Amino Acid Composition, Molecular Weight Distribution and Antioxidant Stability of Shrimp Processing Byproduct Hydrolysate

MATERIALS AND METHODS

Materials and chemicals: Shrimp processing byproduct was purchased at autumn 2010 from local Seafood Market, Zhejiang, China. It was defatted, dried and grounded into fine powder, then stored at -20°C until used. Alcalase (250 U mg^-1) was purchased from Shanghai Chemical Reagent Co. Ltd., China. 1,1-diphenyl-2-picrylhydrazyl (DPPH), vitamin B₁₂, aprotinin, cytochrome C and bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, USA). All other chemicals used in the experiments were from commercial resource and of analytical grade.

Preparation of the SPBH: Alcalase was chosen to hydrolysis the shrimp processing byproduct to produce antioxidative peptides (Huang et al., 2011). The hydrolysis conditions were: temperature of 50.5°C, time duration of 120 min, pH 8.5 and enzyme-to-substrate ratio of 415 U g^-1. A 100 g portion of shrimp processing byproduct powder was homogenized in 1 L of distilled water and the homogenate was preheated for 10 min at 50.5°C, then adding 166 mg alcalase to the mixture to begin hydrolysis. During hydrolysis, the mixture was adjusted to pH 8.5 using 1 N NaOH or 1 N HCl every 15 min. At the end of hydrolysis, the solution was boiled for 10 min to inactivate the enzyme and then centrifuged at 3, 500 g for 10 min at 4°C. The supernatant was collected for further study.

Ultrafiltration of SPBH: The supernatant of SPBH was collected and subjected to ultrafiltration membranes system with Molecular Weight Cutoff (MWCO) of 10, 5, 3 and 1 kDa, respectively. Five peptide fractions with different Molecular Weight (MW) ranges were prepared from SPBH: SPBH-I (>10 kDa), SPBH-II (5-10 kDa), SPBH-III (3-5 kDa), SPBH-IV (1-3 kDa) and SPBH-V (<1 kDa). The peptide fractions were lyophilized and stored at -20°C until use.

Determination of protein content: The protein contents of fractions after ultrafiltration of SPBH were determined by Folin-phenol method (Lowry et al., 1951) using Bovine Serum Albumin (BSA) as standard.

DPPH radical scavenging activity: DPPH Radical Scavenging Activity (DSA) was assayed following the method of Wu et al. (2003) with some modifications. The 0.375 mL of each sample solution at different concentrations was added to 2.0 mL 0.1 mmol L^-1 DPPH dissolved in methanol. The mixture was shaken and left for 30 min in dark at room temperature and the absorbance of the resulting solution was measured at 517 nm. The DSA was calculated as a percentage of DPPH discoloration using the Eq. 1:

(1)

where, A_s, A₀ and A_DPPH represent the absorbance of sample, sample blank and control, respectively.

Determination of amino acid composition and molecular weight distribution: The total amino acid composition was determined according to the method of Heu et al. (2003). The SPBH sample was placed in an ampoule and mixed with 6 N HCl. After sealing the ampoule under vacuum, the SPBH was hydrolyzed at 110°C for 24 h. The sample was diluted, filtered and loaded on a Model S433D automatic amino acid analyzer (Sykam Corp., Eresing, Germany) for amino acid analysis. The Molecular Weight (MW) distribution of the antioxidant hydrolysate was analyzed by Gel Permeation Chromatography (GPC) on a GPC column (5 Diol-120-II, 7.5x600 mm) following the method of Dong et al. (2008) with some modifications. Vitamin B₁₂, aprotinin, cytochrome C and bovine serum albumin were used as MW standards. The mobile phase was 10 mmol L^-1 phosphate buffer solution including 100 mmol L^-1 Na₂SO₄ at a flow rate of 1.0 mL min^-1 and the eluent was monitored at 214 nm.

Antioxidant stability
Thermal and pH stability of antioxidant activity: The thermal and pH stability was analyzed following the method of Binsan et al. (2008).

Effect of food additives and metal ions on the antioxidant activity: The effects of food additives and metal ions on the antioxidant activity of SPBH were determined. Food additives (glucose, sucrose, sodium chloride and sodium benzoate) and metal ions solutions (CuSO₄, ZnSO₄, KCl, CaCl₂ and MgCl₂) were used to study the antioxidant stability of SPBH.

Table 1:	Concentrations of food additives and metal ions used in the antioxidant stability experiments
*The metal ions mean Cu2+, Zn2+, K+, Ca2+ and Mg2+, respectively

Sample of SPBH (100 μL) was added to the food additives or metal ions solution (1.9 mL) with various concentrations to the SPBH and the residual DSA was determined. The final concentrations of food additives and metal ions in the stability experiments were listed in Table 1. Distilled water instead of samples was used for control.

Retention activity: The retention activity was calculated as a percentage of DSA using the Eq. 2:

(2)

where, DSA_s and DSA₀ represented the absorbance of sample and control, respectively.

RESULTS AND DISCUSSION

Amino acid composition: The amino acid composition of SPBH was presented in Table 2. The amino acid compositions of SPBH revealed that they were rich in Glu, Asp, Gly and Leu with values of 16.4, 13.4, 8.2 and 7.5%, respectively. It was in good agreement with the data reported by Synowiecki and Al-Khateeb (2000) using commercially available alcalase to recover the protein and chitin from shrimp processing discards. It was also reported that Glu, Asp and Arg tended to predominate in the hydrolysate during autolysis of shrimp head (Cao et al., 2008). The Glu, Asp, Gly and Ala were flavour enhancers which might account for the good taste (Kirimura and Shimizu, 1969). The SPBH also had an extremely high content of flavour amino acids which accounted 44% of the total amino acids. Nevertheless, the sensitive amino acids, such as Met and Try were not detected in SPBH. Shahidi et al. (1995) reported that sensitive amino acids, such as Met and Try, presented in smaller amounts after hydrolysis of capelin proteins. The essential amino acids of SPBH made up 32.2% of all amino acids which was a little lower than the reference values of 40% recommended by Food and Agriculture Organization (FAO/WHO, 2001). The content of hydrophobic amino acids (Pro, Gly, Ala, Val, Ile, Leu and Phe) in SPBH was 40.4%. The high antioxidant activity of SPBH might be relationship to the high level of hydrophobic amino acids. Rajapakse et al. (2005) reported that for protein hydrolysate and peptides, an increasement in hydrophobicity would increase their solubility in lipid and therefore enhanced their antioxidant activity. Peptides derived from many materials with increased hydrophobicity had been reported to relate with antioxidant activity (Chen et al., 1996, 1998; Li et al., 2008). The high level of hydrophobic amino acids might contribute to the bitterness of the hydrolysate. FitzGerald and O’Cuinn (2006) reported that bitterness of protein hydrolysates was associated with the release of peptides containing hydrophobic amino acid residues.

Table 2:	Amino acids composition of SPBH

Table 3:	The protein distribution and DSA of five fractions by ultrafiltration
The values are Mean±SD of triplicate measurements. Values with different letters in the same row are significantly different (p<0.05)

Ultrafiltration of SPBH: The relative protein contents and DSA of five fractions by ultrafiltration membrane were determined as shown in Table 3. The protein was mainly distributed in SPBH-II and SPBH-III fractions and the SPBH-V fraction had the least quantity of protein. The DSA of five fractions (at 500 μg mL^-1 level) were also detected. From the results (Table 3), it was found that SPBH-IV and SPBH-V showed significantly higher DSA (62.78 and 65.11%, respectively) than the other three fractions at significant level p<0.05. SPBH-V with MW lower than 1 kDa exhibited the highest antioxidative activity of all. SPBH-I with the largest molecular weight exhibited the lowest DSA (<40%). However, the high DSA of SPBH might be thought to be due to the low molecular weight, as well as the sequence of peptides. These findings were in agreement with other studies (Dong et al., 2008; Xie et al., 2008) and supported the fact that functional properties of antioxidant peptides were highly influenced by properties such as molecular mass. Many reports had already shown that the antioxidant activity of hydrolysate was depended on their molecular weight distribution (Moure et al., 2006; Zhu et al., 2006; Dong et al., 2008; Li et al., 2008).

All fractions enriched in peptides obtained following the ultrafiltration steps, were analyzed by gel permeation chromatography in order to evaluate their molecular weight distribution. The molecular weight distribution of five fractions by ultrafiltration membrane was determined by gel permeation chromatography. From the HPLC chromatograms (Fig. 1), it was found that ultrafiltraion had the effectiveness of separation by MWCO. The chromatographic data indicated that SPBH-V with the highest antioxidant activity was composed of four major fractions. The molecular weights of four major fractions were around 1.6 kDa, 800, 500 and 300 Da, respectively. In the future studies, the four major fractions should be further purified and characterized.

Antioxidant stability: The thermal stability of antioxidant activity of SPBH was shown in Fig. 2a. The retention activity was decreased with a rise of temperature but activities of all fractions were more than 90% remained. Arcan and Yemenicioglu (2007) also found that antioxidant activities of water-soluble protein extracts from chickpeas indicated a high free radical scavenging capacity and thermostability.

Fig. 1:

Molecular weight distribution of SPBH fractions by gel permeation chromatography

Fig. 2(a-b):

Thermal and pH antioxidant stability of the SPBH. The values are Mean±SD of triplicate measurements. Values with different letters are significantly different (p<0.05)

The effects of pH on the antioxidant stability of the SPBH were shown in Fig. 2b. The acidic pH mostly affected antioxidative activities of SPBH. It could be found that the antioxidant stability of SPBH decreased slightly with the increase of acidity. Acidic condition might change the antioxidant property. Though the SPBH was more stable in alkaline and neutral environment, the retention of antioxidant activity maintained nearly 70% of the original activity in pH 2.0. Binsan et al. (2008) reported that DPPH radical scavenging activity of water soluble fraction from Mungoong decreased slightly when the pH was above 8. The antioxidant activity of water soluble fraction was more than 80% remained when heated up to 100°C. This study showed that SPBH exhibited good thermal stability and was stable at alkaline conditions.

Slight decreases in DSA at present of glucose and sucrose were observed and no marked changes were observed (Fig. 3a, b). It seemed that glucose caused more changes than that of sucrose at low concentrations. Instead sucrose had a little stronger effect to antioxidant activity of SPBH than that of glucose at high concentrations. At present of glucose or sucrose, the retention activity of about 80% remained. Sodium chloride and sodium benzoate had little or no effect on the DSA (Fig. 3c, d). The effects of metal ions on the antioxidant stability were investigated and showed that the majority metal ions inhibited the DSA to various extents (Fig. 4). The antioxidant activity was inhibited by all metal ions used. The effects of Cu²⁺ and Zn²⁺ were notable while the retention activity was more than 80% in the conditions with high concentration of K⁺, Ca²⁺ and Mg²⁺. Nkhili and Brat (2011) reported that the presence of metal ions in the studied matrix will influence antioxidant stability, thereby leading to the under estimation of their antioxidant properties. So far, the SPBH should avoid contacting with the substance including Zn²⁺ and Cu²⁺ during processing.

Fig. 3(a-b):

Effects of glucose, sucrose, sodium chloride and sodium benzoate on antioxidative activity of SPBH. The values are Mean±SD of triplicate measurements. Values with different letters are significantly different (p<0.05)

Fig. 4:

Effects of metal ions on antioxidative activity of SPBH. The values are Mean±SD of triplicate measurements. Values with different letters in the same metal ion are significantly different (p<0.05)

CONCLUSION

The antioxidant activity of SPBH was related to the amino acids, molecular weight and affected by environment conditions. SPBH had high nutritional value and the amino acid compositions were rich in hydrophobic amino acids which might contribute to the high antioxidant activity and bitterness. The SPBH-V fraction with the MW lower than 1 kDa exhibited the highest activity which composed of four major fractions in chromatogram. This fraction could be further purified and characterized to obtain new potential antioxidant peptides. SPBH had good stability in various conditions but the effects of metal ions, such as Zn²⁺and Cu²⁺ on the antioxidative activity were significant. Therefore, the SPBH avoid contacting with the substance including Zn²⁺ and Cu²⁺ during processing. It might be used as food additives and diet nutrients, further detailed studies in specific food systems and debittering should be applied in order to obtain SPBH with the sensorial acceptability for further applications.

ACKNOWLEDGMENT

The research work was supported by Zhejiang Provincial Natural Science Foundation of China (Project No. Y3110278).