Fungistatic Compounds in Broiler Production

Fungistatic Compounds in Broiler Production. 2. Effect on Feed Microflora1 T. C. CHEN, B. C. DILWORTH, and E. J. DAY Poultry Science Department, Mississippi State University, Mississippi State, Mississippi 39762 A. S. ARAFA, R. D. MILES, and R. H. HARMS Poultry Science Department, University of Florida, Gainesville, Florida 32611 G. L. ROMOSER, V. G. DiFATE, and K. J. SHAVER

(Received for publication December 8, 1978) ABSTRACT Feed samples were examined from five broiler experiments using either sorbic acid, gentian violet, or calcium propionate as fungistatic compounds. Feed samples were taken from storage and from the feed troughs of each pen at 0, 2, 4, 6, and 8 weeks of broiler age. Samples from the same diet and sampling location were composed and mixed. Serial dilutions of these mixed samples were made and plated for aerobic, anaerobic, and mold and yeast counts. The initial feed microflora counts varied greatly with their ingredient sources. In general, the log anaerobic counts were slightly lower than those of the aerobic counts with a difference of less than one log cycle. The addition of the fungistatic compounds had no consistent effect on the aerobic, anaerobic, and mold and yeast counts. After two and four weeks of feeding, a steady increase in total aerobic and anaerobic counts was observed for feed samples obtained from feeder troughs; however, there was no increase in mold and yeast counts. Increasing the moisture content of the feed to approximately 15% had no effect on the microflora counts. No visible mold growth or caking problems were detected for any feeds after four weeks of storage. In one experiment, sorbic acid consistently reduced the coliforms and the lactobacillus counts for feeds sampled from feed troughs. 1979 Poultry Science 58:1451-1455 INTRODUCTION In recent years, various fungal inhibitors have been used in p o u l t r y feed t o c o n t r o l fungal g r o w t h and s u b s e q u e n t mycoses and m y c o t o x i c o s i s in p o u l t r y . Richardson and Halick ( 1 9 5 7 ) assayed various fungistatic c o m p o u n d s for their heat-inhibiting activity with corn meal in which t h e m o i s t u r e ranged from 14 t o 15%. T h e y reported t h a t propionic acid and p r o p i o n i c a n h y d r i d e were effective at a level of . 1 % . D e y o e and Quadri ( 1 9 7 0 ) r e p o r t e d t h a t a dry p r o d u c t with 20% propionic acid 2 as an active ingredient, w h e n applied at a rate of . 1 % , resulted in a significant decrease in m o l d p o p u l a t i o n of pelleted feed. Sorbic acid and its sodium and potassium

'Approved for publication in Journal Article Number 4155 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State, MS 39762. 2 Mold Curb®, Kemin Industries.

salts are established as effective preservatives at low c o n c e n t r a t i o n s for t h e control of m o l d and yeast in m a n y food p r o d u c t s . Sorbates and their salts have b r o a d s p e c t r u m activity against yeast and m o l d . Sorbic acid inhibits t h e dehydrogenase system in m o l d . This inhibition is reversable in t h e presence of high m o l d population and low acid c o n c e n t r a t i o n (Melnick et al, 1954). Sorbic acid and potassium s o r b a t e are generally recognized as safe additives for use in foods u n d e r t h e regulations of t h e US F o o d and Drug Administration (Furia, 1 9 7 2 ) . Sorbic acid is metabolized like o t h e r fatty acids in t h e m a m m a l i a n b o d y . This acid also can b e metabolized b y microorganisms as well (Furia, 1972). Sorbic acid a t a level of . 3 % in corn meal, containing 1 6 . 3 % m o i s t u r e , prevented heating for 4 2 d a y s ; whereas, at a level of . 1 % and .2% heating was delayed for 3 t o 7 days and 33 d a y s , respectively ( S i m m o n , 1 9 7 0 ) . In view of t h e lack of i n f o r m a t i o n concerning feed preservation, this s t u d y was designed

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to evaluate the effects of certain antifungal additives, environment, and storage on microbiological populations of poultry feed. MATERIALS AND METHODS

RESULTS AND DISCUSSION Total Aerobic and Anaerobic Counts. As expected, initial total aerobic and anaerobic microbial counts of poultry feed varied greatly with their ingredient sources. In one study, the total aerobic counts for 12 diets mixed at experimental location M (Mississippi State University) ranged from log counts of 5.90 to 6.88/g, while log counts of 3.72 to 4.08/g were recorded for the six diets mixed at location F (University of Florida). Lower initial aerobic counts, as recorded for the 12 finisher diets mixed at location M, as compared to the starter

FIG. 1. Aerobic counts of poultry feed. S, storage tank; F, feeder trough; f, Florida; m, Mississippi; —, control; - —, with .05% GV-11 at Florida or with .10% Ca-propionate at Mississippi; - - -, with .04% sorbic acid; — , with .02% sorbic acid and .02% adipic acid.

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Feed Samples. Feed samples were obtained from five broiler experiments as described by Dilworth et al. (1979). Sorbic acid, calcium propionate, gentian violet, and adipic acid plus sorbic acid were used as fungistatic compounds. Approximately 200 g of feed from the storage tank and the feeder trough were sampled from each individual pen at 0, 2, 4, 6, and 8 weeks of broiler age. Samples from the same diet and sampling location were composited and mixed. Microbiological Analyses. Ten grams of the well mixed samples were mixed with 90 ml of sterile nutrient broth in a dilution bottle. The mixture was vigorously shaken for 2 min. The serial dilutions of the samples were made and plated with the following media: a) standard method agar (BBL), b) anaerobic agar (BBL), and c) acidified potato dextrose agar (BBL). The duplicated standard method agar and anaerobic agar plates were incubated for 48 hr at 37 C to determine the total aerobic and anaerobic counts, respectively. Mold and yeast were determined on acidified potato dextrose agar; counts were made after 5 days of incubation at 25 C. Total number of coliform organisms was determined by direct plating on violet red bile agar as suggested by Recommended Methods for the Microbiological Examination of Foods (APHA, 1976). Lactobacillus numbers were enumerated with Rogosa SL medium. Moisture and pHMeasurements. Methods for feed moisture and grain pH determinations as described in Methods of Analysis (AOAC, 1975) were followed.

diets mixed at the same location supported the importance of ingredient sources on the total microbial count (Fig. 1). In most cases, the log anaerobic counts of feed samples were slightly lower than those of the aerobic counts with a difference of less than one log cycle (Fig. 2). Occasional anaerobic counts, which were higher than the aerobic counts, were observed, especially when the feed samples were obtained from feeder areas. The addition of selected antifungal additives at the practical levels, as described in the first part of this report series (Dilworth et al, 1979), had no consistent effect on the total aerobic and anaerobic counts. Due to the limitation of space and the similarity of the results, only four selected diet treatments at location M and three treatments at location F were reported. These treatments represent control feeds, feeds with calcium propionate supplement at . 1 % , feeds with GV-11 at .05%, feeds with sorbic acid supplement at .04%, and feeds with .02% sorbic acid and .02% adipic acid. In general, at location M there was no increase in total aerobic and anaerobic counts

FUNGISTATIC COMPOUNDS IN BROILER DIETS. 2.

for feed samples obtained from the storage tank; however, a steady increase in these counts was observed for feed samples obtained from feeders after 2 and 4 weeks of feeding. Results were consistent for all treatment diets as conducted in both experimental locations. This increase in total aerobic and anaerobic counts in feeder feed samples might have been due to the contamination of the feeding environment

from soil, fecal material, feathers, dust, and others. Adding water to the feed for a final moisture of approximately 15% did not affect the total aerobic and anaerobic microbial counts. Moisture content of the feed decreased under experimental storage conditions. For example, the high moisture starter diets had a moisture content of 14.44% after mixing; moisture content was reduced to 13.42% and 12.66% after 2 and 4 weeks of storage, accordingly (Table 1). Similar results were recorded for finisher diets; moisture content was reduced from 15.15% to 14.59% and 13.41% after 2 and 4 weeks of storage in the container. The low moisture diets had moisture contents ranging from 11.06% to 11.84%. As Table 2 indicates, the addition of fungistatic compounds did not influence the moisture content of poultry feed. It was interesting to note that the control and the sorbate treated feed samples had lower pH values at higher feea moisture levels after 4 weeks of storage than the low moisture feeds (Table 3). Increased pH values were observed for feed with . 1 % calcium propionate as a supplement. The slight increase in pH values for 11.06% moisture feed with .04% sorbic acid and .02% each of sorbic and adipic acids might have been due to mixing and/or sampling error. Total Mold and Yeast Counts. As with the total aerobic and anaerobic counts, total mold and yeast counts varied with their ingredient sources. Figure 3 indicated the total mold and yeast counts at experimental location M were higher than those of experimental location F. At experimental location F, a slightly lower mold and yeast count was observed for .04%

TABLE 1. Feed moisture contents of the low and high moisture control diets Percentage feed moisture Broiler age, weeks

Diet

Low moisture diet

High moisture diet

Starter Starter Starter

11.77 + .09 11.23 ± .17 11.06 + .06

14.44 ± .17 13.42 + .16 12.66 ± .02

Finisher Finisher Finisher

11.20 ± .11 11.84 ± .06 11.38 ± .06

15.15 ± .09 14.59 + .14 13.41 ± .13

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FIG. 2. Anaerobic counts of poultry feed. S, storage tank; F, feeder trough; f, Florida; m, Mississippi; —, control; , with .05% GV-11 at Florida or with .10% Ca-propionate at Mississippi; , with .04% sorbic acid; — — , with .02% sorbic acid and .02% adipic acid.

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TABLE 2. Effect of calcium propionate, sorbic acid, adipic acid, and the addition of water on broiler feed following four weeks storage Percentage feed moisture Dietary supplements

Starter feed at 4 weeks

Finisher feed at 8 weeks

Additive

(%)

Control Calcium propionate Sorbic acid Sorbic acid Sorbic acid Sorbic acid plus adipic acid Mean

.1 .02 .03 .04 .02

11.06+ .06 11.36 ± .06 10.99 ± .17 11.07 ± .16 11.26 ± .07 11.05 + .07 11.13

11.38 ± 11.70 ± 11.46 ± 11.50 ± 11.52 ± 11.51 ± 11.51

Control Calcium propionate Sorbic acid Sorbic acid Sorbic acid Sorbic acid plus adipic acid Mean

.1 .02 .03 .04 .02 + .02

12.66 12.92 13.15 13.49 13.18 12.78 13.03

13.41 + .13 13.23 ± 05 13.59 ± .13 13.37 + ,11 13.80 ± .10 13.42 ± ,16 13.47

Coliforms and Lactobacillus Counts. A s t e a d y increase in coliform p o p u l a t i o n in t h e feed from t h e cage trough was observed as t h e

length of feeding t i m e advanced. Addition of sorbic acid at .04% t o t h e feed retarded this increase. This retardation in t h e increase of coliform p o p u l a t i o n m i g h t have resulted in h e a l t h y birds and m o r e sanitary carcasses (Table 4 ) . Consistently lower lactobacillus c o u n t s were observed for p o u l t r y feed with .04% of sorbic acid. T h e biggest difference was observedafter t w o weeks of feeding t i m e . This difference in c o u n t s diminished as t h e length of feeding time increased (Table 4 ) . In conclusion, t h e addition of t h e fungistatic c o m p o u n d s t o feed at practical levels had n o consistent effect on t h e aerobic, anaerobic, and mold and yeast c o u n t s . After t w o and four

TABLE 3. Mean3- pH values of finisher diets pH Dietary supplements Additive

(%)

11.06% moisture

15.15% moisture

Control Calcium propionate Sorbic acid Sorbic acid Sorbic acid Sorbic acid plus adipic acid

.1 .02 .03 .04 .02 + .02

5.97 6.02 5.97 5.94 6.09 6.04

5.48 6.13 5.50 5.49 5.28 5.65

Each mean represents four observations.

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sorbic acid treated samples as c o m p a r e d t o t h e c o n t r o l s ; however, t h e m o l d and yeast c o u n t s for t h e control and t h e treated feed samples were similar at experimental location M. Unlike t h e total aerobic and anaerobic c o u n t s , there was n o drastic increase in total m o l d and yeast c o u n t s for feed sampled from t h e feed t r o u g h . Experimental data indicate t h a t t h e storage t i m e , sampling location, and tested m o i s t u r e variable has n o a p p a r e n t effect o n t h e t o t a l m o l d and yeast c o u n t s in feed. No visible m o l d growth or caking p r o b l e m was d e t e c t e d for t h e tested feed after four weeks of storage.

± .03 + .12 ± .20 ± .19 +- .30 ± .10

.01 .04 ,08 03 01 02

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TABLE 4. Coliform and lactobacillus counts of broiler starter feeds with and •without .04% sorbic acid as additive Log coun ts/g

Type of counts

Treatment

0-wk

1-wk

2-wk

3-wk

4-wk

Coliform

Control .04% Sorbic acid

2.95 2.58

3.74 2.90

4.30 2.96

4.36 2.71

4.68 3.01

Lactobacillus

Control .04% Sorbic acid

3.16 2.20

3.51 2.16

3.48 2.21

3.45 2.83

4.06 3.91

2

4

6

8

2

4

6

8

WEEKS

FIG. 3. Mold and yeast counts of poultry feed. S, storage tank; F, feeder trough; f, Florida; m, Mississippi; —, control; , with .05% GV-11 at Florida or with .10% Ca-propionate at Mississippi; , with .04% sorbic acid; — , with .02% sorbic acid and .02% adipic acid.

e x p e r i m e n t , sorbic acid consistently reduced t h e coliform and t h e lactobacillus c o u n t s for t h e feed samples from feed troughs.

REFERENCES American Public Health Association, 1976. Compendium of methods for the microbiological examination of foods. APHA, Washington, DC. Association of Official Analytical Chemists, 1975. Official methods of analysis. 10th ed. AOAC, Washington, DC. Deyoe, C. W., and S. F. Quadri, 1970. Effects of mold inhibitor in various types of feeds. Feedstuffs 42:58. Dilworth, B. C , T. C. Chen, E. J. Day, R. D. Miles, A. S. Arafa, R. H. Harms, G. L. Romoser, V. G. DiFate, and K. J. Shaver, 1979. Fungistatic compounds in broiler production. 1. Effect on rate of gain and feed utilization. Poultry Sci. 58:1445— 1450. Furia, T. E., 1972. Handbook of food additives. CRC Press, Cleveland, OH. Melnick, D., F. H. Luckman, and C. M. Gooding, 1954. Sorbic acid as a fungistatic agent for food: VI. Metabolic degradation of sorbic acid in cheese by mold and the mechanism of mold inhibition. Food Res. 19:44-58. Richardson, L. R., and J. V. Halick, 1957. Studies on feed spoilage: Heat inhibiting activity of various compounds and commercial products. Texas Agr. Exp. Sta. Bull. 879. Simmon, J., 1970. Chemical control of microorganisms in stored grains. Doctoral dissertation, Kansas State University, Manhattan, KS.

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weeks of feeding, a steady increase in aerobic and anaerobic c o u n t s was observed for feed samples o b t a i n e d from t h e feeder t r o u g h ; however, t h e r e was n o increase in m o l d and yeast c o u n t s . Increasing t h e m o i s t u r e c o n t e n t of t h e feed t o a p p r o x i m a t e l y 15% had n o effect on t h e microflora c o u n t . No visible m o l d growth or caking p r o b l e m s were d e t e c t e d for any feeds after four weeks of storage. In one