Indian Journal of Chemical Technology Vol. 6, May 1999, pp. 117-120

Evaluation of the antioxidant activity of wheat bran

Magda A Abd EI Mageed & Hoda H M Fadel

Department of Flavour and Aromatic Chemistry, National Research Centre, Dokki, Cairo, Egypt

Received 29 April/998; accepted 28 January /999

Wheat bran was extracted successively with hexane, chloroform and ethanol. The antioxidant activity of each extract was measured using the thiobarbituric acid (TBA) test. The ethanol extract showed the highest activity, it caused 90% inhibition on the oxidative degradation of linoleic acid. For further evaluation of its activity, ethanol extract was added at two concentrations (0.02% and 0.05% of the beef fat level) to ground beef containing 20% fat. The aroma concentrates of the cooked beef samples containing the ethanol extracts or those containing commercial antioxidants BHA and BHT were isolated both before and after storage for one week at 4°C. The oxime derivatives of the volatile carbonyl components isolated from· the aroma concentrate of each sample were prepared and subsequently estimated by ultraviolet absorption at wavelength 212 nm for the total carbonyl oximes. The results showed that the ethanol extract has significant (P<0.05) effect in reducing rancidity in the stored beef samples in addition to decreasing the decomposition rate of the unsaturated fatty acids of the beef fat.

During refrigeration of frozen storage of the precooked or partially cooked meat products, warmed over flavour (WOF), developed rapidly with a loss in the desirability of these products l

-4 . The deterioration in meat flavour has been positively correlated with the content of carbonyl compounds formed through lipid autoxidation reaction2

,4,5 The lipid hydro­peroxides, the initial products of lipid autoxidation reaction are unstable and undergo further degradation through free radical mechanisms to form aliphatic aldehydes, alcohols, ketones and hydrocarbons6,7.

These secondary products of lipid autoxidation contribute to off flavour in meats3

,4,8. To overcome this problem, several synthetic phenolic antioxidants, such as butylated hydroxyanisol (BHA) and butylated hydroxytoluene (BHT) have been used successfully to delay WOF in restructured meat products4

,9.

However, there has been a growing demand for natural antioxidants due to reports that BHA and BHT have toxic and carcinogenic effects in animals lO

•

Natural antioxidants from plants may be an alternative source for compounds capable of protecting lipids in foods. The antioxidant properties of spices are apparently related to their phenolic contents 1 1,12, thus their antioxidant action is similar to or greater than BHA and/or BHT13

-17

. Pussayanawin and Wetzel 18 reported that, phenolic compounds are concentrated in the aleurone and bran portion of cereals. They detected a high concentration of phenolic acids especially ferulic acid in the bran of

durum wheat. Similar observations were made by Ohta e/ af. 19. It was also reported that the incorporation of wild rice and its extracts into different meat products could retard the development of rancidity during frozen storage and" improve sensory scores20

,2I. Onyencho and Hettiarachch/ 2

identified different phenolic acids in durum wheat bran among which ferulic, vanillic and p-Coumaric compounds were present in the highest concentrations. However, the relative antioxidant properties of wheat bran in inhibiting both hydroperoxide formation and decomposition are properties that have received little attention in literature, · however decomposition of hydroperoxides and the resulting formation of volatile compounds clearly been shown to have significant impact on the flavour deterioration of precooked meat4

. Though the present work was designed to evaluate the antioxidant activity of the wheat bran extracts, the solvent extraction was conducted in an attempt to isolate the most active components. The protection effect of the most active extract was evaluated by following the changes in the fatty acid cQmposition and carbonyl content of the precooked ground beef stored at 4°C.

Experimental Procedure Materials and Methods--The wheat bran was supplied

by South Cairo Milling Company, Giza, Egypt. Food grade antioxidants, butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT) were obtained

118 INDIAN J. CHEM. TECHNOL., MAY 1999

0.7 ~---------'--------.

-+-Control 0.6 _BHT

~BHA - .,. •. Hexane extnlct

- Chloroform extract - Ethanol extract

0.5

~ 0.4

~ . ~ 0.3

0.2

3 6 9 12

Tlme,Oa18

15 18 21

Fig. I-Antioxidant activities of wheat bran extracts (hexane: - -x - -); chloroform: _._; ethanol : -_e_: BIjA (-D--) and BHT(-6-) (All data points are means of 4 determinations, SD not more than M . I and all samples are significantly different from control, P<O.05)

6 4.89

C' 5 .. ..

.Q

E t>I)

4 0 .:.

~ 3 E .. >.. 2 c

C> .Q .. .. u 1 '3 C> I-

f •• h Stond 0.02 % 0.02 "I. 0.02 % 0.05% control control BHA BHT WBEE WBEE

Fig. 2--{:hanges in the total carbonyls in the beef samples stored for one week at -40 c. (Values are the mean of three determinations, SD not more than

M .I)

from Sigma Chemical Co., St. Lovis, Mo. Thiobarbituric acid (TBA) was purchased from Aldrich Chemical Co. , Milwaukee, Wl . USA. All other chemicals were of analytical grade. Beef (from shoulder) was purchased from local market (within one day after its arrival from the slaughterhouse). After removal of all visible fat the ground beef were prepared by mixing and grinding 8 parts of lean and 2 parts of fat together by using the house meat grinder.

Solvent extraction-Wheat bran ( I 00 g) was subjected to sequential extraction with 500 mL of n-

hexane, chloroform and ethanol, respectively. The extraction period was 12 h per solvent. Following at each solvent extraction stage, the' wheat bran was placed in filter paper (Whatman No.1, 20 cm) to facilitate removal of solvent and finally squeezed to recover most of the solvent prior to addition of the next one. The wheat bran extracts (WBE) were evaporated under reduced pressure, then stored in dark at 5°C until test (within two days).

Antioxidant activity of the wheat bran extracts--The thiobarbituric acid (TBA) test was used to determine the antioxidant activity of each WBE (hexane, chloroform and ethanol) using the methodology of Osawa and Namiki23

• The TBA values were calculated from the absorbance at 532 nm by using a SHIMADZU Corporation spectrophotometer [(UV-1601 [PC]S) 3500563 S2]·(JAPAN) CAT NO. 206-67501-93.

Evaluation of ttie wheat bran ethanol extract (WHEE) antioxidant activity-Five samples of the fresh ground beef were used as the test material in this study. Two concentrations of the WBEE (0.02 and 0.05% of the beef fat level) and BHA and BHT (0.02% of the beef fat level for each) were separated and thoroughly mixed with 200 g of ground beef. The sample containing no' antioxidant represented the control. The five meat samples were cooked at 80°C for 2 h and immediately refrigerated at 4°C for 7 days. The activity of the WBEE was evaluated by determining the changes in the fatty acid composition and carbonyl content of the stored precooked beef samples.

Fatty acid composition-The lipid component of each sample was extracted at room temperature by soaking in n-hexane with subsequent stirring and filtration . The solvent was evaporated under reduced pressure to obtain the lipid. The lipid samples were methylated using the methodology of Luddy et al. 24 and thel!1 subjected to gas chromatography-mass spectrometric analysis by using a Varian 3400 GC equipped with DB-wax capillary column (0.32 mm id x 30 m length, J& W Scientific products, 91 Blue Ravine Roda Folsom, CA 95630-4714 FISONS, USA and coupled to a Finnigan-Mat 55 Q 7000). Analysis was carried out by using the helium as the carrier gas, flow rate 1.1 mLimin . The column temperature was 250°C. The ionization voltage applied was 70 eV . The isolated peaks were identified by comparison with data from the library of the mass spectra (nst), retention time and mass spectra of authentic samples. The

•

ABD EL MAGEED & FADEL: EVALUATION OF ANTIOXIDANT ACTIVITY OF WHEAT BRAN 119

Table I---Changes in the fatty acid composition of BEEF FAT samples stored for one week at 4°C (concentration %)

Fatty acid Treatment Fresh Fresh BHA BHT WBEEI WBEE II

control stored 0.02% 0.02% 0.02% 0.05%

c 14:0 2.90 3.\0 3.00 2.90 2.90 2.90 c 15 :0 1.50 1.50 1.50 1.50 1.50 1.50 c 16:0 26.50 29.04 27.35 27.00 27.07 26.88 c 16:1 3.50 4.00 4.08 3.90 3.90 3.87 c 17:0 1.50 1.50 1.50 1.50 1.50 1.50 c 18:0 22.00 29.40 25.89 24.80 24.81 23 .59 c 18: I 38.50 30 . .5 35.09 36.41 36.32 37. 16 c 18 :2 2.80 0.96 1.48 1.69 1.75 2.11 c 18:3 0.80 0.11 0.30 0.25 0.49

Saturated 54.4 64.54 59.24 57.70 57.78 56.37

Unsaturated 45.4 35.46 40.75 42.30 42.22 43.63

All reported values are the mean of three determinations, SD not more than ±O. I

quantitative determination was carried out based on peak area integration.

Isolation and determination of the volatile carbonyl compounds-The aroma concentrate was isolated by using a modified Likens-Nicl.erson steam distillation­extraction apparatus25 from 100 g of each sample, then the collected aroma concentrate was separately fractionated into carbonyl and noncarbonyl fractions26

. The amount of total carbonyl was determined according to Hebash and Fadel27

. To the carbonyl fraction of each sample, 10 mL of 10% (w/w) aquous solution of hydroxylamine hydrochloride were added. The absorption spectrum for the prepared solutions were recorded from 200 to 400 nm using an appropriate blank solution . n­Hexanal (Aldrich) was used as a calibration standard for determining the wavelength for the absorption maximum corresponding to the total carbonyl. It was analyzed in concentrations of 1-10 ppm.

Statistical Analysis-Statistical analysis was performed by using student I-test.

Results and Discussion Antioxidant activity of the wheat bran extract-Extracts

from wheat bran were obtained with hexane, chloroform and ethanol and the total yie lds of these extracts were 1.5±0.0 I, 1.06±0.02, and 1.7±0.0 I g respectively per 100 g of the bran. Decomposition of hydroperoxides generates aldehydic compounds that can be quantified by TBA test and this method is generally accepted as an effective method for following lipid oxidation28

. Ethanol extract had the strongest inhibition on oxidative degradation of linoleic acid (Fig. I). It showed 90% inhibition

whereas BHA and BHT caused 83% and 87% inhibition, respectively. The hexane and chloroform extracts showed less antioxidant activity. This variation in the antioxidant activity of the WB E's (hexane, chloroform and ethanol) may be due to the nature of the components present in each extract.

In view of the above results, different concentrations of the WBEE (0 .02% and ·0.05% of the beef fat level) were used as natural antioxidants. Their activity in retarding the rancidity of the precooked ground beef was evaluated by following the changes in the fatty acid composition and the total carbonyl content of the beef fat during storage for one week at 4°C.

Fatty acid composition-Changes in the fatty acid composition are shown in Table I . Oleic acid, the major fatty acid in beef fat showed a marked decrease in the control sample during storage. The linoleic acid exhibited similar trend. The antioxidant treated samples showed slight decrease in the concentration of oleic and linoleic acids. The decrease in the concentration of unsaturated fatty acids may be attributed to formation of their hydroperoxide derivatives which can be decomposed to volatile carbonyl compounds27

,29,3o.

A decrease in the total unsaturation was detected in all samples under investigation during storage, however, the control sample exhibited significant loss (P <0.05) as shown in Table I. There are no significant differences (P >0.05) in the total unsaturation among the antioxidant treated samples, however the increase in the added concentration of WBEE led to an increase in its protection effect

120 INDIAN 1. CHEM. TECHNOL., MAY 1999

against the oxidative degradation of unsaturated fatty acid in the beef fat during refrigerated storage.

The volatile carbonyl compounds-The analysis of the absorption spectra of hexanal, used as a calibration standard, showed an absorption maximum. at 212 nm. It has been reported that, hexanal determination could be used as a measure for flavour deterioration than hydro peroxide formation 31

• It has also been used as a marker for the change in the total carbonyl27. The control sample exhibited noticeable increase in the amount of the total carbonyls after storage for one week at 4°C as shown in Fig. 2. This increase in the total carbonyls which are derived from the hydroperoxides of the unsaturated fatty acids is parallel to the decrease in the total. unsaturation (Table I). All of .the antioxidant treated samples exhibited much lower increases in the total carbonyls (P <0.05), compared with the control sample. However, it is obvious that the increase in the added amount of WHEE resulted in best results. Similar effects to those obtained in this recent study were obtained by addition of rosemary oleoresin to beef products during short term (2 days) chill storage in non-modified atmosphere32

,33.

The above mentioned results show that ethanol as a polar solvent is more effective than, chloroform and hexane in the extraction of the antioxidant compounds present in wheat bran. The WBEE exhibited antioxidant properties equal to or grealer than the two synthetic antioxidants which have begun to be restricted because of their toxicity 10. It is well known that the bran portion of cereals are good suppliers of dietary fibre in addition to their physiological function 34

.36

. Therefore to confirm its safety use as an alternative antioxidant, detailed biological and physiological studies on the WBEE will be carried out in future.

References 1 Lai S M, Gray J I, Smith D M , Booren A M, Crackei R L &

Buckley D J, J Food Sci, 56 (1991 ) 616 2 Diumm T D & Spanier AM, ) Agric Food Chern, 39 (1991)

336 3 Jayathilakan K, Vasundhara T S & Kumudavally K V. )

Food Sci Technol, 34 (1997) 128 4 Wu Y, Hamouz F & Schnepf M, ) Agric Food Chern . 4

(1998) 3677 5 Shahid i F, Yun 1, Rubin L J & Wood D F, Food Sci Technol,

20 (1987) 104 6 Lillard D A, Oxidative deterioration in meats poultry and

fish. In warmed-over flavour of meats, edited by St. Angelo A 1 & Bailey M E (Academic Press : Orlando F L), 1987, 41

7 Love J, Mechanizme of.iron Catalysis of lipid autoxidation in warmed-over flavour of meat. In warmed .. over flavour of meat, edited by St. Angelo A 1 & Bailey M E (Academic Press: Orlando F L), 1987, 19

8 St. Angelo A J, Vercebtti J R, Legendre M G, Vinnet C, Kuan J W, James C J & Duppy H P, ) Food Sci, 82 ( 1987) 1163

9 Crackel R L, Gray J I, Pearson A M, Booren A M & Buckley D,) Food Chern, 28 (1989b) 187

10 Ito N, Fukushima S & Fukashima H, CRC Crit Rev Food Technol, 15 (1985) 109

II Frankel E N, Huang S W, AeSchbach R & Prior E, J Agric Food Chern, 41 (1996) 131

12 Aruoma 0 I, Spencer J P, Rosss R, Aeschbach R, Khan A, Mahmood M, Munoz A, Murcfa A. Butler J & Hallrwell B, Food Chern Toxicol, 34 (1996) 449

13 Houlihan G MHO, CT in Flavour Chemistry of Fats and Oils, edited by D B Min & T H Smouse (Am Oil Chern Soc Champign III), 140

14 Farag R S, Badei A Z M A, Hewadi F M & EI Baroty GSA, ) Am Oil Chern Soc, 66 (1989) 792

15 Svoboda K P & Deans S G, Flavour Fragr, 7 (1992) 81

16 Basaga H, Tekkaya C & Hcikel F, Lebensm-Wiss-U Techno!, 30 (1997) 105

17 Terens W & Schwarz K, Ziebensm Mnters Forsch, 20 1 (1995) 548

18 Passayanowin V & Wetzel D L.) Chmateryr, 39 (1987) 243 19 Ohta T, Yamasaki S, Egashira Y & Saneda H, ) Agric Food

Chern, 42 (1994) 653 20 Minerich P L, Addis P B, Epley R J & Bingham C,) Food

Sci, 56 (1991) 1154. 21 Wu K, Zhong W, Addis P B, Epley R J, Salih A M &

Lehrfeld J, ) Agic Food Chern, 42 (1994) 34

22 Onyencho S· N & Hettiarachchy N S, ) Agric Chern , 40 (1992) 1496

23 Osawa T & Namiki M, Agric Bioi Chern, 45 (1981) 732 24 Luddy FE, Barford R A & Riemenschneider R W, ) Am Oil

Chern Soc, 37 (1960) 447 25 Schultz T H, Flath R A, Mon J R, Eggling S B & Teranishi

R, ) Agric Food Chern, 25 (1977) 446 26 Teitelbaum C L, ) Org Chern, 23 (1958) 646 27 Hebash K A & Fadel H M Die, Nahrung, 38 (1994) 278

28 Halliwell H & Chirico S, Am) Clin Nutr, 57 (1993) 7155 29 Selke E & Frankel E N, ) Am Oil Chern Soc, 64 (1987) 749 30 Snyder J M, Frankel E N, Seke E & Warner K, ) Am Oil

Chern Soc, 65 ( 1988) 1617 31 Framkel E N, Huang S W, Kanner J & Germen 1 B, ) Agric

Food Chern, 42 (1994) 1054

32 St Angelo A 1, Crippen K L, Dupuy H P & lames C 1, J Food Sci, 55 (1990) 1501

33 Huisman M, Madsen H L, Skibsted L H & Bertelsen G, Z­Lebensm Unters Forsch, 198 (1994) 57

34 Ebihara K & Tukeuchi M, Biosci Biotechllol Biochem, 56 (1992) 1508

35 Miyasaka H & Sanada H, Aynxoy Biosci Biotechnol Biochem, 56 (1992) 157

36 Ohta T, Semboku N, Kuchii A, Egashira Y & Sanada H. ) Agric Food Chern, 45 (1997) 1644