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An Effective Combination of Carbohydrases That Enables Reduction of Dietary Protein in Broilers: Importance of Hemicellulase

M. Tahir^*,1, F. Saleh, A. Ohtsuka and K. Hayashi

^* Department of Animal Nutrition, NWFP Agricultural University, Peshawar, Pakistan; and Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima-shi 890-0065, Japan

¹ Corresponding author: tahir065{at}yahoo.co.uk

	ABSTRACT

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After observing the effects of purified cellulase (C), hemicellulase (H), pectinase (P), and their combinations on the in vitro digestibility of a corn-soybean meal broiler diet, we examined the associations between pectin breakdown and the digestibilities of CP and DM by using free galacturonic acid (GA) as an index of pectin breakdown. There was no significant effect of the single enzymes except for H. However, the enzyme combinations H + P, C + H, and C + H + P significantly increased CP and DM digestibilities, whereas the combination of C + P was not effective. Because H has activities of both H and P, these enzymes were considered to be important in stimulating digestion. Furthermore, when the enzymes increased CP and DM digestibilities, GA concentration was significantly higher, and clear correlations between CP and DM digestibilities and GA concentration were observed, whereas correlations between the digestibilities and concentration of glucose or xylose + mannose as indices of cellulose and hemicellulose breakdown, respectively, were not significant. From these observations, we hypothesized that a mixture of enzymes could increase the protein digestibility of broiler feed. Thus, in the in vivo experiment, low-protein (19% CP) diets made mainly of corn and soybean meal with or without mixed enzymes were prepared and given to broiler chicks. The birds given the diet containing mixed enzymes showed significantly higher BW gain, with higher CP and DM digestibilities than the birds given the diet without the mixed enzymes. Moreover, the growth rate was same as that of the birds given the normal (21% CP) diet. The results indicate that the mixed enzyme preparation can effectively degrade indigestible cell constituents and thus enable the protein of the broiler feed to become more digestible. Furthermore, the results indicate the importance of H as a rate-limiting factor of cell wall breakdown.

Key Words: carbohydrase • corn-soybean meal • digestibility • pectin • broiler

	INTRODUCTION

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In the poultry industry, profit depends mainly on the cost and nutritive value of the feed. Although corn-soybean meal poultry feed is considered to be favorable because of its high nutritional value, poultry cannot digest 15 to 25% of its nutrients. The main antinutrients that may limit the nutritive value of the feed are the nonstarch polysaccharides of soybean meal (Bedford and Schulze, 1998; Adeola and Bedford, 2004). Half of the nonstarch polysaccharides present in the cell wall matrix of soybean meal are pectic substances that cement the other polysaccharides. They entrap approximately 10% of the protein (on a DM basis) within the cell wall matrix (Chesson, 2001). Galacturonic acid (GA) is the main component of pectin and exists either as the polymer rhamnogalacturonic acid or polygalacturonic acid. Pectinase (rhamnogalacturonase and polygalacturonase; P) opens the cleavages of pectic polysaccharides and thus exposes the entrapped nutrients for further hydrolysis. Free GA was therefore used in the present study as an index of pectin breakdown to elucidate the role of pectin as an inhibitor of digestion.

Protein is the most costly nutrient in the broiler diet required for optimum growth performance. Because of the expense of providing sufficient protein to growing broilers, numerous studies have been carried out to investigate the possibility of reducing dietary protein and energy by using feed enzymes. It has been reported that nitrogen retention and energy utilization are often increased when a cell wall-degrading enzyme is added to a corn-soybean meal broiler diet (Oloffs et al., 1999; Douglas et al., 2000; Kocher et al., 2002).

To enhance the worth of feed enzymes and to clarify their mode of action, enzyme preparations were designed by using purified enzymes and were tested in the current study to determine their digestion-stimulating action in corn-soybean meal broiler diets. In this study, 2 in vitro experiments and 1 in vivo experiment were conducted. In the in vitro experiments, the effects of purified cellulase (C), hemicellulase (H), P (experiment 1), and their combinations (experiment 2) on the digestibility of a corn-soybean meal broiler diet were observed, and the relationship between pectin breakdown and the digestibilities of CP and DM were examined. From the observation of in vitro experiments, we hypothesized that a mixture of enzymes could increase the protein digestibility of broiler feed. Thus, in the in vivo experiment, we compared the effects on broiler performance of low-protein diets (19% CP; CP19) and normal-protein diets (21% CP; CP21) with or without mixed enzymes.

	MATERIALS AND METHODS

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Birds and Housing

The animal experiment was conducted in accordance with the guidelines of Kagoshima University. Male broiler chickens (Cobb) were kindly provided by the Kumiai Hina Center, Kajiki, a commercial hatchery in Kagoshima prefecture, Japan. Chicks were brooded until 12 d of age in an electrically heated battery brooder and were fed ad libitum a commercial starter diet with 21% CP and 3,000 kcal/kg ME, with free access to water. On d 12, a total of 40 male birds were selected and grouped, and each bird was housed individually in wire-floored aluminum cages. The experiment was conducted in a temperature-controlled room in which the birds were exposed to a 14 L:10 D cycle. Birds had free access to feed and water throughout the experiment. Room temperature (24°C) and RH (50 to 70%) were maintained throughout the experiment.

Dietary Treatments and Feeding

The composition of the 2 basal diets, CP21 and CP19, are given in Table 1. The CP21 diet was formulated to meet the minimum nutrient requirements of broilers as recommended by the Japanese Feeding Standard for Poultry (Ministry of Agriculture, Forestry and Fisheries of Japan, 1997). To measure digestibility, an indigestible marker (chromium oxide) was included at a level of 0.3% in the basal diets. The birds were preconditioned for 3 d from 12 to 15 d of age with the basal diet given ad libitum. The enzyme was mixed with the basal diet. The birds were grouped into 4 treatments with 10 replications. Treatment groups were the CP21 and CP19 with or without enzyme with a setup of 2 x 2 factorial arrangements of treatments. The enzymes used were C, H, and P. The levels of C, H, and P used were 0.33, 2, and 2 units (U)/g of feed, respectively. The birds were raised on the experimental diets from 15 to 27 d of age.

The enzymes (P, C, and H) were all purchased from Sigma Chemical (St. Louis, MO). Pectinase was from Aspergillus niger and 1 U would liberate 1.0 µmol of GA from polygalacturonic acid per min at pH 4.0 at 25°C. Cellulase was from Trichoderma viride and 1 U would liberate 1.0 µmol of glucose from cellulose per h at pH 5.0 at 37°C. Hemicellulase was from A. niger and 1 U would liberate 1.0 µmol of D-galactose from hemicellulose per h at pH 5.5 at 37°C. The concentrations of enzymes in the medium used in the present study were previously determined in our laboratory: C, 0.33 U; H, 2 U; and P, 2 U/g of feed (Saleh et al., 2003).

Parameters Measured and Chemical Analysis

Body weight was recorded every 3 d, and feed intake was recorded daily. At the end of the experimental period, all the birds were killed by decapitation, and the carcass weights were determined by removing the feathers, feet, head, and viscera. The birds were dissected and the breast muscles (musculus pectoralis superficialis) were removed. For the ileal digesta sample, the body cavity was opened soon after dressing and the contents of the ileum (from Meckel’s diverticulum to 1 cm above the ileocecal junction) were collected and immediately placed on ice. The digesta samples were frozen at –20°C and freeze-dried before analysis.

Dry matter contents of the diets and ileal digesta sample were determined by oven-drying at 105°C. Nitrogen and ash were determined by the Kjeldahl method and by muffle furnace (600°C for 2 h), respectively. In vitro protein and DM digestibilities were determined by the pepsin-pancreatin method as reported by Saunders et al. (1973), with slight modifications as described by Saleh et al. (2003). Galacturonic acid was determined by a modified carbazole method described by Bitter and Muir (1962). Chromium oxide was determined by the procedure of Dansky and Hill (1952). The ileal digestibilities of CP, DM, and ash were determined by the formula described by Hong et al. (2002).

To measure monosaccharides, the supernatant of the medium (0.5 mL) obtained by the pepsin-pancreatin method (Saunders et al., 1973) was homogenized with 5 mL of 80% ethanol. After heating the solution in a water bath at 100°C for 30 min, the precipitate was removed by centrifuging at 5,800 x g for 15 min. The precipitate was reextracted with 5 mL of 80% ethanol in the boiling water and recentrifuged at 5,800 x g for 10 min and the supernatant was removed. The supernatants were pooled and dried by a rotary evaporator at 40°C. The dried residues were resuspended in 5 mL of distilled water, followed by the gradual addition of 1 mL of 0.3 N barium oxide and 1 mL of 5% zinc sulfate to precipitate protein and pigments in the solution. The solution was then filtered through filter paper (no. 5A) and transferred into a 25-mL volumetric flask. A small amount of this solution was filtered through a 0.45-µm membrane filter, and for the determination of monosaccharides, 25 µL of this solution was injected into an HPLC instrument equipped with a Shim-pack SCR-101P column (Shimadzu, Kyoto, Japan), with distilled water as a mobile phase at a flow rate of 1 mL/min. The monosaccharides were detected by an RI 4974 detector (Hitachi, Tokyo, Japan). Glucose could be separated but xylose, but mannose could not be separated by the method; thus, the total amount of xylose and man-nose was determined.

Statistical Analysis

Data were analyzed by ANOVA with the GLM procedure of SAS (SAS Institute, 1988). Tukey’s multiple range tests were applied to separate the means. A P-value of 0.05 was considered statistically significant. Regression analysis was performed between the digestibilities of CP and DM, and monosaccharide concentrations.

	RESULTS

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In Vitro Experiment 1

The effects of a single enzyme on the in vitro digestibilities of CP and DM and on monosaccharide concentrations in the incubation medium are shown in Table 2. The effect of a single enzyme on CP and DM digestibility was not statistically significant. However, GA concentration in the supernatant was significantly higher when treated with H, whereas P and C had no effect. Glucose was increased slightly but significantly by all the enzyme treatments, showing the effectiveness of the enzymes. Concentration of xylose + mannose was increased by C, indicating that cellulose was cleaved to produce mannose. Because xylose and mannose could not be separated by HPLC, the total amount of the 2 sugars is shown.

The effects of various combinations of the 3 enzymes are shown in Table 3. The digestibilities of CP and DM were improved by H + P, C + H, and C + H + P, and the effect was highest when diets were treated with C + H + P. Galacturonic acid concentration was significantly increased by H + P, C + H, and C + H + P, but not by C + P. The C + H + P group rendered the maximum GA among the treatments. Glucose was increased by all the enzyme combinations. Xylose + mannose concentration was not increased by any of the single enzymes, whereas C + H + P was effective.

View larger version (9K): [in this window] [in a new window]   Figure 1. Relationship between CP digestibility and galacturonic acid concentration in corn-soybean meal diets supplemented with a single carbohydrase and a combination of carbohydrases.

View larger version (8K): [in this window] [in a new window]   Figure 2. Relationship between DM digestibility and galacturonic acid concentration in corn-soybean meal diets supplemented with a single carbohydrase and a combination of carbohydrases.

Table 4 shows BW gain (BWG), feed intake, feed conversion ratio (FCR), carcass yield, and breast muscle weight in broiler-fed diets with or without enzymes. Broilers fed the CP19 + C diet had a lower BWG than the broilers fed the CP21 + C diet. However, supplementation of the enzyme preparation to the CP19 diet caused a significant increase in BWG; thus, differences between the CP19 + E and CP21 + C diets could not be observed. Feed intake was not different between groups, and FCR was clearly improved by the enzyme preparation. Carcass yield was low (P < 0.05) in the CP19 + C diet; however, when supplemented with a mixture of enzymes, the carcass yield was increased and the difference between the CP19 + E and CP21 + C diets disappeared. The highest carcass yield was found in the CP21 diet with enzymes. The effects on the breast muscle were same as those on the carcass yield. The interactions of CP and enzyme on the FCR and carcass yield were significant (P < 0.1).

	DISCUSSION

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Galacturonic acid is the main component of pectin (homogalacturonans and rhamnogalacturonans), and P can cleave these polymers and eliminate GA. Soybean contains rhamnogalacturonan with a backbone of 1,4-linked GA and 1,2-linked rhamnose, with a number of different side chains composed principally of arabinose and galactose (Aspinall and Cottrell, 1971; Siddiqui and Wood, 1972; Bacic et al., 1988; Daveby and Aman, 1993). Hemicellulase contains rhamnogalacturonase activity; thus, the bonds between GA and rhamnose can be cleaved by H. In the present experiment, the effect of the mixed enzymes on GA concentration was significant except for C + P. The effect of C + H + P on GA concentration was greater than those of H + P and C + H, showing that the 3 enzymes used in the current study may act in a coordinated manner to enhance the degradation of the cell-wall polysaccharides of soybean meal and corn.

The linear relationships between GA concentration and digestibilities of CP (R² = 0.75) and DM (R² = 0.97) show that the improvement in nutrient digestibility by enzymes was associated with increased pectin breakdown. By the breakdown of pectin, encapsulated intracellular nutrients may be released. On the other hand, CP and DM digestibilities in the present study were poorly correlated with glucose concentration (y = 2.92x + 56.6, R² = 0.26; and y = 3.17x + 40.02, R² = 0.32, respectively), indicating that the C used in the present study contributed little to CP and DM digestibilities. Similarly, xylose + mannose, an index of hemicellulose (arabinoxylans) breakdown, did not show a significant association with CP and DM digestibilities (y = –0.92x + 78.5, R² = 0.01; and y = –3.69x + 67.4, R² = 0.11, respectively), suggesting that the H used in the present study was lacking xylanase activity. This is in accordance with the company statement indicating that the H from A. niger liberates D-galactose from hemi-cellulose.

On the basis of the improvements in CP and DM digestibilities, it is tempting for the nutritionist to lower the CP level of the diet. A merit to the broiler producer is that a type of diet is used that not only boosts bird performance, but also diminishes nitrogen excretion. Feed enzymes offer the producer the opportunity to reduce both feed cost and nitrogen excretion. Indeed, it was shown in the present study that the carcass yields of broilers fed the CP19 diet supplemented with enzymes were same as those of broilers fed the CP21 diet without enzymes, showing that the enzymes could save dietary protein. Such an effect was not achieved by the single enzymes C or H as shown in our previous experiment (Tahir et al., 2005).

The multienzyme preparation usually improved the birds’ performance. Disruption of the complex cell wall matrix by multicarbohydrase would increase the exposure of encapsulating nutrients to digestive enzymes (Bedford, 2000). By the enzyme preparation used in this study, protein digestion was stimulated, and thus dietary protein content could be successfully reduced.

Enzyme supplementation also resulted in a significant improvement in DM digestibility. Uni et al. (1999) and Noy and Sklan (1995) reported that considerable amounts of encapsulated nutrients may escape from digestion, reach the hindgut, and undergo fermentation by gut microflora, releasing volatile fatty acids and gas with a relatively low energy yield. The ME content of soybean meal is relatively low (2,441 kcal/kg; NRC, 1994) for poultry, which is mainly caused by the poor digestibility of indigestible cell constituents (Pierson et al., 1980). On the basis of the increased DM digestibility caused by the mixed enzymes, broilers might acquire more protein from the diet when it is supplemented with enzymes.

These results indicate that degradation of pectin and H enables protein and energy, especially the protein of broiler feed, to become more digestible. Hence, the level of protein could be reduced from 21 to 19%.

Received for publication August 17, 2007. Accepted for publication January 5, 2008.

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This Article Abstract Full Text (PDF) _Erratum_ A correction has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Tahir, M. Articles by Hayashi, K. Search for Related Content PubMed Articles by Tahir, M. Articles by Hayashi, K.