SYNERGISTIC ACTIVITY OF CAPELIN PROTEIN HYDROLYSATES WITH SYNTHETIC ANTIOXIDANTS IN A MODEL SYSTEM

RYSZARD AMAROWICZ and MAGDALENA KARAMAC

Division of Food Sciences Institute of Animal Reproduction and Food Research Polish Academy o Sciences f 10-718 Olsztyn 5, P.O. Box SS, Poland
AND FEREIDOON SHAHIDI

Department of Biochemistry Memorial University o Newfoundland f St. John S , NF, AlB 3x9,Canada
Received for Publication August 22, 1999 Accepted for Publication October 10, 1999

ABSTRACT Capelin protein hydrolysates (CPHs) were examined in a pcarotene-linoleate model system together with synthetic antioxidants, namely butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butyl hydroquinone (TBHQ). Addition of CPHs was at 0.25 and 1 mgper 5 mL in the above emulsion. BHA and BHT were added at levels of 2.5 and 5 p g and TBHQ at 5 and 10 pg. f Absorbance o model emulsion system at 470 nm was recorded evew 30 rnin for 2 h. CHPs and synthetic antioxidants inhibited oxidation of linoleic acid effectively. In the experiment with BHA, the influence of CPHs was signifcant (p<O.Ol) for the samples incubated for 60, 90, and I20 rnin; and in experiments with BHT and TBHQ during the entire incubationperiod. The influence of added BHA and BHT on inhibition of bleaching of pcarotene in the emulsion system was significant (p <O.OI) during the incubationperiod and afier 60,90 and 120 min when TBHQ was used. A synergistic effect was observed onlyfor CPHs and TBHQ when incubation time was 60, 90, and 120 min.

Journal of Food Lipids 6 (1999) 271-275. All Rights Reserved. 'Copyright I999 by Food & Nutrition Press, Inc., Trumbull, Connecticut.

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R. AMAROWICZ, M. KARAMAC and F. SHAHIDI

INTRODUCTION
Many reports have shown that some amino acids and protein hydrolysates possess antioxidant properties. Considerable antioxidant activity was noticed for histidine and tryptophan in linoleic acid and linoleate model systems (Marcuse 1962). Similar results were found for tryptophan and lysine in butter fat (Menametov and Gadzhieva 1976), methionine in vegetable oils (Simsand Fioriti 1977), histidine, threonine, lysine, and methionine in sunflower oil emulsion (Rissom et al. 1980),and proline in sardine oil (Revankar 1974). Good antioxidant properties were found for autolyzed yeast and hydrolyzed soybean proteins in a freeze-dried model system (Bishov and Henick 1972, 1975). Seher and Loschner (1985) noticed strong antioxidant activity for a fraction containing polar compounds whch was separated fiom krill extract. Shahidi and Amarowicz (1996) and Amarowicz and Shahidi (1997) observed antioxidant activity of protein hydrolysates and peptide fractions of capelin in a P-carotene-linoleate model system. Enzymatic hydrolysis of p-conglycinin (soybean 7 s protein) yielded fractions with antioxidative activity against peroxidation of linoleic acid in an aqueous system. Antioxidative peptides isolated from the hydrolysates were composed of 5- 16 amino acid residues, including hydrophobic amino acids, valine or leucine, at the N-terminal position, and proline, histidine, or tyrosine in their sequences (Chen et al. 1995,1996). In this study, we examined the synergistic effect of capline protein hydrolysate with synthetic antioxidants, namely butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butyl hydroquinone (TBHQ) in a p-carotenelinoleate model system.
MATERIALS AND METHODS

Capelin protein hydrolysates (CPHs) were prepared from capelin (Mallotus villosus), a small silvery fish found in the coastal waters of Newfoundland, according to Shahidi et al. (1995). CPHs were tested together with synthetic antioxidants, namely BHA, BHT, and TBHQ. All antioxidants were obtained from Sigma (St. Louis, MO). A p-carotendlinoleate model emulsion was prepared according to the procedure of Miller (1971). In experiments reported here, CPHs were used as factor A and synthetic antioxidant as factor B. To five milliliters aliquot of the emulsion, CPHs were added at two levels of 0.25 mg (1) and 1 mg (2). BHA and BHT were added at levels of 2.5 pg (1) and 5 pg (2); TBHQ at 5 pg (1) and 10 pg (2). Experimental groups were described as A,B, ,A,B,, A2B,,and A,B, Model emulsions were incubated for 2 h at 50C . Antioxidant activity was evaluated by reading the absorbance values at 470 nm every 30 min. The results

SYNERGISTIC ACTIVITY OF CAPETIN PROTEIN HYWRLYSATES

213

are expressed as mean values from four independent determinations, together with standard deviations. The significance of A and B factors was tested by a two-way analysis of variance using a Statpal 4.0statistical system.

RESULTS AND DISCUSSION

Results reported in Table 1 show that the antioxidant activity in a p-carotenelinoleate model system was moderate with respect to both factors examined: A (CHPs) and B (synthetic antioxidants). In the experiment with BHA, factor A was significant for the samples incubated for 60,90, and 120 min (Table 1). In the case of BHT and TBHQ statistical significance of factor A was also noted after 30 min of incubation (Table 1). The influence of factor B on the results was significant after 30,60,90, and 120 min of incubation with BHA and BHT (Table 1). When TBHQ was used in these experiments, factor B was effective after 60,90, and 120 min of incubation, but not after the first 30 min (Table 1). Results presented in Table 1 also indicate that only the presence of TBHQ resulted in a synergistic effect with CPHs. This effect was observed when incubation time was 60, 90, and 120 min. From literature data it appears that antioxidant activity of BHA and BHT is higher than that of TBHQ in this model system as examined by Millers test (Amarowicz and Karamac 1997) and also in a lard emulsion (Cort et al. 1975). Similar trends were observed using antioxidantlinoleic acid monolayer on silica gel (Porter et al. 1977) and in lyophilized red blood cell membranes (Porter et al. 1978). The synergistic effects of several soybean protein hydrolysates on the antioxidative activities of several synthetic antioxidants and tocopherols have been reported by Chen et al. (1995). In the presence of BHA, and d-&tocopherol each hydrolysate derived from p-conglycinin, glycinin, and 7s basic soybean globulin exhibited synergistic effect, which increased in the order of BHA, tocopherol, and BHT. In the work of Yamaguchi et al. (1979,1980), soybean protein hydrolysates were also shown to act synergistically with d-6-tocopherol. BHA and BHT were among the most effective phenolic antioxidants when combined with the protein hydrolysates (Bishov et al. 1977). In the latter studies, induction period of lipid oxidation was evaluated by monitoring oxygen consumption using gas chromatography. Thus, it is clear that while hydrolyzates may serve as antioxidants in emulsion systems, their synergism with synthetic antioxidants depends on the chemical nature of the compound involved. Furthermore, it should be noted that results from one system cannot be easily extrapolated to other systems, especially when dealing with bulk oil or composite food.

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R AMAROWICZ, M. KARAMAC and F. SHAHIDI

TABLE 1. ANTIOXIDANT ACTION OF CAPLIN PROTEIN HYDROLYSATE AS REFLECTED IN ABSORBANCE AT 470 nm IN A PCAROTENE-LINOLETE MODEL SYSTEM AND ITS SYNERGISM WITH BUTYLATED HYDROXYANISOLE (BHA), BUTYLATED HYDROXYTOLUENE (BHT) AND TERT-BUTYL HYDROQUINONE (TBHQ)
Incub.liOn time (min)
AlBl

AIB2

A2BI

482

FA

Fa

FAX6

B u t y W hydroxyanilak 30
60 90
17 .0

0 . ~ 0 . 0 0 2 0.613i0.007 0.57S0.006 0.553i 0.009

0.538SO.oMI

0.602*0.009

0.607i0.003 0.5M0.004
0.560i 0.006 0.538* 0.009

0.621 14.677.. 17.820** 31.754..

9.399. 19.497** 32.757.r 32.274..

1.726
0.10s

0.S94i0.005 0.577* 0.005

0.562tO.013

0.529i 0.015 0.498i 0.017

0.645 2.100

0 5 I 0.009 .6i

30 60
90

0.638i0.009 0.657* 0.017

0.607i0.012 0.53% 0.015 0.472+ 0.019

0.43W 0.022

0 . 6 2 B 0.012 0.5741 0.018 0.522i 0.024

0 . 4 W 0.025

27.544.' 32.898** 24.310.* 21.482..

9.340** 14.727.'
13.207.'

0.036

0.62Ok0.019 0.57% 0.025 0.545i 0.022

0.62Oi 0.019 0.57% 0.025

0.383
0.295
0.608

120

O.WS* 0.022

I5.359**

30

0.548i0.003 0.524i0.006
0.50SO.010

0.S54iO.004
0.535i 0.004 0.51&k0.005

0.517*0.013 0.461i 0.030

0.415i0.045

0.541*0.007

13.488** 29.261'. 24.310.'

1.243 1S.736,. 13.407..

3.871

60
90

0.513* 0.009
0.4901 0.013

6.372. 6.548.

o . m a0.056 (win+ 0.016 z7.789** I 3.979- G.WV 0 . 4 m 0.01I o m * 0.007 F signillant It 0.05 kvel ;** F signif& a1 0.01 lever: A. a p l i n pmlcin hyddptcat 1.0.25 and 2. 1.00 mg. 8. for BHA and BHTand 10 pg for synthetic llntioxidunrIt I. 2.5 wlor BHA andBHTand 5.0 1~ for TBHQ. .nd2.5
IM

mHQ.

REFERENCES

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SYNERGISTIC ACTIVITY OF CAPETIN PROTEIN HYWRLYSATES

215

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