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Alginate in food
From a regulatory point of view, the FDA recognizes alginate as GRAS. In Europe, alginates are enlisted as food additives in category 4 from E400 to E495, e.g. E400 (alginic acid), E401 (sodium alginate) or E402 (potassium alginate). Their use is versatile and they can be employed as thickeners, stabilizers, gel-producer, film-forming agents in milk puddings, syrups, purees, desserts, dry mixes, icing, and so on. The work of Glicksman examines the several applications of alginate in different food systems.180
Gel formation: the egg-box model
Alginic acid is a natural linear polymer commercially extracted from brown seaweed and often used in the food and pharma industries due to its gel and viscosity properties. Various proportions of covalently linked 1,4-β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues compose alginate. The M/G ratio depends on the source of the alginic acid and vary according to geographic location, species, algae age and harvesting season.181 In terms of structure, alginates are irregularly arranged homopolymeric blocks (MM or GG) or heteropolymeric blocks (MG) (Figure 8).182-185 Its ability to form stable hydrogels results from its crosslinking properties once in aqueous solution containing di- or trivalent cations.186 The network is formed by the electrostatic interactions between the oxygenated functions of the G residues and the ion, forming a so-called egg-box model described for the first time by Grant et al. (Figure 9).187 The gel formation and its viscoelastic properties are linked to the affinity of the ion with the G residues as a result of electrostatic forces and steric hindrance. Therefore, the affinity varies with the molecular weight of alginate and its M/G ratio as well as the concentration and type of the ion in solution. For instance, the strength of the binding is greater with Ca2+ than with Mg2+.188 Thus being the major reason why Ca2+ predominates the description on the literature of the mechanism of gelification of alginate.
The mechanism proposed on Figure 9 happens once the disorderly GG-blocks become orderly by the lateral association of two GG-blocks at their junction zones where a Ca2+ is trapped and the solution gelifies.186 This phenomenon, so-called ‘zipper mechanism’, forms stable junctions for a minimum of 8 consecutive G units in the case of Ca2+. The study of Sikorski et al. allowed the confirmation of the phenomenon via structural analysis using X-ray diffraction.
Figure 9: Egg-box model: Gelation of a homopolymeric block (GG) of α-L-guluronic acid with calcium ions.
Additionally, Fang et al. studied the binding process and formation of the egg-box junctions as a function of the concentration of Ca2+ and the different rations of guluronate/mannuronate. They report three sequential steps with the increase of cations in solution: (i) formation of a monocomplex by the interaction of Ca2+ with a single guluronate unit, (ii) pairing of the monocomplexes and formation of the egg-box and, (iii) lateral association of the egg-boxes (Figure 10). This ascertainment is valid when Ca2+ is introduced slowly and homogeneously to the system. Otherwise, if the solution of alginate is extremely concentrated, the gelation is quick, strong and irreversible, leading to heterogeneous gels.190
Alginate chains disintegration
The stability of alginates will strongly depend on temperature, pH, impurities in solution and the packing of the linked units. The glycosidic bonds are susceptible of rupturing in acid and basic conditions. As an example, if the pH gets below 5, the alginate chains get cut inducing a decline of the viscosity of the solution due to the hydrolysis of the bonds resulting in a shorter chain. In acidic and basic conditions, the decrease in the degree of polymerization follows a first order kinetic reaction:
Haug et al. observed the intrinsic viscosity of an alginate solution over the pH range of 4 to 14. Indeed, the inverse of the viscosity is proportional to the number of broken bonds in the polysaccharide chain with a slower degradation between the pH of 5 to 10.191 Erosion is favored therefore below pH 5 and above pH 10. Another possibility for gel disintegrating is with the addition of chelating agents that sequestrates the Ca2+ out of the box. Two examples of molecules capable of dismantling the gel are ethylenediaminetetraacetic acid (EDTA) and sodium citrate.
Silica
The second most abundant element on earth is silicon that occurs as silicates and silica in fresh water, seawater, soils and stones such as basalt or granite. Many living species, e.g. diatoms or eukaryotic algae, utilize silicon for major metabolic processes or even to build their external structures conferring them mechanical protection while allowing material exchange crucial in cellular growth.192 The phenomenon of deposition of oxides on living matter is known as biomineralization and in nature certain bacteria, an example is Bacillus subtilis, can have a mineral crust (e.g. silica or iron-hydroxide depending on the water composition) formed around themselves protecting against predators or even dehydration. It is on the light of such natural phenomenon that silica was chosen as inorganic material to confer mechanical strength and chemical stability to encapsulated alginate beads containing LGG.
Amorphous silica in food
Plants, and more specifically cereals, contain high amounts of silicon in the form of SiO2 whereas food from animal sources contains rather lower levels. The main readily available source of silicon to humans is orthosilicic acid Si(OH)4 that is widely present in drinking water and beer, and rapidly eliminated by the kidneys when ingested.193 Silicates and amorphous silica are approved as food additives by the European Food and Safety Authorities (EFSA) and are enlisted as E551 (amorphous SiO2), E552 (calcium silicate), E553a(i) (magnesium silicate), E553a(ii) (magnesium trisilicate), E553b (talc) and E554 (sodium aluminosilicate). A consumption of amounts up to 1500 mg per day of silica is assumed to be harmless by the regulating organizations. Its main role in dried food formulations relates to the insurance of long-term storages of the product as a water-activity regulator, a so-called anti-caking agent in food additive terms. Silicon is a structural component of connective tissues and it is key for the healthy maintenance of bones, skin, cartilages, and nails.
In academic research, there have been attempts on using silica as encapsulating agent for food purposes. For instance, hybrid core-shell approaches, such as alginate-silica, were explored by Callone et al. for the confinement of Oenococcus oeni or yeast cells with applications in the wine fermentation process.194 Additionally, mesoporous silica loaded with folic acid was elaborated aiming the enrichment of yogurts195 and fruit juices.
Chemical silica structures
Silica or silicon dioxide is a solid mineral with the chemical formula of SiO2. Generally speaking it can be natural or synthetic, crystalline or amorphous. The silicon atoms in quartz for instance are arranged in a highly ordered microscope structure given to it a crystalline property. Figure 11 illustrates the crystalline and amorphous bulk arrangements of some SiO2 units. In both cases, the bulk of silica is composed of SiO4 tetrahedral units that form siloxane rings of different Si–O sizes. The size of the silica rings present on the silica surface generally ranges from flexible 12-membered rings to strained 4-member Si–O rings, and the distribution of such siloxane rings generally depends on the calcination/activation temperature of silica.
At the surface, different kinds of silanols can be found and they can be classified as isolated (non-H-bonded), geminal, vicinal, and interacting (H-bonded) silanols (Figure 12). In the isolated silanol groups (Q3), silicium forms three covalent bonds with oxygen atoms and a fourth bond with a surface hydroxyl. In the vicinal silanol (Q3), two hydroxyl functions are bond to different silicium atoms and they are close enough to interact via hydrogen bonding among them. In the geminal silanol (Q2), two hydroxyl functions are bond to the same silicium. They are actually two close to establish a hydrogen bond. Such group is usually in minority. In the siloxane bridge, four oxygen atoms are covalently linked to one silicium (Q4).198 In the NMR 29Si spectroscopy, the different silicium atoms are designated as Qn where n represents the number of other silicate structures linked to the one in question. The 29Si chemical shifts (-ppm) for silica structures are in the order of: -90 ppm (Q2), -100 ppm (Q3) and -110 ppm (Q4).
In the case of crystalline silica, only three kinds of silanols can be found at the surface: geminal, vicinal and isolated silanols while in the case of amorphous silica, all kinds of silanols are present. This property explains the highest reactivity of amorphous silica surfaces vs crystalline ones. Moreover, depending on the reaction conditions, such as temperature or condensation degree, the OH density at the surface of the material can be tuned between less than 1 to 7 OH/nm2. There are several types of synthetic amorphous silica and their final properties are mostly related to the different fabrication processes the material goes through. Yet the sol-gel route is the most studied specially in the entrapment of living materials.
Silica formation: the sol-gel process
The sol-gel route could be seen as a biomineralization process. In nature, orthosilicic acid Si(OH)4 is present in water and this molecule gives rise to silica that is formed by the condensation of the silanol groups (Si−OH) of two molecules forming an oxo bridge (Si−O−Si) while releasing a water molecule.
The building blocks of a silica network are formed by four oxygen atoms at the corner of a regular tetrahedron holding a silicon ion in the center (SiO4). The Si−O bond formed at this stage is the most stable of all Si−X bonds. Usually the overall reaction can be written as it follows:
However, the synthesis of synthetic silica are usually undertaken with alkoxide precursors Si(OR)4, where R is an alkyl group instead of Si(OH)4. Figure 13 illustrates the formation mechanism of silica and the release of ethanol during the hydrolysis/condensation reactions of tetraethyl orthosilica (TEOS).
In the present study, the two silica alkoxide precursor used are tetramethyl orthosilica (TMOS) and (3-aminopropyl)trimethoxisilane (APTMS) (Figure 14). Silica is mainly formed in three steps: (i) The precursors are hydrolyzed in the presence of water to orthosilicic acid Si(OH)4 while methanol is released, (ii) spontaneous condensation of silanol groups give rise to a colloidal dispersion of silica particles in the solution known as sol, (iii) and the hydrolysis and condensation progresses simultaneously. The growth of the silica particles and their linking into branched chains gives rise to a 3D network, a continuous solid skeleton within the solution, known as gel.
The sol-gel has been extensively studied in cell encapsulation due to its ability to form hybrid silica materials in aqueous solutions under room temperature. Saccharomyces cerevisiae was a pioneering study dealing with cell immobilization in silica gel.203 It was then expanded to other types of cells ranging from bacterial to eukaryotic. Some examples are pancreatic islets for the obtention of bioartificial organs,204 Escherichia Coli for the study of their enzymatic activity under confinement205 and long-term cell viability,206,207 anaerobic sulfate-reducing bacteria,208 luminous recombinant E. Coli as a whole-cell biosensor,209 Serratia marcescens for the production of prodigiosin, a red pigment exhibiting therapeutic properties,210 mammalian cells for transplantation,211 the fungus Stereum hirsutum for remediation of polluted water212 and L. rhamnosus for lactic acid production.
Even though the biomineralization technique has potential for the encapsulation of a variety of cells, it cannot be seen as a generic method since the specificity of each cell must be taken into account beforehand. For instance, S. cerevisiae is an alcohol-tolerant yeast and therefore is able to withstand a certain level of alcohol release during the hydrolysis step of the process. On the other hand, alkoxi-based matrixes can be very often detrimental to the bacteria survival. Some approaches used in those cases are distilling the alcohol out under vacuum before adding the cells214 or using different precursors of silica such as colloidal silica (Ludox) or sodium silicate.215
In the course of this study, sodium silicate, also known as waterglass, was employed as silica precursor as well. Sodium silicate is a generic name for silica compounds containing various proportions of silica and sodium oxides [(SiO2)n : Na2O where n4]. The molar ratio is defined as the ratio between the silica oxide concentration divided by the sodium oxide concentration, i.e. Rm = [SiO2/Na2O]. With the aid of 29Si NMR, Harris et al. demonstrated that the increase of this ratio favors the polymerization of silicon species present in the solutions of sodium silicate. When Rm > 2, colloidal species were observed in the solution whereas if Rm < 2, there are predominately monomeric species.216 The commercialized precursor used in our studies holds a Rm between 2 and 3 giving a composition of about 20 % of monomers and 80 % of oligomers in the departing sodium silicate solution.
When calcium ions are added to a sodium silicate solution, the equilibrium between the monomeric and oligomeric species is destabilized and aggregates start forming. Nieto et al. demonstrated with the aid of 29Si NMR that there is an augmentation of the connectivity degree when an initial sol reaches the gel state. Such increase implies on the formation of siloxane bonds during gelification.217 The industry’s interest on this precursor relates mainly to the fact that it is environmental-friendly and cost-effective when compared to its alkoxi counterparts.
Surfactant-induced porosity
Surfactants were briefly described in the emulsification section. Yet their use as structure-directing agents, i.e. for the obtainment of porous silica materials, was not described previously. First of all, in the case of the encapsulation of cells, the material porosity plays a fundamental role in the bidirectional diffusion of nutriments and metabolites in and out of the system, which allows the maintenance of cell viability under confinement.
Table of contents :
1. INTRODUCTION
1. GENERAL INTRODUCTION
2. ETAT DE L’ART
2. STATE-OF-THE-ART
2.1. Gastrointestinal microbiota
2.2. Gut microbiome and host health
2.3. Probiotics
2.4. Encapsulation techniques
2.5. Encapsulation materials
2.6. Conclusions and perspectives
2.7. References
3. EXPERIMENTAL SECTION
3.1. Chemicals
3.2. Syntheses methods
3.3. Gastrointestinal passage procedure
3.4. LGG assessment in beverages
3.5. Materials characterization
3.6. Bacterial viability
3.7. LGG characterization
3.8. References
4. RESULTAT ET DISCUSSION
4. RESULTS AND DISCUSSION
4.1. Encapsulation of LGG via emulsification
4.2. Encapsulation of LGG via electrospraying
5. CONCLUSIONS GÉNÉRALES ET PERSPECTIVES
5. GENERAL CONCLUSIONS AND PERSPECTIVES
