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Reyes, L. Polymeric Platforms and Delivery Systems of Probiotics. Encyclopedia. Available online: https://encyclopedia.pub/entry/20653 (accessed on 15 September 2024).
Reyes L. Polymeric Platforms and Delivery Systems of Probiotics. Encyclopedia. Available at: https://encyclopedia.pub/entry/20653. Accessed September 15, 2024.
Reyes, Luis. "Polymeric Platforms and Delivery Systems of Probiotics" Encyclopedia, https://encyclopedia.pub/entry/20653 (accessed September 15, 2024).
Reyes, L. (2022, March 16). Polymeric Platforms and Delivery Systems of Probiotics. In Encyclopedia. https://encyclopedia.pub/entry/20653
Reyes, Luis. "Polymeric Platforms and Delivery Systems of Probiotics." Encyclopedia. Web. 16 March, 2022.
Polymeric Platforms and Delivery Systems of Probiotics
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This entry is adapted from the peer-reviewed paper 10.3390/fermentation8030117

The selection of optimal material for probiotic encapsulation and the appropriate processing route are key parameters to ensure an efficient delivery strategy, where early degradation by GIT stimuli and harsh conditions are largely avoided. Nevertheless, selecting the materials for superior performance in complex physiological environments such as the GIT is a task made challenging not only by the obstacles to be overcome to reach the target site but also by the need to maintain high biological activities and positive responses to the changing surroundings. The fast-growing notion that encapsulates can be composed of active biomaterials should be driven by matching the material’s properties with expected responses through the GIT. Particularly, polymers exhibit versatile molecular moieties that have been widely exploited to fabricate chemical and physical delivery platforms with properties that can be finely tuned by adjusting interchain interactions. Chemical polymeric scaffolds are formed by covalent bonds between adjacent chains, while the physical ones are maintained together by charged polyvalent surfactants or ion interactions. Moreover, their versatile processability schemes facilitate obtaining different morphologies, functionalities, and the possibility to form composites with nanostructured materials in search of an enhanced response when subjected to a stimulus. Typical polymeric encapsulates comprise microparticles, microspheres, microcapsules, hydrogels, and, more recently, nanocomposite 3D matrices.

Nanostructure Hydrogels Polymeric probiotic

1. Introduction

The term “probiotic” has been complimented since its first appearance in the 1960s. It was initially defined as a substance secreted by microorganisms that has beneficial effects on the human body [1][2]. Then, in 1980, some specific characteristics were added to this definition. Additional claims stated that probiotics are “strains that have a beneficial impact, non-toxic, non-allergic, and nonpathogenic, available in large quantities as viable cells, suitable for the environment of the gut, and storable as well as stable” [1]. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) defined probiotics as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [1][2][3][4]. Additionally, both organizations have classified products containing live organisms into four categories: (a) live or active cultures; (b) probiotics in food or supplements without a health claim; (c) probiotics in food or supplements with a specific health claim; and (d) probiotic drugs [2].
Probiotic strains generally belong to the following genera: Lactobacillus, Bifidobacterium, Saccharomyces, Pediococcus, Streptococcus, and Leuconostoc [1]. Lactobacillus and Bifidobacterium are the most common strains. Different aspects need to be considered when selecting probiotic strains, and these include stomach pH and bile tolerance, adherence to epithelial surfaces, capacity for immunostimulation, antagonistic activity against pathogens, and antimutagenic and anticarcinogenic properties [5]. Probiotics have been successfully employed in manufacturing a wide variety of fermented products for daily consumption, including yogurt, kefir, sour pickles, milk, miso soup, and several soft cheeses [6]. The average probiotic consumption for a single person varies from 107 to 109 CFU/mg/day, whereas the significant benefit probiotic content in food must be of the order of 106 CFU/g [1][4]. Major health benefits have been attributed to these microorganisms, which nowadays can be used for the prevention and treatment of ailments such as liver disorders, cardiovascular diseases, dental caries, gastrointestinal inflammation, diarrhea, diabetes, obesity, and irritable bowel syndrome [1][3][6][7].
Probiotics can produce essential metabolites, including enzymes, vitamins, amino acids, peptides, exopolysaccharides, antioxidants, and anti-inflammatory agents. For example, some Bifidobacterium strains can produce B6 vitamin, while L. reuteri can produce cobalamin [8]. Some studies performed on pediatric patients who suffered from some kind of food allergy showed, with moderate certainty, that consumption of probiotics such as L. rhamnosius GG, LC705, L. casei LOCK 0900 and LOCK 0908, L. paracasei LOCK 0919, B. breve Bbi99, Propionibacterium freudenreichii ssp, or Shermanii JS could alleviate the symptoms caused by bovine lactose intolerance. This is because probiotics are thought to induce the production of β-galactosidase and lactase enzymes, helping to metabolize lactose quite effectively [8][9].
However, it is important to keep in mind that first, the strain must reach its site of action, usually the gut, and thus survive the physiological stress met during its ingestion, i.e., acid stomach and gut pH and the presence of biliary salts. Furthermore, its ingestion must not lead to any major risks for the host, maintaining its characteristics and remaining stable during the manufacturing process where it is usually incorporated into a delivery matrix [6]. A few strains can maintain viability after 1 h at a pH of 1, and most of them lose viability after 3 h at a pH of 3. The human stomach pH varies from 1 when fasting to 4.5 after eating. The process can take more than 3 h [10].
Different strategies exist to protect probiotics and their viability by edible carriers such as cheese, drinks, and bread [11][12]. Also, polymeric matrices have attracted significant attention for encapsulation, protection, and probiotic release [13]. Probiotics have evolved in sophistication from the first to the fourth generation. First-generation probiotics are either fresh or lyophilized cells without any coating. This has led to a low survival rate between 7% and 30%. Second-generation probiotics are incorporated into polymeric capsules or tablets with fillers. Usually, these strains show higher survival rates, but low performance due to rapid metabolite degradation. Third-generation probiotics are those encapsulated in natural, semi-synthetic, or synthetic polymers. The microcapsules are designed to be consumed gradually, which helps maintain the metabolites’ activity. Their structure can be a 3D matrix, a crosslinked construct, or have an external coating. Fourth-generation probiotics are those incorporated into biofilms, which improve protection when transiting the gastrointestinal tract [14][15].
Encapsulation techniques are widely used for varied applications in the food industry, including masking and design of flavors, colors, and odors, improving the shelf life of products, protecting some components against nutritional loss, and regulating undesirable oxidative reactions. With probiotics, encapsulation provides protection from media effects and enhanced viability and allows controlled dosing and handling of cells [16]. One of the biggest challenges when encapsulating is selecting effective and safe materials for the capsules’ manufacturing, an efficient release system, and proper production techniques (e.g., extrusion, emulsion, spray-drying, etc.). A much more comprehensive discussion of different methods used in the design of encapsulation microgels is given by McClements [13]. Additionally, it is vital to consider economic, regulatory, and consumption factors to assure scalability and successful market penetration [13][16].
Materials used for capsule manufacturing have diverse origins. The most common ones are derivates from cellulose, proteins, polysaccharides, carrageenan, gelatin, pectin, and alginate [16]. Materials are chosen depending on the characteristic physicochemical and structural features of the capsules, and generally, polysaccharides and proteins are selected due to their versatility. In this regard, natural polymers such as alginate, xanthan gum with divalent cations, casein gels, or gelatins have been chosen due to their ease of crosslinking, and the possibility of combining them to achieve different levels of mechanical resistance. Chitosan, lysine, or whey protein are used for the external coating of structures [15]. Some of the most important physicochemical properties of the materials to consider for a rational design are solubility, gelation mechanisms, degradability, and electric properties [13]. There are different methods to perform encapsulation processes such as injection (e.g., extrusion, atomization, and microfluidic), template techniques (e.g., emulsions), biopolymer phase separation, precipitation, reduction, drying, and more recently, biofilm formation to promote colonization and enhance the permanence of probiotics in the host intestinal mucosa [13][14].
The gastrointestinal tract comprises the mouth, esophagus, stomach, gut, and colon. Its microbiota concentration varies over the tract due to changes in pH and the presence of bile and enzymes. For example, in the stomach, such concentration is low (101 bacteria/g), increasing through the duodenum (103 bacteria/g), the jejunum (104 bacteria/g), and the ileum (107 bacteria/g). The largest concentration of microorganisms is found in the gut and colon, rounding 1011 to 1012 bacteria/g [17]. Such microbiota presents a great phylogenetic diversity, allowing the required metabolic performance. The present microbiota is mainly composed of Bacteroidetes, Firmicutes, and Proteobacteria, and to a lesser extent by Actinobacteria, Clostridium, Enterobacter, Verrumicrobia, bacteriophages, viruses, and several Aspergillus, Candida, Cryptococcus, and Penicillium fungi genres [7][18][19].
The digestion and nutrient absorption processes are carried out by the small intestine, where there is an intestinal barrier composed of a mucosae layer and a cell component (intestinal epithelium and underlying lamina propria) that acts as a physical barrier to the microorganisms present in the gut [3]. Mucosae is composed of an outer loosely adhering layer and a dense inner layer. This last one is the first effective defense mechanism because of its high density, preventing most bacteria from penetrating and adhering [17]. The intestinal epithelium creates a separation between the gut lumen and the lamina propria, and comprises enterocytes, goblet, Paneth, and endoenterocrine cells. In contrast, the lamina propria is formed by dendritic cells, macrophages, and plasma cells that can engulf pathogens and eliminate apoptotic cells and waste [3].
The microbiota existing along the intestine have an immunological vigilance function that allows the detection of pathogens and stimulates the immune system to respond adequately. The pathogen control mechanism comprises four major steps: first, production of bacteriocin and other inhibitors; second, the competitive exclusion by the binding sites; third, stimulation of the immune response; and last, the inhibition of virulent genes or expression of proteins in pathogens [7]. Intestinal homeostasis occurs when the immune system establishes an equilibrium between commensal, mutualistic, and opportunistic bacteria. This happens when the microbiota communicates effectively with the immune system through a healthy intestinal barrier [3].

2. Polymeric Materials in Probiotic Encapsulation

In protected-delivery technologies, a suitable polymeric material should be able to preserve its core from adverse environmental conditions (e.g., reduce the acid-induced degradation of probiotics by gastric fluids in the stomach), exhibit inertness with the encapsulated materials, promote a controlled release of the encapsulate, achieve higher encapsulation efficiencies of bioactive compounds on a per mass basis and, ultimately, favor high levels of absorption into the targeted organs (i.e., the overall efficacy of the compounds) [20][21][22]. All the selected materials must also be biodegradable and biocompatible since they will be in direct contact with various types of cells [20][23].
Among the natural polymers, alginate, a heteropolysaccharide, has been applied successfully as a pH-sensitive material for the encapsulation of probiotic bacteria [24]. Alginate, extracted from algae, is composed of two monosaccharide units: α-L-guluronic acid and β-D-mannuronic acid, linked together by a β (1–4) glycosidic bond. Due to its toxicity, inexpensiveness, ease of processing, and biocompatibility, calcium alginate has been extensively employed in the encapsulation of probiotics [21][25][26][27][28][29]. Yet, calcium alginate encapsulates are chemically susceptible to disintegration in the presence of excess monovalent ions, Ca2+-chelating agents such as phosphate and citrate, and harsh chemical conditions (e.g., low pH) [30][31][32]. To increase the stability of alginate and decrease the loss of encapsulated material, alginate is usually coated with polycationic polymers such as chitosan and poly-L-lysine [20][23][32][33].
Chitosan is a very abundant polysaccharide obtained from chitin and is composed of (1,4)-linked 2-amino-deoxy-b-d-glucan [34]. It also shows high biocompatibility and biodegradability under physiological conditions. For these reasons, it has enabled several encapsulation applications in the food and pharmaceutical industries, including liposome coating, chitosan–alginate coating, controlled delivery of small molecules and biologicals, and the release of bioactive metabolites (e.g., essential oils, probiotics, vitamins, antioxidants, and flavors). The unique cationic character of chitosan allows forming multi-layer systems with anionic alginate for probiotic encapsulation, which can bring efficient protection to cargoes, reduced porosity, stability at various pH ranges, and reduced leakage of the encapsulated probiotic. In experiments where the gastric environment was simulated, chitosan coating was more efficient than poly-l-lysine and alginate coatings in protecting probiotics, which represents a possible route to overcome the challenges of oral delivery [34]. Furthermore, chitosan coating with drying processes can prolong the long-term storage of some probiotics at different temperatures [35].
Another frequently studied material for encapsulation is gelatin, a commercially available denatured protein obtained by the hydrolysis of collagen from the skin and bones of bovines or fish. This protein might be positively or negatively charged, depending on whether an acidic or alkaline method was used for its extraction, which can be exploited to design multifunctional controlled release systems [36]. For example, gelatin-coated alginate capsules and microspheres can protect labile drugs from the stomach’s acidic environment, enabling their release in the target intestinal area, whose environment has a basic pH [37]. Other studies have reported that the addition of fish gelatin significantly raised polymer matrix density and improved the physical integrity of alginate capsules because of a more stable and ordered 3D structure. Some other aspects, such as the survival rate of microorganisms during the GIT’s passage, are enhanced compared to non-encapsulated cells [38].
Poly-l-lysine is a cationic, non-ribosomal, non-toxic, biocompatible, biodegradable, and antimicrobial homopolymer produced by modified strains of Streptomyces albulus. Lysine is frequently used as a preservative and food additive [39], and studied as an alginate bead coating. The marked antimicrobial activity of most cationic polymers poses a major challenge because they tend to inhibit the growth of some microorganism strains, depending on pH and incubation times. That is the case not only of lysine but also of chitosan and polyethyleneimines [40]. However, different in vitro studies that have implemented mixed manufacturing techniques, such as freeze-frying, have shown that poly-l-lysine coatings for alginate capsules are well-suited to maintain cells’ growth and proliferation at low pH values and viability for a storage period of up to 16 weeks at 4 °C [41].
The materials mentioned above correspond to the most popular and studied polymers for survival enhancement and protection of probiotics in hostile environments, such as those found in the compartments of the GIT. Many other materials might be suitable for producing capsules and coatings for this application, including polyethyleneimines, poly(2-dimethyl(aminoethyl)methacrylate), dextran, pectin, Arabic gum, starch, sodium caseinate, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, and succinylated or acylated carrageenan [41][39][42][43].

3. Hydrogels

Hydrogels have drawn particular attention among encapsulation alternatives for probiotics, given their ease of processing, the wide range of materials available for their fabrication, and their capability to form three-dimensional networks that protect the probiotic’s integrity [44]. Remarkably, the inertness of hydrogels in environments with high water activity makes them suitable to entrap molecules and microorganisms, and ensure their integrity and viability in physiological environments [45]. However, one of the main concerns about implementing hydrogels is the proper tuning of mechanical performance, along with optimal porosity to enable microorganism survival while maintaining sufficient cell entrapment and a considerable degree of swelling [46]. This has been addressed by chemically modified polymers through different routes, including the addition of ionizable functional groups, functionalized backbones, and combined polymeric blending [47][48][49].
Hydrogels can be classified mainly according to their (i) composition (homo or copolymers), (ii) network size (macrogels, microgels, nanogels), (iii) electrical charge (non-ionic, cationic, anionic, amphoteric or zwitterionic), and (iv) crosslinking method (physical or chemical) [50][51]. Moreover, recent studies have demonstrated the fabrication of chemically crosslinked platforms oriented toward the encapsulation of probiotics into hydrogel beads crosslinked with the aid of glutaraldehyde, Genipin, calcium chloride (CaCl2), and ferrous sulfate (FeSO4) [52][53][54][55]. Alginate, gelatin, and chitosan are the three main polymers of choice for hydrogel synthesis as they have proven to be effective in protecting probiotic cells from harsh environmental conditions [51][56].
One of the most intensive research areas is the development of strategies for tuning degradation rates and matrix porosities to improve their performance in protecting encapsulated living organisms [57]. This has been achieved by the supramolecular design of monomeric structures and by controlling polymerization reactions with carefully applied light and thermal stimuli [58][59]. Depending on the polymerization scheme selected (i.e., bulk polymerization, solution polymerization, suspension polymerization, emulsion polymerization, and graft polymerization), macroscopic and microscopic properties such as porosity and polymeric mesh size might change significantly [60]. Notably, the capacity of engineering the tortuosity and interconnectivity of hydrogels’ mesh has been reported as critical for the smart release of the encapsulated cargoes [61]. Further control over such a process can be achieved by chemical modifications with hygroscopic polymers such as polyethyleneglycol, which leads to the enhanced mucoadhesiveness of the encapsulates by both physical entanglement and hydrogen bonding with the base encapsulating polymer [62][63]. Therefore, the following section will discuss the fast-growing area of stimuli-responsive hydrogels, emphasizing how they can be activated, de-activated, or re-activated for a particular delivery purpose [64].

4. Microencapsulates

Microparticles, microcapsules, and microspheres usually made of food-grade polymers, such as alginate, chitosan, carboxymethyl cellulose, cellulose acetate phthalate, xanthan gum, starch, carrageenan, gelatin, and pectin [25][65], have demonstrated to be protective barriers of high performance against the GIT’s environmental conditions [66][67][68][69][70]. These microencapsulates’ dimensions usually range between 1 and 1000 µm [71]. According to recent studies, an effective microencapsulation system should maintain the stability of the probiotics during storage, protect them from the harsh conditions of the upper GIT, release them in the colon, and finally, promote their ability to colonize the mucosal surfaces [71][72][73][74].
Microparticles typically consist of a core composed of one to several ingredients surrounded by a wall or barrier of uniform or non-uniform thickness, either single-layered or multi-layered. The design of microencapsulated ingredients requires knowledge of (1) the core, (2) the materials for encapsulation, (3) the interactions between the core, matrix, and the environment, (4) the stability of the microencapsulated ingredients under storage conditions and when incorporated into food matrices, and (5) the mechanisms that control the release of the core [22][25][65]. Matrix degradation, and consequently, the release of its contents, can be controlled to occur at different times. Larger particles generally release encapsulated compounds more slowly and over more extended periods, while particle size reduction favors adhesiveness and therefore prolonged GI transit time, leading to a higher drug bioavailability [22][25][65][75].
Typical technologies employed for probiotic encapsulation include emulsification [76][77][78], emulsification and enzymatic gelation [79][80][81][82], atomization (e.g., spray drying [77][78][83], spray freeze drying [77][78][83][84][85]), coating and agglomeration [78][86][87][88], and extrusion [89][90].

5. Nanostructured Platforms

One of the leading strategies to improve microencapsulates’ tolerance to different GIT environments and ensure their efficient delivery is physical and chemical blending with stimuli-responsive and high mechanical performance nanomaterials [91][92][93]. This approach allows superior control over probiotic delivery due to the unique properties attainable by forming nanocomposites and the possibility of providing multimodal delivery platforms (i.e., including more than one encapsulated component) where survival of probiotics is increased substantially [94][95]. Accordingly, several nanostructured materials have been explored in the fabrication of next-generation delivery platforms, including polymeric and iron oxide-based nanoparticles, nanosheets (e.g., graphene oxide—GO and phyllosilicate clays), nanoliposomes, micelles, and nanoparticles derived from naturally occurring polymers such as nanocellulose and starch nanocrystals [96][97][98].
The dispersion of nanoparticles (NPs) into polymeric arrangements (to form nanocomposites) is attractive mainly due to their intrinsic capacity to fill out pores, therefore avoiding the diffusion of molecules such as hydrogen ions, bile salts, or digestive enzymes that may lead to the undesirable degradation of the encapsulated probiotics [99]. For instance, magnesium oxide (MgO) NPs have been incorporated into alginate–gelatin microgels, which resulted in a more stable encapsulation of probiotics when compared to unmodified microgels. The MgO NPs help neutralize the hydrogen ions present in the gastric fluids, which diminishes the acid-induced degradation of probiotics and maintains a neutral pH inside the microgels [20]. Alternatively, the use of NPs can help improve some physicochemical properties of the encapsulates, such as hardness, compressibility, cohesiveness, and adhesiveness. This is the case of chitosan NPs, which have been reported to enhance the mucoadhesive properties of hydrogels [100]. Through this approach, the interaction with the intestinal mucus is improved by the electrostatic interaction and physical entanglements of the chitosan-containing matrices facilitated by the positive charge of chitosan.
Another approach suggests that combining polymeric platforms with nanocrystals derived from polysaccharides (e.g., cellulose and starch) enhances mechanical stability and shelling properties [101], and increases the surface area for target delivery [102]. For instance, when cellulose nanocrystals (NCs) are combined with alginate during the microencapsulation process, the dissolution time increases while porosity is reduced significantly [103]. Moreover, ionic interaction between the material and the nanocellulose filler reduces the infiltration of gastric fluids, preventing the degradation of probiotics [104]. When starch NCs are implemented as fill-in alginate-based delivery platforms, thicker barrier protection against gastric and intestinal juices provides a stable mechanism to decrease probiotics mortality [96].
Remarkably, novel developments in the fabrication of nanocarbon-based materials (e.g., reduced graphene oxide, graphene quantum dots, graphene nanoribbons), silica-based nanocarriers, and inorganic nanoparticles have enabled the fabrication of emerging nanocomposites with improved mechanical strengths, high drug loading, and reduced toxicity [91][105][106]. These features have been reported to not only favor the controlled release of on-cargo molecules but also increase the survival of encapsulated living organisms (e.g., probiotics and mammalian cells) [107][108]. Similarly, clay mineral silicates have gained popularity given their unique cationic exchange properties that can be exploited to fabricate water barriers due to hydrogen bonding [109][110]. Kim and colleagues reported superior shape integrity for bentonite/alginate-based encapsulates during gastric fluid exposure and appropriate disintegration in the intestinal area upon oral administration in mice [105].

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Luis Reyes
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