Phy Tase

614 JOURNAL OF FOOD SCIENCEVol. 66, No. 4, 2001 2001 Institute of Food Technologists

Sensory and Nutritive Qualities of Food

JFS: Sensory and Nutritive Qualities of Food

Phytase and Citric Acid Supplementation inWhole-Wheat Bread Improves Phytate-phosphorus Release and Iron DialyzabilityJ.M. PORRES, P. ETCHEVERRY, D.D. MILLER, X.G. LEI

ABSTRACT: Conditions were established for maximizing phytate breakdown in whole-wheat flour (wwf) duringbread baking and for assessing the effects of dephytinization on dialyzability of intrinsic and added iron in thebread. Three different sources of phytase (Aspergillus niger, A. fumigatus, and Escherichia coli) with various levels ofcitric acid (0 to 6.25 g/kg wwf) were used. Supplementing citric acid at 6.25 g/kg wwf enhanced phytate degradationcatalyzed by intrinsic phytase from 42% in the untreated bread to 69% (P ,,,,, 0.05). Supplementation of microbialphytase (285 units/kg) plus 3.125 or 6.25 g citric acid/kg wwf further enhanced phytate reduction up to 85%. Com-pared with the untreated bread, citric acid alone and the combination of citric acid and phytase enhanced total irondialyzability by 12- and 15-fold, respectively, while the combination of phytase, citric acid, and ascorbic acidimproved total iron dialyzability in the mixture by 24-fold.

Keywords: whole-wheat bread, phytase, citric acid, iron, phytate.

Introduction

THE WIDESPREAD PREVALENCE OF IRON DEFICIENCY IN MANYareas around the world and its negative health conse-quences in the general population demand constant re-search for ways to improve iron availability from foods.Legumes and cereals are significant sources of iron, buttheir nutritional importance is generally compromised bythe presence of phytate. This compound forms complexeswith di- and trivalent cations at the physiological pH con-ditions of the small intestine, rendering them unavailablefor absorption (Cheryan 1980; Lnnerdal and others 1988).High levels of phytate in whole-wheat bread are associat-ed with nutritional deficiencies of iron and other essentialminerals (Reinhold 1971; Haghshenass and others 1972).Whole-wheat flour and bakers yeast contain intrinsicphytase that can significantly reduce phytate concentra-tion during bread making if pH conditions are favorable(Larsson and Sandberg 1991; Trk and others 1996).

Phytases are a group of enzymes that sequentiallycleave orthophosphate groups from the inositol ring ofphytic acid, increasing the amount of available free phos-phorus and decreasing phytates affinity for different cat-ions (Lei and others 1993). In recent years, we have char-acterized several phytases with different biochemicalproperties and demonstrated their effectiveness in ani-mal feeding (Han and others 1998; Stahl and others1999). But the potential of these phytases for improvingmineral availability of human food such as whole-wheatbread remains to be determined. Other researchers haveused relatively high levels of microbial phytase and or-ganic acids during bread preparation to reduce itsphytate content (Trk and Sandberg 1992). Although theyhave obtained a good reduction of phytate in the bread,there are concerns about the cost associated with highlevels of supplemental phytase and the impact of highlevels of acids on the bread palatability for certaingroups of consumers.

The percentage of total dialyzable iron has been described

to be a reliable indicator of the availability of iron from dif-ferent foods (Schricker and others 1981; Miller and Berner1989; Kapsokefaulou and Miller 1991). Therefore, our objec-tives for this study were: (1) to determine the minimalamounts of extrinsic phytase from three different sources,and the amount of citric acid needed to maximize phytatedegradation and free phosphorus release during whole-wheat bread preparation; and (2) to determine if there was asynergistic effect of phytase and citric and ascorbic acids,two well-documented enhancers of iron absorption, on totaldialyzability of added and intrinsic iron in bread.

Materials and Methods

IngredientsWhole-wheat flour with an extraction rate of 100% [ex-

traction rate is defined as the proportion (%) of the whole-wheat grain obtained as the finished flour], dry yeast (NewHope Mills, Moravia, N.Y., U.S.A.), and other ingredientsused for the bread dough were purchased in local supermar-kets. Ultrapure water (Barnsted Thermolyne Corporation,Dubuque, Iowa., U.S.A.) was used for all the breads pre-pared. Citric acid (granular, monohydrate, FW 210.14) wasfrom J.T. Baker (Phillipsburg, N.J., U.S.A.). Aspergillus nigerphytase was a gift from BASF (Mt. Olive, N.J., U.S.A.) (5000U/g). Aspergillus fumigatus and Escherichia coli phytases(350 and 1,500 U/mL, respectively) were expressed and puri-fied in our laboratory (Rodriguez and others 1999, 2000).One unit of phytase activity (PU) is defined as the amount ofenzyme that liberates 1 mmol of inorganic phosphorus fromsodium phytate per minute at pH 5.5 and 37 8C.

Dough formulation and analysisA standard bread formula was used: whole-wheat flour

(425 g), double-distilled water (315 mL), dry yeast (9 g), salt (6g), and sugar (41 g). Citric acid and phytase were added tothe water prior to mixing all components. Dough was mixed,proofed and baked in a commercial bread maker (Regal

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Phytase and Citrate in Whole-Wheat Bread. . .

Kitchen Pro, Regal Ware, Inc. Kewaskum, Wis., U.S.A.). Wechose the bread machines whole-wheat bread program,which consisted of a primary knead and rise for 10 and 25min, respectively, and a secondary knead and rise for 20 and30 min, respectively, followed by a 30-s punchdown and a fi-nal rise for 70 min. Baking time was 65 min. Temperature in-side the bread dough during the first rise was 30 8C. This roseto 37 8C after the secondary knead and remained constant

through the secondary rise, punch down, and final rise. Tem-peratures in the interior of the loaf reached 100 to 110 8Cduring baking.

After baking, breads were cooled to room temperature,cut into small pieces, frozen, and freeze-dried (LabconcoCorp, Kansas City, Mo., U.S.A.). Freeze-dried breads wereground in a domestic grinder (Oster Corporation, Milwau-kee, Wis., U.S.A.), while samples taken for the time-courseexperiment were ground with a mortar and a pestle. All thesamples were stored at 4 8C until analysis. Dry matter con-tent of the bread was determined by drying for 48 h at105 8C. Dough pH in samples collected at different times ofthe proofing process was measured using an IQ 240 pH / mV/ Thermometer fitted with a model pH16-SS 3.5 mm MicroProbe (IQ Scientific Instruments, Inc., San Diego, Calif.,U.S.A.). Loaf volumes of the different breads were deter-mined using millet displacement.

Phytase and citric acid treatmentsA total of 11 treatments were tested in normally pro-

cessed whole-wheat bread (treatments 1 -11, Table 1). A 12thtreatment was included for a time-course study. Treatments1 to 4 were included to determine the amount of citric acidneeded to provide an optimal pH condition for maximalphytate hydrolysis catalyzed by the intrinsic phytase in theflour. Four levels of citric acid (CA): 0, 3.125, 6.25 and 8 g/kgwhole-wheat flour (wwf) (C, CA/3.125, CA/6.25, CA/8), wereadded to the formulation. Treatment 5 was to test the effectof a high level of supplemental A. niger phytase (1,125 PU/kg)alone on phytate hydrolysis in dough (AN1). Treatments 6 to11 were to determine the effects of three different phytases(A. niger, E. coli, and A. fumigatus) at 285 PU/kg combinedwith two levels of citric acid (3.125 or 6.25 g/kg) (AN2 andAN3, EC1 and EC2, F1 and F2, respectively).

Time-course studyA study was conducted to determine the time-course of

phytate degradation and free phosphorus release by intrinsicand (or) extrinsic phytases at different levels of citric acid.Samples were collected initially and at six different time-points of proofing (15, 30, 60, 90, 120, and 150 min), and as-sayed for phytate and free phosphorus. Five treatments wereincluded: C, CA/6.25, AN3, F1, and F3 (Table 1). TreatmentF3 was to assess the effect of a relatively high dose of phytase(570PU/kg wwf) combined with 3.125 g CA/kg wwf on the ef-ficiency of phytate degradation during proofing time.

Iron dialyzabilityIron dialyzability was assayed according to Kapsokefaulou

and Miller (1991). In all treatments, 279 g Fe/g bread wasadded as the extrinsic iron. The source was from anFe(NO3)3 solution with a concentration of 1,000 mg Fe/L

Table 1Description of the different treatments applied

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)Treatments C CA/3.125 CA/6.25 CA/8 AN1 AN2 AN3 EC1 EC2 F1 F2 F3Phytase source A. niger A. niger A. niger E. coli E. coli A. fumigatus A. fumigatus A. fumigatusPhytase amount 1,125 285 285 285 285 285 285 570

(PU/kg wwf)aCitric acid 3.125 6.25 8 3.125 6.25 3.125 6.25 3.125 6.25 3.125

(g/kg wwf)aPhytase units added per kg of whole-wheat flour.

Figures 1a and 1bTime-course of phytate degradationand free phosphorus release from bread dough (mg/g DM)Each point in a curve is the mean of three different repli-cates. The different treatments are presented in Table 1.Time-repeated measurement analysis showed a time ef-fect (P , 0.0001) and a timetreatment interaction(P , 0.0001)


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(Certified Atomic Absorption Standard , Fisher Sl124-100).Ascorbic acid (Sigma, St. Louis, Mo., U.S.A.) was added at 4.4mg/g bread as an iron dialyzability enhancer. Bread sampleswere digested with pepsin for 2 h in a 50 mL digestion vial at37 8C in a shaking water bath. Afterwards, a dialysis bag con-taining 10 mL of 0.2 N Pipes (pH 7.0) was placed in each di-gestion vial for 30 min. Then, 5 mL of pancreatin-bile mix-ture (2 mg/mL pancreatin, 12 mg/mL bile mixture) (Sigma,St. Louis, Mo., U.S.A.) was added to each digestion vial andincubated for another 2 h. At the end of this incubation, dial-ysis bags were removed from the digestion vials and rinsedby dipping in water. Bag contents were transferred to bea-kers and their contents weighed. Total and ferrous Fe weremeasured in the dialysates using ferrozine according to Kap-sokefalou and Miller (1991). Total dialyzable Fe was ex-pressed as percentage of the total Fe present in each vial.

Phytate and free phosphorus determinationPhytate and free phosphorus content were measured in

bread and dough as described by Latta and Eskin (1980) andChen and others (1956). Samples were extracted with 2.4%HCl for 90 min and centrifuged for 15 min at 2,000g (ModelGS-6KR, Beckman, Palo Alto, Calif., U.S.A.). The superna-tant was diluted with ultrapure water in a ratio 1:2.5 or 1:3depending on phytate concentration, and 15 mL of this di-lution were run through a column packed with DOWEX 14to 400 (200 to 400 mesh) strong basic anion exchange resin.After this, the column was subsequently washed with 15 mLof water and 15 mL of 0.1 M NaCl. All these three washeswere pooled together and free phosphorus was assayed inthat mix. Phytic acid was finally eluted from the column us-ing a 0.7 M NaCl solution and measured using the Wade Re-agent (0.3% sulfosalicylic acid and 0.03% FeCl3 6H2O).

StatisticsData was analyzed using one-way ANOVA (SAS Institute, Inc.

1988). Duncans Multiple Range test was applied to determinesignificance of differences in iron dialyzability, phytate, and freephosphorus content of whole-wheat bread between varioustreatments. Time-repeated measurement analysis was appliedto the time-course data. Significant differences between indi-vidual points of the various bread treatments were analyzed us-ing Duncans Multiple Range test. The level of significance wasset at 0.05.

Results

Effects of citric acid and extrinsic phytasesupplementation

Bread making without supplementation with eithercitric acid or phytase (C) resulted in a 42% reduction inphytate content ( Table 2). Adding different concentra-tions of citric acid to the dough enhanced phytate degra-dation (P , 0.05). Specifically, the level of 3.125, 6.25, and8 g CA/kg wwf (CA/3.125, CA/6.25, CA/8) gave a total of52, 69, and 55% phytate reduction in bread, respectively.Probably due to the inhibition on yeast fermentation bythe lowered pH and (or) high levels of citric acid, 6.25and 8 g CA/kg wwf caused 10.2 and 18.1% decrease in thefinal volume of the bread compared to the control, re-spectively. Supplementation of 1,125 PU/kg wwf in theabsence of citric acid (AN1) reduced the levels of phytatesimilarly to CA/3.125 and CA/8 compared to the untreat-ed bread (C). In contrast, supplementation of 285 PU/kgwwf with either 3.125 or 6.25 gCA/kg wwf (AN2 and AN3)significantly improved phytate degradation. Similarphytate reductions (83.4 against 85%) were produced byA. fumigatus phytase at these two levels of citric acid ad-dition (F1 and F2), whereas A. niger phytase was more ef-fective at 6.25 (AN3) than 3.125 g CA/kg wwf (AN2). At ei-ther level of citric acid, E. coli phytase was less effectivethan the other two phytases in hydrolyzing phytate. Theeffects of all these treatments on free phosphorus releasein the breads were consistent with their effects onphytate degradation.

Table 2Phytate content and free phosphorus released(mg/g DM) from different breads

Phytate % Phytate Phosphorus* (mg/g DM) reduction (mg/g DM)*

WWF 15.26 6 0.75 0% 0.18 6 0.04(1) C 8.84 6 0.37a 42% 1.47 6 0.10a(2) CA/3.125 7.21 6 0.56b 52.8% 1.94 6 0.04bc(3) CA/6.25 4.64 6 0.25e 69.6% 2.05 6 0.06cd(4) CA/8 6.88 6 0.42bc 54.9% 1.87 6 0.02b(5) AN1 6.51 6 0.56c 57.3% 1.85 6 0.11b(6) AN2 4.04 6 0.11f 73.5% 2.24 6 0.02e(7) AN3 2.23 6 0.09g 85.4% 2.69 6 0.06f(8) EC1 5.70 6 0.25d 62.6% 2.08 6 0.08d(9) EC2 3.65 6 0.21f 76.1% 2.33 6 0.06e(10) F1 2.54 6 0.37g 83.4% 2.53 6 0.07g(11) F2 2.28 6 0.20g 85% 2.75 6 0.04f* Values are expressed as means 6 standard deviation (n = 3).a, b, c, d, e, f, g Means within the same column without same superscript aresignificantly different (P , 0.05).

Figure 2Effect of phytase, citric acid, and ascorbic acidon total dialyzable Fe from whole-wheat bread. Each barrepresents the mean 6 SD of three different replicates.Values without sharing a common letter differ (P , 0.05).Abbreviations: HClControl without sample, phytase orFe added. FeControl with the same volume of Fe stan-dard added to all the samples. wwf + Fe, C + Fe, AN1 +Fe, CA/6.25 + Fe, CA/8 + Fe, AN3 + FeAs expressed inTable 1 plus iron standard (279 mg Fe/g bread) addedprior to pepsin digestion. wwf + Fe + AA, C+ Fe + AA,AN3 + Fe + AAAs expressed in Table 1 plus iron stan-dard (279 mg Fe/g bread) and ascorbic acid (4.4 mg AA/g bread) added prior to pepsin digestion. Fe + AACon-trol with the same volume of Fe standard and ascorbicacid as in all other samples.


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Time-course studyThere was a time effect (P , 0.0001) and a timetreatment

interaction (P , 0.0001) on phytate degradation and freephosphorus release from whole-wheat bread catalyzed byphytase in the presence of citric acid (Figures 1a and 1b). Af-ter 90 min, the improvements by phytase and citric acid overthe untreated control were significant. The combination of570 PU of A. fumigatus phytase with 3.125 g CA/kg wwf (F3)(Table 1) was the most effective treatment, and the sharpestreduction of phytate occurred between 60 and 90 min proof-ing. No significant changes in these two parameters were ob-served beyond 120 min. There was an increase of phytase ac-tivity in the dough of untreated (C) and supplemental 6.25 gCA/wwf (CA/6.25) after 2 h leavening. When extrinsicphytases from different sources were supplemented, a con-comitant increase in activity was detected (Table 3).

Iron dialyzabilityTotal iron dialyzability from wwf and untreated bread

(C 1 Fe) was 0 and 1.8%, respectively. Supplementation of1,125 PU in the bread in the absence of citric acid (AN1 + Fe)did not increase dialyzability (Figure 2). When 6.25 or 8 g CA/kg wwf were added to the dough (CA/6.25 1 Fe, CA/8 1 Fe),iron dilalyzability of the bread was 12-fold higher than C 1Fe. The combination of phytase and 6.25 g CA/kg wwf (AN31 Fe) further increased iron dialyzability to a value that was15-fold higher than the untreated bread. Ascorbic acid im-proved (P , 0.05) iron dialyzability in the wwf 1 Fe and C 1Fe groups. The combination of phytase, citric, and ascorbicacid (AN3 1 Fe 1 AA) resulted in a 24-fold increase in irondialyzability compared to C 1 Fe.

Discussion

HIGH LEVELS OF PHYTATE IN WHOLE-WHEAT BREAD (TER-Sarkissian and others 1974) can dramatically inhibit theabsorption of some minerals. This may seriously compro-mise the health status of individuals who rely on it as staplefood (Hagshenass and others 1972; Reinhold and others1973; Brune and others 1992). In recent years, phytases withdifferent biochemical properties have been developed andextensively applied in the field of animal nutrition (Han andothers 1998; Stahl and others 1999). But there are few reportsactually dealing with their applications to human nutrition(Trk and Sandberg 1992; Sandberg and others 1996; Greinerand Konietzny 1998, 1999).

The intrinsic phytase-catalyzed phytate breakdown inbread is increased by citric acid

The improved effectiveness of intrinsic phytase fromwhole-wheat flour in response to acidification of the bread

dough caused by different organic acids has been reportedpreviously (Trk and others 1996). The pH optimum de-scribed for wheat phytase is 5.2 (Peers 1953), although an ef-fective phytate breakdown has been observed between pH4.6 and 5.1 (Larsson and Sandberg 1991; Trk and others1996). In the present study, we conducted a titration studywith various amounts of citric acid added to the dough to es-tablish the pH and citric acid concentrations that optimizethe activity of intrinsic phytase from the whole-wheat flour.The largest reduction in phytate levels was obtained when6.25 g CA/kg wwf were added (CA/6.25), yielding a dough pHof 4.6 to 4.8. Adding more (8 g/kg wwf and a dough pH of 4.3to 4.5) or less (3.125 g/kg wwf and a dough pH of 5.2 to 5.5)citric acid also enhanced phytate reduction, but to a lesserdegree. While pH is clearly an important factor in phytaseactivity, the improvement in phytate breakdown by citricacid supplementation could be partially due to the capacityof the organic acid to complex some of the minerals boundto the phytate molecule, rendering it more susceptible tophytase attack (Maenz and others 1999). Several groups (Re-inhold 1975; Harland and Harland 1980; Harland and Frlich1989; Trk and others 1996; Trk and others 2000) have re-ported that yeast phytase contributes to phytate breakdownduring bread making. In our study, phytase activity wasgreater in the doughs without any extrinsic phytase supple-mentation than phytase activity in the whole-wheat flourused (Table 3). But it is unclear whether this increment ofphytase activity was from the yeast or from the activation ofintrinsic phytase.

Extrinsic phytase is needed to maximize phytatedegradation in bread dough

Trk and Sandberg (1992) have demonstrated the poten-tial of microbial phytase for phytate breakdown in whole-wheat bread. However, the high concentrations of phytaseand lactic acid used in their study may limit the possible ap-plications of this approach. Thus, we were interested in sup-plementing with lower concentrations of both organic acidand extrinsic phytase in the bread that still produce signifi-cant reductions in its phytate levels.

Our results clearly indicate that this goal is achievable byusing the appropriate combination of microbial phytase andcitric acid. Supplementation with high levels of phytase in theabsence of citric acid (AN1) yielded a phytate breakdownthat was only slightly better than the untreated bread (C).Therefore, supplementation of bread dough with organic ac-ids is required to achieve maximal activity of extrinsicphytase, as is the case with intrinsic phytase. This is not sur-prising in view of the high pH of bread doughs (5.7 to 6.8)which is not optimal for the phytase present in the flour orthe added extrinsic phytases. Phytase from A. fumigatus wasthe most efficient enzyme at both concentrations of citricacid. This enzyme has a pH optimum closer to the condi-tions for bread making used in this study than the other twophytases, and is more effective in the hydrolysis of inositolphosphates with lower degrees of phosphorylation (Wyssand others 1999). In contrast, the pH optimum of E. coliphytase is more acidic than the lowest dough pH in ourstudy, precluding its full action.

Maximal degradation of phytate by phytase requires2 h of leavening

The effects of phytase and citric acid on phytate break-down and phosphorus release were time-dependent. Thecombination of 570 PU (A. fumigatus) and 3.125 g CA/kg wwf

Table 3Phytase activity present in whole-wheat flour anddifferent bread doughs after 2 h leavening (Units/kg DM)

Treatment Phytase activity*

WWF 505.95 6 18.73Bread doughs

C (1)** 706.53 6 10.04CA/6.25 (3) 678.94 6 4.92

AN3 (7) 972.41 6 45.77F1 (10) 1047.06 6 70.25F3 (12) 1435.66 6 102.00

* Values are expressed as mean 6 standard deviation (n = 3).

** See Table 1 for an explanation of the numbered treatments.


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(F3) was the most effective treatment, and no appreciabledifference was observed for phytate hydrolysis or free phos-phorus release between time-points 120 and 150 min. Thissuggested that leavening time of 120 min was adequate formaximal hydrolysis of phytate by phytase.

Differences in free phosphorus release between F1 andAN3 observed during the last points of the time-course studyare most probably due to the higher affinity of A. fumigatusphytase for inositol phosphates with lower degree of phos-phorylation (Wyss and others 1999). This characteristic of A.fumigatus phytases may be used to produce cooperative orsynergistic phytate hydrolysis with other types of phytases.

Phytase, citric acid, and ascorbic acid collectivelyimprove iron dialyzability

Iron deficiency is a widespread nutritional problem inmany areas of the world (Fairbanks 1999) and can have veryserious consequences for the welfare and health status of thegeneral population. To study how our treatments could af-fect the availability of iron from whole-wheat bread, we haveused an in vitro technique based on the expected improve-ment of iron absorption when the solubility of this mineral isincreased. Citric acid improves iron dialyzability under ourexperimental conditions. This positive effect of citric acid hasbeen previously described not only for iron but also for oth-er trace minerals (Clydesdale 1983; Hazell and Johnson 1987;Anand and Seshadri 1995; Walter and others 1998). With itscapacity to form complexes with minerals at pHs commonin the lumen of the small intestine, citric acid may helpmaintain iron in a soluble form. Phytate present in foods ofvegetable origin is a well-recognized inhibitor of nonhemeiron absorption (Gillooly and others 1983; Stahl and others1999), and its reduction has consistently improved the avail-ability of this mineral from bread (Brune and others 1992).Phytate hydrolysis is also desirable to increase the availabilityof other nutritionally important minerals like zinc and phos-phorus (Lei and others 1993; Han and others 1998). The abili-ty of phytase supplementation to further reduce phytatepresent in the bread supplemented with 6.25 g CA/kg wwf(AN3 + Fe) gave rise to a significant increase in iron dialyz-ability compared to (CA/6.25 1 Fe), and reached a value 15-fold higher than (C 1 Fe).

The beneficial effects of ascorbic acid as an enhancer ofiron absorption (Cook 1983; Monsen 1988; Kapsokefalou andMiller 1991) are clearly observed in our experiments. Addingascorbic acid caused a significant increase in the dialyzabilityof iron from all the breads studied. Most importantly, thecombination of citric acid, ascorbic acid, and phytase offersa great potential in preventing nutritional iron deficiency, asit dramatically improves iron dialyzability without the needof costly or technologically advanced food processing opera-tions. These results should, however, be corroborated withan in vivo study.

In conclusion, the combination of phytase and citric acidyields a significant reduction of phytate in whole-wheatbread. Supplementation with phytase, citric acid, and ascor-bic acid greatly improved iron dialyzability from this meal,and could be a promising approach to prevent nutritionaliron deficiency and anemia.

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Ms. 20000720

The authors thank Deborah A. Ross, Orlena Cheng, Daniel Omar Maizon, Carol Roneker,William A. House, and Wilson G. Pond for their valuable assistance. Dr. Porres was funded

in part by a grant from the University of Granada, Spain. The project was supported by theCornell Biotechnology Program.

Authors Porres and Lei are with the Department of Animal Science, andauthors Etcheverry and Miller are with the Department of Food Science,Cornell University, Ithaca, N.Y. Please direct inquiries to X.G. Lei, Dept. ofAnimal Science, 252 Morrison Hall, Cornell Univ., Ithaca, NY 14853 (e-mail:[email protected]).

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