Structural Stability and Viability of Microencapsulated Probiotic Bacteria: A Review

Authors

  • Rocío I. Corona-Hernandez,

    1. Departamento de Ciencias Químico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez. Anillo Envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez 32310, Chihuahua, México
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  • Emilio Álvarez-Parrilla,

    1. Departamento de Ciencias Químico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez. Anillo Envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez 32310, Chihuahua, México
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  • Jaime Lizardi-Mendoza,

    1. Coordinación de Tecnología de Alimentos de Origen Animal, Centro de Investigación en Alimentación y Desarrollo, AC. Carretera a la Victoria km. 0.6, AP 1735, Hermosillo 83000, Sonora, México
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  • Alma R. Islas-Rubio,

    1. Coordinación de Tecnología de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y Desarrollo, AC. Carretera a la Victoria km. 0.6, AP 1735, Hermosillo 83000, Sonora, México
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  • Laura. A. de la Rosa,

    1. Departamento de Ciencias Químico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez. Anillo Envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez 32310, Chihuahua, México
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  • Abraham Wall-Medrano

    Corresponding author
    1. Departamento de Ciencias Químico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez. Anillo Envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez 32310, Chihuahua, México
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Abstract

Probiotics are live microorganisms that confer a number of health benefits when consumed in adequate amounts, mostly due to improvement of intestinal microflora. Bacterial strains from the genera Lactobacillus, Bifidobacterium, and Bacillus have been widely studied and are used to prepare ready-to-eat foods. However, the physicochemical stability and bioavailability of these bacteria have represented a challenge for many years, particularly in nonrefrigerated foodstuffs. Microencapsulation (ME) helps to improve the survival of these bacteria because it protects them from harsh conditions, such as high temperature, pH, or salinity, during the preparation of a final food product and its gastrointestinal passage. The most common coating materials used in the ME of probiotics are ionic polysaccharides, microbial exopolysaccharides, and milk proteins, which exhibit different physicochemical features as well as mucoadhesion. Structurally, the survival of improved bacteria depends on the quantity and strength of the functional groups located in the bacterial cell walls, coating materials, and cross-linkers. However, studies addressing the role of these interacting groups and the resulting metabolic impacts are still scarce. The fate of new probiotic-based products for the 21st century depends on the correct selection of the bacterial strain, coating material, preparation technique, and food vehicle, which are all briefly reviewed in this article.

Introduction

Probiotics are defined by the World Health Organization as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO 2001; Gawkowski and Chikindas 2013). Currently, there are many studies investigating probiotic bacteria and their benefits to human health. Several public organizations, including the International Dairy Federation (IDF), state that a food product must contain a minimum of 107 colony-forming units per gram of food (CFU/g) to assure sufficient bioavailable bacteria to exert a functional effect within the body (IDF 1999; Mortazavian and others 2007).

Dairy products are the main carriers of probiotics and have led the market for many years. This relationship has existed for more than centuries, when people consumed large amounts of fermented milks such as kefir, kurut, and yogurt (Cruz and others 2010; Fontana and others 2013), products which up to date are considered as healthy foods (Cruz and others 2012). Yogurts are fortified milk products made with specific bacterial cultures [mainly lactic acid bacteria (LAB)] and marketed as ready-to-eat gels (Marafon and others 2011) or viscous beverages (Castro and others 2013a,b) with or without modifications on their original nutrient composition (Ibarra and others 2012) while kefir grains are mixed cultures used for centuries for the production of the traditional fermented milk drink consisting of LAB (Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus delbrueckii, and so on), yeasts (Kluyveromyces sp., Candida sp., Torulopsis sp., and Saccharomyces sp.), streptococci (Streptococcus salivarius), lactococci (Lactobacillus lactis ssp., Lactobacillus thermophilus, Lactobacillus cremoris, Lactobacillus mesenteroides, and so on) and occasionally acetic acid bacteria (Simova and others 2006). Other dairy foods, such as cheeses (Escobar and others 2012; Lollo and others 2012; Karimi and others 2012a,2012b; Minervini and others 2012; Alves and others 2013), milk protein beverages (Akalin and others 2012; Castro and others 2013a,b; Fontana and others 2013), and ice creams (Kebary and others 1998; Di Criscio and others 2010), have been used to incorporate probiotics into our meal. Other nondairy foods with added probiotics include baked goods (Altamirano-Fortoul and others 2012; Trujillo-de Santiago and others 2012; Zanjani and others 2012), mayonnaise (Fahimdanesh and others 2012), beverages (Gawkowski and Chikindas 2013), and soups made from legumes and cereals (Barbosa and others 2012).

However, in spite of the growing popularity of probiotics, which account for a global market of more than 19 billion dollars in 2011 (BCC Research 2011), the viability of the bacterial strains involved and the quality of the products that contain them are questionable. A simple exploration of the global market shows that the currently employed probiotic strains exhibit little or no survival in final goods, and the food vehicles that carries them are still scarce (Ross and others 2005). Also, from a marketing perspective, the scientific support for permissible health statements in several countries (van Loveren and others 2012; Shimizu 2012) has limited the type of bacterial strains that are generally regarded as safe (GRAS; Allen and others 2012) or can be employed as foods for specified health uses (FOSHU; Duran and Valenzuela 2010; Shimizu 2012).

Due to the adverse physicochemical conditions that probiotic bacteria are exposed to during the preparation and gastrointestinal (GI) passage of a food product, their survival is at risk (Prakash and others 2011). Therefore, in the characterization of a probiotic food, both the viability (within foods) and bioavailability (within the host) of the microorganism involved must be examined, among other parameters (Figueroa-Gonzalez and others 2011). Fortunately, there are several technologies that help to improve the survival of these bacteria during the manufacturing process. Microencapsulation (ME) by means of electrostatic, extrusion/coacervation, or emulsification (freeze-, fluidized-, or spray-drying) treatments is one method used to improve survival (Vidhyalakshmi and others 2009). However, the addition of microencapsulated live bacteria to prepared foods is a relatively new alternative, although it presents huge potential within the probiotic market (Heidebach and others 2012). The bacterial survival rate also depends on the bacterial strain involved and the chemical nature of the artificial or natural matrix in which it will be contained (Del Piano and others 2006; Gawkowski and Chikindas 2013; Gebara and others 2013). Lastly, whatever technology is used to increase the viability of probiotic strains in prepared foods, the resulted products must be subjected to several resistance tests (such as against temperature, pH, salinity, and bile salts) to demonstrate their potential bioavailability (Ding and Shah 2007; Riaz and Masud 2013).

In this article, we discuss the positive influence of ME on resistance to these physicochemical factors for the most common probiotic strains reported in the scientific literature and found in the global probiotic market. Well-studied materials for ME as well as some physicochemical and structural aspects related to interacting functional groups located in bacterial cell walls, covering materials and cross-linkers, are also discussed. Although there are several comprehensive reviews on ME of probiotics, to our knowledge, there are still very few articles that address the structural relationships established during their preparation, the influence of several physicochemical factors in these relationships, and the potential impact at physiological level.

Survival of Probiotic Bacteria in Processed Foods

During food processing and storage, probiotic bacteria are exposed to several challenges that compromise their survival. Oxidative stress, temperature, acid-base changes, and molecular entrapments are just few examples. Information on the influence of these and many other factors has been largely studied in dairy foods. Fermented dairy formulations are highly susceptible to spoilage because of their high water activity (Aw) and nutrient availability (Trujillo-de Santiago and others 2012). Oxidative stress caused by oxygen diffusion of fluid yogurts during storage, since the anaerobic nature of several bacterial strains is compromised (Cruz and others 2010). To overcome this problem, several technological alternatives have been proposed that include the addition of chemical compounds (such as nitrogen), enzymatic treatments (Cruz and others 2012), and the use of adequate plastic packaging systems (Cruz and others 2013), although these technologies could increase the production costs of these dairy foods and so, a careful selection of strains nonsusceptible to redox changes is preferable. For example, L. acidophilus (LA) and Bifidobacterium spp. are both capable of mounting a cellular response against oxygen, and NADH oxidase and peroxidase appear to play an important role in the oxygen-scavenging mechanism. Finally, it has been well documented that probiotic viability rapidly decreases within dairy foods above refrigeration temperatures and therefore a rigorous cold chain is mandatory to ensure their quality and functionality, entailing high costs and a complicated logistic for their distribution (Trujillo-de Santiago and others 2012).

Cheeses provide a valuable alternative compared to fermented dairy as vehicles for probiotic delivery (Karimi and others 2012a,2012b). They have been shown to be a favorable environment for probiotic bacteria during both storage and GI transit, since their solid matrix and fat content protects probiotics from harsh conditions (Escobar and others 2012). However, not all cheeses behave on the same way as probiotic carriers because both compaction level and pH has a strong influence on the viability, release, and metabolic impact of certain probiotics (Escobar and others 2012; Lollo and others 2012). Also, in case of nonrefrigerated foodstuffs such as baked goods (Altamirano-Fortoul and others 2012; Trujillo-de Santiago and others 2012; Zanjani and others 2012), and beverages (Gawkowski and Chikindas 2013), the thermoresistance of probiotic strains has severely limited their incorporation within these foods.

In conclusion, due to the several physicochemical changes occurring during the preparation and storage of probiotic foods and due to the complex metabolism implicit to each strain, several food technologies should be employed to guarantee the survival of probiotics within dairy (Gomes and others 2011; Ibarra and others 2012) and nondairy formulations (Granato and others 2010a,b). Among them, immobilized cell technologies (ICT), such as ME, may reduce the contact between these bacteria and their physicochemical stressors (Selmer-Olsen and others 1999; Heidebach and others 2012).

Methods for Bacterial Immobilization

The loss of probiotic viability within food products (especially fermented ones) and in the acidic-bile conditions of the GI tract has encouraged researchers to find new efficient methods for improving bacterial viability (Mortazavian and others 2007). The chemical nature and physicochemical properties of a particular coating material intrinsically dictate the most convenient entrapment method. The selection of an entrapment method depends on many factors, including the potential for large-scale production, cost, particle shape and resistance and, most importantly, the resulting viable bacterial count. A comprehensive decision pathway for selecting a convenient immobilization technique is depicted in Figure 1. Freeze- and spray-drying methods applied to direct probiotic cultures maintain their viability within foodstuffs and pharmaceutical formulations and, in some cases, improve the resistance of the microorganisms during their passage through the GI tract (Dolly and others 2011; Figueroa-Gonzalez and others 2011). The low temperatures and water sublimation that occur during freeze-drying are less harmful to microorganisms than the higher temperatures used in spray-drying, and the bacterial viability is improved when drying with coating materials (Oliveira and other 2007). However, not all strains can be subjected to freeze- and spray-drying methods without dying in the process (Leja and others 2009; Ghandi and others 2013).

Figure 1.

Cell immobilization techniques.

Other alternatives for increasing probiotic viability are ME and other immobilization techniques. ME is the process of forming a continuous coating around an inner matrix that is wholly contained within a capsule wall (Figure 2), while immobilization refers to the trapping of materials throughout a particular matrix (Vidhyalakshmi and others 2009). ME is commonly used to pack solids, liquids, or gases in micrometric beads consisting of various materials, usually via extrusive/coacervation methods when immobilizing microorganisms. For example, alginate beads can be formed by both extrusion and emulsion methods (Krasaekoopt and others 2003). These beads can release their content at a controlled rate and enable a gradual interaction of the inner materials with the environment under certain physicochemical conditions, such as for bacteria within the GI milieu (Parra 2010). Recently, the viability (within foods) and bioavailability (within the host) of probiotic bacteria were shown to be 5 times higher when using ME (Del Piano and others 2010), leading to an extended shelf-life of food products. Moreover, multistage coating further increases bacterial survival (Mokarram and others 2009). However, there are still significant hurdles with respect to currently available methods for probiotic ME . This is mainly due to the fact that important physicochemical characteristics of microcapsules (such as particle size and type of covering material) appear to be in conflict with the requirements arising from their application in certain food products (Heidebach and others 2012). Lastly, other immobilization techniques include ultrasonic spray-drying (Semyonov and others 2011), fluidized bed coating (Goderska and Czarnecki 2008), electrostatic (Anal and Singh 2007; Ding and Shah 2009), pan- and air suspension-coating (Semyonov and others 2012), vibrational nozzle, and in-situ polymerization methods. Excellent reviews addressing these methods have been published (Vidhyalakshmi and others 2009; Cook and others 2012).

Figure 2.

Microencapsulation of probiotic bacteria. Dotted circles represent bacteria–bacteria and bacteria-coating material interactions.

Selection of Probiotic Bacteria

The design of a probiotic food begins with the function-specific selection of the strain (Islam and others 2010). Several health protection mechanisms of probiotic microorganisms are exerted within the gut and internal organs (Prakash and others 2011): (a) production of pathogen-inhibitory substances, (b) competition with pathogenic bacteria for epithelial adhesion sites, (c) nutrient competition and production, (d) degradation of toxins and toxin receptors, and (e) modulating immune and nonspecific host responses. From a clinical standpoint, the expected functionality is generally associated with the prevention and/or treatment of GI and immunological disorders associated with a great number of organic disturbances, ranging from a simple decrease in intestinal motility (Itsaranuwat and others 2003) to more complex immunological difficulties (Gill and Prassad 2008). In particular, the benefits of probiotics related to allergic disorders, such as atopic eczema (Kalliomaki and others 2001) or acute rhinitis (Giovannini and others 2007), and intestinal disorders, such as constipation and diarrhea (Allen and others 2012), irritable bowel syndrome (Hun 2009), and Crohn's disease (Shen and others 2009), have been supported by numerous studies, with species/strain-specific patterns being observed (Prakash and others 2011).

The genera Lactobacillus, Bifidobacterium, and Bacillus have been widely used to prepare ready-to-eat foods and tested in controlled trials (Gawkowski and Chikindas 2013). However, the expected functional effect of the bacteria is determined by the probiotic strain and other factors, such as the timing of the intervention, the stage of disease progression (Kalliomaki and others 2001), and the host's predominant microflora (Prakash and others 2011) and metabolic condition (Thomas and Greer 2010).

In a systematic PubMed search performed in March 2013, Lactobacillus and Bifidobacterium were found to be the genera that were most commonly reported as “probiotic” (MESH; J02.500.456.500; 10506 entries), returning 4500 and 1881 entries, respectively. Both genera consist of Gram-positive lactic acid producers that colonize the human gut at birth and during breastfeeding (Moore and others 2011; Prakash and others 2011), but nearly disappear in adulthood (Gavini and others 2001). Lactobacillus is the main genus found in marketed foodstuffs (BCC Research 2011), and several species have been patented. Several species and strains belonging to this genus have been shown to prevent antibiotic-, rotavirus-, and Clostridium difficile-associated diarrhea (Guandalini 2011), in addition to conferring other health benefits. Among Lactobacillus species, LA (894 PubMed entries) is one of the main probiotic species colonizing animal and human guts (Moore and others 2011) and when consumed in a sufficient amount, they are capable of creating a balance between beneficial and potentially harmful microflora (Šušković and others 2001). The metabolic activity of LA in fermented foods results in the production of characteristic organoleptic properties and inhibits food spoilage (Jafarei and Tajabadi 2011). Comparative genomic analysis between Lactobacillus species has identified unique gene targets in LA that are responsible for functional properties such as adhesion to mucin and intestinal epithelial cells, acid and bile tolerance, and quorum sensing (Klaenhammer and others 2008). Lactobacillus rhamnosus (880 PubMed entries) and L. casei (635 PubMed entries), 2 other strains commonly found in marketed foods, differ in their genomic content, metabolic and mucus-binding capacities, and host-signaling capabilities (Douillard and others 2013).

Bifidobacterium lactis (403 PubMed entries) and Bifidobacterium longum (354 PubMed entries) are the most frequently mentioned probiotics in PubMed from the genus Bifidobacterium, which includes 32 species of Gram-positive anaerobic, ramified bacteria with no motility. Studies addressing the distribution of bifidobacteria in feces from infants and adults have shown a typical life-cycle adaptation (Gavini and others 2001). As in the case of Lactobacillus spp., Bifidobacterium spp. also inhibit intestinal colonization by harmful bacteria, display antidiarrheal properties, and produce bioactive compounds, such as conjugated linoleic acid (CLA) isomers (Russell and others 2011). Lactobacillus spp. and Bifidobacterium spp. are usually found together within commercial foodstuffs (mainly in yogurt-like and fermented commodities) with GRAS and/or FOSHU recognition (Allen and others 2012). However, the efficacy of these probiotic foods is limited by their short shelf-life. As mentioned in a previous section, oxygen toxicity is widely considered to be responsible for bacterial death (Talwalkar and Kailasapathy 2004): as the oxygen concentration rises, the levels of lactic acid produced by LA and the lactate:acetate ratio produced by bifidobacteria is reduced.

Another group of bacteria with confirmed probiotic effects in vitro and in vivo is the endospore-forming, microaerophilic Gram-positive bacteria that belong to the Bacillus, Sporolactobacillus, and Brevibacillus genera (Jurenka 2012). When sporulated, these bacteria are metabolically inactive but are extremely resistant to harsh treatments such as extreme heat, drying, freezing, chemical treatments, and radiation (Furukawa and others 2005), although they are unstable in food systems with high Aw such as fruit purees (Cerruti and others 2000). Due to their dormancy and resistance, these spores can survive for years in the absence of nutrients, but when a proper stimulus is detected by germinant receptors (GRs), they can rapidly germinate and grow (Ramirez-Peralta and others 2012).

Bacillus coagulans (BC, formerly Bacillus sporogenes), a thermotolerant facultative anaerobe lactic acid bacterium, is the most common species of the genus Bacillus in manufactured goods. BC (37 PubMed entries) has not been studied as much as Bacillus subtilis or Bacillus cereus, although it has resulted in the greatest number of pharmaceutical and prepared food developments (including bread and low-calorie sweeteners) and it is not considered harmful (Bora and others 2009; Jurenka 2012). The complete genome sequence of BC strain 36D1 was recently published by Rhee and others (2011), and its predicted proteome shows a dual phylogeny with Bacillus spp. and Lactobacillus spp. It is noteworthy that the development of novel food products containing BC coincide with the publication of controlled trials supporting its probiotic potential (Baron 2009; Drago and De Vecchi 2009). In particular, Ganeden BC30 ™ (patent name for BC GBI-30, 6086; 9 PubMed entries) is commonly found in the international pharmaceutical market and has recently been commercialized as a food ingredient. From a technological perspective, GanedenBC30 withstands high-temperature processes such as baking and boiling, low-temperature processes such as freezing and refrigeration, and high-pressure applications like extrusion and roll forming. From a clinical perspective, this microorganism is nonpathogenic, nontoxigenic, and is recommended for the treatment of some immunological and inflammatory disorders (Jensen and others 2010; Kimmel and others 2010) due to its capacity to support the maturation of mononuclear phagocytes toward both macrophage and dendritic cell phenotypes and to reduce the numbers of CD14+ CD16+ pro-inflammatory cells (Benson and others 2012). Ganedenbc30™ is currently on track for getting the GRAS approval by the U.S. Food and Drug Administration (FDA) for use in food and beverages (Basiotis and others 2011).

Selection of Coating Materials

Another aspect to consider when microencapsulating probiotics is the chemical nature of the coating material. It has been extensively shown that ME techniques increase the viability of probiotics both within foods and during their passage through the GI tract (Chandramouli and others 2004). However, coating materials behave in structurally different ways and, therefore, their capacity to protect living microorganisms and/or deliver bioactive substances also varies (Riaz and Masud 2013). Also, the effectiveness of any material depends not upon its capsule-forming capability, strength, and enhancing viability but also on its cheapness, availability, and biocompatibility. Many materials have been used to immobilize bacteria, such as gelling polysaccharides (such as starch, cellulose, alginate, pectin, carrageenan, and chitosan), proteins (soy, whey, casein, gelatin, and β-lactoglobulin), and lipids (waxes), all of them extensively studied (Reid and others 2007; Imran and others 2010; Rokka and Rantamäki 2010). The most commonly used materials in the ME of Lactobacillus spp. and Bifidobacterium spp. are ionic polysaccharides (specially alginate and chitosan), microbial exopolysaccharides (for example gellan and xanthan gums), and milk proteins (Truelstrup-Hansen and others 2002; Islam and others 2010), while several different lipid emulsions have been used for ME of Bacillus sp. This strain-specific material selection is a result of several factors, most notably the accumulated evidence regarding the physicochemical properties of new materials or the molecular interactions between these materials, the bacteria, and the host's GI epithelial tissue (Alli and others 2011; Prakash and others 2011).

In addition to the protection capacity of coating materials preventing bacterial degradation along the GI tract, selection of materials with convenient biochemical features is highly desirable. For example, excellent muco-adhesive properties are typical of hydrophilic polymers such as alginate and chitosan, and this property is useful for enhancing the in situ delivery of bacteria along the GI tract (Gombotz and Wee 2012; Chen and others 2013). Hydrophilic polymers possess charged and/or nonionic functional groups capable of forming hydrogen bonds with mucosal surfaces (Dhawan and others 2004; Khutoryanskiy 2011). ME with alginate, an anionic/acidic diheteroglycan [(1-4)-linked-β-d-mannuronate (M) and its C-5 epimer α-l-guluronate (G); Figure 3] extracted from the cell wall of brown algae, increases the odds of survival for several species from the Bifidobacterium and Lactobacillus genera by up to 80% to 95% (Krasaekoopt and others 2004; Prakash and others 2011). Strains microencapsulated in alginate have been included in food formulations such as ice cream, frozen yogurt, and mayonnaise (Kebary and others 1998; Khalil and Mansour 1998; Krasaekoopt and others 2003). From a molecular stand point, alginate in the presence of Ca2+ produces a particularly strong molecular framework. As a result, cold-prepared, thermoirreversible, and freeze-thaw-stable microcapsules can be obtained. However, to form a gel with calcium, alginates need to contain sufficient amounts of G-monomers, a certain proportion of which must occur in G-rich blocks. Ca2+ fits into G blocks such as “eggs in an egg box” (Onsøyen 2001), binding alginate polymers together by forming junction zones (Figure 4). The alginate-to-calcium concentration and the ionic strength of the medium influence the viscoelastic properties of alginate beads (Ouwerx and others 1998), while the muco-adhesion force of alginate increases with its concentration, showing pseudoplastic behavior (Kesavan and others 2010). However, phosphate, citrate, and chelating agents compete for calcium ions, thus weakening alginate gels.

Figure 3.

Alginate: α-l-guluronic acid (G), β-d-mannuronic acid (M) monomers and block types.

Figure 4.

Alginate requires G-G blocks to form a gel, which is known as the “egg box” model.

Chitosan, a linear cationic/basic homoglycan [β-(1-4)-linked d-glucosamine and N-acetyl-d-glucosamine], mainly obtained from crustacean exoskeletons, modifies the gut microbiota (with the exception of Bifidobacterium strains; Koppová and others 2012) through its antibacterial activity (Goy and others 2009). The first reports addressing the use of chitosan to immobilize lactic bacteria originated in the beginning of the 20th century (Le-Tien and others 2004). Improved stability under simulated GI conditions has been observed for L. casei (Koo and others 2001), Bifidobacterium breve (Cook and others 2011), L. gasseri and Bifidobacterium bifidum (Chávarri and others 2010) following ME using alginate and chitosan. Structural studies have shown highly stable polyelectrolyte complexes (Figure 5) between alginate (anion) and chitosan (cation), either in the form of multilayer films (Lawrie and others 2007) or rough microspheres (Lucinda-Silva and Evangelista 2005), which explains the observed resistance to harsh GI conditions and colon delivery of L. rhamnosus and LA (Sohail and others 2011). However, the amount of chitosan and the method used to produce microcapsules modify the gut muco-adhesion properties of chitosan (Dhawan and others 2004) and alginate-chitosan particles (Wittaya-areekul and others 2006). Additionally, ME of LA through the layer-by-layer self-assembly of chitosan and carboxymethyl cellulose (3 nanolayers) not only enhances its survival rates in simulated gastric (SGF) and intestinal fluids (SIF) but also serves to reduce viability losses during freezing and freeze-drying (Priya and others 2011).

Figure 5.

Alginate G-monomer (A)-chitosan glucosamine residue (B) interactions.

Microbial polymers, proteins, and peptides have also been used as coating materials for colon-targeted delivery of probiotics (Jimenez-Pranteda and others 2012a). Alginate combined with gellan gum, a linear acidic triheteroglycan [d-Glc(β1→4) d-GlcA(β1→4) d-Glc(β1→4)l-Rha(α1→3)] exopolysaccharide obtained from Sphingomonas paucimobilis (Fialho and others 2008), improves the thermotolerance of B. bifidum (Chen and others 2007). When alginate is combined with xanthan gum, an improved survival of LA (LA14) and B. lactis (BI-07) in acidic conditions was observed (Albertini and others 2010), while gellan combined with xanthan gum in a 0.75:1 ratio improved the survival of Lactobacillus plantarum and L. rhamnosus against simulated bile (Jimenez-Pranteda and others 2012b). Alginate combined with whey protein in a stable proportion (40%/60%) produces a highly cross-linked matrix (Figure 6) that protects microparticles from acid digestion (pH 1.2) but dissolves at pH 7.5, which is a phenomenon that is highly desirable for the delivery of drugs (Hebrard and others 2013) and bifidobacteria (Picot and Lacroix 2004; Hebrard and others 2010). This phenomenon is also observed for B. lactis (Bi 01) and LA (LAC 4) when they are ME in a casein/pectin complex (Oliveira and others 2007). It is noteworthy that, among the basic amino acids, only histidine possesses the ability to form polyelectrolyte complexes with alginate near a physiological pH (pKR = 6.0). However, only 2 histidines are present in the 178 amino acids (19.9 Da) of β-lactoglobulin (β-LG), which is the major whey protein found in the milk of ruminants. Additionally, polyelectrolyte complexes with poly-l-lysine (PLL) have been obtained, which confer mechanical stability, selective permeability, and bacterial immunoprotection (Prakash and others 2011).

Figure 6.

Hypothetical interactions between basic amino acids (P) and alginate (G-monomer). Exposed R-groups from the tetrapeptide Arg-R-His-Lys (outside gray circle).

Lastly, prebiotic ingredients have also been used as encapsulating agents. Their advantages over other covering materials rely on the fact that, besides being nondigestible carbohydrates, they also have beneficial effects for the host, by selectively stimulating the growth and/or activity of probiotic bacteria (prebiosis) within the colon (Fritzen-Freire and others 2012). Formulations with pre and probiotics are known as Synbiotics which have been shown to enhance the host`s immune response upon regular consumption (Gourbeyre and others 2011; Jirillo and others 2012). Synbiotics have been successfully included in cheese (Alves and others 2013), yogurts (Malpeli and others 2012), and ice creams (Han and others 2012). However, although functional drinks, and other probiotic functional foods, are available in a relatively high number, the market of prebiotics (including Synbiotic formulations) is still small and fragmented (Bigliardi and Galati 2013).

Inulin [highly polymerized fructose (PF)], oligofructose (low PF), and resistant starch are three of the most commonly used prebiotics for ME (Escobar and others 2012; Fritzen-Freire and others 2012). All 3 have been scarcely used alone but there are several studies that report their combination with other dietary carbohydrates (Martin and others 2013; Okuro and others 2013) or proteins (Bedani and others 2013; Fritzen-Freire and others 2013) with which they form stable molecular complexes, leading to different ME products with specific physicochemical and physiological features. For instance, Raftilose P95 when added at 1.5% (w/v) to yogurt improves the viability of Lactobacillus spp. and Bifidobacterium spp. by 1.42 log during 4 wk of storage at 4 °C (Capela and others 2006), spray-dried microcapsules made with different combinations of reconstituted skim milk and inulin or oligofructose showed higher protection for Bifidobacterium BB-12 under simulated GI conditions and under extreme heat conditions (Fritzen-Freire and others 2013), and ME in calcium alginate plus resistant starch increased the survival rate of L. acidophilus La5 in Iranian white brined cheese after 6 mo of storage (Mirzaei and others 2012).

Cell Adhesion and Functional Group Interactions

The nature of all of the possible physicochemical interactions between the bacterial cell wall, the host's intestinal mucosa, and the coating material is another important consideration. While adhesion of the cell walls of probiotics (Van Tassell and Miller 2011, Figure 7c) and complex carbohydrates (Khutoryanskiy 2011; Figure 7d) to the host's intestinal epithelial mucosa (Figure 7b) has been well documented in many scientific reviews, this triad as a whole (Figure 7e) has not been studied in depth. From a structural standpoint, improved bacterial survival and delivery within the colon depend on the type, number, and bonding strength of all functional groups located in the bacterial cell wall, the coating material, and the cross-linkers. However, physicochemical studies addressing the role of these interacting groups are still scarce, although this aspect is of particular importance for the efficient delivery of probiotic bacteria at different stages during their GI passage (Anal and Singh 2007).

Figure 7.

Hypothetical interactions between the bacterial cell wall, a coating material (chitosan) and intestinal mucin. (A) Gray circles represent intestinal epithelial cells, and white circles represent goblet cells. (B) Mucin-containing proteins (MUC) are characterized by tandem repeats of Thr, Pro, and Ser residues with O-linked-(N-GalNAc) oligosaccharides (broken lines) and cysteine-rich regions (Cis-Cis blocks), which promote intermolecular disulfide bonding. (C) Bacterial adhesion and biofilm formation over the intestinal mucosa depends on many interactions between the ionic and hydrophobic species present in mucin and complementary adhesion groups located in bacterial cell wall murein (gray circle), including teichoic acid (TA), lipoteichoic acid (LTA), mucin-binding proteins (MBD), and cell wall lectins (CWL). Some of these interactions are cross-linked with calcium ions. (D) Chitosan (D1, cation) binds to glycosylated mucin residues such as sialic acid (D2, anion) through noncovalent interactions (electrostatic and hydrogen bonds). (E) Ionizable groups located in each of the 3 components of the triad (bacteria-coating material-mucin) interact with each other in a competitive manner.

The cell wall of Gram-positive bacteria is a dynamic structure. Several enzymes coordinately assemble, transport, polymerize, and cross-link all of the cell wall precursors (Morata de Ambrosi and others 1998). Protein and carbohydrate moieties embedded in the bacterial cell wall promote the adherence to the intestinal mucosa (van Tassell and Miller 2011; Yasuda and others 2011) and luminal carbohydrates and participate in biofilm formation (Ambalam and others 2012). The major structural component of the bacterial cell wall is peptidoglycan (a.k.a. murein; Figure 7c, gray circle), a complex polymer made up of glycan strands of repeating disaccharide residues cross-linked via short peptides and organized in a “helical cabling mesh,” which is responsible for producing the closed-bag configuration of the bacterial cell wall and its viability (Hayhurst and others 2008; Jafarei and Tajabadi 2011). The cell walls of Lactobacillus spp., Bifidobacterium spp. and Bacillus spp. contain 30 to 40 layers of murein associated with covalently bound polymers derived from teichoic acid (TA, an anionic phosphate-rich polymer, accounting for 50% to 60% of the cell wall weight), lipoteichoic acid (LTA, a macroamphiphile polyglycerol-phosphate polymer derived from TA), teichuronic acid (a polysaccharide consisting of d-glucose and N-acetyl-d-mannosaminuronic acid), and other “non classical” polysaccharides (Jafarei and Tajabadi 2011; Potekhina and others 2011). Two important features of TA that contribute to cell adhesion, biofilm production, and immunoreactivity are the branching of TA from the cell wall and beyond and the presence of d-alanine residues; both contribute to the cell wall polarization, TA binding capacity, electromechanical properties, and the resistance to antimicrobial cationic peptides (Neuhaus and Braddiley 2003). Murein, TA, and LTA form a polyanionic matrix that exhibits functions related to the elasticity, porosity, tensile strength, and electrostatic steering of Gram-positive cell walls (Neuhaus and Braddiley 2003).

However, TA is quite diverse in its structure and abundance, depending on the strain, rate of growth, pH of the medium, carbon source, and availability of phosphate ions (Jafarei and Tajabadi 2011). For example, in BC (AHU 1638) the main linkage oligomer is Glc(β1→4)GlcNAc, and TA is conjugated to poly(galactosyl(1→2)glycerol phosphate) (Potekhina and others 2011), while other polyglycerol-phosphate derivates of LTA are more prominent in B. bifidum and L. rhamnosus ATCC 7469. In L. lactis, the distribution of d-alanyl esters correlates with the random distribution of α-d-galactopyranosyl residues of LTA (Neuhaus and Braddiley 2003). It is noteworthy that bile salts upregulate genes from the dlt operon in L. rhamnosus GG (Koskenniemi and others 2011) and L. plantarum (Bron and others 2006), including those involved in d-alanylation of LTA; this transcriptional regulation increases the net charge and hydrophobic protein content of the bacterial cell walls, but reduces the hydrophobicity of LTA, thus modifying the bacterial capacity to produce biofilms (Ambalam and others 2012) and to interact with the intestinal epithelium (Figure 7a) and its mucin layer (Figure 7b), which are hydrophobic (Ambalam and others 2012).

Intestinal mucin-associated glycoproteins polymerize, forming a framework involving extensive disulfide bonding between cysteine-rich regions of protein cores (Figure 7b). It creates the characteristic viscoelastic mucus (van Tassell and Miller 2011). Lactobacilli spp., Bifidobacterium spp., and Bacillus spp. can adhere to this mucus by cell wall murein polysaccharides (Morata de Ambrosini and others 1998), mucin-binding proteins (MBP, van Tassell and Miller 2011), and lectins (CWL, Yasuda and others 2011). While TA and its d-alanyl residues (among other functional groups) drive the adhesive electrostatic force, LTA contributes to the hydrophobic interactions between bacterial cell walls and mucus. Additionally, as mentioned earlier, mucoadhesion of complex polysaccharides is also necessary for targeted GI delivery of entrapped bioactive substances (Thongborisute and others 2006). As a consequence, the mucoadhesion of bacterial cell walls and luminal polysaccharides results from concerted passive, electrostatic, hydrophobic, and steric interactions (Dianawati and Shah 2011).

Based on the above information, the existence of physicochemical interactions between bacterial cell walls and ME coating materials is not surprising (Figure 7e). In fact, these interactions are the basis for the immobilization of bacteria for soymilk fermentation (Lye and others 2012), functional sweetener production (Xu and others 2012), and bacterial protease release (Mrudula and Shyam 2012). For instance, interactions with cationic materials such as chitosan are mediated by electrostatic forces with negatively charged moieties (specifically TA) present in the cell wall, presumably due to competing with available calcium ions (Figure 7c). Because this interaction results in cell wall distortion-disruption and exposes bacteria to osmotic shock, such cationic polysaccharides are considered potent (<200 ppm) antimicrobials against Gram-positive bacteria, such as Staphylococcus aureus and Listeria monocytogenes, and, at higher concentrations, against Lactobacilli (Goy and others 2009), while oleoyl-chitosan nanoparticles (a more lipophilic particle) inhibit Escherichia coli and S. aureus by damaging the cell membrane and other intracellular targets (Xing and others 2009). Triantafilou and others (2012) demonstrated that blood proteins such as apolipoproteins, LDL, and transferrin inactivate LTA, reducing its proinflammatory potential. This finding may indicate that dietary proteins can also bind to LTA, reducing or facilitating bacterial release within the GI tract. However, the number of studies investigating the role of other coating materials is insufficient to draw further conclusions regarding the nature of their interaction with probiotic cell walls and their metabolic impact within the host.

Thermotolerance of ME Live Bacteria

Once the critical requirements are established and immobilized probiotic bacteria have been obtained, the shelf-life (viability within foods) and resistance to GI conditions (bioavailability within the host) must be analyzed. Temperature is a critical factor for the viability of probiotic bacteria in refrigerated and nonrefrigerated foodstuffs (Altamirano-Fortoul and others 2012). At temperatures ≤4 °C, the survival of LA and some bifidobacteria improves for several weeks (Sakai and others 1997), but freeze-drying at lower temperatures results in a lower survival rate (Dianawati and Shah 2011). Mosilhey (2003) found that microencapsulated LA was stable up to 15 wk at 5 °C, which makes it ideal for use in refrigerated foods, such as dairy products (Shah 2000). However, the viability of LA is largely dependent on the type of coating material employed: a greater loss of viability occurs under refrigerated storage conditions when using acacia gum (AG) or gelatin (GE) as a coating material compared to AG combined with protein (SP) or soymilk (SM).

In contrast, the number of reports addressing the effects of ME on bacterial tolerance at high temperatures is limited. In theory, the viability of encapsulated bacteria increases in direct proportion to the amount of coating material (Chen and others 2007). Mosilhey (2003) determined the effect of different temperatures on LA microencapsulated with different combinations of materials. Non-ME LA showed a decrease in viability from 1010 to 102 CFU/g between 45 and 65 °C for 30 min. Using 65 °C as a reference temperature, the loss of viability in LA microencapsulated in AG, SP, SM, gelatin (GE), and whey protein (WP) was evaluated using LA suspensions of 109 to 1010 CFU/g. Coating LA with GE or AG was found to increase its resistance to temperature-inactivation resulting in a viability of 103 to 104 CFU/g. Similar results were observed by Kim and others (2008) for LA (ATCC 43121) microencapsulated in alginate and by Mandal and others (2006) for L. casei (NCDC-298) microencapsulated in alginate, though with slight variations in the temperature range. The most important contribution reported by Mosilhey (2003) was the observation that the formation of protein–carbohydrate crystallizable complexes (AG + SM, SP, and WM) improved LA viability in thermal treatments conducted at 60 °C (104 to 106 CFU/g) and 63 °C (104 to 105 CFU/g) for 30 min; AG + SM was the only combination that resisted a temperature of 65 °C (102 CFU/g). During the process of AG (anion) coacervation with WP or SP (cations), the number of electrostatic interactions (Figure 6) is directly related to the viscoelastic properties of the resulting AG + WP colloidal complex (Salazar-Montoya and others 2012), increasing its thermotolerance.

Recoating of “primary” microcapsules with additional materials can help to prevent the exposure of probiotics to oxygen during storage and improve their stability at a low pH and high temperature. Some of the materials employed for recoating include chitosan, poly-l-lysine, alginate, and several starches, gums, and gelatins (Krasaekoopt and others 2004; Mortazavian and others 2007, 2008). However, no significant differences were found in the viability of microencapsulated and nonencapsulated LA 43121 in prebiotics such as fructooligosaccharides, lactulose, and raffinose when polyvinyl acetate phthalate was used as a coating material (second layer) in an aquose system (Eun and others 2007). Nevertheless, double-ME increased bacterial tolerance at 55 °C for 180 and 240 min as compared to single-ME, indicating that heat tolerance can be improved by using a double layer of prebiotics as a coating material. In this regard, Chen and others (2007) evaluated the survival of B. bifidum treated with heat (75 °C for one min), gastric juices, and bile salts after 2 mo of storage using prebiotics containing different concentrations of the bacterium and 2% sodium alginate mixed with 1% gellan gum as coating materials. Following these treatments, the probiotic count remained at 105 to 106 CFU/g for the microcapsules. However, in the studies mentioned above, the temperatures did not exceed 75 °C, and the exposure time tended to decrease as the temperature increased.

Spore-forming Bacillus bacteria, like BC, are highly resistant to physical threats such as heat, drying, radiation, and chemical agents such as hydrogen peroxide (Furukawa and others 2005). Under controlled culture conditions, sporulation can be induced in solution or solid cultures. Sporulation confers protection against sterilization at high temperatures in an aqueous environment, enables the formation of biofilms in a solid environment (Kolari and others 2001), and creates true bridges of communication between the vegetative and growing states (Shank and others 2011). Furukawa and others (2005) described how sporulation during heat treatment (85 °C for 30 min) increased the heat resistance and survival of BC, B. cereus, and Bacillus licheniformis. The relative hydrophobicity of the spore surface was shown to be increased during the treatment, contributing to the formation of spore clusters. Rosemberg and others (2005) observed that incubation at 70 °C for 25 min eliminated all vegetative forms of BC. In spite of the apparent resistance of BC to temperature and pH, the Aw in finished goods should be taken into account to ensure BC availability, as its vegetative state (spore) is lost at Aw > 0.9 (Cerrutti and others 2000). Based on these findings, heat treatment of spores prior to ME may increase their thermal resistance (Bora and others 2009) and water permeability. However, in a systematic PubMed search conducted in March 2013, no studies were found addressing the ME of BC (MESH, B03.510.100.100.218).

Finally, a few investigators have attempted to incorporate probiotics into food under high temperature (including pasteurized or baked products). Most probiotic foods on the market are either refrigerated (yogurts and milk-derived products) or are stored/consumed at room temperature (mayonnaise), and even novel foods consisting of fermented cereal substrates (Rathore and others 2012), such as emmer beverages (Coda and others 2011), are produced and stored at low temperatures. ME of LA in calcium alginate has been found to improve heat tolerance under temperatures of 55 to 65 °C for 20 min (Selmer-Olsen and others 1999). Soares and others (2004) evaluated the thermal breakdown of alginic acid (HAlg) and sodium alginate (NaAlg) in the presence of N2 and air (90 mL/min air pressure) using differential scanning calorimetry (SCD) and observed 2 and 3 breakdown phases, respectively. The first phase (endothermic) observed in the acid corresponded to a dehydration reaction occurring at 80 °C (N2) or between 75 and 96 °C (air), whereas the first phase occurred at higher temperatures for NaAlg. Despite the thermotolerance of alginate, its use has some disadvantages, as capsules composed of this material are more sensitive to acidity (Mortazavian and others 2008). Krasaekoopt and others (2004) demonstrated that LA in alginate microcapsules coated with chitosan shows increased survival in bile salts as compared to uncoated bacteria.

The probiotic resistance to temperatures between 65 and 100 °C observed when LA is microencapsulated in alginate, with or without copolymerization with other materials (for example proteins), may represent an area of opportunity for the possible incorporation of this microorganism into baked and/or pasteurized products. Altamirano-Fortoul and others (2012) recently assessed the viability of LA microencapsulated in a complex matrix of WP, CMC, pectin, inulin, and fresh agave syrup via spray-drying, exposed at 65 °C, 2 bar of pressure, for 135 min. Subsequently, the microcapsules were resuspended in various starch solutions (1% to 2%, w/v), sprayed evenly (118 cm2) over a cold dough (0.1 to 0.2 g/bread), and the bread was baked for 16 min at 180 °C. Before baking, the bread crust contained 108 CFU/g of LA, which was reduced to 107 and 106 CFU/g after baking and 24 h of storage, respectively. From the data presented above, it is clear that additional layers and materials with a different chemical nature can provide improved thermotolerance to microencapsulated probiotics.

Resistance of ME Live Bacteria to GI Conditions

The resistance of various probiotic bacteria, with or without ME, to simultaneous changes in pH and in the presence of gastric juices has also been examined. Mokarram and others (2009) studied the influence of the number of layers of alginate on the survival of LA (PTCC1643) and L. rhamnosus (PTCC1637) in simulated GI conditions. In this study, ME with a double layer of alginate (about 100 μm) was found to provide the best protection against a loss of viability for both bacteria in gastric (pH 1.5, 2 h) and intestinal (pH 7.25, 2 h) juices. Chandramouli and others (2004) and Kim and others (2008) also showed that ME with alginate increased the survival of LA CSCC 2400 and ATCC 43121, respectively, in GI conditions. Lyer and Kailasapathy (2005) reported that LA microencapsulated in 1 or 2 layers of chitosan and resistant starch showed a loss of viability of 1 × 102 or 3 × 103 CFU/g, respectively, in simulated GI conditions, while in non-microencapsulated LA the observed loss of viability was 1 × 105 CFU/g.

Mandal and others (2006) found that the viability of L. casei (NCDC-298) improved as the concentration of alginate increased at pH 1.5 for 3 h, and Li and others (2010) confirmed this result in L. casei (ATCC 393) microencapsulated in alginate-chitosan-carboxymethyl chitosan. Similar results were reported by Sun and Griffiths (2000) for Bacillus infantis ATCC 15697 microencapsulated in gellan gum (0.75%) and xanthan (1%) compared to the non-microencapsulated bacteria at pH 1.5 to 2.5. Sun and Griffiths (2000) also found that microencapsulated B. infantis added to pasteurized yogurt survived 5 wk under refrigeration. However, Trindade and Grosso (2000) reported that ME of B. lactis and LA with calcium alginate was ineffective in protecting cells exposed to bile salts at concentrations of 2% and 4%, respectively.

Studies describing GI resistance when several bacteria are examined simultaneously have highlighted a species-specific trend. Ding and Shah (2009) evaluated the effect of acid (pH 2.0) on the viability (1010 CFU/g) of LA, L. rhamnosus, B. longum, L. salivarius, L. plantarum, L. paracasei, and B. lactis (type BI-O4 and Bi-07) microencapsulated in guar and xanthan gums, carob, alginate, and carrageenan. LA was one of the microorganisms that was most tolerant (105 to 108 CFU/g) to acidity (pH 2.0, 2 h) and to bile salts (taurocholic acid), especially when it was microencapsulated in alginate, xanthan gum, and carrageenan.

Resistance to bile salts also depends on the type of coating material applied. Murata and others (1999) coated chitosan with alginate and observed that chitosan has the ability to bind bile salts. This finding could indicate that the effectiveness of alginate in protecting a probiotic within its matrix may be decreased due to the incorrect selection of a second material, as in the case of chitosan. However, the formation of an insoluble complex between chitosan and bile salts takes place on the surface of chitosan-alginate microparticles. Therefore, the diffusion of bile salts into the matrix core can be limited, protecting the probiotic bacteria from interacting with the bile salts (Koo and others 2001; Yu and others 2001). Lucinda-Silva and Evangelista (2005) prepared alginate-chitosan microparticles and observed a reduction in their electrical conductivity, indicating a high level of coordination between these materials at a ratio of 1:1.5 to 1:1.7. Thus, a balanced ratio of coating materials is essential to reduce the numbers of unconjugated functional groups that are free to bind bacterial cell walls.

Sensory aspects of ME in probiotic foods

It is well known that the addition of probiotics to dairy and nondairy foods, modify their sensory attributes. Their addition to cheese in a suitable culture composition, does not considerably change the flavor and/or other sensory characteristics of the final product, when compared to control cheeses (Karimi and others 2012a). However, the impact of probiotic bacteria on cheese flavor is dependent on the bacterial strain used and its metabolic activity during cheese production and storage. In particular, excessive proteolysis and over acidification during cheese ripening decreases its acceptability by consumers (Michaelidou and others 2003; Milesi and others 2009; Karimi and others 2011). The selection of different probiotic strain has profound effects on the sensory attributes: Escobar and others (2012) studied the effect of probiotics and fava bean starch added to panela cheese (a soft cheese), observing that starchless cheese supplemented with L. rhamnosus GG (1 × 108 CFU/g) showed greater consumer acceptance in terms of cheese compactness, hardness, moisture, and softness, whereas that supplemented with B. breve scored better in creamy and milky flavor attributes. In probiotic ice creams, it has been observed that the original milk fat level, pH, probiotic strain used, overrun (incorporation of air) level, and the use of stabilizing hydrocolloids are critical quality parameters that modify consumer's sensory perception and preference (Soukolis and others 2010; Cruz and others 2012; Ferraz and others 2012). Nondairy foods (like probiotic fruit beverages) also change their sensory attributes upon addition and/or fermentation with probiotic bacteria (de Souza and others 2012; Hassan and others 2012). Although the generation of new sensory characteristics in probiotic-supplemented foods might be desirable in some cases, food habits of some consumers may find these characteristics unpleasant.

ME of probiotic strains can avoid many of the problems associated with their addition in free form. Undesirable characteristics, such as those mentioned above, in both dairy (Özer and others 2008, 2009) and nondairy formulations can be avoided by a convenient selection of a probiotic strain and an entrapment method. As mentioned in previous sections, the confinement of probiotic bacteria within semipermeable polymeric microspheres (1 to 1000 μM) enables the physical isolation of bacteria from the external environment while maintaining a hospitable internal microenvironment (Rathore and others 2013). The addition of microencapsulated L. casei has no significant effect on the sensory properties of nonfermented ice cream (Homayouni and others 2008) and cream-filled cakes (Zanjani and others 2012) and dry sausage batter containing either nonencapsulated or microencapsulated (in alginate) Lactobacillus reuteri show the same sensory profile (Muthukumarasamy and Holley 2006); interestingly, the ME of L. casei and B. bifidum improved the sensory characteristics of mayonnaises (Fahimdanesh and others 2012). However, it is mandatory to consider the shape and size of final microspheres do to their possibility to affect the palatability of products containing them. The smoothness of yogurt supplemented with microencapsulated (in alginate-starch) L. acidophilus and B. lactis (Kailasapathy 2006), the flavor of fermented liquid porridges (like mahewu) supplemented with B. lactis in gellan-xanthan gum (Kokott 2004), and the perception when swallowing fruit juices with L. casei microencapsulated in alginate (Krasaekoop and Kitsawad 2010), are just few examples of products in which the palatability is modified.

In conclusion, the commercial success of a probiotic food on the market necessarily implies that it meets the sensory needs of its target consumer, it is safe, and that its nutritional quality is guaranteed. Sensory evaluation is also mandatory before marketing since the product type and storage conditions might influence the sensory properties of the product due to organic acid production by overproduction of probiotic bacteria. (Céspedes and others 2013). In this sense, the addition of ME of probiotic bacteria to several prepared foods, while maintaining their viability in the food and during intestinal transit, avoids undesirable sensory modifications that are commonly seen when they are added in free form or when fermented with them.

Conclusions

Probiotic bacteria are live microorganisms that help to improve the GI health of consumers. However, it must be taken into account that these microorganisms easily lose their viability and stability due to various physical and physiological conditions to which they are subjected. In this context, ME is a technique that has proven effective in increasing the viability of probiotic bacteria because it protects them from harsh environments and is even involved in symbioses, as some of the coating materials employed are also used as prebiotics. Nevertheless, there is little available research addressing the structure and thermoresistance of probiotics when applied to nonrefrigerated commodities other than fermented milk beverages, yogurts, and pharmaceutical products. There are a wide variety of potential coating materials, although alginate is utilized most frequently, and bacterial viability depends largely on the type and concentration of the coating material and type of bacterium (sporulated or nonsporulated) involved. The selected ME technique will have a large influence on the viability of the associated probiotic bacteria, as will the coating of the capsules with another coating material (double encapsulation). Finally, despite advances in ME techniques and their applications, studies involving sporulated probiotic bacteria are scarce, although these species are inherently thermotolerant.

Acknowledgment

The authors thank the faculty improvement program (PROMEP) for partial funding of this work through the project “Evaluation of antioxidant and antimicrobial potential of extracts obtained from agro industrial by-products and their use in functional foods.” The authors declare no conflicts of interest and are not involved with any private or public organization that represents a conflict with the information provided here.

Author Contributions

All authors contributed equally in the conception, planning, and writing of this article. Corona-Hernandez compiled data, Wall-Medrano drafted the preliminary manuscript, Alvarez-Parrilla and Lizardi-Mendoza made all the original artwork while Islas-Rubio and de la Rosa performed the final editing and proofreading. All authors reviewed the final version of the manuscript.

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