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Process Biochemistry 67 (2018) 19–28

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Process Biochemistry
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Influence of enzymatic hydrolysis conditions on the degree of hydrolysis and T

functional properties of protein hydrolysate obtained from Chinese sturgeon
(Acipenser sinensis) by using papain enzyme
Anwar Nomana,b, Yanshun Xua, Wedad Q. AL-Bukhaitia, Sherif M. Abeda,c,

Abdelmoneim H. Alia,d, Abuubakar H. Ramadhana, Wenshui Xiaa,
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
Department of Agricultural Engineering, Faculty of Agriculture, Sana’a University, Sana’a, Yemen
Food and Dairy Science and Technology Department, Faculty of Environmental Agricultural Science, El-Arish University, 43511 El-Arish, Egypt
Department of Food Science, Faculty of Agriculture, Zagazig University, 44511 Zagazig, Egypt


Keywords: In this study, protein hydrolysate was prepared from the muscles of Chinese sturgeon (Acipenser sinensis). The
Sturgeon effects of different conditions on the degree of hydrolysis (DH) by using papain were investigated. The DH of
Fish protein hydrolysate 24.89% was attained under the optimum conditions including solid-to-liquid mixing ratio of 1:1, en-
Degree of hydrolysis zyme–substrate ratio of 3%, pH 6, temperature of 70 °C, and incubation time of 6 h. The yield of protein hy-
Functional properties
drolysate was 17.47%, in which the protein content was 79.67% and amino acid content was 96.35%. The
molecular weight of peptides decreased with the progress in hydrolysis time. Protein hydrolysate solubility
ranged between 86.57% and 98.74%, emulsifying activity index was 11.0–13.27 m2/g, emulsion stability index
was > 94% at different pH levels, water holding capacity was 1.93 g water/g protein, oil holding capacity was
2.59 g oil/g protein, and foam capacity was 76.67%. The obtained fish protein hydrolysate contains improved
functional properties and has potential applications in food industries.

1. Introduction enzymes to extract protein gives a better product in terms of nutritional

value and functional properties [6]. Currently, the enzymatic hydrolysis
Chinese sturgeons are considered as one of the largest anadromous technique of proteins is employed to recover the physiologically and
fish species in the world. They are widely distributed along China's nutritionally important peptides that result during the production of
coastline and in the estuaries of the large rivers. Currently, the Yangtze fish protein hydrolysates (FPHs). Several enzymes such as alcalase,
river is the only river where Chinese sturgeons live [1]. Adult Chinese papain, pepsin, flavourzyme, neutrase, protamex, trypsin, a-chymo-
sturgeons range between 2 and 5 m in total length, and weigh between trypsin, protease N, protease A, pancreatin, pronase, bromelain, cryotin
200 and 500 kg, ranking them among the largest sturgeons in the world F, orientase, validase, and thermolysin have been used to hydrolyze the
[2]. Among the different species of sturgeons, the Chinese sturgeon fish proteins for the production of FPH [5]. Papain is a plant proteolytic
Acipenser sinensis has the fastest growth rate. It is a species of the pro- enzyme and belongs to the cysteine proteinase family. It naturally exists
tected fish and classified as a kind of migratory or semi-migratory fish in papaya (Carica papaya L.) but manufactured from the latex of raw
species [3]. Recently, aquaculture of sturgeon has increased in many papaya fruits. Papain breaks down proteins, which are made up of
countries including China, which contributed more than 44,200 t in amino acids, known as polypeptides [7]. Enzymatic hydrolysis de-
2011 equivalent to about 86% of the world sturgeon meat production, creases the peptide size, thereby making FPH the most available amino
and was statistically ranked in the second order in terms of the number acid source in human and animal food. FPH can be used as a protein
of sturgeon farms until 2013 [4]. source owing to their good functional properties [5]. Compared with
Protein hydrolysates are small fractions of peptides that contain autolytic hydrolysis, enzymatic hydrolysis presents better control con-
several amino acids [5]. They can be obtained by using conventional ditions for the process in terms of the reaction speed and the high
chemical methods, strong chemicals, and solvents. These chemicals quality of the obtained products [8]. The use of enzymatic hydrolysis is
make the products unsuitable for use in food industry, while the use of often considered as an appropriate and useful method for improving the

Corresponding author.
E-mail address: (W. Xia).
Received 22 September 2017; Received in revised form 15 January 2018; Accepted 16 January 2018
Available online 19 January 2018
1359-5113/ © 2018 Elsevier Ltd. All rights reserved.
A. Noman et al. Process Biochemistry 67 (2018) 19–28

functional properties of proteins and maintaining their nutritional value AOAC method [18]. The moisture content was examined by the eva-
[9]. The process depends on several factors including enzyme type, poration method under 105 °C until an unchanged weight was re-
substrate, and hydrolysis conditions such as enzyme concentration, corded. Crude protein content was determined by the Kjeldahl method
temperature, pH, and time. These factors cooperatively affect the en- (N × 6.25). Fat content was estimated by the Soxhlet method with
zyme activity and thus make the hydrolysis process more controllable petroleum ether. The total ash content was determined using the in-
[10,11]. To increase the solubility of proteins and improve the func- cinerator at 550 °C until the sample was completely turned to ash.
tional properties of fish proteins, enzymatic hydrolysis can be used Carbohydrate content was estimated by subtracting the total contents of
because it is the most efficient method to achieve this purpose [9]. moisture, protein, fat, and ash from 100%. All experiments were per-
Proteins obtained by enzymatic hydrolysis with control of reaction formed in triplicate.
conditions can be modified to enhance their quality and functional
properties such as solubility, oil holding capacity (OHC) and water 2.2.2. Amino acid analysis
holding capacity (WHC), emulsification, foaming properties, and sen- Amino acids were determined according to the AOAC method [18]
sory properties [12,13]. The functional properties of FPH are affected with some modifications. Three hundred milligrams of the solid sample
by the substrate and enzyme used and the degree of hydrolysis (DH) were digested with 8 mL of 6 M HCl at 110 °C for 22 h under nitrogen
[12,14]. FPHs can be used as healthy foods, functional foods, dietary atmosphere. After cooling, 4.8 mL of 10 M NaOH was added, the vo-
supplement, and nutraceuticals, and they have potential applications lume was made up to 25 mL with distilled water, then filtered through
for the prevention and the treatment of multiple conditions including two layers of filter paper No. 40, and finally centrifuged at 10,000g for
gastrointestinal syndromes. In addition to the incorporation into dif- 10 min. Amino acids were analyzed by using the reverse-phase high-
ferent foods such as desserts, crackers, and products of cereals and performance liquid chromatography (Agilent 1100 HPLC; Agilent Ltd.,
meat, FPH can also be used as an excellent food source for some species Palo Alto, CA, USA). Each sample (1 μL) was injected into a Zorbax,
of fish [5]. In the same context, the essential nutrients and bioactive 80 A C-18 column (column size: 4.0 × 250 mm, 5 μm particle size;
peptides present in the FPH prepared from different fish species gained Agilent, USA) at 40 °C with detection at 338 nm. The mobile phase A
interest in food and pharmaceutical industries [15]. Recently, the en- was 7.35 mM/L of sodium acetate/triethylamine/tetrahydrofuran
zymatic hydrolysis of proteins has attracted widespread attention (500:0.12:2.5, v/v/v), adjusted to pH 7.2 using acetic acid, while the
owing to the high quality of the produced FPH, which can be used as a mobile phase B (pH 7.2) was 7.35 mM/L of sodium acetate/methanol/
raw material for the production of bioactive peptides for the treatment acetonitrile (1:2:2, v/v/v). The amino acid composition was expressed
of various diseases [16]. as grams of amino acids per 100 g of protein.
Two different fractions can be produced as a result of the enzymatic
hydrolysis of fish proteins, namely, soluble and insoluble proteins. The
2.2.3. Fatty acid profile analysis
insoluble part has a potential application in animal feed [12]. The so-
Total lipids were extracted from the Chinese sturgeon muscles
luble fraction of the hydrolyzed protein can be converted into a nutrient
(1.0 g) with chloroform:methanol (2:1, v/v). Fatty acid methyl esters
component and used as a nitrogen source for maintaining the growth of
(FAMEs) were prepared according to the method of Chalamaiah,
microorganisms [17]. The resulting powder from the dehydration of the
Jyothirmayi, Bhaskarachary, Vajreswari, Hemalatha, and Kumar [15].
soluble hydrolysate is known as FPH, which is considered stable during
FAMEs were analyzed by using a gas chromatography (GC-14B, Shi-
the storage period and possesses higher content of protein [8,12].
madzu, Tokyo, Japan), equipped with a flame ionization detector and a
To the best of our knowledge, this is the first study that deals with
fused-silica capillary column (PEG–20 M, 30 m × 0.32 mm × 0.5 mm).
the preparation of FPH of Chinese sturgeon from Yangtze River by using
The column was initially held at 100 °C for 4 min, followed by tem-
the papain enzyme. The current study aimed to investigate the effects of
perature programming to 180 °C at the rate of 15 °C/min, then held at
solid–liquid (S/L, w/v) ratio, enzyme–substrate (E/S, w/w) ratio, pH,
180 °C for 4 min, and increased to 215 °C at the rate of 4 °C/min. The
hydrolysis temperature, and incubation time on the DH and subse-
temperatures of injector and detector were set at 250 °C. GC peaks were
quently evaluate the functional properties of the obtained product.
identified by comparing their retention times with those of the re-
ference standards and expressed as percentages. The analysis was car-
2. Materials and methods
ried out in triplicate, and mean values were reported.
2.1. Materials
2.2.4. Preparation of protein hydrolysates
2.1.1. Samples To obtain the optimal enzymatic hydrolysis conditions of Chinese
Chinese sturgeon (Acipenser sinensis) was obtained from the Yangtze sturgeon muscles by using papain, a single-factor test and multiple
River by Hua Da Aquatic Products Science and Technology Industry experiments (S/L, E/S, pH, temperature, and time) were applied
Co., Ltd. The fish was transported directly to the laboratory. It was then (Table 1). Protein hydrolysate was prepared according to the method by
cleaned, and the viscera were removed and kept frozen at −20 °C until Ovissipour, Abedian, Motamedzadegan, Rasco, Safari, and Shahiri [19]
use. Before the enzymatic hydrolysis process, the samples were trans- with some modifications. The minced substrate was mixed with 25 mM
ferred to the refrigerator at 4 °C for 12 h.
Table 1
2.1.2. Enzyme and chemicals The parameters and their levels used to obtain the optimum hydrolysis conditions of
sturgeon muscle by using papain enzyme.
Enzyme papain (Enzyme activity/1000 casein, pH 6.0, 40 °C:
≥6000/(USP-U/mg)) was purchased from Ourchem Co., Ltd, Factors Units Symbol Levels
Guangdong Province, China. The enzyme was directly stored at 4 °C. All
chemicals and reagents used in the experiments were of high purity and 1 2 3 4 5 6 7 8 9
analytical grade. Solid/Liquid ratio S/L 1:1 1:2 1:3 1:4 – – – – –
Enzyme/ % E/S 0.5 1 2 3 4 5 – – –
2.2. Methods Substrate
pH pH pH 5.5 6 6.5 7 – – – – –
2.2.1. Proximate chemical composition
Temperature °C T 35 40 45 50 55 60 65 70 75
The chemical compositions of sturgeon fish samples and FPH ob- Time hour t 0.25 0.5 1 2 3 4 6 8 –
tained by enzymatic hydrolysis were determined according to the

A. Noman et al. Process Biochemistry 67 (2018) 19–28

Fig 1. Scheme of the hydrolysis process of sturgeon muscles

by using papain enzyme.

sodium phosphate buffer to keep the pH constant throughout the in- 2.2.6. Yield
cubation time. The hydrolysis process steps are outlined in Fig. 1. The The yield of protein hydrolysate was calculated by weighing the
reactions were performed in jacketed 250 mL glass vessels equipped FPH produced as percentage of substrate used for the enzymatic hy-
with a stirrer (IKA C-MAG HS 4 S 25) and connected to a circulating drolysis process according to Romadhoni, Afrianto, Pratama, and
water bath (Shanghai Blue pard Yiheng Technical Co., Ltd) to control Grandiosa [21] by using the following equation:
the temperature, which was kept constant throughout the hydrolysis
% Yield = [weight of FPH (g)/weight of raw material (g)] × 100 (3)
time. To deactivate enzyme activity, the mixture was heated to 95 °C for
20 min in a water bath, and then it was cooled to room temperature and
centrifuged at 8000g at 4 °C for 20 min. After removing the surface oil 2.2.7. Determination of the molecular weight distributions
layer using a micropipette, the supernatant was collected and then Molecular weight distributions of Chinese sturgeon hydrolysates
freeze-dried at −50 °C and under vacuum (SCIENTZ-10ND, Ningbo were determined by gel permeation chromatography using a high-
SCIENTZ Biotechnol Co., Ltd., China) to obtain FPH, and finally it was performance liquid chromatography system (Waters 1525, USA) fol-
stored at −20 °C until further analysis. lowing the method of Liu, Gu, Lin, Lu, Yi, Ma, Dong, and Cai [22]. A
TSK gel 2000 SWXL (300 × 7.8 mm) column (Tosoh, Tokyo, Japan)
2.2.5. Determination of the degree of hydrolysis was equilibrated with acetonitrile:water (40:60, v/v) in the presence of
The DH was estimated by formal titration according to the method 0.1% trifluoroacetic acid. The samples were eluted at a flow rate of
of Taylor [20] with some modifications; 1.5 g of protein hydrolysate 0.5 mL/min and monitored at 220 nm and a temperature of 30 °C. Cy-
sample was added to 50 mL of distilled water. The pH of the mixture tochrome C (12384 Da), bacitracin (1422 Da), Gly-Gly-Try-Arg
was adjusted to 7.0 using 0.1 N NaOH. Then 10 mL of 38% (v/v) for- (451 Da), and Gly-Gly-Gly (189 Da) were used as molecular weight
maldehyde solution was added to the mixture and left for 5 min at room standards.
temperature, and then titration was continued to the end point at pH
8.5 with 0.1 N standard NaOH solution. The volume of NaOH was used 2.3. Functional properties of FPH
to calculate the amount of free amino groups. The total nitrogen (TN)
was determined following the Kjeldahl method [18]. The content of the 2.3.1. Solubility
free amino groups and the DH were calculated as follows: The solubility of the FPH was determined according to the method
reported by Jain and Anal [23]. The FPH sample (200 mg) was dis-
% free amino groups = [A × B × 14.007/C × 1000] × 100 (1) solved in 20 mL of distilled water, and the pH was adjusted from 2 to 10
using 0.1 M HCl or 0.1 M NaOH. Mixtures were incubated at 30 °C with
DH = [% Free amino groups/% total nitrogen] × 100 (2)
stirring at 100 rpm for 30 min and then centrifuged at 7500g for 15 min.
where:A = mL of NaOH used.B = the concentration of the solution After appropriate dilution, the protein content of the supernatant and
used for titration (0.1 M NaOH).C = amount of sample (g). sample was determined following the Kjeldahl method [18]. Finally,

A. Noman et al. Process Biochemistry 67 (2018) 19–28

Table 2 at 20,000 rpm for 2 min. The foam volume was noted immediately after
Proximate chemical composition of sturgeon muscles and FPH (n = 3, mean ± SD). 2 min. FS was determined by measuring the fall in the volume of the
foam after every 1 min. FC and FS were calculated using the following
Components (%) Wet weight Dry weight FPHa
Moisture 74.93 ± 0.29 – 6.06 ± 0.78
Protein content 15.23 ± 0.71 64.65 ± 0.42 79.67 ± 1.10 Foaming capacity = [V2 − V1/V1] × 100 (8)
Crude fat 3.21 ± 0.11 23.62 ± 0.35 0.33 ± 0.06
Ash 1.01 ± 0.03 3.66 ± 0.14 6.58 ± 0.14
where:V1 = Volume before whippingV2 = Volume after whipping
Carbohydrates 5.62 ± 1.18 8.07 ± 0.42 –
Foam stability = [Foam volume after time/Initial foam volume] × 100
FPH used in the estimation of proximate composition was obtained under optimal (9)
hydrolysis conditions.

the solubility was calculated using the following equation: 2.3.6. Statistical analysis
All experiments were performed in triplicate, and the results were
Solubility% = [Protein content in supernatant/Total protein content in reported as mean ± SD. The data were analyzed by the one-way
sample] × 100 (4) ANOVA test using statistical analysis software (version 6.4; CoStat,
Monterey, CA, USA). The results were considered statistically sig-
nificant for P values less than 0.05.
2.3.2. Emulsifying properties
Emulsifying activity index (EAI) and the emulsion stability index
(ESI) were determined using the methods of Nalinanon, Benjakul, 3. Results and discussion
Kishimura, and Shahidi [24] and Pacheco-Aguilar, Mazorra-Manzano,
and Ramírez-Suárez [25] with slight modifications. FPH solution 1% 3.1. Proximate chemical composition
was mixed with 10 mL of edible corn oil and homogenized at
20,000 rpm for a minute. After the formation of the emulsion, 50 μL was The proximate composition of the raw material based on the wet
taken from the bottom and diluted with 5 mL of 0.1% sodium dodecyl weight and the dry weight and FPH obtained by using enzymatic hy-
sulfate solution. The absorbance of the solutions was measured at drolysis are shown in Table 2. Based on the wet weight, the moisture
500 nm after 0 and 10 min by using UV 1000 spectrophotometer content was 74.93% ± 0.29%. These results were in the range of
(Techcomp, China). EAI and ESI were calculated as follows: 74%–80% consistent with moisture content in some freshwater fish,
which was found by Jabeen and Chaudhry [28]. Furthermore, based on
EAI (m2/g) = 2 × 2.303 × A × DF/IФC (5) the wet weight, the fat content in the sturgeon samples studied was
ESI (min) = A × Δt/ΔA (6) 3.21% ± 0.1%. The fat content was less than 5%; this percentage is
considered lower than the fat content percentage of some fish types that
ESI (%) = 100 − [EAIA0 − EAIA10/EAIA0] × 100 (7) can therefore be classified as low-fat fish species [28]. Based on the dry
weight, fat content in sturgeon samples was 23.62% ± 0.35%, which is
where:A = Absorption at 500 nm, DF = dilution factor (100), I = path
considered higher than the fat content of those reported by Abii,
length of cuvette (m), Ф = oil volume fraction (0.25), and C = protein
Afieroho, and Nnamdi [29] who found that the fat content ranged be-
concentration in aqueous phase (g/m3).ΔA = A0 − A10Δt: 10 min.
tween 7.5% and 17.5% based on the dry weight.
The crude protein content in Chinese sturgeon was
2.3.3. Water holding capacity 15.23% ± 0.71% based on the wet weight and 64.65% ± 0.42%
WHC was estimated by the centrifuge method according to the based on the dry weight. The content of protein was higher than the
study of Wasswa, Tang, Gu, and Yuan [26] with some modifications. results obtained by Jabeen and Chaudhry [28] for three freshwater fish
The sample 0.5 g of FPH was dissolved in 10 mL of distilled water in species, where the content of the crude protein ranged between 10.6%
centrifuge tubes and vortexed for 30 s. The dispersion was allowed to and 11.3% (wet weight) and 39.8% and 57.3% (dry weight).
stand at room temperature for 6 h, and it was then centrifuged at 5000g The content of ash was 1.01% ± 0.03% based on the wet weight.
for 30 min. The supernatant was then filtered using a Whatman No. 1 Puwastien, Judprasong, Kettwan, Vasanachitt, Nakngamanong, and
filter paper, and the volume recovered was accurately measured. The Bhattacharjee [30] reported that the ash content of some raw and
difference between the initial volume of distilled water added to the cooked freshwater fish ranged between 0.9% and 12.1%. Also, based on
protein hydrolysate sample and the volume of the supernatant was the dry weight, the ash content was 3.66% ± 0.14%. Jabeen and
determined, and the results were reported as milliliters of water ab- Chaudhry [28] stated that the ash content ranged between 6.4% and
sorbed per gram of FPH protein sample. 12.1% based on dry weight in three species of freshwater fish. This
decrease in the ash content is due to the increase in content of protein
2.3.4. Oil holding capacity and fat in the dried sample under study. The percentage of carbohy-
OHC was determined by the centrifuge method according to the drates in Chinese sturgeon samples was 5.96% ± 1.18% and
method reported by Wasswa, Tang, Gu, and Yuan [26] with slight 8.07% ± 0.42% based on wet and dry weights, respectively. These
modifications. The sample 0.5 g of FPH was added to 10 mL of corn oil values are higher than those obtained by Njinkoue, Gouado, Tchoum-
in centrifuge tubes and vortexed for 30 s in a vortex mixer in duplicate. bougnang, Ngueguim, Ndinteh, Fomogne-Fodjo, and Schweigert [31]
The oil dispersion was then centrifuged at 2800g for 30 min. The vo- who reported that the content of carbohydrates in two species of fish
lume of oil separated from the hydrolysate was measured, and OHC was was 0.19% and 0.83% based on wet and dry weights, respectively. In
calculated as the grams of oil absorbed per gram of FPH protein sample. general, these differences may be attributed to the nutritional condi-
tions and some other factors such as species, size, sexual maturity, food
2.3.5. Foaming capacity and foam stability sources, fishing season, water salinity, and temperature.
Foaming capacity (FC) and foam stability (FS) were measured ac- The proximate analysis of FPH obtained from Chinese sturgeon by
cording to the methods of Sathe and Salunkhe [27] with some mod- using papain enzyme is shown in Table 2. Protein content was
ification. 2 g of the sample was placed in 100 mL distilled water. The 79.67% ± 1.10%, which is considered higher than that reported for
sample solution was dissolved at room temperature in volumetric cy- FPH from Acipenser persicus [19], while it was lower than that of FPH
linder 500 mL capacity and the foam was prepared using a homogenizer from Pacific whiting (Merluccius productus) muscles [32]. The fat

A. Noman et al. Process Biochemistry 67 (2018) 19–28

Table 3 Table 4
Composition of amino acids in sturgeon muscles and FPH obtained by using papain en- Fatty acid composition of lipids from sturgeon muscles.
zyme (n = 3, mean ± SD).
Fatty acid Content%
Amino acids Amount (g/100 g protein)
Myristic acid (C14:0) 2.27 ± 0.01
Sturgeon muscles FPHa Palmitic acid (C16:0) 17.03 ± 0.04
Palmitoleic acid (C16:1, n − 7) 2.19 ± 0.36
Essential amino acids Stearic acid (C18:0) 3.13 ± 0.02
Histidine 1.483 ± 08 1.031 ± 0.07 Oleic acid (C18:1, n − 9) 31.84 ± 0.15
Threonine 2.348 ± 0.16 2.772 ± 0.08 Lenoleic acid (C18:2, n − 6) 25.54 ± 0.19
Arginine 11.97 ± 0.12 12.364 ± 0.09 α-Linolenic acid (C18:3, n − 3) 2.92 ± 0.03
Tyrosine 7.311 ± 0.10 1.972 ± 0.08 Behenic acid (C22:0) 3.18 ± 0.03
Valine 5.211 ± 0.17 3.718 ± 0.14 Eicosanoic acid (C20:1, n − 9) 1.37 ± 0.02
Methionine 2.806 ± 0.19 2.987 ± 0.09 Eicosadienoic acid (C20:2, n − 6) 0.89 ± 0.05
Phenyl alanine 3.006 ± 0.17 2.748 ± 0.17 Eicosatrienoic acid (C20:3, n − 6) 0.58 ± 0.06
Isoleucine 4.635 ± 0.18 3.314 ± 0.16 Arachidonic acid (C20:4, n − 6) 0.58 ± 0.01
Leucine 6.211 ± 0.16 7.147 ± 0.17 Eicosapentaenoic acid (C20:5, n − 3) 2.35 ± 0.01
Lysine 4.209 ± 0.22 9.491 ± 0.21 Docosapentaenoic acid (C22:5, n − 3) 1.07 ± 0.01
Docosahexaenoic acid (C22:6, n − 3) 5.12 ± 0.03
Non-Essential amino acids
ΣSaturated fatty acids 25.61 ± 0.10
Asparagine 11.944 ± 0.25 7.180 ± 0.13
ΣMonounsaturated fatty acids 35.40 ± 0.53
Glutamine 18.111 ± 0.39 15.312 ± 0.16
ΣPolyunsaturated fatty acids 39.05 ± 0.39
Serine 1.099 ± 0.09 3.656 ± 0.25
Glycine 2.501 ± 0.16 7.491 ± 0.20
Alanine 0.869 ± 0.01 2.428 ± 0.19
Cysteine-s 0.173 ± 0.01 3.282 ± 0.17 3.3. Fatty acid composition of Chinese sturgeon oil
Proline 5.585 ± 0.24 9.456 ± 0.24
∑AAs 89.472 96.350 The composition of fatty acids in sturgeon muscles is shown in
∑EAAs 49.19 47.544
Table 4. The most abundant fatty acids found were oleic acid
∑N-EAAs 40.282 48.805
∑EAAs/∑AAs (%) 54.98 49.35 (31.84% ± 0.15%), followed by linoleic acid (25.54% ± 0.19%),
∑EAAs/∑N-EAAs 1.221 0.97 palmitic acid (17.03% ± 0.04%), and docosahexaenoic acid
(5.12% ± 0.03%). The contents of saturated (SFAs), monounsaturated
FPH used in the estimation of amino acids was obtained under optimal hydrolysis (MUFAs), and polyunsaturated (PUFAs) fatty acids were 25.61%,
35.40%, and 39.05%, respectively. The high levels of PUFAs, in parti-
cular docosahexaenoic and eicosapentaenoic acids, are of great re-
content in the obtained FPH was 0.33% ± 0.06%. The ash content levance for dealing with or preventing cardiovascular diseases [39].
(6.58% ± 0.14%) was lower than that of Sardinella aurita (12.10% to The content of PUFAs in the present study was higher than that re-
14.81%) reported by Souissi, Bougatef, Triki-Ellouz, and Nasri [33]. ported in the study of Njinkoue, Gouado, Tchoumbougnang, Ngueguim,
Ndinteh, Fomogne-Fodjo, and Schweigert [31] who found that the
contents of PUFAs in Pseudotolithus typus and Pseudotolithus elongatus
3.2. Amino acid analysis were 33.41% ± 0.23% and 29.57% ± 0.54%, respectively.

The composition of amino acids in muscles and FPH of Chinese

3.4. Optimization of enzymatic hydrolysis conditions
sturgeon is presented in Table 3. The major amino acids in the muscle
sample were glutamine, arginine, and asparagine making up
3.4.1. Effect of solid-to-liquid ratio
18.11 ± 0.39, 11.97 ± 0.12, and 11.94 ± 0.25 g/100 g protein, re-
The influence of S/L mixing ratio on the DH was assessed within the
spectively. The major amino acids in FPH were glutamine, arginine, and
range of 1:1 to 1:4, and the results are displayed in Fig. 2A. Practically,
lysine making up 15.31 ± 0.16, 12.36 ± 0.09, and 9.49 ± 0.21 g/
it was not acceptable to use a mixing ratio lower than 1:1 of sodium
100 g protein, respectively. According to the results in Table 3, it is
phosphate buffer; this probably ascribed to the adhesion of samples on
quite obvious that the content of the total amino acids in FPH (96.35 g/
the glass. The optimum S/L for the DH was found to be 1:1. At this
100 g protein) was higher than the content of those in sturgeon muscle
mixing ratio, the obtained DH was 23.35%. However, with a further
sample (89.47 g/100 g protein), which might arise from the effect of
increase in the mixing ratio from 1:1 to 1:4, the DH was substantially
enzymatic hydrolysis by using papain. Furthermore, during the pre-
decreased to 12.63%. Benjakul and Morrissey [40] found that the op-
paration of FPH, the insoluble proteins were discarded by the cen-
timum S/L was 1:1 for producing FPH from Pacific whiting solid waste
trifugation process. The total amino acids detected in the muscle sample
by using different mixing ratios. Also, Bhaskar, Benila, Radha, and
were lower than those reported by Oluwaniyi, Dosumu, and Awolola
Lalitha [41] used the same mixing ratio (1:1) with water for the opti-
[34] in four species of marine fish. Also, it was reported that the total
mization of enzymatic hydrolysis of catla (Catla catla) by using a
amino acids in Atlantic mackerel flesh was 96.23 g/100 g protein [35].
commercial protease. The differences between the DH exhibited in the
However, the total essential amino acids in the Chinese sturgeon
present study and that reported in the previous literature may be due to
muscles under study (49.19 g/100 g protein) were higher than those of
the variation in fish species, enzymes, and experimental conditions
the Atlantic mackerel flesh (33.79 g/100 g protein).
The total amino acids in the FPH of Chinese sturgeon were lower
than those in the FPH of silver carp and red salmon (Oncorhynchus
nerka) found by Dong, Zeng, Wang, Liu, Zhao, and Yang [36] and Sa- 3.4.2. Effect of enzyme–substrate ratio
thivel, Smiley, Prinyawiwatkul, and Bechtel [37] while they were The effect of E/S ratio on the DH was investigated, and the results
higher than those in the FPH of Persian sturgeon obtained by Ovissi- are displayed in Fig. 2B. As observed, at a low enzyme concentration of
pour, Abedian, Motamedzadegan, Rasco, Safari, and Shahiri [19]. The 0.5% (w/w), the DH was only 16.07%, mostly due to the insufficient
differences between our results and those reported in the previous lit- catalytic sites required to enhance the hydrolysis process. As the en-
erature may be attributed to several factors such as species/organs, zyme–substrate ratio increased from 0.5% to 3% (w/w), the DH was
location, age, feeding, and season [38]. gradually increased from 16.07% to 23.35%. The maximum DH was
attained at 3% (w/w) of E/S. However, with a further increase in the

A. Noman et al. Process Biochemistry 67 (2018) 19–28

Fig. 2. Effects of different conditions on the degree of hydrolysis: (A) S/L; (B) E/S; (C) pH; (D) temperature; and (E) time of incubation. Means in the same form with various characters
have significant differences (p ˂ 0.05). Data are expressed as mean ± S.D. of triplicate determinations.

enzyme–substrate ratio beyond 3% (w/w), a slight decrease in the DH further increasing pH to 6.5 and 7, an obvious decrease in the DH was
was observed. Such a decline in the DH may be reasonably ascribed to observed. Such a decline in the DH probably attributed to the dena-
the increase in amino acids and smaller peptides that existed in the turation of protein structure of the enzyme or the disturbance of the
hydrolysate because some of the peptides released were highly hydro- ionic character of the substrates, which both could reduce the ability of
lyzed when the concentration of papain increased [42]. It was found the substrate to bind the enzyme [43]. Similar results were reported by
that increasing the enzyme concentration over the optimal concentra- Damrongsakkul, Ratanathammapan, Komolpis, and Tanthapanicha-
tion has no significant effect on the DH. This finding is probably at- koon [44] who revealed that the optimum hydrolysis pH was in the
tributed to the enzyme aggregation, which leads to an increase in range of 6–7 by using papain enzyme.
substrate diffusion inhibition, thereby causing the saturation of the
reaction rate. Therefore, 3% (w/w total reactants) of enzyme con- 3.4.4. Effect of temperature
centration was selected for the further investigations. The reaction temperature has a great impact on the DH of Chinese
sturgeon muscles. The reaction temperature was set at 35, 40, 45, 50,
3.4.3. Effect of pH 55, 60, 65, 70, and 75 °C to elucidate the effect of reaction temperature
The effect of pH on the DH was evaluated within the pH levels of on the DH, and the results are shown in Fig. 2D. When the reaction
5.5, 6, 6.5, and 7. As shown in Fig. 2C, the DH was increased from temperature increased from 35 °C to 70 °C, the DH was significantly
19.33% to 23.35% as the pH increased from 5.5 to 6. However, by increased from 15.31% to 23.35%. However, by further increasing the

A. Noman et al. Process Biochemistry 67 (2018) 19–28

reaction temperature to 75 °C, a slight decline in the DH was observed. 3.7. Functional properties
Such a reduction in the DH probably resulted from the thermal dena-
turation of enzyme, thus leading to a decrease in the DH [45]. Ac- 3.7.1. Solubility
cordingly, in the present study, 70 °C was chosen as the optimum re- Solubility has an important role in protein function because it
action temperature. The obtained results agreed with those of controls the utilization of the product in many applications such as
Damrongsakkul, Ratanathammapan, Komolpis, and Tanthapanicha- emulsions, gels, and foams [49]. Fig. 4A shows the solubility of the
koon [44] who found that the maximum DH of rawhide fish was at- obtained product under different pH values. Significant differences
tained at 70 °C with higher yield by using papain enzyme. were observed with different pH levels (p < 0.05) in agreement with
the finding of dos Santos, Martins, Salas-Mellado, and Prentice [13]. All
3.4.5. Effect of time enzymatic hydrolysis in the pH range of 2–10 showed high solubility,
The effect of reaction time on the DH was assessed within the time and the highest rate of solubility was observed at pH 6 (higher than
range of 0.25–8 h. From the results presented in Fig. 2E, it can be ob- 98%), while the lowest solubility was observed at pH 10 (less than
served that the DH gradually increased with the progress in the reaction 87%). These results were higher than those reported by Naqash and
time. Haslaniza, Maskat, Wan Aida, and Mamot [46] reported that a Nazeer [50] who found that the solubility of the protein obtained from
longer incubation time would allow enzyme to act more extensively on pink perch (Nemipterus japonicus) muscle was 74% by using the papain
the protein, thus resulting in an increment in the DH. When the reaction enzyme and 77% by using pepsin. On the other hand, it was fairly
time increased from 0.25 to 6 h, the DH was substantially raised from compatible with the result obtained by Nalinanon, Benjakul, Kishimura,
14.93% to 24.89%. However, upon prolonging the incubation time and Shahidi [24] for the solubility of FPH from skipjack tuna by using
beyond 8 h, no significant increase in the DH occurred. Based on the pepsin at multiple pH. The increase in solubility may be attributed to
obtained results, 6 h was chosen as the suitable reaction time. The ob- the decrease in the molecular size and the formation of smaller peptides
tained results were compatible with those reported by Dong, Zeng, [49] and also the formation of carboxylic and amine groups from amino
Wang, Liu, Zhao, and Yang [36] who prepared protein hydrolysates acids, which increased the hydrophilicity of the FPH [13]. In addition,
from silver carp by using the alcalase enzyme with 6 h of incubation. On centrifugation excluded most of the insoluble proteins.
the other hand, the DH presented was to a considerable extent higher
than that previously reported for Alaska pollock frame by using papain
enzyme under optimal conditions (temperature 45 °C, hydrolysis time 3.7.2. Emulsifying properties
300 min, E/S of 1.2 g/100 g, liquid-to-solid ratio 6:1, and initial pH 8.0) FPH is a surface-active material that contains both hydrophilic and
[47]. hydrophobic groups; therefore, it promotes the formation of an oil-in-
water emulsion. Protein ability can be measured through the formation
and stabilization of the emulsion by giving units of area of the interface
3.5. Yield
that is stabilized per unit weight of protein (m2/g) and its continuity
over time through EAI and ESI [51]. It was estimated by the turbidity in
The obtained yield is displayed in Table 5. As shown, the FPH yield
in this study was 17.47% ± 1.03%, which is considered higher than the emulsion at a wavelength of 500 nm. The EAI was affected by pH as
that found by Romadhoni, Afrianto, Pratama, and Grandiosa [21] who shown in Fig. 4B because significant differences were observed at var-
used different solvents to produce protein concentrate from Snakehead ious levels of pH (p < 0.05), where EAI at pH 6 was higher than that of
fish. The observed increase in the obtained yield as compared to the other levels, and the minimum EAI was observed at pH 4 because of the
previous investigations may be due to the increment of incubation time increase in protein solubility at pH 6. By using a 1% concentration of
[48]. protein, our results were not notably different from those obtained by
Thiansilakul, Benjakul, and Shahidi [49]. The EAI decreased with in-
creasing concentration of FPH from skipjack tuna by using pepsin [24].
3.6. Molecular weight distribution
On the other hand, Fig. 4C revealed that all ESI results were above
94% during the experimental time, where the maximum value was
The molecular weight distribution of protein hydrolysates obtained
observed at pH 6 and the minimum at pH 4. This result is clearly as-
from Chinese sturgeon with papain enzyme under the optimal condi-
sociated with the solubility of protein and other factors, and it is
tions is presented in Fig. 3. The results showed that the protein hy-
compatible with the result obtained by Klompong, Benjakul, Kanta-
drolysates obtained at 1, 3, and 6 h contained a mixture of small pep-
chote, and Shahidi [52] who reported that the enzyme type, DH, and
tides. The molecular weights ˂1000 Da fractions were more than 97%
pH affected the ESI of the yellow stripe trevally protein hydrolysates
during different hydrolysis periods. The main proportion of FPH was
prepared by using alcalase and flavourzyme with different DH and pH
made up of peptides in the molecular weight between 180 and 500 Da
and was more than 57%, while the proportion of molecular weights
˂180 Da were 20.61%, 25.91%, and 30.64% for 1, 3, and 6 h (hydro-
lysis time), respectively, which means that increase in the DH was
3.7.3. Water and oil holding capacity
observed with progress in hydrolysis time. Chromatographic determi-
The solubility of protein affects both WHC and OHC because high
nation of silver carp protein hydrolysates obtained using alcalase at 1.5
solubility indicates smaller molecular size, which leads to decrease in
and 4 h showed that the relative proportion of FPH < 1000 Da fraction
the absorption of water and oil. As shown in Table 5, the values of WHC
was more than 60% [36]. The lower molecular weight of peptides may
and OHC in FPH obtained using the optimal conditions were
be attributed to the increase in the DH. The dietary proteins rich in low-
1.93 ± 0.37 and 2.59 ± 0.12 g/g, respectively. WHC value was lower
molecular-weight peptides would have a higher nutritional value [47].
than that reported by dos Santos, Martins, Salas-Mellado, and Prentice
[13] who evaluated the functional properties of protein hydrolysates
Table 5
obtained using alcalase and flavourzyme from microbial sources. The
Yield and functional properties of FPH obtained under optimal hydrolysis
conditions. decrease in the amount of water may be attributed to the effect of the
concentration of polar groups such as COOH and NH2 that resulted
Yield (%) 17.47 ± 1.03 from enzymatic hydrolysis [12]. The OHC for grass carp skin hydro-
WHC(g water/g protein) 1.93 ± 0.37
lysates ranged between 2.4 and 3.6 mL oil/g FPH [26]. The variation in
OHC (g oil/g protein) 2.59 ± 0.12
Foam capacity (%) 76.67 ± 6.24 the absorbed amounts of water and oil may be attributed to the hy-
drophilic polar side chains.

A. Noman et al. Process Biochemistry 67 (2018) 19–28

Fig. 3. Molecular weight distribution profiles of Chinese sturgeon protein hydrolysates during different hydrolysis time, respectively, by using papain enzyme: (A) 1 h (DH: 17.99%), (B)
3 h (DH: 21.59%), and (C) 6 h (DH: 24.89%).

3.7.4. Foaming capacity and foam stability 4. Conclusion

The foaming properties strongly depend on the transportation, pe-
netration, and rearrangement of molecules at the air–water interface. FPHs were successfully prepared from Chinese sturgeon. Under the
FC and FS of FPH are shown in Table 5 and Fig. 4D, respectively. FC of optimum conditions (S/L, 1:1; E/S, 3%; pH, 6; temperature, 70 °C; time,
FPH was 76.67% ± 3.86%. Elavarasan, Naveen Kumar, and Shama- 6 h), the highest DH (24.89%) was obtained. The results indicated that
sundar [53] reported that FC of the FPH obtained from freshwater carp the obtained FPH had appropriate contents of protein and amino acids,
by using different proteases ranged between 75% and 130%. Regarding high solubility, and good properties of emulsification. In addition, the
FS, it was 26.67% after 1 min and 0.1% after 4 min at pH 6, and the FPH possessed good WHC and OHC. The resulting FPH can be used in
foam faded afterwards. Results of foaming properties were fairly close several food industries as an alternative source of protein. Further
to the results of Naqash and Nazeer [50] who studied the functional studies are required to investigate the fractionation and potential ap-
properties of protein hydrolysates from pink perch muscles by using plications of FPHs.
papain and other enzymes. Low FS possibly arises from the formation of
free amino acids during hydrolysis [54]. The decrease in FC and FS may Conflict of interest
be attributed to the aggregation of proteins that interfere in the inter-
actions between proteins and the water needed for the formation of The authors declare no conflict of interest.
foam [55]. The results obtained in the present study agree with the
finding of Hammershøj, Nebel, and Carstens [56] who revealed that Acknowledgments
gravitational forces drain the liquid out of the foam, thus reducing the
stability of the foam. The effect of gravity can be reduced by gradually This research was financially supported by the Natural Science
increasing the surface tension of the protein film surrounding the air Foundation of Jiangsu Province (BK20150152), the earmarked fund for
bubbles in the foam. China Agriculture Research System (CARS-45-26), and the program of

A. Noman et al. Process Biochemistry 67 (2018) 19–28

Fig. 4. Functional properties of FPH at different pH: (A) solubility; (B) emulsion activity index; (C) emulsifying stability index; and (D) foam stability with the passage of time. Means in
the same form with various characters have significant differences (p ˂ 0.05). Data are expressed as mean ± S.D. of triplicate determinations.

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