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Effect of excipients concentration on a lactobacillus rhamnosus GG probiotic powder after spray drying, resorting to a DoE analysis
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Nos últimos anos, os probióticos têm sido extensivamente estudados, em todo o mundo, devido aos seus benefícios e à sua utilização em várias condições clínicas e na saúde humana em geral. Os probióticos são microrganismos vivos não patogénicos, com a capacidade de regenerar o equilíbrio bacteriano em vários sistemas do organismo. É importante explorar processos de fabrico que preservem a viabilidade da bactéria, mantenham a eficácia, qualidade e segurança, e que tenham custos financeiros e ambientais reduzidos. A técnica de secagem por pulverização é económica, rápida e contínua com um conjunto de vantagens quando comparada com outros métodos mais tradicionais, como a liofilização. No entanto, apresenta alguns desafios que têm de ser ultrapassados, como as altas temperaturas de secagem.
Este projeto foca-se na análise do efeito da concentração dos excipientes da formulação, com o objetivo de melhorar o processo de fabrico e obter um produto final otimizado. Foi estudado e analisado o impacto da variação da concentração dos excipientes na sobrevivência das bactérias, na estabilidade do pó, na rapidez do processo, na facilidade de manuseamento e nos custos económicos e ambientais associados. Os parâmetros estudados foram a redução logarítmica, que avalia a sobrevivência bacteriana, a atividade da água, que está ligada à estabilidade do pó e ao crescimento bacteriano, o rendimento, que se relaciona com a rapidez do processo e os custos económicos e ambientais, e o tamanho da partícula, ligado à performance, biodisponibilidade e sobrevivência bacteriana no pó probiótico.
A análise foi realizada em duas vertentes. A viabilidade bacteriana da Lactobacillus rhamnosus GG com a adição de l-leucina foi avaliada com recurso a contagem de unidades formadoras de colónias, valores de redução logarítmica e análise estatística, e a técnica de experimentação fatorial completa foi realizada para aferir o efeito em variar a concentração dos excipientes, trehalose, celulose microcristalina e l-leucina, em quatro parâmetros ou respostas, redução logarítmica (Y1), atividade da água no pó (Y2), rendimento do processo (Y3) and tamanho da partícula no pó (Y4). A trealose tem um efeito protetor e estabilizador contra o stresse causado pela desidratação, ajudando a sobrevivência em condições de secagem extremas, aumentando a viabilidade celular. A celulose microcristalina, sob a forma de pó, pode atuar como um ligante e preencher os espaços vazios na formulação, reforçando-a. A l-leucina pode ajudar na dispersão do pó e na proteção contra a humidade e, em formulações secas por pulverização, pode reduzir o efeito da humidade nos pós. Este último excipiente pode, também, aumentar a aerossolização das partículas por adaptação da sua morfologia superficial e densidade. Todas estas características podem contribuir para um processo de fabrico melhorado e, consequentemente, para um produto final otimizado.
Com a análise da viabilidade bacteriana da L. rhamnosus GG, concluiu-se que as diferenças entre as médias dos valores de redução logarítmica não são significativas, que não é possível associar um maior tamanho de partícula a uma maior taxa de sobrevivência e que a adição de l-leucina na concentração de 1% (m/v) não influenciou, estatisticamente, a sobrevivência da L. rhamnosus GG.
Relativamente à técnica de experimentação fatorial completa, a principal conclusão foi que nenhum dos modelos obtidos se ajusta às respostas, nem nenhum dos modelos obtidos é estatisticamente significativo. No entanto, foi possível aferir que a trealose é o excipiente com maior impacto em atingir os valores pretendidos para as quatro respostas e que a l-leucina acaba por ter um efeito negativo ou não ter nenhum efeito com impacto em muitas das respostas.
In the last few years, probiotics are being widely studied around the world due to their benefits and uses in several clinical conditions and human heath in general. Probiotics are live non-pathogenic micro-organisms with the capacity of restoring microbial balance in many organ systems. It is important to explore manufacturing processes that preserve the viability of the bacteria, maintains the safety, quality and efficacy, and with lower financial and environmental costs. The spray drying technique is an affordable, fast and continuous process with a series of advantages when compared to other traditional drying methods, such as freeze drying. However, spray drying presents a few challenges that must be overcome, such as the high drying temperatures. This project focus on analysing the effect of varying the excipients concentration to improve the manufacturing process and obtain an optimised final product. It was studied and analysed the impact in varying excipients concentrations on results that can reflect aspects such as the bacterial survival, stability of the powder, process speed, easier handling, and financial and sustainable costs. The studied parameters were log-reduction, which evaluates the bacterial survival, water activity that is connect to the powder’s stability and microbial growth, yield that relates to the speed of the process and the financial and sustainable costs, and the particle size, connected with performance, bioavailability and bacterial survival in the probiotic powder. The analysis was performed in two separate parts. The bacterial viability of Lactobacillus rhamnosus GG with the addition of l-leucine was assessed with resource to CFU counting, log-reduction values and a statistical analysis, and a full factorial design analysis was executed to assess the effect in varying the concentrations of the excipients, trehalose, MCC and l-leucine, on four parameters or responses, log-reduction (Y1), water activity of the powder (Y2), process’ yield (Y3) and particle size in the final powder (Y4). Trehalose has a protective and stabilising effect against dehydration stress helping the survival in extreme dried conditions, increasing of the cell viability. MCC in its powder form can act like a binder and filler, reinforcing the formulations. L-leucine can help powder dispersion and moisture protection and, in spray-dried formulations, can reduce the moisture effect on the powders. This last excipient can also increase the aerosolization of the particles by adapting their surface morphology and density. All these characteristics can contribute to a better manufacturing process and, therefore, an optimised final product. With the assessment of bacterial viability, the conclusions taken were that the difference between the average values of the log-reduction was not significant, that it was not possible to associate a bigger particle to a higher survival rate, and the addition of l-leucine, in the concentration of 1% (m/v) did not influence, statistically, the survival of L. rhamnosus GG. Regarding the full factorial design analysis, the main conclusion was that none of the models fit the responses, nor do they have statistical significance. However, it was established that trehalose was the excipient with more impact at reaching the target values for all four responses and that l-leucine ends up having a negative effect or no impactful effect in many of the responses.
In the last few years, probiotics are being widely studied around the world due to their benefits and uses in several clinical conditions and human heath in general. Probiotics are live non-pathogenic micro-organisms with the capacity of restoring microbial balance in many organ systems. It is important to explore manufacturing processes that preserve the viability of the bacteria, maintains the safety, quality and efficacy, and with lower financial and environmental costs. The spray drying technique is an affordable, fast and continuous process with a series of advantages when compared to other traditional drying methods, such as freeze drying. However, spray drying presents a few challenges that must be overcome, such as the high drying temperatures. This project focus on analysing the effect of varying the excipients concentration to improve the manufacturing process and obtain an optimised final product. It was studied and analysed the impact in varying excipients concentrations on results that can reflect aspects such as the bacterial survival, stability of the powder, process speed, easier handling, and financial and sustainable costs. The studied parameters were log-reduction, which evaluates the bacterial survival, water activity that is connect to the powder’s stability and microbial growth, yield that relates to the speed of the process and the financial and sustainable costs, and the particle size, connected with performance, bioavailability and bacterial survival in the probiotic powder. The analysis was performed in two separate parts. The bacterial viability of Lactobacillus rhamnosus GG with the addition of l-leucine was assessed with resource to CFU counting, log-reduction values and a statistical analysis, and a full factorial design analysis was executed to assess the effect in varying the concentrations of the excipients, trehalose, MCC and l-leucine, on four parameters or responses, log-reduction (Y1), water activity of the powder (Y2), process’ yield (Y3) and particle size in the final powder (Y4). Trehalose has a protective and stabilising effect against dehydration stress helping the survival in extreme dried conditions, increasing of the cell viability. MCC in its powder form can act like a binder and filler, reinforcing the formulations. L-leucine can help powder dispersion and moisture protection and, in spray-dried formulations, can reduce the moisture effect on the powders. This last excipient can also increase the aerosolization of the particles by adapting their surface morphology and density. All these characteristics can contribute to a better manufacturing process and, therefore, an optimised final product. With the assessment of bacterial viability, the conclusions taken were that the difference between the average values of the log-reduction was not significant, that it was not possible to associate a bigger particle to a higher survival rate, and the addition of l-leucine, in the concentration of 1% (m/v) did not influence, statistically, the survival of L. rhamnosus GG. Regarding the full factorial design analysis, the main conclusion was that none of the models fit the responses, nor do they have statistical significance. However, it was established that trehalose was the excipient with more impact at reaching the target values for all four responses and that l-leucine ends up having a negative effect or no impactful effect in many of the responses.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2024, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Lactobacillus rhamnosus GG Spray drying Design of experiments Bacterial viability Trehalose l-leucine Mestrado Integrado - 2024