Production and stability of a multi-strain Bacillus based probiotic product for commercial use in poultry.

Probiotics can be effective alternatives to the prophylactic use of antibiotic growth promoters (AGPs) in response to industry and consumer concerns around their use in poultry. Studies on the suitability of Bacillus probiotics are emerging and showing benefits, but information on the production technology is limited. We developed the production process for a novel probiotic product previously shown to be effective in field trials. All strains were cultivated to a spore concentration exceeding 1 × 1010 CFU. mL-1. The spores of each strain were harvested, processed into a powder intermediate and formulated into an end product with 100 % recoveries and a shelf life stability >1 year. The probiotic was shown to be incorporated into broiler feed exceeding the desired concentration of 1 × 106 CFU. g-1. Using efficient process technology and lower cost materials, this study presents a commercially relevant case for the potential adoption of probiotic products by the poultry industry.

Isolation, selection and evaluation of Bacillus spp. as potential multi-mode probiotics for poultry.

Bacillus based probiotics are becoming relevant as alternatives to antibiotics used in poultry production and in other animal husbandry. This study describes the isolation of 48 Bacillus spp. candidates, from chickens and chicken environments, for use as potential probiotics in poultry production. These isolates, plus a further 18, were tested in a comprehensive in vitro screening regime that was specifically designed to select the best isolates that satisfied multiple modes of action desirable for commercial poultry probiotics. This screening programme involved the evaluation of the ability of the isolates to survive and grow in the limiting conditions of the chicken gastrointestinal tract. Only 11 of the isolates fulfilled these criteria; hence, they were further evaluated for the ability to adhere to epithelial cells, produce extracellular enzymes, and to demonstrate antagonistic activity against selected pathogens of significant importance in poultry production. Of these, a total of 6 isolates were selected, due to their all-round probiotic capability. Identification by 16S RNA sequencing confirmed these isolates as B. subtilis and B. velezensis, identities which are generally regarded as safe. The Bacillus isolates reported in our study exhibit strong all-inclusive probiotic effects and can potentially be formulated as a probiotic preparation for poultry production.

A novel Bacillus based multi-strain probiotic improves growth performance and intestinal properties of Clostridium perfringens challenged broilers.

There is a necessity for the implementation of in-feed probiotics in the poultry production industry, following strict regulations around the use of antibiotic growth promoters (AGP). Bacillus spp. are becoming an attractive alternative because of their functionality and stability. This study aims to evaluate the effect of a novel multi-strain Bacillus based probiotic on growth performance and gut health in male Ross 308 broiler chickens challenged with Clostridium perfringens Type A. Broilers on a 4 phase feeding program were fed diets containing either a standard metabolizable energy (ME) (100%) or a reduced ME (98%) level. The test probiotic was compared to an un-supplemented negative control and a commercial benchmark product as positive control over a 35 D feeding trial, using a 2 × 3 factorial experimental design. Chicks were inoculated with a once-off dose of C. perfringens on day 14. Growth performance was measured weekly to calculate body weight (BW), feed intake (FI) and feed conversion ratio (FCR). Villi histomorphology, gut lesions, and liver weight were assessed at day 35. Broilers fed the reduced ME diet with the test probiotic achieved higher final BWs (P = 0.037) and FCR (P = 0.014) than the negative control. Broilers fed the standard ME diet with the test probiotic showed improved (P = 0.001) FCR than the negative control from day 21 onwards. Increased duodenal villi height (P = 0.012) and villi height to crypt depth ratio in the duodenum (P < 0.0001) and jejunum (P = 0.0004) were observed in broilers fed the reduced ME diet containing the test probiotic. Additionally, the test probiotic resulted in significantly reduced relative liver weights in both ME groups. Consequently, the results suggest that the novel multi-strain Bacillus based probiotic enhanced broiler performance and improved gut health and is thus attractive as an alternative to AGP's in broiler production.

Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris.

A gene encoding a lipolytic enzyme amplified from the alkaliphilic bacterium Bacillus halodurans LBB2 was cloned into the pPICZalphaB vector and integrated into the genome of the protease deficient yeast strain Pichia pastoris SMD1168H. This previously undescribed enzyme was produced in active form, and cloning in frame with the Saccharomyces cerevisiae secretion signal (alpha-factor) enabled extracellular accumulation of correctly processed enzyme, with an apparent molecular mass of 30 kDa. In shake-flask cultivations, very low production levels were obtained, but these were significantly improved by use of a "batch-induced" cultivation technique which allowed a maximum enzyme activity of 14,000 U/l using p-nitrophenyl butyrate (C-4) as a substrate and a final extracellular lipolytic enzyme concentration of approximately 0.2 g/l. Partial characterization of the produced enzyme (at pH 9) revealed a preference for the short-chain ester (C-4) and significant but lower activity towards medium (C5-C6) and long (C16 and C18) fatty acid chain-length esters. In addition, the enzyme exhibited true lipase activity (7,300 U/l) using olive oil as substrate and significant levels of phospholipase activity (6,400 U/l) by use of a phosphatidylcholine substrate, but no lysophospholipase activity was detected using a lysophosphatidylcholine substrate.

Effect of postinduction nutrient feed composition and use of lactose as inducer during production of thermostable xylanase in Escherichia coli glucose-limited fed-batch cultivations.

Escherichia coli is a microorganism routinely used in the production of heterologous proteins. The overexpression of a xylanase (Xyn 10 A Delta NC), which originated from the thermophile Rhodothermus marinus cloned under the control of the strong T7/lac promoter in a defined medium (mAT) using a substrate-limited feed strategy, was however shown to impose a significant metabolic burden on host cells. This resulted in a decreased cell growth rate and ultimately also a decreased target protein production. The investigation hence centers on the effect of some selected nutrient feed additives (amino acid [Cys] or TCA-intermediates [citrate, succinate, malate]) used to relieve the metabolic burden imposed during the feeding and postinduction phases of these glucose-limited fed-batch cultivations. The use of either succinic acid or malic acid as feed-additives resulted in an increase in production of approximately 40% of the heterologous thermostable xylanase. Furthermore, use of lactose as an alternative inducer of the T7/lac promoter was also proven to be a suitable strategy that significantly prolonged the heterologous protein production phase as compared with induction using isopropyl beta-D-thiogalactopyranoside (IPTG).

The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus.

A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3gl(-1) xylanase with a maximum activity of 3130Ul(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T(m)) was 78 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris.