Introduction

Probiotic is a term originated from Greek words meaning “for life” and the definition has been evolving since over time (Hill et al. 2014). More than a century ago, the concept of probiotic was introduced by Metchnikoff who stated that intake of Lactic acid bacteria would promote longevity. Since then, a term probiotic was often coined and used as an antonym of antibiotics (Lilly and Stillwell 1965). It was also suggested that feeding probiotics provide health benefit by modulating the microbial balance in the body (Fuller 1989). In the present, World Health Organization/Food and Agriculture Organization (WHO/FAO) defined that probiotics are live organisms that, when administrated in adequate amount, confers a health benefit to the host. Commonly used probiotic strains includes Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium breve and Bifidobacterium bifidum (Macfarlane and Cummings 1999).

Over the past decades, the market size of probiotics has greatly increased as modern consumer concern about health-promoting effect of nutraceuticals (Augustin and Sanguansri 2015). Since probiotic-containing products in general do not require for Food and Drug Administration approval, they are commonly available in the market in various food formats such as fermented milk, cheese, yogurt and juice (Sanders 2010). In recent years, probiotics have been extensively studied as a treatment option of various diseases such as obesity (Chen et al. 2014), diabetes (Lindsay 2015), cancer (Serban 2014), human immunodeficiency virus infection (Monachese et al. 2011), irritable bowel syndrome (Claes et al. 2010).

For probiotics to exert beneficial activities, a sufficient amount of probiotics should be alive and functionally active at the site of action as well as in a product (Cook et al. 2012). Probiotics are recommended to be present at a minimum level of 6 log colony forming unit (CFU)/g in a food product (Doleyres and Lacroix 2005) or 7 log CFU/g at the point of delivery (Lee and Salminen 1995). Due to the vulnerability of probiotics to harsh conditions during manufacturing, storage and passage through the gastrointestinal (GI) tract, however, it is difficult that viable probiotics successfully exert beneficial activities. During manufacturing and/or storage, the viability of probiotics can be negatively affected by several factors such as temperature, water activity and other food ingredients. Specifically, high temperature during manufacturing processes is a main reason for reduced viability because most probiotics have low thermo-resistance (Vesterlund et al. 2012). Maintaining viability in the stomach is another difficult task for probiotics to reach the target site because most of probiotics die or lose their functionality at acidic conditions. Next, survived probiotics should be released at the target site of action which is usually small or large intestine. Therefore, an ideal probiotic delivery system should protect probiotics from adverse conditions during fabrication and storage and in the acidic gastric environment so that the sufficient amount of probiotics is available in the site of action (Fig. 1).

Fig. 1
figure 1

Proposed ideal probiotic delivery

Full size image

In this review, we provide an overview of probiotic delivery, focusing on health benefits of probiotics, environmental factors affecting the probiotic viability and materials that are widely used for microencapsulation technology. Recently developed probiotic delivery technologies are also discussed.

Health benefits

The role of gut microbiota in human health has gained increasing attention. A number of studies found that human gut is colonized by diverse groups of bacteria species whose composition is strongly linked to GI health of each individual (DuPont and DuPont 2011). There are also growing evidences that administration of probiotics contributes to the microbial ecosystem which exerts a variety of health benefits including a prevention and/or treatment of diseases (Gareau et al. 2010). Recently the human microbiome project was launched to explore correlations of microbiomes with human health (Proctor and IHiR Network 2014). In this section, we briefly review the interaction of probiotics in a gut and their related health benefits. The potential mechanism of probiotics in gut is illustrated in Fig. 2.

Fig. 2
figure 2

Potential mechanisms of action of probiotics. 1 Probiotics inhibit pathogens by competing for nutrition and binding site, or by secreting anti-bacterial agents. 2 Probiotics enhance tight junction and promote secretion of mucins. 3 Probiotics contribute to intestinal homeostasis by immunomodulation effect

Full size image

Probiotics are able to inhibit pathogens by competing for nutrition and binding site and by secreting antimicrobial factors. For the competition with pathogens, adhesion of probiotics to epithelial cells is crucial for antibacterial activity (Schluter et al. 2014). For example, the adhesion of Escherichia coli to Caco-2 cells reduced when it was co-cultured with Lactobacillus plantarum (Anderson et al. 2010). Similarly, Lactobacillus fermentum also inhibited adhesion of E. coli and G. Vaginalis to HeLa and HT-29 cell lines (Kaewnopparat et al. 2013). In another study, bifidogenic strains showed anti-Salmonella activity by inhibiting adhesion on HT29-MTX cell layers (Zihler et al. 2011). Saccharomyces boulardii, which is a probiotic yeast, protected mice from invasive property of Salmonella enterica (Martins et al. 2010). It was also found that five important pathogens, L. monocytogenes, Salmonella spp., C. jejuni, E. coli 0157:H7 and B. cereus were inhibited by antimicrobial property of Lactobacillus rhamnosus yoba (Mpofu et al. 2016).

An intestinal barrier plays an important role in keeping electrolytes and water not leaking into the intestinal lumen and in preventing permeation of harmful agents from an outer environment. Probiotics are known to strengthen epithelial barrier function by tightening the junctions between epithelial cells, modulating cell proliferation efficacy and promoting secretion of mucus (Saxelin et al. 2005). Lactobacillus rhamnosus GG also regulated intestinal epithelial homeostasis in a mouse colitis model by activation of the epidermal growth factor receptor and Akt pathway (Yoda et al. 2014). Escherichia coli Nissle 1917-derived protein increased the expression of tight junction proteins (Hering et al. 2014).

Probiotics are also related to immunomodulation. It was found that probiotics contribute to intestinal homeostasis by an interplay with innate or adaptive immune system (van Baarlen et al. 2013). Therapeutic effect of Lactobacillus lactis was assessed in a Crohn’s disease mouse model (del Carmen et al. 2011). This study found that Lactobacillus rhamnosus GG and Streptococcus thermophilus induced interleukin-10, an anti-inflammatory cytokine, and stimulated a cytokine signaling suppressor (Latvala et al. 2011). In another study, Lactobacillus plantarum attenuated the symptoms of colitis in a germ free interleukin-10 knock out mouse model (Schultz et al. 2002).

As discussed above, probiotics are generally considered advantageous in both healthy and diseased conditions. However, the probiotic-conferred effects varies depending on probiotic strains, environmental factors and each individual. Further studies are needed to elucidate the effect and mechanisms of probiotics in the body (Marco and Tachon 2013; van Baarlen et al. 2013).

Factors that affect viability of probiotics

Although the viability of probiotics is essential for functioning of probiotics, it is a difficult task to maintain the viability from fabrication/storage to the target site in the GI tract. For this reason, a majority of probiotic delivery studies focus on how to improve the probiotic viability. This section discusses factors that affect probiotic viability during manufacturing, storage and passage through the GI tract.

Thermal stress

The integrity of probiotics can be damaged by thermal stress during a long-term storage as well as commonly applied manufacturing processes such as drying and pasteurization (Burns et al. 2008). It is well known that probiotics, when exposed to a high temperature, are inactivated by denaturation of protein and subsequent cell damages (Perdana et al. 2012). Lactobacillus spp. were examined for heat tolerance at 60 °C for 5 min and the result showed that the viability decreased by 6 log cycle depending on their thermosensitivities (Paéz et al. 2012). In another study, more than 7 log cycle reduction of Lactobacillus rhamnosus was observed with incubation at 60 °C for 150 s (Ananta and Knorr 2009).

Oxidative stress

Since many of probiotic strains are anaerobes or microaerophiles, the viability of probiotics can be deteriorated by the existence of oxygen. Reactive oxygen species are generated under oxidative condition and they interact with probiotic components such as proteins, lipid or nucleic acid (Santivarangkna et al. 2008). A study showed that the growth rate of Bifidobacterium spp. were inhibited in the presence of oxygen (Simpson et al. 2005). In another study, oxygen concentration dependent toxicity was observed in Lactobacillus acidophilus and Bifidobacterium species (Talwalkar and Kailasapathy 2004).

Osmotic shock

Osmotic shock also impairs the viability of probiotics during a drying process. Dehydration that happens during a drying process leads to efflux of water from a probiotic cell, which causes the osmotic shock by increased intracellular molarity in probiotic cells, resulting in damaged cell functions (Poolman 2002). For example, decreased viability of Lactobacillus plantarum was enumerated due to air drying in a desiccator and a spray dryer (Perdana et al. 2012). The viability of Saccharomyces cerevisiae was decreased with increasing hyperosmotic shock (Beney et al. 2000).

Gastric juice

After intake of probiotics, the first and biggest barrier for maintaining the viability of probiotics is the harsh environment in the stomach, more specifically the gastric juice, which is extremely acidic. The pH of stomach is commonly ranged between 1 and 2.5 (Evans et al. 1988) and the gastric emptying time is around 2 h (Hellmig et al. 2006). Probiotics cannot survive under the acidic conditions for 2 h owing to disruption in metabolic and cytoplasmic activities (Hutkins and Nannen 1993). Since the passage through the stomach is inevitable for probiotic to reach the target site, acid resistance is considered an indispensable property of a effective probiotic delivery system. Acid resistance can be tested in vitro using a simulated gastric juice which possesses characteristics of human stomach fluid, such as buffer capacity, osmolality and surface tension (Charteris et al. 1998; Fredua-Agyeman and Gaisford 2015).

Materials for encapsulating probiotics

The probiotic-conferred benefits strongly depend on the ability of microorganisms to survive and multiply in the host. Microencapsulation is a technology to encapsulate probiotics into microparticles or beads and has long been utilized as a key strategy to maintain the viability of probiotics during storage and in an acidic condition of stomach in GI tract. Probiotics within encapsulating materials can be protected from adverse environments such as low pH and osmotic pressure. The objective of probiotic encapsulation, is not only to protect the cells against adverse environments, but also to liberate probiotics to the target intestine in a viable and functional state (Picot and Lacroix 2004). The viability of encapsulated probiotics depends on the physicochemical properties of the encapsulating material (Chen and Chen 2007). In this section, we describe commonly used materials for microencapsulation of probiotics.

Alginate

Alginate, a natural polysaccharide derived from brown algae or bacteria, has been widely used as an encapsulating material for probiotics due to biocompatibility and an easy gelling process by an ionic gelation with Ca2+ (Krasaekoopt et al. 2003). Two common methods to encapsulate probiotics in alginate are extrusion and emulsion (Cook et al. 2012). In the extrusion method, the mixture of aqueous alginate and concentrated probiotics is extruded through a syringe and dripped into a hardening solution containing divalent ion such as Ca2+ (Lee et al. 2015). The size of alginate beads is dependent on the diameter of the needle and free fall height to the surface of the alginate solution through the syringe needle. Extrusion is a relatively facile method to encapsulate probiotics with a low cell loss and small deviation; however, it is not an appropriate method to make the size of hundreds micrometer and is not easy to scale up (Anal and Singh 2007). On the other hand, emulsion can be used to make the hundreds micrometer size of alginate hydrogel particles and is easy to scale up. In the emulsion method, the mixture of alginate, probiotic cells and CaCO3 is added to an oil phase with agitation (Song et al. 2013). Due to the shearing force, the water phase containing alginate, probiotics and CaCO3 becomes a discrete phase. Organic acids such as acetic acid are subsequently added to liberate Ca2+ from CaCO3, resulting in the formation of alginate microcapsules. Then Ca2+ ion is liberated from CaCO3 as pH decreased. However, alginate also has some disadvantages such as an uncontrollable swelling behavior and susceptibility to the acid pH. To resolve this problem, additional coating materials such as chitosan or mixing with starch have be utilized (Krasaekoopt et al. 2003, Cook et al. 2011). Recent technologies that overcome the problems will be discussed in detail in the “Recent trends of probiotic delivery system” section.

Gums

Xanthan, an exopolysaccharide derived from Xanthomonas campestris, is the most commonly used gum and is composed of glucose, mannose and glucuronic acid (Garcia-Ochoa et al. 2000). Xanthan gum is known to possess resistance to a wide range of pH and thermal stress (Leela and Sharma 2000). Ding et al. evaluated effectiveness of xanthan gum based microencapsulation (Ding and Shah 2009). In the study, microcapsules were produced by an emulsion method in which the discrete water phase, containing xanthan gum was cross-linked with calcium chloride whilst suspended in oil. Gum acacia has also been used to protect probiotics (Desmond et al. 2002, Lian et al. 2003). The result demonstrated that microencapsulation of probiotics, such as Lactobacillus casei and Bifidobacterium spp., with gum acacia by a spray drying provide resistance to an acidic environment. Guar gum, locust bean gum, and carrageenan are other gums used as encapsulating matrices, all of which showed protective effect, to some extent, for the 10 strains of probiotic bacteria investigated (Ding and Shah 2009). Carrageenan, especially, used for microencapsulating Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 was effective in keeping the numbers of probiotic cells higher than the level of the therapeutic minimum (7 log CFU/g) while the counts of free cells declined approximately 3 log cycle during cheese manufacturing process (Özer et al. 2009).

Proteins

Proteins also can be used as a protective material for probiotics and become a popular choice in recent years. Probiotics are encapsulated into proteins by an enzymatic or chemical cross-linking or temperature-dependent gelation (Cook et al. 2012). Amphiphilic nature of the proteins provides unique property for the probiotic delivery system. Several proteins such as gelatin (Annan et al. 2008), whey protein (Doherty et al. 2011) and casein (Heidebach et al. 2009) have been used for microencapsulation of probiotics (Livney 2010, Poulin et al. 2011).

Gelatin is a protein composed of glycine proline and 4 hydroxyproline, which is derived from hydrolysis of collagen (Tabata and Ikada 1998). It can form a gel with a thermos-reversible property and an amphoteric nature (Burgain et al. 2011). Because of its low rigidity, gelatin-based microencapsulation needs cross-linking agent (Annan et al. 2007).

Milk proteins such as whey protein and casein has also been used for encapsulating probiotics (Cook et al. 2012). Whey is a protein derived from milk by extraction of cheese or yogurt. Whey-based microencapsulation is based on acid-induced gelation and heat-induced gelation. Doherty et al. demonstrated that microbeads made by whey protein increased the survival rate of Lactobacillus rhamnosus GG exposed to ex vivo porcine gastric contents (Doherty et al. 2011). Casein is able to form water insoluble matrix in acidic conditions (at below pH 6), indicating that casein-based microencapsulation could be used for protecting probiotics during the gastric transit. It was reported that Lactobacillus paracasei and Bifidobacterium lactis were successfully encapsulated into casein by transglutaminase-induced caseinate gelation and were protected from simulated gastric juice (Heidebach et al. 2009).

Synthetic polymer

Synthetic polymer such as poly (D,L-lactic-co-glycolic acid) (PLGA), polyvinyl alcohol (PVA) and polyacrylamide has been employed as an encapsulating material. PLGA is a FDA-approved biocompatible material which used for time-dependent release. Probiotics containing PLGA microparticles were produced using a water-in-oil-in-water double emulsion method with solvent evaporation (Della Porta 2012). However, use of synthetic polymers as an encapsulating material is still challenging due to involvement of organic solvents during fabrication, which causes cell damages. Preparation methods and solvents for the polymers should be carefully considered when developing synthetic polymer-based probiotic delivery systems.

Recent trends of probiotic delivery system

As described in the previous section, encapsulating probiotics into carrier materials had been a common strategy for probiotic delivery until recently. However, challenges still exist for effective protection of probiotics from tough conditions during a manufacturing process, a long-term storage and a transit in the GI tract in order to obtain a sufficient number of viable bacteria in the target site. This has propelled development of new strategies in probiotic delivery. In this section, we describe recent advancement in probiotic delivery systems. Some of them are summarized in Table 1.

Table 1 Overview of recent probiotic delivery systems
Full size table

Alginate has been the most extensively studied encapsulating material; however, a protective effect of bare alginate is not enough to obtain a sufficient number of viable probiotics in target sites due to a porous nature and an uncontrollable swelling behavior, which could allow H+ ion penetration and make the alginate system susceptible to acids. In addition, cell leakage by low mechanical durability in storage is a potential problem of alginate (Kim et al. 2014). Recent studies have employed various coating technologies to overcome the limitation by providing an additional protection to the surface of alginate microparticles or beads.

Chitosan coating on alginate beads has been used to provide probiotics for protection from acids by reducing pore size of alginate beads. Cook et al. evaluated the viability of probiotics from chitosan-coated alginate beads and compared to bare alginate beads (Cook et al. 2013b). After 60 min of incubation in an acidic pH, chitosan-coated alginate beads showed a higher viability by delayed H+ penetration as compared to bare alginate beads. In another work, chitosan-coated alginate beads were used for encapsulating Bifidobacterium breve, resulting in over 6 log CFU/ml of cells survived, while no viable cells were observed with non-coated in detectable range (Cook et al. 2011). As the number of chitosan-alginate coating increased, the protective effect for probiotics were also enhanced. That was verified with chitosan-alginate single and double-coated beads that encapsulate Lactobacillus plantarum (Nualkaekul et al. 2012). As compared to single-coated beads, the double-coated counterpart showed higher survivability, which was more than one log cycle after incubation with simulated gastric fluid. The protective effect of chitosan/alginate coating was also confirmed with multi-layer coated alginate beads encapsulating Lactobacillus plantarum (Cook et al. 2013a, b).

Polydopamine coating on alginate beads was used to encapsulate Saccharomyces cerevisiae (Kim et al. 2014). It was found that polydopamine coating enhanced mechanical durability of alginate beads. Unlikely to bare alginate beads, polydopamine coated alginate beads effectively prohibited cell leakages for up to 25 h in the presence of monovalent ions. Since monovalent ions can break alginate gel network which is formed by divalent calcium ions, disintegration of alginate beads was accelerated. In the study, polydopamine coating prevented bead swelling, enzymatic degradation and UV radiation, resulting in a good protection during storage and encapsulation process (Kim et al. 2014).

For enhanced target delivery of encapsulated probiotics to the target intestinal area, protamine was formulated with alginate. Inner alginate core containing Lactobacillus casei was surrounded by composite shell of alginate and protamine. Since protamine is digested by proteases in the GI tract, a rapid and selective release of probiotics was observed in the small intestine area (Mei et al. 2014).

Enteric-coating materials have been used for targeted delivery of probiotics. Eudragit L 100 55 was used with ethylcellulose to protect Bifidobacterium breve from gastric juice (de Barros et al. 2014). In the study, the results showed only less than 0.5 log reduction after 2 h of incubation in the simulated gastric fluid. When Eudragit L 100 and alginate was formulated to a tablet form to protect Lactobacillus fermentum, only 1 log cycle was decreased for 2 h of incubation at pH 1 (Villena et al. 2015a, b). Eudragit was also used as a coating material for Lactobacillus rhamnosus-containing microsphere (de Barros et al. 2015). The Eudragit-coated microparticles released more than 8 log log CFU/dose within 1 h incubation of the simulated intestinal fluid following 2 h exposure to simulated gastric juice.

To maximize probiotic-conferred health benefits, prebiotics such as galactooligosacchride and chicory have been added to probiotic delivery systems. Prebiotics is a non-dietary fiber that can selectively boost probiotic strains and confer synergistic effects (Kolida and Gibson 2011). When galactooligosaccharide-loaded PLGA particles were encapsulated in alginate beads with Bifidobacterium breve, the viability of Bifidobacterium breve increased up to 8 log log CFU/mL (Cook et al. 2014). Synergistic effect by prebiotics not only provides health beneficial effect, but also increases gastro resistance and thermos tolerance of probiotics. Alginate beads which encapsulate chicory and Staphylococcus succinus showed 95 % of survival while the viability of free cells decreased to 77 % after 35 day of storage (Sathyabama et al. 2014). In another paper, Bifidobacterium BB-12 was encapsulated in milk proteins blended with oligofructose-enriched inulin (Fritzen-Freire et al. 2012). In the study, more than 10.5 log CFU/g of cells were survived after 180 days at 4 °C storage (Fritzen-Freire et al. 2012).

Manufacturing processes can influence viability of probiotic bacteria (Grzeskowiak et al. 2011). Since many of probiotics can be exposed to high temperatures for pasteurization and spray-drying process and low temperatures for a freeze-drying process, maintaining viability during the manufacturing processes is also of importance (Tripathi and Giri 2014, Broeckx et al. 2016). Various technologies have been incorporated into formulations to improve the survival rate of probiotic bacteria during manufacturing processes and in storage. To enhance a survival rate of Lactobacillus reuteri in a heated condition, aluminum carboxymethyl cellulose-rice bran microcapsules were fabricated (Chitprasert et al. 2012). After 25 s of exposure to 85 °C, more than 8 log CFU/g of cells survived, while free cells survived less than 4.8 log CFU/g (Chitprasert et al. 2012). In another study, bacterial nanocellulose that encapsulate probiotics with pectin protected the probiotics well under a microwave drying and during a long-term storage at a variety of temperatures (Khorasani 2016). Nanocellulose was also used to decrease cell damage of Lactobacillus plantarum at freeze-drying processes by adhesion to the surface of probiotics (Nahr et al. 2015).

Recently, non-microencapsulation-based probiotic delivery systems have been attempted. For examples, tablet-based systems have been investigated as a probiotic delivery system. Govender et al. developed bi-layered mini-tablet-in-tablet system to deliver Lactobacillus acidophilus to both small intestine and colon (Govender et al. 2015). Ovalbumin which is known to possess gastro-resistant properties was used to prepare mini tablets where probiotics were incorporated. The ovalbumin mini tablets were then place inside each layer. Major excipient of two layers was lactose and Eudragit S100 which was chosen to deliver probiotics to intestinal and colon targeting, respectively. The tablets showed effective site-specific delivery of Lactobacillus acidophilus as intended. In another study, Eudragit L100–sodium alginate tablets were shown to improve the survival of Lactobacillus fermentum CECT 5716 when exposed to an acidic medium as compared to free cells, resulting in the survival of 9 log CFU/tablet after 2 h of incubation. The tablets also protected cells during storage at 4 °C for over 6 months (Villena et al. 2015a, b).

Cell surface engineering has also emerged as a non-microencapsulation-based technology to protect probiotics from gastric conditions. Cell permeability can be modulated by adhesion of polymer molecules on the cell surface (Fakhrullin et al. 2012). Pepsin is an enzyme with a large molecular weight which is present in gastric juice. Inhibition of pepsin penetration to a probiotic wall is of important for probiotic viability. By introducing chitosan and carboxymethyl cellulose onto bacterial surface, the penetration of large molecular weight enzyme was effectively inhibited while leaving a small molecular nutrition freely flow in and out (Priya et al. 2011). As a result, more than 8 log CFU/g of Lactobacillus acidophilus survived whereas the viability of non-coated cells decreased to less than 3.5 log CFU/g. The protective effect of chitosan and dextran sulfate coated Saccharomyces cerevisiae was also reported (Ben Thomas et al. 2014).

Conclusion

The field of probiotics has been growing due to well-documented health benefits. Since only viable probiotics can confer the health benefits, keeping the viability of probiotics up until reaching the site of action is of great importance in probiotic delivery. The viability can be affected by several environmental factors such as gastric pH, temperature and osmotic pressure. Microencapsulation has been widely used to improve the survival rate of probiotics. In general, alginate, gums or proteins have been used as an encapsulating material which provides sufficient protection to probiotics during storage and delivery to the target site. Recently, an array of novel technologies, such as coating systems, prebiotics and microencapsulation with newly developed materials, have been developed to enhance the viability. Another novel aspect of probiotic delivery is a controlled release of probiotics at the target site. Despite all the efforts, however, most delivery systems still suffer from loss of viable probiotics and a need for an ideal probiotic delivery system has yet to be met. Another issue that needs to be addressed in probiotic delivery is lack of tools for in vivo evaluation of probiotic viability and functioning. In conclusion, different aspects of this review may open new avenue for extensive research in the field of probiotic delivery.