Evaluation of keratinase stability in pelleted broiler diets1

Evaluation of keratinase stability in pelleted broiler diets1

©2009 Poultry Science Association, Inc. Evaluation of keratinase stability in pelleted broiler diets1 C. R. Stark,*2 B. E. Spencer,* J. C. H. Shih,* ...

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©2009 Poultry Science Association, Inc.

Evaluation of keratinase stability in pelleted broiler diets1 C. R. Stark,*2 B. E. Spencer,* J. C. H. Shih,* C. G. Chewning,* and J. J. Wang

Primary Audience: Feed Manufacturers, Nutritionists, Researchers SUMMARY Versazyme (VZ) is an approved keratinase-based feed additive produced naturally as a fermentation product of Bacillus licheniformis PWD-1. The experiment was designed to test the recovery and efficacy of VZ using typical feed industry pelleting parameters. The pelleted diets were conditioned to 85°C for 30 s and pelleted using a 4 × 32 mm die, cooled, and crumbled. A total of 192 male broiler chicks were fed a corn-soybean meal starter diet formulated at 18.5% CP to 21 d of age. The 4 treatments used in the feeding study were as follows: VZ 0.0%-mash, VZ 0.0%-pellet, VZ 0.1%-mash, and VZ 0.1%-pellet. Body weight and feed consumption were determined at 21 d of age. The recovery of VZ in feed was 93% in the mash diet and 66% in the pelleted diet. There was a 55-g increase in BW gain and 0.17 improvement of FCR when birds were supplemented with 0.1% VZ. Pelleting improved BW gain (P < 0.05) but did not significantly improve the FCR. Growth performance results indicate that pelleting did not affect the efficacy of VZ. Key words: pelleting, dietary protein, protease, keratinase 2009 J. Appl. Poult. Res. 18:30–33 doi:10.3382/japr.2008-00038


Versazyme (VZ) is a keratinase preparation produced from the fermentation of Bacillus licheniformis PWD-1 [1, 2], which is a Generally Recognized As Safe bacterium approved for animal feed. The bacterium was discovered and isolated from a thermophilic anaerobic digester [3, 4]. The thermophilic bacterium produced a heat-stable keratinase that could break down feather keratin [1]. The discovery of keratinase and the development of keratinase technology have been reviewed in 2 recent publications [5, 6]. In cage studies, VZ supplementation in corn1

soybean meal broiler diets resulted in improved growth performance and FE in adequate or marginal CP diets [7]. In pen trials and commercial tests with broilers raised to market age, VZ demonstrated positive results in corn-soybean meal mash diets [8] and pelleted diets [9]. Low VZ recovery rates have been observed in field samples after pelleting. A series of samples (n = 10) were collected at a commercial feed mill after the pelleting process. The pelleting process significantly lowered the recovery of enzyme activity, but without compromised performance [10]. Pelleted feed samples ranged from 228 to

Use of trade names in this publication does not imply endorsement by the North Carolina Agriculture Research Service or the North Carolina Cooperative Extension Service of the products mentioned. 2 Corresponding author: [email protected]

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*Department of Poultry Science, North Carolina State University, Raleigh 27695; and †BioResource International Inc., Raleigh, NC 27560

Stark et al.: KERATINASE STABILITY IN PELLETS Table 1. Broiler starter diet Item

% 61.61 28.41 5.20 1.18 2.08 0.20 0.24 0.01 0.47 0.10 0.20 0.20 0.10 3,200 18.50 1.00 0.50 0.20 1.18 0.82 0.73 0.75 0.87 1.25


The vitamin premix supplied the following per kilogram of feed: vitamin A, 13,200; cholecalciferol, 3,960 IU; niacin, 110 mg; pantothenic acid, 22 mg; riboflavin, 13.2 mg; pyridoxine, 7.9 mg; menadione, 4 mg; folic acid, 2.2 mg; thiamin, 4 mg; and biotin, 0.25 mg. 2 The mineral premix supplied the following per kilogram of feed: Zn, 120 mg; Mn, 120 mg; Fe, 80 mg; Cu, 10 mg; I, 2.5 mg; Co, 1.0 mg. 3 Selenium premix provided 0.3 ppm Se. 4 Analyzed CP = 18.3%; gross energy = 3,780 kcal/kg.

260 U/g in enzyme activities and 72 to 83% recoveries, as compared with the mash feed. However, VZ has not yet been tested or compared in the same trial using the same diet in mash and pellets. The objective of this study was to test the efficacy of VZ pelleted under normal feed manufacturing conditions.

MATERIALS AND METHODS Broiler Management The experiment was conducted in Alternative Design [11] battery cages in an environmentally controlled room. The care of the birds used in the trial conformed to the Guide for the Care and Use of Agriculture Animals in Agricultural Research and Teaching [12]. A total of 192 male

1-d-old broiler chicks (Ross 244 × Ross 508 FS) [13] were grouped by weight and randomly assigned to 24 cages, for a similar starting weight. There were 6 replicates per treatment. The experimental unit was the pen of 8 birds. Birds had ad libitum access to feed and water; room temperature started at 32°C on d 1 and gradually decreased to 24°C on d 21. Feed Formulation and Manufacturing A corn-soybean meal starter diet was formulated at 18.5% CP, 1.18% Lys, and 0.82% Met + Cys. The diets were isonutritive, but the protein and amino acids did not meet the suggested NRC [14] requirements (Table 1). A common basal diet was prepared and subdivided into 4 lots. A commercially available keratinase enzyme product (VZ) [15] was added at 0.1% to 2 of the mash sublots and remixed to produce the VZ treatment. The calculated keratinase concentration in the finished feed was 380 U/g. The pelleted diets were conditioned to 85°C for 30 s and pelleted using a 4 × 32 mm die [16]. Feeder rate was held constant to ensure similar conditioning parameters during the pelleting process. The pellets were cooled and fed as crumbles. Data Collection Initial BW was recorded at 1 d of age. Body weights and feed consumption were determined at 21 d of age. Dead birds were removed daily to calculate mortality. Feed conversion ratios were adjusted for mortality by adding the weights of dead birds to final pen weights for all treatments. Analytical Methods Moisture content was determined on the preconditioned mash, conditioned mash, and cooled pellets [17]. Samples of the feed were analyzed for CP [18] and gross energy [19]. All treatment feeds were assayed for VZ activity before the growth trial. In short, feed samples were ground and suspended in 0.1 M phosphate buffer (pH 8.0; shaking incubator, 200 rpm) at room temperature for 5 min and then centrifuged (5 min, 4,000 × g). Resulting supernatant was collected and assayed using the azo-casein hydrolysis method [20].

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Ingredient   Corn   Soybean meal (48% CP)   Poultry fat   Limestone   Dicalcium phosphate   dl-Met   l-Lys HCl   Thr   NaCl   Vitamin premix1   Choline Cl, 60%   Trace mineral premix2   Se premix3 Calculated composition4   ME, kcal/kg   CP, %   Ca, %   Available P, %   Na, %   Lys, %   Met + Cys, %   Thr, %   Ile, %   Val, %   Arg, %


JAPR: Research Report


Table 2. Body weight gain, feed conversion, and feed intake from 1 to 21 d of age for birds fed mash and pelleted/ crumbled diets with and without enzyme supplementation Item

BW gain, g

Feed intake, g

743 798 823 878 19.2

1.81 1.57 1.65 1.55 0.06

1,343 1,253 1,328 1,362 41.9

771 850 13.6

1.69 1.60 0.04

1,289 1,345 29.7

783 838 13.6 0.0005 0.0099 0.9925

1.73 1.56 0.04 P-value 0.1440 0.0083 0.2422

1,336 1,308 29.7 0.2729 0.5138 0.1545


Values represent the feed conversion after being corrected for mortality. Versazyme (VZ) [15] concentration in the meal diet was 354 U/g (93% recovery). 3 Versazyme [15] concentration in the pelleted diet was 249 U/g (66% recovery). 2

Data Analysis The experiment was a randomized completed block, with a 2 × 2 factorial arrangement of treatments: VZ supplementation (VZ 0.0% vs. VZ 0.1%) and feed form (mash vs. pellet). Cage means were used to derive performance data. Data were analyzed using the GLM procedure of SAS [21]. The least squares means procedure was used to separate treatment means; significance was set at P < 0.05.

RESULTS AND DISCUSSION Feed Formulation and Processing The pelleted diets were manufactured using typical industry conditioning parameters. The conditioning temperature was 85°C, production rate was 655 kg/h, and hot pellet temperature was 80°C. The final moisture content of the pellets was within 1 to 2% of the mash before the pelleting process. The laboratory feed assay of keratinase activity recovered 354 U/g (93%) from the mash diets and 249 U/g (66%) from the pelleted diets. The low recovery rate of enzyme activity in the pellets was similar to results observed in field samples. The cause for the low

recovery has not been determined. Because the beneficial effect of VZ was sustained in the pelleted diet and VZ is based on a relatively heatstable keratinase [1], it is believed that the low recovery is largely due to the poor extractability of the enzyme from the pellets into the buffer solutions, where the enzyme activities were determined. The conditioning and pelleting process that occurred for 30 s at 85°C did not change the benefit of the enzyme. It is also possible that pelleting may bind and stabilize the enzyme onto the feed granules, which makes the enzyme hard to extract and more resistant to heat. Whatever the reason, the retention of the beneficial effect of VZ in the diet was clearly demonstrated in bird performance. Growth Study Overall bird mortality was 2.1%. The inclusion of VZ in low-protein diets resulted in greater BW gain (P < 0.01; Table 2). Feed intake was not influenced by diet form or enzyme supplementation. The FCR was also improved (P < 0.01) when birds were fed the VZ supplemented to low-protein diets. Results are in agreement with Wang et al. [22], who found VZ supplementation improved 21-d BW gain when diets contained

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Feed form × enzyme   Meal   Meal + VZ (0.1%)2   Pellet/crumble   Pellet/crumble + VZ (0.1%)3   SEM Feed form   Meal   Pellet   SEM Enzyme   0.0%   0.1%   SEM Source of variation   Feed form   Enzyme   Form × enzyme




1. The beneficial effect of VZ on early growth and feed utilization was demonstrated in both mash and pelleted diets. 2. Pelleting improved BW gain of birds in comparison with birds fed mash. 3. The recovery rate of the keratinase enzyme from pelleted feed was low with the current feed assay, although the beneficial effect in broiler performance was retained.

REFERENCES AND NOTES 1. Lin, X., E. S. Casale, C. G. Lee, and J. C. H. Shih. 1992. Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis. Appl. Environ. Microbiol. 58:3271–3275. 2. Wang, J. J., and J. C. H. Shih. 1999. Fermentation production of keratinase from Bacillus licheniformis PWD1 and recombinant B. subtilis FDB-29. J. Ind. Microbiol. Biotechnol. 22:608–616. 3. Williams, C. M., and J. C. H. Shih. 1989. Enumeration of some microbial groups in thermophilic poultry waste digesters and enrichment of a feather-degrading culture. J. Appl. Bacteriol. 67:25–35. 4. Williams, C. M., C. S. Richter, J. M. MacKenzie Jr., and J. C. H. Shih. 1990. Isolation, identification, and characterization of a feather-degrading bacterium. Appl. Environ. Microbiol. 56:1509–1515. 5. Shih, J. C. H., and J. J. Wang. 2006. Keratinase technology: From feather degradation and feed additive, to prion destruction. CAB Rev.: Perspect. Agric. Vet. Sci. Nutr. Nat. Resour. 1. No. 42, 1–6. http://www.cababstractsplus.org/cabreviews Accessed October 2006. 6. Shih, J. C. H., Y. M. Guo, J. J. Wang, and J. D. Garlich. 2008. Keratinase, a new feed enzyme that improves

protein nutrition. Proc. World Poult. Congr. 2008, Brisbane, Australia. Watt Publishing, Mt. Morris, IL. 7. Odetallah, N. H., J. J. Wang, J. D. Garlich, and J. C. H. Shih. 2003. Keratinase in starter diets improves growth of broiler chicks. Poult. Sci. 82:664–670. 8. Odetallah, N. H., J. J. Wang, J. D. Garlich, and J. C. H. Shih. 2005. Versazyme supplementation of broiler diets improves market growth performance. Poult. Sci. 84:858– 864. 9. Wang, J. J., J. D. Garlich, and J. C. H. Shih. 2006. Beneficial effects of Versazyme, a keratinase feed additive, on body weight, feed conversion, and breast yield of broiler chickens. J. Appl. Poult. Res. 15:544–550. 10. Wang, J. J. 2008. BioResource International Inc., Raleigh, NC. Personal communication. 11. Alternative Design, Siloam, AR. 12. Federation of Animal Science Societies. 1999. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. 1st rev. ed. Fed. Anim. Sci. Soc., Savoy, IL. 13. Aviagen Inc, Huntsville, AL. 14. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Sci., Washington, DC. 15. BioResource International Inc., Raleigh, NC. 16. Master Model HD, California Pellet Mill Co., Crawfordsville, IN. 17. NTFA. 2002. Laboratory dry matter by oven drying for 3 h at 105°C. National Forage Testing Assoc., Omaha, NE. 18. AOAC. 2006. Kjeldahl. AOAC Official Method 984.13 (A-D). AOAC Int., Washington, DC. 19. Merrill, A. L., and B. K. Watt. 1973. Energy Value of Foods—Basis and Derivation. Agriculture Handbook No. 74. US Government Printing Office, Washington, DC. 20. Sarath, G., R. S. De La Motte, and F. W. Wagner. 1989. Protease assay methods. Pages 25–55 in Proteolytic Enzymes. A Practical Approach. R. J. Benson and J. S. Bond, ed. IRL Press, Oxford, UK. 21. SAS Institute. 1996. SAS/STAT User’s Guide: Statistics. Release 6.11. SAS Institute Inc., Cary, NC. 22. Wang, H., J. Guo, and J. C. H. Shih. 2007. Effects of dietary supplementation of keratinase on growth performance, nitrogen retention and intestinal morphology of broiler chickens fed diets with soybean and cottonseed meals. Anim. Feed Sci. Technol. 140:376–384. 23. McKinney, L. J., and R. G. Teeter. 2004. Predicting effective caloric value of nonnutritive factors: I. Pellet quality and II. Prediction of consequential formulation dead zones. Poult. Sci. 83:1165–1174. 24. Lemme, A., P. J. A. Wijtten, J. vanWichen, A. Petri, and D. J. Langhout. 2006. Responses of male growing broilers to increasing levels of balanced protein offered as coarse mash or pellets of varying quality. Poult. Sci. 85:721–730. 25. Moritz, J. S., R. S. Beyer, K. J. Wilson, and K. R. Cramer. 2001. Effect of moisture addition at the mixer to a corn-soybean-based diet on broiler performance. J. Appl. Poult. Res. 10:347–353. 26. Nir, I., and R. Hillel. 1995. Effect of particle size on performance. 3. Grinding pelleting interactions. Poult. Sci. 74:771–783.

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soybean meal and cottonseed meal. This study confirmed similar performance improvements observed in a 48-d commercial study in which diets were supplemented with VZ and pelleted [7]. Researchers consistently report greater BW gain in birds fed pelleted diets [23–25]. Pelleting improved BW gain (P < 0.01); however, no difference in FCR (P > 0.05) was detected in the 21-d growth study. Nir and Hillel [26] reported no difference in 21-d FCR; however, Moritz et al. [25] reported improved FCR at 21 d of age in birds fed pellets. Although the in vitro assay of enzyme activity was lower in the pelleted diets, performance responses were still seen in greater BW gain and improved FCR.