascorbic acid when pH
>4.
19. A method as claimed in claim 16, wherein the acid component is
one selected from a group consisting of a formic acid, an acetic
acid, a propionic acid, a butyric acid, a malic acid, a fumaric
acid, a lactic acid, a citric acid and the phosphoric acid, and
either of the organic base and the phosphate is one of an alkali
salt and an alkali earth metal salt.
20. A method as claimed in claim 19, wherein the organic acid is
the citric acid, and the organic base is one of a sodium citrate
and a potassium citrate.
21. A method as claimed in claim 16, wherein the ascorbic acid has
a concentration range from 60 ppm to 1000 ppm.
22. A method as claimed in claim 16, wherein the salt of the
ascorbic acid is one selected from a group consisting of a sodium
salt, a potassium salt, a calcium salt, a magnesium salt and a
combination thereof.
23. A method as claimed in claim 16, wherein the ascorbic acid is
an L-ascorbic acid.
Description
FIELD OF THE INVENTION
[0001] 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.
BACKGROUND OF THE INVENTION
[0002] After 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.
[0003] Gastric juice contains gastric acid and pepsinogen secreted
by the stomach wall is activated into pepsin by gastric acid. As
food passes through the stomach to the duodenum, in the meantime,
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.
[0004] To enhance the digestion and absorption in the digestive
tract and promote the growth of animals, antibiotic growth
promoters have been widely used in animal feeds in the animal
husbandry industry since the 1950s. However, with the rise of
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. In recent years, 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. However, 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.
[0005] In view of the above-mentioned drawbacks of the existing
antibiotic growth promoters and acidulants, 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.
SUMMARY OF THE INVENTION
[0006] The main object of the present invention is to increase
digestion and/or absorption efficacies in humans or animals. In
order not to disturb the normal physiology of the gastrointestinal
tract, 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.
[0007] Different from the existing feed additives, 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). To achieve the above-mentioned effects, 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
[0008] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Other objectives advantages and efficacies of the present
invention will be described in detail below taken from the
preferred embodiments with reference to the accompanying
drawings.
[0010] FIG. 1 shows the effect of pH on the activities of three
digestive enzymes in the gastrointestinal tract.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0017] When the multiple pHs buffer formulation composition of the
present invention is designed, the pH environment of the
gastrointestinal tract should be taken into consideration. Please
refer to FIG. 1, which is described in Essentials of Human
Physiology (2017). As shown in FIG. 1, each of the three main
digestive enzymes in the gastrointestinal tract has its own optimal
pH environment. For example, pepsin has the optimal activity at pH
around 2, and trypsin has the optimal activity at pH around 8. In
fact, in the digestion process of the gastrointestinal tract, the
pH of the food varies rather than being constant. When the food
enters the upper part of the stomach, its pH is about 4.0 to 6.5,
and when it enters the lower part of the stomach, its pH is about
1.5 to 4.0. Similarly, 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. Although there is a better proteolysis
efficiency in the optimum pH environment of pepsin and trypsin, 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.about.7.5), especially at the pH 5.about.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).
[0018] 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.
[0019] 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.
TABLE-US-00001 TABLE 1 Organic acids Number of carbon pKa1 pKa2
pKa3 (1) formic acid 1 carbon atom 3.75 -- -- (2) acetic acid 2
carbon atoms 4.76 -- -- (3) propionic acid 3 carbon atoms 4.88 --
-- (4) butyric acid 4 carbon atoms 4.82 -- -- (5) malic acid 4
carbon atoms 3.4 5.1 -- (6) fumaric acid 4 carbon atoms 3.02 4.38
-- (7) lactic acid 3 carbon atoms 3.83 -- -- (8) citric acid 6
carbon atoms 3.13 4.76 6.4 (9) ascorbic acid 6 carbon atoms 4.1
11.8 -- (10) phosphoric acid 0 carbon atom 2.12 7.21
12.32/12.66
[0020] 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.
[0021] 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 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. In a preferred
embodiment, the organic base is sodium citrate or potassium
citrate.
[0022] 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.
[0023] The protein digestion enhancer used in the present invention
is an ascorbic acid, a salt of the ascorbic acid or a combination
thereof. Preferably, 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.
[0024] The ascorbic acid (L-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.
[0025] In another aspect, 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.
[0026] According to the present invention, 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. In one embodiment, the protein digestion
enhancer is distributed in the buffer formulation. In another
embodiment, the buffer formulation is evenly mixed with the protein
digestion enhancer.
[0027] In the present invention, the protein digestion enhancer is
ascorbic acid, its sodium salt, potassium salt, calcium salt,
magnesium salt or a combination thereof. In a preferred embodiment,
the protein digestion enhancer is ascorbic acid, especially
L-ascorbic acid, which has a concentration ranges from 60 ppm to
1000 ppm. Preferably, the concentration of the ascorbic acid ranges
from 60 ppm to 240 ppm, and more preferably 240 ppm. Of course, 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).
[0028] According to the present invention, the dosage form of the
above-mentioned composition includes powder, particle, tablet,
micron-particle, liquid or capsule. Preferably, the composition is
manufactured as a delayed or extended-release formula.
EMBODIMENTS
I. Experimental Methods
[0029] 1. Experiment of the Proteolysis of Casein by Pepsin
(pH=2.0)
[0030] 1.1 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.degree. 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 .mu.L of the supernatants taken from
the above-mentioned test tubes at four time points were put into
four new test tubes. 2.4 mL 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, .mu.g/mL).
[0031] 1.2 Experimental groups (60 ppm ascorbic acid): Prepare four
experimental solutions containing casein (3.85 mg/mL), 60 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0032] 1.3 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0033] 1.4 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 2.0 citric acid buffer
solution and put them into four test tubes. The four test tubes
were respectively incubated at 37.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0034] 2. Experiment of the Proteolysis of Casein by Pepsin
(pH=5.0)
[0035] 2.1 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.degree. 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 .mu.L of the supernatants taken from the
above-mentioned test tubes at four time points were put into four
new test tubes. 2.4 mL 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 the 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
TAAE (.mu.g/mL).
[0036] 2.2 Experimental groups (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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0037] 2.3 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.degree. 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 .mu.L
of the supematants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0038] 2.4 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0039] 3. Experiment of the Proteolysis of Casein by Pepsin
(pH=6.0)
[0040] 3.1 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.degree. 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 .mu.L of the supernatants taken from the
above-mentioned test tubes at four time points were put into four
new test tubes. 2.4 mL 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 the 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
TAAE (.mu.g/mL).
[0041] 3.2 Experimental groups (60 ppm ascorbic acid): Prepare four
experimental solutions containing casein (3.85 mg/mL), 60 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0042] 3.3 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 6.0 citric acid buffer
solution and put them into four test tubes. The four test tubes
were respectively incubated at 37.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0043] 3.4 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0044] 4. Experiment of the Proteolysis of Casein by Trypsin
(pH=5.0)
[0045] 4.1 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.degree. 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 .mu.L of the supernatants taken from the
above-mentioned test tubes at four time points were put into four
new test tubes. 2.4 mL 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 the fluorescence
spectrometer. The obtained fluorescence intensity was calculated as
the amount of tyrosine according to the tyrosine calibration line
established in advance at the same pH value, and represented as
TAAE (.mu.g/mL).
[0046] 4.2 Experimental groups (60 ppm ascorbic acid): Prepare four
control solutions containing casein (3.85 mg/mL), 60 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.degree. 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 .mu.L of the
supernatants taken from the above-mentioned test tubes at four time
points were put into four new test tubes. 2.4 mL 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
the fluorescence spectrometer. The obtained fluorescence intensity
was calculated as the amount of tyrosine according to the tyrosine
calibration line established in advance at the same pH value, and
represented as TAAE (.mu.g/mL).
[0047] 4.3 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0048] 4.4 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0049] 5. Experiment of the Proteolysis of Casein by Trypsin
(pH=6.0)
[0050] 5.1 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.degree. 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 .mu.L of the supernatants taken from the
above-mentioned test tubes at four time points were put into four
new test tubes. 2.4 mL 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 the fluorescence
spectrometer. The obtained fluorescence intensity was calculated as
the amount of tyrosine according to the tyrosine calibration line
established in advance at the same pH value, and represented as
TAAE (.mu.g/mL).
[0051] 5.2 Experimental groups (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.degree. 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 .mu.L of the
supematants taken from the above-mentioned test tubes at four time
points were put into four new test tubes. 2.4 mL 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
the fluorescence spectrometer. The obtained fluorescence intensity
was calculated as the amount of tyrosine according to the tyrosine
calibration line established in advance at the same pH value, and
represented as TAAE (.mu.g/mL).
[0052] 5.3 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0053] 5.4 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL).
[0054] 6. Experiment of the Proteolysis of Casein by Trypsin
(pH=7.5)
[0055] 6.1 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.degree. 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 .mu.L of the supernatants taken from the
above-mentioned test tubes at four time points were put into four
new test tubes. 2.4 mL 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 the fluorescence
spectrometer. The obtained fluorescence intensity was calculated as
the amount of tyrosine according to the tyrosine calibration line
established in advance at the same pH value, and represented as
TAAE (.mu.g/mL).
[0056] 6.2 Experimental groups (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.degree. 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 .mu.L of the
supernatants taken from the above-mentioned test tubes at four time
points were put into four new test tubes. 2.4 mL 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
the fluorescence spectrometer. The obtained fluorescence intensity
was calculated as the amount of tyrosine according to the tyrosine
calibration line established in advance at the same pH value, and
represented as TAAE (.mu.g/mL).
[0057] 6.3 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.degree. 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 .mu.L
of the supernatants taken from the above-mentioned test tubes at
four time points were put into four new test tubes. 2.4 mL 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 the fluorescence spectrometer. The obtained fluorescence
intensity was calculated as the amount of tyrosine according to the
tyrosine calibration line established in advance at the same pH
value, and represented as TAAE (.mu.g/mL). 6.4 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 7.5 citric acid buffer solution and put them into
four test tubes. The four test tubes were respectively incubated at
37.degree. 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 .mu.L of the supernatants taken from
the above-mentioned test tubes at four time points were put into
four new test tubes. 2.4 mL 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 the fluorescence
spectrometer. The obtained fluorescence intensity was calculated as
the amount of tyrosine according to the tyrosine calibration line
established in advance at the same pH value, and represented as
TAAE (.mu.g/mL).
[0058] Results
[0059] Please refer to Table 2 and FIG. 2A, which 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.
TABLE-US-00002 TABLE 2 Time (hr) pH 2.0 Group 0.1 1 2 3 Pepsin
Control -9.8.sup.a 19.3 50.6 30.7 Pepsin + 60 ppm 3.8 44.9 70.9
44.9 ascorbic acid 120 ppm 6.7 45.1 73.0 58.7 240 ppm 5.6 54.6 76.4
38.2 .sup.athe numbers shown in the above table are represented as
total amino acid equivalent (TAAE) .mu.g/mL
[0060] Please refer to Table 3 and FIG. 2B, which 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.
TABLE-US-00003 TABLE 3 Time (hr) pH 5.0 Group 0.1 1 2 3 Pepsin
Control -3.6.sup.a 31.1 31.5 38.9 Pepsin + 60 ppm 6.4 32.7 58.6
42.0 ascorbic acid 120 ppm 19.5 22.1 55.4 38.1 240 ppm 49.5 81.1
92.2 72.6 .sup.athe numbers shown in the above table are
represented as TAAE (.mu.g/mL)
[0061] Please refer to Table 4 and FIG. 2C, which 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.
TABLE-US-00004 TABLE 4 Time (hr) pH 6.0 Group 0.1 1 2 3 Pepsin
control -12.8.sup.a 7.8 19.3 10.1 Pepsin + 60 ppm 4.2 47.9 40.4
27.5 ascorbic acid 120 ppm -6.7 43.9 24.4 24.2 240 ppm 1.8 31.5
57.4 34.5 .sup.athe numbers shown in the above table are
represented as TAAE (.mu.g/mL)
[0062] Please refer to Table 5 and FIG. 3A, which 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.
TABLE-US-00005 TABLE 5 Time (hr) pH 5.0 Group 0.1 1 2 3 Trypsin
Control -2.2.sup.a 22.3 34.3 29.0 Trypsin + 60 ppm 11.5 30.4 37.8
45.2 ascorbic acid 120 ppm 15.3 38.0 39.0 49.1 240 ppm 38.9 37.5
87.9 89.3 .sup.athe numbers shown in the above table are
represented as TAAE (.mu.g/mL)
[0063] Please refer to Table 6 and FIG. 3B, which 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.
TABLE-US-00006 TABLE 6 Time (hr) pH 6.0 Group 0.1 1 2 3 Trypsin
Control 33.1.sup.a 60.9 55.0 54.0 Trypsin + 60 ppm 57.7 86.6 90.4
93.7 ascorbic acid 120 ppm 54.2 85.6 91.3 94.8 240 ppm 57.9 102.4
103.1 91.5 .sup.athe numbers shown in the above table are
represented as TAAE (.mu.g/mL)
[0064] Please refer to Table 7 and FIG. 3C, which show the
proteolytic efficiencies of trypsin (control group) and
experimental groups containing different concentrations of ascorbic
acid at pH 7.5. Because the optimal environment for the trypsin is
at pH 7.5, 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.
TABLE-US-00007 TABLE 7 Time (hr) pH 7.5 Group 0.1 1 2 3 Trypsin
Control 61.5.sup.a 79.1 90.4 69.0 Trypsin + 60 ppm 82.6 107.0 103.8
105.1 ascorbic acid 120 ppm 78.4 116.3 107.3 99.0 240 ppm 60.4 91.1
100.5 86.8 .sup.athe numbers shown in the above table are
represented as TAAE (.mu.g/mL)
[0065] While comparing the relative concentrations of the
environment of various pH values, it is found that the measured
concentration of amino acid is the highest in the optimal
environment, and the concentration gradually decreases with the
worsening of the environmental conditions, whether the pepsin or
the trypsin. Under the environment of pH 2-6, it was observed that
the ascorbic acid has a significant effect on the proteolysis
efficiency of the pepsin under the environments of pH 5, pH 6 and
pH 2, where the control group with the optimum pH (pH 2) was used
as a comparison indicator. Under the environments of pH 5-7.5, it
was observed that the ascorbic acid has a significant effect on the
proteolysis efficiency of the trypsin at the environments of pH 5,
pH 6 and pH 7.5, where of the control group with the optimum pH (pH
7.5) was used as a comparison indicator.
[0066] Because the 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.
[0067] It is understood, that this invention is not limited to the
particular embodiments disclosed, but is intended to cover all
modifications which are within the spirit and scope of the
invention as defined by the appended claims, the above description,
and/or shown in the attached drawings.
* * * * *