University of Leeds (UoL):
BMG were characterized by analysing the diffusion of fluorescein through them by UV and by cryo-SEM and SEM.
Prawns were bought frozen and the meat prawn was donated to Saint George Crypt, a charity in Leeds that offers meal to homelessness. Chitin was isolated and deacetylated to yield very low degree of acetylation (DA) and high molecular weight (MW) CS. The reacetylation (RAc) of the CS obtained was attempted in the UoL although there was no time for its characterization prior moving to University of Murcia (UM).
Only one squid pen (SP) supplier company was found worldwide. The SP transport through the border was not allowed due to Brexit rules and SP got spoiled. As a contingency strategy, a small amount of chitin leftover obtained from SP in a previous project in which the candidate had participated in was used to produce high MW and low DA CS.
All the research material was sent through the border by a courier. Due to the Brexit restrictions, the material was kept in the border for one month before being delivered.
University of Murcia
RMN analyses revealed that RAc was unsuccessful. Despite applying the protocols reported previously in literature, no appropriate DA was obtained. Not until changing the CS solubilisation conditions, were the CS successfully reacetylated. The prawn CS obtained in UoL was depolymerized to two different degrees prior of being reacetylated. Finally, three different MW CS with four DA each were prepared. A new methodology was developed for the characterization of the CS MW. Not until the end of the period of the project were the CS produced. As a contingency strategy, commercially available CS that show different MW, DA and origins were used. Namely: Low MW and Medium MW CSs from Sigma Aldrich, CS HMC 70/200, 80/200 and 90/200 from Heppe Medical CS, Ultra Low MW from Glentham Life Sciences, CS from Aspergillus niger and mushroom from Chibio Ltd.
The biocompatibility of mucin type III from Sigma Aldrich, CS and BMG were analysed by MTT on SAF-1 and SAB-1 cell lines obtained from Sparus aurata. The reactive oxygen species production, the cell viability and the effects on the mitochondria were analysed by Seahorse technology from Agilent. The presence of cells within BMG after a week of incubation was qualitatively analysed by confocal microscopy.
The authorisation for in vivo experiments using S. aurata specimens was obtained from the authorities. This part of the project will be carried out as soon as possible.
University of Zaragoza
The interaction between mucin and CS by Dynamic Light Scattering allowed to determine the number of charges in mucin. This data was used for performing the Langmuir films (Lf) at mucin to CS ratio of equal number of charges. The mucin to CS interaction were analysed by Lf of the mixtures and analysing the surface pressure and potential. The changes in the surface of the Lf were analysed by Brewster Angle Microscope. Langmuir Blodgett films were prepared on mica and were analysed by Atomic Force Microscopy. Finally, mucin Lf was prepared at a specific surface pressure and then CS were added in the subphase. The position of the barriers to keep the surface pressure was recorded.
The results obtained in this work has been published in Fish and Shellfish Immunology (2022) 130:1-8. Doi: 10.1016/j.fsi.2022.08.030
The results of this project has been communicated in several congresses and conferences, such as 16th International Meeting on Mucins in Health and Disease and 2nd International Symposium Mucosal Health in Aquaculture, among others.
Also this project has been disseminated in several events such as 6th Virtual International Conference on Education, Innovation and ICT, EDUNOVATIC 2021, IV Science and Technology Fair of Cieza, Marie Sklodowska-Curie actions day MSCA 2022 from the University of Murcia, 1st International Forum on Blue Economy and Growth, European Mednight 2022, Science and Technology Week (SeCyt’22), 4th World Symposium on Sustainability Science and Research, among others. Also the webpage www.mariachit.com has been created.
