Encapsulation of probiotics envisaged for folate production in human intestine environment

Malnutrition is a problem that still affects one in three Human Beings (International Food Policy Reasearch Institute, 2015). There are different reasons that can explain this problem, although the lack of some micronutrients, such as folate, can be associated with malnutrition in pregnant women, children, elderly people and in people consuming a limited diet (WHO, 2012; Who, 2014a, 2014b). This work is thus focused on the construction and characterization of a coated microcapsule system capable to be a continuous folate producer, once into the intestine. This system is mainly constituted by alginate capsules, coated with different materials through layer-bylayer assembly (LbL) and smaller than 100 µm. The coated microcapsules are meant to: a) host probiotic bacteria; b) pass as intact as possible through the gastrointestinal system; c) adhere to the intestine mucus layer; and d) exchange nutrients (probiotic activation) and products (mostly folates) in the intestine, by changing the porosity of a membrane induced by pH, whilst retaining probiotics. With this, we believe that: 1) the residence time of probiotics in the intestine will increase; 2) a possible inflammatory response, due to direct contact with new bacteria, will be avoided or at least reduced; and 3) natural folate will be directly produced and assimilated (increased bioavailability comparing with supplementation with synthetic forms such as folic acid). To construct a microcapsule smaller than 100 µm (to avoid the modification of sensorial effects on the food where it might be incorporated), extrusion and emulsification techniques were tested, but just the last technique was able to produce capsules smaller than 100 µm and capable to encapsulate probiotics. Different alginates were tested for probiotics protection and it was concluded that on the one hand alginate CR8223 would be fit as a more permeable microcapsule core, and on the other hand alginate CR8133 would be more suitable for an increased protection (less permeability), as a second coating. Furthermore, the design of the first and third coating layers was planned to: limit the continuous swelling of the alginate microcapsules that could lead to their total destruction, using Ɛ-PLL (2nd coating); resist to acidic media, to protect probiotics and adhere to epithelial cells in order to increase the residence time of the coated microcapsule, using chitosan (3rd coating). In order to predict the interaction of these materials, turbidity tests were performed and showed the existence of interactions between the polymers in a range of pH values that would be interesting for the work (pH = 2 and 7, i.e. stomach and intestine). After the coated microcapsule construction, these tests were complemented by FTIR and confocal imaging aiming at proving the adhesion of the several coatings. These tests showed the consecutive adhesion of each material, proving the establishment of interactions between them. The alginate|Ɛ-poly-Llysine| alginate|chitosan microcapsules (APACM) was demonstrated to be stable during in vitro gastrointestinal tests, as shown by confocal imaging. The capsules were also subjected to in vitro gastrointestinal tests, which showed that the APACM had the capacity to protect Lactococcus lactis cremoris (LLC) when in contact with acidic media. In fact, the free LLC cells just survived during 90 min, having a total loss of viability after that. Once encapsulated, however, there was a decrease of viability during the first hour but 6 Log CFU of LLC cells were kept viable during the rest of the experiment. The characterization of the different coated microcapsules’ sizes, alginate|Ɛ-poly-Llysine microcapsules (APM), alginate|Ɛ-poly-L-lysine|alginate microcapsules (APAM) and APACM, during contact with KCl-HCl and PBS solutions showed that they had an average diameter of 20 µm (app.) and 40 µm (app.) in each medium, respectively. These results proved that there was a swelling degree of approximately 2-fold, in APACM between an acidic and a neutral medium, which might influence the porosity of the system, creating a more permeable structure at neutral pH. Regarding the release kinetics results it was proved that the differently coated microcapsules behaviour was adequately described by a LSM model and the mechanisms involved in folic acid release were composed by Fick diffusion ( MF) and two or more relaxation processes ( MR). Free LLC folate production showed that the highest amount achieved by cumulative quantification was at 10 h, reaching 95.25 ± 26 µg.L-1. In order to validate the nutrients exchange capacity of APACM, these capsules containing LLC were tested, although there was no sufficient production of any compound (folate or lactic acid) to validate the system, possibly due to the lack of viability of the cells. Free L. plantarum was also tested and proved to be able to produce high amounts of lactic acid. The test to validate the APACM’s nutrients exchange capacity when containing L. plantarum, showed a production of 2 g.L-1 of lactic acid by encapsulated L. plantarum after 20 h. These results proved that the APACM was able to allow the entrance of nutrients, L. plantarum activation and the release of lactic acid. The study of APACM adhesion to the cells showed an adhesion of 38 % to HT-29 cells and 33 % to Caco2 cells. In conclusion, APACM characterization proved that all designed functions were working effectively and because of that we believe that this system might be a significant step forward to deal with folate malnutrition, e.g. being used to substitute the normal supplementation of foods with folic acid.

Name thesis:degree_name

PhD in Chemical and Biological Engineering

Grantor

Universidade do Minho

Year dc:date.issued

2016

Author dc:creator

• Ramos, Philippe Emmanuel Cruz

Advisors dc:contributor.advisor

• Vicente, A. A.
• Teixeira, J. A.

Statement dc:rights

• restrictedAccess

Language dc:language.iso

por

Handle dc:identifier.uri

https://hdl.handle.net/1822/42561

OAI record oai:identifier

http://repositorium.sdum.uminho.pt/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arepositorium.uminho.pt%3A1822%2F42561