Administration of mushroom extract to broiler chickens for bifidobacteria enhancement and Salmonella reduction

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1 2009 Poultry Science Association, Inc. Administration of mushroom extract to broiler chickens for bifidobacteria enhancement and Salmonella reduction W. L. Willis,* 1 K. King,* O. S. Iskhuemhen, and S. A. Ibrahim * Department of Animal Sciences, Department of Natural Resources and Environmental Design, and Department of Family and Consumer Sciences, North Carolina Agricultural and Technical State University, Greensboro, NC Primary Audience: Nutritionists, Broiler Producers, Researchers SUMMARY Two experiments were conducted to evaluate the administration of mushroom extract on bifidobacteria and Salmonella populations in broiler chicken feces. In the first experiment, a total of 100 female broiler chicks were assigned to 1 of the following treatment conditions, with 20 chicks per treatment: 1) control (no additive), 2) mushroom extract, 3) vinegar via water, 4) mushroom substrate extract, or 5) milk + water. In experiment 2, a total of 120 female broiler chicks were assigned to 1 of 4 treatment groups, with 30 chicks per treatment: 1) control (no additive), 2) experimental human bifido-strain, 3) mushroom extract, or 4) PrimaLac (probiotic). Fecal samples were collected from 5 pastured birds per treatment on d 49. Results of experiment 1 showed that broilers receiving the mushroom extract had a significant increase in the population of bifidobacteria in the fecal samples and no significant reduction in Salmonella fecal populations. Results of the second experiment showed that significantly higher bifidobacteria and lower Salmonella populations were observed after mushroom extract was withdrawn at d 21 from pastured broilers evaluated at d 49. Overall, continual administration of mushroom extract to broiler chickens enhanced fecal shedding of bifidobacteria and early intermittent administration of the extract decreased Salmonella populations. Key words: broiler chicken, bifidobacteria, mushroom extract, Salmonella 2009 J. Appl. Poult. Res. 18 : doi: /japr DESCRIPTION OF PROBLEM Intestinal microflora constitute a dynamic ecosystem that is essential to the health of the chicken. Moreover, foodborne pathogens such as Salmonella are associated with widespread human illness from poultry consumption. Salmonellosis is one of the most common foodborne diseases, with an estimated 1.4 million illnesses and approximately 600 deaths annually in the United States [1]. Therefore, developing preslaughter intervention strategies to reduce this source of infection is warranted. Many Salmonella reduction programs exist, but no overall solution has been found. Recently, new approaches using probiotics to decrease Salmonella and other pathogens are drawing considerable attention in the research arena. Bifidobacterium spp. 1 Corresponding author: willisw@ncat.edu

2 Willis et al.: MUSHROOM AND BIFIDOBACTERIA 659 and Lactobacillus spp. are 2 major species of the intestinal microflora in chickens that have beneficial effects on the host [2]. Bifidobacteria are anaerobic, rod-shaped, gram-positive bacteria that are normally present in the intestinal flora of animals and humans [3]. Increasing the numbers of bifidobacteria in the intestine of broilers is believed to induce immunostimulation, compete with pathogenic bacteria for adhesion sites, and produce essential volatile fatty acids for energy [4]. The common use of antimicrobials in animal rearing has increased public interest because of newly emerging antibiotic-resistant strains of pathogens [5]. Therefore, alternatives to promote intestinal health and product safety in broilers are being seriously investigated. One approach that is receiving attention is the use of pre- and probiotics. Probiotics are live microbial dietary supplements that could possibly benefit the host by improving its intestinal microbial balance [2]. Recently, certain plant polysaccharides have been recognized as having a prebiotic effect. Prebiotics are nondigestible food ingredients that induce the host to produce certain bacterial species in the colon [6]. Of the polysaccharides, the shiitake mushroom (Lentinus edodes) has been demonstrated to increase the resistance of the host to bacterial and viral infections [7]. Several compounds extracted from this mushroom have been demonstrated to have antifungal and antibacterial activities [8, 9]. Mushrooms are rich sources of natural antibiotics that have immunomodulatory properties in the cell wall glucans and in extracellular secretions by the mycelium that combat bacteria [10, 11]. Guo et al. [12] reported that supplementation with mushroom and herb extracts resulted in enhancement of both cellular and humoral immune responses in Eimeria tenellainfected chickens. Moreover, they found that increased numbers of bifidobacteria and lactobacilli reduced Bacteroides spp. and Escherichia coli numbers. In another study, Willis et al. [13] reported substantial continual shedding of bifidobacteria in broiler chicken feces after chickens received a shiitake mushroom extract. The antimicrobial activity of Portuguese wild edible mushroom methanolic extracts was studied by Barros et al. [14]. They observed inhibited growth of gram-positive bacteria (Bacillus cereus, Bacillus subtilis), whereas E. coli bacteria were resistant to these mushrooms. As the poultry industry moves away from the use of antibiotics, the face of health and product safety is bound to change, with more interest being drawn to gut microflora and the environment of the bird. In this context, the ability of mushrooms to restore and maintain the digestive balance, which provides protection against pathogens or the effects of stress, offers great potential for broiler production. In the present study, 2 substances (vinegar and milk) were provided to the chicks to create a favorable and an unfavorable growth environment for bifidobacteria in chickens. The objective of these experiments was to evaluate the effect of administering mushroom extract on bifidobacteria and Salmonella fecal population growth in broiler chickens. MATERIALS AND METHODS Birds and Housing Two experiments were conducted to evaluate different methods of administering mushroom extract and other additives to broiler chickens and to examine their influence on the recovery of fecal bifidobacteria and Salmonella populations from feces. In each experiment, newly hatched Avian Avian [15] female broiler chicks were obtained from a commercial hatchery and housed in Petersime battery starter cages ( cm) [16]. All broilers were vaccinated at the hatchery for infectious bronchitis, Newcastle disease, and Marek s disease. Each cage was equipped with 1 feeder and water trough. All broilers were fed a commercial starter-grower diet free of medication, except for PrimaLac [17] in 1 treatment. In both experiments, feed and water with or without additives were provided ad libitum, and light was maintained constant at 24 h. The thermostat for the room heating system was set at 31 C and was reduced each week by 3 C. After 3 wk, broilers were moved to finisher cages (68.58 cm 2 ) in the first experiment and to a pasture house in experiment 2. All bird procedures were reviewed and approved by the university Institutional Animal Care and Use Committee.

3 660 Experiment 1 Experiment 1 was conducted using Petersime battery brooder starter and finisher cages [16] in an environmentally controlled room. One hundred day-of-hatch female broiler chicks were obtained from a local commercial hatchery, randomized, weighed, and assigned to 1 of the following treatment conditions: 1) control (no additive), 2) mushroom extract, 3) vinegar via water, 4) mushroom substrate extract, and 5) milk + water. Each treatment used 20 chicks, with 5 each placed in 4 different cages. Shiitake mushrooms grown in a sawdust substrate block were obtained from the Mushroom Biology and Fungal Biotechnology Laboratory at North Carolina Agricultural and Technical State University Farm (Greensboro, NC). A known weight (100 g) of dried shiitake mushrooms or the mushroom substrate (medium containing 80% sawdust and 20% wheat-based carbohydrate supplement, on which the mushrooms were grown) was used in the extraction. A fine dried shiitake powder (1- mm sieve, 100-g sample) was extracted by stirring with 1,000 ml of sterile deionized water at 25 C and centrifuged at 1,073 g for 16 h. The mixture obtained was centrifuged for 30 min at 2,147 g, and the supernatant was concentrated to 150 ml in a rotary evaporator at 55 C. The mixture was held at 4 C before use as an additive in the water given to broilers. A commercially available white apple cider vinegar containing 5% acetic acid was administrated at 1 oz/gal (29.57 ml/3.79 L) of water in treatment 3 of this experiment. The diluted vinegar was used in small quantities to provide fresh mixtures regularly. Broilers subjected to treatment 5 were given the opportunity to consume tap water and raw, whole unpasteurized milk separately. The milk was obtained daily from the university dairy parlor and replaced daily. At the conclusion of the 7-wk period, fecal samples from each replicate group were collected and analyzed for bifidobacteria and Salmonella. Experiment 2 In experiment 2, 120 day-of-hatch female broiler chicks were obtained from a local commercial hatchery. They were randomly divided into 4 treatment groups with 30 chicks in each treatment, with 15 each in 2 different cages as follows: 1) control (no additive), 2) experimental human bifido-strain, 3) mushroom extract, and 4) PrimaLac (probiotic) [17]. Broilers in treatments 2 and 3 were given 0.5 ml of additive orally at 1, 7, 14, and 21 d. The bifido concentration was cfu/ml for the 0.5 ml administered to the chicks. The PrimLac in treatment 4 was administrated via the feed for 3 wk. After 3 wk, broilers from all treatments were wing-banded, placed in an outside Clear Span Chick-Inn Chicken Hutch (3.05 m 2 ; 10 ft 2 ) [18], and given access to a mixed clover-grass pasture during the day. All broilers had access to a Clear Span Pastured Poultry Chick-Inn Coop (3.66 m 2 ; 12 ft 2 ) [18] within the open pasture for shade and shelter. All treatment broilers were naturally infected with Salmonella and were allowed to share the same housing, unmedicated feed, and pasture together for 4 wk. On d 49, five broilers per treatment that had previously received treatments were collected and placed in separate cages, where overnight fecal matter droppings were pooled per cage, placed into a cooler, and transported to the laboratory for bifidobacteria and Salmonella analyses. Bifidobacteria and Salmonella Culture Methods JAPR: Research Report The population of bifidobacteria in fecal samples was determined using the standard laboratory method [19, 20]. Fecal samples (11 g) were diluted with 99 ml of sterilized 0.1% peptone water and homogenized using Stomacher 400 Laboratory System 4 [21] for 2 min, and 100 μl of appropriate dilution was plated onto modified B1M 24 agar [19]. The level was determined at a serial dilution of Plates were incubated at 37 C for at least 3 d to allow for bifidobacteria cell growth and a total count of the bacterial population. Further, the gram-stain technique [22] was used to facilitate microscopic examination of morphological characteristics of bifidobacteria. Fructose 6-phosphate phosphoketalase activity was measured to confirm the identity of bifidobacteria. The population of Salmonella in fecal samples was determined by blending in a Stomacher 400 Laboratory System [21] for serial dilution examination. Samples were serially diluted (1:10) in 0.1% peptone [23]. One hundred

4 Willis et al.: MUSHROOM AND BIFIDOBACTERIA 661 microliters from each dilution tube was placed onto a xylose lysine deoxycholate agar [23], and spread evenly on the agar, and all plates were incubated for 24 h at 37 C. The number of colony-forming units of Salmonella was expressed exponentially as log 10 Salmonella per gram of feces. Confirmation of Salmonella was conducted by microscopic observation and by biochemical screening of Salmonella colonies from selective agar onto triple sugar iron [23] agar slants. The commercial microbial cultures of probiotics contained sources of live naturally occurring microorganisms that were incorporated into the feed ration. The analysis was a minimum of cfu/g (Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, Enterococcus faecium). The PrimaLac [17] was incorporated into the ration at a level of g/t of feed as purchased from a commercial vendor. Statistical Analysis Data were statistically analyzed using the GLM procedure of SAS [24]. The means were compared using Duncan s multiple range test and Fisher s protected least significant difference test [25]. Statements of significance were based on P < 0.05 unless otherwise indicated. RESULTS AND DISCUSSION Experiment 1 The effect of mushroom extract on bifidobacteria and Salmonella fecal populations is shown in Table 1. Bifidobacterial populations from broilers given the mushroom extract (treatment 2) were significantly higher than the respective population values for broilers in the control group (treatment 1) and for treatments 4 (mushroom substrate extract) and 5 (milk + water), but not in treatment 3 (vinegar via water). The mushroom substrate extract did not enhance the bifidobacterial population in the feces as was observed with mushroom extract (treatment 2). Bifidobacteria are major species components of the chicken gut microflora [26] that may quantitatively and qualitatively influence the intestinal microflora. There are reports in the literature of using bifidobacteria as probiotics; however, there are very few reports on the use of mushroom extract to promote bifidobacteria in chickens. Recent findings indicate that mushroom and herb polysaccharide extracts stimulate beneficial bacteria (bifidobacteria and lactobacilli) while reducing the number of harmful bacteria [12]. In another study, an increase in bifidobacteria was observed in broilers for which the mushroom extract supplement was withdrawn after 3 wk [13]. A newly developed compound derived by fermentation, isomaltooligosaccharide, has been used successfully to enrich cecal bifidobacterial populations and reduce colonization levels of Salmonella in the ceca of broiler chickens [27]. In this experiment, no significant reduction in Salmonella was observed from the mushroom extract treatments, but Salmonella were numerically lower than in the control treatment. Broilers given the vinegar had the lowest fecal Salmonella population count when compared with those in other treatments. Interestingly, the bifidobacterial population of vinegar-treated broilers was on the same level as those given mushroom extract, which indicates a balance of beneficial and detrimental bacteria in their systems. The optimal growth requirement of bifidobacteria is slightly acidic; therefore, vinegar with a ph of approximately 2.4 functions as an acidifier in the drinker water. No scientific reports were cited in which vinegar was used to lower the ph or of its effect on the bifidobacterial population in poultry. There were reports of using sodium bisulfate and other substances on specific parameters, but not on bacterial populations. The efficacy of apple cider vinegar for poultry has not been studied frequently, but it is clear that the lower ph and ionic form of acetic acid create an undesirable environment for some microbes. A large body of literature indicates wide use of vinegar in the water of free-range flocks, and this study adds to the body of knowledge regarding the positive attributes of vinegar in poultry production. In another study, the administration of B. bifidum to chickens increased the number of cecal bifidobacteria and resulted in a nonsignificant decrease in the number of aerobes, coliforms, and clostridia, demonstrating that B. bifidum may reduce the populations of potentially pathogenic bacteria under commercial rearing conditions [28].

5 662 JAPR: Research Report Table 1. Mean log colony-forming units of bifidobacteria and Salmonella populations in the feces of broiler chickens subjected to various additives 1 Treatment Bifidobacteria, log 10 cfu Salmonella, log 10 cfu Control (water) 7.47 ± 0.11 b 5.98 ± 0.16 a Mushroom extract ± 0.09 a 5.81 ± 0.05 ab Vinegar ± 0.08 a 5.12 ± 0.18 c Mushroom substrate extract ± 0.08 b 5.63 ± 0.01 ab Milk + water ± 0.14 b 5.53 ± 0.19 b a c Means within a column with common superscripts do not differ significantly (P 0.05). 1 Values are means of 4 replicates (n = 4). 2 The mushroom extract used 100 g of dry mushrooms in 1 L of water. 3 White distilled apple cider vinegar with 5% acetic acid and a ph of Mushroom substrate material consisted of 80% sawdust and 20% wheat-based carbohydrate supplement. 5 Whole, unpasteurized cow s milk protein concentrations of mg/ml. Experiment 2 The results of the second experiment are shown in Table 2. As observed in experiment 1, the mushroom extract resulted in bifidobacterial populations that were significantly higher than the respective values for the control group. Moreover, the bifidobacterial population values for experiment 2 suggest that administering mushroom extract, even in a noncontinuous manner, also increases these beneficial organisms in broiler chickens. The results suggest that continuous administration of mushroom extract is superior to withdrawal in promoting bifidobacterial growth and shedding in the feces. As seen in the experimental strain of bifidobacteria treatment, the bifidobacterial population values were comparable to those in the mushroom treatment. The PrimaLac [17] treatment also had significantly higher levels of bifidobacteria than the control treatment but significantly lower levels than the experimental bifidobacteria strain and the mushroom treatments. It should also be acknowledged that the PrimaLac treatment resulted in the lowest fecal Salmonella population in this experiment. The mechanism by which mushroom extract stimulates bifidobacterial growth and survival in the gut of chickens is not known. However, it is sufficient to assume that the shiitake mushroom extract, which is known to be rich in β-glucans (polysaccharides), has components that may either act as quality and specific nutritional factors or create a buffered physical chemical environment in which bifidobacteria can survive and multiply compared with the levels in control samples. The intermittent administration of mushroom extract to broilers early in the production cycle of this experiment decreased the number of Salmonella population significantly (P 0.05; Table 2). A reduction in the Salmonella population in broilers given the intermittent dose of mushroom extract was associated with less mushroom extract as compared with more in experi- Table 2. Mean log colony-forming units of bifidobacteria and Salmonella populations in the feces of broiler chickens subjected to various additives 1 Treatment Bifidobacteria, log 10 cfu Salmonella, log 10 cfu Control (water) 6.49 ± 0.17 c 5.34 ± 0.03 a Bifidobacteria human strain 2 (water) 7.87 ± 0.12 a 4.78 ± 0.13 b Mushroom extract 3 (water) 7.80 ± 0.11 a 4.59 ± 0.05 bc PrimaLac 4 (feed) 7.18 ± 0.10 b 4.34 ± 0.11 c a c Means within a column with common superscripts do not differ significantly (P 0.05). 1 Values are means of 5 replicates (n = 5). 2 Bifidobacteria were cfu/ml for 0.5 ml administered to the birds. 3 The mushroom extract used l00 g of dry mushrooms in 1 L of water. 4 PrimaLac [17] was incorporated into the feed at a level of g/t, with a minimum of cfu/g (Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, Enterococcus faecium).

6 Willis et al.: MUSHROOM AND BIFIDOBACTERIA 663 ment 1. A similar trend was recently observed in a report that showed less Salmonella reduction with greater isomaltooligosaccharide levels in supplemented diets [27]. These findings suggest that more research is needed to determine the level of mushroom extract or powder to be administered to broilers to achieve bifidobacterial stimulation while suppressing Salmonella or other pathogenic growth in the gut of chickens. These results are interesting because scientists are continually seeking alternative supplements to enhance broiler health. Mushroom extract and vinegar exhibit potential because they seem to stimulate health-enhancing bifidobacteria, thereby competitively reducing the Salmonella population. This study adds to the body of research suggesting that mushroom extract could prove helpful in the fight against pathogenic organisms colonizing broiler chickens. Administering the optimal level of mushroom extract may help reduce Salmonella colonization or proliferation in chickens. This topic therefore merits more research. CONCLUSIONS AND APPLICATIONS 1. The results of the present study suggest that supplementation with mushroom extract for a short or long duration increases the population of bifidobacteria in the fecal matter of broiler chickens but that a long, continuous administration results in a higher level in the feces. 2. Vinegar exhibited a positive effect on bifidobacteria enhancement and Salmonella reduction when used in the water. The PrimaLac [17] dietary treatment also exerted a positive effect on bifidobacteria enhancement and Salmonella reduction. 3. By increasing the number of bifidobacteria in the gastrointestinal tract of the chicken, the potential exists to reduce the numbers of pathogenic bacteria, especially Salmonella, in the intestines and environment with naturally pastured poultry. 4. Further studies are needed to assess the dose and economics of administering mushroom extract or vinegar to commercial or pastured broiler chickens to enhance health, product safety, and bird well-being. REFERENCES AND NOTES 1. Mead, P. S., L. Slutsker, and V. Dietz Food-related illness and death in the United States. J. Emerg. Infect. Dis. 5: Fuller, R Probiotics for farm animals. Pages in Probiotics: A Critical Review. G. W. Tannock, ed. Horizon Scientific Press, Hethersett, Norwich, UK. 3. Scardovi, V Genus Bifidobacterium Orla-Jensen, AL. Pages in Bergey s Manual of Systematic Bacteriology. Vol. 2. Williams and Wilkins, Baltimore, MD. 4. Williams, C. H., S. A. Witherly, and R. K. Buddington Influence of dietary neosugar on selected bacterial groups of human faecal microbiota. Microb. Ecol. Health Dis. 7: McDermott, P. F., J. W. Zhao, X. Wagner, D. D. Simjee, R. D. Walker, and D. G. White The food safety perspective of antibiotic resistance. Anim. Biotechnol. 13: Gibson, G. R., and M. B. Roberfroid Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. J. Nutr. 125: Jong, S. C., and J. M. Birmingham Medicinal and therapeutic value of shiitake mushroom. Adv. Appl. Microbiol. 39: Morita, K., and S. Kobayashi Isolation structure and synthesis of lenthionine and its analogs. Chem. Pharmac. Bull. 15: Yasumoto, K., K. Iwami, and H. Mitsuda A new sulfur containing peptide from Lentinus edodes acting as precursor for lenthionine. Agric. Biol. Chem. 35: Benedict, R. G., and L. R. Brady Antimicrobial activity of mushroom metabolites. J. Pharmacol. Sci. 61: Kupra, J., T. Anke, G. Oberwinkler, G. Schramn, and W. Steglich Antbiotics from basidiomycetes, V11 Crinipellin, a new antibiotic from the basidiomycetous fungus Crinipellin stipitaria. J. Antibiotics 32: Guo, F. C., B. A. Williams, R. P. Kwakkel, H. S. Li, X. P. Li, J. Y. Luo, W. K. Li, and M. W. Verstegen Effects of mushroom and herb polysaccharides, as alternatives for an antibiotic, on the cecal microbial ecosystem in broiler chickens. Poult. Sci. 83: Willis, W. L., O. S. Isikhuemhen, and S. A. Ibrahim Performance assessment of broiler chickens given mushroom extract alone or in combination with probiotics. Poult. Sci. 86: Barros, L., R. C. Calhelha, J. A. Vaz, I. C. F. R. Ferreira, P. Baptista, and L. M. Estevinho Antimicrobial activity and bioactive compounds of Portuguese wild edible mushrooms methanolic extracts. Eur. Food Res. Technol. 225: Pilgrams Pride, Staley, NC. 16. Petersime Incubation Company, Gettysburg, OH. 17. Star Labs, St. Joseph, MO. 18. FarmTek, Dyersville, IA.

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