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Authors
Abstract(s)
A doença celíaca afeta 1% da população, que sofre de inflamação intestinal
desencadeada pela ingestão de glúten, em particular pelo seu elevado conteúdo de péptidos
imunogénicos, onde está incluída a gliadina. (1) O glúten é formado por variadas proteínas
estruturais presentes em alguns cereais. Ele pode ser utilizado para cozer pão e também como
um aditivo alimentar para melhorar as características reológicas, por exemplo, da carne.
Indivíduos com doença celíaca, doença inflamatória intestinal e distúrbios relacionados
com a ingestão de glúten têm dificuldade em evitar o consumo deste componente e manter uma
dieta saudável. Os substitutos do glúten não possuem as mesmas características que o original,
o que afeta o bem-estar social e psicológico dos indivíduos que o devem evitar.
As bactérias ácido-láticas, como o Lactococcus lactis, são microrganismos bem
conhecidos, utilizados como fábricas celulares na indústria alimentar. Também são utilizadas
na indústria farmacêutica para tratar e prevenir doenças – Lactococcus lactis geneticamente
modificado pode expressar e libertar proteínas de interesse para alvos terapêuticos. Além disso,
esta bactéria tem benefícios para a saúde humana, sendo por isso considerada “geralmente
reconhecida como segura”.
O objetivo deste trabalho foi produzir gliadina menos imunogénica, para agir como
substituto ao glúten convencional na indústria alimentar. O gene da gliadina, ao qual foram
adicionadas sequências de restrição, foi amplificado por PCR, para depois ser clonado no
plasmídeo pNZ814, através da restrição e ligação. As bactérias Lactococcus lactis foram
transformadas com o plasmídeo recombinante e as proteínas foram expressas a nível
intracelular com o sistema NICE.
O gel desnaturante de poliacrilamida (SDS-PAGE) e análise por “western blotting”
mostraram que as bactérias transformadas expressaram a gliadina mutada. As proteínas foram
purificadas por cromatografia de afinidade com iões imobilizados (IMAC) e analisadas num
SDS-PAGE, em que a gliadina foi identificada, embora uma parte estivesse ou degradada, ou
formou oligopeptídeos. Também observamos que as bactérias transformadas para expressar a
gliadina mutada cresceram mais rapidamente e secretaram mais proteína, em comparação com
o controlo negativo e o “wild type”.
Neste trabalho foram dados os primeiros passos para a produção de um potencial
substituto do glúten que pode vir a ser utilizado na indústria alimentar, como alternativa aos
produtos sem-glúten convencionais.
Celiac disease affects 1% of the population, and those affected suffer from chronic intestinal inflammation triggered by the consumption of gluten, and its high content in immunogenic peptides, including gliadin. (1) Gluten is made of variable structural proteins present in some cereals, such as wheat. It is used in bread making and as a food additive to enhance rheological properties, for example in meat products. Patients with celiac disease, inflammatory bowel disease and gluten-related neurologic disorders struggle to avoid wheat and maintain a healthy varied diet. Gluten substitutes do not provide the desired qualities that gluten provides, which affects the social and psychological well-being of those who must avoid it. Lactic acid bacteria, such as Lactococcus lactis, are well-known as safe microorganisms used as cell-factories in the food industry. They are also used in the pharmaceutical industry to treat and prevent diseases – genetically engineered Lactococcus lactis can express or deliver desired proteins to therapeutic targets. This bacterium has beneficial properties for human health, which earned it the status of “generally recognized as safe”. The aim of this work was to produce less-immunogenic gliadin as a substitute for conventional gluten in the food industry. The gliadin gene construct was amplified by PCR, and restriction sites were added to allow molecular cloning into plasmid pNZ8148, using ligation and restriction. Lactococcus lactis bacteria were transformed with the recombinant plasmid and proteins were expressed intracellularly using the NICE system. Further analysis with denaturant polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis showed that the transformed bacteria expressed mutated gliadin. Proteins were purified by immobilized metal affinity chromatography (IMAC) and analysed by SDS-PAGE, and gliadin was identified, although some was either degraded or in oligomer form. We also observed that bacteria transformed to express the mutated gliadin grew faster and expressed more protein in comparison to the negative control and the wild-type gliadin. Altogether, in this work an expression system was developed that have the potential to allow the production of a potential substitute for gluten that could be used in the food industry and help people on a gluten-free diet.
Celiac disease affects 1% of the population, and those affected suffer from chronic intestinal inflammation triggered by the consumption of gluten, and its high content in immunogenic peptides, including gliadin. (1) Gluten is made of variable structural proteins present in some cereals, such as wheat. It is used in bread making and as a food additive to enhance rheological properties, for example in meat products. Patients with celiac disease, inflammatory bowel disease and gluten-related neurologic disorders struggle to avoid wheat and maintain a healthy varied diet. Gluten substitutes do not provide the desired qualities that gluten provides, which affects the social and psychological well-being of those who must avoid it. Lactic acid bacteria, such as Lactococcus lactis, are well-known as safe microorganisms used as cell-factories in the food industry. They are also used in the pharmaceutical industry to treat and prevent diseases – genetically engineered Lactococcus lactis can express or deliver desired proteins to therapeutic targets. This bacterium has beneficial properties for human health, which earned it the status of “generally recognized as safe”. The aim of this work was to produce less-immunogenic gliadin as a substitute for conventional gluten in the food industry. The gliadin gene construct was amplified by PCR, and restriction sites were added to allow molecular cloning into plasmid pNZ8148, using ligation and restriction. Lactococcus lactis bacteria were transformed with the recombinant plasmid and proteins were expressed intracellularly using the NICE system. Further analysis with denaturant polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis showed that the transformed bacteria expressed mutated gliadin. Proteins were purified by immobilized metal affinity chromatography (IMAC) and analysed by SDS-PAGE, and gliadin was identified, although some was either degraded or in oligomer form. We also observed that bacteria transformed to express the mutated gliadin grew faster and expressed more protein in comparison to the negative control and the wild-type gliadin. Altogether, in this work an expression system was developed that have the potential to allow the production of a potential substitute for gluten that could be used in the food industry and help people on a gluten-free diet.
Description
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2023, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Lactococcus lactis Recombinant protein Gliadin Gluten-free Mestrado Integrado - 2023