Thu hồi protein và lipit từ đầu cá ngừ bằng enzyme protease công nghiệp

J. Sci. & Devel., Vol. 11, No. 8: 1150-1158 Tạp chí Khoa học và Phát triển 2013, tập 11, số 8: 1150-1158 
www.hua.edu.vn 
1150 
PROTEIN AND LIPID RECOVERY FROM TUNA HEAD USING INDUSTRIAL PROTEASE 
Nguyen Thi My Huong 
Faculty of Food Technology - Nha Trang University 
Email: huongdhts@yahoo.com 
Received date: 28.09.2013 Accepted date: 22.12.2013 
ABSTRACT 
Protein and lipid recovery from yellowfin tuna heads by enzymatic hydrolysis was studied. Hydrolysis of tuna 
head was carried out using 0.5% Protamex at 45°C without pH control for 120 minutes with a water/material ratio of 
1:1 (ml/g). Nitrogen recovery, amino acid composition of protein hydrolysate, lipid recovery and fatty acid composition 
of fish oil obtained from hydrolysis of tuna heads were determined. Results showed that after 120 minutes of 
hydrolysis, the nitrogen and lipid recoveries were 70.3% and 65.4% respectively. Protein hydrolysate from yellowfin 
tuna heads had of 80% protein, 1.3% lipid and 7.9% ash. Protein hydrolysate had high content of essential amino 
acids (33.67%). The fish oil obtained from hydrolysis of tuna heads was rich in omega-3 fatty acids (18.99%), 
especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The content of omega-6 fatty acids was 
4.37%. Fatty acids with high contents in tuna head oil were palmitic acid (29.75%), oleic acid (16.76%) and 
docosahexaenoic acid (14.56%). 
Key words: Enzymatic hydrolysis, protein hydrolysate, protein and lipid recovery, tuna head. 
Thu hồi protein và lipit từ đầu cá ngừ bằng enzyme protease công nghiệp 
TÓM TẮT 
Sự thu hồi protein và lipit từ đầu cá ngừ vây vàng theo phương pháp thuỷ phân bằng enzyme đã được nghiên 
cứu. Sự thuỷ phân đầu cá ngừ được thực hiện bằng Protamex 0,5% ở nhiệt độ 45°C, không điều chỉnh pH, trong 
thời gian 120 phút, tỉ lệ nước/nguyên liệu là 1/1 (ml/g). Hiệu suất thu hồi nitơ, thành phần axit amin của sản phẩm 
thuỷ phân protein, hiệu suất thu hổi lipit và thành phần axit béo của dầu cá thu được từ sự thuỷ phân đầu cá ngừ đã 
được xác định. Kết quả đã chỉ ra rằng sau 120 phút thuỷ phân, hiệu suất thu hồi nitơ và lipit lần lượt là 70,3% and 
65,4%. Sản phẩm thuỷ phân protein từ đầu cá ngừ vây vàng có hàm lượng protein 80%, lipit 1,3% và tro 7,9%. Sản 
phẩm thuỷ phân protein có hàm lượng axit amin không thay thế cao (33,67%). Dầu cá thu được từ sự thuỷ phân đầu 
cá ngừ giàu axit béo omega 3 (18,99%), đặc biệt axit docosahexaenoic (DHA) và eicosapentaenoic (EPA). Hàm 
lượng axit béo omega 6 là 4,37%. Các axit béo có hàm lượng cao trong dầu đầu cá ngừ là palmitic (29,75%), oleic 
(16,76%) và docosahexaenoic (14,56%). 
Từ khoá: Đầu cá ngừ, sản phẩm thuỷ phân protein, thu hồi protein và lipit, thuỷ phân bằng enzyme. 
1. INTRODUCTION 
Tuna is one of the most economically 
important pelagic species. The main products 
from tuna are frozen tuna, canned tuna, smoked 
tuna and tuna sashimi, etc. The fish processing 
industry generates more than 60% by-products, 
which includes head, viscera, trimmings, 
frames, skin, liver and roes, etc (Chalamaiah et 
al., 2012). Tuna heads generated from tuna 
processing contain not only valuable proteins, 
lipids as well as vitamins and minerals, but are 
also an important source of environmental 
contamination. Today, they are seen as a 
potential resource instead of a waste. There is a 
great potential in marine bioprocessing industry 
Nguyen Thi My Huong 
1151 
to convert and utilize these by-products to 
value-added products. Enzymatic hydrolysis is 
one of the most efficient methods to recover 
proteins and lipids from fish by-products and to 
produce the protein hydrolysates with a high 
content of amino acids. Enzymatic hydrolysis of 
fish by-products was studied by several 
scientists (Liaset et al., 2002; Aspmo et al., 2005; 
Sathivel et al., 2005; Slizyté et al., 2005; Mbatia 
et al., 2010; Nguyen et al., 2011). The results 
showed that the protein hydrolysates produced 
from fish by-products had high content of protein, 
and were rich in essential amino acids. The fish 
oil obtained from hydrolysis of fish by-products 
was a good resource of omega-3 fatty acids. The 
potential use of fish protein hydrolysate and fish 
oil in food and feed was also reported by several 
authors (Yu and Tan, 1990; Berge and 
Storebakken, 1996; Refstie et al., 2004; Tang et 
al., 2008; Nguyen et al., 2012). The possibility of 
tuna head use for the production of fish protein 
hydrolysate and fish oil could potentially 
generate significant revenue for fish processing 
industry and environment. 
The purpose of this work was to study 
protein and lipid recovery from yellowfin tuna 
head by enzymatic hydrolysis as well as to 
analyse the amino acid compositions in tuna 
head protein hydrolysate and the fatty acid 
compositions in tuna head oil. 
2. MATERIALS AND METHODS 
2.1. Yellowfin tuna heads 
Yellowfin tuna (Thunnus albacares) caught 
in the Pacific Ocean was transported to 
processing plant (Hai Vuong company, Khanh 
Hoa, Vietnam). The heads were taken from the 
frozen fish and transported to the laboratory at 
Nha Trang University. Upon arrival, the frozen 
tuna heads were thawed, cut and ground in 
a grinder through 3mm plate. Ground tuna 
heads were frozen in the plastic bags and stored 
at - 20°C until use. 
2.2. Enzyme 
Protamex is a Bacillus protease complex 
developed for the hydrolysis of food proteins. 
Protamex was produced by Novozymes A/S, 
(Bagsvaerd Denmark) and complies with the 
recommended purity specifications for food-grade 
enzyme issued by the Joint FAO/WHO Expert 
Committee on Food Additives (JECFA) and the 
Food Chemicals Codex (FCC). The pH of 5.5-7.5 
and temperature range of 35-60°C are optimal working 
conditions for Protamex.. Protamex has a declared 
activity of 1.5 Anson Units/g. 
2.3. Hydrolysis process of yellowfin tuna 
heads 
Hydrolysis process of yellowfin tuna heads 
is shown in Figure 1. 
The ground and frozen tuna heads were 
thawed overnight in the refrigerator at 4°C. Five 
hundred grams of ground tuna head were mixed 
with 500 mL of distilled water. The hydrolysis 
was performed in a glass vessel. The mixture 
was stirred at 300 rpm with an impeller. The 
enzymatic hydrolysis was started when the 
temperature of the mixture reached 45°C by 
adding 0.5% Protamex (by weight of tuna head). 
The hydrolysis was carried out for 30, 60, 90 
and 120 minutes at 45°C without pH control 
(initial pH = 6.5). After each hydrolysis period, 
the enzyme was inactivated by heating at 95°C 
for 15 minutes in a water bath. Then, the 
mixture was filtered through a mesh to remove 
the bones. The filtrate was centrifuged at 10000 
rpm at 4°C for 30 minutes. After centrifugation, 
the following three fractions were collected: the 
oil fraction on the top; the liquid protein 
hydrolysate (water-soluble compounds) in the 
middle; and the sludge (non-water-sobuble 
part) at the bottom. The liquid protein 
hydrolysate was then freeze-dried to obtain fish 
protein hydrolysate. The experiments were 
carried out in triplicates. 
2.4. Chemical analyses 
The moisture content was determined by 
drying the samples in an oven at 105°C until 
constant weight. Ash content was measured by 
Protein and Lipid Recovery from Tuna Head Using Industrial Protease 
1152 
incinerating the samples in a furnace at 600°C. 
Total nitrogen content was determined by the 
Kjeldahl method. Crude protein content was 
estimated by multiplying total nitrogen content 
by 6.25. Lipid content was determined after 
extraction of lipids from the samples according 
to Folch et al. (1957). The nitrogen recovery 
(NR) in the hydrolysate was calculated 
according to Liaset et al. (2002) as follows: 
NR = (Total nitrogen in the hydrolysate/ Total 
nitrogen in 500g of ground tuna head) x 100. 
Amino acid composition was determined 
using the EZ: faast TM procedure described by 
Kechaou et al. (2009). The lipid recovery (LR) in 
the oil fraction was calculated as follows: 
LR = (Lipid in the oil fraction / Lipid in 500 
g of ground tuna head) x 100. 
Fatty acid composition in the tuna head oil 
was determined by gas chromatography 
according to Noriega-Rodríguez et al. (2009). 
2.5. Statistical analyses 
The statistical program SPSS (SPSS Inc., 
Chicago, IL, USA) was used for data processing 
and statistical analysis. Data were subjected to 
analysis of variance (ANOVA). Means were 
separated by using Duncan’s multiple range 
test (Tang et al., 2008). Differences in treatment 
means were considered significant at P<0.05. 
Figure 1. Scheme of the enzymatic hydrolysis of tuna heads 
Inactivation of the enzyme 
 by heating at 95°C, 15 min. 
Enzymatic hydrolysis 
(at 45°C, 30, 60, 90 and 120 min) 
Mesh 
Centrifugation 
(at 10000 rpm at 4°C for 30 minutes) 
Protamex 
Filtrate 
Oil Liquid protein hydrolysate Sludge 
 Freeze -drying 
Ground tuna heads 
Fish protein hydrolysate 
 Bones 
Water 
Nguyen Thi My Huong 
1153 
3. RESULTS AND DISCUSSION 
3.1. Chemical compositions of yellowfin 
tuna heads and protein hydrolysate 
The chemical compositions of yellowfin tuna 
heads and protein hydrolysate obtained from 
yellowfin tuna head after 120 minutes of 
hydrolysis are shown in Table 1. 
The yellowfin tuna heads had 14.8% protein 
and 13.5% lipid, indicating that the yellowfin tuna 
heads are a good source of protein and lipid which 
can be recovered by enzymatic hydrolysis. 
The protein content of tuna head protein 
hydrolysate was 80%, which was higher than 
those reported for salmon head protein 
hydrolysates (62.3% to 64.8%) by Sathivel et al. 
(2005) but lower than that reported for herring 
head protein hydrolysate (85.2%) by Sathivel et 
al (2003). The high protein content was a result 
of the solubilization of protein during 
hydrolysis, the removal of insoluble undigested 
non-protein substances and removal of lipid 
after hydrolysis (Benjakul and Morrissey, 1997). 
The lipid content of tuna head protein 
hydrolysate was low (1.3%). This result was 
similar to that reported for herring head protein 
hydrolysate (1.2%) (Sathivel et al., (2003). The 
removal of the oil layer after hydrolysis caused 
a low lipid content in the protein hydrolysate 
(Benjakul and Morrissey, 1997). The low lipid 
content might enhance stability of the 
hydrolysate towards lipid oxidation. The ash 
content of tuna head protein hydrolysate was 
7.9%, which was higher than those reported for 
salmon head protein hydrolysates (6.9% to 
7.7%) (Sathivel et al., (2005) but lower than that 
shown for herring head protein hydrolysate 
(10.1%) (Sathivel et al., (2003). 
3.2. Nitrogen recovery 
According to Benjakul and Morrissey 
(1997), the nitrogen recovery (protein recovery) 
reflects the yield that can be recovered in the 
protein hydrolysate from the hydrolysis process. 
Nitrogen recovery was used as an index of 
nitrogen solubilization to describe the 
hydrolysis yield (Guérard et al. 2002). Nitrogen 
recovery indicates the percentage of nitrogen 
solubilized into hydrolysate to total nitrogen in 
the raw material and is presented in Figure 
2.The results indicated that the nitrogen 
recovery increased with increasing hydrolysis 
time. These results are in accordance with those 
reported in previous studies on fish by-products 
(Liaset et al., 2002; Aspmo et al., 2005). The 
nitrogen recovery in protein hydrolysate after 
30 minutes of hydrolysis of tuna head was 
52.8% while nitrogen recovery of 70.3% was 
obtained after 120 minutes of hydrolysis. There 
were significant differences in nitrogen recovery 
among the samples with different hydrolysis 
time. The break of peptide bonds under the 
action of Proteome during hydrolysis resulted in 
the amount of soluble nitrogen in the protein 
hydrolysate. The hydrolysis of the fish protein 
was characterized by an initial rapid phase. 
Thereafter, the rate of enzymatic hydrolysis 
decreased. A reduction in the reaction rate 
might be due to limitation of the enzyme 
activity by formation of reaction products 
(Guérard et al., 2002). Shahidi et al. (1995) 
reported that considerable soluble protein was 
released during initial phase. The rate of 
hydrolysis and nitrogen recovery was reduced 
with high concentration of soluble peptides in 
the reaction mixture. 
Table 1. Chemical compositions of yellowfin tuna head and protein hydrolysate 
 Moisture (%) Crude protein (%) Lipid (%) Ash (%) 
Yellowfin tuna head 59.0 ± 1.1 14.8 ± 0.1 13.5 ± 0.1 11.8 ± 1.1 
Protein hydrolysate 8.2 ± 0.4 80 ± 0.8 1.3 ± 0.3 7.9 ± 0.3 
Protein and Lipid Recovery from Tuna Head Using Industrial Protease 
1154 
52,8
a
61,6
b 67,8
c 70,3
d
0
20
40
60
80
30 60 90 120
Hydrolysis time (min)
N
itr
og
en
 re
co
ve
ry
 (%
)
Figure 2. Nitrogen recovery in the tuna head protein hydrolysate. 
Values reported are means of three replicates. Mean values with 
different superscript letter are significantly different (P<0.05) 
3.3. Amino acid composition of the protein 
hydrolysate from yellowfin tuna heads 
 Table 2 shows the amino acid composition 
of protein hydrolysate from hydrolysis of 
yellowfin tuna heads for 120 minutes. 
The protein hydrolysate from yellowfin 
tuna heads had a total amino acid content of 
60.43% and total essential amino acid content of 
33.67%. The tuna head protein hydrolysate was 
rich in aspartic acid, leucine, glycine, histidine, 
Table 2. Amino acid composition of the protein hydrolysate from yellowfin tuna head 
Amino acids Content (% of dry matter) 
Arginine 4.03 ± 0.08 
Histidine 5.06 ± 0.06 
Isoleucine 4.17 ± 0.12 
Leucine 6.25 ± 0.11 
Lysine 3.01± 0.18 
Methionine 2.10 ± 0.07 
Phenylalanine 2.42 ± 0.06 
Threonine 3.15 ± 0.09 
Valine 3.48 ± 0.11 
Alanine 4.42 ± 0.16 
Aspartic 7.53 ± 0.10 
Glutamic 2.35 ± 0.08 
Glycine 5.13 ± 0.13 
Hydroxyproline 1.36 ± 0.06 
Proline 1.45 ± 0.10 
Serine 2.36 ± 0.07 
Tyrosine 2.16 ± 0.10 
Total essential amino acids (TEAA) 33.67 ± 0.35 
Total non-essential amino acids (TNEAA) 26.76 ± 0.33 
Total amino acids (TAA) 60.43 ± 0.30 
TEAA/TAA (%) 55.72 ± 0.54 
TEAA/TNEAA 1.26 ± 0.03 
Nguyen Thi My Huong 
1155 
alanine, isoleucine and arginine. The ratio of 
essential amino acids to total amino acids was 
55.72% and the ratio of essential amino acids to 
non-essential amino acids was 1.26. Both values 
exceeded the reference values of 40% and 0.6 for 
human, which are recommended by World 
Health Organization (WHO)/Food and 
Agriculture Organization (FAO) (FAO/WHO 
1990). The results of the present study showed 
that the protein hydrolysate from yellowfin tuna 
heads was found to have high nutritional value 
and could be used in human and animal diets. 
3.4. Lipid recovery 
 In addition to the protein hydrolysate 
obtained after the hydrolysis of tuna head with 
Protamex, the fish oil was also extracted during 
hydrolysis. According to Dumay et al. (2006), 
the disruption of tissues increased oil liberation 
during hydrolysis of fish by-product with enzyme. 
The lipid recovery in the oil fraction from 
hydrolysis of tuna head is shown in Figure 3. 
The results indicated that the lipid recovey 
from tuna head increased within the first 90 
minutes of hydrolysis. The prolonged hydrolysis 
(90 -120 minutes) did not increase the lipid 
recovery further. Decrease in oil yield after 90 
minutes could be due to interaction of more 
lipids with the hydrolysed proteins. The reduced 
release of lipid may result from the formation of 
lipid-protein aggregates as pointed out by 
Šližyte et al. (2005). These results are in 
accordance with those reported by Mbatia et al. 
(2010) who showed that the prolonged 
hydrolysis did not improve the oil yield further 
but resulted in a colour change of hydrolysate 
solution to brow. 
After 120 minutes of hydrolysis of tuna 
head with 0.5% Protamex at 45°C, the lipid 
recovery in the oil fraction was 65.4%. Daukšas 
et al. (2005) showed that the lipid recovery in 
the oil fraction after hydrolysis of different by-
products ranged from 36.4% to 82.8%. Batista et 
al. (2009) reported that the lipid recovery was 
35% after 1h of hydrolysis of sardine by-product 
with Protamex and only a small increase of 5% 
was observed in the next 3 hours. The previous 
studies indicated that the oil liberation was 
dependent on the raw material and hydrolysis 
conditions such as the temperature, the pH, the 
hydrolysis time and the enzyme ratio (Daukšas et 
al., 2005; Batista et al., 2009; Mbatia et al., 2010). 
3.5. Fatty acid composition in the oil 
recovered from yellowfin tuna head 
Fatty acid composition in the oil recovered 
from hydrolysis of yellowfin tuna heads for 120 
minutes is shown in Table 3. 
65,4
c69,1
d
57,2
b
36,8
a
0
20
40
60
80
30 60 90 120
Hydrolysis time (min)
Li
pi
d 
re
co
ve
ry
 (%
)
Figure 3. Lipid recovery in the oil fraction from hydrolysis of yellowfin tuna head. 
Values reported are means of three replicates. Mean values with 
different superscript letter are significantly different (P<0.05) 
Protein and Lipid Recovery from Tuna Head Using Industrial Protease 
1156 
Table 3. Fatty acid composition in the oil recovered from yellowfin tuna head 
Fatty acids Content (% total fatty acids) 
C14:0 (Myristic) 3.52 ± 0.10 
C16:0 (Palmitic) 29.75 ± 0.35 
C18:0 (Stearic) 10.28 ± 0.10 
C24:0 (Linoceric) 1.35 ± 0.09 
C16:1 (ω-7) (Palmitoleic) 5.54 + 0.13 
C18:1 (ω-9) (Oleic) 16.76 + 0.16 
C18:1 (ω -7) (Vacenic) 3.19 ± 0.06 
C20:1 (ω -9) (Adoleic) 2.13 ± 0,09 
C22:1 (ω -9) (Erucic) 2.32 ± 0.07 
C24:1 (ω -9) (Nervonic) 1.80 + 0.05 
C18:2 (ω -6) (Linoleic) 1.99 ± 0.04 
C18:3 (ω -3) (Linolenic) 0.75 ± 0.03 
C18:4 (ω -3) (Stearidonic) 0.94 ± 0.07 
C20:4 (ω -6) (Arachidonic) 1.52 + 0.06 
C22:4 (ω -6) (Docosatetraenoic) 0.86 ± 0.07 
C20:5 (ω -3) (Eicosapentaenoic, EPA) 2.44 ± 0.06 
C22:5 (ω -3) (Docosapentaenoic) 0.30 + 0.02 
C22:6 (ω -3) (Docosahexaenoic, DHA) 14.56 ± 0.14 
Total saturated fatty acids (SFA) 44.90 ± 0.33 
Total monounsaturated fatty acids (MUFA) 31.74 ± 0.16 
Total polyunsaturated fatty acids (PUFA) 23.36 ± 0.25 
ω -3 PUFA 18.99 ± 0.23 
ω -6 PUFA 4.37 ± 0.17 
The results indicated that the content of 
saturated fatty acids in the oil recovered from 
yellowfin tuna heads was 44.9% of total fatty 
acids. Palmitic acid was the highest among the 
saturated fatty acids with the content of 29.75% 
followed by stearic acid with 10.28%. The 
content of monounsaturated fatty acids was 
31.74%. The most abundant monounsaturated 
fatty acid was oleic acid with 16.76%. The 
content of polyunsaturated fatty acids was 
23.36% of total fatty acids. Docosahexaenoic 
acid (DHA) was the highest among the 
polyunsaturated fatty acids with 14.56% 
followed by eicosapentaenoic acid (EPA) with 
2.44%. DHA and EPA are known as essential 
fatty acids for human. DHA has an important 
role in the development of the brain and the 
nervous system. EPA plays an important role in 
the prevention of cardiovascular diseases 
(Stansby et al., 1990). The tuna head oil had an 
omega-3 fatty acid content of 18.99% and 
omega-6 fatty acid content of 4.37%. The results 
in this study indicated that the fatty acids with 
high contents in tuna head oil were palmitic 
acid, oleic acid and docosahexaenoic acid. 
Previous studies have shown that the content of 
DHA in the tuna was higher than that in most 
other fish species and that the content of DHA 
was higher than the content of EPA (Stansby et 
al., 1990; Shimada et al., 1997). 
Nguyen Thi My Huong 
1157 
4. CONCLUSIONS 
The tuna head generated from tuna 
processing industry can be utilized as a good 
source for recovery of both proteins and lipids. 
Enzymatic hydrolysis of tuna head with 
Protamex for 120 minutes is suitable for 
production of high quality products that can be 
used for different applications. The tuna head 
protein hydrolysate produced was rich in 
essential amino acids and could be applied in 
food for human or feed for shrimp and fish. The 
produced tuna head oil was rich in DHA and 
EPA. Tuna head oil can be used in aquaculture 
feeds or could be used as an ingredient in food 
industries after when refined. 
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