Amino Acids Production from Fish Proteins Hydrolysis in Subcritical Water*

Chinese Journal of Chemical Engineering, 16(3) 456ü460 (2008)

Amino Acids Production from Fish Proteins Hydrolysis in Subcritical Water* ZHU Xian (ᅋຩ)**, ZHU Chao (ᅋб), ZHAO Liang (ვ४) and CHENG Hongbin (ё‫܀‬μ)

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 subcritical water reactor without catalysts were investigated in a reactor with volume of 400 ml under the conditions of reaction temperature from 180320ºC, pressure from 526 MPa, and time from 560 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 temperature, pressure and time on amino acids yield. The optimum conditions for high yield are obtained from the experimental results. It is found that the nitrogen and carbon dioxide atmosphere should be used for leucine, isoleucine 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 0.0017ˈ0.0045 and 0.0097 at 220ºC, 240ºC and 260ºC respectively. The activation energy is 145.1 kJǜmolˉ1. Keywords biomass, subcritical water, hydrolysis, reaction kinetics, amino acids

1

INTRODUCTION

China is the largest market of fishery in the world, and there is approximately 40% ocean marine products processed in China [1], but the fish proteins utilization ratio is less 30%. Besides, 40%45% wastes can be produced in fishery processing, which means that a large amount of biomass is discarded as waste. These wastes also contain a lot of proteins and bio-active matter [2]. The chemical properties of super(sub)critical water are similar with acetone, and its ionic product is over thousand fold that of normal water. So, it plays the role of catalyst as acid or alkali without any environmental pollution [37]. The biomass can be hydrolyzed into high value industrial raw material: amino acid, unsaturated fatty acid (DHA, EPA, etc.), oil, polysaccharide and so on. Yoshida et al. [8] studied hydrolysis of fish for producing amino acids by using a set of stainless steel tube with 5 ml capacity under protection of argon. In this article, we investigated hydrolysis of fish proteins in a super(sub)critical water reactor with 400 ml capacity to produce amino acid. These hydrolysis experiments were studied under the atmosphere of air, nitrogen or carbon dioxide instead of argon to reduce the cost of industrial production. Under the condition of water excess, the macroscopic reaction kinetics were obtained for fish proteins hydrolysis. These results are very useful for industrialization. 2

EXPERIMENTAL

2.1

Materials (1) Fish meat: purchased from market.

(2) 18 kinds of pure amino acid reagent (biochemical reagent grade): Shanghai Kangda Amino Acid Factory. (3) Hydrochloric acid 36% (by mass) AR grade. (4) AAA-Direct amino acid analysis apparatus: DIONEX Co., USA. (5) HL-F (0.2L+1.5MG)/30MPa-IIA super-critical water equipment: Hangzhou Huali Pump Co. (Fig. 1); reaction temperature from room temperature to 550°C; reaction pressure, 035MPa; capacity, 2001300 ml. (6) Electronic scale AB104N: Mettler Toledo Co., Shanghai. 2.2

Subcritical water hydrolysis

The experimental flow chart is depicted in Fig. 1. The reactor was filled by chosen reaction atmosphere (nitrogen, air or carbon dioxide) at 0.15 MPa. Then put quantitative deionized water (about 200 ml) into reactor and set reaction temperature for thermostat. The fish meat emulsion was prepared with a colloidal mill to get the homogeneous milky sample at the concentration of 100 g meat per liter. When the temperature and pressure of reactor reached to the preset values, fish proteins emulsion sample was injected into reactor by high pressure metering pump rapidly. Although no stirring was applied, the mixture was in boiling-like status under the subcritical state. The timer started after injection, and sampling was conducted at regular interval for analysis. 2.3

Hydrochloric acid hydrolysis 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: [email protected]

Chin. J. Chem. Eng., Vol. 16, No. 3, June 2008

457

Figure 1 Flow chart of sub-critical water hydrolysis experimental apparatus 1,2üfeeding vessel; 3üreaction atmosphere bottle; 4,5üpump; 6,7üwater tank; 8üpressure reactor; 9üfeeding funnel; 10üsampling device; 11ücooling device; 12ücollector

Figure 2 Compare of amino acid chromatogram between standard and sample hydrolysate of fish proteins aüarginine; bülysine; cüalanine; düthreonine; eüglycine; füvaline; güproline; hüserine; iüisoleucine; jüleucine; kümethionine; lühistidine; müphenylalanine; nüglutamic acid; oüaspartate; pücystine; qütyrosine; rütryptophan

for 28 h in 20% (by mass) HCl solution. The total amino acid yield in hydrolysate was taken as the theoretical 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 chromatogram between 18 kinds of amino acid standard samples and hydrolysate sample of fish proteins was shown in Fig. 2. 3 3.1

Figure 3 Effect of reaction temperature on amino acid yield (5 MPa, 30 min) Ż tyrosine; Ź arginine;ƹalanine; cystine;Ƶisoleucine; ƽ leucine;Ʒhistidine;ͩphenylalanine

RESULTS AND DISCUSSION Reaction temperature

Figure 3 shows that the relationship of amino acid 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 with increasing temperature at first, then decreases, except cystine whose yield seems very low and inde-

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 corresponding temperature is different from each other. 3.2

Reaction time Figure 4 shows that the yield of amino acids in

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Chin. J. Chem. Eng., Vol. 16, No. 3, June 2008

Figure 4 Effect of reaction time on amino acid yield in hydrolysate (5 MPa, 260°C) Ż tyrosine; Ź arginine;ƹalanine; cystine;Ƶisoleucine; ƽ leucine;Ʒhistidine;ͩphenylalanine

Figure 5 Effect of pressure on amino acid yield in hydrolysate (260°C, 30 min) Ż tyrosine; Ź arginine;ƹalanine; cystine;Ƶisoleucine; ƽ leucine;Ʒhistidine;ͩphenylalanine

hydrolysate rises with increasing reaction time at first, then decreases a little, except cystine which is like independent with reaction time.

tyrosine and phenylalanine may be in air. It is found that amino acids could be produced in 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 improvement can help in industrial conversion of biomass into a useful resource.

3.3

Reaction pressure

Figure 5 shows that the effect of pressure on yield of amino acids in hydrolysate is not very marked as compared with temperature and time. 3.4 Contrast of different atmosphere results Figure 6 shows that the effect of different reaction atmosphere on different amino acid yield in hydrolysate is different. No matter whatever atmosphere 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 in atmosphere of nitrogen or carbon dioxide, while

(a) Leucine

4

HYDROLYSIS KINETICS

Biomass hydrolysis kinetics in super (sub)-critical water have been studied [1012]. Hydrolysis kinetics of fish proteins in sub-critical water was researched in this article. 4.1

Kinetics formula of fish proteins hydrolysis

It is very difficult to analyze the fish protein, but very easy to determine the total yield of amino acids

(b) Tyrosine

(c) Histidine

(d) Isoleucine (e) Phenylalanine Figure 6 The amino acid yield in hydrolysate of fish proteins versus temperature under nitrogen (Ƶ), air (ƽ), carbon dioxide (Ʒ) atmosphere respectively

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Chin. J. Chem. Eng., Vol. 16, No. 3, June 2008

in hydrolysate at different reaction time by using AAA-Direct. The amino acid yield rate X at any time can be defined as: X

(1)

M (a)t / M  a 0

where M(a)t is the total amount of amino acids in hydrolysate 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: K

fish proteins + water o amino acid other products (2) So, the hydrolysis kinetic equation may be expressed as a

b

 K 1  X > H 2 O @

d 1  X / dt

Figure 7 ( 1  X ) changing with reaction time under different temperatures Ƶ 220°C;ƽ240°C;Ʒ260°C Table 2

(3) T

in which t is the reaction time (s), K the hydrolysis rate constant, and a, b are the reaction order. In this experiment, the water is much more excessive, so [H2O]b can be set as a constant to be incorporated into K. So Eq. (3) can be turned into Eq. (4): d 1  X / dt

 k 1  X a 1/(1 a )

1  >1  k (1  a)t @

(5)

According to the Arrhenius equation :

ln k

k/min

ˉ1

lnk

1/ RT

220°C

0.0017

ˉ6.37713

0.000202

240°C

0.0045

ˉ5.40368

0.000196

260°C

0.0097

ˉ4.63563

0.000190

(4)

Integrating Eq. (4) leads to Eq. (5): X

The values of k, lnk and 1/RT under different temperatures

 Ea / RT  ln A

values under different temperature are in Table 2. The relationship between lnk and 1/ RT is shown in Fig. 8. ˉ Ea is 145.1 kJǜmol 1 and the pre-exponential factor is ˉ1 0.615 ˉ1 9 9.476×10 (mg·g ) ǜs .

(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 effect of reaction time on (1  X ) at different temperatures is showed in Fig. 7. Table 1

( 1  X ) values changing with reaction time under different temperatures

t/min

1 X 220 °C

240 °C

260 °C

1

0.933

0.848

0.728

3

0.888

0.798

0.548

5

0.812

0.701

0.383

10

0.809

0.677

0.348

15

0.729

0.650

0.251

20

0.712

0.623

0.239

25

0.706

0.549

0.150

It is found that the hydrolysis reaction order is 1.615, and the reaction rate constant k, lnk and 1/RT

Figure 8 5

lnk versus ( 1/RT )

CONCLUSIONS

(1) Different amino acid shows different relationship between reaction temperature and amino acid yield, even under the same reaction time and pressure. There is a maximum yield for each amino acid, but the corresponding temperature is different from each other. (2) Reaction atmosphere may be carbon dioxide, nitrogen and air. Leucine, histidine and isoleucine should be hydrolyzed in atmosphere of nitrogen or carbon dioxide. The others can be hydrolyzed in atmosphere of air. (3) The experimental results show that the hydrolysis reaction order is 1.615 and the velocity conˉ stants are 0.0017ˈ0.0045 and 0.0097 min 1 at 220ć, 240ć and 260ć respectively. The activation energy ˉ is 145.1 kJǜmol 1 and the Arrhenius pre-exponential ˉ ˉ factor is 9.476×109(mg·g 1)0.615ǜs 1.

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Chin. J. Chem. Eng., Vol. 16, No. 3, June 2008

REFERENCES 1 2 3 4 5 6

Zhao, Z.X., Zhu, T.Y., Yang, X.H., “The actuality and expectation of marine lives manufacture in China”, J. Hehai Univ., 15 (4), 3034 (2001). (in Chinese) Yoshida, H., Terashima, M., Takahashi, Y., “Production of organic acids and amino acids from fish meat by sub-critical water hydrolysis”, Biotechnol. Prog., 15, 10901094 (1999). Saphier, D., Raymond, P., “Design of highly moderated pressurized water reactor based on critical heat flux considerations”, Nucl. Eng. Des., 163, 263271 (1996). Yutaka, I., “Fundamental properties of supercritical water”, J. Japan Soc. Corros. Eng., 3 (49), 117121 (2000). Zhang, L.L., Chen, L., Zhao, X.F., Yu, J.L., Tian, Y.L., “Super-critical water: Its properties and applied”, J. Chem. Ind. Eng., 20 (1), 3436 (2003). (in Chinese) Yang, J.C., Shen, Z.Y., “Technology of super-critical fluids and its applied in biochemical engineering”, Progr. Chem. Eng., (4), 3438 (1997). (in Chinese)

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Wang, Q., Zhu, X., “Toluene oxidization to benzaldehyde in sub-critical water”, J. Chem. Eng. Chin. Univ., 19 (4), 503506 (2005). (in Chinese) Yoshida, H., Takahashi, Y., Terashima, M., “A simplified reaction model for production of oil, amino acid, and organic acids from fish meat by hydrolysis under sub-critical and supercritical conditions”, J. Chem. Eng. Jpn., 36, 441448 (2003). Sato, N., Armando, T.O., Kang, K., Daimon, H., Fujie, K., “Reaction kinetics of amino acid decomposition in high-temperature and high-pressure water”, Appl. Chem., 43, 38 (2004). Minowa, T., Inoue, S., Hanaoka, T., “Hydrothermal reaction of glucose and glycine as model compounds of biomass”, J. Jpn. Inst. Energy, 10 (83), 794798 (2004). Tim, R., Herrmann, S., Brunner, G., “Production of amino acids from bovine serum albumin by continuous sub-critical water hydrolysis”, J. Supercrit. Fluids, 36, 4958 (2005). Khuwijitjaru, P., Fujii, T., Adachi, S., Kimura, Y., Matsuno, R., “Kinetics on the hydrolysis of fatty acid esters in subcritical water”, J. Chem. Eng., 99, 14 (2004).

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