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THOÂNG BAÙO KHOA HOÏC
ENZYMATIC HYDROLYSIS OF TRASH FISH (HERRING FISH ARDINELLA GIBBOSA) USING FLAVOURZYME ENZYME
NGHIÊN CỨU ỨNG DỤNG ENZYME FLAVOURZYME THỦY PHÂN CÁ TRÍCH
(SARDINELLA GIBBOSA)
Nguyễn Thị Vân1, Đỗ Thị Thanh Thủy2
Ngày nhận bài: 10/3/2014; Ngày phản biện thông qua: 11/3/2014; Ngày duyệt đăng: 02/6/2014
ABSTRACT
The hydrolysis of herring for production of protein hydrolysate was studied. The hydrolysis process was carried
out using Flavourzyme enzyme at natural pH with a water/material ratio of 1:1. The results show that the appropriate
hydrolysis conditions were: temperature 550C, the ratio of enzyme-substrate 38.96 LAPU/g protein, hydrolysis time is 6
hours. The hydrolysis degree, the nitrogen recovery and total evaporation bases nitrogen obtained in the hydrolysis protein
solution are respectively 50.58%, 63.38% and 0.92 g/l. The resulting product contains 11.79 g/l amino acid nitrogen, have
the potential applications in many fields.
Keywords: Enzymatic hydrolysis, protein hydrolysis, herring fish, Flavourzyme
TÓM TẮT
Dịch đạm hòa tan đã được nghiên cứu sản xuất từ protein cá trích bằng enzyme Flavourzyme ở pH tự nhiên với tỷ
lệ nước/nguyên liệu là 1/1. Kết quả nghiên cứu cho thấy điều kiện thủy phân thích hợp là: nhiệt độ 55oC, tỷ lệ enzyme - cơ
chất 38.96 LAPU/g protein, thời gian 6 giờ. Độ thủy phân, hiệu suất thu hồi nitơ và tổng nitơ bazơ bay hơi trong dịch thủy
phân thu được lần lượt đạt là 50.58%, 63.38% và 0.92 g/l. Sản phẩm thu được chứa hàm lượng nitơ a xít amin 11.79 g/l,
có tiềm năng ứng dụng trong nhiều lĩnh vực.
Từ khóa: thủy phân, dịch đạm thủy phân, cá trích, Flavourzyme
I. INTRODUCTION
Herring is a species of trash fish that caught in
large amount - account for about 16.46% of the total
trash fish exploitation. Currently, herring used only to
eat fresh, produced dried products, used in animal
feed, a few exported as frozen bait, create low
economic efficiency. Research to create value-added
products from plentiful source of herring fish to
improve the efficiency of natural resource, bring
more income to the people are very necessary.
There is a great potential in marine bioprocess
industry to convert this kind of trash fish into
value-added products. Enzymatic hydrolysis is one
of the most efficient methods to recover proteins from
trash fish and to produce the protein hydrolysates.
1
The research to find out the appropriate mode
hydrolysis to produce soluble protein fluid rich in
amino acids from herring is a feasibility idea. The
potential use of fish protein hydrolysate in food and
feed was also reported by several authors (Refstie
et al., 2004; Nguyen et al., 2012). Production of
protein hydrolysate produced from herring
protein could generate significant revenue for fish
processing industry.
II. MATERIALS AND METHODS
1. Materials
1.1. Herring
The herring fish (Sardinella Gibbosa) are
purchased in Hon Ro ports - Nha Trang City.
ThS. Nguyễn Thị Vân, 2 ThS. Đỗ Thị Thanh Thủy: Khoa Công nghệ thực phẩm – Trường Đại học Nha Trang
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Fish was fresh, bright, no stains lesions, size 19 - 20
units/kg. Fish are washed, removing impurities,
storage and transport to the laboratory in insulation
foam box at 0 - 400C. At the laboratory, to ensure
uniformity, fish were washed, drained, minced,
mixed thoroughly, divided, and packaged in vacuum
PA bags, frozen and stored at -20 ± 20C
1.2. Flavourzyme Enzyme
Flavourzyme protease enzyme was produced by
Novozymes A/S (Bagsvaerd Denmark). Flavourzyme
Herring fish
Hydrolysis
Inhibit enzyme
Dregs
Filter
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including the activity of endopeptidase and
exopeptidase but mostly exopeptidase, derived from
Aspergillus oryzae (Kamnerdpetch et al., 2007).
The optimal working conditions for Flavourzyme
are reported to be at pH of 5.0 - 7.0 and at a
temperature of 50 - 550C. Flavourzyme has a
declared activity of 500 LAPU (Leucine
aminopeptidase Units)/g.
2. Research Methodologies
2.1. Process diagram and experimental design
Hydrolysis conditions
of Flavourzyme enzyme
Determine the effect of temperature:
45 ÷ 65oC, δ = 5oC
Determine the effect of E/S:
9.74 ÷ 48.7 LAPU/g protein
Determine the effect of time: 3 - 7
hours, δ = 1 hour
Natural pH of the fish, W/NL =1/1
The filtrate
Lipid
Suspended solids
Centrifugal
Dregs
Hydrolysis solution
Figure 1. Process diagram and experimental design
- Materials: the samples were collected and
processed as Section 1.1, defrost at 0 - 40C, in
15 hours.
- Identify appropriate hydrolysis temperature:
set th = 45 - 650C, δ = 50C and the ratio of
enzyme-substrate (E/S) = 9.74 LAPU/g protein,
hydrolysis time is 3 hours, W/NL = 1/1, the natural
pH of the fish.
- Identify the ratio of enzyme-substrate: set
E/S = 9.74 - 48.7 LAPU/g protein, δ = 9.74 LAPU/g
protein, the temperature found in previous
experiments, the time 3 hours, W/NL = 1/1, the
natural pH of the fish.
- Identify suitable hydrolysis time: set T= 3 - 7
hours, δ = 1 hour; temperature, E/S found in two
previous experiments, W/NL = 1/1, the natural pH
of the fish.
- The enzyme was inactivated by heating at
950C for 15 minutes in a water bath.
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2.2. Chemical analyses
Water content: TCVN 3700-90; Ash content:
calcined at 6000C methods; lipid content: according
to TCVN 3703:2009, the total nitrogen content:
TCVN 3705-90; NH3 and TVB-N content: TCVN
9215:2012; amino acid nitrogen: formol method;
amino acid composition: HPLC, hydrolyzsis degree
DH: DNFB method; The nitrogen recovery in protein
hydrolysate was calculated according to Liaset et
al., 2002.
2.3. Statistical analyses
Each experiment was performed three times
in parallel, each time test three samples. Data
were processed with SPSS 16.0 statistical
software, the Microsoft Office Excel 2007
software was used to calculate results. Differences
in treatment means were considered significant
at p < 0.05.
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III. RESULTS AND DISCUSSION
1. Chemical compositions of herring
Chemical compositions of herring are shown in
table 1.
Table 1. Chemical compositions of herring
Component
Percentage (%) compared to wet weight
Water
73.87 ± 0.01
Protein
19.25 ± 0.04
Lipid
2.53 ± 0.03
Ash
0.96 ± 0.01
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Table 1 showed that the herring have high total
protein content (19.25%) higher than cuttlefish
(17 - 21%), much higher than some other species
such as oysters (8 - 9%), squil (13 - 16%) and sea
snail (11 - 12%), low lipid content (2.53%), classified
as lean fish and are suitable for the production of
hydrolysed protein solution.
2. Determine the appropriate parameters for
the hydrolysis of herring protein by Flavouyme
enzyme
2.1. Affect of the temperature to efficiency of the
hydrolysis
Figure 2. Effect of temperature on the hydrolysis degree
(DH) and nitrogen recovery efficiency (NRE)
Figure 3. Effect of temperature
to the rate of Naa/NT and TVB-N of hydrolysed protein
Figures 2 and 3 show that, when the temperature
rose from 450C to 550C, then 3 indicators DH, NRE
and Naa/NT increased and peaked at 55°C; if the
temperature continues to rise to 650C, the measured
indices were reduced. But TVB-N concentration
decreased continuously as the temperature increases
from 450C to 650C. This can be explained as follows:
when the temperature increases from 450C to 550C,
the activity of Flavourzyme enzyme increased due
to the activation energy of the reaction is enhanced,
then further increase in temperature up to 65oC, the
activity of Flavourzyme enzyme decreased because
high temperature inhibits the activity of Flavourzyme
enzyme. When the temperature increases from
45 - 650C, the activity of microbial flora reduced
because the microbiota in herring active at topt less than
450C, when the temperature increases from 45 - 650C
their activity are inhibited, leading to the TVB-N
concentration decreases with hydrolysis temperature.
At 550C (DH, NRE, Naa/NT and TVB-N
corresponding in the order of 32.08%, 51.00%,
44.94% and 0.53 g/l) achieved the best results in
experimental conditions and should be selected as
appropriate temperature to proteolytic herring.
2.2. Affect of the ratio of enzyme-substrate (E/S) to
hydrolysis efficiency of Flavourzyme enzyme
Figure 4. Affect of the rate of enzyme - substrate to the
hydrolysis degree (DH) and nitrogen recovery efficiency (NRE)
Figure 5. Affect of the rate of enzyme - substrate to the ratio
of Naa/NT and TVB-N of hydrolysed protein
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Effect of the rate of E/S to the efficiency of the
Motamedzadegan et al., 2010). This can be
hydrolysis shows in figures 4 and 5, the indicators
explained: When increasing E/S from 0 - 38.96
DH, NRE and Naa/NT of hydrolysed protein
LAPU/g protein, protein hydrolysed (disconnected
solution increased when the rate of E/S increases from
0 - 38.96 LAPU/g protein, continue to increase E/S to
48.70 LAPU/g protein, the indicators did not increase
significantly, according to the statistical analysis, the
difference was not significant (p > 0.05). For TVB-N
indicator, E/S increases from 0-29.22 LAPU/g
protein, the TVB-N increased strongly, then did not
increase significantly when further increasing E/S
to 48.70 LAPU/g protein. The previous study also
showed that the soluble nitrogen under the action
polypeptide) occurs strongly by the excess substrate,
leading to increasing DH, pulling NRE and Naa/NT
increased. Then continue to increase E/S, the
velocity of hydrolysis is little changed since the
concentration of enzyme saturation with substrate
concentration. Ratio E/S = 38.96 LAPU/g protein
(corresponding to DH = 60.17%; NRE = 60.17%;
Naa/NT = 54.65% and TVB-N = 0.69 g/l) gave the
most satisfying results of the conditions set out
of enzymes in the hydrolysis and nitrogen recovery
so should be selected as appropriate rate for the
rate of hydrolysis products, as well as the severing of
hydrolysis of herring protein.
peptide links increases when the enzyme concentr
2.3. Effect of time to the efficiency of the hydrolysis
tion increase (Wachirattanapongmetee et al., 2009;
by the Flavourzyme enzyme
Figure 6. Effect of the time to the hydrolysis degree (DH)
and nitrogen recovery efficiency (NRE)
Figure 7. Effect of the time to the rate of Naa/NT and TVB-N
of hydrolysed protein
Figure 6 and 7 showed that, when the hydrolysis time increased from 3 to 7 hours, all indicators
DH, NRE, Naa/NT, TVB-N increased. However, after 6 hours, the mentioned indicators did not increase
significantly (p < 0.05). For TVB-N, this indicator increased with hydrolysis time. The previous study also
showed that DH increased over time hydrolyzed (Souissi et al., 2007; Chun et al., 2006; Amiza et al.,
2012; Wachirattanapongmetee et al., 2009; Ovissipour et al., 2010; Shamloo et al., 2012). This is
explained as follows: Time hydrolytic should be able to ensure for enzyme cut the link in the substrate,
creating the
desired end products met the target to the research. Prolonged duration of action, the
enzyme hydrolyzed herring protein thoroughly But, if prolonged hydrolysis process will facilitate the microbial
activities that cause rotten produce more low-end products such as NH3, H2S, indole, scaptol... that affecting
product quality.
6 hours of hydrolysis time shows best results, satisfy the set conditions so should be chosen as the
appropriate time to proteolytic herring by Flavourzyme enzyme, corresponding to DH = 50.57%; NRE = 62.32%;
Naa/NT = 69.55% and TVB-N = 0.90 g/l.
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3. Determine the effectiveness of the hydrolysis mode
Apply appropriate hydrolysis mode, results are shown in table 2 and 3.
Table 2. Evaluation the quality of hydrolysed protein of herring
No.
1
2
Indicators
Sensory
Chemistry
Results
Colors
Pale yellow
Odor
Characteristic odor of hydrolyzed protein solution, pleasant
Taste
Slightly bitter
Status
Pure fluid
Naa
11.79 g/l
NT
16.84 g/l
DH
50.58%
NRE
63.38%
TVB-N
0.92 g/l
Table 2 shows that, the color of protein hydrolysate solution is pale yellow, paler than that of fish sauce
produced under traditional methods, pleasant smell, TVB-N/NT (5.46%). Compared with the Vietnamese
standards 5107:2003 of special fish sauce product about ammonia nitrogen content and total soluble protein,
the hydrolysed protein can fully studied and put to use in the food sector.
Table 3. Amino acid composition of the protein hydrolysate solution
Amino acids
Content (g/l)
Amino acids
Content (g/l)
Aspartic
1.13
Tyrosine
0.44
Serine
0.91
Valine*
0.76
Glutamine
0.35
Methionine*
0.65
Glycine
0.26
Lysine*
1.38
Histidine*
0.82
Isoleucine*
0.12
Arginine
1.20
Leucine*
0.71
Threonine*
0.73
Phenylalanine*
0.75
Alanine
0.56
TAA
11.02
Proline
0.11
TEAA
5.71
Cysteine
0.14
TEAA/TAA
51.81
(*) Essential amino acids, TAA (Total amino acids), TEAA (Total essential amino acids)
Results of amino acid composition analysis in Table 3 show that herring protein hydrolysates solution have
high nutritional value, rich in essential amino acids (51.81%). The amino acid accounts for high content in
herring protein hydrolysates solution is: Lysine (1.38 g/l), Arginine (1.20 g/l), Aspartic (1.13 g/l), this result
coincides with research of scad hydrolysis by Flavourzyme (Chun et al., 2006). A number of other studies on
hydrolyzed tuna head (Nguyen et al., 2012) and hydrolysis salmon head (Sathivel et al., 2005) also showed that
essential amino acids content in hydrolyzed protein obtained from these studied were quite high.
IV. CONCLUSION
Herring fish could be hydrolysed by Flavourzyme enzyme at appropriate conditions: hydrolysis temperature
(550C); ratio of E/S 38.96 LAPU/g protein; and hydrolysis time is 6 hours. Enzymatic hydrolysis of herring fish is
suitable for production of protein hydrolysate that can be used for different applications. The resulting product
can be well applied in the food industry to produce more type sauce and nutritional supplements for a variety
of other foods.
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