D e a c t i v a t i o n of T a n n i n in H igh T a n n i n Mi lo by T r e a t m e n t w i th Urea1, 2

R. W. RUSSELL 3 and J. R. LOLLEY 4 Department of Animal and Dairy Sciences

Auburn University, AL 36849

ABSTRACT

Experiments were conducted to deter- mine the effectiveness of urea in deacti- vating tannin in high tannin milo. High tannin milo (Pioneer B 815, 3.4 + .3% tannin) was reconstituted with aqueous urea solutions to give combinations of 26, 30, and 34% moisture with 2, 3, and 4% urea (percentage of urea per dry weight of milo). All treatments were maintained at 25°C and were effective in deactivating tannin with no differences among mois- ture or urea content. The average rate of tannin deactivation was 68 + 2% d -1. Temperature affected rate of tannin deac- tivation in milo reconstituted to 30% moisture and 3% urea when stored at 25 or 60"C. Rate of tannin deactivation was 44 + 5 and 89 + 18% d -1 at 25 and 60°C, respectively. Tannin in high tannin milo can be deactivated rapidly and com- pletely by reconstitution with aqueous urea. These studies demonstrate that tan- nin is deactivated completely under con- ditions where urea is an effective preser- vative of high moisture milo.

INTRODUCTION

Milo is an important cereal grain crop, rank- ing third in US production and fifth in world production (2). Tannin content of milo varies

widely (.2 to 6,9%) according to variety (3). Tannin content is associated positively with many agronomic attributes but is associated negatively with nutritional quality of the grain. Tannins reduce bird depredation (9), preharvest germination (4), preharvest molding (5), and increase resistance to pathogens and insects (6). Tannins, however, also reduce both feed intake and digestibility in animals; cause histopatho- logical changes in intestine, liver, spleen, and kidney; and are associated with fatal constipa- tion (6). The reduced performance of animals fed feeds containing tannins appears to be due to both inhibition of digestive processes and direct toxic properties of tannin metabolites that are absorbed (1, 14).

Tannin content of milo can be reduced by reconstitution followed with anaerobic storage (10, 13), by malting or formaldehyde treatment (8), and by treatment with acids (13) or alkali (12, 13). The nonspecificity of alkali (NH4OH, NaOH, K2CO3, CaO) in deactivation or modi- fication of tannin suggests that other alkalis also would be effective. Treatment of high moisture milo with urea increases grain pH due to hydrolysis of urea to NH3 and has been shown to be effective in preservation of stored grain (15). This trial was designed to determine if conditions effective in preserving stored, high moisture milo by urea treatment also would destroy tannin during storage.

MATERIALS AND METHODS

Received September 29, 1988. Accepted March 30, 1989. 1 J o u m a i Article Number 4-881681P of the Alabama Ag-

ricultural Experiment Station, Auburn 36849. Project Num- ber 00655.

2Research was partially supported by Columbia Nitrogen Corporation, Augusta, GA, and Alabama Farmers Federa- tion~ Montgomery, AL.

JPresent address: Division of Animal and Veterinary Sci- ences, West Virginia University, Morgantown 26506-6108. To whom reprint requests should be addressed.

4present address: USDA Parasitology Laboratory, Au- burn, AL.

Experiment 1

Pioneer B 815 milo grown at the Wiregrass Substation, Headland, AL, during the summer of 1987 and stored at 12% moisture was used in the experiments. Foreign material was re- moved by a commercial grain cleaner. Grain was stored in a 250-L drum after thorough mixing. All subsamples were taken directly from the 250-L drum.

Triplicate subsamples (27 total subsamples) of 500 g were reconstituted to 26, 30, or 34%

1989 J Dairy Sei 72:2427-2730 2427

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water with urea solutions such that urea con- tents were 2, 3, or 4% urea per milo dry weight. Urea solutions were made from feed-grade urea. The reconstituted milo was stored at 25°C in 600-ml plastic beakers covered with clear plastic wrap 5 to minimize moisture loss. Sam- ples were taken by probe from approximately the midpoint of each container at 3, 7, 14, and 21 d after reconstitution. These samples were freeze-dried overnight, ground using a Cyclotec 6 mill, and assayed immediately for tannin content (11).

Deactivation of tannin was evaluated using analysis of variance (16) of the amount of assayable tannin remaining in treated milos as a 3 x 3 x 4 factorial [moisture (26, 30, 34%) x urea (2, 3, 4%) x time (3, 7, 14, 21 d)]. The model included the main effects in addition to moisture x urea, moisture x time, urea x time, moisture x urea x time interactions, and tripli- cate within moisture and urea. Effects of mois- ture, urea, and moisture x urea were tested against replicate within moisture and urea. The rate of tannin deactivation was determined us- ing a single component, first-order nonlinear model; Y = Ae -kt, iterating with the multivari- ate secant method (16). In this situation, nonlin- ear regression weights data as they asymptoti- cally approach zero more appropriately than linear regression of natural logarithms.

Experiment 2

Triplicate 7.2-kg subsamples were reconsti- tuted to 30% moisture and 3% urea (dry weight basis) and stored at 60 or 25°C in 15-L plastic buckets covered with plastic wrap. Samples were removed from each container at 1, 2, 3, 4, 7, 10, and 14 d after reconstitution and pro- cessed as in Experiment 1.

Deactivation of tannin was evaluated using analysis of variance (16). The model included temperature, time, replicate (n = 3) within tem- perature, and the time x temperature interac- tion. Effect of temperature was tested against replicate within temperature. The rate of tannin deactivation was determined by nonlinear re- gression (16).

5Astor Products, Inc. Jacksonville, FL 32203. 6Fisher Scientific, Norcross, GA 30091.

RUSSELL AND LOLLEY

RESULTS AND DISCUSSION

Experiment 1

Tannin content of the Pioneer B 815 milo averaged 3.4 + .3% tannin (expressed as cate- chin equivalents). Tannin content in the recon- stituted milo decreased rapidly after reconstitu- tion and tannin was virtually undetectable 7 to 14 d after reconstitution (Table 1). All of the concentrations of moisture and urea tested were effective in total deactivation of tannin. There were no significant differences due to moisture or urea content or in the moisture x urea inter- action (Table 2). Because there were no differ- ences in moisture x time, urea x time, or mois- ture x urea x time interactions, all data were pooled after calculating means within replicates to estimate the first-order rate of tannin deacti- vation. Tannin deactivation rate was 68 + 2% d -1. Tannin deactivation in milo reconstituted to 25% moisture without urea addition ranged from 4.5 to 13.7% d -1 depending on variety (10). Mitaru et al. (10) varied moisture content from 15 to 25%, and rate of tannin deactivation increased with increasing moisture content.

It is unlikely that the marked difference in tannin deactivation rates is due to difference in milo variety used in the present study versus those varieties used by Mitaru et al. (10). Within variety (BR-54), reconstitution with aqueous alkali (NH4OH, NaOH, or K2CO3) markedly increased rate of tannin deactivation compared with reconstitution with water alone (12).

The alkali generated by hydrolysis of urea apparently enhanced the rate of tannin deactiva- tion relative to what would be expected from reconstitution with water alone as was observed for other alkalis. Reconstitution with water alone was attempted, but at these moisture con- tents without any effort to maintain anaerobio- sis the grain rotted, rendering data unreliable. Although tannins may inhibit urease within the gastrointestinal tract (7), the pH response and presence of ammonia odor were similar to that observed when low tannin milo is reconstituted with urea solutions (15).

Experiment 2

Experiment 1 demonstrates that reconstitu- tion of high tannin milo with urea solutions

Journal of Dairy Science Vol. 72, No. 9. 1989

PRODUCTION TECHNICAL NOTE

TABLE 1. Effect of moisture and urea content of reconstituted milo on deactivation of tannin. 1

2429

Time (d)

Moisture Urea 0 3 7 14 21

(% Tannin remaining) 2

26 2 100 10.4 2.5 .6 3.7 26 3 100 18.6 2.1 .2 .3 26 4 100 7.7 .6 0 .1 30 2 100 14.0 1.6 .2 0 30 3 100 8.5 0 0 2.0 30 4 100 14.7 1.4 .4 .2 34 2 100 13.7 1.6 .5 0 34 3 100 17.5 0 0 0 34 4 100 13.7 0 0 .6

1pooled SEM = 2.2. 2Initial tannin concentration was 3.4%.

were effective in tannin deactivation at 25*C. In field trials of preserving high moisture (30% moisture) milo with urea, grain temperature within the stack reaches approximately 60°C. Experiment 2 was designed to determine the effect of temperature on tannin deactivation.

Data are presented in Figure 1. Temperature had a significant effect on tannin deactivation (Table 3). Rate constants for tannin deactiva- tion were 44 + 5 and 89 + 18% d -1 for 25

TABLE 2. Statistical summary of effect of moisture, urea, and time on assayable tannin in reconstituted milo. 1

Sum of Probability Source df squares > F

Moisture 2 .0031 .91232 Urea 2 .0182 .58802 Moisture 4 .0789 .35142

x urea Replicate 18 .2999 .74233

(moisture urea) Time 3 3.5863 .00013 Moisture 6 .0594 .84393

x time Urea × 6 .0415 .92773

time Moisture x 12 .2462 .52753

urea x time Residual 54 1.1954

1percentages of moisture and urea were 26, 30, and 34 and 2, 3, and 4, respectively. Tannins were completely de- activated at all combinations of moisture and urea.

2Tested using replicate (moisture urea) mean square. 3Tested using residual mean square.

and 60"C, respectively. Tannin in milo stored at 60"C virtually was eliminated by 2 d after reconstitution. Thus, this regression is based primarily on three data points. More data be- tween d 0 and 2 would be required to reduce the error on the estimate of deactivation rate constant. This effect of temperature on rate of tannin deactivation is consistent with data of Mitaru et al. (10) where increasing the tempera- ture from 25 to 45°C of milo reconstituted with water to 25% moisture increased the rate of tannin deactivation from 4.5 to 66.6% d -1. There were only small differences, however, at

1.0

"~ .8

~ .6

e- - ~ .4

o

.2 .o

r~. 0

2 3

-X

: X

'. o~\ i O \

- - - 25 +, predicted . . . . . 60 °, predicted

• 25 °, observed o 61) +, observed

• . \ \

• o \ • \

\

? ' o . . . . . . . . o" . . . . . ~- - - ,~ I | I I I I

5 10 Time (Days)

- , - 7 - F - ~

Figure 1. Effect of temperature on rate of tannin deacti- vation in milo reconstituted with aqueous urea. Data with zero y values were omitted from the regression.

Journal of Dairy Science Vol. 72, No. 9, 1989

2430 RUSSELL AND LOLLEY

TABLE 3. Statistical summary of effect of temperature on tanmn deactivation in milo reconstituted with aqueous urea.

Sum of Probability Source df squares > F

Temperature 1 .129 .0(3011 Replicate 4 .002 .98382

(temperature) Time 7 5.235 .00012 Temperature 7 .276 .00012

x time Residual 28 .127

lTested using replicate (temperature) mean square. 2Tested using residual mean square.

4 and 52"C in t ann in deac t iva t i on rate in mi lo r econs t i t u t ed wi th N H 4 O H , N a O H , or K2CO3 (12).

In conc lus ion , t ann in in h igh t ann in m i l t s can be deac t iva ted rap id ly and comple t e ly by r econs t i t u t ion wi th aqueous urea. T a n n i n a lso is deac t iva t ed comple t e ly by urea u n d e r condi - t ions where urea is an e f fec t ive p re se rva t ive o f h igh moi s tu re milo .

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13 Reichert, R. D., S. E. Fleming, and D. J. Schwab. 1980. Tannin deactivation and nutritional improvement of sorghum by anaerobic storage of H20-, HCI-, or NaOH-trealed grain. J. Agric. Food Chem. 28:824.

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16 SAS Institute, Inc. 1987. SAS/STAT Guide for personal computers, version 6 ed. Cary, NC.

Journal of Dairy Science Vol. 72, No. 9, 1989