Stability of bacitracin methylene disalicylate
and chlortetracyline in combination in a
pelleted pig feed
$S.E. Watkins
a, P.W. Waldroup
a,*, J.P. Peters
b, A.D. Desai
b aPoultry Science Department, University of Arkansas, Fayetteville, AR 72701, USAbAlpharma Inc., One Executive Drive, Fort Lee, NJ 07024, USA
Received 13 May 1998; received in revised form 5 January 1999; accepted 24 May 1999
Abstract
A study was carried out to determine if the presence of bacitracin methylene disalicylate (BMD) interfered with the assay of chlortetracycline (CTC) and if the presence of CTC interfered with the assay of BMD when both are combined in a pig feed. In addition, the potential influence of steam pelleting on active concentrations of BMD and CTC was determined. Further, the stability of BMD in the presence of CTC and the stability of CTC in the presence of BMD was determined in pelleted pig feed. Finally, the homogeneity of BMD and CTC during normal mixing procedures and the extent of segregation during shipping were determined. The results of the study demonstrated that when present in pig feeds at levels anticipated for actual use, BMD and CTC can be assayed without interference, that each of these antibiotics remains within specifications for up to 13 weeks of storage and that standard feed mixing results in homogenous dispersion of the antibiotics which remain dispersed following transport.#1999 Elsevier Science B.V. All rights reserved.
Keywords: Antibiotics; Stability; Interference; Feed segregation
1. Introduction
To meet regulatory requirements, it is essential that persons utilizing antibiotics in animal feeds periodically monitor dietary levels to insure compliance. Antibiotics are
81 (1999) 35±42
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Published with the approval of the Director, Arkansas Agricultural Experiment Station. Manuscript no. 98042.
*Corresponding author. Tel.: 001-501-575-2065; fax: 001-501-575-3474.
often fed in combination with other feed additives which may cause interference with assay of one or both products. Chlortetracycline (CTC) has been shown to be difficult to separate from virginamycin and bacitracin by chemical separation approaches (Ragheb et al., 1989).Micrococcus luteusATCC 10240 is the primary test microorganism for the measurement of bacitracin in feed samples; this strain is also sensitive to CTC (Mathers and Desai, 1995). Using a variant strain developed from Micrococcus luteus ATCC 10240, Mathers and Desai (1995) were able to measure bacitracin activity with no interference from 40 ug/ml added CTC.
As a requirement for obtaining clearance for the use of a combination of bacitracin methylene disalicylate (BMD) and CTC in pig feeds, a study was carried out to determine if the presence of BMD interfered with the assay of CTC and if the presence of CTC interfered with the assay of BMD when both are combined in a pig feed. In addition, the potential influence of pelleting on active concentrations of BMD and CTC was determined. Further, the stability of BMD in the presence of CTC and the stability of CTC in the presence of BMD was determined in pelleted, compound pig feed. Finally, the homogeneity of BMD and CTC during normal mixing procedures and the extent of segregation during shipping were determined.
2. Materials and methods
2.1. Mixing equipment
Mixing of the diets was carried out at the University of Arkansas Poultry Research Feed Mill, Fayetteville, AR. Primary mixing of the basal diet was carried out using a 908 kg capacity vertical mixer (Weigh-Tronics, Fairmont, MN). Mixing of the test diets was carried out using a 454 kg capacity horizontal mixer (H.C. Davis Sons Manufacturing, Bonner Springs, KS). Premixes used in preparing the test diets were prepared in a 22 kg capacity stainless-steel cross-flow V-mixer (Patterson-Kelley, East Stroundsburg, PA). These mixers had been evaluated previously for proper mixing times using the salt dispersion method (McCoy, 1994).
2.2. Mixing of test diets
One batch (908 kg) of unmedicated pig grower feed (160 g CP/kg) was prepared in a cleaned vertical mixer.1 After mixing for 15 min to allow for complete dispersal of nutrients, the feed was removed and placed in bags weighing22 kg each; actual weight of the bags was determined. The bags were numbered sequentially as they were filled. This feed was then divided into two lots using odd-numbered bags for the first lot and even-numbered bags for the second lot.Feed from the first lot (454 kg) was placed into a cleaned horizontal mixer. After mixing for 15 min, an initial representative sample
1The pig grower feed contained (per kg) 763.64 g ground maize, 178.56 g dehulled soyabean meal, 32.33 g poultry oil, 2.01 g salt, 8.7 g limestone, 9.47 g dicalcium phosphate, 0.29 g L-lysine HCl, and 5.0 g vitaminÿmineral premix.
(2.27 kg) of the unmedicated, complete mash feed was obtained by taking feed from four evenly-spaced quadrants of the mixer. From the remaining feed, 4.54 kg was removed and blended in a stainless-steel V-mixer with 75.3 g of bacitracin methylene disalicylate premix2to form a premix; this premix was returned to the mixer to yield a concentration of 11 mg BMD/kg in the finished feed. After an additional 15 min mixing, a 2.27 kg sample of feed was taken from the mixer as described previously. From the remaining 445 kg of complete feed, 1798 g of feed was removed and discarded. Another 4.54 kg of feed was removed and blended in the V-mixer with 1798 g of chlortetracycline premix3. This premix was then returned to the mixer to yield a concentration of 440 mg/ kg of CTC. After an additional 15 min mixing, another 2.27 kg sample of feed was taken; this sample was calculated to contain 11 mg BMD/kg and 440 mg CTC/kg. The remainder of the feed was bagged for homogeneity/segregation and feed stability portions of the study. The mixer was then thoroughly cleaned and flushed.
Feed from the second lot (454 kg) was then placed in the horizontal mixer. After mixing for 15 min, an initial representative sample (2.27 kg) of unmedicated, complete mash was removed as described previously. From the remaining feed, 1807 g of feed was removed and discarded. Additional feed (4.54 kg) was removed and blended with 1807 g of CTC 50 premix in the V-mixer to form a premix. This premix was then returned to the horizontal mixer to yield a concentration of 440 mg CTC/kg. This feed was allowed to mix for an additional 15 min and a 2.27 kg sample was removed from the mixer. From the remaining feed, 899 g was removed and discarded. Then another 4.54 kg of feed was removed and blended with 899 g of BMD 30 in the V-mixer to form a premix; this premix was returned to the horizontal mixer to yield a concentration of 132 mg BMD/kg and 440 mg CTC/kg. This feed was mixed again for 15 min and a final sample of 2.27 kg was taken. The remainder of the feed was used in the subsequent feed stability portion of the study.
2.3. Homogeneity/segregation studies
Feed was prepared as described above to contain 11 mg BMD/kg and 440 mg CTC/kg. This feed was dispersed into bags that held22 kg of feed. Nine evenly-spaced 2.27 kg samples were obtained from the sequence of bags and were numbered as H1 to H9 in the order that the mash feed was removed from the mixer. These nine samples were used to determine the homogeneity of mixing.
Additionally, one bag was filled with22 kg of mash feed. To ensure initial uniformity of the feed, a sample of2.4 kg was taken from each of nine bags, the composite mixed, and placed in a paper feed bag. This bag was sealed, placed in an upright position in a truck, and hauled for 85 km. The bag was then opened to expose the top layer of feed. Then one sample of1 kg was taken from each opposing end of the top layer of feed. The top one-third of feed was carefully removed and two more such samples taken from the middle layer of feed. Another one-third of the feed was removed and two more such
2BMD 30 premix (bacitracin methylene disalicylate equivalent to 66 g bacitracin per kg), Alpharma, Inc., One Executive Drive, Fort Lee, NJ 07024.
samples taken from the bottom layer of the bag. These six samples of1 kg each were numbered S1 through S6 in the order taken, left to right, top to bottom of the bag.
2.4. Feed stability study
Two mixtures of a complete pig mash feed prepared as described above were used in this portion of the study. The first mixture was calculated to contain 11 mg BMD/kg, its lowest approved level, and 440 mg CTC/kg, its highest approved level. This mixture was used to determine the stability of BMD. The second mixture contained 132 mg BMD/kg, considered as a high level, and 440 mg CTC/kg; this mixture was used to evaluate the stability of CTC. Each feed was pelleted using a 4 mm die (Master Model 30 hp pellet mill, California Pellet Mill Company, Crawfordsville, IN) with ca. 9 kg of each batch used to flush the die. The feeds were pelleted under normal commercial conditions with pellet temperatures of 82±888C. The pellets were thoroughly cooled, randomly sampled, put into feed storage bags (22 kg per bag), and sealed. A sample of each batch of the feed, both medicated and unmedicated, was taken prior to pelleting.
The pelleted medicated feed was stored at ambient humidity at either ambient temperature or in a room where the temperature was elevated (to 378C). As a consequence of a heat wave that occurred during the time of this study, the maximal ambient temperatures were similar to the minimal temperatures observed in the elevated temperature area.
2.5. Analysis of feed samples
Concentrations of bacitracin in the feed were determined by the microbiological method usingMicrococcus luteusstrain EN5 as described by Mathers and Desai (1995). Concentrations of chlortetracycline in the feed were determined by the method described by AOAC (1990). All assays were conducted in duplicate.
2.6. Statistical analysis
Data were analyzed using the general linear models (GLM) option of the Statistical Analysis System (SAS Institute, 1991). Statements of probability are based onp0.05.
3. Results and discussion
3.1. Assay non-interference
Table 1 presents a summary of analyzed concentrations of BMD and CTC in mash feeds. When a high level of BMD (132 mg/kg) was present with CTC (440 mg/kg), the assayed CTC concentration was 0.93 of claimed value. When CTC (440 mg/kg) was present with a low level of BMD (11 mg/kg), the assayed bacitracin concentration was 0.96 of claim. Assay limits for complete feeds containing BMD are30% of claim and for CTC are also30% of claim. Therefore, the presence of one antibiotic did not cause interference with assay of the other antibiotic in this study.
3.2. Stability during pelleting
The effects of the pelleting process on the analyzed concentrations of BMD and CTC are shown in Table 2. When a low level of BMD (11 mg/kg) was present with CTC (440 mg/kg) the assayed bacitracin concentration before pelleting was 0.96 of claimed value and 0.91 after pelleting. The assayed CTC concentration in this diet was 0.98 of claim value prior to pelleting and 0.86 after pelleting. When a high level of BMD (132 mg/kg) was present with CTC (440 mg/kg) the assayed bacitracin concentration before pelleting was 1.05 of claimed value and 0.86 after pelleting. The assayed CTC concentration in this diet was 0.93 of claim before pelleting and 0.97 after pelleting. All of these values fall within the allowable variation of 30% of claim. Thus, steam pelleting of the diets did not adversely affect the concentration of BMD or CTC in the pig feed.
3.3. Stability during storage at ambient and elevated temperature
The effects of storage at ambient and elevated temperatures on assayed values of BMD and CTC are shown in Table 3. Temperatures at ambient conditions fluctuated between 18.38and 37.88C while temperatures at elevated conditions ranged from 36.78to 43.38C. Regression analysis indicated that storage under ambient or elevated conditions did not result in significant reduction in activity of bacitracin or chlortetracycline (p0.05). Loss of product by drying throughout the study were as expected and did not appear to Table 1
Initial concentrations of bacitracin methylene disalicylate (BMD) and chlortetracycline (CTC) in mash pig feed
Mash Claim (mg/kg) Assayed (mg/kg) Loss on drying
BMD CTC BMD CTC
1 0 0 <1.37 <2.64 12.8
2 11 0 10.56 <2.64 13.7
3 11 440 10.56 430.9 12.7
4 0 440 1.52 422.9 13.1
5 132 440 139.2 408.2 12.3
Table 2
Stability of bacitracin methylene disalicylate (BMD) and chlortetracycline (CTC) in a pig feed during the pelleting process
Claim level (mg/kg) Diet form BMD (mg/kg) CTC (mg/kg)
BMD OTC Assayed % of claim Assayed % of claim
11 440 Mash 10.6 0.96 430.9 0.98
Pellet 10.0 0.91 378.4 0.86
132 440 Mash 139.2 1.05 408.2 0.93
impact the interpretation of the concentrations of BMD or CTC (data not shown). Therefore, it can be concluded that, when in combination in finished pig feed, BMD and CTC remain within their assay limits for up to 13 weeks at ambient temperature and for up to four weeks at elevated temperature, the maximal time periods evaluated in this study.
3.4. Homogeniety and segregation
Results for the homogeneity of the feed with 11 mg/kg BMD and 440 mg/kg CTC are shown in Table 4. The mean concentration of bacitracin for the nine samples was 11.5 mg/kg which was 1.05 of claim with a coefficient of variation (CV) of 6.36%. The industry standard for uniform mixing is a CV of 10% or less (personal communication, Dr. Keith Behnke, Kansas State University, Manhattan, KS). The mean concentration of chlortetracycline for the nine samples was 435.6 mg/kg which was 0.99 of claim with a CV of 6.1%. Thus, the homogeneity was considered as acceptable as there were no systematic variations of concentration of BMD or CTC. All feeds were within the claimed specifications, supporting the fact that these antibiotics uniformly disperse in pelleted pig feeds.
Results for the segregation of feed containing 11 mg/kg BMD and 440 mg/kg CTC are presented in Table 5. Feed for this segment of the trial was transported 82 km prior to sampling. The mean concentration of bacitracin for the six samples was 12.8 mg/kg which was 1.16 of claim with a CV of 9.1%. The mean concentration of chlortetracycline was 376.5 mg/kg which was 0.86 of claim with a CV of 1.5%. Therefore, both BMD and CTC appear to remain dispersed in pelleted pig feeds subjected to transport.
Segregation of microingredients in pig concentrate feeds has been described as troublesome (Anonymous, 1976). Feed manufacturing practices such as finer grinding of dietary components and the addition of liquid fats, as was done in the present study, are suggested as aids in reducing this segregation. Inadequate mixing times are considered Table 3
Stability of bacitracin methylene disalicylate (BMD) and chlortetracycline (CTC) in pelleted pig feed at ambient and elevated temperature with ambient humidity
Week Bacitracin (mg/kg) Chlortetracyline (mg/kg)
11 mg/kg BMD with 440 mg/kg CTC 440 mg/kg CTC with 132 mg/kg BMD
Ambient Elevated Ambient Elevated
0 10.0 10.0 427.2 427.2
2 11.2 10.3 nda 373.7
3 nda 11.4 nda 383.2
4 12.3 11.9 412.4 nda
6 9.0 nda nda nda
9 9.6 nda 425.7 nda
11 8.9 nda nda nda
13 10.6 nda 387.3 nda
aNot determined.
one of the prime causes of lack of homogeneity (Anonymous, 1976). Poultry feeds that have high variability due to inadequate mixing may result in reduced performance (McCoy et al., 1994). In the present study, the mixer was operated for a time previously determined to optimize mixing of microingredients.
Although feed manufacturers recognize that proper mixing of feed ingredients is important, there have been comparatively few studies that address the problems of stability and homogeniety of feed additives during the process of mixing, pelleting, and transportation. Several studies have addressed the problems of maintaining stability of various vitamins (Charles and Huston, 1972; Jones, 1986; Nir et al., 1978, 1981), Table 4
Homogeneity of bacitracin methylene disalicylate (11 mg/kg) and chlortetracycline (440 mg/kg) in mash pig feed
Sample Assayed value
BMD CTC
H1 11.2 400.0
H2 12.9 483.1
H3 11.8 445.8
H4 10.8 421.0
H5 10.7 437.4
H6 11.7 430.4
H7 11.6 430.2
H8 11.8 455.6
H9 12.1 417.2
Mean 11.6 435.6
SD 0.67 24.2
CV% 6.36 6.1
Minimum 10.7 400.0
Maximum 12.8 483.1
Table 5
Segregation of bacitracin methylene disalicylate (11 mg/kg) and chlortetracycline (440 mg/kg) in mash pig feed
Sample Assayed value (mg/kg)
BMD CTC
S1 13.8 376.8
S2 12.3 380.9
S3 12.9 375.9
S4 12.5 384.3
S5 14.2 369.0
S6 10.9 372.1
Mean 12.8 376.5
SD 1.2 5.6
CV% 9.1 1.5
Minimum 10.9 369.0
enzymes (Willingham et al., 1961; Inborr and Bedford, 1994), lysine (Dale, 1992), and fungicides (Paster et al., 1985). However, studies on stability of antibiotics to pelleting appear to be limiting. McGinnis and Stern (1953) fed procaine penicillin, diamine penicillin, and chlortetracycline in mash and pellet form to turkeys and concluded on the basis of growth response that the antibiotics were not sufficiently destroyed by pelleting to affect growth rate. The results of the present study demonstrated that when present in combination in pig feeds at levels anticipated for actual use, BMD and CTC can be assayed without interference, are not affected by pelleting, that each of these antibiotics remains within specifications for up to 13 weeks of storage in a pelleted diet, and that standard feed mixing practices result in homogenous dispersion of the antibiotics which remain dispersed following transport.
Acknowledgements
The laboratory support of Norbert Fedor and Linda Panozzo is gratefully acknowl-edged.
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