Effects of a high-beef diet on bowel flora: a preliminary report 2

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1 Effects of a high-beef diet on bowel flora: a preliminary report 2 Bruce R. Maier,3 Ph.D., Margaret A. Fiynn,4 Ph.D., Gienna C. Burton,5 Ph.D., Robert K. Tsutakawa,6 Ph.D. and David J. Hentges,7 Ph.D. ABSTRACF Our laboratory has undertaken a study to determine whether the quantitative and/or qualitative bowel flora of man could be perceived as changing as a function of a controlled, systematic dietary sequence which included a high-beef regimen. The microbiological flora from each diet regimen was analyzed by both facultative and anaerobic studies performed on fecal specimens in order to test whether known carcinogen-producing species increased as a function of a high-beef protein diet. Initial untested data show that under the meatless diet coliforms rose in number. A subsequent high-beef diet cauied elevated Bacteroides populations which persisted in a subsequent normal diet regimen. Am. J. Clin. Nutr. 27: , The correlation of colon/rectal cancers and diet has been extensively investigated in recent years (1). A causative relationship between the intake of animal protein and the frequency of bowel carcinogens was first postulated by Gregor and co-workers (2-3). Additional perspective to the dietary hypothsis of bowel cancers was added by Hill et al. (4) who documented that various anaerobic bacterial species of the bowel may produce carcinogens. Our laboratory therefore undertook a study to determine whether the quantitative and/or qualitative bowel flora of man gould be perceived as changing as a function of a controlled, systematic dietary sequence which included a high-beef regimen. The microbiological flora from each diet regimen was analyzed by both facultative and anaerobic studies performed on fecal specimens in order to test whether known carcinogen-producing species increased as a function of a high-beef protein diet. Materials and methods Subjects and diet Ten male subjects were selected from medical school and graduate student volunteers between the ages of 21 and 30. Medical examinations and psychological screening were applied to assure successful adherence to the dietary sequence. The experiment was divided into two sequential periods utilizing five subjects for each run. The diets administered were composed of regular American food and meal types. The foods were purchased in lot amounts for control of constancy in nutrient composition. Table 1 summarizes the nutrient content qf the four administered diets. Theoretical analysis was calculated from USDA food tables while actual chemical analyses of various daily diets were performed and are tabulated. The meatless diets contained protein from vegetable sources with protein supplement from dairy and fish protein, whereas all protein in the high-meat diet was beef in origin. An attempt was made to keep constituent fiber, fat, and carbohydrates equal in all four diets. The lower actual fat content as compared to theoretical calculations in the normal is not readily explainable although the actual fat content of milk used may have been lower than USDA published values. The foods were fed as constant diets. That is, the same kind and amount of food was ingested daily over a period of 4 weeks per diet. Intake by all subjects of caffein, outside food or drink, and drugs of any kind was prohibited during the study. Tobacco smoking was permitted. Water was taken ad libitum and recorded by the subjects. Weight was monitored throughout the dietary sequence and needed calories were added by the additional of readily assimilated sugar. The sequence and order of administration was as fouows: 1 month each of the normal diet, the meatless diet, the high-meat diet, and fmally a normal diet. Three fecal specimens per subject were collected 1 From the University of Missouri School of Medicine, Columbia, Missouri This work was supported by National Institutes of Health Contract N01CF Assistant Professor of Microbiology. Associate Professor of Food and Nutrition. Research Associate. Associate Professor of Statistics. 7Professor of Microbiology TheAmerican Journal ofclinicalnutritiois 27: DECEMBER 1974, pp Printed in U.S.A.

2 TABLE 1 Calculated and actual diets administered for 4 weeks each EFFECTS OF A HIGH-BEEF DIET 1471 Normal Meatless High-Meat Normal Theory Actual Theory Actual Theory Actual Theory Actual Protein Fat CHO Fiber Ash Weeks per dietary regimen, grams per day. during the last (4th) week of each diet regimen. No time interval was allotted between the diet regimens, and therefore collection of fecal specimens represented a 3-week acclimatization period to the current administered diet. Specimen collection and workup Fecal specimens were collected in a specially built commode which allowed a strategically located Gas- Pak Jar (BBL) to be purged with oxygen-free nitrogen during the natural passage of each fecal specimen. With a continuing nitrogen purge of the collection jar, the standard Gas-Pak lid was locked into place to produce a totally anaerobic collection system. The specimens were taken immediately to the bacteriology laboratory and entered into an anaerobic glove box isolator system (Coy type B) where emulsification, apportionment for other analytical procedures and tenfold serial dilutions were performed. Aliquots were plated onto a battery of differential and selective anaerobic media including enriched brucella base agar with blood, veilonella media with neomycin (Difco) modified FM media (Difco) and kanamycin-vancomycin agar. Facultative plates were prepared from MacConkeys, Rugosa, Mycophyll, Todd Hewitt, and thallous acetate agars. Eighty degree heat shock for 20 min was performed to select for spore-forming organisms with subsequent plating on Naglar egg yolk agar. Trypticase soy-blood agar was used for total facultative counts. After 4 days incubation, and total-count enumeration, colonies were picked at a frequency of 40 different clones per fecal specimen. Twelve anaerobic colonies were selected for immediate identification in the ratio with which the colonial type was found on the dilution plates. All other selected colonies were enumerated, recorded, and stored at -70 C. All facultative organisms were picked from the differential and selective media and identification procedures were begun. Identification of anaerobics was a composite protocol utilizing high concentration antibiotic sensitivity discs (Finegold et al. (5)), biochemical and carbohydrate fermentation reactions using BBL Taxos discs in basal growth media, and gas-liquid chromotography. Taxonomic identification criteria were modeled after the VPI manual for anaerobes (6) Results Figure 1 shows a comparison between total facultative and total anaerobic counts for all ten subjects. These mean avenges show that total anaerobic counts were elevated during the high-meat and final normal diets, but statistical analysis at this time indicates these differences are not significant. A comparison of total optical counts as compared with total recovered plate counts indicate that approximately 70% of the optically counted cells were recovered in anaerobic enumeration. Comparative enumeration of individual species as compared with total viable counts is yet to be done. The Fig. 2 shows the family groups of facultative micoorganisms recovered from five subjects during the first complete experimental protocol. The mean averages plotted in the figure show what appears to be an increase in coliforms and lactobacilli during the meatless diet regimen, followed by an apparent decrease in lactobadilli during the high-meat diet. All diets seem to revert to normal base lines during the final normal diet regimen. The high degree of variation in staphylococci and fungi reflect average values for these species as recovered from five subjects. The individual variability, however, between fecal specimens from the same individual, and variability between individuals on the same diet, raises questions as to the statistical significance of the apparent mean averages shown in Fig. 2. A nonparametric analysis is currently being performed in order to establish the significance of the recovery. Dominant anaerobic groups of bacteria recovered from five subjects are plotted in Fig. 3. These data show that the mean average of Bactemi4es species decreased from a normal

3 1472 MAIER ET AL. value of 2 X 1O #{176} to 7 X lo organisms per gram of dry weight during the meatless diet regimen. Both high-meat and final normal diets caused Bacteroides to be elevated to almost 10 cells per gram dry weight, a significant increase over the meatless diet. Similarly, Fusobacteria can be judged as having increased during the meatless and high-meat diet approximately on log over normal diet regimens. Propionibacterla, Eubacteria, and Bifidobacteria appeared to be stable throughout the various diets. Ciostridia on the other hand, was depressed one log from 10 to 1 0 cells per FIG. 1. Total facultative and anaerobic mean counts of all subjects in each diet. L Dry Wt gram dry weight, by the high-meat diet, and this population depresssion sustained toward another tenfold reduction within the normal diet. Other species routinely recovered within the anaerobic protocol include Veiionella, Actimonycetes, and Arachnia. Selected mean averages from five subjects are plotted in Fig. 4, where Bacteroides, Bifidobacteria and coliforms are plotted together in order to visually test the hypothesis that a high-meat diet increases coliform counts and simultaneously depresses Bacteroides in the bowel. This postulate of bowel cancer etiology is supported by the data of Fig. 4 which have been subjected to more sophisticated weighted correction for sample size and recovery frequency of the three genera plotted. Analyses of these data show that Bacteroides and Bifidobacteria have an inverse population relationship and that as the coliforms increased during a meatless diet regimen. Bacteroides populations were depressed, but not to levels of statistical significance. Coliforms were recovered from the high-meat diet in these five subjects at the same population density as in the meatless diet, whereas Bacteroides increased to a level of 9 X 10 #{176}organisms per gram dry weight of feces. hi meat FIG. 2. Mean counts of facultative groups isolated from five subjects.

4 EFFECTS OF A HIGH-BEEF DIET 1473 Lo Dry LO DryWt Wt 11 IC ( j P g #{163} 1 normal meatless hi meat normal FIG. 3. Mean counts of anaerobic genera isolated from five subjects. c:-.:-_ ,- -., C normal meatless hi meat normal FIG. 4. Statistically corrected comparison of Bacteroides, Bifidobacteria, and coliforms isolated from five subjects.

5 1474 MAIER ET AL. This increase may be statistically significant with respect to Bifidobacteria, but further analysis is necessary before the relationship of coliforms to Bacteroides can be accurately interpreted. Discussion Dominant anaerobic and facultative genera are reported from five of the ten experimental subjects. The data reported from these five may well be amplified, modified, and/or made more significant as calculations from the last five subjects are processed. In addition, complete speciation of all bacteria genera is underway and may give elaboration to the averages of genera tabulated in this report. The additional information gained by taxonomic identification of between 30 and 40 species per fecal specimen will certainly add to our knowledge of the normal gut flora under controlled dietary condtions. Conclusions are very tenuous at this time. Available data show wide individual variations between the quantitative and qualitative bacteriological counts from fecal specimens produced by the same person during one diet regimen, as well as extreme variation between different individuals on the same diet. These ranges have the net effect of making statistical significance very questionable. This position of analysis may change with the advent of an expanded sample base using all ten subjects, and with the incorporation of nonparametric analysis which tends to deemphasize variations between subjects and give better credibility to changes within the diet sequence as the diet affected the flora of each individual subject. Our analysis on data from five subjects indicates that the genus Bacteroides may be statistically elevated under the conditions of a high-meat diet, while the average coliform population was elevated during the meatless diet and yet did not drop under the effect of a high-meat regimen. This series of observations tends to support the literature in which population from low-meat dietary cultures contained more coliforms and less anaerobes where as population in areas of high-meat diet tended to have higher anaerobic counts (7). Our data must at this time, however, be interpreted with much reserve until all entry and analysis from the entire experiment can be reported. At this time we have reservations as to whether 4 weeks of each dietary regimen were sufficient to allow the bacterial flora to become modified by the environmental influence. Changes which may have statistical as well as epidemiological significance, such as elevated coliform counts under the meatless diet did not change in four subsequent weeks of high-meat diet. Similarly, the elevated Bacteroides populations under the high-meat diet did not revert to the initial normal population when the final diet sequence follpwed the high-meat regimen. Thus we may be observing a sequence effect in which the bacterial flora of the gut changed, or began to change, but sustained enough inertia to resist subsequent diet impact within a 4-week period. I! The authors express their thanks for the capable assistance of Mr. Paul Rexroad for biochemical analysas of the diets, and Ms. Pam Greasor for statistical assistance. References 1. BERG, J. W., M. A. HOWELL AND S. J. SIL- VERMAN. Dietary hypothesis and diet-related research in the etiology of colon cancer. Health Services Rept. 88: 10, 915, GREGOR, 0., R. TOMAN AND F. PRUSOVA. Gastrointestinal cancer and nutrition. Gut 10: 1031, GREGOR, 0., R. TOMAN AND F. PRUSOVA. Relation of gastrointestinal cancer mortality in general. Scand. J. Gastroenterol. 6: 79, HILL, M. J., J. S. CROWTHER, B. S. DRASAR, G. HAWKSWORTH, V. ARIES AND R. E. WIL- LIAMS. Bacteria and etiology of cancer of large bowel Lancet 1: 95, FINEGOLD, S. M., J. E. ROSENBLATF, V. L. SUTFER AND H. R. ATrEBERY. Scope Monograph on Anaerobic Infections. Kalamazoo: Upjohn Press, HOLDEMAN, L. V., AND W. E. C. MOORE (editors). Anaerobe Laboratory Manual. Blacksburg, Va.: Virginia Polytechnic Institute and State Univ., ARIES, V., J. S. CROWTHER, B. S. DRASAR, M. J. HILL AND R. E. 0. WILLIAMS. Bacteria and the aetiology of cancer of the large bowel. Gut 10: 334, 1969.