(12) MALAYSIAN PATENT (11) MY-195701-A

(21) Application No. : PI2020000218 (56) Prior Art:

CHANG SUI KIAT ET AL: "Antioxidant
(22) Filing Date : 14 JANUARY 2020 peptides purified and identified from the
oil palm (Elaeis guineensis Jacq.) kernel
(47) Date of Publication and Grant: protein hydrolysate", Journal of
06 February 2023 Functional Foods, April 2015, vol. 14,
pages 63-75, DOI:
(30) Priority Data : 10.1016/j.jff.2015.01 .011 *see whole
None document
NG. K. L. ET AL: "Enzymatic
(51) Classification, INT CL : preparation of palm kernel expeller
A61K 36/889 protein hydrolysate (PKEPH)",
A61K 38/01 International Food Research Journal,
2012, vol. 19, no. 2, pages 721-725
(73) Patent Owner: *see whole document
MALAYSIAN PALM OIL BOARD LAU BENJAMIN Yll CHUNG ET AL:
No. 6, Persiaran Institusi, "Method development to extract proteins
Bandar Baru Bangi, from oil palm chromoplast for proteomic
43000 Kajang Selangor analysis", SpringerPlus, 2015, vol. 4, no.
Malaysia 791, DOI: 10.1186/s40064-015-1576-4
*see whole document
(74) Agent: WO 2014/189356 A1 (SIME DARBY
Mohana Murali A/L Kodivel MALAYSIA BERHAD) 27 November
Adastra Intellectual Property Sdn. Bhd. 2014 *see whole document

(72) Inventors :
Benjamin Lau Yii Chung
Abrizah Othman

(54) Title : Method of Extraction of Bio-Active Peptides From Oil Palm Mesocarps

(57) Abstract:

The present embodiment relates to a method of extraction of a plurality bio-active peptides

from oil palm materials such as mesocarps. The method of extraction includes the extraction
of a total protein by precipitation of the total protein using phenol and methanol containing
ammonium acetate. The total protein is hydrolyzed by using pepsin or trypsin, wherein the
enzyme hydrolysis generates the plurality of bio-active peptides. The plurality of bio-active
peptides are fractionated and the sequence of the plurality of bio-active peptides is
determined. The plurality of bio-active peptides possess anti-oxidant and anti-fungal
properties. Fig. 1

Fig. 1
 1

METHOD OF EXTRACTION OF BIO-ACTIVE PEPTIDES FROM OIL PALM

MESOCARPS

FIELD OF THE INVENTION

5
The present embodiment relates to a method of protein extraction, and more
particularly relates to a method of extraction of bio-active peptides from the
mesocarps of oil palm.

10 BACKGROUND OF INVENTION

Palm mesocarp oil is one of the largest revenue for countries like Malaysia and
Indonesia. Oil palm is an incredibly efficient crop, producing more oil per land area
than any other equivalent vegetable oil crop. Globally, palm oil supplies 35% of the
15 world’s vegetable oil demand on just 10% of the land. Oil palm is also known to
possess anti-microbial and anti-oxidant properties.

Currently, various methods are being employed for the extraction of bio-active
peptides from the mesocarps of oil palm by using a combination of approaches like
20 chemical and enzymatic hydrolysis. Also, there are many ongoing studies for
identifying the sequence of the proteins, known to confer anti-microbial and anti­
oxidant properties to the oil palm. However, researchers are unable to identify the
sequence conferring the anti-microbial and anti-oxidant properties with the bio-active
peptides.
25
Therefore, it is required to extract and identify the peptides conferring the anti­
microbial and anti-oxidant properties to the oil palm.

SUMMARY OF THE INVENTION

30
In view of the foregoing, the embodiment herein provides a method for extraction
and identification of the bio-active peptides from the mesocarps of oil palm.

In an aspect, a method for extraction and isolation of the plurality of bio-active

35 peptides is provided. The method for the extraction and isolation of the plurality of
 2

bio-active peptides includes: extracting a total protein from oil palm mesocarps by
using combination of solvent washes and phenol extraction; hydrolyzing the total
protein using enzymes such as trypsin and pepsin but not limited to, for the
production of the plurality of bio-active peptides; fractionation and isolation of the
5 plurality of bio-active peptides through sequential elution with C18 membrane and
acetonitrile. The plurality of bio-active peptides are sequenced to identify the amino
acid sequence. The plurality of bio-active peptides are sequenced to have a
sequence ID No.: 1-8.

10 The extraction of the total protein from the oil palm mesocarps comprises
homogenizing and washing the oil palm mesocarps in trichloroacetic acid and
dithiothreitol. The homogenized oil palm mesocarps are further centrifuged, washed
with acetone and methanol containing ammonium acetate. The oil palm mesocarp is
further re-suspended in an extraction buffer, sonicated and is mixed with equal
15 volume of tris-saturated phenol. The homogenized oil palm mesocarps are then
centrifuged and the oil palm mesocarp total protein is recovered in the phenol
phase. The total protein is precipitated by using ammonium acetate-saturated
methanol. The total protein obtained, is further hydrolyzed by using trypsin and
pepsin hydrolysis for generating the plurality of bio-active peptides having sequence
20 ID No.: 1-8. The plurality of bio-active peptides are purified with C18 membrane and
fractionated using acetonitrile. The plurality of bio-active peptides are analyzed and
assayed to possess anti-fungal and anti-oxidant properties.

The preceding is a simplified summary to provide an understanding of some aspects

25 of embodiments of the present invention. This summary is neither an extensive nor
exhaustive overview of the present invention and its various embodiments. The
summary presents selected concepts of the embodiments of the present invention in
a simplified form as an introduction to the more detailed description presented
below. As will be appreciated, other embodiments of the present invention are
30 possible utilizing, alone or in combination, one or more of the features set forth
above or described in detail below.

35
 3

BRIEF DESCRIPTION OF THE DRAWINGS

The above and still further features and advantages of embodiments of the present
invention will become apparent upon consideration of the following detailed
5 description of embodiments thereof, especially when taken in conjunction with the
accompanying drawings, and wherein:

Figure 1 illustrates a method (100) for extraction and isolation of a plurality of bio­
active peptides, according to an embodiment herein;
10
Figure 2 illustrates an anti-oxidant activity of the plurality of bio-active peptides,
according to an embodiment herein; and

Figure 3 illustrates an anti-fungal activity of the plurality of bio-active peptides,

15 according to an embodiment herein.

DETAILED DESCRIPTION

As used throughout this application, the word "may" is used in a permissive sense
20 (i.e., meaning having the potential to), rather than the mandatory sense (i.e.,
meaning must). Similarly, the words “include”, “including”, and “includes” mean
including but not limited to.

The phrases “at least one”, “one or more”, and “and/or” are open-ended expressions
25 that are both conjunctive and disjunctive in operation. For example, each of the
expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of
A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” means A alone, B
alone, C alone, A and B together, A and C together, B and C together, or A, B and C
together.
30
The term “a” or “an” entity refers to one or more of that entity. As such, the terms “a”
(or “an”), “one or more” and “at least one” can be used interchangeably herein. It is
also to be noted that the terms “comprising”, “including”, and “having” can be used
interchangeably.
35
 4

Oil palm mesocarps are used for the extraction and isolation of a plurality of bio­
active peptides as disclosed herein. As used herein, the term “mesocarp” refers to
the pulp of the fruit of the oil palm. In an embodiment, the ripe oil palm mesocarps
are used for the extraction and isolation of the plurality of bio-active peptides. The oil
5 palm mesocarps are sliced and stored in liquid nitrogen for the extraction of the
plurality of bio-active peptides.

Figure 1 illustrates a method (100) for the extraction and isolation of the plurality of
bio-active peptides from the oil palm mesocarp. The method includes extracting
10 (102) total amount of protein from the oil palm mesocarp, hydrolyzing (104) the total
protein as extracted for producing the plurality of bio-active peptides, purification and
fractionation of the plurality of bio-active peptides, sequencing the plurality of bio­
active peptides.

15 At step 102, a total amount of all proteins is extracted and precipitated from the oil
palm mesocarps. The oil palm mesocarps contain a diverse group of secondary
compounds like, but not limited to, phenolics, lipids, pigments, organic acids and
carbohydrates. The total protein of the oil palm mesocarps is separated from the
secondary compounds for the extraction of the plurality of bio-active peptides.
20
In an embodiment, the total protein is extracted by using an organic compound and
by using physical methods like agitation and sonication. In an embodiment, the total
protein from the oil palm mesocarps is extracted by employing, but not limited to,
trichloroacetic acid (TCA)-acetone precipitation, tris buffered phenol extraction and
25 hybrid technique of TCA-acetone/phenol-SDS.

In a preferred embodiment, the total protein from the oil palm mesocarps, but not
limited to, is washed with trichloroacetic acid containing dithiothreitol, acetone and
methanol containing ammonium acetate. Trichloroacetic acid reduces the
30 electrostatic forces and exposes the hydrophobic structure of the proteins. Acetone
reduces the solubility of the protein and dithiothreitol reduces the di-sulfide bonds of
the proteins. In an embodiment, the oil palm mesocarps are homogenized by
grinding and sonication in the extraction buffer having sucrose, tris-HCl, NaCl,
dithiothreitol (DTT), EDTA and Roche protease inhibitors. The oil palm mesocarp
35 pellet re-suspension is sonicated and filtered for removing the non-macerated plant
 5

material. In an embodiment, the oil palm mesocarp pellet re-suspension is filtered

through, but not limited to, a membrane and a filter paper. The oil palm mesocarp
pellet re-suspension is mixed with tris-saturated phenol having a pH in the range of
7-9. The oil palm mesocarp pellet re-suspension is centrifuged and the total protein
5 in the upper phase is precipitated by using cold ammonium acetate-saturated
methanol. The mixture is incubated for a night. In an embodiment, the total protein
pellet is incubated for a night at a temperature of -10°C to -30°C. The precipitated
total protein is centrifuged and the supernatant is discarded. The total protein pellet
is washed with ammonium acetate-saturated methanol and acetone. The total
10 protein pellet is air dried and used for the protein hydrolyzation for generating the
plurality of bio-active peptides.

At step 104, the total protein is hydrolyzed for generating the plurality of bio-active
peptides. In an embodiment, the hydrolysis of the total proteins can be done by
15 chemical and enzymatic methods. In an embodiment of the present disclosure, the
hydrolysis of the total protein is carried out by the enzymatic method. In an
embodiment, the enzyme hydrolyzes the proteins at the optimum temperature and
pH and target specific peptide cleavage bond, resulting in the generation of peptides
of varying size.
20
In a preferred embodiment, the hydrolysis of the proteins is carried out by
solubilizing the total protein pellet in a buffer and by the addition of enzymes. The
total protein pellet obtained from step 102 is solubilized in the buffer having tris-HCl
and KCl and an enzyme selected from a group of trypsin and pepsin. The
25 solubilization of the total protein pellet in the buffer and the enzymes generates a
total protein mix.

In an embodiment, the buffer has a pH in the range of 7-9. In an embodiment, the

enzymes used for the hydrolysis of the total protein are proteolytic enzymes. In an
30 embodiment, the enzymes are modified with customized cleavage sites for targeting
the specific cleavage bonds of the total protein. In an embodiment, the cysteine
residues in the total protein is modified and carboxymethylated. The modification of
the cysteine residues prevents the formation of disulfide bonds. In another
embodiment, the methionine is oxidized, amide group of asparagines and glutamine
35 is removed or converted into other functional group (deamidation). In yet another
 6

embodiment, the acetyl N-terminal modifications are carried out in the total protein.
The modifications effectively enhance the hydrolysis reaction of the total protein by
breaking the total protein into the plurality of bio-active peptides.

5 The total protein mix is incubated and agitated for the effective hydrolysis of the total
protein. The enzymes break the peptide bonds of the total protein and generate the
plurality of bio-active peptides. The total protein hydrolysis is terminated by heating
the total protein mix at a higher temperature. In an embodiment, the total protein
hydrolysis is terminated by heating the total protein mix at a temperature in the
10 range of 90-110°C for duration of 5-15 minutes. In an embodiment, the heating of
the total protein pellet mix inactivates the enzymes. In an embodiment, the heating
of the total protein mix inactivates the enzymes and the hydrolysis reaction is
terminated.

15 At step 106, the plurality of bio-active peptides are extracted and isolated by
fractionation. In an embodiment, the plurality of bio-active peptides may be
fractionated by using diverse techniques like, but not limited to, sequential
fractionation, chromatographic techniques and subcellular fractionation. In an
embodiment of the present disclosure, the plurality of bio-active peptides are
20 fractionated and isolated by using C18 membrane and sequential elution using
acetonitrile. In an embodiment, C18 disk columns are used in the reverse phase
liquid chromatography. In an embodiment, C18 disks in the reverse phase liquid
chromatography constitute the stationary phase.

25 The plurality of bio-active peptides are separated by liquid chromatography coupled

to the mass spectrometry (LC-MS). The liquid chromatography coupled to the mass
spectrometry (LC-MS) is used for the identification of polar, high molecular weight
organic compounds and allows semi-quantitative determination of concentrations.
The liquid chromatography separates the organic compounds on the basis of
30 differential retention with a stationary phase and a mobile phase. In a preferred
embodiment, reverse phase liquid chromatography is employed for the separation
and identification of the plurality of bio-active peptides.

The plurality of bio-active peptides are extracted and isolated by using reverse
35 phase liquid chromatography. The reverse phase liquid chromatography column is
 7

equilibrated with formic acid and acetonitrile. In an embodiment, the formic acid and
the acetonitrile constitute the mobile phase.

The fractions obtained from step 104 having the plurality of bio-active peptides are
5 loaded into the reverse phase liquid chromatography column. The elution rate of the
reverse phase liquid chromatography columns is adjusted. In an embodiment, the
term “elution rate” as used herein refers to the volume of mobile phase passed
through the reverse phase liquid chromatography column per minute. The plurality
of bio-active peptides are retained and separated based on their interaction with the
10 stationary phase and the mobile phase.

The fractions of the plurality of bio-active peptides obtained from the reverse phase
liquid chromatography are ionized using electrospray ion source. Every fraction of
the plurality of bio-active peptides is converted into ions by electrospray ionization.
15 In an embodiment, the separated plurality of bio-active peptides are converted into
ions by using a spray voltage of 1800-2500 Volts. In an embodiment, every fraction
of the plurality of bio-active peptides is a charged species.

The charged species are analyzed with quadrupole and orbitrap mass analyzers
20 based on mass to charge ratio (m/z). The resolving power and m/z isolation window
are adjusted in the mass spectrometer. The plurality of bio-active peptides having a
mass in the range of 110-1800 and a charge of 2-7 are analyzed in the mass
spectrometer.

25 At step 108, the plurality of bio-active peptides obtained from step 106 are
sequenced for identifying the particular sequence of the amino acids in the plurality
of bio-active peptides. In an embodiment, the plurality of bio-active peptides can be
sequenced by various methods, but not limited to, Sanger’s method, Edman
degradation, peptide mass fingerprinting, de novo sequencing and shotgun
30 sequencing. In a preferred embodiment, the sequence of the plurality of bio-active
peptides are determined by using shotgun sequencing. The plurality of bio-active
peptides are broken down into smaller fragments and the sequence of the plurality
of bio-active peptides is determined by reading the overlapping sequences.
 8

The plurality of bio-active peptides obtained from the trypsin hydrolysis have the
sequence ID No.: 1-4. The sequence of the plurality of bio-active peptides obtained
from the trypsin hydrolysis are:

5 Sequence ID No.: 1
EPHPNEFVGLM

Sequence ID No.: 2
SPPEQLGKSFNF
10
Sequence ID No. 3
SIWGDIGQGVGKAAYWVGKAMGNMSDVNQASRINRKKKH

Sequence ID No.: 4
15 NKGCAICSIGAACLVDGPIPDFEIAGATGLFGLWG

The sequence of the plurality of bio-active peptides obtained from the trypsin has
amino acid residues in the range of 11-39. The plurality of bio-active peptides having
sequence ID No.: 1-4 have hydrophobic amino acid residues. The plurality of bio­
20 active peptides having sequence ID No.:1 have 36% hydrophobic amino acid
residues. The plurality of bio-active peptides having sequence ID No.: 2 have 23%
hydrophobic amino acid residues. The plurality of bio-active peptides having
sequence ID No.:3 have 35% hydrophobic amino acid residues. The plurality of bio­
active peptides having sequence ID No.:4 have 54% hydrophobic amino acid
25 residues.

The plurality of bio-active peptides obtained from the pepsin hydrolysis have the
sequence ID No.: 5-8. The sequence of the plurality of bio-active peptides obtained
from the pepsin hydrolysis are:
30
Sequence ID No.:5
RMRRSKSGKGSGGSKGSGSKGSKGSKGSGSKGSGSKGGSRPGGGSSIAGGGS
KGKGGTOTA

35
 9

Sequence ID No.:6
CLAGRLDKQCTCRRSQPSRRSGHEVGRPSPHCGPSRQCGCHMD

Sequence ID No.:7
5 RQRDPQQQYEQCQKHCQRRETEPRHMQTCQQRCERRYEKEKRKQQKRYEEQQ
REDEEKYEERMKEEDN

Sequence ID No.:8
KRDPQQREYEDCRRRCEQQEPRQQHQCQLRCREQQRQHGRGGDMMNPQRGG
10 SGRYEEGEEEQS

The sequence of the plurality of bio-active peptides obtained from the pepsin has
amino acid residues in the range of 48-68. The plurality of bio-active peptides having
sequence ID No.: 5-8 have repeating amino acid residues. The plurality of bio-active
15 peptides having sequence ID No.:5 have 6% hydrophobic amino acid residues. The
plurality of bio-active peptides having sequence ID No.:6 have 25% hydrophobic
amino acid residues. The plurality of bio-active peptides having sequence ID No.:7
have 8% hydrophobic amino acid residues. The plurality of bio-active peptides
having sequence ID No.:8 have 11% hydrophobic amino acid residues. The plurality
20 of bio-active peptides having sequence ID No.: 5-8 have lower hydrophobic activity
than the plurality of bio-active peptides having sequence ID No.: 1-4.

EXAMPLES

25 EXAMPLE 1: EXTRACTION OF TOTAL PROTEINS

The oil palm mesocarps were sliced and stored in liquid nitrogen at a temperature of
-80°C. The oil palm mesocarps were washed by using acetone, 5-15%
trichloroacetic acid and 0.5-1.5 mM dithiothreitol, generating delipidated mesocarps.
30 The mesocarps were further mixed with 50-100% methanol containing 0.05-0.5 M
ammonium acetate. The mixture was then centrifuged at 10,000-15,000 g for a
duration of 5-15 minutes at a temperature of 2-6 °C. The supernatant was discarded
and the mesocarp pellet was washed with 50-100% acetone. The oil palm mesocarp
pellet was further centrifuged at 10,000-15,000 g for a duration of 5-15 minutes at a
35 temperature of 2-6 °C. The mesocarp pellet was re-suspended in the extraction
 10

buffer having 0.5-1.5 M sucrose, 0.05-1.5 M tris-HCl, 1-10 M NaCI, 25-100 mM

dithiothreitol (DTT), 0.5-1.5 mM EDTA and 1-2 tablets of Roche protease inhibitors.
The oil palm mesocarp pellet was sonicated and was filtered for removing the non­
macerated plant material. An equal volume of fresh 25-75 mM tris-phenol was
5 added to the oil palm mesocarp pellet re-suspension. The oil palm mesocarp pellet
re-suspension was centrifuged at 10,000-20,000 g for a duration of 10-20 minutes at
a temperature of 2-6°C. The total proteins in the upper phase were precipitated by
using 1-10 volumes of cold ammonium acetate-saturated methanol. The precipitated
total proteins were again centrifuged at 10,000-20,000 g for a duration of 10-20
10 minutes at a temperature in the range of 2-6°C and the supernatant was discarded.
The oil palm mesocarp pellet was separated and incubated for a night at a
temperature of -10°C to -30°C. The total protein pellet was washed 1-5 times with
acetone having a concentration of 50-100%. The total protein pellet was then air
dried and used for the protein hydrolyzation for generating the plurality of bio-active
15 peptides.

EXAMPLE 2: PROTEIN HYDROLYZATION

The total protein pellet was solubilized in 100-1000 μL buffer having 10-100 mM tris-
20 HCl. An enzyme having a concentration 0.5-50 mg, in a buffer having a pH in the
range of 1-5 was added to the total protein pellet. The enzymes having a
concentration of 0.5-50 mg were added to the total protein pellet, generating a total
protein mix. The enzymes had a pH in the range of 1-5. The total protein pellet mix
was incubated for a duration of 6-48 hours with an agitation rate of 100-500 rpm for
25 effectively carrying out the hydrolysis of the total proteins. The total protein
hydrolysis was terminated by heating the total protein pellet mix at a temperature of
90-110°C for a duration of 5-15 minutes.

In an embodiment, trypsin having a concentration of 0.5-50 mg is added to the total

30 protein pellet generating a total protein pellet mix. In an embodiment, trypsin has a
pH in the range of 1-5.

In other embodiment, pepsin having a concentration of 0.5-50 mg is added to the

total protein pellet generating the total protein pellet mix. In an embodiment, pepsin
35 has a pH in the range of 1-5.
 11

EXAMPLE 3: FRACTIONATION AND ISOLATION

The plurality of bio-active peptides were purified and fractionated with C18
membrane and sequential elution with 2-100% acetonitrile. The plurality of bio-active
5 peptides were further separated with a reverse phase liquid chromatography column
comprising 50-100% acetonitrile and 0.05-0.15% formic acid. The elution rate of the
reverse phase liquid chromatography column was adjusted at 200-350 nL min-1. The
plurality of bio-active peptides were converted into a charged species by passing
through an electrospray ion source. Ions were generated by electrospray ionization
10 using a spray voltage of 1800-2500 V. The charged species were fed and analyzed
by the mass spectrometer. The mass spectrometer was adjusted to have a resolving
power of 50,000-70,000 and m/z isolation window of 0.5-2.0. The plurality of bio­
active peptides having a mass in the range of 110-1800 and a charge of 2-7 were
analyzed in the mass spectrometer.
15
EXAMPLE 4: SEQUENCING OF PLURALITY OF BIO-ACTIVE PEPTIDES

The plurality of bio-active peptides were sequenced by shotgun sequencing and the
sequence of the plurality of bio-active peptides were identified using software. The
20 plurality of bio-active peptides were sequenced to have sequence ID No.: 1-8.

EXAMPLE 5: ANTI-OXIDANT ASSAY

The plurality of bio-active peptides were analyzed for the presence of anti-oxidant
25 activity. The anti-oxidant activity was analyzed by assessing 2, 2-Diphenyl-1-
picrylhydrazyl (DPPH) free radical scavenging activity assay. The 1-100 ml of DPPH
solution was prepared by mixing 0.5-1.5 mg DPPH and 10-100% methanol. In an
embodiment, DPPH constitutes a blank sample for analyzing the anti-oxidant activity
of the plurality of bio-active peptides. A gallic acid having a concentration of 0-10
30 μg/mL is used as an assay standard solution. The assay standard solution was
prepared by mixing 5-30 mg gallic acid and 1-10 ml double distilled water. 5-25 μL
of the assay standard solution and the 5-25 μL of plurality of bio-active peptides
samples were mixed with DPPH and absorbance was measured at 505-520 nm
after an interval of every 10-25 seconds. In an embodiment, the absorbance of the
35 blank solution was represented by the symbol ABSblank. In an embodiment, the
 12

ABSblank was the absorbance value of the blank solution at a time interval of 195
seconds. In an embodiment, the absorbance of the plurality of bio-active peptides
was represented by the symbol ABSsample.

5 The antioxidant activity of the plurality of bio-active peptides was expressed as

percentage of DPPH free radical scavenging activity. In an embodiment, the
percentage of DPPH free radical scavenging activity was represented by the symbol
%DPPHSC. The percentage of DPPH free radical scavenging activity was calculated
by using the formula:
10

%DPPHsc = (ABSblank - ABSsample) / (ABSblank X 100%)

The graph indicating the antioxidant activity of the plurality of bio-active peptides
was plotted (shown in figure 2).
15
In an embodiment, the plurality of bio-active peptides obtained from the pepsin
hydrolysis have greater anti-oxidant property than the plurality of bio-active peptides
obtained from the trypsin hydrolysis. In an embodiment, the plurality of bio-active
peptides obtained from pepsin hydrolysis have a scavenging activity of 22.3% after
20 3 minutes (shown in figure 2). In other embodiment, the plurality of bio-active
peptides obtained from trypsin hydrolysis have a scavenging activity of 17.3% after
3 minutes (shown in figure 2).

EXAMPLE 6: ANTI-FUNGAL ASSAY

25
The plurality of bio-active peptides were analyzed for the presence of anti-fungal
activity against a fungus, Ganoderma boninense. 5-10 μL of the plurality of bio­
active peptides was mixed with 50-150 μL of potato dextrose broth in the micro-titer
plate. In an embodiment, the potato dextrose broth was prepared by suspending
30 sliced, unpeeled potatoes in distilled water. The mixture was filtered and was mixed
with dextrose and was autoclaved for duration of 15 minutes. pH of the potato
dextrose broth was adjusted by using tartaric acid. The plug of the fungus
Ganoderma boninense having a diameter of 2.5-7.5 was added to the micro-titer
plate. The micro-titer plate was incubated in dark at a temperature of 25-30°C and
35 the growth of the fungus was observed. In an embodiment, the plurality of bio-active
 13

peptides inhibit the growth of Ganoderma boninense by disrupting the cell wall
structure of the fungal membranes.

The plurality of bio-active peptides obtained from trypsin hydrolysis having sequence
5 ID No.: 1-4 possess anti-fungal properties. The eluted fractions 25% and 35% of the
trypsin hydrolysis inhibit the growth of Ganoderma boninense at day 2 and 3 (shown
in figure 3). The plurality of bio-active peptides obtained from pepsin hydrolysis
having sequence ID No.: 6-8 possess anti-fungal properties. The eluted fractions
10% and 30% of the pepsin hydrolysis inhibit the growth of Ganoderma boninense
10 at day 2 and 3 (shown in figure 3).

In an embodiment, the present embodiment provides the plurality of bio-active

peptides having anti-oxidant and anti-fungal properties. In an embodiment, the
plurality of bio-active peptides having sequence ID No. 1-8 are used in formulating
15 compositions for the inhibition of fungal and microbial growth. In an embodiment, the
plurality of bio-active peptides having sequence ID No.: 1-8 are used for preventing
the damage due to free radicals.

The foregoing discussion of the present invention has been presented for purposes
20 of illustration and description. It is not intended to limit the present invention to the
form or forms disclosed herein. In the foregoing Detailed Description, for example,
various features of the present invention are grouped together in one or more
embodiments, configurations, or aspects for the purpose of streamlining the
disclosure. The features of the embodiments, configurations, or aspects may be
25 combined in alternate embodiments, configurations, or aspects other than those
discussed above. This method of disclosure is not to be interpreted as reflecting an
intention the present invention requires more features than are expressly recited in
each claim. Rather, as the following claims reflect, inventive aspects lie in less than
all features of a single foregoing disclosed embodiment, configuration, or aspect.
30 Thus, the following claims are hereby incorporated into this Detailed Description,
with each claim standing on its own as a separate embodiment of the present
invention.

Moreover, though the description of the present invention has included description
35 of one or more embodiments, configurations, or aspects and certain variations and
 14

modifications, other variations, combinations, and modifications are within the scope
of the present invention, e.g., as may be within the skill and knowledge of those in
the art, after understanding the present disclosure. It is intended to obtain rights
which include alternative embodiments, configurations, or aspects to the extent
5 permitted, including alternate, interchangeable and/or equivalent structures,
functions, ranges or steps to those claimed, whether or not such alternate,
interchangeable and/or equivalent structures, functions, ranges or steps are
disclosed herein, and without intending to publicly dedicate any patentable subject
matter.
10
 15

CLAIMS

1. A method for extraction and isolation of bio-active peptides from oil palm
mesocarp, the method comprising:
5 - extracting a total protein from oil palm mesocarps using phenol and
methanol containing ammonium acetate;
- hydrolyzing the total protein using an enzyme combination of pepsin and
at least one hydrolytic enzyme in buffer for generating said bio-active
peptides; and
10 - isolation and fractionation of the plurality of bio-active peptides through
sequential elution by reverse phase liquid chromatography using C18
membrane with acetonitrile to obtain bio-active peptides;
- wherein said bio-active peptides obtained from isolation and fractionation
are bio-active peptides having antimicrobial and antioxidant properties, or
15 said obtained bio-active peptides are selected from the group consisting
of
a bio-active peptide having a sequence of SEQ ID NO: 1,
a bio-active peptide having a sequence of SEQ ID NO: 2,
a bio-active peptide having a sequence of SEQ ID NO: 3,
20 a bio-active peptide having a sequence of SEQ ID NO: 4,
a bio-active peptide having a sequence of SEQ ID NO: 5,
a bio-active peptide having a sequence of SEQ ID NO: 6,
a bio-active peptide having a sequence of SEQ ID NO: 7, and
a bio-active peptide having a sequence of SEQ ID NO: 8.
25
2. The method for extraction and isolation as claimed in claim 1 further comprises
sequencing the plurality of bio-active peptides.

3. The method for extraction and isolation as claimed in claim 1, wherein the
30 extraction of the total protein from oil palm mesocarps further comprises:
- washing and centrifuging of homogenized oil palm mesocarps with
methanol, acetone, tricholoroacetic acid, dithiothreitol and ammonium
acetate;
- mixing and sonicating the washed oil palm mesocarps in sucrose, tris-
35 HCL, sodium chloride, dithiothreitol, EDTA and protease inhibitor;
 16

- mixing the homogenized oil palm mesocarps with the methanol containing
ammonium acetate and centrifuging the homogenized oil palm
mesocarps;
- filtering the oil palm mesocarp pellet using a membrane;
5 - mixing of equal volume of tris-saturated phenol; and
- precipitation of the total protein using cold ammonium acetate-saturated
methanol.

4. The method for extraction and isolation as claimed in claim 3, wherein the
10 concentration of trichloroacetic acid lies in the range of 5-15%.

5. The method for extraction and isolation as claimed in claim 3, wherein the
concentration of dithiothreitol lies in the range of 0.5-1.5 mM.
6. The method for extraction and isolation as claimed in claim 3, wherein the
15 concentration of tris-HCl lies in the range of 10-100 mM.

7. The method for extraction and isolation as claimed in claim 1, wherein the
enzymes are selected from trypsin and pepsin.

20 8. The method for extraction and isolation as claimed in claim 1, wherein the
concentration of the enzymes lies in the range of 0.5-50 mg.

9. The method for extraction and isolation as claimed in claim 7, wherein

hydrolysis of the total protein by trypsin generates bio-active peptides having
25 the peptide sequence of sequence ID No.: 1-4.

10. The method for extraction and isolation as claimed in claim 7, wherein
hydrolysis of the total protein by pepsin generates bio-active peptides having
the peptide sequence of sequence ID No.: 5-8.
30
11. The method for extraction and isolation as claimed in claim 1, wherein the
concentration of acetonitrile lies in the range of 2-100%.
 17

ABSTRACT

METHOD OF EXTRACTION OF BIO-ACTIVE PEPTIDES FROM OIL PALM

MESOCARPS
5
The present embodiment relates to a method of extraction of a plurality bio-active
peptides from oil palm materials such as mesocarps. The method of extraction
includes the extraction of a total protein by precipitation of the total protein using
phenol and methanol containing ammonium acetate. The total protein is hydrolyzed
10 by using pepsin or trypsin, wherein the enzyme hydrolysis generates the plurality of
bio-active peptides. The plurality of bio-active peptides are fractionated and the
sequence of the plurality of bio-active peptides is determined. The plurality of bio­
active peptides possess anti-oxidant and anti-fungal properties.

15 Fig. 1
 1/2

100

Fig. 1

-^DPPH (blank) —^Gallic acid (std)

-•-Pepsin 10 ul avg -o-Trypsin 10 ul avg

Fig. 2
 2/2

-veM^F5J10 Fl5 E20 F25 E30 F35 F40F50

· 'i ·1 ·' λ Λ A · .&■ ■ Trypsin
- V'W ' v \ ' \ -1 y;kV

ΟΆ. k^· * 4 J , ■ Pepsin

Trypsin

Pepsin

Day 7

Fig. 3