Antioxidant Properties of Milledrice Co-products and Their Effects on Lipid Oxidation in Ground Beef

F.F. SHIH AND K.W. DAIGLE

Food Chemistry and Toxicology

ABSTRACT: Antioxidant properties were analyzed for methanolic extracts of rice seeds, milled-rice co-products, and other selected plant seeds, including cottonseed, soybean, and corn. Values of antioxidant effectiveness ranged from 45% to 86%, based on 100% activity at no change in color during the -carotene bleaching test. A correlation exists between the antioxidant activity and the total phenolic content of the rice ingredient extracts (r2 = 0.81). When selected extracts were applied to ground beef, the lipid oxidation was inhibited by, in relative effectiveness, rice hull > rice bran > brown rice. When applied directly to the beef, both defatted brown rice flour and rice bran strongly retarded the lipid oxidation. Keywords: antioxidant, rice bran, rice hull, plant seeds, lipid oxidation

Introduction

ancidity of food because of lipid oxidation is a serious problem because it not only produces off-flavors but also decreases the nutritional quality and safety of the food. Benefits of antioxidants in food storage have been studied extensively in recent years (Minerich and others 1991; Kim and Godber 2001; Bekhit and others 2003). Synthetic antioxidants, such as butylated hydroxylanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), and tert-butylhydroquinone ( TBHQ), have been commonly used to suppress the formation of free radicals, preventing lipids from oxidation and food spoilage. Although these synthetic reagents are efficient and relatively cheap, special attention has been given to natural antioxidants because of a worldwide trend to avoid or minimize the use of synthetic food additives. Plant seeds contain phytochemicals, which are natural antioxidants (Pratt 1992). These natural antioxidants are attracting further interest because of their clear benefits as anticarcinogenic agents and as inhibitors of biologically harmful oxidation reactions in the body (Frankel 1998; Basu and others 1999). Rice is one of the major staple foods in the world, and the processing, characterization, and utilization of rice has been investigated extensively. Rice ingredients are recognized to be nutritious, hypoallergenic, and healthy for human consumption. However, only limited information is available on their capacity as antioxidant materials. Pigments in rice were shown to be associated with antioxidant components (Choi and Oh 1996; Chung and others 2000). Rice bran and rice oil are particularly rich in potent antioxidants, such as oryzanol and tocopherol (Lloyd and others 2000; Qureshi and others 2000; Gopala-Krishna 2002). Ramarathnam and others (1989) identified in rice hull a c-glycosyl flavonoid, isovitexin, which was as potent as BHT in inhibiting lipid oxidation. Methanolic extracts of wild rice or wild rice hull were reported to contain

phytic acid, and they showed appreciable antioxidant activities when added to ground beef (Wu and others 1994). Kim and Godber (2001) reported that when rice bran oil was incorporated into restructured beef roast, both oxidative stability and vitamin E levels of the product improved. Rice hull and rice bran are particularly attractive as sources of antioxidants because these milled-rice coproducts are plentiful and cheap. Rice ingredients such as rice flour and rice bran have been used in foods to provide texture and body. It is desirable to investigate whether they also play a role in inhibiting lipid oxidation and enhancing the stability of the product. In this article, we compared the antioxidant properties of methanolic extracts of rice ingredients and other selected plant seeds. We studied possible correlation between the antioxidant activity and the total phenolic content of the extracts. Finally, we incorporated selected extracts of the rice ingredients or the rice ingredients directly into beef patties and investigated their effects on the lipid oxidation that was involved.

Materials and Methods

Materials
Long-grain brown rice, raw rice bran, rice oil, and rice hull were provided by Riceland Foods Inc. (Stuttgart, Arkansas, U.S.A.). Rice wax was from Cyvex Nutrition (Irvine, Calif., U.S.A.), soybean seeds (Asgrow 5098) from A.E. Staley Co. (Galesbury, Ill., U.S.A.), glandless Acala cottonseed from Texas A&M Univ. (College Station, Tex., U.S.A.), and field dent corn (Pennington seed, Lot 232), a gift from Dr. Mike Dowd, was grown and harvested at SRRC (New Orleans, La., U.S.A.). -Carotene, thiobarbituric acid (TBA), tetraethoxypropane (TEP), BHT, and Folin-Ciocalteau reagent were purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.).

Methanol extraction
Fifty grams of seeds or rice hull were frozen in liquid nitrogen, finely ground in a Waring blender (St. Louis, Mo., U.S.A.), and then extracted in a Soxhlet extractor (St. Louis, Mo., U.S.A.) with 1 L of 90% methanol for 17 h. The methanolic extract was concentrated

MS 20030389 Submitted 7/9/03, Revised 9/8/03, Accepted 9/26/03. Author Shih and Daigle are with the Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, LA 70124. Direct inquiries to author Shih (Email: fshih@srrc.ars.usda.gov).

Antioxidant milled-rice co-products . . .

on a rotary evaporator at 40 C. The extract was freeze-dried to recover the extract containing mostly phenolic compounds. In another series of experiments, raw rice bran (50 g) was 1st defatted by extraction in a Soxhlet extractor with l L petroleum ether for 17 h. The crude oil in the petroleum ether extract was recovered by concentration on a rotary evaporator at 40 C to remove the solvent. The residue was air-dried at room temperature for 16 h and then extracted with l L 90% methanol in a Soxhlet extractor for 17 h. The methanolic extract, which was oil-free and contained phenolic compounds, was recovered by evaporation as described previously. samples (11.5 g) were homogenized with 40 mL of the TCA solution in an Osterizer blender (Sunbeam Corp., Milwaukee, Wis., U.S.A.) for 3 min. The meat slurry was centrifuged at 10000 g for 5 min. The supernatant was recovered and filtered through a Whatman microfiber glass filter grade C (Whatman, Hillsboro, Oreg., U.S.A.). A 2.0-mL aliquot of the filtrate was reacted with 2.0 mL of 88 mM TBA for 15 min at 94 C in a capped tube in a heat block. After adjusting the pH to approximately 7 by NaOH and phosphate buffer (pH 8), the solution was loaded onto a Sep-Pak cartridge (Waters, Milford, Mass., U.S.A.). Before use, the cartridge was washed with 10 mL absolute methanol and then 10 mL deionized water, each at a flow rate of approximately 20 mL/min. The malondialdehyde and thiobarbituric acid (MDA-TBA) complex was recovered by eluting with 4.0 mL absolute methanol, and the eluent was read at 525 nm. TBARS were calculated from a standard curve of malondialdehyde (MDA), freshly prepared by acidification of tetraethoxypropane (TEP), and the TBA number was recorded as mg MDA/kg sample. Samples were analyzed in triplicate unless indicated otherwise. Standard deviations were calculated by MS Excel (Microsoft Corp., Redman, Wash., U.S.A.).

Antioxidant activity by the

-carotene method

The -carotene method used was based on the method of Marco (1968). A -carotene solution was prepared by dissolving 10 mg of the reagent in 200 mL chloroform. Two grams of Tween 40 and 250 mg linoleic acid was added to 30 mL of the -carotene solution. An aliquot of 3.0 mL of the resulting well-mixed mixture was subjected to a stream of nitrogen gas to remove the chloroform. The condensed residue was added and mixed well with 25 mL water that had been oxygenated by bubbling through it oxygen at 50 C for 30 min. After 3 min, to 3.0 mL of the emulsified solution was added 100 L sample extract, 0.8 mg, or methanol as control. After equilibrating for 2 min in a cuvette, the solution was read at 470 nm every 30 s for 15 min on a Shimadzu 160A spectrophotometer (Shimadzu, Houston, Tex., U.S.A.) with the temperature of the chamber maintained at 50 C. The percentage of antioxidant activity was calculated from the reactions rates as follows: % antioxidant activity = 100 (rate of control rate of sample)/rate of control

Results and Discussion

Antioxidant activities of extracts
Methanolic extracts of plant materials were analyzed for their antioxidant activities by the -carotene method. Typically, as shown in Figure 1, the intensity of the color complex formed because of -carotene and lipid peroxidation with or without the ex-

Total phenolic analysis

Total concentration of phenolics in the methanol extract was determined as follows: Samples were dissolved in methanol and water (60:40) containing 0.3% HCl to a concentration of 2.0 mg/mL. Test solutions (100 L) were added to 2.0 mL 2% sodium carbonate. After 2 min, 100 L 50% Folin-Ciocalteau reagent was added and allowed to stand at room temperature for 30 min. Absorbance was measured at 750 nm. The blank consisted of all reagents and solvents without the sample, and a calibration curve was prepared using gallic acid as the standard.

TBA method for analysis of lipid peroxidation in beef

Lean ground beef (approximately 15% fat) was purchased from a local supermarket; 100 g of the meat was thoroughly mixed with various rice ingredients, using a KitchenAid food processor (St. Joseph, Mich., U.S.A.) for about 30 s. The rice ingredients included brown rice flour, defatted rice bran, and crude rice oil at 15 g each, or the extract of rice bran, brown rice, and rice hull at 200 mg each in 20 mL ethanol. In the rice extract experiments, 20 mL ethanol was used as the control. In the rice ingredient experiments, 15 mL of water was used as the control. Test samples were prepared by taking 11.5 g of the mixture in 50-mL beakers. The sample beakers were covered with aluminum foil and cooked at 78 C for 2 h in an oven. The cooked beef was stored tightly covered at 4 C until analysis at 3 to 16 d for the rice extract experiments and at 2 to 15 d for the rice ingredient experiments. Thiobarbituric acid-reactive substances (TBARS) were determined for the stored beef samples using a slightly modified method of Raharjo and others (1992). A Trichloroacetic acid(TCA) solution containing a small amount of BHT was prepared by adding 18 mg BHT in 12 mL absolute ethanol to 225 mL 5% TCA solution. Meat

Figure 1Effects of rice ingredients on the rate of -carotene oxidative bleaching control, ; rice oil, ; rice wax ,*; rice hull, ; brown rice, X; and rice bran, .

Antioxidant milled-rice co-products . . .

tracts was recorded as a function of time. The resulting rate of reaction was used to calculate the antioxidant activity. While 100% activity is assigned to the situation with no change in rate, 0% activity was obtained when, using the solvent methanol as a control, -carotene was fully bleached under oxidative conditions. When the extracts were added, antioxidant activities were shown ranging from 45% to 86% (Figure 2). Selected milled-rice co-products including rice bran, rice oil, and rice hull appeared to contain ingredients that are effective antioxidants. Particularly, rice hull extract, with 86% antioxidant activity, is on top of the list. Similar findings on rice hull and wild rice hull being antioxidant-rich have been reported in the literature (Ramarathnam and others 1989; Wu and others 1994).
Table 1Yield and total phenolic content of various seeds and milled-rice co-products Total phenolics, mmoles gallic acid standard (per g extract) 0.23 0.59 0.08 0.04 0.57 0.18 0.16 0.16 0.002 0.019 0.010 0.010 0.015 0.003 0.008 0.005 (per kg plant material) 0.096 0.001 0.457 0.015 0.190 0.005 0.158 0.003 0.188 0.009 0.169 0.005

Sample Brown rice Rice bran Crude rice oil Rice wax Rice hull Cottonseed Soybean Corn

Extract yield (g) (per 100 g plant material) 4.25 7.79 3.3 8.90 11.85 10.65

Food Chemistry and Toxicology

Total phenolic content

Phenolic compounds are common natural antioxidants in plant seeds. Table 1 shows the yields and total phenolic content in the methanolic extracts of milled-rice co-products and selected seeds. Phenolic compounds serve to protect plant seeds from oxidative reactions and are mostly stored in the outer bran and hull. Rice bran and rice hull are processed products and, as expected, have the highest concentration of phenolic compounds in their extracts, substantially higher than the flours from the whole seeds. Other coproducts in the milling of rice, such as rice oil and rice wax, have lower phenolic contents because of the removal of the phenolic-rich components during processing. Based on data from Figure 2 and Table 1, coefficient of determination can be calculated and a good correlation is shown between the antioxidant activities of the rice

ingredient extracts to their phenolic contents (r2 = 0.81). However, the correlation is poor when all the extracts are included (r2 = 0.49), indicating that different plant sources may also contain various amounts of extractable antioxidant ingredients that are not phenolic compounds. Table 1 also shows the total phenolic content per kg of the extracted material, which depends on both the yield and the concentration of the phenolics in the extract. Rice bran has by far the greatest total phenolic content per kg of the extracted material.

Figure 2Antioxidant activities of methanolic extracts of various seeds and milled-rice co-products as determined by the -carotene method.

Figure 3Effects of methanolic extracts on lipid oxidation in beef determined by the TBA method for control, ; and milled-rice co-products of brown rice, ; rice bran, ; rice hull ,*; and BHT, .

Antioxidant milled-rice co-products . . .

Effect of extracts on lipid oxidation in ground beef
Extracts of rice bran, rice hull, and brown rice were investigated for their ability to inhibit lipid oxidation in ground beef. They were chosen because, in addition to having high antioxidant activities, they are readily available and, especially for rice bran and rice hull, cheap. The reaction was measured by the formation of MDATBA complex, which increased with increased incubation time, and the results are shown in Figure 3. In the control ground beef sample without the extract, lipid oxidation was indicated from day 3 and increased sharply during the rest of the 16-d incubation. At lower levels of oxidation in decreasing order are profiles of samples with the extract of brown rice, rice bran, and rice hull. The extract of rice hull was almost as effective as BHT. Extracts of milled-rice co-products showed potential to be useful as natural antioxidants in inhibiting lipid oxidation in beef and thus prolonging the storage stability of beef products. Antioxidants appear to be promising value-added products from these surplus milled-rice commodities.

Effect of rice ingredients on lipid oxidation in ground beef

Rice ingredients, such as rice flour, rice oil, and rice bran, have been used in foods and recognized for their nutritional and functional benefits. The added advantage of their being rich in antioxidants, and potentially beneficial in food storage, has not been fully studied or appreciated. Thus, experiments were conducted to apply brown rice flour, rice oil, and rice bran directly to ground beef and investigate the changes in the subsequent lipid oxidation. A substantial amount of the rice ingredient (15% by weight) was incorporated into the beef sample to ensure the introduction of not only antioxidant benefit but also body and other textural characteristics to the product. Figure 4 shows the profiles of lipid oxidation as a function of time for beef samples in the absence and presence of rice ingredients. In the absence of rice ingredient, the control beef sample showed a steady and sharp increase in oxidation throughout the 15-d incubation. When rice bran or brown rice flour was applied, the reaction was almost totally retarded during the same incubation period. Rice oil was less effective and substantial oxidation was indicated particularly at the later part of the 15-d incubation.

Conclusions

ethanolic extracts of milled-rice co-products, including rice bran and rice hull, were found to be effective antioxidants. A good correlation was also noticed between antioxidant activities and phenolic content of the rice ingredient extracts. When incorporated into ground beef, the extracts of brown rice, rice bran, and rice hull, or the flours of brown rice and rice bran, inhibited lipid oxidation effectively and thus prolonged storage stability of the product.

References
Basu TK, Temple NJ, Garg ML, editors. 1999. Antioxidants in human health and disease. New York: CABI Publishing. 450 p. Bekhit AED, Geesink GH, Ilian MA, Morton JD, Bickerstaffe R. 2003. The effects of natural antioxidants on oxidative processes and metmyoglobin reducing activity in beef patties. Food Chem 81:17587. Choi HC, Oh SK. 1996. Diversity and function of pigments in colored rice. Korean J Crop Sci 41:19. Chung IM, Kim KH, Ahn JK, Lee JO. 2000. Varietal variation in antioxidative activity of rice grain by DPPH and TBA methods. Korean J Crop Sci 45:2616. Frankel E. 1998. Lipid oxidation. Dundee, Scotland: The Oily Press. 303 p. Gopala-Krishna AG. 2002. Nutritional components of rice bran oil in relation to processing. Lipid Technol 14:804. Kim JS, Godber JS. 2001. Oxidative stability and vitamin E levels increased in restructured beef roasts with added rice bran oil. J Food Qual 24:1726. Lloyd BJ, Siebenmorgen TJ, Beers KW. 2000. Effects of commercial processing on antioxidants in rice bran. Cereal Chem 77:5515. Marco GJ. 1968. A rapid method for evaluation of antioxidants. J AOCS 45:594 8. Minerich PL, Addis PB, Epley RJ, Bingham C. 1991. Properties of wild rice/ground beef mixtures. J Food Sci 56:11547. Pratt DE. 1992. Natural antioxidants from plant material. In: Huang IVT, Ho CT, Lee CY, editors. Phenolic compounds in food and their effects on health. New York: Am Chem Soc. p 54-71. Qureshi AA, Mo H-B, Packer L, Peterson DM. 2000. Isolation and identification of novel tocotrienols from rice bran with hypocholesterolemic, antioxidant, and antitumor properties. J Agric Food Chem 48:313040. Raharjo S, Sofos JN, Schmidt GR. 1992. Improved speed, specificity, and limit of determination of an aqueous acid extraction thiobarbituric acidC18 method for measuring lipid peroxidation in beef. J Agric Food Chem 40:21825. Ramarathnam N, Osawa T, Namiki M, Kawakishi S. 1989. Chemical studies on novel rice hull antioxidants. 2. Identification of isovitexin, a c-glycosyl flavonoid. J Agric Food Chem 37:3169. Wu K, Zhang W, Addis PB, Epley RJ, Salih AM, Lehrfeld J. 1994. Antioxidant properties of wild rice. J Agric Food Chem 42:347.

Figure 4Effects of rice ingredients on lipid oxidation in beef determined by the TBA method for control, ; and milled-rice co-products of brown rice, ; rice oil, ; and rice bran, .