Effect of Antibiotics on Intestinal Microflora and Flavor of Broiler Meat

MARKETING AND PRODUCTS Effect of Antibiotics on Intestinal Microflora and Flavor of Broiler Meat B. W. SHELDON1 and E. O. ESSARY

Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 (Received for publication March 24, 1981)

INTRODUCTION Many factors are thought to contribute to poultry flavor including the age and sex of poultry, diet, the influence of intestinal microorganisms, freezing and storage conditions, carcass chilling, food preparation, and chemical constituents. In considering these factors, the influence of intestinal microorganisms on flavor has received limited consideration, yet bacteria account for 25 to 42% of the bulk of fecal matter (March, 1977). Historically, poultry was processed without removing viscera at the time of slaughtering (New York dressed). Poultry processed in this manner developed a characteristic flavor resembling a "visceral taint" or "gamey" flavor (Shrimpton, 1966). Concurrent with the development of this off-flavor, a "green struck" phenomenon developed as illustrated by carcass greening (Barnes and Shrimpton, 1957; Shrimpton, I960; Pennington and Sherwood, 1922). Greening was caused by bacteriallyproduced hydrogen sulfide diffusing from the gastrointestinal tract and reacting in the pres-

1

Present address: Department of Food Science, North Carolina State University, Raleigh, NC 27650.

ence of air with the muscle pigment myoglobin. The green pigment produced was sulfmyoglobin (Pennington and Sherwood, 1922; Nickerson and Fitzgerald, 1939). The gamey flavor characteristic of New York dressed birds was suggested by Shrimpton (1966) to be associated with the metabolic activities of intestinal microorganisms with the subsequent absorption of microbial metabolites by skeletal tissue. Shrimpton and Grey (1965) studied the nature and origin of flavor precursors in raw and cooked chicken breast muscles. Sixteen of 23 volatile flavor components isolated from breast tissue were identified in the ceca of cannulated birds. The concentration of breast tissue isolates increased in uneviscerated birds held at 15 C following processing. Accompanying these volatile increases was a noticeable gamey flavor in the breast tissue. Harris et al. (1968) used a triangle taste test to compare the flavor of germfree, gnotobiotic (in contact with Clostridium perfringens, Escherichia coli, and Streptococcus faecalis) and conventionally reared birds. A highly significant difference in flavor was noted between germfree and conventionally reared chicken meat, a lower significant difference between gnotobiotic and conventional meat, and no difference between gnotobiotic and germfree

280

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ABSTRACT Variations in broiler meat broth flavor, intestinal microflora, and bacterial metabolites resulting from the administration of dietary antibiotics were investigated. Hubbard X Hubbard cross chicks were divided into four groups and presented one of the following acidified aqueous solutions for 7 weeks: 1) combination of 1.0 mg/ml streptomycin and bacitracin and .1 mg/ml mycostatin; 2) 1.0 mg/ml penicillin G; 3) .0011 mg/ml flavomycin; 4) acidified water. Treatments were evaluated by sensory panels, gas chromatographic analyses of flavor volatiles and organic acids, and intestinal bacterial cultures. Significant differences in flavor scores, organic acids, flavor profiles, and bacterial populations were noted between the experimental treatments and broilers receiving acidified water without antibiotics. Birds that received the penicillin and combination antibiotics exhibited several germfree-like characteristics including distended ceca, fluid cecal contents, and thinner intestinal walls. Results of this study demonstrated that dietary antibiotics significantly modified the intestinal microflora and metabolites which resulted in a significant change in broiler meat broth flavor. (Key words: broiler flavor, antibiotics, intestinal microflora) 1982 Poultry Science 61:280-287

INFLUENCE OF INTESTINAL BACTERIA ON BROILER FLAVOR TABLE 1. Composition of corn-soybean ration (g/kg)

Ground yellow corn Dehulled soybean meal (49% protein) Menhaden fish meal Alfalfa meal Meat and bone scrap (50% protein) Iodized salt Feeding oil (stabilized) Calcium carbonate Dicalcium phosphate Manganese sulfate Trace minerals mix a DL-methionine Ethoxyquin (66.6% pure) Vitamin premix 0 Choline chloride

576.2 299.0 25.0 25.0 25.0 5.0 20.0 7.5 7.5 .3 1.0 .5 1.5 5.0 1.5

Total

1,000.0

The trace mineral mix was guaranteed to contain: 15% manganese, 10% zinc, 7% iron, 1% copper, .22% iodine, and .08% cobalt. The following quantities in milligrams of vitamin supplements were supplied per kilogram of complete diet by the vitamin and feed additive premix: vitamins A - D 3 (650,000 IU/g, 325,000 ICU/g), 15; thiamine HC1, 100; niacin, 100; riboflavin, 16;D-calcium pantothenate, 100; vitamin B 12 , 22; pyridoxine HC1, 6; Dbiotin, (3.6 mg/g); folic acid, 4; vitamin E, 60; menadione dimethyl-pyrimidol bisulfite complex, 30; inositol, 100; sucrose, (4.446 g/kg).

reared chicken meat. Conventionally reared chicken had a stronger and more characteristic chicken flavor than germfree chickens. These findings clearly indicated the association of metabolic activities of poultry intestinal microorganisms with flavor development. Shrimpton (1958) demonstrated that broad spectrum antibiotics administered in water to chickens shortly before death significantly influenced the production of microbial metabolites and delayed the onset of carcass greening in uneviscerated birds. The present study investigated broiler flavor as affected by alteration of the intestinal microflora and microbial metabolites through dietary antibiotics. MATERIALS AND METHODS Chickens and Facilities. One hundred straight-run, day-old chicks (Hubbard X Hubbard cross) were purchased from a commercial hatchery and divided equally among four

groups. Birds were reared in cages and processed between 7 to 8 weeks. Carcasses were held frozen at —28.8 C prior to evaluation. Diet and Antibiotic Variables. Each group was assigned to a commercial corn-soybean broiler ration (Table 1). Antibiotic treatments were prepared following the procedures of Van der Waaij (1968) and G. Bruckner (1978, personal communication). Aqueous antibiotic solutions (Table 2) were prepared in 20 liter glass carboys and acidified to pH 4.5 with 12N HC1. Each group of birds were randomly assigned an antibiotic treatment and administered the preparation as drinking water for seven weeks. Treatment no. 4 birds received acidified drinking water (pH 4.5) without antibiotics. Intestinal Metabolic Products. Eight broilers (two per treatment) were sacrificed by cervical dislocation at 2, 4, 6, and 7 weeks of age and their alimentary tracts examined for microbially-produced organic acids. Four sections of the alimentary tract were analyzed including the gizzard, a 3 in (7.6 cm) portion of small intestine anterior from the ileocecal junction, the ceca, and a section posterior from the ceca to the vent (colon). Two 1 g samples of the intestinal contents were transferred from each section into preweighed 12 x 75 mm tubes, weighed, and stoppered. The procedures of Holdeman et al. (1977) were followed to extract, identify, and quantitate organic acids. To aid the extraction procedure, 1 ml of distilled water was added to the samples prior to acidification and extraction. Data were collected and normalized with a Hewlett Packard Model 3380A recorder-

TABLE 2. Antibiotics added in drinking water and administered to five groups of broilers

Treatment 1 1. Streptomycin Bacitracin Mycostatin 2. Penicillin G 3. Flavomycin (.44% pure) 4. Antibiotic free, acidified water 5. Antibiotic free, nonacidified water

(mg/ml acidified water) 0 1.0 1.0 .1 1.0 .25 0 0

Antibiotics from Sigma Chemical Co., St. Louis, MO. b

Adjusted to pH 4.5 with 12N HC1.

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Ingredients

281

282

SHELDON AND ESSARY

integrator. Acids were identified and quantitated using standards as prepared by Holdeman et al. (1977). Concentrations of organic acids were expressed as milliequivalents per 100 ml of contents. Statistical differences between chromatograms were calculated by a stepwise discriminant analysis as outlined by Powers (1968) and Powers and Keith (1968). Isolation of Flavor Volatiles. Two 8-weekold carcasses per treatment were thawed, hand deboned into groups of breast meat, dark meat including thighs and drumsticks, and skin with adhering fat. Samples were ground separately in a Kitchenmaid model 4-C grinder with a no. 10 plate. A slurry of 255 g of ground raw poultry meat and 510 g of triple distilled, deionized water was placed in a 1000 ml round bottom flask and assembled to a modified Buchler flash evaporator (Fig. 1). A gas inlet tube connected to the sample reservoir provided a capillary leak of prepurified compressed air at a flow rate of 50 cc/min. The function of the air purge was to simulate oxidative cooking conditions.

TABLE 3. Ingredients used in preparing chicken broth for evaluation of broiler flavor Ingredient

(g)

Ground meat Boiling water Iodized salt Monosodium glutamate

172.00 666.44 8.50 3.06

Total

850.00

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FIG. 1. A modified rotary flash evaporator used for extracting broiler meat flavor volatiles. A = meat slurry flask; B = trap 1 immersed in slush ice; C = spiral condensor (—20 C); D = compressed air capillary leak valve; E = vacuum gauge; F = trap 2 immersed in slush ice; G = vacuum control valve.

The slurry was distilled 1 hr at 60 C, 460 mm Hg; 1 hr at 75 C, 289 mm Hg; and 1 hr at 75 C, 260 mm Hg. The final distillate volume was approximately equal to the initial volume of added water. Condensates were combined, transferred to a 1000 ml separatory funnel and extracted four times using portions of nanograde diethyl ether equal to 12.5% of the distillate volume. Prior to extraction, 100 ;ul of internal standard, methylnonadecanoate (Applied Science Labs, State College, PA) (1 mg/ml, w/v) were added to the condensate. The ether extractions were transferred to a 250 ml Kuderma Danish flask (PGC Scientific Corp., Rockville, MD) fitted with a 5 ml graduated centrifuge tube and a 29.8 cm Snyder column (ACC Glass Inc., Vineland, NJ). Ether volumes were adjusted to 8 ml with a rotary flash evaporator (20 C, 560 mm Hg), chilled to —30 C to remove water, decanted, and concentrated to 2 5 111 under a stream of prepurified nitrogen. Fractionation of Flavor Isolates. Volatiles were separated on a Bendix model 2600 gas chromatograph with dual flame ionization detectors and fitted with a 6.1 m X 2 mm i.d. glass column packed with 3% SP 2100 on 100— 120 mesh Supelcoport (Supelco Inc., Bellefonte, PA). The helium carrier gas flow rate was 60 cc/min with the column temperature programmed from 50 to 250 C at a rate of 2 C/ min and held isothermally at 250 C for 54.5 min. Detector and injector temperatures were 250 C and 185 C, respectively. Chromatograms were recorded on a 1 mV full scale strip chart recorder and integrated with a Spectra Physics autolab minigrator. Normalization and Plotting of Chromatograms. Retention times were normalized relative to the internal standard, whereas peak areas were expressed as a log percentage of the total log area excluding the solvent peak. Normalized

INFLUENCE OF INTESTINAL BACTERIA ON BROILER FLAVOR

283

TABLE 4. Influence of antibiotics in drinking water on microbially produced organic acids in the gastrointestinal tract of broilers Antibiotic treatments 4 Combination

Sections

Penicillin

Flavomycin

Treatment

Treatment

4

5

Organic acids 0 .275 1.404 6.378 2.158 2.554 -54.0 -72.4

.341 1.973 4.894 2.305 2.378 -57.1 -74.3

.499 2.873 8.089 3.371 3.708 -33.2 -59.9

.391 1.335 8.014 12.454 5.548

.119 6.393 21.665 8.825 9.250

-40.0

Combination containing 1.0 mg/ml streptomycin and bacitracin, .1 mg/ml mycostatin; 1.0 mg/ml penicillin G; .25 mg/ml flavomycin; Treatment 4, acidified water without antibiotics; Treatment 5, nonacidified water without antibiotics. Milliequivalents/100 ml; average of 2, 4, 6, and 7 week values. Average of four sections. Percent difference from Treatment 4. Percent difference from Treatment 5.

retention times and areas were submitted to a computer histogram plotting program with the Y ordinate representing log percent areas and the X ordinate representing relative retention times. Discriminant Analysis of Chromatograms. Chromatographic data were analyzed with a computer discriminant analysis program with

the differences between chromatograms expressed as a percentage. Percentages were calculated by dividing the two profiles being compared into equidistant time slices. Time slice intervals were chosen as the shortest retention time between two adjacent peaks. Corresponding time slice log areas were compared between chromatograms and their log differences calcu-

TABLE 5. Stepwise discriminant analysis of the cecal fermentation of 2, 4, and 6-week-old broilers

products

Level of significance

F Value entered

Wilks' lambda

114.0078 34.3686 29.5739 12.1095

.01156 .00033 .00001 <.00001

<.001 <.001 <.001 .002

Weeks

Step

Peaks used to form ratio

2

1 2 3 4

Succinic/propionic Acetic/propionic Butyric/propionic Iso valeric/valeric

4

1 2 3 4

Isobutyric Butyric Isovaleric Isobutyric/propionic

23.7738 10.0739 30.3124 89.9856

.05311 .00480 .00010 <.00001

.005 .003 .001 .003

6

1 2 3 4

Acetic/propionic Isobutyric/propionic Isovaleric/isobutyric Propionic/succinic

7.9693 133.3076 57.0795 6.1919

.14333 .00107 .00001 •C00001

.037 <.001 <.001 .004

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Proven triculus-gizzard Ileum Ceca Colon Avgc % Differenced % Difference6

284 100

SHELDON AND ESSARY

o en 2Io Q-CD

O 1

^ O

LD O

r-

1

"

I

d o LU 1 ^ CC O=o 3* 1—

l 1

Z - Ml UJ^ LJ QC _ Hi

,

L1J0 Q_C\J

c

i if '

1 il Hi .1

.2

.3

.6

.7

.8

NORMALIZED

.9

1.0

1.1

1.2

1.3

TIME

—T" 1.4

1.5

FIG. 2. Gas chromatogram of breast meat volatiles extracted from 8-week-old broilers given streptomycin, bacitracin, and mycostatin in drinking water.

lated. Profile differences were calculated by dividing the total sum of log area differences between the two profiles by the total log areas of both profiles times 100. Triangle Taste Panels. A panel of 10 judges were chosen by the preliminary screening method of Pippen et al. (1954). Panelists were chosen on their ability to distinguish between broth samples which differed in strength by 17%. Color variations between samples were eliminated by using tan colored lights. Chicken Broth Preparation. Two carcasses per treatment were thawed and hand deboned into breast and dark meat (thigh and drumstick) groups. Meat was ground as previously oudined. Preparation variations were reduced by canning all samples concurrently. Broth ingredients (Table 3) were dispensed into no. 401—411 enamel coated tin cans. Filled cans were exhausted in a steam tunnel for 6 min, reaching an internal product temperature of 87.8 C, sealed, processed for 1 hr at 121.1 C (16 PSIG), and refrigerated at 4.4 C. Prior to evaluation, sealed cans were heated in boiling water for 20 min. Broth was decanted through a no. 80 USA standard testing

sieve to remove meat particles and then transferred to a 1000 ml separatory funnel from which the aqueous phase was decanted. Bacteriological Examination of the Alimentary Tract. Eight broilers (two per treatment) were sacrificed by cervical dislocation at 2 , 4 , and 6 weeks of age. Identical alimentary tract sections as described under the Intestinal Metabolic Products section were aseptically excised and their contents cultured for total aerobic and anaerobic microorganisms. Serial dilutions were prepared in prereduced anaerobically sterilized (PRAS) salts solution. Anaerobes were cultured in a PRAS modified E agar as described by Holdeman et al. (1977). Aerobes were cultured on modified E agar without resazurin and cysteine. Both media were modified as suggested by Barnes and Impey (1970) by substituting equal portions of a chicken fecal extract for rumen fluid (75 ml). Anaerobic roll tube cultures were incubated at 37 C for 5 days, while aerobic pour plates were incubated at 37 C for 3 days. RESULTS AND DISCUSSION

Intestinal

Metabolic

Products.

The most

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_J°

11

285

INFLUENCE OF INTESTINAL BACTERIA ON BROILER FLAVOR

Stepwise Discriminant Analyses of Organic Acid Data. A summary of the classification analyses is presented in Table 5. Only four steps were required to correctly discriminate between treatment chromatograms. As the number of steps increased, the fit of the classification, as measured by decreasing Wilks' lambda values, became more nearly perfect. The ratios of suc-

cinic to propionic, isobutyric, and acetic to propionic were chosen as the most discriminating for 2, 4, and 6-week-old broilers, respectively. Isolation of Flavor Volatiles. Figure 2 shows the complex breast meat volatile profile from broilers treated with the antibiotic combination. Volatile profiles were equally as complex for the thigh-drumstick and skin-fat composites. Additionally, considerable visual differences were noted between tissue types and treatment chromatograms. Results of the discriminate analyses are presented in Table 6. The average percent profile difference for the three tissue types across treatments was 88.1, 79.0, and 74.5% for breast meat, dark meat, and skin-fat chromatograms, respectively. Results of these analyses suggest that changes in the intestinal flora or metabolic products resulting from dietary antibiotics may influence the volatile flavor components of broilers. Moreover, this discriminant analysis technique appears to be an effective tool for comparing complex chromatograms. Taste Panels. Panelists were better able to distinguish between white meat broths than dark meat broths (Table 7). Significant broth

TABLE 6. Discriminant analyses of meat volatiles extracted from broilers receiving antibiotics in acidified drinking water Percent difference 1 Treatment 0 Treatments

Tissue type°

Combination Penicillin G Flavomycin Treatment 4

Breast

Combination Penicillin G Flavomycin Treatment 4

Dark

Combination Penicillin G Flavomycin Treatment 4

Skin + fat

a

Combination

Penicillin G

Flavomycin

Treatment 4

0

97.2

97.2 88.0 83.3

0

88.0 92.2

92.2 88.5

0

83.3 88.5 79.2

79.2

0

.9

82.0

82.0 69.9 84.0

0

69.9 83.3

83.3 72.8

0

84.0 72.8 81.5

81.5

0

0

94.9

94.9 87.5 90.8

0

87.5 69.6

69.6 58.8

0

90.8 58.8 45.3

45.3

0

Sum of differences between chromatograms/total area of both chromatograms X 100. Breast and dark meat (thigh and drumstick) without skin and adhering fat. Combination, 1.0 mg/ml streptomycin, 1.0 mg/ml bacitracin, .1 mg/ml mycostatin; penicillin G, 1.0 mg/ml; flavomycin, .25 mg/ml; Treatment 4, antibiotic-free, acidified water; all antibiotics dissolved in p'H 4.5 water.

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active fermentations occurred in the cecum followed by the colon, small intestines, and proventriculus-gizzard sections (Table 4). Broilers treated with penicillin yielded the smallest concentration of organic acids over 6 weeks (2.378 meq) followed by the combination antibiotics, flavomycin, and Treatment no. 4 groups. Organic acid levels decreased by 57.1, 54.0, and 33.2%, respectively, for penicillin, combination, and flavomycin treatments when compared to Treatment 4 broilers. In comparison to broilers reared without antibiotics or acidified water (Treatment 5), significant reductions in organic acid concentrations were found in the intestinal tract of Treatment 4 broilers. These results illustrated that both antibiotics and acidification of drinking water were effective in altering the organic acids produced by the intestinal microflora.

286

SHELDON AND ESSARY TABLE 7. Triangle taste test results of chicken broths prepared from broilers White meat

Treatments 3

Dark meat Total

Correct

Total

7' 9 4 4 3 4

10 10 10 10 10 10

10 10 10 10 10 10

a Antibiotic dosages in acidified water (pH 4.5), combination (1 mg/ml streptomycin, 1 mg/ml bacitracin, .1 mg/ml mycostatin); 1 mg/ml penicillin G; .25 mg/ml flavomycin; Treatment 4, antibiotic-free, acidified water.

*P<.05. **P<.01.

birds receiving the two higher antibiotic dosages. Bacteriological Examination of the Alimentary Tract. Intestinal aerobic counts recovered from Treatment 5 birds are included so that comparisons can be made of the effects of water acidification on the aerobic microflora (Table 8).

flavor differences were noted between Treatment 4 broilers and broilers that received the combination and penicillin treatments. Differences were noted between broths from birds reared on high (combination, penicillin G) and low (flavomycin) levels of antibiotics, but no difference was noted between the broths from

TABLE 8. Aerobic and anaerobic intestinal microflora counts of 6-week-old broilers :

Log number of bacteria per gram ± log standard deviation i

Proventriculus and gizzard Treatments 1

Aerobic VC2

6 Weeks Combination Penicillin Flavomycin Treatment 4 Treatment 5

<4.0 <4.0 <4.0 4.4 (.17) 7.7 NSD 3

Anaerobic VC

8.2 5.9 5.5 6.8

(.03)3 (.ll)c (.00) c (.31) b

Ileum Aerobic VC

4.5 4.2 5.3 6.9 8.9

Anaerobic VC

(.28) c (.39) c (,62) b (.ll)a NSD

8.7 (.07)3 8.0(1.2) a b 6.8 (.44) b 7.6 (.08) a b Colon

6 Weeks Combination Penicillin Flavomycin Treatment 4 Treatment 5

6.7(.17) a b 5.9(l.l)b 6.2 (.18) b 7.4 (.69) a 10.2 NSD 3

9.6 10.1 9.9 9.9

(.15) b (.21)3 (.26)3 (.11)3

6.6 (.27) c 5.0 (.00) d 8.1 (.21)3 7.4(.51) b 9.7 NSD

9.3 9.1 9.0 9.1

(.07)3 (.06) a (.11)3 (.60)3

' ' ' Treatments with different superscripts in the same column differ significantly at the (P-C05) level of significance. 'Combination antibiotic contained 1.0 mg/ml streptomycin and bacitracin, .1 mg/ml mycostatin; 1.0 mg/ml penicillin G; .25 mg/ml flavomycin; Treatment 4, antibiotic-free, acidified water (pH 4.5); Treatment 5, antibiotic-free, nonacidified water. 2

VC, viable count.

3

NSD, no standard deviation.

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Treatment 4 X combination antibiotic Treatment 4 X penicillin G Treatment 4 X flavomycin Combination antibiotic X penicillin G Combination antibiotic X flavomycin Penicillin G X flavomycin

Correct

INFLUENCE OF INTESTINAL BACTERIA ON BROILER FLAVOR

Salanitro et al. (1978) demonstrated that intestinal anaerobic counts generally exceed aerobic counts by a factor of 10 2 . However, in this study, a reduction in the anaerobic flora in Treatments 1 to 4 was noted when compared to the aerobic flora of Treatment 5 birds. These results indicate that antibiotics administered in acidified water were responsible for a significant reduction of both aerobic and anaerobic bacteria. Physical observations of the intestinal tract of these broilers were similar to those reported on germfree-like broilers treated with antibiotics (Gordon and Pesti, 1971; Wostmann et al, 1973; Gordon, 1975). The ceca content of the combination and penicillin treated birds were more fluid, larger, and more distended, and had thinner walls than those of Treatment 4 and flavomycin treated birds. The cecal content of Treatment 4 birds was pasty. This study demonstrated significant variations resulting from antibiotic supplements and acidified water in broth flavor, the population of intestinal microorganisms, and the nature and quantity of organic acids of the alimentary tract. From these variations in microflora and organic acids, we anticipate other microbial metabolites of potential flavor importance to be altered, thus leading to a change in broiler flavor.

REFERENCES Barnes, E. M., and C. S. Impey, 1970. The isolation and properties of the predominant anaerobic bacteria in the caeca of chickens and turkeys. Brit. Poultry Sci. 11:467-481. Barnes, E. M., and D. H. Shrimpton, 1957. Causes of

greening of uneviscerated poultry carcasses during storage. J. Appl. Bacteriol. 20:273-285. Gordon, H. A., 1975. The role of the intestinal flora in absorption: a comparative study between germfree and conventional animals. Page 237 in Intestinal absorption and metabolism. T. Z. Csaky, ed. Raven Press, New York, NY. Gordon, H. A., and L. Pesti, 1971. The gnotobiotic animal as a tool in the study of host microbial relationships. Bacteriol. Rev. 35:390—429. Harris, N. D., D. H. Strong, and M. L. Sunde, 1968. Intestinal flora and chicken flavor. J. Food Sci. 33:543-547. Holdeman, L. V., E. P. Cato, and W.E.C. Moore, 1977. Anaerobe laboratory manual. 4th ed. Virginia Polytechnic Institute Anaerobe Lab., Blacksburg, VA. March, B. E., 1977. Intestinal microflora and nutrition. Anim. Nutr. Health. March:18-22. Nickerson, J.T.R., and G. A. Fitzgerald, 1939. Problems arising during holding of poultry prior to evisceration and freezing. Proc. 7th World's Poultry Congr., Cleveland, OH, Waverly Press, Baltimore, MD. Pennington, M. E., and C. M. Sherwood, 1922. The greening of poultry. Poultry Sci. 1:114—124. Pippen, E. L., A. A. Campbell, and I. V. Streeter, 1954. Origin of chicken flavor. J. Agr. Food Chem. 2:364-367. Powers, J. J., 1968. Computers, statistics and gas chromatography: toward objective evaluation of food flavor. Food Technol. 22:39-44. Powers, J. J., and E. S. Keith, 1968. Stepwise discriminant analysis of gas chromatographic data as an aid in classifying the flavor quality of foods. J. Food Sci. 33:207-213. Salanitro, J. B., I. G. Blake, P. A. Muirhead, M. Maglio, and J. R. Goodman, 1978. Bacteria isolated from the duodenum, ileum, and cecum of young chicks. Appl. Environ. Microbiol. 35:782-790. Shrimpton, D. H., 1958. Studies on the metabolism of the caecal flora in situ in live fowls. Proc. 11th World's Poultry Congr., Mexico City, Mexico. Shrimpton, D. H., 1960. Control of greening in undrawn hens. Agriculture 67:20—25. Shrimpton, D. H., 1966. Metabolism of the intestinal microflora in birds and its possible influence on the composition of flavour precursors in their muscles. J. Appl. Bacteriol. 29:222-230. Shrimpton, D. H., and T. C. Grey. 1965. Speculations on the origin and nature of flavour precursors in chicken meat. World's Poultry Sci. J. 21:180. Van der Waaij, D., 1968. The persistent absence of Enterobacteriaceae from the intestinal flora of mice following antibiotic treatment. J. Infect. Dis. 118:32-38. Wostmann, B. S., B. S. Reddy, E. Bruckner-Kardoss, H. A. Gordon, and B. Singh, 1973. Page 261 in Germfree research: biological effect of gnotobiotic environments. J. B. Heneghan, ed. Academic Press, New York, NY.

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The use of antibiotics and acidified water exerted a pronounced effect on the aerobic intestinal microflora. Penicillin was the most effective antibiotic in reducing aerobic counts. In comparison to Treatment 4, penicillin reduced the aerobic counts after 6 weeks by .4, 2.7, 1.5, and 2.4 logs in the proventriculusgizzard, ileum, cecum, and colon, respectively. Furthermore, acidification of the drinking water to pH 4.5 yielded a 3.3, 2.0, 2.8, and 2.3 log reduction of aerobes in the proventriculusgizzard, ileum, cecum, and colon, respectively, when comparing Treatments 4 and 5.

287