Volume 62, No. 2, 1997—JOURNAL OF FOOD SCIENCE—281 Soybean Flour Lipoxygenase Isozymes Effects on Wheat Flour Dough Rheological and Breadmaking Properties B. CUMBEE, D.F. HILDEBRAND, and K. ADDO ABSTRACT The effects of soy flour from LOX null mutant isolines and purified LOX isozymes on the rheological and breadmaking properties of a com- mercial hard wheat flour were investigated. Wheat flours were fortified with either 3% soy mutant flours alone, or in the presence of 0.2% and 1% linoleic acid. Purified LOX 2 isozyme had the greatest effect among LOX isozymes on dough extensibility and strength. Linoleic acid sub- strate addition reduced dough extensibility and strength. The L2L3 null isoline mutant for L1 resulted in the largest increase in bread volume. A reduction in bread firmness occurred after 5 day storage for all three single null mutant-containing samples in the presence of 1.0% linoleic acid substrate. Key Words: soy, lipoxygenase, isozymes, rheology, breadmaking INTRODUCTION THE MAIN USES of enzyme-active soy flour in the commercial production of white breads are, to improve wheat flour dough rheological, baking and nutritional properties (Frazier, 1979; Hoover, 1979; Faubion and Hoseney, 1981; Gardner, 1988). However, enzyme-active soy flour is used only up to 0.5% in wheat flour-based breads due to adverse effects including for- mation of volatiles that cause off-flavors (Wolf, 1975; Hoover, 1979). Soy flour contains three distinct lipoxygenase isozymes designated as L1, L2 and L3. These have been isolated and characterized in seeds of commercial soybean cultivars (Arai et al., 1970; Axelrod et al., 1981; Rackis et al., 1979; Wolf, 1975). A few attempts have been made to isolate the different iso- zymes of lipoxygenase, and to investigate rheological and bak- ing properties of wheat flours fortified with them. Shiiba et al. (1991) fractionated and purified the three major endogenous wheat lipoxygenase isozymes. They reported an increase in foaming activity, and overall improvement in breadmaking qual- ity of wheat flour supplemented with the wheat L3 isozyme. Experiments reported by van Ruth et al. (1992) on the effects of stored soybean preparations, showed that soy L2 and/or L3 were primarily responsible for bleaching of dough and formation of volatile compounds. Later studies by Addo et al. (1993) showed that soy L2 was mostly responsible for production of undesirable aroma compounds in bread doughs. However, the impact of these specific individual soy lipoxygenase isozymes on other breadmaking qualities of wheat flours has not been extensively investigated. The objective of our present study was to characterize the effect of soy mutant isolines and purified soy lipoxygenase iso- zymes L1, L2, L3 (with or without an added linoleic acid sub- strate) on wheat flour dough rheological and breadmaking properties. MATERIALS & METHODS Soybean samples The commercial soybean cultivar ‘Century’ and the lipoxygenase mu- tant isolines back crossed to Century (Davies and Nielsen, 1987) were Authors Cumbee and Addo are affiliated with the Dept. of Nutri- tion & Food Science, and author Hildebrand is with the Dept. of Agronomy, Univ. of Kentucky, Lexington, KY 40506. Direct in- quires to Dr. K. Addo. grown on the University of Kentucky Agronomy research farm at Lex- ington, KY in 1990. The lines were designated according to the lipox- ygenase isozyme present: L2/L3 = lipoxygenase 1 null; L1/L3 = lipoxygenase 2 null; L1/L2 = lipoxygenase 3 null. Soy flour preparation Commercial and mutant soy beans were processed into full-fat, en- zyme-active soy flours. Seeds were cracked using a standard, screw-type, household grinder with coarse blades, and then dehulled. The meal was first ground in a Wiley Mill (0.5 mm sieve) and then in a household coffee mill (Mouliner Model 505. Mouliner Regal Inc. Virginia Beach VA) and sieved for uniformity before using. Full-fat flours were stored at -10°C until used. Purification of lipoxygenase isozymes Lipoxygenases 1, 2 and 3 (L1, L2, L3) were purified from -L2L3, -L1L3 and -L1L2 lines respectively, as described by Hildebrand et al. (1990). Lipoxygenase activities were determined by spectrophotometric measurement of the formation of conjugated dienes at 235 nm as de- scribed by Hildebrand et al. (1991). The activities of L2 and L3 were determined using linoleic acid as the substrate at pH 6.8, and L1 activity was determined using linoleic acid at pH 9.0. Wheat flour and mutant soy flour mixtures Mixtures consisting of a commercial untreated hard wheat flour (Car- gill Flour Milling Co., Chattanooga, TN), and 3% full-fat soy flour were formulated for the commercial Century flour and all mutant flours. The soy flour was thoroughly mixed with part of the wheat flour to ensure uniform distribution. This mixture was then added to the larger amount of wheat flour and again thoroughly mixed. For flour mixtures containing linoleic acid (Sigma Chemical Co., St. Louis, MO), the exact amount of fatty acid was placed in a vial and dissolved in a small amount of pe- troleum ether (PE). The mixture was then carefully added with a Pasteur pipette to the wheat flour and thoroughly mixed. Flour mixture was placed under fume hood for 1 hr to remove any residual PE, followed by the addition of soy flour as described above. Wheat flour and purified enzyme mixtures Purified soy lipoxygenase isozymes (4000 μM/sec) in 400 mL of 10 mM phosphate buffer (pH 6.8) were incubated and shaken with the wheat flour and/or wheat flour and substrate mixture at 40°C for 4 hr. After incubation, the slurry was frozen immediately and lyophilized, ground, and sieved by a cyclone mill with a 0.5 mm filter (Udy Cor- poration, Fort Collins, CO). The control flour was treated the same way but with no enzyme (Shiiba et al., 1991). These flours were used for the alveograph rheological studies involving the purified isozymes. Alveograph testing Alveograph measurements were performed under conditions of con- stant dough water content and mixing times using standard Method 54- 30 (AACC 1983). For data recording and storage, the modified procedure of Addo et al. (1990) was used. The following alveograph parameters were automatically recorded by a computer software pro- gram: maximum over-pressure needed to blow the dough bubble, P, an index of resistance to extension; average abscissa at bubble rupture, L, an index of dough extensibility; and deformation energy, W, an index of dough strength.