ANTIOXIDANT ACTIVITY OF PROTEIN HYDROLYZATES FROM BY-PRODUCTS OF THE FOOD INDUSTRY

ANTIOXIDANT ACTIVITY OF PROTEIN HYDROLYZATES FROM BY-PRODUCTS OF THE FOOD INDUSTRY

E. FLACZYK', R. AMAROWICZ2s3 and J. KORCZAK'

'Department of Human Nutrition and Food Technology Agricultural University in Poznan

Al . Wojska Polskiego 31 Poland

'Department of Food Sciences Institute of Animal Reproduction and Food Research

Polish Academy of Sciences ul. Tuwima 10 P.O. Box 55

10-718 Olszvn, Poland

Received for Publication October 28,2002 Accepted for Publication January 3 1,2003

ABSTRACT

Hydrolyzate was obtained from cracklings (CEH) using Alcalase. Hydrolyzates were also prepared from cracklings (CAH) and chicken feathers (FAH) using hydrochloric acid. Antioxidative activity of the above hydrolyzates was evaluated by spectroscopic methods. The oxidative stability of lard with the addition of hydrobzates was tested using a Rancimat and an Oxidograph apparatus set at 11 OC. Hydrobzates were analyzed using RP-HPLC method. The highest antioxidative eficacy of hydro&zates examined was found for CEH at an addition level of 0.25 and 0.50% N,, usingspectroscopic methods. The antioxidant efect of BHT at a level of 0.01 % N,,, was stronger than that of all Hydrolyzates tested. The strongest antioxidant efects evaluated using the Rancimat method were noted for both acid hydrolyzates applied at level of 0.5%N,,. Antioxidant activity of BHT, added to lard at a 0.02% level, was lower than those of CEH, CAH and GAH at a level of 0.5% N,o,. Superior antioxidant activity determined under conditions of the Oxidograph test was observed for CAH at a level of 0.5% Nlo,. Antioxidant activities observed from synthetic antioxidants added to lard at a 0.02% level were higher than those from any of the three hydrobzates tested. RP-RP-HPLC chromatograms of the acid hydrolyzates (CAH and FAH) were

Corresponding author. TEL: (48-89)523-4675; FAX: (48-89)524-0124; EMAIL: [email protected]

Journal of Food Lipids 10 (2003) 129-140. A l l Righis Reserved. 0 Copyright by Food & Nutrition Press, Inc., Trumbull, Connecticut. 129

130 E. FLACZYK, R. AMAROWICZ and J. KORCZAK

similar. A completely diferent looking chromatogram was obtained for the enzymatic hydrolyzate (CEH). It was characterized as having many, not well- separated peaks originating from peptides.

INTRODUCTION

Many reports have confirmed that certain amino acids, peptides and protein Hydrolyzates possess antioxidative activity against peroxidation of lipids or fatty acids. It is belived that antioxidant activity of amino acids is due to the reaction of amino or sulfur groups with lipid hydroperoxides resulting in the formation of imines, sulfides, thiosufinates and sulfoxides (Pokomy and Korczak 200 1). Several amino acids, such as methionine, histidine, threonine, lysine and tryptophan, have been shown to act as antioxidants in different model systems (Marcuse 1960, 1962; Merzametov and Gadzhieva 1976; Revankar 1974; Rissom et al. 1980). Some amino acids, when dissolved in water at concentrations of 0.5 and 0.05 mM, partially inhibited the damage to deoxyribose in the presence of ferric-EDTA and H,O, (Martinez-Tome et al. 2001). An antioxidative effect was observed in vivo for taurine, hypotaurine, carnosine and anserine (Aruoma et al. 1988,1989). Dipeptides containing ahme, tyrosine, histidine andmethionine at theN- terminus exhikited a higher antioxidative activity than the constituent amino acid mixture (Yamaguchi et al. 1975; Kawashimaet al. 1979). Carnosine, which is a htidine- containing dipeptide, can act as an endogenous skeletal muscle antioxidant (Chan and Decker 1994). Protein hydrolyzates were reported to possess antioxidant properties (Flaczyk 1997a, b, c). Autolyzed yeast and hydrolyzed soybean proteins investigated in a freeze-dried model system showed good antioxidant properties. The same properties were noted by Shahidi and Amarowicz (1996) and Amarowicz and Shahidi (1997) in the case of protein hydrolyzates and peptide fractions of capelin, when assayed with a 0-carotene-linoleate model system. Korczak et al. (1995) investigated and characterized antioxidant properties of soybean and casein hydrolyzates. Chen et al. (1995 and 1996) reported the antioxidant activity of enzymatically hydrolysed reaction products of soybean 7 s protein. Tsaldci et al. (1999) postulated that peptides may be contributing to the overall antioxidant activity of lupin seed flour. The antioxidative activity of pepsin-solubilized elastin (PSE) and acid-solubilized elastin (ASE) were noted by Hattori et al. (1998). Whey protein isolate that was hydrolyzed by the commercial crude proteases exhibited antioxidant activity (Pena-Ramos and Xiong 2001). The synergistic effect of several soybean protein hydrolyzates on the antioxidative activity of several synthetic antioxidants and tocopherols was reported by Chen et al. (1995). In the works of Yamaguchi (1979,1980), soybean protein hydrolyzates were also shown to act synergistically with d-6-tocopherol.

131 ANTIOXIDANT ACTIVITY OF PROTEIN HYDROLYZATES

A synergestic effect was observed by Amarowicz et al. (1999) for capelin protein hydrolyzates and TBHQ.

The aim of present study was to measure the antioxidant activity of protein hydrolyzates obtained fiom the food indushy byproducts (crackhgs and feathers) using a spectroscopic method and Rancimat and Oxidograph tests.

MATERIALS AND METHODS

Materials

Cracklings and feathers from chickens were obtained from the local food industry in Poznan (Poland). The content of total nitrogen (NtoJ in the cracklings and feathers was determined to be 13.1% and 14.1%, respectively. The microbial enzyme Alcalase 2.4 L was obtained from Novo Nordisk Co. (Bagsvaerd, Denmark). Alcalase is a microbial protease from the bacterium Bacillus lichenformis with endopeptidase activity and has a declared activity of 0.6 Anson units (AU) per gram.

Enzymatic Hydrolysis

A hydrolyzate from cracklings (CEH) was prepared using the enzyme Alcalase. The hydrolysis was carried out in a 250 mL thermostated vessel equipped with a stirrer and pH-meter, set at a temperature of 55rtlC for 1 h at pH 8.5, at an enzyme to substrate ratio of 0.15 AU/g (Shahidi et al. 1995). After hydrolysis, the enzyme was inactivated by heating the mixture at 80C for 5 min. Nonhydrolyzed material was precipitated from the solution using chloroform and then removed by centrifugation at 2000 x g. The pH of the supernatant was adjusted to 7, and the hydrolyzate was lyophilized.

Acid Hydrolysis

Acid hydrolysis was carried out using 4.75 M HCl at 110C for 4 h (cracklings) or 12 h (feathers), at a material to HC1 solution ratio of 3 to 7. After hydrolysis, solutions were neutralized and adjusted to pH = 5.7 with a powder of sodium carbonate. The mixtures were shaken with activated charcoal (1.8% ( d v ) during heating at 60-70C for 15 min), filtered through Whatman No. 3 filter paper, stored at 4C for 4 weeks and then finally spray dried. A Mini Spray Dryer B-191 (Buchi, Switzerland) was used for this purpose. Five hundred milliliters of sample were spray-dried over a period of 30 min at an inlet temperature of 16C; an outlet temperature of 70C was employed.


Chemical Analysis

The content of total nitrogen (N,J in the hydrolyzates was determined using the Kjeldahl method. a-Amino nitrogen (NNHZ) and the degree of hydrolysis (DH) of hydrolyzates were determined using the TNBS (trinitrobenzenesulfonic acid) method (Panasiuk et al. 1998).

Reversed-phase HPLC

Hydrolyzate solutions were prepared at a concentration of 2 mg/mL in 40% acetonitrile (v/v) containing 0.1% of trifluoroacetic acid (TFA); these solutions were then filtered through a 0.45 km membrane. A sample (20 pL) was injected into an HPLC (Shimadzu Corp. Kyoto, Japan) system equipped with a Shimadzu LC-IOAD pump, SCTL 10A system controller, SPD-M 1OA photodiode array UV-VIS detector, CTO-IOAS column oven and CLASS-VP. A C,, Hypersil Bakerbond BDC, 4.6 x 250 mm, 5 p column (Mllincrodt Baker Inc., Phillipsburg, NJ) was used for the separation of peptides. A flow rate of 1 mL/min, and gradient elution of 0.1% TFA in water (solvent A), 0.1% TFA in 40% (v/v) acetonitrile (solvent B), 0 to 30 min solvent B from 0 to 100% were employed. Temperature of separation was 25C and detection of peptides was monitored at 220 nm.

Antioxidant Activity Evaluated by a Spectroscopic Method

In this study, antioxidant activity was evaluated by the spectroscopic method described by Lingnert and Enksson (1980). Briefly, hydrolyzates were added to 2.2 mL of a 10 mM linoleic acid emulsion at levels of 0.1,0.25 and 0.50% of N,,, in relation to linoleic acid. The emulsion was prepared using 0.1 M phosphate buffer, pH 7.2, containing 0.3% (v/v) of Tween 20. Experimental samples, the control and a sample containing 0.02% of BHA were incubated at 37C for 19 h. Absorbance values at 234 nm were read using a Carl Zeiss Specord 40 UV- spectrophotometer (Jena, Germany). Antioxidant efficacy (AE) was calculated as follow:

d C - & A

(L1C A E =

where dAC is the increase of absorbance of the control, and dAA is the increase of absorbance of the sample with antioxidant added.

ANTIOXIDANT ACTIVITY OF PROTEIN HYDROLYZATES 133

Stability Against Oxidation at Higher Temperatures

The oxidative stability was tested using a Rancimat (Methrom, Switzerland) and an Oxidograph (Mikrolab, Denmark) apparatus set at 110C. Hydrolyzates were added to lard at a level of 0.1,0.25, and 0.50% of the N tot. Results were expressed as protection factors, defined as the ratio of induction time of the sample with antioxidant to that of the control.

Statistical Analysis

All experiments and measurements were replicated three times. Analysis of variance and Tukey ’s studentized range test were used to evaluate the significance of differences among mean values (Freund et al. 1988).

RESULTS AND DISCUSSION

The chemical composition of the hydrolyzates is presented in Table 1. The highest content of N,, was determined for CEH. This hydrolyzate was characterized as having the lowest level of & (1.72%) and the lowest degree of hydrolysis (DH). The content of N,,, in CAH was 7.94% and the DH was approximately 54%. When FAH was compared to CAH, its content of N,,, was slightly higher, but its DH lower. The DH for CEH was in range of values reported in the literature for various protein sources hydrolyzed by Alcalase. For rapeseed proteins, the DH as determined by the pH-stat method was ca. 9% (Gwiazda et al. 1994), for soybean protein isolate ca. 17% (Kim et al. 1990), for oat flours 7.6% (Ponnampalam et al. 1987), for herring (Clupea harengus) ca. 40% (Hoyle and Merritt 1994) and for harp seal (Phoca groenlandica) 19.5% (Shahidi et al. 1994).

TABLE 1. CHEMICAL CHARACTERISTIC OF INVESTIGATED HYDROLYZATES

CEH CAH FAH

12.32 7.94 8.21

1.72 3.75 3.79

14.0 53.8 46.2


[-

-?---A x E P n w

L s


Elution profiles of hydrolyzates separated by reversed-phase HPLC are depicted in Fig. 1. Chromatograms of the acid hydrolyzates (CAH and FAH) were similar. The majority of compounds (i.e., small peptides or amino acids) were eluted between 10 and 20 min. The retention time of the highest peak was 12.51 min for both hydrolyzates. A completely different looking chromatogram was obtained for the enzymatic hydrolyzate (CEH). It was characterized as having many not well-separated peaks originating from peptides. No baseline separation was achieved. This type of chromatogram is typical for enzymatic proteins hydrolyzates. For example, similar FW-HPLC chromatograms of peptides obtained from soybean using a protease from Bacillus subtilis were presented by Chen et al. (1995).

The antioxidant efficacy of hydrolyzates examined using the spectroscopic method is presented in Table 2. All hydrolyzates exhibited antioxidant activity in a concentration-dependent manner. The highest efficacy was found for CEH. For this hydrolyzate, the difference in results obtained at an addition level of 0.25 and 0.50% was not significant (I' > 0.05). In the case of CAH and FAH, the differences were significant ( P < 0.05), but only between results noted for the lowest and highest levels of addition. The antioxidant efficacy of FAH was weaker than that of CAH. The antioxidant effect of BHT at a level of 0.01% N,, was stronger than that of all hydrolyzates tested. The antioxidant activity of protein hydrolyzates and peptide fractions were reported by Shahidi and Amarowicz (1996) and Amarowicz and Shahidi (1997) using a P-carotene- linoleate model system, which is similar to the spectroscopic method employed in the present study.

TABLE 2. ANTIOXIDATIVE EFFICACY OF HYDROLYZATES INVESTIGATED BY A

SPECTROPHOTOMETRIC METHOD

Hydro1 ysate 0.1 % Nt0, 0.25% N,ot 0.5% N,,,

CER 0.55f0.02a 0.64+0.03ah2 0.74+0.0Ib2 CAI3 0.32_+0.02a2 0.52f0.03b 0.67+0.02c FAI3 0.13f0.02a ' 0.25+0.03ah I 0.39?0.06b I

BHT 0.01% 0.9220.06

The means with the same column or raw followed by the same superscript letter or number, respectively, are not significantly (P > 0.05) different.

136 E. FLACZYK. R. Ah4AROWICZ and J. KORCZAK

Table 3 summarizes the results of the antioxidant activity measured using the Rancimat method. Using this technique, changes in conductivity caused by ionic volatile organic acids, mainly formic acid, from oxidative oils are automatically and continuously measured (Shahidi and Wanasundara 2002). The antioxidant activity of all hydrolyzates evaluated using the Rancimat method was compared against lard oxidation. The strongest antioxidant effects were noted for both acid hydrolyzates employed at a level of 0.5% N,. Antioxidant activity of BHT, added to lard at a 0.02% level, was lower than those of CEH, CAH and GAH at a level of 0.5% N,,,.

TABLE 3. ANTIOXIDATIVE EFFICACY OF HYDROLYZATES INVESTIGATED BY THE

RANCIMAT METHOD

Hydrolysate 0.1 % N,t 0.25% N,,, 0.5% N,,,

CEH 1 . 1 8f0.01a ' 1.28f0.02' ' 1 .80f0.04b CAH 1 .42+0.03a3 1.42?0.02' 1 .54+0.02b ' FAH 1 .31+0.02a 1 .67+0.05b ' 1.78f0.OSC2

BHT 0.02% 1.42+0.02

The means with the same column or raw followed by the same superscript letter or number, respectively, are not significantly (P > 0.05) different.

Results of the antioxidant activity of hydrolyzates determined under conditions of the Oxidograph test are presented in Table 4. Regarding the Oxidograph method, as a sample of oil absorbs oxygen, the pressure change in the reaction vessel is measured electronically by means of pressure transducers (Shahidi and Wanasundara 2002). In our experiments, a protective effect was noted for all hydrolyzates. In general, protection factors correlated well with the quantity of hydrolyzate added to the lard. Superior activity was observed for CAH at a 0.5% N,, level. Antioxidant activities observed for synthetic antioxidants added to lard at a 0.02% level were higher than those from any of the three hydrolyzates tested. Further studies on sensory analyses of lard, to which these protein hydrolyzates were added, are in progress.

ANTIOXIDANT ACTIVITY OF PROTEIN HYDROLYZATES 137

TABLE 4. ANTIOXIDATIVE EFFICACY OF HYDROLYZATES INVESTIGATED BY THE

OXIDOGRAPH METHOD

Hydrolysate 0.1 % Ntot 0.0.25% N,o, 0.5% A’,,,

GEH 1.2 1 k0.02” ’ 1 .32f0.0Sab 1 .53f0.05ab ’ GAH 1.22f0.0Ia ’ 1 .64+0.02b 1.85+0.02‘ ’ FAH 1.32f0.02” 1 .68+0.04b ’ 1 .67+0.04b

BHT 0.02% 3.40+0.05

The means with the same column or raw followed by the same superscript letter or number, respectively, are not significantly (P 5 0.05) different.

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ANTIOXIDANT ACTIVITY OF PROTEIN HYDROLYZATES FROM BY-PRODUCTS OF THE FOOD INDUSTRY

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