The effects of different plant extracts on bile salt hydrolase activity of Lactobacillus strains isolated from the gastrointestinal tract of poultry

Abstract

The bile salt hydrolysis (BSH) enzyme weakens fat metabolism through bile salt deconjugation and reduces poultry performance, in order to cope with the antibacterial properties of the bile. Therefore, reducing the activity of this enzyme through the use of feed additives is probably a promising alternative to antibiotics for improving poultry performance. Plant extracts have long been used as feed additives for promoting poultry growth. In the current experiment, five Lactobacillus strains including Lactobacillus animalis, Lactobacillus acidophillus, Lactobacillus gallinarum, Lactobacillus lactis, and Lactobacillus returi were obtained from the poultry hindgut and were used as the probiotic application. A plate test and two-step enzymatic reaction method were used for deconjugation activity determination of the Lactobacillus strains. Further, four plant extracts (i.e., the aerial parts of Rosemary (Rosmarinus officinalis), Roselle calyx (Hibiscus sabdariffa), Berberis vulgaris root, and Green tea) were examined in terms of BSH enzyme inhibitors using the cell-free extracts as the potential antibiotic alternative. Furthermore, the gallbladders of the broilers were freshly collected from the poultry slaughterhouses, and their contents were extracted. The results showed that all Lactobacillus strains could hydrolyze the taurocholate acid (TCA) and chicken bile salt mixture (CBSM) to unconjugated bile acid. Moreover, ethanolic extracts of B. vulgaris root and Green tea relatively reduced the activity of the BSH enzyme that could potentially be investigated as an appropriate alternative in poultry feed in vivo. In conclusion, all five Lactobacillus strains were resistant to bile salts (i.e. TCA and CBSM) by BSH activity, and the addition of Green tea and B. vulgaris root extracts to the bacterial medium demonstrated inhibitory effects against the BSH enzyme. key words: lactobacilli; bile salt hydrolase; plant extracts; antibiotic growth promoters _____________________________________________________________________________________________ DOI: 10.24099/vet.arhiv.0887 ________________ *Corresponding author: Assoc. Prof. Dr. Mohsen Daneshyar, Department of Animal Science, Faculty of Agriculture, Urmia University, P.O. Code: 5715944931, Urmia, Iran, Phone and Fax: +98 443 2942 341; E-mail: [email protected] various ideas exist to clarify the principle of antibiotic-mediated growth improvement. The initial concept of AGP is associated with their antibacterial mode which decreases the total number of gut microbiota (FRANCOIS, 1961; VISEK, introduction For decades, antibiotic growth promoters (AGP) have been supplemented in poultry feed to improve the growth rate and feed conversion efficiency. Although the exact impacts of these supplementations are not completely recognized, A. Dibamehr et al.: Plant extracts on Lactobacillus strains in poultry Vet. arhiv 91 (1), 89-99, 2021 90 for reducing the level of cholesterol in serum through its direct impact on the host’s bile salt metabolism (BEGLEY et al., 2006). For instance, BSH produced by lactobacilli, deconjugates the taurine and glycine of bile acids (LANGHOUT et al., 1999; BEGLEY et al., 2006), which decreases the formation of micelles (MACDONALD et al., 1983) and the performance of broilers subsequently (SHARIFI et al., 2012). According to the literature, the population of Lactobacillus strains, the major BSH producers in the chicken intestine, decreased in reaction to AGP (KNARREBORG et al., 2002; DUMONCEAUX et al., 2006; GUBAN et al., 2006; BEGLEY et al., 2006; ENGBERG et al., 2000). Therefore, AGPs may promote chicken performance by reducing BSH activity, as it is an enzyme that exerts a negative effect on host fat digestion and metabolism. Accordingly, the current study sought to evaluate the BSH activity of different Lactobacillus strains isolated from the chicken hindgut, as well as the inhibitory impact of different plant extracts as potential alternatives for AGP on the BSH activity of L. acidophillus. The decrease in the BSH activity of lactobacilli strains as probiotics is believed to promote their efficiency. Materials and methods Bacterial strains and culture conditions. Five Lactobacillus strains, including L. animalis, L. acidophillus, L. gallinarum, L. lactis and L. returi, were obtained from a microbiology laboratory (Urmia University, Iran), having been previously isolated from the gastrointestinal tract of the native chickens. The Lactobacillus strains were separately cultured in de Man, Rogosa and Sharpe (MRS) broth (Scharlau, Spain) and stored in 10% glycerol at -20 °C before use. Preparation of CBSM. The gallbladders of broilers were freshly collected from poultry slaughterhouses, and the contents of the gallbladder were extracted, stored in an oven at 40 °C, dried for 24 hours, and finally, powdered and autoclaved before use in bacterial cultures as CBSM. Detection of bacterial BSH activity using the plate assay method. The bacteria were cultured in sterile MRS broth and incubated for 20 hours at 37 °C prior to checking the strains’ BSH activity. The 1978), leading to reduced competition for nutrients and microbial substances such as bile catabolism, which improves growth rate (FEIGHNER and DASHKEVICZ, 1987; GASKINS et al., 2002; KNARREBORG et al., 2004). However, there is concern regarding the development of antibiotic-resistant strains of bacteria that could be considered as a potential hazard to humans and animals (MARON et al., 2013). The World Health Organization strongly supports restraint in AGP use and the European Union banned them entirely in 2006 (MARSHALL and LEVY, 2011). Therefore, researchers are interested in finding alternatives to AGP with a similar impact for poultry production (REID and FRIENDSHIP, 2002; COX and PAVIC, 2010). Although there are several groups of alternatives to AGP in the poultry industry, including probiotics, prebiotics, symbiotics, organic acids, enzymes and phytogenics (medicinal plants), a perfect alternative should have the same beneficial impacts of AGP and ensure the optimization of animal growth and performance (HUYGHEBAERT et al., 2011). Phytogenics, as an interesting group of feed additives, may be a potential alternative to AGP. The biological or therapeutic activity of a phytogenic is completely related to its bioactive compounds and properties, which are variable according their derivatives. On the basis of the plant’s biological activity, its antioxidant properties, gut microflora manipulation and immune system improvement are the main modes of action by which phytogenics have a positive impact on the growth performance and health of animals (HASHEMI et al., 2009a, b; GUO et al., 2004). Probiotic strains, such as Lactobacillus spp., are cultures of live microbes that have a beneficial impact on poultry performance by affecting the intestinal microbiota population (FULLER, 1989; SHOKRYAZDAN et al., 2017; HUANG et al., 2004). However, a limited number of research experiments have investigated the microbiota products or enzymes that influence growth performance. Lactobacillus and Bifidobacterium as potent probiotics contain the BSH enzyme which catalyzes primary bile salt to secondary, and changes the host lipid metabolism. It is also known A. Dibamehr et al.: Plant extracts on Lactobacillus strains in poultry Vet. arhiv 91 (1), 89-99, 2021 91 deconjugation of bile salt in Lactobacillus strains was qualitatively determined by the direct plate assay method with a slight modification (WANG et al., 2012). The MRS agar medium was enriched with bile salts [0.5% w/v, taurocholic acid, Fulka; 0.5% w/v CBSM and CaCl2 (0.37 g/L)] and autoclaved before use. Next, bacterial cells were streaked onto the MRS agar plates and incubated at 37 °C for 5 days anaerobically. MRS agar medium plates were used as the control without supplementing TCA and CBSM. The presence of precipitated unconjugated bile acid around the bacterial colonies (opaque halo) for CBSM, and the formation of opaque granular shiny colonies on the agar indicated BSH activity specific for TCA. Preparation of cell-free extracts (CFEs). BSH activity in the CFEs of each Lactobacillus strain was determined (LIONG and SHAH, 2005; TANAKA et al., 2000; DONG et al., 2012) with some modifications. First, the resting cell suspensions harvested from an overnight culture were centrifuged (10,000×g, 10 min at 4◦C) and washed twice with 0.1 M sodium phosphate buffer containing 10 mM dithiothreitol (Merck; pH 7), and then resuspended in the same buffer to obtain a suspension with an optical absorbance (A600 nm) of 3.0. Next, the cell pellet of each Lactobacillus strain was submitted to an ultrasonic homogenizer (Hielscher, Germany) for 5 min with a 50% duty circle at level 5 using 75% amplitude and constant cooling. Finally, the mixture was centrifuged for 10 min (20,000 g at 4 °C) and the supernatant was stored as a CFE at -20 °C. Colorimetric BSH assay. A two-step standard BSH assay (TANAKA et al., 2000) was performed to determine the BSH activity quantitatively with some modifications by the amount of amino acid released from the conjugated bile salts (TCA and CBSM) using Lactobacillus strains of CFEs. Briefly, 10 μL of CFEs, 10 μL of conjugated bile salts (100 mM TCA and 3% w/v CBSM) were added to 180 μL of reaction buffer (0.1 M sodiumphosphate, with a pH of 6.0). Then, the reaction mixture was conducted at 37 °C for 30 min. Next, 200 μL of 15% (w/v) trichloroacetic acid was immediately added to stop the reaction, and the sample was centrifuged to remove the precipitates. For the second reaction, the supernatant (100 μL) was completely mixed with 1.9 mL of ninhydrin reagent (0.5 mL of 1% (w/v) ninhydrin in 0.5 M sodium-citrate buffer with a pH of 5.5, 0.2 mL of 0.5 M sodium-citrate buffer with a pH of 5.5, and 1.2 mL of glycerol), and the mixture was vortexed and boiled for 14 min. After subsequent cooling for 3 min in tap water, the absorbance at 570