Selection of Probiotic Bacterium Capable of GABA Production for the Application as Feed Supplement in Laying Hens

Abstract

Background and Objectives : Main problems affecting the raising of laying hens in Thailand, especially on the growth and efficiency of egg production are the epidemics and heat stress. Supplementation of viable probiotic cells to poultry diet has been widely accepted as an effective treatment in the reduction of infection and promoting poultry health instead of the use of antibiotics treatment. Nowadays, the antibiotics supplementation in the livestock production is restricted and controlled in many countries including Thailand. In addition, the gamma – aminobutyric acid (GABA) substance plays a significant role in mitigation of heat stress impacts on growth and egg production efficiency of laying hens. Thus, the discovery of GABA - producing probiotic strain, a dual - functional feed supplement, with high specificity to poultry gastrointestinal tract is a promising strategy for the laying hens raising and egg production. The objectives of this research were then to screen the probiotic strains capable of GABA production from the digestive tract of laying hens for the application in feed supplement purposes, especially reduction of the impacts from above mentioned problems and to investigate the survivability of the probiotic cells after the formulation of product powder.

Methodology : Isolation of lactic acid bacteria from the cloaca of laying hens was conducted on de Man Rogosa and Sharpe (MRS) agar. Those isolates were subjected to the preliminary in vitro tests of their probiotic properties. The tests included the investigation of hemolytic activity (alpha -, beta - and gamma - hemolysis) on sheep blood agar, the growth ability under the acid conditions (pH 2.0, 2.5, 3.0 and 3.5) and the presence of bile (0.3 and 1.0% (w/v)), the cell surface hydrophobicity (%CSH) assessed by the auto-aggregation ability of cells after toluene addition, the antibiotic susceptibility on 13 antibiotics interpreted according to the standard protocol of the Clinical and Laboratory Standards Institute (CLSI), and the antimicrobial activity against some enteric pathogens; i.e. Escherichia coli, Salmonella Typhi and Staphylococcus aureus, using the spot on lawn technique. Subsequently, the ability of the selected isolates on GABA production in MRS broth supplemented by 0.5% (w/v) monosodium glutamate was studied. GABA was quantitatively and qualitatively determined by using spectrophotometry method and thin layer chromatography (TLC), respectively. Finally, species identification of the selected isolate by the analysis of 16S rRNA gene nucleotide sequencing and used to query GenBank via BLAST was investigated. Furthermore, probiotic powder was formulated by mixing with rice bran and drying by freeze-drying process for further study of the cell survivability when stored at 4^oC and room temperature for 12 weeks in the sealed aluminum foil bag.       

Main Results : The results showed that 42 isolates of lactic acid bacteria were obtained from the feces samples collected from laying hens cloaca. They were Gram-positive bacteria, non-catalase producing bacteria and capable of acid production. Moreover, it was found that their morphology of cell shapes were coccus and rod (5 cocci- and 37 rod-shape isolates). After the preliminary test of probiotic properties, it was investigated that isolate LH5, a coccus shape bacterium that occurs in pairs and short chains, exhibited the highest potential of probiotic properties when tested under the simulated conditions of poultry digestive tract. It could grow at all tested pH levels (2.0–3.5) and high bile concentration of 1.0% (w/v). Its cell surface hydrophobicity was at the moderate level with the cell surface hydrophobicity value of 57%. The isolate LH5 was susceptible to penicillin, ampicillin, vancomycin, cefoxitin, chloramphenicol, clindamycin, ceftriaxone, ciprofloxacin, cephalothin and rifampicin, while resisted to three antibiotics, i.e. gentamycin, erythromycin and nalidixic acid. This isolate possessed the antimicrobial activity against two pathogenic bacteria, i.e. S. Typhi, a harmful pathogen causing Salmonellosis outbreak in poultry, and S. aureus. The isolate LH5 did not show a hemolytic activity on sheep blood agar (gamma-hemolysis). Subsequently, the isolate LH5 was identified as Enterococcus faecium with the similarity of 100%. It could also produce GABA of 39.05 mg/L and GABA could be obviously detected on silica gel - coated aluminum sheet with high intensity.  In addition, the formulation of probiotic powder was achieved. The survival rate of E. faecium LH5 in probiotic powder was high at 95.39 + 0.07 and 94.15 + 0.19 % after the 12 weeks of storage at 4^oC and room temperature (approximately 30^oC), respectively, and not significantly different (p>0.05) comparing between both temperatures at each week.

Conclusions : Enterococcus faecium LH5 bacterium was a potential GABA producing probiotic. Its cells were stable during the formulation process into a powder form and the storage at room temperature. Therefore, it was a promising probiotic strain for further utilization as the feed additive in laying hens. However, a study on the effect of E. faecium LH5 supplementation on growth performances and the efficiency of egg production under the challenge of either heat stress or pathogenic infection is necessary to be further investigated before applying as a feed supplement.