Encapsulation Of Probiotic Bacteria Thesis Statements

Methods allowing the stable and controlled delivery of bacteriophages are of great value in phage therapy. One such method is encapsulation, which in farm animals protects orally administered bacteriophages from the harsh environment of the stomach and facilitates their retention during passage through the intestinal tract to ensure a successful therapeutic effect24,25,26. By maintaining bacteriophage stability, micro- or nano-encapsulation enables not only their oral administration through feed or water but also their administration in other forms, such as inhalation, thereby assuring an adequate dose of the therapeutic phage. Furthermore, encapsulation can overcome other problems related to the application of bacteriophages in food industry processes. The materials used in bacteriophage encapsulation have been examined in several studies12,13 and include alginate, alone or in combination with other materials8,10,16,17,18. However, few studies have described the in vivo use of alginate-encapsulated bacteriophages21,22,27. Thus, our study is the first to test a cocktail of three alginate/CaCO3-encapsulated virulent bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87)11,23 as oral therapy in Salmonella-infected poultry under farm-like conditions. Phages prepared according to this method were shown to be effective in protecting broilers against infection for up to 15 days.

The encapsulation methodology described herein allows the encapsulation of bacteriophages with different morphologies, without jeopardizing infectivity. The encapsulation efficiency values obtained in this study were ~99%, similar to the percentages reported by other authors8,10,16,17,25. Moreover, the alginate/CaCO3 encapsulated bacteriophages showed excellent stability when stored at 4 °C for 6 months, with minor losses determined only for UAB_Phi20 and UAB_Phi78. Also, the encapsulated phages were stable at room temperature at least for two weeks, period of time that is sufficient for the administration to animals in drinking water.

Another promising feature of the alginate/CaCO3 microcapsules was their size (124–149 μm), which was almost ten times smaller than other types of capsules described in the literature8,10,16,17,25 and facilitated their potential commercial applications. All this is the result of various systematic studies aimed at optimizing the alginate concentration (1.8%) and the posterior curing time of the capsules in the bath of CaCl2 (90 min).

Phages orally administered to broilers must withstand the low pH of the stomach contents of the chickens, which is typically in the range of 2.1–3.628,29. Under acidic conditions, the alginate/CaCO3-encapsulated phages proved to be much more stable than their non-encapsulated counterparts (Fig. 2) (p < 0.05), resulting in titre losses after 60 min of incubation in SGF that were around 5 times lower for phages UAB_Phi78 and UAB_Phi87. There was no loss of encapsulated UAB_Phi20. It is reported that the incorporation of CaCO3 slows the gelation rate of alginate capsules, whereas CO3 ions dissociated from CaCO3 diffuse into the medium and slightly increase the pH as a role of antacid, thus protecting the bacteriophages10,19. Our results are in accordance with those obtained by10, who also reported a good protection effect on the bacteriophage K once encapsulated in a mixture of alginate/CaCO3 capsules of a diameter of ~900 μm. However, in our case, this protection effect was achieved even lowering the size of the capsules down to ~150 μm of size.

Another important feature of alginate/CaCO3-encapsulated bacteriophages is their release in the animal intestine, where the host pathogen is located. In this study, the three phages exhibited slightly different in vitro release kinetics. Thus, whereas almost a complete release of bacteriophage UAB_Phi87 was observed after 20 min of incubation in SIF, UAB_Phi20 and UAB_Phi78 were released more slowly, being this release completed after 40 min (Fig. 3). A potential explanation of the faster release of UAB_Phi87 is that this phage has a larger size than the other two phages, which could provoke the formation of alginate capsules with a lower reticular or cross-linked structure and therefore, a faster release of the encapsulated phages. In addition, a comparison of our release results with those of other authors is difficult, since the size and composition of the microcapsules, the composition of the SIF and the incubation conditions (pH and temperature) differed8,10,16,18.

However, bacteriophage release kinetics will undoubtedly differ in vivo. Currently, whether phages adhere to the intestinal epithelium30 or their presence becomes insignificant in the absence of the bacterial host is unclear11,31. The mucoadhesive properties of alginate32 could prolong the presence and the effect of bacteriophages used in oral phage therapy. Our in vivo results of residence time demonstrate that alginate/CaCO3 encapsulation enables the significant intestinal retention of the bacteriophages even in the absence of host. Thus, bacteriophages were detected in 71.4% of the chickens 72 h after oral administration of a single dose of the cocktail of encapsulated bacteriophages compared to 9.5% of the chickens treated with the non-encapsulated phages (p < 0.001). The percentage obtained with the alginate/CaCO3 capsules was better than obtained in a previous in vivo study of liposome-encapsulated bacteriophages24, perhaps due to the higher encapsulation efficiency achieved with the former.

The cocktail composed of the three alginate/CaCO3-encapsulated bacteriophages demonstrated long-term efficacy when used in commercial broilers chickens infected with Salmonella, mimicking real farm conditions. Colonization by Salmonella was effectively reduced by administering a phage cocktail composed of alginate/CaCO3-encapsulated bacteriophages to the poultry 1 day prior to infection with the bacterium and for an additional 7 days during treatment. Moreover, the protective effect was significantly maintained for 1 week after treatment was stopped (on day 15-post infection). Differently, the non-encapsulated bacteriophages quickly loosed their effect once the treatment was stopped. During the first 6 days post-infection, the encapsulated and non-encapsulated cocktails were of similar efficacies, with maximum reductions in Salmonella counts of 3.1 and 2.8 log10, respectively. Only on the first day of treatment was the non-encapsulate cocktail significantly more effective (reduction of 2.9 vs. 1.3 log10; p < 0.05; Table 2). This may have reflected the additional time required for the encapsulated phages to accumulate to an effective therapeutic concentration following their release. Then, it is likely that the phage release in vivo would be slower than in vitro SIF studies. However, the effect of the non-encapsulated cocktail was abolished 1 day after the cessation of treatment (day 7 post-infection) whereas the encapsulated cocktail maintained its effectiveness until the end of the experiment.

This is the first study to report the use of alginate/CaCO3-encapsulated bacteriophages as an in vivo oral therapy against Salmonella infections in poultry. While our results are similar to those achieved with liposome encapsulation24, preparation of the alginate/CaCO3 capsules is simpler and provides much higher encapsulation rates. Although treatment with the alginate/CaCO3 cocktail did not remove Salmonella totally, the concentration of Salmonella used in the experimental poultry infection was very high (~6 log10/g of caecum). Further studies should seek to ascertain the threshold Salmonella concentration in poultry farming and if the encapsulated cocktail could eliminate Salmonella from the chickens completely in those conditions.

Another important aspect in the success of phage therapy is the relationship between the phages and their bacterial hosts. According to the literature on bacteriophage-bacterial dynamics in vitro, the number of phage increases only when the cell density is sufficient, so that the probability of an encounter between the bacteriophages and the bacteria and the subsequent infection of the latter exceeds the probability of phage death33. Therefore, the success of phage therapy is largely determined by the relationship between the concentration of the bacteriophages and that of their bacterial host34. However, the in vivo dynamics are presumably much more complex because multiple external factors (e.g., rapid clearance of the bacteriophages by passive/active host immunity, spatial refuges, and intestinal mucous) influence treatment success35.

In our in vivo study, the dynamics of the non-encapsulated and encapsulated bacteriophages in controlling Salmonella differed which agree with a previous study performed by us with liposome-encapsulated bacteriophages24. Thus, with the daily administration of non-encapsulated bacteriophages, their uptake together with the new phage progeny produced in the intestinal tract led to a marked decrease in the Salmonella concentration (~50%) 1 day post-infection. Thereafter, until almost day 8 post-infection, the Salmonella concentration increased slightly but with significant therapeutic effect. Once the phage uptake was stopped, the equilibrium between phages and bacteria was disrupted and the Salmonella concentration increased significantly with respect to day 1 post-infection (days 8 and 10, p < 0.05; day 15 post-infection, p < 0.001; Table 2). The requirement for continuous administration of bacteriophages along the time to achieve a low population of Samonella has also been suggested by other authors11,36. Several factors as partial emergence of bacteriophage-resistant Salmonella, bacterial phenotypic changes, physical refuges or slow growth rate of bacterial cells could explain this fact35,36. By contrast, when the encapsulated bacteriophage cocktail was administered, the Salmonella concentration decreased gradually during all the experiment, regardless of whether treatment was ongoing or had stopped. Therefore, the encapsulation of bacteriophages abolished the need for a continuous treatment to achieve a low bacterial concentration. In this case, the mucoadhesiveness of the capsules and the release kinetics of the bacteriophages from them must be the most important features for this effect. It is remarkable that when the bacteriophage uptake was stopped the bacteriophage concentration remained nearly constant (Table 3) in both treatments, but the Salmonella concentration in the gut was higher in the non-encapsulated bacteriophage treatment than in encapsulated one. At this respect, it has been proposed that there is a threshold density of bacteria that must be present in order for the bacteriophage concentration to increase33. Further works are needed to identify the in vivo mechanism(s) underlying this fact.

In summary, this study demonstrated the utility of a simple, efficient, and inexpensive encapsulation method that can be used with bacteriophages of different morphologies. The small size of the resulting microcapsules (124–149 μm) enables their use in diverse applications in phage therapy. Moreover, alginate/CaCO3 encapsulation confers excellent protection against the deleterious effects of gastric juice and promotes greater intestinal retention of the bacteriophages. The results presented here, together with those from our previous investigation of liposome-encapsulated bacteriophages24, show that encapsulation is important for a prolonged and successful oral phage therapy in commercial broilers infected with Salmonella.

Abbreviations

PET

probiotic encapsulation technology

M

mannuronic acid

G

guluronic acid

FDA

food and drug administration

FAO

food and agricultural organization

WHO

world health organization

CAP

cellulose acetate phthalate

ASM

american society of microbiology

SDS-PAGE

sodium dodecyl sulphate polyacrylamide gel electrophoresis

FTIR-ATR

fourier transformer infra red-attenuated total reflectance

SEM

scanning electron microscope

TEM

transmission electron microscope

1. Introduction

Probiotic survival in products is affected by a range of factors including pH, post-acidification during products fermentation, hydrogen peroxide production and storage temperatures [1]. Providing probiotic living cells with a physical barrier against adverse conditions is an approach currently receiving considerable interest [2].

Probiotic encapsulation technology (PET) is an exciting field of biopharmacy that has emerged and developed rapidly in the past decade. Based on this technology, a wide range of microorganisms have been immobilized within semipermeable and biocompatible materials that modulate the delivery of cells. The terms immobilization, entrapment and encapsulation have been used interchangeably in most reported literature [3]. While encapsulation is the process of forming a continuous coating around an inner matrix that is wholly contained within the capsule wall as a core of encapsulated material, immobilisation refers to the trapping of material within or throughout a matrix [3]. Encapsulation tends to stabilize cells, potentially enhancing their viability and stability during production, storage and handling. An immobilized environment also confers additional protection to probiotic cells during rehydration. As the technique of immobilization or entrapment became refined, the cell immobilization technology has evolved into cell encapsulation technology [3], which we refer to here as PET.

The best application of PET in biopharmacy is the controlled and continuous delivery of cells in the gut. The potential benefit of this therapeutic strategy is to maintain greater cell viability despite the acidity into the stomach. In their viable state, probiotics may exert a health benefice on the host [4,5]. One research group showed that alginate could pass through the stomach without any degradation. Gel beads formed from this biomaterial were visualized in the human gut by nuclear magnetic resonance imaging [6]. The choice of the biomaterial is crucial because it determines the effectiveness of the protective device. Beyond this protection, the device must withstand during the passage through the stomach, disintegrate in the gut to release the cells. Probiotics are currently encapsulated in polymer matrices for various applications. The physical retention of cells in the matrix and their subsequent separation is the consequence of the encapsulation technology used.

Selecting the encapsulation technology is very important. Whereas probiotics are living cells, the conditions for implementation of this technology are designed to maintain cell viability, and solvents involved in the encapsulation technology must be non-toxic. Furthermore, assess the release conditions of encapsulated probiotics in a gastrointestinal tract model is an essential approach, which would give an idea of the cells’ behavior.

This paper reviews the methodological approach of probiotics encapsulation including biomaterials selection, choice of appropriate technology, in vitro release studies of encapsulated probiotics, and highlights the challenges to be overcome in this area.

2. Selecting the Biomaterials for Microencapsulation

The concept of biomaterials usually results in various definitions. A definition often accepted in the field of biology and medicine is “any natural material or not, which is in direct contact with a living structure and is intended to act with biological systems” [7]. The biomaterials used for probiotics encapsulation include natural polymers and synthetic polymers [7]. The terms biocompatible and biodegradable are associated with many of these biomaterials. Biomaterials for probiotics encapsulation are in direct contact with the living cells.

After microencapsulation, the protective device-based biomaterial is intended to be in contact with the digestive tract of the host. For all these reasons, much of the general criteria developed for choosing biomaterials can be applied. Issues involved when selecting biomaterials for probiotics encapsulation are: (a) physicochemical properties (chemical composition, morphology, mechanical strength, stability in gastric and intestinal fluids; (b) toxicology assay; (c) manufacturing and sterilization processes.

Biomaterials are inorganic or organic macromolecules, consisting of repeated chain of monomers linked by covalent bonds. Their chemical structure and the conformation of the monomer chains give them specific functionality such as ability to form gels [8]. The most common biomaterial used for probiotics encapsulation is alginate [9,10,11]. Other supporting biomaterials include carrageenan, gelatin, chitosan, whey proteins, cellulose acetate phthalate, locust bean gum and starches [11].

Alginate is a linear polymer of heterogeneous structure composed of two monosaccharide units: acid α-L-guluronic (G) and acid β-D-mannuronic (M) linked by β (1–4) glycosidic bonds [12,13]. The appearance of G and M monomers in the alginate chains occurs in blocks of alternating sequences, not randomly. This arrangement of chains is widely described in the literature and varies from one structure to another [13,14,15,16]. The M/G ratio determines the technological functionality of alginate. The gel strength is particularly important that the proportion of block G is high. Temperatures in the range of 60 °C to 80 °C are needed to dissolve alginate in water. Alginate gels are known to be insoluble in acidic media [17]. The success of the use of alginate in microencapsulation of probiotics is due to the basic protection against acidity it provides to the cells [18,19,20].

Carrageenan are polymers of linear structure consisting of D-galactose units alternatively linked by α(1–3) and β(1–4) bonds. Three types of carrageenan are known: kappa (κ) carrageenan, iota (ι) carrageenan and lambda (λ) carrageenan [21]. κ-Carrageenan (monosulfated) and ι-carrageenan (bisulfated) have an oxygen bridge between carbons 3 and 6 of the D-galactose. This bridge is responsible for conformational transitions. It is also responsible for the gelation of κ-carrageenan and ι-carrageenan. The λ-carrageenan (trisulfated) that does not have this bridge is unable to gel [22]. Carrageenan gelation is induced by temperature changes. A rise in temperature (60 to 80 °C) is required to dissolve it and gelation occurs by cooling to room temperature [22,23]. Carrageenan is commonly used as food additive; its safety has been approved by several government agencies including FDA, codex alimentarius and the joint FAO/WHO food additives [24]. The use of carrageenan in microencapsulation of probiotics is due to its capacity to form gel that can entrap the cells. However, the cell slurry should be added to the heat-sterilized suspension between 40 and 45 °C, otherwise the gel hardens at room temperature [25].

Whey proteins are usually used because of their amphoteric character. They can be easily mixed with negatively charged polysaccharides such as alginate, carrageenan or pectin [25,26]. When the pH is adjusted below their isoelectric point, the net charge of the proteins becomes positive, causing an interaction with the negatively charged polysaccharides [17,27,28].

Gelatin is frequently used in the food and pharmaceutical industries. It is a protein derived by partial hydrolysis of collagen of animal origin. Gelatin has a very special structure and versatile functional properties, and forms a solution of high viscosity in water, which sets to a gel during cooling [29]. It does not form beads but could still be considered as material for microencapsulation.

Chitosan is a positively charged polysaccharide formed by deacetylation of chitin. Its solubility is pH-dependent. It is water insoluble at a pH higher than 5.4 [30]. This insolubility presents the drawback of preventing the complete release of this biomaterial into the gut which pH is greater than 5.4 [30]. However, studies have reported the effectiveness of chitosan as a coating agent of alginate gel beads [30,31,32]. Chitosan can form a semipermeable membrane around a negatively charged polymer [29]. Whey proteins, gelatin and chitosan are usually used to develop capsules [9] or to coat gel beads to improve their stability [11].

Cellulose acetate phthalate (CAP) is a polymer insoluble at a pH below 5 but and soluble when the pH is greater than 6 [9,11]. This property is essential for probiotics encapsulation because the biomaterial must not dissolve into the stomach, but only into the gut. The disadvantage of CAP is that it cannot form gel beads by ionotropic gelation; only capsules have been developed by emulsification using this biomaterial. CAP is widely used as a coating agent.

Locust bean gum and starches are usually mixed with alginate or carrageenan to develop gel beads or capsules. It appears that specific interactions occur during mixing. The ratio between the proportions of each biomaterial before mixing is essential [9].

Selecting the appropriate biomaterial is a preliminary study which requires a rigorous methodological approach. For probiotics encapsulation, biomaterials such as proteins and polysaccharides must be stable in acidic environment and unstable in environment with a pH above 6. This pH is the minimum pH found in the intestinal lumen, usually at the beginning of the duodenum [18]. For example, the stability of proteins under varying conditions of pH can be assessed by electrophoresis (SDS-PAGE). For polysaccharides and other biomaterials treated under various conditions of pH, FTIR-ATR can be used to study their stability by determining the any change in its initial structure. Publications have referred to the mixture of biomaterials (proteins-polysaccharides or polysaccharide-polysaccharide) to encapsulate probiotics [1,2]. However, it would be interesting to elucidate the interactions between these biomaterials [17]. Once the biomaterial has been used to develop the protective device, it would also be interesting to elucidate the mechanism of resistance of this device in an acidic medium, and its disintegration or dissolution in environment with a pH above 6. Searching for new encapsulation materials will be of paramount importance in the near future. These materials must meet the requirements of non-toxicity, resistance to gastric acidity and compatibility with respect to probiotic cells. Several challenges are faced in this area.

3. Selecting the Microencapsulation Technology

Most of the reported literature on PET was based on small-scale laboratory procedures. PET requires techniques that are gentle and non-aggressive towards the cells. The first encapsulation techniques developed to improve the shelf-life of probiotics were to transform cells cultures into concentrated dry powder. The techniques of spray-drying, freeze-drying or fluidized bed drying have shown their limitations because the cells encapsulated by these techniques are completely released into the product. Thereby, the cells are not protected towards the food matrix environment and in the presence of gastric fluid or bile [33]. However, probiotics in dried or freeze-dried form exhibit compatibility with traditional starter culture such as milk or cheese and have a longer shelf-life compared to their cell slurry form [29].

With specific reference to spray-drying, recent publications make reference to its effectiveness in protecting probiotic cells [34,35]. This technique commonly used in food industry involves atomization of an aqueous or oily suspension of probiotics and carrier material into a drying gas, resulting in rapid evaporation of water [29]. Water evaporation is defined as the difference between air inlet temperature and air outlet temperature. The spray-drying process is controlled by these temperatures, but also by the product feed and the gas flow [29]. Despite the advantages of spray-drying technique, the high temperatures needed to facilitate water evaporation reduce the probiotics viability and their activity in the final product. The minimum air inlet temperature reported in the literature for probiotic encapsulation is 100 °C while the maximum is 170 °C. The air outlet temperature vary between 45 °C and 105 °C [29]. At these temperatures, it is unlikely that the cells retain all their probiotic activity. Probiotic activity must be differentiated from probiotic survival. Probiotic activity takes into account the ability of cells to resist to gastrointestinal environment and to adhere to intestinal mucosa [36], so it is important that the encapsulation technique does not reduce cell survival and does not inhibit their subsequent activities.

Providing probiotics with a physical barrier against adverse conditions is an approach receiving considerable interest. For this, other techniques have been introduced to further improve the protection of probiotics. These techniques were intended to develop gel beads or capsules which were made from hydrocolloids by means of extrusion or emulsification techniques [37,38]. Hydrocolloids are aqueous dispersion of biomaterials (natural or synthetic polymers).

The encapsulation process of these two techniques is summarized in Figure 1.

Figure 1. Diagram of the encapsulation process of probiotics by extrusion technique (a) and by emulsification technique (b).

Figure 1. Diagram of the encapsulation process of probiotics by extrusion technique (a) and by emulsification technique (b).

In extrusion technique (a), the hydrocolloid is mixed with probiotics. The resulting mixture is fed into an extruder, typically a syringe. Pressure exerted on the syringe plunger drops the contents of the syringe into a gelling solution, with gentle stirring. The size and shape of the drops depend on the diameter of the needle, and the distance between the needle and the gelling solution. Extrusion is a simple and easy implementation, allowing the retention of a high number of cells. Automated processes exploiting this principle are available today [39].

In the emulsification technique (b), the mixture represents the discontinuous phase. This phase is dispersed in a large volume of vegetable oil (continuous phase). The water-in-oil emulsion being formed is continuously homogenized by stirring. The stirring speed is a critical step because it affects the size and the shape of the droplets formed [40]. Once the emulsion has been broken, the droplets are collected by settling. The use of this technique for probiotics encapsulation has been described in the literature [40,41].

Emulsification generates oily or aqueous droplets commonly named capsules, while the extrusion gives gelled droplets called beads. The core of the capsule is liquid while the core of the bead presents a porous network [7]. The capsules have sizes that are at least 100 times lower than those of the beads [9]. The difference between capsules and beads is shown in Figure 2. Capsules have unequal size and shape compared to beads whose shape is uniform.

Figure 2. (a) Photographs of alginate gel beads and (b) Photographs of alginate capsules [39].

Figure 2. (a) Photographs of alginate gel beads and (b) Photographs of alginate capsules [39].

Extrusion is much easier to realize compared to emulsification. Emulsification is more expensive because it requires additional raw materials such as vegetable oil and emulsifiers to stabilize the emulsion. Emulsification also presents difficulties in implementation including emulsion instability, need for vigorous stirring which can be detrimental to cells survival, random incorporation of cells into the capsules, and inability to sterilize vegetable oil if you have to work under conditions of strict asepsis.

From these two techniques are introduced changes to improve beads or capsules stability. Among these improvements are coating with others biomaterials [32], cross-linking with organic solvents [42], or adding additives or cryoprotectants in the mixture [43]. In the literature, rare are the studies in which authors have shown photographs of probiotics entrapped in capsules. Electron microscopy (SEM or TEM) is an effective technique to provide evidence of the presence of probiotics in capsules or beads and to assess the bacterial loading [20].

4. Selecting the in Vitro Conditions for Cells Release

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