Microencapsulation of <i>S. cerevisiae</i> to improve the thermal inactivation kinetics during the production of broiler chicken feed pellets
Creators: name: Turner, John
Publisher: name: Loughborough University
Year: 2024
Subjects: classificationCode: 400499
subject: Chemical engineering not elsewhere classified

description: Anti-microbial resistance is a significant threat to global health and food security and is estimated to cause 700,000 deaths per year. (Ruckert et al., 2020) Antibiotics have been heavily restricted in agriculture which has created an opportunity for the use of probiotics as growth promoters. Animal feed pellets are produced by high temperature extrusion that reduces the viability of probiotics (Amerah et al., 2013). Microencapsulation could allow for the inclusion of live probiotics in animal feed pellets by improving the thermal inactivation kinetics. <i>Saccharomyces cerevisiae</i> was selected to encapsulate because it is already used on a global scale and is recognised as safe by the key regulatory bodies. However, the viability of <i>S. cerevisiae</i> was reported to decrease rapidly at temperatures above 50<sup>o</sup>C (López-Malo, Guerrero and Alzamora, 1999). The temperature of animal feed pelleting systems was reported to reach over 90<sup>o</sup>C (Ebbing et al., 2022). Coating the animal feed pellets with probiotics after cooling was called top dressing and reportedly resulted in inconsistent coverage and poor storage stability (Aguirre-Guzmán, Ricque-Marie and Cruz-Suárez, 2002). Time lost at the feed mill for the top dressing process was costly. The aim from the sponsor, AB Vista was to include viable <i>S. cerevisiae</i> in a microencapsulated formulation that would be mixed with bulk feed ingredients and pelleted, overcoming problems with top dressing and delivering probiotics on a wider scale. There is a clear need for a system to improve the thermal inactivation kinetics of probiotics so that they can be included in animal feed pellets. Storage stability of microencapsulated <i>S. cerevisiae</i> should be investigated to allow for the formulation to be pelleted, stored for long periods of time and transported before being digested by the farm animal. The gastrointestinal stability of microencapsulated <i>S. cerevisiae</i> should be investigated to deliver viable <i>S. cerevisiae</i> to the small intestine of the host animal.Various encapsulation materials and methods were evaluated in the literature review. The reagents and encapsulation system were chosen because were commercially available and easily scalable. The most suitable encapsulation method was chosen to be extrusion using a Buchi B390 encapsulator system. Beads and core shell capsules were produced by extruding a solution into a laminar jet that broke into monodispersed droplets and fell into a hardening bath. Various encapsulation materials were compared, the most suitable encapsulation material was chosen to be alginate because of its benign cross-linking mechanism, was non-toxic with <i>S. cerevisiae</i> and was widely reported for use in microencapsulation (Santos and Machado, 2021).A high titre of <i>S. cerevisiae</i> was fermented using a 5L bioreactor system and concentrated to 5x10<sup>9</sup> CFU/mL for use in formulations. <i>S. cerevisiae</i> ascospores were sporulated from vegetative <i>S. cerevisiae</i> and concentrated to 2x10<sup>8</sup> CFU/mL. A Buchi B390 encapsulator was used to produce 8 encapsulated formulations and 2 non-encapsulated formulations. The two main assays were thermal inactivation kinetics and gastrointestinal stability. Each formulation was characterised by scanning election microscopy and digital scanning calorimetry. Significant developments for the improvement of <i>S. cerevisiae</i> thermal inactivation kinetics were the sporulation of ascospores from vegetative <i>S. cerevisiae</i>, the production of hydrogenated palm kernel oil (HPKO) beads using a cold water bath at close distance and analysis of the inactivation behaviour with the Weibull model.Both vegetative <i>S. cerevisiae</i> and ascospores were encapsulated in alginate beads. Alginate beads had an average diameter of ~900μm, a titre of 1x10<sup>8</sup> CFU/g vegetative <i>S. cerevisiae</i> and an encapsulation efficiency of 90%. Ascospores in alginate beads had an average diameter of ~800μm, a titre of 8x10<sup>6</sup> CFU/g S. cerevisiae ascospores and an encapsulation efficiency of 82%.The thermal inactivation kinetics were analysed using the Weibull model. This produced the scale and shape parameters, decimal reduction time (<b>D</b> value) and thermal resistance constant (<b>Z</b> value). Non-encapsulated <i>S. cerevisiae</i> ascospores had better heat stability than non-encapsulated vegetative <i>S. cerevisiae</i> with a thermal resistance constant of 9.8<sup>o</sup>C compared to 8.8<sup>o</sup>C. However, encapsulation of ascospores in alginate beads actually decreased the thermal resistance constant to 7.8<sup>o</sup>C. Vegetative <i>S. cerevisiae</i> in alginate beads had a thermal resistance constant of 9.9<sup>o</sup>C. Encapsulation of vegetative <i>S. cerevisiae</i> in alginate beads allowed CFUs to be detected at 65<sup>o</sup>C, where there were none for non-encapsulated <i>S. cerevisiae</i>.Vegetative <i>S. cerevisiae</i> were emulsified in a neutral oil, Miglyol 840. The emulsion actually performed worse than non-encapsulated <i>S. cerevisiae</i> with the lowest scale value at 65<sup>o</sup>C and thermal resistance constant of any formulation in the thesis. The emulsion was encapsulated in core shell capsules with an alginate shell. The core shell capsules performed well at 60<sup>o</sup>C with scale and shape values higher than non-encapsulated <i>S. cerevisiae</i>, but the benefits were lost at 65<sup>o</sup>C which brought down the thermal resistance constant to 8oC.<i>S. cerevisiae</i> ascospores were emulsified in molten HPKO and extruded into solid beads using the Buchi B390 heated nozzle system. The beads had an average diameter of ~700μm and a titre of 3x10<sup>6</sup> CFU/g. The thermal resistance constant for ascospores in HPKO beads was 10.1<sup>o</sup>C. This was higher than for the alginate based formulations and similar to non-encapsulated ascospores and vegetative <i>S. cerevisiae</i> in alginate beads. However, these formulations did not retain CFUs at the target temperature of 70<sup>o</sup>C, unlike the HPKO beads and core shell capsules. This made ascospores in HPKO beads the preferred formulation for pilot scale pelleting trials.HPKO core shell capsules were produced using the Buchi B390 concentric heated nozzle system. They consisted of a 100% HPKO shell and a core of 70% HPKO 25% <i>S. cerevisiae</i> ascospores and 5% PGPR. HPKO core shell capsules had an average diameter of ~450μm and a titre of 6x10<sup>5</sup> CFU/g. The thermal resistance constant was 11.4<sup>o</sup>C, the highest of any formulation. However, this formulation was held back by the lower initial titre due to the higher content of HPKO on a per gram basis.This thesis provides a blueprint for the improvement of thermal inactivation kinetics and the gastrointestinal stability of probiotics. The principles of using a hydrophobic barrier and a phase change material were successful in improving thermal resistance, compared to non-encapsulated <i>S. cerevisiae</i>. The target for thermal resistance was to retain a titre of 1x10<sup>6</sup> CFU/g after 120s at 90<sup>o</sup>C. However, no CFUs were detected above 70<sup>o</sup>C and further work should aim to achieve that.Future work should aim to develop an assay for shear resistance once the importance of particle diameter is understood. Observations suggested that the core shell capsules were especially soft with their liquid emulsion core. An assay that physically tested the shear resistance of the formulations could direct the investigation in a more promising direction. The most accurate test for these formulations is the industrial pelleting process and pilot tests are ongoing with the sponsor, AB Vista. These results may be interesting because they are industrially relevant. Furthermore, storage stability tests could have been accelerated by incubating at higher temperatures to increase the rate of decay.