Recombinant production of a citrus miraculin in the Pichia pastoris expression system for the development of industrial sweeteners

• Grant number: 20/05532-4
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Start date: September 01, 2021
• End date: May 31, 2022
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Daniela Morilha Néo Justino
• Grantee: Daniela Morilha Néo Justino
• Company: DMN Justino Ltda
• CNAE: Fabricação de produtos alimentícios não especificados anteriormente ; Fabricação de refrigerantes e de outras bebidas não-alcoólicas
• City: São Carlos
• Pesquisadores principais: Andrea Soares da Costa Fuentes
• Associated scholarship(s): 21/10846-0 - Recombinant production of a citrus miraculin in a Pichia pastoris expression system for the development of industrial sweeteners, BP.PIPE

Abstract

In recent years there has been a significant increase in the consumption of highly calorie, more specific or sugar foods. With the passage of time or the consumption of sugar in Brazil and in the world, it has been causing and bringing consequences for human health, leading patients to overweight and the development of diseases such as obesity, diabetes and metabolic syndrome. In the search for alternatives that aim to reduce sugar consumption, research related to the development of caloric and non-caloric additives has been carried out with compounds of natural or artificial origin. Among the natural compounds there is a group of proteins that have the property of modifying or flavoring food. These proteins are called flavor modifying proteins (PMS). Thus, there is a growing interest in this class of molecules, more specific in the protein miraculin, extracted from the plant Richardella dulcifica, which has the ability to modify the bitter taste in the sweet taste. However, the cost of extracting the native miraculin protein is quite high, which makes its inclusion in commercial products unfeasible. An alternative in this matter would be the production of a recombinant citrus miraculin, using the expression system Pichia pastoris, with the advantage of the protein being secreted for the induction medium, or that makes the production process simpler and economically more economical. However, it is necessary to verify if the recombinant protein used in Pichia pastoris is presented as original properties of the miraculin Richardella dulcifica native. To this end, evaluate the taste-modifying capacity of citrus miraculin and also evaluate the inclusion of this protein in products such as, for example, juices or other foods in order to define the best commercial vehicle for sugar substitution. For that, experiments of sensory analysis with protein will be carried out and then mass spectrometry tests to carry out tests of inclusion of proteins in foods. In later stages, the cytotoxicity of citrus miraculin in mammalian cell culture will be evaluated, in order to prove its safety, an essential step for inclusion in commercial products. These studies provide important information for future applications of citrus miraculin as a sugar substitute in acidic beverages, providing an alternative use for a natural substance as harmful. (AU)