*3.2. Biopolymers in Probiotic Encapsulation and Delivery*

Due to their special properties, biopolymers have been used to encapsulate probiotics. Probiotics are living organisms with benefits on the hosts' health if ingested in adequate amounts [164]. According to the International Scientific Association for Probiotics and Prebiotics (ISAPP), a sufficient amount of probiotics with a beneficial effect on the hosts' health involves ingesting 1 <sup>×</sup> <sup>10</sup><sup>9</sup> CFU per serving [165]. Unlike probiotics, prebiotics are nutrients, usually high-fiber foods, providing the substrate that is selectively utilized by the hosts' microorganisms, conferring a health benefit [165]. Most probiotics in the human body form the commensal intestinal microbiota with a role in increasing resistance to infections and boosting host immune system, glucose and lipid metabolism, degradation of complex carbohydrates, and synthesis of vitamins and bile acid [166]. Although the effects of probiotics on various diseases is still debatable, several studies showed beneficial effects in the treatment and prevention of infectious diseases. For example, strains of *Lactobacillus plantarum*, *Lactobacillus casei*, or *Lactobacillus paracasei* had antifungal, antibacterial, and antioxidant effects. Other strains have been shown to have anti-inflammatory effects, to lessen the risk of osteoporosis, maintain cholesterol levels, and prevent the proliferation of cancer cells [167]. Therefore, due to their beneficial effects and health claims, there has been a worldwide explosion of probiotic-based health products in the form of dietary supplements [168]. As such, the global probiotic market is soon reaching USD 50 billion and, with that, the range of probiotics-containing products and associated health claims continue to expand rapidly. Currently, in Europe, the probiotic market is subject to regulatory

requirements and compliance with rules and regulations in order to meet certain standards for product registration and use [169].

The use of encapsulation technologies of probiotics has been intensely studied in order to increase probiotics' viability throughout manipulation, storage, commercialization, and incorporation in food and pharmaceutical products so that these cells are viable during their transit and residence in the gastrointestinal tract. Therefore, improving probiotic survival and resistance to adverse conditions through encapsulation is paramount to their effectiveness in health and disease conditions. To prove their effectiveness, encapsulated probiotic strains have been incorporated into a wide range of food products such as yoghurt [170], cheeses [171], frozen dairy desserts [172], beverages [173], and meat products [174], increasing their therapeutic effects [168]. Once encapsulated, probiotics embedded in food matrices maintained their viability even two months under refrigeration [175]. They can also be mixed in a single microcapsule or in dual core capsules with separation microcompartments [176] and a combination of at least two strains can improve their effect [174]. In order to scale up production, several industrial partnerships between food producers and probiotic companies have been formed. For example, Christian Hansen and Dos Pinos developed probiotic ice cream, Balchem Encapsulates and Rosell Institute developed probiotic raisins and bars, and Dannon uses probiotics encapsulated in their products [177]. Encapsulated probiotics have been effective in irritable bowel syndrome [178], colitis, abdominal pain [179], and other gut or metabolic conditions characterized by microbiota dysbiosis [180].

Several encapsulation methods have been developed and used.


(7) Layer-by-layer is technology based on alternating coating layers of cationic (e.g., chitosan) with anionic (e.g., alginate) biopolymers on cells via electrostatic interaction [184]. It has the advantage of enhanced bacterial viability throughout the gastrointestinal tract, along with the survival of probiotic cells against acidic and bile salt insults, mucoadhesion and growth on intestinal tissues, and in vivo survival [179].

In addition to ensuring cell viability along the gastrointestinal tract, the stability of probiotics during storage is also very important. In this regard, encapsulation has proved to be an effective method. For this, the material used to encapsulate microorganisms is the first and most important factor in maintaining their viability [185]. It improves the survival of probiotics during manufacturing processes, especially heat processing [186] and storage [187]. An important aspect of this process is cytotoxicity. According to ISO 10993-5 [188], a material used for encapsulation is potentially cytotoxic when cell viability decreases below 70% after exposure [189]. In this regard, polysaccharides such as alginate, starch, chitosan, and cellulose, as well as other biopolymers or chemicals, have low or no toxicity and do not affect cell viability. On the contrary, they maintained cellular stability for a long time, particularly when kept in refrigerated or frozen conditions.

Although the encapsulation method has many advantages, there are still several aspects that must be consider. These are: (i) biosafety concerns preventing clinical translation of the cell microencapsulation; (ii) concerns regarding the manipulation and extraction procedures that must be refined in order to be as minimally invasive as possible; (iii) concerns regarding the optimization of cost effectiveness; and (iv) concerns regarding the consideration of internationally accepted regulations for the use of probiotics. Therefore, applications of biopolymers for the coating of encapsulated strains for the purpose of protection in the intestinal gastrointestinal tract or as carriers for direct encapsulation of microorganisms should involve procedures that facilitate high bacterial viability.
