LWT - Food Science and Technology 98 (2018) 18–24

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LWT - Food Science and Technology

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Characterization of sacha inchi protein hydrolysates produced by crude T

papain and Calotropis proteases
Saroat Rawdkuena,∗, Nurdalila Rodzia,b, Suttiporn Pinijsuwana,∗∗
a
Unit of Innovative Food Packaging and Biomaterials, School of Agro-Industry, Mae Fah Luang University, Chiang Rai, 57100, Thailand
b
Department of Food Technology, Faculty of Applied Sciences, University Teknologi Mara (UITM), Shah Alam, 40450, Selangor, Malaysia

A R T I C LE I N FO A B S T R A C T

Keywords: Sacha inchi is widely used as a raw material in the edible oil industry. The de-oiled pressed cake has high
Amino acid profile amounts of proteins, which makes it highly desirable for industrial use due to their value-added products. The
Bioavailability objectives of this study were to produce and characterize protein concentrate (PC) and its hydrolysates (PH)
Oil seed meal hydrolyzed by crude papain and Calotropis proteases. The proximate compositions of sacha inchi used in this
Plukenetia volubilis
study included protein 459, carbohydrate 361, fat 67, ash 59, crude fiber 58, and moisture 53 g/kg (wb). PC
Sequential digestion
hydrolyzed by crude papain (PH-P) and Calotropis proteases (PH-C) had a degree of hydrolysis (DH) of 2.7% and
11.2%, respectively. PH-P contained a higher amount of essential amino acids (474 g/kg) than PH-C (410 g/kg).
The protein pattern of PC and PHs were determined by SDS-PAGE, and the molecular weights were clearly
observable between < 8 and 57 kDa. The antioxidant properties, such as DPPH scavenging and FRAP showed a
rising PH when increasing the DH. Sacha inchi protein hydrolysates can be produced in a cost-effective way by
using crude enzyme extracts.

1. Introduction
Sacha inchi (Plukenetia volubilis L.) or “Inca peanut” is an oleaginous
plant found under the Euphorbiaceae family. Sacha inchi plants are
star-shaped green fruits, and they yield edible dark brown seeds that are
slightly enlarged in the center and squashed towards the edges. They
rich in oil (35–60 g/100 g) and proteins (27 g/100 g), and they contain
heat-labile substances that produce a bitter taste (Fanali et al., 2011).
They are also a source of macronutrients and bioactive compounds
(Brajesh, Kumari, Luis, & Alexis, 2014).
Sacha inchi oil contains high amounts of polyunsaturated fatty acids
(PUFA) including 47–51 g/100 g of alpha-linolenic acid (omega-3) and
34–37 g/100 g of linoleic acid (omega-6) (Fanali et al., 2011; Guillén,
Ruiz, Cabo, Chirinos, & Pascual, 2003). These are the main essential
unsaturated fatty acids obtained from vegetable oils (Follegatti-Rome,
Carla, Renato, & Fernando, 2009). Reportedly, there are advantages for
human health derived from PUFA. It is understood to prevent cancer,
arthritis, coronary heart disease, diabetes, hypertension, inflammatory
skin disease, and attention deficit hyperactivity disorder (Gogus &
Smith, 2010), so for these reasons sacha inchi has received great at-
tention from edible oil manufacturers. Pressed cake is a necessary by-
product of the de-oiling process of sacha inchi, and it can be in the
amount of more than 50% (depending on the oil extraction technique
applied).
The protein content of the seeds is also relatively high (ca. 33 g/
100 g) with the main component being 3S storage protein, a water so-
luble albumin (Hanssen & Schmitz-Hübsch, 2011). It has high potential
to be used in protein concentrate production since it still contains high
amounts of proteins. Moreover, the derived protein concentrate can be
further used for producing protein hydrolysate due to its high level of
cysteine, tyrosine, threonine and tryptophan, which have a major effect
on antioxidant activities (Rawdkeun, Murdayanti, Ketnawa, &
Phongthai, 2016). Protein hydrolysate involves the breakdown of pro-
tein by enzymes into small fractions that can be easily absorbed in the
human body, unlike proteins that are intact. However, enzymes ex-
tracted from animals pose a major problem for certain consumers who
oppose animal-use on ethical, moral, or religious grounds. Due to the
increased demand for natural food products, the use of plant-source
enzymes, such as crude papain extract from papaya latex and Calotropis
protease from Calotropis procera, could be viable alternatives and be
very helpful for use in protein hydrolysate preparation.
The objectives of this study were to prepare protein concentrate
from sacha inchi pressed cake and its hydrolysates by using crude pa-
pain extract from papaya latex and Calotropis protease. In addition, the

∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: saroat@mfu.ac.th (S. Rawdkuen), suttiporn.pin@mfu.ac.th (S. Pinijsuwan).

https://doi.org/10.1016/j.lwt.2018.08.008
Received 28 July 2017; Received in revised form 2 August 2018; Accepted 3 August 2018
Available online 04 August 2018
0023-6438/ © 2018 Elsevier Ltd. All rights reserved.
S. Rawdkuen et al.
derived protein samples were also determined for their chemical and
biological properties. The results obtained will be useful for sacha inchi
oil seed producers and manufacturers.
2. Materials and methods
2.1. Materials
L-cysteine, sodium dodecyl sulphate (SDS), trichloroacetic acid
(TCA), and bovine serum albumin (BSA) were obtained from Fluka
(Buchs, Switzerland). Trolox (( ± )-6-Hydroxy-2, 5, 7, 8-tetra-
methylchromane-2-carboxylic acid) and DPPH (2, 2-diphenyl-1-picry-
hydrazyl) were purchased from Sigma Aldrich (Steinem, Germany).
Tris-(hydroxymethyl)-aminomethane, trypsin from bovine pancreas
(EC.232.650.8), pepsin from porcine gastric mucosa (EC.232.629.3)
were purchased from Merck (Darmstadt, Germany). Sacha inchi was
purchased from Thai CMS Company (Chiang Rai, Thailand). Papaya
latex was obtained from locally grown Carica papaya in Chiang Rai,
Thailand. Calotropis procera latex was obtained from Nayong, Trang,
Thailand.
2.2. Crude enzyme preparation and determinations
Carica papaya latex was collected by making longitudinal incisions
on the unripe fruit with a stainless steel knife. The latex from the
Calotropis procera stems was collected in a clean tube by breaking the
stems. The exuded latex was allowed to run down and drip into col-
lecting devices attached around the trunk. The latex then was trans-
ferred to a plastic bottle. The collected latex was diluted with distilled
water at a ratio of 1:1 (mL/mL). The mixture was then mixed well and
centrifuged at 15,000 g at 4 °C for 10 min. The supernatant was col-
lected and filtered through Whatman paper No. 1. The sample was
referred to as “crude enzyme extract” and then put aside for use in
further study. The protein content was determined by using the Biuret
method (Robinson & Hodgen, 1940), and bovine serum albumin (BSA)
was used as a protein standard.
2.3. Proteolytic determinations
The proteolytic activity of the crude papain extracts was executed
according to the method described in Chaiwut, Nitsawang, Shank, and
Kanasawud (2007). Approximately 0.1 mL of extract was mixed with
0.30 mL of buffer solution (pH 7), and then added with 0.10 mL of
activating agent (40 mmol/L cysteine in 20 mmol/L EDTA disodium
salts). The mixture then was incubated at 37 °C for 5 min. The reaction
was initiated by adding 0.5 mL of 1 g/100 mL casein solution. It was
then left for 10 min. The reaction was stopped by adding 1.5 mL of 5 g/
100 mL cold TCA. The supernatant of the mixture was separated and
collected by centrifugation at 15,000 g for 5 min (Sorvall model RC-B
Plus centrifuge, Newtown, CT, USA), and the absorbance was read at
275 nm.
The proteolytic activity of the Calotropis proteases was executed by
following the method described in Rawdkuen, Pinthathong, Chaiwut,
and Benjakul (2011) with slight modification. Approximately 0.1 mL of
enzyme solution was mixed with 1.1 mL of 1 g/100 mL casein in
0.1 mol/L Tris-HCI (pH 8) containing 12 mmol/L cysteine. The mixture
was incubated at 37 °C for 10 min, and the reaction was stopped by
adding 1.8 mL of 5 g/100 mL cold TCA. The supernatant was separated
and collected by centrifugation at 15,000 g for 5 min. The absorbance
was measured at 275 nm. One caseinolytic unit (katal) is defined as the
amount of enzymes needed to catalyse the transformation of one mol of
casein per second at the assayed condition.
2.4. Proximate analysis of sacha inchi meal
The moisture, protein, ash, crude fiber, and fat content of the sacha
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LWT - Food Science and Technology 98 (2018) 18–24
inchi pressed cake were all determined according to AOAC (2005).
2.5. Extraction of sacha inchi protein
Extraction of sacha inchi protein was performed according to the
method described in Alashi et al. (2014) with some modifications.
Approximately 20 g of sacha inchi pressed cake was suspended in
200 mL of 1 mol/L NaOH at a solid-liquid ratio of 1:10 (pH 11.0). The
mixture was stirred for 1 h at 50 °C in a water bath at 160 rpm and then
centrifuged at 15,000 g for 30 min (Sorvall model RC-B Plus centrifuge,
Newtown, CT, USA). Two additional extractions of the residue from the
centrifugation process were carried out with the same volume of 1 mol/
L NaOH. The supernatant was then pooled, and the pH was adjusted to
the isoelectric point (pH 4.5) using 1 mol/L HCl, and it was then cen-
trifuged (8000 g for 10 min). The precipitate was collected and solu-
bilized with distilled water, and then the pH was adjusted to 7.0 using
1 mol/L of NaOH and freeze-dried. The resultant sacha inchi protein
concentrate was stored at −20 °C until required for further analysis.
2.6. Preparation of sacha inchi protein hydrolysates
Sacha inchi concentrate was hydrolyzed with enzymes at fixed ac-
tivity of 0.03 katals/mg. The beaker contained the mixtures of protein
concentrate and deionized water (5 g/100 mL). It was placed in a
thermostatically controlled water bath and agitated constantly. The
following hydrolysis condition was used: crude papain (pH 7.0 at 40 °C)
and Calotropis proteases (pH 8.0 at 60 °C). During hydrolysis, the pH of
each mixture was maintained using 1 mol/L of NaOH. The amount of
NaOH added to keep the pH constant was recorded and used for cal-
culating the degree of hydrolysis (DH). The reaction was terminated by
immersing the reaction vessel in boiling water for 10 min. The resultant
slurry was centrifuged at 15,000 g at 4 °C for 20 min (Sorvall model RC-
B Plus centrifuge, Newtown, CT, USA). The supernatant was collected
and freeze dried. The yield obtained was calculated as follows:
Weight of powdered protein hydrolysate
Yield (%) = × 100
Weight of sacha inchi meal
The DH was calculated by using the pH-stat method according to
Adler-Nissen (1986, p. 427). The DH was based on the amount of NaOH
used to maintain the constant pH. DH was calculated according to
following equation:
DH (%) = [(β × Nb)/(α × Mp × htot)] × 100
Where β is volume of 4 mol/L NaOH (mL), Nb is molarity of NaOH
(4 mol/L), α is average degree of dissociation of NH2 released during
hydrolysis (1.13), Mp is mass of substrate (N × 6.25), and htot is total
number of peptide bonds in the protein substrate.
2.7. Colour
The colour of sacha inchi meal, concentrate, and hydrolysate were
all measured by using a portable Hunterlab instrument (10° standard
observers, illuminant D65, Hunter Associates Laboratory; Virginia,
USA). The chromameter was standardized by using white tile. L*, a*
and b* parameters indicate lightness, redness, and yellowness, respec-
tively.
2.8. Chemical properties of sacha inchi protein hydrolysates
2.8.1. Amino acid analysis
Protein sample were analyzed for their amino acid profiles by the
Central Laboratory (Thailand), Co. Ltd, Chiang Mai, Thailand using an
in-house method based on AOAC Official Method 994.12.988.15
(2000). They were detected by GC-MS (Agilent technologies Model
6890 N, Germany-Agilent technologies Model 5973 inert, USA)
S. Rawdkuen et al.
equipped with Phenomenex Zebron ZB-AAA 10 m × 0.25 mm, 0.25 u
film thickness, USA.
2.8.2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE)
SDS–PAGE was carried according to the method of Laemmli (1970),
using 15 g/100 mL separating and 4 g/100 mL stacking gels. The sam-
ples were mixed with the sample buffer (0.5 mol/L Tris–HCl, pH 6.8,
0.5 g/100 bromophenol blue, 10 g/100 mL glycerol, and 2 g/100 mL
SDS) with and without betamercaptoethanol at a ratio of 1:1 for re-
ducing and non-reducing conditions, respectively. The mixture was
boiled for 3 min, and 4 μg of protein was loaded in each well and then
subjected to separation at 15 mA/gel by using Mini Protean Tetra Cell
units (Bio-Rad Laboratories, Inc, Richmond, CA, USA). Staining was
carried out by using Coomassie Brilliant Blue R-250 and then destained
with a methanol-acetic acid solution.
2.8.3. Sequential in vitro digestion
Sequential in vitro digestion (pepsin-trypsin) was done according to
the method of Xia et al. (2012) with some modifications. Protein dis-
persion (1 g/100 mL, pH 1.5) was mixed with pepsin (enzyme: protein
of 1:100, g/g), and then gently stirred at 37 °C for 120 min. The sam-
pling was done by taking 1 mL of the mixture and immersed in boiling
water for 3 min to deactivate the enzyme at 0, 1, 5, 10, 30, 60, and
120 min. Protein determination was done by using the Biuret method
(Robinson & Hodgen, 1940). The mixture was then neutralized with
1.0 mol/L of NaOH in order to stop the pepsin digestion. The trypsin
digestion was performed by adding trypsin (enzyme: protein of 1:100,
g/g) into the neutralized pepsin-digested mixture. The sampling was
done in the same method as pepsin digestion for protein determination
until 240 min. After incubation at 37 °C for 120 min, protein dispersions
were heated in boiling water for 10 min to stop the trypsin digestion.
Protein digestibility (%) was calculated as follows:
Protein content at T0 − Protein content at Tt
Protein digestibility (%) =
Protein content of initial protein solution
× 100
Where T0 is digestion time at 0 min and Tt is digestion time at t min.
2.9. Biological properties of sacha inchi protein concentrate and
hydrolysates
2.9.1. Ferric reducing antioxidant power (FRAP)
The ability of samples to reduce ferric ion (Fe3+) was evaluated
according to the method of Benzie and Strain (1996) with slight mod-
ification. FRAP reagent (a freshly prepared mixture of 2.5 mL of
10 mmol TPTZ solution, 2.5 mL of 300 mmol/L acetate buffer, 2.5 mL of
20 mm ferric chloride) was mixed and incubated at 37 °C for 4 min prior
to being mixed with 150 μL of sample. The reaction mixture was al-
lowed to stand in the dark for 30 min at room temperature and then the
absorbance was measured at 593 nm. The result was expressed as μmol
FeSO4/g sample.
2.9.2. DPPH radical scavenging activity
DPPH radical scavenging activity was determined according to the
method of Zhao, Yan, Mouming, Jiaoyan, and Bao (2011). DPPH re-
agent was prepared by dissolving 0.1 mmol/L DPPH in 95 mL/100 mL
ethanol. Protein solution (0.5 mL) was added with 2.0 mL of DPPH re-
agent. The mixture was allowed to stand for 30 min in the dark at room
temperature. The absorbance of the resulting solution was measured at
517 nm. The result was expressed as μmol Trolox/g sample.
2.9.3. TCA-soluble peptide
Determination of TCA-soluble peptide content was done according
to the method of Rawdkuen, Sai-Ut, Khamsorn, Chaijan, and Benjakul
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LWT - Food Science and Technology 98 (2018) 18–24
(2009). Two grams of sample was mixed with 18 mL of 5 g/100 mL TCA
and the mixture was homogenized and centrifuged using a Mikro 20
centrifuge (Hettich Zentrifugen, Tuttlingen, Germany). TCA-soluble
peptides in the supernatant were measured by using the Lowry method,
and the results were expressed as μmol tyrosine/g sample.
2.10. Statistical analysis
Experiments (protein hydrolysates preparation) were run in dupli-
cate and in 3 measurements. Statistical analysis was carried out by
using an SPSS package (SPSS 16.0 for window, SPSS Inc, Chicago, IL).
When ANOVA identified differences among groups, the differences in
mean values were compared using Tukey's HSD test at a 5% level of
significance (P < 0.05). Data are presented as mean ± standard de-
viation.
3. Results and discussion
3.1. Enzyme activity determinations
Enzyme activity was determined prior to the production of protein
hydrolysate. It is shown in Table 1. Casein was used as a substrate for
crude papain and Calotropis proteases for determining the proteolytic
activity for both enzymes. Proteolytic activity is used to measure the
concentration of enzymes during a specific activity in order to de-
termine the purity or the quality of enzyme. Proteolytic activity and the
protein content of crude papain were 4.2 katal/mL and 81 mg/mL,
whereas Calotropis proteases had activity and protein content of 1
katal/mL and 43 mg/mL, respectively. Obviously, Crude papain has
higher proteolytic and protein content than Calotropis proteases. Caro,
Villeneuve, Pina, Reynes, and Graille (2000) reported that the protein
content and proteolytic activity of crude papain were 60.7% and
11.02–3842 units/g. Meanwhile, the total protein content and total
activity of Calotropis procera crude latex in the study of Khadpoon,
Pintathong, Chaiwut, and Rawdkuen (2010) were reported as 228.5 mg
and 7665 units, respectively. The different activity of the enzymes may
be due to the purity and the preparation processes.
3.2. Proximate compositions of sacha inchi meal
According to the proximate analysis, sacha inchi meal was com-
posed of 3 major components, which included protein (459 g/kg),
carbohydrate (361 g/kg), and fat (67 g/kg). The minor compounds are
ash (59 g/kg), crude fiber (58 g/kg), and moisture (53 g/kg) (wb), re-
spectively. Fanali et al. (2011) reported that the meal of sacha inchi
after oil extraction retained 27 g/kg of protein. The content of carbo-
hydrate inside the sacha inchi meal is not too high, as sacha inchi plant
already has a high amount of fat and protein. The fat content of sacha
inchi was quite low due to the pressing process and the extraction of
sacha inchi oil. Therefore, sacha inchi can be considered as a proper
raw material for protein concentrate production because it has a high
amount of protein and is low in unwanted compounds.
Table 1
Proteolytic activity, specific activity, and protein content of crude enzymes use
for protein hydrolysates preparation.
Type of enzymes Proteolytic activity Protein content Specific activity
(katal/mL) (mg/mL) (katal/mg)
Crude papain 4.2 ± 0.1a 81 ± 1a 0.06 ± 0a
Calotropis 1.0 ± 0.1b 43 ± 1b 0.03 ± 0b
proteases
The results are given as mean ± SD from triplicate determination.
Different letter in the same row indicate significantly differences (p < 0.05).
S. Rawdkuen et al.
Table 2
Protein yield and content, DPPH, FRAP, and TCA soluble peptide of sacha inchi
protein concentrate and hydrolysates.
Sample Yield (%) Protein DPPH (μmol FRAP (μmol Total soluble
content Trolox/g) FeSO4/g) peptide
(mg/ml) (μmol/g)
PC 7 ± 0.6b 45 ± 0.2a 18 ± 2c 12 ± 0.6c 34 ± 0c
PH-P 50 ± 3a 24 ± 0c 23 ± 0.4b 26 ± 2b 964 ± 0b
PH-C 51 ± 3a 37 ± 3b 27 ± 1a 76 ± 1a 1023 ± 0a
The results are given as mean ± SD from triplicate determinations. Different
letter in the same row indicate significantly differences (p < 0.05).PC: protein
concentrate; PH-P: sacha inchi protein hydrolysates produced by crude papain;
PH-C: sacha inchi protein hydrolysates produced by Calotropis proteases.
3.3. Sacha inchi protein extraction and hydrolysates
The extraction from sacha inchi meal produced a protein yield and
protein content of 7% and 45 mg/mL, respectively. When the protein
concentrates were hydrolyzed with papain and Calotropis proteases, an
increase of protein yield or a decrease of protein content was observed.
Sacha inchi protein concentrate was used to produce sacha inchi pro-
tein hydrolysate with the fixed proteolytic activity at 0.03 katals/mg of
crude papain and Calotropis proteases. The yield and protein content of
PHs are presented in Table 2. The extraction yield of PH-P (50%) and
PH-C (51%) were not significantly different (p > 0.05). The amount of
peptide bond or protein content was higher in PC (45%) followed by
that of PH-C (37%) and PH-P (24%). The lower amount of protein
content in both sacha inchi protein hydrolysates was due to hydrolysis
process, which breaks the peptide bond, resulting in the release of
peptides and amino acids.
3.4. Sacha inchi protein hydrolysate properties
Crude papain and Calotropis at the same proteolytic activity (0.03
katals/mg) were used for producing sacha inchi protein hydrolysates.
The degree of hydrolysis (DH) was calculated by the pH stat method. It
was found that the DH increased as the hydrolysis time was prolonged.
A rapid hydrolysis was observed within 40 min; thereafter, the rate of
hydrolysis was increase slowly until 60 min. PH-C had higher DH (11%)
than PH-P (3%). The higher DH obtained by PH-C was due to the ex-
tensive hydrolysis of Calotropis proteases, which is more effective than
crude papain even though these enzymes are in the same group of cy-
steine protease. The difference in DH may be due to the conformation of
protein, which causes the difference in the number of accessible peptide
bonds that alter the hydrolysis rate (Li, Youravong, & H-Kittikun,
2010). The different range of DH obtained from different types of en-
zymes and substrates has been reported. The commercial papain pro-
duced chickpea and yellow pea protein hydrolysates with 31–44% DH
(Barbana & Boye, 2010). Whey protein hydrolysate hydrolyzed by Al-
calase, Neutrase and Flavourzyme had DH values of 20.5, 12.5, and
11.4%, respectively (O'Keeffe & FitzGerald, 2014). Amaranthus and
Buckwheat protein was able to hydrolysate with 11.6–17.0% DH ob-
tained from pepsin hydrolysis (Bejosano & Corke, 1999). The difference
in DH is related to the variation of the mechanisms of peptide bond
cleavage between amino acid sequences and the difference between
protein structure and/or protein composition (Barbana & Boye, 2010).
Colour parameters of sacha inchi meal, concentrate, and hydro-
lysates, are all presented in Table 3. The highest value of L* was found
in PH-C (81) while the lowest values were from sacha inchi meal. Sacha
inchi meal has the highest values of a* (3) and b* (20) and lowest value
of L* (73). The lower L* values of sacha inchi meal were indicated by a
darker colour, while the highest value of a* and b* was indicated by a
red and yellow colour. The lowest a* and b* values were observed for
PH-C and PH-P, respectively. Hydrolysis of sacha inchi meal produced
protein powder, which is in white to yellow colour ranges. Among the
21
LWT - Food Science and Technology 98 (2018) 18–24
Table 3
Colour attributes in terms of L*, a* and b* values of sacha inchi meal, sacha
inchi protein concentrate and sacha inchi protein hydrolysates.
Sample Appearance L* a* b*
Meal 73 ± 0d 3 ± 0a 20 ± 0a
PC 77 ± 0c 0.4 ± 0b 14 ± 0c
PH-P 79 ± 1b −0.5 ± 0c 10 ± 0d
PH-C 81 ± 1a −1.3 ± 0.9c 14 ± 0b
The results are given as mean ± SD from triplicate determination. Different
letter in the same row indicate significantly differences (p < 0.05).PC: protein
concentrate; PH-P: sacha inchi protein hydrolysates produced by crude papain;
PH-C: sacha inchi protein hydrolysates produced by Calotropis protease.
hydrolyzed PC, PH-C showed the highest brightness and yellowness
compared to PH-P, which is due to the high degree of hydrolysis.
However, the result of this study is inconsistent with the study done by
Wani, Sogi, Shivhare, and Gill (2015) that showed that kidney bean
protein isolate is brighter when compared to kidney bean protein hy-
drolysate. The difference in colour may be due to the different type of
protein substrate used in the study. The changes in colour that occur in
sacha inchi hydrolysate also depend on the type of enzyme used. Dif-
ferent types of enzyme produce a different colour of hydrolysate, which
will directly effect the colour of the original products.
3.5. Chemical properties of sacha inchi protein concentrate and its
hydrolysates
Sacha inchi protein hydrolysates were analyzed for amino acid
compositions. The results are shown in Table 4. The major amino acid
in PH-P is composed of lysine (110 g/kg) followed by leucine (76 g/kg),
phenylalanine (73 g/kg), and glutamic acid (69 g/kg). The predominant
amino acids in PH-C were lysine (109 g/kg), followed by glutamic acid
(72 g/kg), phenylalanine (61 g/kg), and tyrosine (57 g/kg). The low
amount of arginine, hydroxylysine, and hydroxyproline were observed
in both hydrolysates. PH-C contained a high amount of glutamic acid
(69 g/kg) and aspartic acid (34 g/kg) when compared to PH-P (69 g/kg,
33 g/kg). According to the study of Udenigwe and Aluko (2011), glu-
tamic acid and aspartic acid have strong antioxidant activities due to
the presence of excessive electrons that can act as electron donors to
free radicals. Trypsin and phenylalanine can act as proton donors to
electron deficient radicals (Ajibola, Fashakin, Fagbemi, & Aluko, 2011).
Some amino acids such as tyrosine (PH-C: 57 g/kg; PH-P: 61 g/kg),
leucine (PH-C: 49 g/kg; PH-P: 76 g/kg), histidine (PH-C: 46 g/kg; 52 g/
kg), and Lysine (PH-C: 110 g/kg; PH-P: 109 g/kg) can also be related to
the antioxidant capacity of hydrolysate (Erdmann, Cheung, & Scröder,
2008). Hydrophobic amino acids can be related to antioxidant capacity
in compounds such as phenylalanine (PH-C: 61; PH-P: 73 g/kg), alanine
(PH-C: 12; PH-P: 13 g/kg), isoleucine (25 g/kg), and leucine (PH-C: 49;
PH-P: 76 g/kg). The results of this study show that PH-P contained a
high amount of hydrophobic amino acid. It can acts as an antioxidant
compared to PH-C. Methionine and cysteine also have the ability to
S. Rawdkuen et al. LWT - Food Science and Technology 98 (2018) 18–24

Table 4 with MWs between 42 and 57 kDa and containing disulfide bond was
Amino acid profile of sacha inchi protein hydrolysates prepared by using crude reduced, allowing protein sub-units below 30 kDa to be detected. In
papain and Calotropis proteases. addition, the protein profile drastically changed after the hydrolysis
Amino acid Content (g/kg) process. Enzymatic breakdown during the hydrolysis process involved a
major structural change, in which the protein is slowly degraded into
PH-P PH-C smaller peptide units. Sacha inchi protein hydrolysates had protein
bands ranging from MW < 8–24 kDa.
Essential amino acid
Cysteine 21 12 The in vitro digestibility of sacha inchi protein concentrate was done
Tyrosine 61 57 by using trypsin and pepsin at the same condition as used in gastro-
Threonine 5 6 intestinal digestion. The digestibility of sacha inchi protein concentrate
Valine 27 25
during the hydrolysis by pepsin and trypsin is presented in Fig. 2. The
Methionine 4 5
isoleucine 32 25 digestibility of sacha inchi protein concentrate was determined through
Leucine 76 50 the amount of protein content in the sample during the sampling pro-
Lysine 110 109 cess in accordance with the biuret method. At the initial stage, the di-
Histidine 52 46 gestibility (0–15 min) of the protein rapidly increased to 15%. It then
Phenylalanine 73 61
levelled off at a constant until 60 min. After hydrolysis by pepsin for
Tryptophan 13 14
Total 474 410 120 min, the hydrolysis was continued with trypsin. The same pattern
Non-essential amino acid could be observed. The digestibility of sacha inchi protein concentrate
Glycine 12 12 by trypsin was increased at 121–130 min, and then increased by more
Glutamic acid 69 72
than 50% from 120 to 240 min.
Aspartic acid 33 34
Arginine < 0.05 < 0.05
To further investigate the contribution of polypeptide hydrolysis by
Alanine 13 12 pepsin and trypsin, a reducing and non-reducing SDS PAGE was used. It
Proline 17 15 was obviously observed that the protein band at 0 min was different.
Serine 9 9 Proteins with a MW between 42 and 57 kDa may contain disulfide
Total 153 154
bonds, because this band disappeared in the presence of a reducing
PH-P: sacha inchi protein hydrolysates produced by crude papain. agent, resulting in protein sub-units below 30 kDa. The protein band of
PH-C: sacha inchi protein hydrolysates produced by Calotropis proteases. sacha inchi during the hydrolysis by pepsin and trypsin is shown in
Fig. 2b and c. The protein pattern of sacha inchi concentrate hydrolyzed
donate their sulfur hydrogen to acts as radical scavengers (Rajapakse, by pepsin at 1–120 min showed a MW of 31–57 kDa, and completely
Mendis, Byun, & Kim, 2005). PH-P (472 g/kg) contained a high amount hydrolyzed by trypsin after 120–240 min. Compared to protein hydro-
of essential amino acid as compared to PH-C (408 g/kg). lysate, the band of highest MW was at 57 kDa, which was similar to the
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- digestion of pepsin on sacha inchi protein concentrate. This result re-
PAGE) was used to determine the molecular weight of sacha inchi veals that the hydrolysis of sacha inchi can contribute to a rapid in-
protein concentrate and hydrolysates. The result is shown in Fig. 1. crease in digestibility by use of pepsin and trypsin. Protein hydrolysate
Sacha inchi protein concentrate showed protein bands ranging from 15 involves with the protein, which breaks down into smaller peptides that
to 93 kDa. Four of the most intense bands were observed for sacha inchi can be digested easily when compared to proteins that are intact.
protein concentrate at the MWs of 15, 18, 31 and 57 kDa. The protein
pattern of PC was different in the presence of beta-mercaptoethanol 3.6. Biological properties of sacha inchi protein concentrate and
(reducing agent). This might be due to the fact that sacha inchi protein hydrolysates

Different methods were used to evaluate antioxidant activity due to

Fig. 1. Protein patterns of sacha inchi protein concentrate and hydrolysates in
15% separating polyacrylamide gel. (M) Protein marker, (a) sacha inchi meal
(44.60 mg/mL), (b) sacha inchi protein hydrolysates produced by crude papain
(36.50 mg/mL), and (c) sacha inchi protein hydrolysate produced by Calotropis
proteases (23.80 mg/mL).
the diversity of antioxidant actions and processes. The DPPH radical
scavenging activity of sacha inchi concentrate and hydrolysate is shown
in Table 2. DPPH radical is an oil-soluble free radical that will become
stable once it accepts the electron or hydrogen from an antioxidant. The
highest DPPH activity belonged to PH-C (27.46 μmoL Trolox/g) fol-
lowed by PH-P (23.15 μmoL Trolox/g) and PC (17.80 μmoL Trolox/g).
The results show that PH-C had a higher ability to act as hydrogen
donor than sacha inchi protein concentrate (p < 0.05). It was found
that DPPH activity was positively related to the DH. According to the
study of Jamdar et al. (2010), DPPH radical scavenging ability also
increased with increasing DH. Ghribi et al. (2015) noted that DPPH
radical scavenging activity of chickpea protein hydrolysate was higher
than non-hydrolyzed form. According to the study of Ajibola et al.
(2011), the DPPH radical scavenging activities of the peptides are de-
pendent on molecular size. The smaller peptides can contribute to
better DPPH radical scavenging activities. DPPH radical scavenging
activity is also related to hydrophobic content. There were some reports
that have indicated that DPPH values or other radical scavenging ac-
tivities directly depended on the amount of hydrophobic peptides (Li,
Jiang, Zhang, Mu, & Liu, 2008; Pownall, Udenigwe, & Aluko, 2010).
Ferric reducing antioxidant power (FRAP) measures the ability of
the antioxidant to reduce ferric (Fe3+) to ferrous (Fe2+) through the
donation of an electron. In Ajibola et al. (2011), African yam bean seed
showed that smaller peptides had a better reducing power than that of
high MW. This result was also supported by the study of Ji, Sun, Zhao,

22
S. Rawdkuen et al.
Fig. 2. In vitro digestibility (%) of sacha inchi protein concentrates by using pepsin and subsequent trypsin digestion at 37 °C for 4 h (a) and protein patterns of
sequential in vitro digestibility of sacha inchi protein hydrolysates under reducing (b) and non-reducing (c) conditions.
Xiong, and Sun (2014), where the reducing power assay of low MW
(MV < 3 kDa peanut protein isolate hydrolysate) had stronger anti-
oxidant abilities compared to an isolated one. In this study, the lowest
FRAP value was obtained from PC (11.58 μmoL FeSO4/g) followed by
PH-P (26.44 μmoL FeSO4/g) and PH-C (75.54 μmoL FeSO4/g). It was
found that higher DH contributes to a higher value of reducing power.
This is due to the highest amounts of peptides that can contribute to the
antioxidant properties of protein hydrolysate. During the hydrolysis
process, a wide variety of smaller peptides and free amino acids were
produced. Changes in size, level, and composition of free amino acids
and small peptides, all affect antioxidative activity (Wu, Cher, & Shiau,
2003).
Total soluble peptide determination is used to measure the soluble
peptide inside the sample. The total soluble peptide in PH-C was
1022.94 μmoL/g, followed by PH-P (963.87 μmoL/g), and PC (33.64
μmoL/g). Sacha inchi protein hydrolysate contained a high amount of
TCA-soluble peptide when compared to sacha inchi concentrate. This is
due to the hydrolysis process that released peptide and amino acids.
The higher DH produced a high amount of soluble peptides.
23
LWT - Food Science and Technology 98 (2018) 18–24
4. Conclusion
Sacha inchi meal contains protein as its highest component. The
sacha inchi protein hydrolysates produced by using crude papain and
Calotropis proteases could be useful for further application. Both hy-
drolysates showed effective bioactivities as well as high nutritional
value (Lys, Phe, Tyr) after in vitro gastrointestinal digestion. Thus, crude
papain and Calotropis proteases can be used as alternative natural en-
zymes for production of protein hydrolysates by using sacha inchi meal
as a starting material.
Acknowledgements
The authors would like to express appreciation and thank to Mae
Fah Luang University, Thailand MFU23/2559 and Faculty of Applied
Sciences Universiti Teknologi Mara, Malaysia for supporting this re-
search both financially and with their facilities. Thanks to all scientists
and students who were involved in completing this research. This
manuscript was edited for grammatical accuracy by Matthew Robert
Ferguson of Mahidol University International College, Bangkok,
Thailand.
S. Rawdkuen et al.
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