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Chinese Journal of Chemical Engineering, 16(3) 456—460 (2008)

Amino Acids Production from Fish Proteins Hydrolysis in


Subcritical Water*

ZHU Xian (朱宪)**, ZHU Chao (朱超), ZHAO Liang (赵亮) and CHENG Hongbin (程洪斌)
Department of Chemical Engineering, School of Environment and Chemical Engineering, Shanghai University,
Shanghai 201800, China

Abstract The hydrolysis technology and reaction kinetics for amino acids production from fish proteins in sub-
critical water reactor without catalysts were investigated in a reactor with volume of 400 ml under the conditions of
reaction temperature from 180-320ºC, pressure from 5-26 MPa, and time from 5-60 min. The quality and quantity
of amino acids in hydrolysate were determined by bioLiquid chromatography, and 17 kinds of amino acids were
obtained. For the important 8 amino acids, the experiments were conducted to examine the effects of reaction tem-
perature, pressure and time on amino acids yield. The optimum conditions for high yield are obtained from the ex-
perimental results. It is found that the nitrogen and carbon dioxide atmosphere should be used for leucine, isoleu-
cine and histidine production while the air atmosphere might be used for other amino acids. The reaction time of 30
min and the experimental temperature of 220ºC, 240ºC and 260ºC were adopted for reaction kinetic research. The
total yield of amino acids versus reaction time have been examined experimentally. According to these experimental
data and under the condition of water excess, the macroscopic reaction kinetic equation of fish proteins hydrolysis
was obtained with the hydrolysis reaction order of 1.615 and the rate constants being 0.0017,0.0045 and 0.0097 at
220ºC, 240ºC and 260ºC respectively. The activation energy is 145.1 kJ·mol-1.
Keywords biomass, subcritical water, hydrolysis, reaction kinetics, amino acids

1 INTRODUCTION (2) 18 kinds of pure amino acid reagent (bio-


chemical reagent grade): Shanghai Kangda Amino
China is the largest market of fishery in the world, Acid Factory.
and there is approximately 40% ocean marine products (3) Hydrochloric acid 36% (by mass) AR grade.
processed in China [1], but the fish proteins utilization (4) AAA-Direct amino acid analysis apparatus:
ratio is less 30%. Besides, 40%-45% wastes can be DIONEX Co., USA.
produced in fishery processing, which means that a large (5) HL-F (0.2L+1.5MG)/30MPa-IIA super-critical
amount of biomass is discarded as waste. These wastes water equipment: Hangzhou Huali Pump Co. (Fig. 1);
also contain a lot of proteins and bio-active matter [2]. reaction temperature from room temperature to 550°C;
The chemical properties of super(sub)critical reaction pressure, 0-35MPa; capacity, 200-1300 ml.
water are similar with acetone, and its ionic product is (6) Electronic scale AB104N: Mettler Toledo Co.,
over thousand fold that of normal water. So, it plays Shanghai.
the role of catalyst as acid or alkali without any envi-
ronmental pollution [3-7]. The biomass can be hydro- 2.2 Subcritical water hydrolysis
lyzed into high value industrial raw material: amino
acid, unsaturated fatty acid (DHA, EPA, etc.), oil, The experimental flow chart is depicted in Fig. 1.
polysaccharide and so on. Yoshida et al. [8] studied The reactor was filled by chosen reaction atmosphere
hydrolysis of fish for producing amino acids by using (nitrogen, air or carbon dioxide) at 0.15 MPa. Then
a set of stainless steel tube with 5 ml capacity under put quantitative deionized water (about 200 ml) into
protection of argon. In this article, we investigated reactor and set reaction temperature for thermostat.
hydrolysis of fish proteins in a super(sub)critical water The fish meat emulsion was prepared with a colloidal
reactor with 400 ml capacity to produce amino acid. mill to get the homogeneous milky sample at the con-
These hydrolysis experiments were studied under the centration of 100 g meat per liter. When the tempera-
atmosphere of air, nitrogen or carbon dioxide instead ture and pressure of reactor reached to the preset val-
of argon to reduce the cost of industrial production. ues, fish proteins emulsion sample was injected into
Under the condition of water excess, the macroscopic reactor by high pressure metering pump rapidly. Al-
reaction kinetics were obtained for fish proteins hydroly- though no stirring was applied, the mixture was in
sis. These results are very useful for industrialization. boiling-like status under the subcritical state. The
timer started after injection, and sampling was con-
2 EXPERIMENTAL ducted at regular interval for analysis.

2.1 Materials 2.3 Hydrochloric acid hydrolysis

(1) Fish meat: purchased from market. The fish proteins hydrolysis was carried at 108°C

Received 2007-09-25, accepted 2008-03-01.


* Supported by the National Natural Science Foundation of China (50578091) and Shanghai Leading Academic Discipline Project
(T-105).
** To whom correspondence should be addressed. E-mail: xzhu@staff.shu.edu.cn
Chin. J. Chem. Eng., Vol. 16, No. 3, June 2008 457

Figure 1 Flow chart of sub-critical water hydrolysis experimental apparatus


1,2—feeding vessel; 3—reaction atmosphere bottle; 4,5—pump; 6,7—water tank; 8—pressure reactor; 9—feeding funnel;
10—sampling device; 11—cooling device; 12—collector

Figure 2 Compare of amino acid chromatogram between standard and sample hydrolysate of fish proteins
a—arginine; b—lysine; c—alanine; d—threonine; e—glycine; f—valine; g—proline; h—serine; i—isoleucine; j—leucine;
k—methionine; l—histidine; m—phenylalanine; n—glutamic acid; o—aspartate; p—cystine; q—tyrosine; r—tryptophan

for 28 h in 20% (by mass) HCl solution. The total


amino acid yield in hydrolysate was taken as the theo-
retical total amino acids yield after entirely hydrolyzed.

2.4 Amino acid analysis

The quantitative determination of the amino acids


was determined by BioLC (Amino Acid Analyzer,
DIONEX, USA). Comparison of amino acid chroma-
togram between 18 kinds of amino acid standard sam-
ples and hydrolysate sample of fish proteins was Figure 3 Effect of reaction temperature on amino acid
yield (5 MPa, 30 min)
shown in Fig. 2. ◄ tyrosine; ► arginine; ◆ alanine; cystine; ■ isoleucine;
● leucine; ▲ histidine; ▼ phenylalanine

3 RESULTS AND DISCUSSION


3.1 Reaction temperature pendent with temperature. This is perhaps because of
decomposition of amino acid in high temperature [9].
There is a maximum yield for each amino acid, but the
Figure 3 shows that the relationship of amino acid corresponding temperature is different from each other.
yield with reaction temperature is different for different
kinds of amino acid under the same reaction time and
pressure. The yield of amino acid in hydrolysate rises 3.2 Reaction time
with increasing temperature at first, then decreases,
except cystine whose yield seems very low and inde- Figure 4 shows that the yield of amino acids in
458 Chin. J. Chem. Eng., Vol. 16, No. 3, June 2008

Figure 4 Effect of reaction time on amino acid yield in Figure 5 Effect of pressure on amino acid yield in hydro-
hydrolysate (5 MPa, 260°C) lysate (260°C, 30 min)
◄ tyrosine; ► arginine; ◆ alanine; cystine; ■ isoleucine; ◄ tyrosine; ► arginine; ◆ alanine; cystine; ■ isoleucine;
● leucine; ▲ histidine; ▼ phenylalanine ● leucine; ▲ histidine; ▼ phenylalanine

hydrolysate rises with increasing reaction time at first, tyrosine and phenylalanine may be in air.
then decreases a little, except cystine which is like It is found that amino acids could be produced in
independent with reaction time. air, nitrogen or carbon dioxide, and it is much cheaper
than other methods of hydrolysis for breaking down
biomass which require expensive argon gas. This im-
3.3 Reaction pressure provement can help in industrial conversion of bio-
mass into a useful resource.
Figure 5 shows that the effect of pressure on
yield of amino acids in hydrolysate is not very marked 4 HYDROLYSIS KINETICS
as compared with temperature and time.
Biomass hydrolysis kinetics in super
3.4 Contrast of different atmosphere results (sub)-critical water have been studied [10-12]. Hy-
drolysis kinetics of fish proteins in sub-critical water
Figure 6 shows that the effect of different reac- was researched in this article.
tion atmosphere on different amino acid yield in hy-
drolysate is different. No matter whatever atmosphere 4.1 Kinetics formula of fish proteins hydrolysis
is used, there is a given temperature for maximum
yield of amino acid in hydrolysate. Fig. 6 suggest that
leucine, histidine and isoleucine should be hydrolyzed It is very difficult to analyze the fish protein, but
in atmosphere of nitrogen or carbon dioxide, while very easy to determine the total yield of amino acids

(a) Leucine (b) Tyrosine (c) Histidine

(d) Isoleucine (e) Phenylalanine


Figure 6 The amino acid yield in hydrolysate of fish proteins versus temperature under nitrogen (■), air (●), carbon dioxide
(▲) atmosphere respectively
Chin. J. Chem. Eng., Vol. 16, No. 3, June 2008 459

in hydrolysate at different reaction time by using


AAA-Direct. The amino acid yield rate X at any time
can be defined as:
X = M (a)t / M ( a )0 (1)
where M(a)t is the total amount of amino acids in hy-
drolysate at different reaction time, M(a)0 the total
amount of amino acids in hydrolysate of fish proteins
entire hydrolysis by using hydrochloric acid. So, the
fraction of remainder fish proteins at any time is 1 − X .
The hydrolysis of fish proteins is as follows: Figure 7 ( 1 − X ) changing with reaction time under dif-
ferent temperatures
fish proteins + water ⎯⎯→ amino acid + other products
K
■ 220°C; ● 240°C; ▲ 260°C
(2)
So, the hydrolysis kinetic equation may be expressed as Table 2 The values of k, lnk and −1/RT under
d (1 − X ) / dt = − K (1 − X ) [ H 2 O ]
a b different temperatures
(3)
T k/min
-1
lnk −1/ RT
in which t is the reaction time (s), K the hydrolysis
rate constant, and a, b are the reaction order. 220°C 0.0017 -6.37713 0.000202
In this experiment, the water is much more ex- 240°C 0.0045 -5.40368 0.000196
cessive, so [H2O]b can be set as a constant to be in-
corporated into K. So Eq. (3) can be turned into Eq. (4): 260°C 0.0097 -4.63563 0.000190

d (1 − X ) / dt = − k (1 − X )a (4)
Integrating Eq. (4) leads to Eq. (5):
values under different temperature are in Table 2. The
X = 1 − [1 − k (1 − a)t ]
1/(1− a )
(5) relationship between lnk and −1/ RT is shown in Fig. 8.
Ea is 145.1 kJ·mol 1 and the pre-exponential factor is

According to the Arrhenius equation : 9 -1 0.615 -1


9.476×10 (mg·g ) ·s .
ln k = − Ea / RT + ln A (6)
where k is the hydrolysis rate constant, Ea the active
energy, and A the pre-exponential factor.
The values of a and k can be obtained by non-linear
numerical fitting of experimental data to Eq. (5). Ea and
A may be obtained from linear plot of lnk versus 1/T.

4.2 Kinetics parameters

(1 − X ) values changing with reaction time under


different temperature are showed in Table 1. The ef-
fect of reaction time on (1 − X ) at different tempera-
tures is showed in Fig. 7. Figure 8 lnk versus ( −1/RT )

Table 1 ( 1 − X ) values changing with reaction time under


5 CONCLUSIONS
different temperatures
1− X (1) Different amino acid shows different rela-
t/min tionship between reaction temperature and amino acid
220 °C 240 °C 260 °C
yield, even under the same reaction time and pressure.
1 0.933 0.848 0.728 There is a maximum yield for each amino acid, but the
3 0.888 0.798 0.548 corresponding temperature is different from each other.
(2) Reaction atmosphere may be carbon dioxide,
5 0.812 0.701 0.383
nitrogen and air. Leucine, histidine and isoleucine
10 0.809 0.677 0.348 should be hydrolyzed in atmosphere of nitrogen or
15 0.729 0.650 0.251 carbon dioxide. The others can be hydrolyzed in at-
20 0.712 0.623 0.239
mosphere of air.
(3) The experimental results show that the hy-
25 0.706 0.549 0.150 drolysis reaction order is 1.615 and the velocity con-
stants are 0.0017,0.0045 and 0.0097 min 1 at 220℃,

240℃ and 260℃ respectively. The activation energy


is 145.1 kJ·mol 1 and the Arrhenius pre-exponential

It is found that the hydrolysis reaction order is
factor is 9.476×109(mg·g 1)0.615·s 1.
- -
1.615, and the reaction rate constant k, lnk and −1/RT
460 Chin. J. Chem. Eng., Vol. 16, No. 3, June 2008

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