Hydrolysis of Nyamplung

(Calophylluminophyllum) protein and their

antioxidant activity
Cite as: AIP Conference Proceedings 1755, 140010 (2016); https://doi.org/10.1063/1.4958571
Published Online: 21 July 2016

Rini Yanti, Pudji Hastuti, Harry Sulistyo, and Chusnul Hidayat

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© 2016 Author(s).
 Hydrolysis of Nyamplung (Calophylluminophyllum) Protein
and Their Antioxidant Activity
Rini Yanti,1,b)Pudji Hastuti,1) Harry Sulistyo,2) ChusnulHidayat,1,a)
1
Department of Food and Agriculture Product Technology, Faculty of Agriculture Technology, Universitas Gadjah
Mada, Jl.Flora, Yogyakarta,55281, Indonesia.
. 2Departement of Chemical Engineering, Faculty of Engineering, Universitas Gadjah Mada, Jl.Teknika Utara
Yogyakarta,55281, Indonesia
a)
Corresponding author: chusnul@gadjahmada.edu
b)
riniyanti@ugm.ac.id

Abstract. The seedcake of nyamplung seeds still contains high protein. Recent studies show that protein hydrolysate has
many useful effects, such as an antioxidant. The protein was isolated by the isoelectric precipitation method, further on
hydrolysed into short chain peptides. The chemical composition of seedcake and the protein content of extract were
analysed. The hydrolysis of the extract was carried out in a various condition such as substrate concentration, pH, and
temperature for a different length of hydrolysis and the degree of hydrolysis was determine by TNBS method. The
protein isolation process performed on the defatted seedcake produced 52.84 ± 0.07% of protein extract with a brownish
colour. The degree of hydrolysis (DH) was increased by increasing the length of hydrolysis and showed the optimum DH
with 6.15mg/mL substrate concentration. The increase of temperature also increased the DH, however at 70°C with 240
min time reaction, the DH tended to decrease. The pH of reaction between papain showed the similar DH among various
pH of the reaction, with pH 7 showed the stable increasing DH till 240 min of reaction. The antioxidant activity showed
an increase to 90% of inhibition by the addition of the hydrolysate to the system until 1300 ppm. However, the addition
of more than 1300 ppm showed a reduction activity

INTRODUCTION
The use of nyamplung seeds for oil resources produces seedcake as a waste, which still contains high protein. It
could be used as a source of protein hydrolysates. Hydrolysis of proteins is carried out for (i) improving nutritional
characteristics, such as biologically active peptides and antioxidant activity and (ii) obtaining better functional
properties, such as solubility, foaming, coagulation, and emulsifying capacities. Enzymatic hydrolysis has long been
used to modify the functional and nutritional properties of the protein in the manufacture of traditional foods, such
as cheese and fermented foods from plant materials.
The properties of protein hydrolysates depend on the source of protein and enzyme as a catalyst and reaction
conditions. Alcalase, an enzyme alkaline produced by Bacillus licheniformis, and papain from the plant (Carica
papaya) have been known as an enzyme that is best for the preparation of protein hydrolysates function of fish [1-3].
Hydrolysis alters the increase of the functional properties of proteins. It is highly dependent on the degree of protein
was hydrolysed. The degree of hydrolysis (DH) is useful to control the degradation of excess/bond termination. DH
is defined as the percentage of the total number of peptide bonds that have been released during hydrolysis [4].
Hydrolysis with protease possessed antioxidant activity [5,6]. The amino acid sequence of the peptide might play an
important role in its activity. The molecular weight of protein hydrolysates that is ranging from 551 Da up to 3 k Da
had the antioxidant activity [7-10].
The fact that the large biomass from the nyamplung’s oil extraction industry has high protein content, nyamplung
offers the potential source of protein. The purpose of this study was to get basic information about the yield of the

Advances of Science and Technology for Society

AIP Conf. Proc. 1755, 140010-1–140010-6; doi: 10.1063/1.4958571
Published by AIP Publishing. 978-0-7354-1413-6/$30.00

140010-1
method of isolation and characteristic of nyamplung extract, the effect of enzymatic hydrolysis in various factors,
and the antioxidant activity of hydrolysates.

MATERIAL AND METHODS

Material
The seedcake of nyamplung (Calophyllum inophyllum L.) was obtained from biodiesel processing industry in
Jawa Tengah. Papain was obtained from Merck KGaA (Darmstadt, Germany). NaOH, hexane, HCl, Na2CO3,
ammonium persulfate, SDS, glycine, follincoacalteu, Coomassie brilliant blue G-250, petroleum ether, 1, 1-
diphenyl-2-picrylhydrazyl (DPPH), methanol were obtained from Sigma-Aldrich (USA). ß-mercaptoethanol and
bromophenol blue were obtained from Chroma.

Protein Extraction
Seedcake was defatted using hexane and followed by drying. The defatted seedcake was mixed with water at
ratio powder: water about 1:20 (w/v). pH was adjusted with 1N NaOH until the pH reached 11. The mixture was
separated by centrifugation at 4000 g for 30 min. Soluble protein in the supernatant was precipitated at pH 3, and the
natant was separated at 4000 g for 30 min. Protein was dried using freeze dryer and stored in -20 °C until to be used.
Content of water, protein, fat, and ash, and carbohydrate of both the nyamplung seedcake and the defatted seedcake
were analyzed according toAOAC (1995) [11]. The purity of protein extract was also analysed by AOAC(1995)
[11].

Effect of Initial Substrate Concentration on Degree of Hydrolysis

Nyamplung protein concentrate was dissolved in buffer pH 7 (1.43, 2.17, 6.15, 7.66, and 8.22 mg/mL) and stirred
for 30 min. About 7.4 unit/mL papain was added. The mixture was further incubated at 60°C for 240 min. One mL
of sample was taken ata certain time.The enzyme was inactivated by heating at 100°C for 5 min. DH of the sample
was determined. DH is defined as the percentage of the hydrolysed protein (h) to the total number of peptide bonds
before hydrolysis (h total). The DH was measured by TNBS method [12].

Effect of pH on Degree of Hydrolysis

The protein hydrolysis was carried out at various pH of phosphate buffer (pH 5,6,7). The initial protein
concentration was 6.15 mg/mL.About 7.4 U/mL papain was added to the mixture, and the mixture was incubated at
60°C for 240 min.The DH was measured by TNBS method [12].

Effect of Temperature on Degree of Hydrolysis

The protein hydrolysis was carried out at various temperatures(40, 50, 60, and 70°C). The protein was dissolved
in phosphate buffer pH 7 (6.15 mg/mL). About 7.4 unit/mLpapain was added to the mixture. The DH was measured
by TNBS method [12].

Antioxidant Activity Analysis

The antioxidant activity of protein hydrolysates was analysed by DPPH Radical-Scavenging Assay method
according to the method of Shimada et al., (1992) [13].

RESULTS AND DISCUSSION

The chemical compositions of the pressed cake (PC) and defatted (DF) cake were presented in Fig. 1. The
defatted process of PC increased the rate of oil reduction after pressing. As much as 67.01 % of oil inPC could be

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removed. The protein content of PC nyamplung was high enough (22%) as a potential source for protein isolate or
concentrate. Oil extraction of PC resulted in an increase in protein concentration to 27.32% due to lowering oil
content. The protein content in DF flour of nyamplung was lower compare to DF of castor beans 65.60% [14]. But it
was higher than DF of kenaf seeds (26.19%) [15]. A removing of oil content resulted in a significant increase in the
protein content. Differences levels of protein defatted flour were produced from a variety of sources for the different
levels of protein in the seeds of each plant. If the seeds with high protein content, it will produce the defatted flour
with high protein content is also associated with the process of oil extraction by solvents. The extracted Nyamplung
protein produced yield about 22.09 ± 0.86%,inwhich protein content was 52,84 ± 0.07% with a brown colour.

60
50
40
(%)

30
Pressed cake
20
Defatted cake
10
0
water lipid ash protein fiber carbo

FIGURE 1. Chemical composition of Nyamplung seedcake

The Effect of Initial Substrate Concentration on Degree of Hydrolysis

Protein Hydrolysis was affected by initial substrate concentration (Fig. 2). An increasein the initial substrate
concentration tended to increase DH. An increase in substrate concentration from 1.43 mg/mL to 2.17mg/mL and
6.15 mg /mL resulted in an increase in DH about 1.9 times and 2.8 times, respectively. Further increase in substrate
concentration to 7.66 mg/mL and 8.22 mg/mL resulted in a decrease in DH about 2.76 times and 4.4 times. It is
suggested that high protein concentration became as a substrate in hibitor. The similar result was obtained by Ruan
et al (2010) [16], it showed a decrease in DH at an increase in initial substrate concentration. According to Lescovac
(2004) [17], the substrate concentration was the limiting factor. Thus, the enzymatic reaction rate will increase when
substrate concentration increases. However at higher concentrations, the substrate will often act as an inhibitor.

12
Degree of hydrolysis

1.43mg/ml
10
2,17mg/ml
8
(%)

6 6.15mg/ml

4 7.66mg/ml
2 8.22mg/ml
0
0 50 100 150 200 250
Time of hydrolysis (min)

FIGURE 2. The effect of initial substrate concentration on degree of hydrolysis by papain, to = 7.4 unit/mL, T=60oC, pH 7

The Effect of pH on Degree of Hydrolysis

DH of nyamplung protein, which was hydrolysed by papain under different pH values, is shown in Fig. 3. The
hydrolysed protein rates increased with an increase in pH value from 5 to 6 at the first two h reaction. It was not

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significantly different between pH 6 and 7. An increase in reaction time to 4 h resulted in a decrease in protein
hydrolysis. It is suggested that the reaction reached the equilibrium. Beside the stability of the enzyme at low pH
may also the reason on lower enzyme activity for longer reaction time. Denaturation of proteins at low pH is an
event or sequence of events that lead to structural changes and in most cases these changes alter the biological
function of proteins, which in the case of an enzyme means a loss in catalytic activity [18]. Whitaker (2000) [19]
state each enzyme has an appropriate interval of pH that helps to maintain its tree-dimensional structure in the active
site and provide the essential ionisable group.
Papain activity has the range activity at pH 4-7.0 and it depends on the nature of the substrate. This experiment
showed that the optimum activity of papain was at pH 7. According to Mangaroni (2003) [20], the activity of an
enzyme is profoundly affected by pH. Usually, enzymes display a bell-shaped activity versus pH profile. A decrease
in activity on either side of the pH optimum can be due to two general causes. First, pH may affect the stability of
the enzyme, causing it to become irreversibly inactivated. Second, pH may affect the kinetic parameters of the
enzymatic reaction: It may affect the stability of the ES complex, the velocity of the rate-limiting step, or both.

12
Degree of Hydrolysis

10
8
(%)

6 pH5

4 pH6

2 pH7
0
0 50 100 150 200 250
Time of Hydrolysis (min)

FIGURE 3. The effect of pH on degree of hydrolysis by papain, eo= 7.4 unit/mL, T = 60oC, So = 6.15 mg/mL

The Effect of Temperature on DH

DH of the hydrolysed nyamplung protein at various temperatures is shown in Fig. 4. It increased very fast at the
first 60 min of reaction. Further increase in reaction time resulted in a decrease in DH rate. On the other hand, DH
increased 1.6 times, 2.7 times, and 3.3 times with an increase in temperature from 40 qC to 50 qC, 60 qC and 70 qC
after 180 min of reaction, respectively. The rate of the reaction showed the highest level of DH at 70°C. However,
DH did not increase significantly after 180 min of reaction. It is suggested that enzymes are labile protein complexes
and biocatalysts activity depend on process conditions, in which the original properties can be altered significantly.
The temperature will have a major impact not only on the enzyme activity but also on the stability of the enzyme.
An increase in temperature resulted in an increase in the rate of catalytic reactions, while it also increased the rate of
enzyme inactivation [21].

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 12

Degree of hydrolysis (%)

40°C
10
50°C
8 60°C
6 70°C
4
2
0
0 50 100 150 200 250
Time of hydrolysis (min)

FIGURE 4.The effect of pH on degree of hydrolysis by papain e0 = 7.4 unit /mL, S0= 6.15 mg/mL, pH=7

Antioxidant Activity of NyamplungHydrolysate

The antioxidant activity of protein hydrolysates is presented in Fig.5. In general, an increase in the concentration
of hydrolysate from 60 to 1300 ppm resulted in an increase in the antioxidant activity. Further increase in the
hydrolysate concentration resulted in a decrease in antioxidant activity. For an extract of protein hydrolysate from
initial protein concentration of 0.05 g/mL, the RSA inhibition was 13.9% at a concentration of 60 ppm. While the
RSA inhibition was 14.4% at a concentration of 72.5 ppm from the initial protein concentration of 0.75 g/mL
extract. An increase in the concentration of protein hydrolysate to1300 ppm resulted in an increase in antioxidant
activity 6.3 times. The addition of protein hydrolysate more than 1300 ppm showed a decrease in antioxidant
activity. It is suggested that higher protein hydrolysate concentration resulted in changing the property of protein
hydrolysate from antioxidant to pro-oxidant. Similarly results were also reported that antioxidant agent changed to
the pro-oxidant activity at high protein hydrolysate concentration, such as resveratrol [22], lipolic acid and
dihydrolipic acid [23]. Beside it is also reported that antioxidant of protein hydrolysates had molecular weight range
from551 Da up to 3kDa [7-10]. Therefore the protein of nyamplung has to be hydrolysed to breakdown the peptide
binding to short chain peptide to obtain the antioxidant activity.

100
DPPH scavenging activity (%)

0,05 g extract/ml
80
0,075 g extract /ml
60

40

20

0
0 2000 4000 6000 8000 10000 12000
Concentration of protein hydrolysate (ppm)

FIGURE 5.DPPH scavenging activity of nyamplung hydrolysate

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 CONCLUSION
The by-product of the extraction oil of nyamplung could be processed to obtain protein extract. The isolation of
protein showed the protein content about 52.84 ± 0.07%. The optimum condition of hydrolysis with a high and
stable degree of hydrolysis was initial substrate concentration 6.15 mg /mL, pH 7 and temperature 60°C. To increase
antioxidant activity in the system, adding the hydrolysate until 1300 ppm is suggested, however, more than 1300
ppm showed the pro-oxidant characteristic.

ACKNOWLEDGEMENTS
This study was partially supported by Kementerian Pendidikan dan Kebudayaan Republik Indonesia under BPPS
programme.

REFERENCE
1. S.Benjakul and M.T. Morrissey, J. Agric. Food Chem 45, 3423-3430 (1997).
2. F. Shahidi, J.Synowiecki, and J. Balejko, J. Agric. Food Chem. 42, 2634-2638 (1994).
3. K. Sugiyama, M. Egawa, H. Onzuka, and K. Oba, Nippon Suisan Gakkaisi 57, 475-479 (1991).
4. D. Spellman, E. McEvoy, G.O'Cuinn,and G.R.J. FitzGerald, Int. Dairy J., 13, 447-453 (2003).
5. P.W.M.I.H.K. Marambe, P.J. Shand, and J.P.D. Wanasundara, J Am Oil ChemSoc 85, 1155-1164 (2008).
6. R. Darmawan, N.A. Bringe and E. Gonzalez De Mejia, Plant Foods Hum. Nutr. 65, p. 233-240 (2010).
7. Y. Sun, S. Hayakawa, M. Ogawa, S. Naknukool, Y. Guan, and Y. Matsumoto. Food Sci. Biotechnol. 20,
303-310 (2011).
8. R.J. Ganesh, R.A. Nazeer, and N.S. Sampath-Kumar,Food Sci. Biotechnol. 20, 1087-1094 (2011).
9. C. Chen, Y-J. Chi, and W. Xu, Food and Bioprocess Technology 5, 2342–2352 (2012).
10. S.Y Naqash,. and R.A. Nazer, International journal of Food Science and Technology 46, 37-43 (2011).
11. AOAC, Official Methods of Analysis of The Association of Official Analytical Chemistry, 16th edition,
(AOAC International, Virginia, 1995).
12. J. Adler-Nissen, Am. Chem. Soc, 1256-1525 (1979).
13. K.Shimada, K.Fujikawa, K. Yahara, andT. Nakamura,Journal of Agricultural and Food Chemistry 40, 945–
948 (1992).
14. D.M. Marrufo-Estada, M.R. Segura-Campos, L.A. Chel-Guerrero, and D.A. Betancur-Ancona, Food
Chemistry 136,77-83 (2013).
15. S.K.C. Chang, “Protein Analysis. In Nielsen” in S.S. Food Analysis (Plenum Publisher, New York, 2013).
16. C.Q. Ruan, Y.J. Chi, and R.D. Zhang, Czech J. Food Sci 28, 355-363 (2010).
17. V.Leskovacs, ComprehensiveEnzymeKinetics(KluwerAcademicPublishers, New York, 2014), p 192
18. J.M.Broering and A.S. Bommarius, J. Phys. Chem. B 109, 43, 20612-9 (2005).
19. J.R. Whitaker, “Enzyme catalysed reactions experimental factors that affect rates.” in Handbook of Food
Enzymology (Marcel Dekker, Inc., New York, 2000), p43- 48.
20. A.G. Mangaroni,Enzyme Kinetics. A Modern Approach(John Wiley & Son, Inc Publication, 2003).
21. A. Illanes, Enzyme Biocatalysis. Principles and Applications (Springer, 2008).
22. C. de La Lastra andI. Vilegas,Biochemical Society Transactions 35, 1156-1160 (2007).
23. H. Moini, L. Pakker, and N.E.L. Saris, Toxicology and Applied Pharmacology 182, 84-90 (2002).

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