The Effect of Enzyme Treatment of Soybean Meal on Oligosaccharide

7/31/2019 The Effect of Enzyme Treatment of Soybean Meal on Oligosaccharide

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The Effect of Enzyme Treatment of Soybean Meal on OligosaccharideDisappearance and Chick Growth Performance

K. K. Graham,* M. S. Kerley,*,1 J. D. Firman,* and G. L. Allee*

*Department of Animal Sciences, CAFNR, University of Missouri, Columbia, Missouri 65211

ABSTRACT Research was conducted to determine theeffects of enzymatically hydrolyzing raffinose and stachy-ose from soybean meal (SBM) on fecal oligosaccharideconcentration and growth performance of chicks fed acorn-SBM diet. The -galactosidase treatment was opti-mized for oligosaccharide degradation. Enzyme treat-ment degraded raffinose and stachyose in SBM by 69 and54%, respectively, compared to untreated soybean meal(USBM). Diets containing enzyme-treated soybean meal(ESBM) resulted in excreta raffinose and stachyose con-centrations reduced to below measurable levels ( 0.05) in growth performance among treatments.Chicks fed the ESBM diet had an increased (P < 0.05)fecal neutral detergent fiber (NDF) content in one of twostudies. A fourth experiment was conducted to determineif heating, used to enhance enzyme treatment, woulddecrease lysine availability. Heating significantly (P 0.05) improvedby enzyme treatment.

This research was conducted to determine if treatingSBM with-galactosidase prior to mixing the diet wouldreduce raffinose and stachyose concentrations in the ex-creta. Growth performance effects and TME of thetreated SBM were also measured in the chicks.

MATERIALS AND METHODS

Chick Studies

Experiment 1 consisted of three treatment diets allot-ted randomly to six pens, with five birds per pen foreach diet. Experiment 2 used the same three treatmentdiets but had eight pens allotted randomly to each dietwith five birds per pen. All studies followed the experi-mental guidelines discussed below with diets fed for 14d. Diets fed to chicks in Experiments 1 and 2 (Table 1)comprised 55% corn and 36% untreated soybean meal(USBM), or water-treated soybean meal (WSBM), or -

galactosidase-treated soybean meal (ESBM). WSBM wasused to determine that effects of enzyme treatment wereattributable to the enzyme and not the aqueous solution.Diets fed in Experiment 3 consisted of 49.6% corn and

Abbreviation Key: ADFI = average daily feed intake; ESBM = en-zyme-treated soybean meal; NDF = neutral detergent fiber; SBM = soy-

bean meal; USBM = untreated soybean meal; WSBM = water-treatedsoybean meal.


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ENZYME TREATMENT OF SOYBEAN MEAL 1015

TABLE 1. Ingredient composition of the diets fedin Experiments 1, 2, and 3

Experiments 1 and 2 Experiment 3Ingredient (% as fed) (% as fed)

Ground corn 55.20 49.60Soybean meal 35.90 39.40Corn oil 5.20 6.00Dicalcium phosphate 1.90 2.60Limestone 0.85 1.20Salt 0.41 0.50DL-Methionine 0.21 0.35Trace mineral1 0.10 0.10Vitamin premix2 0.07 0.10Choline chloride 0.07 0.10L-Lysine HCl 0.05 . . .Selenium premix3 0.03 0.02Copper sulfate 0.01 0.01Crude protein (%)4 21.0 24.2Lysine (%)4 1.3 1.3Stachyose 1.4 1.5Raffinose 0.4 0.4

1Trace mineral premix contained 2.5% calcium (calcium carbonate)minimum, 3.5% calcium maximum, 6% iron (ferrous sulfate), 2.68%magnesium (magnesium oxide), 11% manganese (manganese sulfate),11% zinc (zinc sulfate), and2,000ppm iodine (ethylenediamine dihydri-odide).

2Provided per kilogram of premix; 17,637 KIU vitamin A, 7,716 KIUvitamin D3, 27,558 IU vitamin E, 110,231 mg niacin, 33,069 mg D-panto-thenicacid, 11,023 mg riboflavin,4,409 mg pyridoxine,3,307 mg menadi-one, 2,756 mg folic acid, 2,205 mg thiamine,441 mg biotin,22 mg vitaminB12, 2% flow agent (rice hulls).

3Selenium premix contained 38% calcium (calcium carbonate) and600 mg/kg selenium.

4Calculated from NRC (1994).

39.4% WSBM or ESBM. All diets were formulated tomeet or exceed the requirements according to the NRC(1994). Experiment 3 diets were formulated to meet 120%of the NRC amino acid requirement.

All procedures involving animals were approved bythe University of Missouri Animal Care and Use Com-mittee. Day-old male broiler chicks were obtained froma commercial hatchery. Chicks were fed a commercialchick starter crumble diet,2 until the start of the trial. At3 d of age, chicks were individually weighed and allottedrandomly to pens. Birds were housed in raised-wirefloor, stainless steel chick batteries within a temperature-controlled room. Temperature was 32.2 C for the first 5d and then reduced to 26.7 C over 2 d for the remainderof the study. The chicks were exposed to 24 h of light.Feed and water were offered ad libitum throughout the

experiment. At the conclusion of the experiments, birdswere weighed individually. Total gain was calculatedby subtracting the beginning weight of the chicks fromthe final weight. Daily feed intake was calculated bydividing the total pen intake by the number of birds inthe pen by the number of days fed experimental diets.If a pen contained a dead bird, feed intake was calculated

2MFA, Inc., Columbia, MO 65202.3Arthur H. Thomas Co., Philadelphia, PA 19102.4Gelman Sciences, Inc., Ann Arbor, MI 48103.5Beckman Coulter, Inc., Fullerton, CA 92835.

for that bird by dividing the total feed intake up untilthe day the bird died by the number of animals in thepen. Gain to feed was calculated by dividing the averagedaily gain by the average daily feed intake of the pen.Total feces were collected on the last 3 d of Experiments1, 2, and 3, and stored in ziplock freezer bags at 0 C untilanalyzed for dry matter, oligosaccharides, and neutraldetergent fiber (NDF).

A fourth chick experiment was conducted to deter-mine if drying (55 C) the ESBM resulted in reducedlysine availability. The same feeding and handling pro-cedures as the first studies were followed, except that

birds (five birds per pen, six pens per treatment) beganreceiving experimental diets at 7 d of age. The studywas conducted over 14 d. Basal diets in this experiment(Table 2) consisted of three increasing levels of lysine,added as L-lysine HCl. By using a slope ratio technique(Littell et al., 1995), USBM and ESBM were each fed attwo dietary levels to determine lysine availability. Ly-sine percentages of diets were measured.

SBM Preparation

All SBM used in these studies was taken from thesame batch load. The WSBM was prepared by sprayingtap water 1:5 (vol/wt) on 5,000 g of SBM in a 15-L plastic

jug, using a hand-held sprayer. The jug was then rolledfor 1 h on a ball mill roller to facilitate complete coverageof the SBM. The -galactosidase treatment was accom-plished by spraying a 4% (vol/wt) enzyme solution ontothe SBM. The -galactosidase enzyme (49.6 mg protein/mL) used in these studies released 0.1 M galactose permin/mL from stachyose. After mixing for 1 h, the SBMwas spread out on wire screens covered in cheesecloth

and air-dried at room temperature for 3 d (88% drymatter). In Experiment 4 only, ESBM was placed into adrying oven (55 C) for 48 h after application of -galac-tosidase.

Analyses

The TME analysis of the SBM was conducted withadult roosters following the method of Sibbald (1986).The TME value for each SBM treatment was the averageof data from six roosters. Fecal samples were dried ina55Covenfor3dandgroundinaWileyMill,3 througha 1-mm screen. Raffinose and stachyose were extractedusing 85% methanol. A 2.5-g subsample of dried, groundfecal material was extracted in 25 mL of 85% methanolfor 2 h. The liquid fractions of the samples were thenfiltered through glass microfiber filter paper with a poresize of 1.5 m. The retentate was extracted two addi-tional times. The filtrate was then brought to a totalvolume of 100 mL in volumetric flasks using 85% metha-nol. One milliliter of the total volume was evaporatedto dryness under a stream of nitrogen, and 5 mL ofdistilled water was then added. One milliliter of thediluted sample was filtered through a 0.2-g Acrodisc,4

and analyzed using a Beckman HPLC5 equipped with


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GRAHAM ET AL.1016

TABLE 2. Ingredient composition of the diets fed in Experiment 4 1

Basal Basal12 Basal22 USBM32 USBM62 ESBM32 ESBM62

Soybean meal . . . . . . . . . 3.3 6.6 3.3 6.6Starch 46.9 46.8 46.6 43.7 40.4 43.7 40.4Corn oil 1.0 1.0 1.0 1.0 1.0 1.0 1.0Peanut meal 46.2 46.2 46.2 46.2 46.2 46.2 46.2Dicalcium phosphate 2.3 2.3 2.3 2.2 2.1 2.2 2.1Limestone 1.1 1.1 1.1 1.2 1.2 1.2 1.2L-Lysine HCl . . . 0.1 0.3 . . . . . . . . . . . .Premix3 2.5 2.5 2.5 2.5 2.5 2.5 2.5

1All numbers expressed on as-fed percentage of the diet.2Basal1 and Basal2 = basal diet with 0.1 and 0.2% added lysine, respectively; SBM3 and SBM6 = basal diet

with 3.3 and 6.6% SBM added, respectively; ESBM3 and ESBM6 = basal diet with 3.3 and 6.6% ESBM added.3Contents of premix based on percentage of the diet; 0.50% methionine, 0.50% NaCl, 0.39% threonine, 0.29%

isoleucine, 0.27% valine, 0.14% choline chloride, 0.10% trace mineral premix [2.5% calcium (calcium carbonate)minimum, 3.5% calcium maximum, 6% iron (ferrous sulfate), 2.68% magnesium (magnesium oxide), 11%manga-nese (manganese sulfate), 11% zinc (zinc sulfate), and 2,000 ppm iodine (ethylenediamine dihydriodide)], 0.10%vitamin premix [provided per kg of premix: 17,637 KIU vitamin A, 7,716 KIU vitamin D 3, 27,558 IU vitamin E,110,231 mg niacin, 33,069 mg D-pantothenic acid,11,023mg riboflavin, 4,409 mg pyridoxine,3,307 mg menadione,2,756 mg folic acid, 2,205 mg thiamine, 441 mg biotin, 22 mg vitamin B12, 2% flow agent (rice hulls)], 0.03%tryptophan, 0.003% selenium premix, 0.0003% copper sulfate, 0.18% lysine HCl.

a Dionex pulsed amperometric detector6 to determine

the concentration of fecal raffinose and stachyose. Flowrate was 0.9 mL (50 mM NaOH)/min for 20 min. ADionex Carbopac,6 column was used to separate oligo-saccharides. The NDF (Goering and Van Soest, 1970)was determined in feces by using the ANKOM 200 fi-

ber analyzer.7

In Experiment 3, feces were collected daily from eachpen on Days 8 through 10 of the experiment. Feed intakeduring the collection period was measured 24 h in ad-vance of fecal collection for each pen. After collection,feces were immediately dried in a 55 C oven for 3 dand then ground through a Wiley Mill (1-mm screen).Samples were composited by pen and dry matter and

calculated by drying feces at 105 C for 24 h. Percentageapparent digestibility was determined by dividing thefeed dry matter apparently digested by the feed drymatter intake. The feed dry matter digested was calcu-lated by subtracting the total dry matter excreted duringthe 3-d collection period from the feed dry matter con-sumed during that period.

Statistics

The TME data were analyzed as a completely random-ized design using the general linear models procedureof SAS software (SAS Institute, 1996). Individual animaldata were the experimental unit for TME data. The re-sults in Experiments 1, 2, and 3 were analyzed as acompletely randomized design by using the general lin-ear models procedure of SAS software (SAS Institute,1996). Pens were the experimental units in Experiments1, 2, and 3, and least squared means are reported. Differ-ences among treatment means were determined bymeanwise comparison using least squared difference

6Dionex Corporation, Sunnyvale, CA 94085.7ANKOM, 1997; Fairport, NY 14450.

procedure of SAS software (SAS Institute, 1996). The

results in Experiment 4 were analyzed as a completelyrandomized design in a 3 2 factorial plus a basal diet.Regression analysis of basal, SBM, and ESBM was con-ducted by regressing total gain on added lysine intake.The methods of Littell et al. (1995) were used to deter-mine curvature of the individual slopes, intersection atthe y-axis, and differences between slopes. Means(GLM) were separated using the least squared differenceprocedure of SAS software. Statistical significance wasaccepted at P 0.05.

RESULTS AND DISCUSSION

Chick Experiments

Maximum hydrolysis of raffinose and stachyose wasachieved by a 1:5 (vol/wt) ratio of 0.4% enzyme solutionand SBM (data not reported). To ensure maximum re-moval of the oligosaccharides in SBMfed in these experi-ments, concentration of the enzyme solution used totreat SBM was increased 10-fold. This level of enzymesolution was shown in preliminary experiments toachieve maximum oligosaccharide hydrolysis. Treat-ment of SBM with -galactosidase reduced (P < 0.05)raffinose and stachyose by 65 and 50%, respectively.Fecal raffinose and stachyose in the ESBM diets werealso significantly reduced (P < 0.05) in chick feces (Table3). These data illustrated that -galactosidase treatmentof the SBM effectively removed raffinose and stachyosein the feces. Because these galactosides are the primarycause of fecal hydroscopic properties in poultry, theirremoval should alleviate the consequences associatedwith sticky droppings.

Leske et al. (1991) observed that chicks fed SBM sup-plemented with raffinose or SBMthat hadbeen extractedwith ethanol produced excreta that appeared wetter(sticky droppings) than that of chicks fed SBM treatedwith -galactosidase. Sticky droppings are attributed to


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TABLE 3. Growth performance and fecal oligosaccharide composition of chicks fed enzyme treatedand untreated soybean meal (Experiments 1, 2, and 3)1

Initial BW ADFI2 Fecal FecalDiet2 wt (g) gain (g) (g/d) G/F raffinose (%) stachyose (%)

Experiment 1

USBM 75 514.8 45.9 0.80 0.43a 1.36a

WSBM 74 539.8 47.8 0.81 0.48a 1.51a

ESBM 75 512.0 45.8 0.80 0b 0b

SEM 0.6 13.4 0.71 0.01 0.05 0.18

Experiment 2Initial BW ADFI Fecal Fecal Fecalwt (g) gain (g) (g/d) G/F raffinose (%) stachyose (%) NDF (%)

USBM 67 511.9 44.1 0.83 0.15a 0.33a 28.39b

WSBM 68 517.0 43.4 0.86 0.09b 0.34a 29.24b

ESBM 66 525.4 45.8 0.82 0c 0b 30.92a

SEM 0.07 10.6 1.0 0.02 0.02 0.07 0.39Experiment 3

Initial BW ADFI Fecalwt (g) gain (g) (g/d) G/F NDF (%) APDig (%)

WSBM 72 502.5 45.0 0.80 24.90 71.39ESBM 73 525.4 46.0 0.82 25.32 72.07SEM 0.5 10.4 0.85 0.01 0.003 0.004

acValues within columns within experiments with unlike subscripts are significantly different (P < 0.05).

1ADFI = Average daily feed intake per bird; G/F = gain to feed ratio; NDF = neutral detergent fiber; APDig= apparent digestibility expressed on a dry matter basis as a percentage of intake.

2Untreated soybean meal (USBM), water-treated soybean meal (WSBM), and enzyme-treated soybean meal(ESBM).

several problems in the poultry industry such as meatdiscoloration, foot problems, and increased disease. Wetdroppings can also cause an increase in ammonia pro-duction, reducing air quality in poultry houses and at-tract flies and rodents (Choct, 1996).

A decreased oligosaccharide concentration would po-tentially reduce viscosity of the digesta, leading to

slower digesta passage rate (increased retention time),greater access of digestive enzymes to substrates, andmore rapid diffusion of absorbable nutrients to the intes-

FIGURE 1. Effect of lysine intake per bird on body weight gain of chicks fed diets containingsoybean meal that washeated during-galactosidasetreatment. Equation for the chick growth response to the basal, untreated, and treated soybean meal diets was y = 751.34 (lysine intake) + 75.80(r2 = 0.8576), y = 736.98 (lysine intake) + 74.15 (r2 = 0.7914), and y = 745.91 (lysine intake) + 54.12 (r2 = 0.6993), respectively.

tinal mucosa. The result would be an increased digest-ibility of the diet. Supporting this theory was an increase(dry matter basis) in true metabolizable energy due to-galactosidase treatment. USBM had a TME of 2,974 35.8 kcal/g and ESBM had a TME of 3,328 145.4 kcal/g. If the metabolizable energy density of the diet is in-creased, efficiency of growth should be increased. The

raffinose and stachyose may be the primary cause oflower TME values for SBM in chickens due to the poordigestibility of the complex sugars (Leske et al., 1991).


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TABLE 4. Chick performance data and lysine intake of Experiment 4 1

Basal Basal1 Basal2 USBM3 USBM6 ESBM3 ESBM6 SEM

Initial wt (g) 154.6 154.8 155.8 154.7 154.3 156.6 155.5 5.7BW gain (g) 341.1e 442.9b,c 510.3a 423.2c,d 497.3a,b 358.5d,e 439.9a,b 20.3ADG (g/d) 28.05 36.48 42.21 34.33 40.85 31.76 35.76 1.18ADFI (g/d) 46.87c 51.58b 58.80a 51.42b 57.68a 49.30b,c 50.48b,c 1.54G/F 0.60d 0.73a 0.72a 0.68b,c 0.72a,b 0.66c 0.72a 0.01DLI (g/d) 0.37d 0.46c 0.59a 0.46c 0.58a 0.44c 0.50b 0.01

aeUnlike superscripts within rows are significantly different (P < 0.05).1Basal1 and Basal2 = basal diet with 0.1 and 0.2% added lysine, respectively; SBM3 and SBM6 = basal diet

with 3 and 6% SBM added, respectively; ESBM3 and ESBM6 = basal diet with 3 and 6% ESBM added; ADG =average daily gain; ADFI = average daily feed intake; G/F = gain to feed ratio; DLI = daily lysine intake.

Addition of raffinose and stachyose to a broiler dietsignificantly reduced TME values compared to controls(Leske et al., 1993), but removal of raffinose and stachy-ose from SBM by using 80% ethanol extraction resultedin an increased TME for broiler chicks and roosters(Coon et al., 1990; Leske et al., 1991; Leske and Coon,1999). These results were similar to results of our studies

using-galactosidase to hydrolyze the oligosaccharides.The growth parameters gain, ADFI, and gain/feedamong diets of Experiments 1, 2, and 3 (Table 3) didnot differ significantly (P > 0.05) due to -galactosidasetreatments, although in Experiments 2 and 3, there wasa trend (P < 0.15) for the ESBM diet to support a greater

body weight gain over 14 d than diets containing nonen-zyme-treated SBM and WSBM (Experiment 3). Similarto our data, chick performance was not improved withthe addition of -galactosidase to a corn-soy diet in anexperiment done by Irish et al. (1995). The higher aver-age daily gain in Experiment 2 could be attributed toan increased feed intake. The bird has more potential

to grow if it takes in more nutrients to support thatgrowth. Even though a numerically improved growthperformance was seen in Experiments 2 and 3, it wasnot concluded that the increased growth response wasconsistent.

Fiber analysis performed on the feces of chicks inExperiment 2 and 3 showed an increased NDF percentfor the ESBM diet. The increase in NDF was due to theremoval of raffinose and stachyose from the feces (non-NDF constituents) causing the percentage of hemicellu-lose, cellulose and lignin (NDF constituents) to be ele-vated when compared to the other two diets. Increasingthe digestibility of a diet should increase the amount of

nutrients the bird can utilize for growth. Reduction ofraffinose and stachyose using -galactosidase could im-prove digestibility by reducing viscosity of the digesta,or hydrolyzing the oligosaccharides into monosaccha-rides the bird can utilize. In these experiments, therewas no significant difference (P > 0.05) in apparent drymatter digestibilities between treatments. Based uponthe reduction of raffinose and stachyose in the feces, anincreased dry matter digestibility was expected. How-ever, the lack of a response in digestibility explains whygrowth parameters were not increased due to -galac-tosidase treatment. The lack of growth response due to

-galactosidase treatment is in agreement with Irish etal. (1995).

Heating (55 C) compared to not heating SBM after-galactosidase application further decreased raffinoseand stachyose concentrations from 35 and 50% to 7 and16%, respectively. This result may have occurred be-cause heating increased enzyme activity. Experiment 4

was conducted to determine if heating reduced lysineavailability of -galactosidase treated SBM. Based uponstatistical differences in slopes at a 95% confidence inter-val, heating SBM in Experiment 4 produced a lower gainfor chicks fed ESBM compared to chicks fed the USBMand basal diets (Table 4). Daily lysine intake and ADFIwas also lower in the ESBM diet. Lysine bioavailabilityof USBM was 98.7% of the basal diet measured by re-gressing lysine intake on body weight gain (r 2 = 0.7914).Lysine bioavailability of -galactosidase treated SBM,determined using the same procedure, was 75.9% of the

basal diet (r2 = 0.6993; Figure 1). The birds fed the ESBMdiets did not consume enough lysine to meet their

growth needs due to the decreased bioavailability,which explains the smaller average daily gain. HeatingESBM decreased further the oligosaccharide concentra-tion in SBM and increased the concentration of free sug-ars. These sugars contain a carbonyl group that can bindto free amino acids or to the amino group of lysine,forming a Maillard product that is intestinally unavail-able. This conversion may have been the reason thatlysine availability was reduced in heat-treated com-pared to nonheat-treated ESBM in this study.

These studies indicated that addition of the enzyme-galactosidase to poultry diets without heat treatmentcould eliminate raffinose and stachyose in feces, whichmay help improve growth parameters in young chicksand may also reduce digesta viscosity and, therefore,reduce occurrence of sticky droppings. If heat treatmentis used to enhance oligosaccharide removal from SBM,consideration should be given to effects on lysine avail-ability.

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The Effect of Enzyme Treatment of Soybean Meal on Oligosaccharide

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