Field of the Invention
The present invention provides a novel proteolytic enzyme composition more particularly
an orally administered proteolytic enzyme composition comprising of one or more acid
proteases, one or more alkaline protease and one or moreplant proteases. More particularly, the
composition comprises of microbial (fungal, bacterial or other microbes) protease enzymes,
proteases from plant and animals proteases thereof.
Background of the Invention
Proteolytic enzyme, also called protease, proteinase, or peptidase, or a group of
enzymesthat break the long chain proteinsinto shorter fragments (peptides) and eventually into
their components, the amino acids. Protease enzymes isolated from microbial sources (various
strains of fungi, bacteria, etc)have been used as additives in the food processing industry for
almost a century (Underkofler, L.A., et al., “Microbiological process report- Production of
microbial enzymes and their applications,” Applied Microbiology Vol.6, page 212-221 (1958)).
Proteases/enzymes like pepsin, trypsin and pancreatin extracted from animals have been used
since decades as an aid for digestion in humans.
Proteolytic enzymes break down dietary protein and protein-based foreign bodies. They
function as digestive aids in the gut. It is well established that optimal nutrition and digestion are
paramount for improving athletic performance.In addition to health benefits, research suggests
that digestive enzyme supplementation may aid athletic performance. To increase the lean
muscle mass, athletes rely on various protein supplements to get ample amount of amino acids
which are the structural components of muscles. Whey protein is one of the popular protein
supplements among athletes and is considered one of the best sources of proteins available.
US 2004/0057944, titled “Microbial Enzyme Mixtures useful to treat digestive disorders”
describes mixture of microbial enzymes for the treatment of maldigestion in humans. The
enzyme mixture comprises of a concentrated lipase of Rhizopusdelemar, a neutral protease of
Aspergillusmelleus and an amylase of Aspergillusoryzae.
US 20080199448, titled “Enzyme Composition for improving food digestion” discloses a
combination of digestive enzymes and L-Glutamine as active ingredients which have therapeutic
effects on food absorption and digestion. The combination further comprises of a lipase, an
amylase and a protease. The portion of each enzyme is enteric coated.
3
US 5,387,422, titled “Proteolytic Fungal Enzyme Food Supplement Composition”
discloses a proteolytic enzyme food supplement composition wherein the proteolytic enzyme is
obtained from fungal source only.
Research suggests that in order to increase or maintain the lean muscle mass, protein
intake of 1.5-2.8 g/kg/day is effective for individuals. Protein intake as high as 2.8 g/kg/day (3.5
times the current recommended daily allowance) has been reported to have no adverse effects on
renal function. Supplementing the diet with processed whey proteinis one of the popular choices
to achieve these levels of daily protein consumption. Some products have a suggested per day
serving size as high as 50g of protein. However, due to inability of endogenous digestive
enzymes to digest such a large quantity of protein and their rapid transit time in the
gastrointestinal tract, approximately only 15g of the protein is digested and absorbed. The
collective effect of these factors may contribute to incomplete digestion, thereby limiting the
absorption rate of protein before it reaches the ceacum and is eliminated as waste. It stated that
the undigested protein causes issues related to indigestion such as gas bloating, belching and
sometimes vomiting and loose motions. Thus, the incomplete digestion reduces the desired
positive therapeutic effects, such as increased lean muscle mass, increased N2 retention, and
positive cardiovascular effects such as reduced levels of C-reactive protein (CRP). (Anderson, et
al., “An open label study to determine the effects of an oral proteolytic enzyme system on whey
protein concentrate metabolism in healthy males,” Journal of the International Society of Sports
Nutrition,2008, 5:10).
The objective of the present invention, is to develop a novel proteolytic enzyme
composition which increases the absorption rate of whey protein in vivo, produces a positive N2
balance and decrease the CRP Levels effectively. As a result of extensive research, the inventors
have developed a proteolytic enzyme composition which effectively increases the protein
digestibility.
Summary of the Invention
The present invention relates to a proteolytic enzyme composition. More particularly it
relates to a blend of proteolytic enzymes which can be obtained from microbial sources (fungi,
bacteria,etc), plant and animals sources and are added to protein supplements.
Accordingly, in a first aspect, the present invention provides a proteolytic enzyme
composition comprising of:
4
a) one or more of an acid protease;
b) one or more of an alkaline protease;
c) one or more of a plant protease.
In an embodiment of the above aspect the proteolytic enzyme composition comprises of:
a) an acid protease;
b) an alkaline protease;
c) a plant protease.
In another embodiment of the above aspect, the proteolytic enzyme composition
comprises of:
a) an acid protease;
b) an alkaline protease;
c) two plant proteases.
In one embodiment of the above aspect, the proteolytic enzyme composition comprises
of:
a) acid protease enzyme selected from fungal enzyme or bacterial enzyme or a
combination of both fungal enzyme and bacterial enzyme.
b) alkaline protease enzyme selected from fungal enzyme or bacterial enzyme or a
combination of both fungal enzyme and bacterial enzyme.
c) plant protease enzyme selected from kiwi, papaya, fig , mango and pineapple plants.
In another embodiment of the above aspect, the proteolytic enzyme composition
comprises of:
a) acid protease fungal enzymeselected from the group consisting of Rhizopusoryzae or
Rhizopusniveus or Aspergillusniger or Aspergillusoryzaeor the acid protease bacterial enzyme
selected from the group consisting of Bacillus subtilis or Bacillus licheniformis;
b) alkaline protease fungal enzyme selected from the group consisting of Rhizopusniveus
or Rhizopusoryzae or Aspergillusniger or Aspergillus oryzaeor alkaline protease bacterial
enzyme is selected from the group consisting of Bacillus subtilis or Bacillus licheniformis;
5
c) plant proteases selected from actinidin, papain, ficin and bromelain.
In another embodiment of the above aspect, the acid protease is present in the
concentration ranging from about 5% to about 40% w/w; alkaline protease is present in the
concentration ranging from about 20% to about 45% w/w; andplant protease enzyme is present
in the concentration ranging from about 5% to about 30% w/w of the composition.
In a further embodiment of the above aspect, the concentration of plant proteases
actinidin ranges from about 1% to about 10% w/w, the concentration of papain ranges from
about 2% to about 15% w/w, the concentration of ficin ranges from about 0.5% to about 5% w/w
and the concentration of bromelain ranges from about 7% to about 25% w/w of the composition.
In a yet another embodiment of the above aspect, the weight ratio of acid protease to
alkaline protease in the composition ranges from about 1: 0.5 to about 1: 6. The weight ratio of
acid protease to plant protease ranges from about 1:0.1 to about 1:2. The weight ratio of alkaline
protease to plant protease ranges from about 1:0.1 to about 1:2. The ratio of actinidin to ficin in
the composition ranges from about 1: 0.5 to about 1: 2 The ratio of papain to bromelain in the
composition ranges from about 1:0.5 to about 1:4. Preferably, the weight ratio of acid protease to
alkaline protease to plant protease ranges from about 1:0.5:0.1 to about 1:6:2.
In anotherembodiment of the above aspect, the proteolytic enzyme composition
mayadditionally comprise a neutral protease, a pancreatin, trypsin, pepsin or a combination
thereof. The neutral protease fungal enzyme may beselected from the group consisting of
Rhizopusoryzae or Rhizopusniveus or Aspergillusniger or Aspergillusoryzae and pancreatin,
trypsin and pepsin may beselected from the group consisting of animal pancreas and porcine
gastric mucosa.Further, the neutral protease may be present in the concentration ranging from
about 5% to about 25% w/w of the composition.The weight ratio of acid protease to neutral
protease in the composition ranges from about 1: 0.25 to about 1: 2. The concentration of
pancreatin, trypsin and pepsin (in a combination) ranges from about 1.5% to about 12% w/w of
the composition.
In yet another embodiment of the above aspect, there is provided a proteolytic enzyme
composition which provides an additional aid to endogenous enzyme system to digest the
consumed protein to the maximum extent.
6
In still another embodiment of the above aspect, the proteolytic enzyme composition
works in a broad pH range, ranging from pH2 to pH 9.
In yet another embodiment of the above aspect, the composition of the present invention
increases the digestion and bioavailability of the protein supplement by digesting protein in the
wide gastrointestinal pH range of the human digestive system.
In still another embodiment of the above aspect, the proteolytic enzyme composition is
administered orally as capsules or as powder with protein supplements.
The aforementioned aspects and embodiments, and other aspects, features and advantages
of the present invention will be apparent from the following detailed description.
Brief Description of The Drawings
Figure-1 illustrates the effect of different enzyme treatments on degree of protein hydrolysis.
Figure -2 illustrates Whey Protein Bioavailability Enhancement of IAAs.
Figure- 3 illustrates Bioavailability Enhancement of BCAAs.
Figure- 4 illustrates Average 24 hour nitrogen excretion between Treatment A/B and Control
Group.
Figure- 5 illustrates Average C-reactive protein (CRP) levels in Control Group and Treatment
A/B.
Detailed Description of the Invention
As used herein the following definitions apply unless clearly indicated otherwise. It
should also be noted that the singular forms “a” “an” and “the” include plural reference unless
the context clearly dictates otherwise.
The proteolytic enzyme composition described herein relates to a blend of proteolytic
enzymes which can be obtained from microbial sources (fungi, bacteria, etc) and plant source
and are added to protein supplements. The proteolytic enzyme composition comprises one or
more acid proteases, one or more alkaline proteases, and one or more plant proteases. All these
enzymes are the essential component of the present invention. The proteolytic enzymes from
animal origin may be additionally added. Further, the source of acid protease and alkaline
protease used, in accordance with the present invention is vital. “Protease enzyme” and
“proteolytic enzyme” are used interchangeably herein. All protease enzymes suitable for use in
7
the present invention are commercially available from known sources andcan be accessed by a
person skilled in the art.
The term “Acid Protease Enzyme” is defined as a protein-digesting enzyme, which
exhibits maximum activity and stability in acidic conditions, at pH range of 2.0 to 5.0. Acid
proteases have a low isoelectric point and are low in basic amino acids. The acid protease fungal
enzymes which can be utilized include those obtained from RhizopusoryzaeorRhizopusniveus or
AspergillusnigerorAspergillusoryzae. The acid protease bacterial enzyme which can be utilized
include those obtained from Bacillus subtilis or Bacillus licheniformis.the acid protease may be
present in the concentration ranging from about 5% to about 40% w/w, particularly from about
20% to about 40% w/w of the composition.These enzymes can be obtained commercially and
would be known to the person skilled in the art.
The term” Alkaline Protease Enzyme” refers to an endoprotease enzyme capable of
hydrolyzing broad range of peptide bonds. The alkaline protease enzymes are active at neutral as
well as alkaline pH.The alkaline protease fungal enzymes which can be utilized include those
obtained from Rhizopusniveus or Rhizopusoryzae or Aspergillusniger or Aspergillus oryzae. The
alkaline protease bacterial enzyme which can be utilized include those obtained from Bacillus
subtilis or Bacillus licheniformis. Thealkaline protease may be present in the concentration
ranging from about 20% to about 45% w/w of the composition.These enzymes can be obtained
either by known methods or can be purchased commercially from the available sources.
The term “plant protease” refers to enzymes selected from the group consisting
ofactinidin, papain, ficin and bromelain. These enzymes may be obtained from kiwi, papaya, fig
and pineapple plants respectively and actinidin is selected from the group consisting of kiwi and
mango.The plant protease enzyme may be present in the concentration ranging from about 5% to
about 30% w/w of the composition.The concentration of actinidin ranges from about 1% to about
10% w/w, the concentration of papain ranges from about 2% to about 15% w/w, the
concentration of ficin ranges from about 0.5% to about 5% w/w and the concentration of
bromelain ranges from about 7% to about 25% w/w of the composition. These enzymes can be
obtained commercially and would be known to the person skilled in the art.
The term “actinidin” refers to a proteolytic enzyme extracted from either Kiwi fruit or
Mango fruit having activity in the pH range of 6-7.
8
The term “ficin” refers to a proteolytic enzyme extracted from fig having activity in the
pH range of 5.5-7.5.
The term “papain” refers to a proteolytic enzyme extracted from papaya plant with pH
range of 3-9.
The term “bromelain” refers to a proteolytic enzyme extracted from pineapple juice and
stem and is active at pH range of 5-8.
The term” Neutral Protease Enzyme” refers to an endoprotease enzyme capable of
hydrolyzing peptide bonds. The neutral protease enzymes are active at neutral pH.The neutral
protease fungal enzymes which can be utilized include those obtained from Rhizopusniveus or
Rhizopusoryzae or Aspergillusniger or Aspergillus oryzae. The neutral protease bacterial enzyme
which can be utilized include those obtained from Bacillus subtilis or Bacillus licheniformis.
Further, the neutral protease may be present in the concentration ranging from about 5% to about
25% w/w of the composition.The weight ratio of acid protease to neutral protease in the
composition ranges from about 1: 0.25 to about 1: 2. These enzymes can be obtained either by
known methods or can be purchased commercially.
In yet another embodiment of the present invention, the composition is made by
combining an effective amount of acid protease enzyme, alkaline protease enzyme, plant
proteases, and if desired additional enzymes useful in enhancing the digestive activity of proteins
like pepsin, pancreatin and trypsin; and a pharmaceutically acceptable carrier, excipient, adjuvant
and/or vehicle.In yet another embodiment, the composition of the present invention besides the
preferred enzymes, may include but not limited to additional protease enzymes in the
composition which are preferably selected from the group consisting of keratinase, renin,
cathepsin, chymotrypsin, subtilisin, thermolysin, endoproteinase, dipase, collagenase and
combinations thereof. The concentration of pancreatin, trypsin and pepsin (in a combination)
ranges from about 1.5% to about 12% w/w of the composition.
Exemplary excipients include, for example, magnesium stearate, stearic acid, acacia gum,
fructose, modified cellulose gum, colloidal silicon dioxide, gelatin, glutens, artificial colours,
microcrystalline cellulose, dibasic calcium phosphate, aspartame, and natural flavouring agents.
Carriers may be prepared from a wide range of materials. Without being limited thereto,
such materials include diluents, binders and adhesives, lubricants, plasticizers, disintegrants,
9
colorants, bulking substances, flavouring agents, sweeteners and miscellaneous materials such as
buffers and adsorbents in order to prepare a particular composition,
The proteolytic composition further comprises a carrier material, such as maltodextrin,
dextrin, starch, calcium salts, and combinations thereof.
Examples of binding agents suitable for use in the present invention include, but are not
limited to croscarmellose sodium, povidone, dextrin, and combinations thereof.
The proteolytic enzyme compositions may preferably be designed to be ingested with
protein in order to convert the ingested dietary protein into free amino acids in the
gastrointestinal system, which then may be used by the body for muscle synthesis.
Preferably, the composition comprises a solid form such as, for example, a powder, a
tablet, a capsule, a caplet, a sachet, or an encapsulated liquid. Particularly preferably, the
composition is in the form of a capsule or powder or powder filled in a capsule.
The composition disclosed in U.S.Patent No. 5,387,422 is a blend of digestive proteases
from fungus Aspergillusniger and Aspergillusoryzae from Triarco Industries. Aspergillusniger is
an acid protease fungal enzyme and Aspergillusoryzae is a semi-alkaline protease fungal
enzyme.
The method of component incorporation for the proteolytic enzyme compositions, in
accordance with the present invention is described by way of examples. As shown by performing
an Application study for degree of protein hydrolysis using enzymes, a combination of acid
protease, alkaline protease with plant proteases are very effective in improving the protein
digestibility.
In one embodiment, the composition is to be administered orally and added in or taken
with protein supplements to improve protein digestion and make it available for muscle building
process during anabolic cycles.
According to another embodiment, the compositions works at a broad pH range, ranging
from pH 2 to pH 9. The composition is thus active in the digestion window of gastrointestinal
tract, wherein protein digestion takes place with a pH as low as pH 2 of the stomach to pH as
high as pH 8 of the intestine. The composition is thus constantly active throughout the protein
digestive window associated with human digestive system, therefore the amount of amino acid
released is increased as compared to the amount generated only by the natural digestive process.
10
According to yet another embodiment, the composition relates to increasing the digestion
and bioavailability of the protein supplement by digesting protein in the wide or broad
gastrointestinal pH range of the human digestive system.
Experimental Section
All the components were weighed. The enzymes were sieved together and blended for 20
minutes at 15 rpm. Maltodextrin, sodium starch glycolate, microcrystalline cellulose, silicon
dioxide, croscarmellose sodium were sieved and blended for 20 minutes at 15 rpm. Both the
blends were mixed. Magnesium stearate was sieved and was added to the blend. The formulation
was blended for 5 minutes at 15 rpm. Theblend was filled in capsules.
The present invention will be further described having reference to the following
examples, by way of illustration only.
Examples
Example 1:
Components Amount (mg)
Acid Protease 56.50
Alkaline Protease 90.50
Neutral Protease 14.50
Pancreatin 7.50
Papain 17.80
Ficin 2.00
Trypsin 12.00
Pepsin 20.00
Maltodextrin 15.00
Sodium Starch Glycolate 10.00
Microcrystalline Cellulose 47.70
Silicon Dioxide 1.75
Croscarmellose Sodium 2.75
Magnesium Stearate 3.00
Total 300.00
11
Example 2:
Components Amount (mg)
Acid Protease 78.00
Alkaline Protease 102.00
Papain 19.00
Ficin 6.00
Bromelain 25.00
Actinidin 13.00
Microcrystalline Cellulose 49.00
Silicon Dioxide 1.00
Croscarmellose Sodium 4.00
Magnesium Stearate 3.00
Total 300.00
Example 3:
Components Amount (mg)
Acid Protease 72.00
Alkaline Protease 93.00
Actinidin 14.00
Papain 12.00
Bromelain 24.00
Trypsin 15.00
Maltodextrin 26.00
Sodium Starch Glycolate 10.00
Microcrystalline Cellulose 29.50
Silicon Dioxide 1.50
Magnesium Stearate 3.00
Total 300.00
Example 4:
Components Amount (mg)
Acid Protease 102.00
Alkaline Protease 70.00
Neutral Protease 46.20
Papain 22.00
12
Pepsin 5.00
Maltodextrin 13.00
Microcrystalline Cellulose 32.50
Silicon Dioxide 1.80
Croscarmellose Sodium 4.50
Magnesium Stearate 3.00
Total 300.00
Example 5:
Component Amount (mg)
Acid Protease 74.00
Alkaline Protease 85.00
Neutral Protease 29.80
Actinidin 8.20
Bromelain 28.40
Trypsin 17.00
Ficin 6.00
Maltodextrin 15.60
Microcrystalline Cellulose 28.50
Silicon Dioxide 2.80
Croscarmellose Sodium 1.70
Magnesium Stearate 3.00
Total 300.00
Example 6:
Component Amount (mg)
Acid Protease 109.50
Alkaline Protease 97.00
Papain 15.00
Bromelain 28.00
Microcrystalline Cellulose 48.50
Silicon Dioxide 2.50
Croscarmellose Sodium 3.50
Magnesium Stearate 3.00
Total 300.00
13
Example 7:
Component Amount (mg)
Acid Protease 20.00
Alkaline Protease 108.00
Neutral Protease 33.00
Pancreatin 22.00
Papain 5.00
Actinidin 10.00
Trypsin 21.00
Maltodextrin 19.00
Microcrystalline Cellulose 54.50
Silicon Dioxide 1.25
Croscarmellose Sodium 3.25
Magnesium Stearate 3.00
Total 300.00
Example 8:
Component Amount (mg)
Acid Protease 86.50
Alkaline Protease 97.00
Papain 23.00
Bromelain 45.00
Microcrystalline Cellulose 40.00
Silicon Dioxide 2.50
Croscarmellose Sodium 3.00
Magnesium Stearate 3.00
Total 300.00
Example 9:
Component Amount (mg)
Acid Protease 72.00
Alkaline Protease 101.00
Ficin 15.00
Papain 15.00
Bromelain 14.50
14
Trypsin 11.00
Maltodextrin 23.50
Sodium Starch Glycolate 8.00
Microcrystalline Cellulose 35.00
Silicon Dioxide 2.00
Magnesium Stearate 3.00
Total 300.00
Example 10:
Component Amount (mg)
Acid Protease 92.00
Alkaline Protease 80.00
Neutral Protease 35.00
Papain 35.00
Pepsin 12.00
Maltodextrin 15.00
Microcrystalline Cellulose 23.50
Silicon Dioxide 1.50
Croscarmellose Sodium 3.00
Magnesium Stearate 3.00
Total 300.00
Example 11: The objectives of this study were to hydrolyze whey proteins using proteolytic
composition comprising of proteases as herein disclosed to evaluate the degree of hydrolysis
(DH) using Ninhydrin method for free alpha-amino nitrogen (FAAN).
Method:
1. About 50 g whey protein mixed with 400 ml water was incubated with enzyme
composition at 37C for 2 hours
2. Samples at 0, 15, 30, 60, 90, and 120 min were taken and filtered.(Pipette out different
volumes(0.1ml-1ml) of test/control solution to the respective labelled test tubes.)
3. Added distilled water in all the test tubes to make up the volume to 4ml.
4. Added 4ml of distilled water to the test tube labelled blank.
15
5. Ninhydrin reagent (1 mL) was added to all the test tubes including the test tubeslabelled
'blank' and 'unknown'.
6. The contents of the tubes were vortexed.
7. The mouths of the tubes were covered with aluminum foil.
8. The test tubes were placed in boiling water bath for 15 minutes.
9. The test tubes were removed from boiling water, cooled and 1ml of ethanol was added to
each test tube and mixed well.
10. The absorbance was recorded at 570nm for each solution using a colorimeter.
Experimental set up Details:
Set up Parameters Value Unit
Whey Protein 50 g
Water Added (RO) 400 ml
Batch size 450 ml
Retention time 120 minutes
Temperature 37 °C
Table 1: Effect of different enzyme treatments on degree of protein hydrolysis (example 8,
example 6, prior art product as per U. S. Patent No. 5,387,422).
Application Mode
FAAN (Free Alfa-Amino Nitrogen) in sample (ppm)
0
Minute
15
Minute
30
Minute
60
Minute
90
Minute
120
Minute
Untreated Control 190±5 190±5 190± 5 190±5 190±5 190±5
Example 6 190±5 400±5 430± 5 445±5 485±5 560±5
Example 8 190±5 415±5 420± 5 435±5 475±5 500±5
Competitor 190±5 265±5 285± 5 305±5 315±5 322±5
Based on the observed results, it can be appreciated thatExample 6 and Example 8 treated whey
protein samples showed higher degree of protein hydrolysis and filtration rate as compared to the
prior artproduct(Figure-1).
Example 12:

A study was performed in 24 healthy, lean, males age 20–36 years, each with a body
mass index (BMI) ranging from 20 to 24 under fasting conditions. The study was approved by
Independent Ethics Committee (IEC) and was enrolled with the Clinical Trials Registry – India
(CTRI/2019/02/017731). Each subject was pre-informed of the purpose, methods, and probable
risks associated with the study, and informed consent was signed by each participant.
All the subjects were divided into three equal groups comprising eight subjects in each
group. The groups were assigned to either Treatment-A or Treatment-B or Control Group. All
subjects received a 50 gram whey protein with 360 mL water either alone (Control Group) or
with treatment A (One capsule containing the proteolytic enzyme compositionas per Example
8,(commercially available as MB EnzymePro®
) or treatment B [Two capsules of MB
EnzymePro®
] after an overnight fasting of at least 12 hours. All the participants followed a
specified, balanced diet of 2200 Kcal/day during the stay at the clinic facility.
The objective of this clinical study was to establish, if MB EnzymePro®
(Enhanced
Absorption Formula, EAFTM) would significantly affect the amount of whey proteinmetabolized
in-vivo and if any effect would be adequate to significantly alter nitrogen (N2) balance and Creactive protein (CRP) levels as compared to the control group. Comparing levels of total serum
amino acids (TSAA) after ingestion, with and without MB EnzymePro®
, would be an indicator if
the composition of the invention was effective in increasing the amount of digested and absorbed
whey protein.
TSAA levels
Analysis of TSAA kinetic profiles of all treatment groups showed a progressive, timedependent increase for about 1.5 hours and then a constant decrease till 4 hours. Statistical
comparison of means between treatment groups and control group showed differences between
groups and all time points to be significant (p = 0.05). The results demonstrated that
postprandial TSAA levels were significantly increased over control from a 50gram dose of whey
protein co-administered with MB EnzymePro®
. This showed that supplementation with MB
EnzymePro®
in treatment A and B increased the digestion of whey protein leading to increase in
its digestion and absorption rate and is additionally supported by significant (1.53 and 1.57
times) increases in the sum of area under the curve (AUC) of individual amino acids (IAAs)
(Figure-2) as compared to the control group. After baseline subtraction, no significant
17
differences were found between the AUC of each treatment A and B representing that the WP
absorption rate may be close to maximum in treatment A.
Individual amino acid levels
There was an overall increase in individual amino acids (IAA) level for the seventeen
amino acids analysed in the treatment groups. There were statistically significant differences
between control and treatment groups for cysteine, isoleucine, leucine, lysine, phenylalanine,
proline, threonine, tyrosine, methionine and valine (10 of 17). Alanine, glycine, histidine,
arginine aspartic acid, glutamic acid, and serine were the amino acids that were not significantly
different from the control group. The sum of AUC was significantly greater in each treatment
group as compared to the control group (p = 0.05). There was a significant increase of 163 %
and 167 % in the AUC of branched amino acids (BCAAs) (sum of AUC of isoleucine, leucine
and valine) for treatment group A and B respectively as compared to the control group (Figure3).
MB EnzymePro® had a profound increase in the level of amino acids especially the
BCAAs. BCAAs are of special importance for bodybuilders and athletes because these amino
acids are used by the body to build up the protein for muscle synthesis and muscle repair.
BCAAs are essential amino acids that are not produced by the body, therefore, they must be
acquired from the diet or from nutritional supplements. BCAAs are metabolized in the muscle
rather than in the liver. Consequently, the effect of these BCAAs is much quicker and efficient
than that of any other amino acid. If the diet is balanced, BCAAs will be used for protein
synthesis, essential for endurance athletes and strenuous workouts. BCAAs are used to reduce
fatigue in both anaerobic and endurance sports.
Nitrogen excretion
The average amount of N2 excreted in 24 hours was determined as urea for both
treatment groups and the control group. In the treatment groups A and B, the average amount of
N2 excreted over 24 hours was 7.65 g and 7.60 g respectively. In the control group, the average
amount of N2 excreted over 24 hours was 9.59 g (Figure-4). The total nitrogen excretion
decreased significantly for both treatment groups A and B with MB EnzymePro®
. This array of
evidences offers additional support that whole body protein metabolism was decreased in each
treatment group. These findings also suggest increased protein utilization.
C-reactive protein
CRP was measured at 0.0 hour and at 4 hours in each treatment group and control group
Significant reductions(p = 0.003) were found between 0.0 hour and 4 hour in each treatment
group as compared to the control group. Treatment group A showed a reduction of 16.15% and
treatment group B showed a reduction of 23.61% as compared to the control group at 4 hours
(Figure 5). This may be related to the differences in the amount of whey proteindigested and
absorbed between the control group and treatment groups. Supplementing whey proteinwith MB
EnzymePro® may contribute to significantly reducing CRP levels by producing bioactive
peptides in-vivo.

We claim:
1. A proteolytic enzyme composition comprising of:
a) one or more of an acid protease;
b) one or more of an alkaline protease;
c) one or more of a plant protease.
2. The proteolytic enzyme composition according to claim 1, wherein the proteolytic
enzyme composition comprises of:
a) an acid protease;
b) an alkaline protease;
c) two plant proteases.
3. The proteolytic enzyme composition according to claim 1, wherein the proteolytic
composition comprises:
a) acid protease enzyme selected from fungal enzyme or bacterial enzyme or a
combination of both fungal enzyme and bacterial enzyme.
b) alkaline protease enzyme selected from fungal enzyme or bacterial enzyme or
a combination of both fungal enzyme and bacterial enzyme.
c) plant protease enzyme selected from kiwi, papaya, fig , mango and pineapple
plants.
4. The proteolytic enzyme composition according to claim 3, wherein the proteolytic
composition comprises:
a) acid protease fungal enzymeselected from the group consisting of
Rhizopusoryzae or Rhizopusniveus or Aspergillusniger or Aspergillusoryzaeor
the acid protease bacterial enzyme selected from the group consisting of
Bacillus subtilis or Bacillus licheniformis;
b) alkaline protease fungal enzyme selected from the group consisting of
Rhizopusniveus or Rhizopusoryzae or Aspergillusniger or Aspergillus
oryzaeor alkaline protease bacterial enzyme is selected from the group
consisting of Bacillus subtilis or Bacillus licheniformis;
c) plant proteases selected from actinidin, papain, ficin and bromelain.
5. The proteolytic enzyme composition according to claim 4, wherein the proteolytic
composition comprises acid protease in the concentration ranging from about 5% to about
20
40% w/w; alkaline protease in the concentration ranging from about 20% to about 45%
w/w; and plant protease enzyme in the concentration ranging from about 5% to about
30% w/w of the composition.
6. The proteolytic enzyme composition according to claim 4, wherein the concentration of
plant proteases actinidin ranges from about 1% to about 10% w/w; the concentration of
papain ranges from about 2% to about 15% w/w;the concentration of ficin ranges from
about 0.5% to about 5% w/w and the concentration of bromelain ranges from about 7% to
about 25% w/w of the composition.
7. The proteolytic enzyme composition according to claim 4, wherein the weight ratio of
acid protease to alkaline protease in the composition ranges from about 1: 0.5 to about 1:
6; the weight ratio of acid protease to plant protease ranges from about 1:0.1 to about 1:2;
the weight ratio of alkaline protease to plant protease ranges from about 1:0.1 to about
1:2; the ratio of actinidin to ficin in the composition ranges from about 1: 0.5 to about 1:
2; the ratio of papain to bromelain in the composition ranges from about 1:0.5 to about
1:4.
8. The proteolytic enzyme composition according to claim 4, wherein the weight ratio of
acid protease to alkaline protease to plant protease ranges from about 1:0.5:0.1 to about
1:6:2.
9. The proteolytic enzyme composition according to claim 1, wherein the proteolytic
enzyme composition additionally comprisesenzymes selected from thegroup consisting of
neutral protease, a pancreatin, trypsin, pepsin or a combination thereof.
10. The proteolytic enzyme composition according to claim 1, wherein the proteolytic
enzyme composition works in the pH rangeof pH 2 to pH 9.
11. The proteolytic enzyme composition according to claim 1, wherein the composition
increases the digestion and bioavailability of the protein supplement by digesting protein
in the wide gastrointestinal pH range of the human digestive system.
12. The proteolytic enzyme composition according to claim 1, wherein the proteolytic
enzyme composition is administered orally as capsules or as powder with protein
supplements.