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Int.J.Curr.Microbiol.App.Sci (2018) 7(4): 1432-1438 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 04 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.704.162 Evaluation of Antimicrobial Effect of Emblica officinalis Fruit Powder on Intestinal Micro-biota in Broilers Chicken Rajesh Dalal*, Parveen Kumar Ahlawat, Sonu, Vinus, V.S. Panwar, B.S. Tewatia and Nancy Sheoran Department of Animal Nutrition, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125004, Haryana, India *Corresponding author ABSTRACT Keywords E. coli, Lactobacilli, Micro-biota, Phytobiotics Article Info Accepted: 12 March 2018 Available Online: 10 April 2018 To study the effect of dietary supplementation of amla fruit powder on intestinal microbiota of broiler chicken, 300 commercial broiler chicks were randomly distributed into six treatments having five replicates consisting of ten birds each. The chicks fed with standard basal diet in two different growth phases i.e. starter (0-28d) and finisher (29-42 d). The first group was kept as control (T 1) and given the basal diet without antibiotic, while second group (T 2) was given basal diet with antibiotic. In third (T 3), fourth (T4), fifth (T5) and sixth (T6) groups, basal diet was supplemented with amla fruit powder @0.25%, 0.50%, 0.75% and 1%, respectively. The birds were weighed fortnightly to calculate performance parameters viz. feed intake, body weight change and FCR. Then one bird from each replicate was slaughtered and ileal contents were collected aseptically and serially diluted upto six dilutions and 0.1 ml of each dilution was poured and spread uniformly on MacConkey lactose agar for E. coli and MRS for lactobacilli and incubated at 37ºC for 24 hours. The average number of colonies was multiplied by reciprocal of the dilution factor and expressed as log Cfu/g of contents. Due to supplementation of phytogenic feed additive microbial load of gram negative E. coli decreased and beneficial gram positive Lactobacilli increased significantly at higher level of amla supplementation as compare to control group. Feeding diets containing phytobiotics may result in inhibition of growth and colonization of entero-pathogenic microbes in the digestive tract and favours growth of beneficial bacteria i.e. Lactobacilli, thus contributing to the balance of gut microflora. Introduction The practice of feeding livestock with sub therapeutic levels of antibiotics has been in use for over fifty years. Antibiotics improve the production performance and utilization of nutrients in meat producing chicks. However, the use of antibiotics is being placed under more and more pressure, as consumers increasingly fear that their use in the rations of poultry leads to the formation of resistance against bacteria which are pathogenic to humans (Langhout, 2000). Recently, the emphasis is being directed towards the search of herbal formulations which could be effective for amelioration of stress and leads 1432 Int.J.Curr.Microbiol.App.Sci (2018) 7(4): 1432-1438 to increase in production of birds. Several Indian herbs are reported to possess adaptogenic, antistress and immunomodulator properties (Sapcota et al., 2005; Wadhwa et al., 2007). Herbs and their bio-active constituents possess a broad antimicrobial activity (Dorman and Deans et al., 2000). Herbs and their extracts stimulate the growth of beneficial bacteria and minimize pathogenic bacterial activity in the gastrointestinal tract of poultry (Langhout, 2000; Wenk, 2000). Herbs or phytochemicals can influence selectively the microorganisms by an antimicrobial activity or by a favourable stimulation of the eubiosis of the microflora. The mechanism by which the majority of herbal feed additives exert their antibacterial effect is by acting on the bacterial cell wall structure, denaturing and coagulating proteins. The amla fruit due to presence of saponins, phenols and tannins have potent antimicrobial activity against both Gram positive and Gram negative bacteria. Materials and Methods Ethical approval The animal experiment was conducted in accordance with guidelines approved by the Institutional Animal Ethics Committee, 12/CPCSEA Dated 6.2.2017 in the Department of Animal Nutrition, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar. Experimental design Completely Randomized Design (CRD) was used as experimental design at uniform and standard management practices. Birds and management A total of 300 commercial broiler chicks (Ven Cobb strain) were randomly distributed into six treatments having five replicates consisting of ten birds each. The chicks fed with standard basal diet in two different growth phase i.e. starter (0-28d) and finisher (29-42 d). The first group was kept as control (T1) and given the basal diet without antibiotic, while in second group (T2) basal diet was given with antibiotic. The diet in third (T3), fourth (T4), fifth (T5) and sixth (T6) groups were supplemented with amla fruit powder @0.25%, 0.50%, 0.75% and 1%, respectively. The birds were weighed fortnightly to calculate growth performance parameters viz. feed intake, body weight gain and FCR. Then, one bird from each replicate was slaughtered and ileal contents were collected aseptically, samples were weighed (1 gm), transferred to sterile tubes and homogenized with sterile 0.9% normal saline solution. Then the solutions were mixed on vortex and serial dilutions of samples were made up to six dilutions, 0.1 ml of each dilution was poured and spread uniformly on MacConkey lactose agar for E. coli and MRS for Lactobacilli and incubated at 370C for 24 hours. The average number of colonies was multiplied by reciprocal of the dilution factor and expressed as log Cfu/g of contents. Figuring and composition of diets Basal ration was formulated as per BIS (2007) to fulfill the metabolizable energy (ME) and crude protein requirements of birds. Level of crude protein in starter (0-4weeks) and finisher (4-6weeks) ration was 22 percent and 20 percent, respectively. The respective ME content was 3000 and 3200 KCal/kg are presented in Table 1. All feed ingredients, additives and supplements used in the experiment were procured in one lot before the start of the experiment. The ingredients, additives and supplements used in the diet 1433 Int.J.Curr.Microbiol.App.Sci (2018) 7(4): 1432-1438 formulation were maize, soybean meal, vegetable oil, fish meal, mineral mixture, vitamins, coccidiostat, lysine, DL- methionine and amla fruit powder. The sources, composition and mixing rate of additives/supplements used in ration formulations are presented in Table 2. The experimental chicks were reared under deep litter system. The floor of the pens was thoroughly cleaned, disinfected before scattering of the bedding material. Well chopped dry wheat straw was used as bedding material to form the litter. The straw was evenly spread upto 5 cm thickness. The litter was regularly racked to avoid any lump formation. Wooden brooders fitted with bulb in the centre were used in each pen for brooding. The feeding programme consisted of a starter diet until 28 days and a finisher diet from 29 to 42 days of age. Weighed amount of feed was offered on paper sheets for first 3 days and thereafter, in the automatic feeders up to 28 days of age. Afterwards, the feeds were offered through hanging feeders maintained at appropriate heights. The chicks were provided ad libitum clean drinking water through the plastic waterers during first two weeks of the experiment. Thereafter, bigger plastic waterers were used till the end of the experiment. Individual body weight of chicks was recorded at 0 day age and thereafter fortnightly. At the end of the experiment, one bird from each replicate was slaughtered ethically by mechanical stunning followed by exsanguinations. Statistical analysis Data was analysed statistically as described by Snedecor and Cochran (1994). Analysis of variance was used to study the differences among treatment means and they were compared by using Duncans Multiple Range Test (DMRT) as modified by Kramer (1956). Results and Discussion Gut flora Data pertaining to E. coli and Lactobacilli count of ileal content of the experimental birds under different dietary treatments are presented in Table 3. Log value for E. coli (log cfu/g) ranged from 4.60 to 6.89 among different dietary treatments. Significant reduction in the E. coli count was observed inT2 and T6 group. Other treatment groups also showed significant reduction in the E. coli count as compared to control group (T1). Total Lactobacilli (log cfu/g) ranged from 4.84 (T2) to 7.08 (T5) and significantly higher value was recorded in the T5 and T6 group supplemented with 0.75% and 1% amla powder. Our study was in consonance with finding of Bostami et al., (2017) who revealed that, there was negative impact of medicinal herb supplementation on the gram negative microbes and E. coli and positive impact on the Lactobacillus counts. Therefore, it was observed that medicinal herb Emblica officinalis supplementation significantly suppress (P<0.05) the pathogenic E. coli and non-pathogenic Lactobacillus count was significant higher in amla supplemented groups as compared to control group. Jamroz et al., (2005) reported that plant extract supplement also significantly increased the Lactobacillus numbers following an application of natural plant extract. Similarly, Siddiqui et al., (2015) studied the effect of different dietary levels of Nigella sativa seed powder on Escherichia coli and total viable bacterial count in excreta of broilers and found E. coli and total bacterial counts were significantly decreased by Nigella sativa seed powder supplemented diets irrespective of inclusion levels. Castillo et al., (2006) reported that different herbs are able to enhance the growth of Lactobacilli this indicates that herbs have the ability to increase the beneficial bacteria. 1434 Int.J.Curr.Microbiol.App.Sci (2018) 7(4): 1432-1438 Table.1 Chemical composition of feed ingredients used in ration formulation Ingredient Maize Soybean meal Fish meal CP (%) 9.11 45.15 47.40 CF (%) 2.44 3.93 1.79 EE (%) 3.44 3.16 5.16 TA (%) 2.25 8.47 26.62 Lysine* (%) 0.18 2.57 1.42 Methionine* (%) 0.15 0.76 1.42 ME* (kcal/kg) 3300 2230 2210 *Calculated values (Singh and panda 1992) Table.2 Ingredient composition of experimental diets during different phases of growth Ingredient (kg /100 kg of feed) Maize Soybean meal Fish meal Vegetable oil Mineral mixture Feed additives (g/100 kg feed) Spectromix Spectromix BE Veldot Choline chloride Lysine DL-methionine 0-4wks 58 30 7 3 2 4-6 wks 60 25 7 6 2 10 20 50 50 50 150 10 20 50 50 50 150 Composition, sources and rate of mixing of feed additives/supplements Spectromix: Powder (Ranbaxy Animal Health, New Delhi). Each gm. contained VitaminA-82,500 IU, Vit D312000 IU, Vit B2-50 mg and Vit.K-10mg.Mixing rate: 10 g/100Kg of feed. SpectromixBE: Powder (Ranbaxy Animal Health, New Delhi). Each gm. Contained Vit.B1- 8mg, Vit.B6- 16mg, Vit.B12- 80mg, niacin-120mg, calcium pentothenate-80mg, Vit. E-160 mg, Lysine hydrochloride-10 mg, DLmethionine-10 mg and calcium 260 mg. Mixing rate: 20g/100kg of feed. Veldot: Venkeys- Dinitro-O-Toluamide (Coccidiostat). Mixing rate: 50g/100kg of feed. Choline chloride: Contain 60 percent choline. Mixing rate: 50g/100kg of feed. Lysine: Contained 98% lysine. Mixing rate: 50g/100kg of feed. DL-methionine: Contained 98% methionine. Mixing rate: 150g/100kg of feed. Table.3 E. coli (log cfu/g) and total Lactobacilli (log cfu/g) count of the ileal content of the experimental birds under different dietary treatments Treatments T1 T2 T3 T4 T5 T6 E. coli (log cfu/g) 6.89a±.09 4.60d±.19 6.56a±.08 5.84b±.26 5.19c±.15 4.88cd±.17 Lactobacilli (log cfu/g) 5.96c±.35 4.84d±.23 6.19bc±.23 6.77ab±.18 7.08a±.02 7.03a±.03 Means bearing different superscripts in a column differ significantly (P<0.05) 1435 Int.J.Curr.Microbiol.App.Sci (2018) 7(4): 1432-1438 The antimicrobial activity of plant secondary metabolites has been attributed to a number of terpenoid and phenolic compounds (Helander et al., 1998; Chao et al., 2000). The antimicrobial mode of action is supposed to be due to the hydrophobicity of these phenolic compounds and due to the fact that these compounds can enter the bacterial cell membrane leading to disintegration of cell membrane, leakage of ions and eventually cell death (Burt, 2004). Feeding diets containing phytobiotics may result in inhibition of the growth and colonization of entero-pathogenic microbes in the digestive tract, thus contributing to the balance of gut microflora (Harris et al., 2001) and promoting the growth performance and health of birds (Adibmoradi et al., 2006). Savage et al., (1996) reported supplementation of oligosaccharides may have a prebiotic effect through an increase in production of lactic acid, thus increasing the proliferation of beneficial bacteria and reducing the presence of Gram-negative bacteria, in vitro and in vivo studies has confirmed that phytobiotic in animal nutrition can stimulate feed intake, antimicrobial, coccidiostatic and immune-stimulatory action (Panda et al., 2006). Enhancement of intestinal activities of trypsin, lipase and amylase (Lee et al., 2004) and improved gut morphological characteristics (Jamroz et al., 2003) are the major mechanisms through which phytoadditives exert their beneficial effect on the nutrient digestibility. The beneficial influence of the phytogenic feed additives on improved performance and feed conversion ratio could be also explained due to the antioxidant activity of bioactive compounds such as carvacrol, thymol, cineol and pinene (Faleiro et al., 2005; Hazzitet al., 2006) as well as from improved enzyme activity in the alimentary tract, stimulation of useful and inhibition of pathogenic microflora. This eventually resulted in improved absorption and utilization of nutrients (Windisch et al., 2008; Frankic et al., 2009). Based upon the above study, it can be concluded that amla fruit powder can be effectively supplemented as an alternative to antibiotics as growth promoter in poultry ration and best results were obtained at 0.75% amla supplementation level (T5) regards to gut ecology. Antibacterial effects of amla on gram negative bacteria and beneficial effect on gram positive bacteria results in production of more lactic acid by Lactobacilli in gut which further leads to decreased pH in intestine thereby at low pH Coliforms are not able to colonise themselves within intestinal mucosa, which interns cause less sloughing of tissue due to decreased toxin produced by them. It also results in favourable environment due to production of short chain fatty acids by Lactobacilli which cause effective migration of enterocytes along the tip of villi, resulted in enhanced growth of intestinal villi and thus leading to improved nutrient absorption and better utilization. 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Use of phytogenic products as feed additives for swine and poultry. Journal of animal science 86(14_suppl): E140-E148. How to cite this article: Rajesh Dalal, Parveen Kumar Ahlawat, Sonu, Vinus, V.S. Panwar, B.S. Tewatia and Nancy Sheoran. 2018. Evaluation of Antimicrobial Effect of Emblica officinalis Fruit Powder on Intestinal Micro-biota in Broilers Chicken. Int.J.Curr.Microbiol.App.Sci. 7(04): 1432-1438. doi: https://doi.org/10.20546/ijcmas.2018.704.162 1438