Effects of feeding the pomegranate pomace silage and dry pomegranate pomace on the intestinal microflora of Mehraban male lambs

The antibacterial and antifungal effects of pomegranates peel and seeds are associated with the presence of phenolic compounds (flavonoids and tannin). These compounds accumulate in the skin and pomegranate juice and account for 92% of the antioxidant activity of pomegranates (Abid et al. 2017). Total tannins for pomegranate peel and pulp were reported to be 9.73 and 0.66%, respectively (Delavare et al. 2014). The different species of pomegranate have antibacterial and antifungal properties that can influence on a wide range of microorganisms (Carlton et al. 2000). Phenolic compounds in plants and foods by changing the gut microbial population can increase the amount of useful bacteria and reduce harmful bacteria (Katiyar 2002). It has been found that intestinal microflora plays a critical role in the health of the digestive tract and is dependent on the ration as the final source for metabolism of organic compounds. (Choct et al. 1996). To our knowledge, the effects of pomegranate by-products have been investigated on livestock and rumen function, however their effects on the intestinal microflora in ruminants have not been addressed. Therefore, due to the high concentration of tannins and phenolic compounds in the pomegranate by-products and their effect on intestinal microflora, the aim of this study was to investigate the effects of pomegranate pomace silage and pomegranate air-dried pomace on intestinal microflora in Mehraban fattening lambs.

Nine male lamb of Mehraban breed (mean weight of 27.03±3.5 kg and mean age of 187.8±1.4 d), were fed on three iso-nitrogenous and iso-caloric diets. Diets were balanced according to NRC (2007) recommendation including control diet, diet contain 27% pomegranate pulp silage (mixture of seed and pulp at equal ratio) and diet contain 31% air-dried pomegranate seed pulp. All three diets were fed for 60 d after 3 weeks for adaptation in individual pens with free access to salts lick and water. At the end of experiment all lambs were slaughtered and after than for enumeration of intestinal fluoromicrobes, one gram freshly digested specimens of ileum and cecum were collected. Samples were spread on the surface of agar medium. Colonies were counted by ophthalmic count and bacterial count was calculated as CFU/g (number of colonies per gram). The MRS agar and MacConkey (MC) medium were used for identification and enumeration of Lactobacillus spp and Escherichia coli respectively. All samples were incubated at 37ºC for 24 hours. All colonies were enumerated and recorded as CFU/g of culture suspension. For confirmation of Escherichia coli detection on MacConkey agar medium, polymerase change reaction (PCR) was conducted as DNA extraction using commercial kit (Bioneer, Sout Korea), polymerase change reactions, and electrophoresis of PCR products. Detection of molecular bacteria was done using the primers of 23S rRNA gene PCR. The PCR process was initial denaturation at 94 ºC for 2 minutes and totally 35 cycles, denaturation at 94 ºC for 45 seconds, and extension at 72 ºC for 2 minutes. All data was analyzed as a complete randomized design using SPSS software. Significant difference for means was considered at 0.05 level of differences.

The results of this study showed that in MRS medium either in ilium or cecum, the number of lactobacillus bacteria in all groups were not statistically significant. The mean number of Escherichia coli decreased due to feeding of pomegranate by-products (P<0.05), while the type of pomegranate by-product has not significant effect on number of Escherichia coli. The importance role of gut microflora is well recognized in GIT health, although population of gut microbes has been influenced by diet (Choct et al 1996). In contrast of useful effects of lactobacillus on GIT, Escherichia coli damages the intestine of animals and produces lipopolysaccharide (Munyaka et al. 2012). Tannins are considered as a toxin to microorganisms; these compounds in the soluble environment produce some stable complexes, mainly with protein and to a lesser extent with carbohydrates or some physiological ions elements such as iron and copper (Chung and Chou 1998). The pomegranate peel extract at different levels has antimicrobial effect against microorganisms such as Staphylococcus aureus, Escherichia coli, Candida tropicalis and Candida albicans (Ahmed et al. 2013). The phenolic materials in pomegranate fruit, are responsible for the antimicrobial properties of pomegranates (Seeram et al. 2006). In the present study, reduction of Escherichia coli population in lambs fed pomegranate by-products can be attributed to the adverse effect of phenolic substances in the pomegranate byproducts on Escherichia coli population. Several mechanisms have been introduced for antimicrobial properties of phenolic compounds in the pomegranate. Phenolic substances, with high molecular weight proteins, form complexes and by these complexes can react to the some cytoplasmic and membrane enzymes after absorption (Seeram et al. 2006). These complexes can also prevent cell surface receptors from attachment of harmful microorganisms (Cowan 1999). Phenolic compounds can react with the cellular proteins of microorganisms, alter cell wall structure and function (Hugo and Bloomfield 1971), reducing cell wall permeability and reducing substrate transport to cells (Goel et al. 2005). In addition, phenolic compounds can alter or denature some microbial enzymes, and also form complexes with certain nutrients and remove them from microorganisms (Hugo and Bloomfield 1971). The decline of Escherichia coli can also be attributed to the increase in the number of Lactobacillus; because by increasing the Lactobacillus and consequently increasing the production of lactic acid and creating an acidic environment, the population of Escherichia coli is reduced due to sensitivity of Escherichia coli to acidic environment (Hammer et al. 1999).

Feeding of the pomegranate pomace silage and air-dried pomegranate pomace in fattening lambs, decreased the population of Escherichia coli in ileum and cecum, although lactobacillus bacteria was not affected by pomegranate by-products. It seems that the tannins and phenolic compounds present in the pomegranate can effect on Escherichia coli population in ileum and cecum.