The effects of bee pollen extracts on the broiler chicken’s gastrointestinal microflora Miroslava Kac ˇániová a,⇑ , Katarína Rovná b , Henrieta Arpášová c , Lukáš Hleba a , Jana Petrová a , Peter Hašc ˇík d , Juraj C ˇ ubon ˇ d , Adriana Pavelková d , Róbert Chlebo c , Alica Bobková e , Michal Stric ˇík f a Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovak Republic b Department of Green’s Biotechnics, Horticulture and Landscape Engineering Faculty, Slovak University of Agriculture, Nitra, Slovak Republic c Department of Poultry Science and Small Animal Husbandry, Slovak Agricultural University, Nitra, Slovak Republic d Department of Animal Products Evaluation and Processing, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovak Republic e Department of Food Hygiene and Safety, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovak Republic f Department of Economy, University of Economics in Bratislava, The Faculty of Business Economics with Seat in Košice, Slovak Republic article info Article history: Received 26 June 2012 Accepted 27 February 2013 Keywords: Intestinal microflora Antimicrobial effect Enterococci Lactobacilli Enterobacteriaceae genera Pathogens abstract The aim of this study was to investigate the effects of bee pollen ethanolic extracts on the in vivo gastro- intestinal tract microflora colonization of broiler chickens. A completely randomized experiment based on six treatments (different concentrations of bee pollen – 0, 5, 15, 25, 35 and 45 g kg À1 diet) was used during 7 weeks. The highest count of faecal Enterococci was found in the experimental group with the addition of 15 g of pollen (8.85 ± 0.87 log CFU g À1 ) per 1 kg of feed mixture. The highest count of Lacto- bacilli was detected in the experimental group with 35 g of pollen per 1 kg of feed mixture and the high- est number of the Enterobacteriaceae genera count was found in the control group (8.43 ± 0.15 log CFU g À1 ). Moreover, the MALDI TOF MS Biotyper identified the following genera: Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, as well as Lactobacillus acidophilus, L. crispatus, L. fermentum and L. salivarius from the Lactobacilli group and Enterococcus avium, E. casseliflavus, E. cecorum, E. faecalis, E. faecium, E. gallinarum, E. hirae and E. malodoratus from the Enterococci group. Additionally, the in vitro antimicro- bial activities of pollen against five bacteria species isolated from gastrointestinal tracts of chickens were tested. The best antimicrobial effect of the pollen extract was detected against K. oxytoca. Ó 2013 Elsevier Ltd. All rights reserved. Introduction Natural supplements as a part of complementary and alterna- tive medicine have been used increasingly in the general human population, not only for their health and well-being aspects, but also for their potential therapeutic effects on certain diseases or conditions (Kennedy, 2005; Stanciu et al., 2009). However, many of these therapies are not recognized or supported by conventional health care providers. One of the main reasons for their disap- proval by the conventional medicine is the lack of science-based data from animal experiments and double – blind controlled hu- man studies. Bee pollen is one of the widely used natural supple- ments. It contains many essential nutritional elements important for growth and development of animals and humans (Bell et al., 1983; Orzaez Villanueva et al., 2002; Hašc ˇík et al., 2011; Capcarova et al., 2012; Petruška et al., 2012). Bees use pollen as their nutri- tional source of proteins (25–30%), carbohydrates (30–55%), lipids, including fatty acids and sterols (1–20%), vitamins and minerals. Furthermore, pollen is rich in carotenoids, flavonoids, phytos- terols, polyphenols and other beneficial compounds (Baltrusaityte et al., 2007; Moreira et al., 2008). Stored pollen undergoes chemical changes (Roulston, 2005), which are commonly associated with microorganisms supposedly playing a role in the conversion of pollen into a more digestible food for the bees (Human and Nicolson, 2003). This association may contribute to the pollen’s acquisition of certain organoleptic properties, which are specific for each bee species (Fernandes de Silva and Serrao, 2000). Currently, there are no data regarding the effects of bee pollen on the gastrointestinal microflora of broilers. Therefore, our study suggests that the addition of bee pollen into poultry feed could re- duce the number of the Enterobacteriaceae genera and increase the quantity of Enterococci and Lactobacilli. Eventually, these expected changes could help to improve welfare, health status and perfor- mance of broiler chickens. Materials and methods Animals Caecums of 49-day-old chickens (n = 196) were used for the quantitative determination of the selected individual groups of 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.02.022 ⇑ Corresponding author. Address: Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. Andreja Hlinku 2, 949 76 Nitra, Slovak Republic. E-mail address: miroslava.kacaniova@gmail.com (M. Kac ˇániová). Research in Veterinary Science 95 (2013) 34–37 Contents lists available at SciVerse ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc