Microencapsulation of Probiotic Cells for Food ...

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Microencapsulation of Probiotic Cells for Food Applications a

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Thomas Heidebach , Petra Först & Ulrich Kulozik

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ZIEL Research Center for Nutrition and Food Science, Institute for Food Process Engineering and Dairy Technology, Technische Universität München, Weihenstephan Weihenstephaner Berg 1, 85354, Freising-Weihenstephan, Germany Published online: 14 Feb 2012.

To cite this article: Thomas Heidebach , Petra Först & Ulrich Kulozik (2012): Microencapsulation of Probiotic Cells for Food Applications, Critical Reviews in Food Science and Nutrition, 52:4, 291-311 To link to this article: http://dx.doi.org/10.1080/10408398.2010.499801

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Critical Reviews in Food Science and Nutrition, 52:291–311 (2012) C Taylor and Francis Group, LLC Copyright ISSN: 1040-8398 / 1549-7852 online DOI: 10.1080/10408398.2010.499801

Microencapsulation of Probiotic Cells for Food Applications ¨ THOMAS HEIDEBACH, PETRA FORST, and ULRICH KULOZIK

Downloaded by [University of Leicester] at 01:16 02 June 2013

ZIEL Research Center for Nutrition and Food Science, Institute for Food Process Engineering and Dairy Technology, Technische Universität München, Weihenstephan Weihenstephaner Berg 1, 85354, Freising-Weihenstephan, Germany

The addition of microencapsulated probiotic cells to food products is a relatively new functional food concept. Most of the published scientific research in this field is not older than ten years. However, the technological background reaches back to the 1980s, where lactic acid bacteria were microencapsulated within the concept of the so-called immobilized cell technology (ICT). Target applications of ICT were continuous fermentation processes and improved biomass production. The methods adopted from immobilized cell technology were applied for the microencapsulation of probiotics, often optimized towards specific requirements associated with the protection of probiotic cells in food applications. However, there are still significant hurdles with respect to currently available methods for probiotic cell microencapsulation. This is mainly due to the fact that important characteristics of microcapsules based on ICT appear to be in conflict with the requirements arising from an application of probiotic microcapsules in food products, with particle size and inappropriate matrix characteristics being the most prominent ones. Based on this situation the aim of this review is to give a critical overview of the current approaches regarding the microencapsulation of probiotic cells for food applications and to report on emerging developments. Keywords Functional foods, immobilization, entrapment, Lactobacillus, Bifidobacterium

ANNOTATIONS ICT: Immobilized cell technology CFU: Colony forming units EY: Encapsulation yield Probiotic microcapsules: Microcapsules that contain probiotic cells as core-material NGYC: medium non-fat milk, glucose, yeast-extract, and cysteine medium MRS: medium de Man, Rogosa, Sharpe medium

INTRODUCTION An important trend in the food industry in recent years is the demand for health promoting foods from which the concept of “functional foods” emerged. The term has been Address correspondence to Thomas Heidebach, ZIEL Research Center for Nutrition and Food Science, Institute for Food Process Engineering and Dairy Technology, Technische Universität München, Weihenstephan Weihenstephaner Berg 1, 85354, Freising-Weihenstephan, Germany. E-mail: [email protected]

coined to describe foods fortified with ingredients capable of producing health benefits (Stanton et al., 2001). In this context, the addition of living probiotic microorganisms to food is a prominent way to create functional foods (Rodgers, 2008). Various health related properties of different probiotic strains are well documented and living lactic acid bacteria with probiotic activity are believed to play a beneficial role in the ecosystem of the human intestinal tract (Jia et al., 2008; Naidu et al., 1999; Tamime et al., 2005). Recently, genome-based studies started to provide insights regarding the mechanistic functions of probiotics in the ecosystem of the human gut (Ventura et al., 2009). However, the loss of bioactivity, that is the loss of living probiotic cell numbers during processing, storage, and gastrointestinal transit caused by various stress factors is an important issue and has to be avoided (Mattila-Sandholm et al., 2002; Shah, 2000; Siuta-Cruce and Goulet, 2001). Hence the protection of living probiotic cells became an important issue. In this context, microencapsulation is the most prominent technique for providing a protective environment for microorganisms under adverse conditions (Augustin, 2003; Champagne et al., 2005; Ross et al., 2005; Parada and Aguilera, 2007). Microencapsulation consists of coating or entrapment of a core material into capsules in the size range of a few micrometers up to a few millimeters (Kirby, 1991).

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In food systems microencapsulation can have various aims. A prevalent objective is to protect the core material from degradation by reducing its reactivity to environmental conditions (Gibbs et al., 1999; Schrooyen et al., 2001). This is mainly achieved by control of the mass transfer between the core and the external environment by using the shell material as a physical barrier (Champagne and Fustier, 2007; Desai and Park, 2005; Kailasapathy, 2002; Lopez-Rubio et al., 2006). A possible design commonly used for encapsulation of microbial cells involves the so-called matrix capsule, where the living cells as core material are embedded and immobilized randomly in a continuous matrix, which often is a hydrogel (Desai and Park, 2005). Hence, the terms “immobilization” and “encapsulation” are used as synonyms in most reported works about the microencapsulation of probiotics (Anal and Singh, 2007; Krasaekoopt et al., 2003). The motivation for microencapsulation of living probiotic cells is to decrease the unavoidable drop of living cell numbers from the first addition of the probiotic concentrate to the food, until they reach their final destination in the human gut. Along with this, a complete release of the probiotic cells from the capsule into the human gut should be ensured, because colonization of the gastrointestinal tract is seen as an important requirement to exert probiotic effects (Naidu et al., 1999). Furthermore, the capsules must be sufficiently small to avoid a negative sensorial impact on the functional food product they have been added to. However, until today many approaches regarding probiotic encapsulation have significant flaws when it comes to delivering these key features for food applications. As a result there are still many apparent technological hurdles associated with the currently available solutions for the microencapsulation of probiotic cells. Many of them can be explained by the fact that the currently used probiotic encapsulation techniques and matrix-materials emerged from the technological background of immobilized cell technology (ICT). However, some important characteristics of microcapsules based on ICT appear to be in contrast to the requirements arising from adding microcapsules containing probiotic cells (later on referred to as probiotic microcapsules) directly to a food product, with particle size and inappropriate matrix characteristics being the most prominent ones. Based on this perception this paper critically reviews the current approaches for microencapsulation of probiotics for food applications and resulting future perspectives.

IMMOBILIZED CELL TECHNOLOGY (ICT) ICT is applied in biotechnology and involves the entrapment of living cells in spherical gel beads (Kailasapathy, 2002). It was successfully used in different areas of fermentation, with the main utilization of this technology settled in the dairy industry (De Giulio et al., 2005), in applications such as continuous inoculation of milk for yogurt or cheese making, lactic acid production, and optimized biomass production within the matrix

of the beads (Champagne et al., 1994; Lacroix et al., 2005; Prevost et al., 1985). At the time when protection of probiotics became an issue in food applications, ICT-methods for the entrapment of lactic acid bacteria and its applications in the dairy industry were already well studied and established. Therefore, it occurred as an immediate near-in solution for the emerging problems of probiotic cell protection to use already existing ICT-techniques. However, besides physical protection of the entrapped cells, some of the main objectives of ICT-based applications differ from the above-mentioned targets of probiotic encapsulation, as outlined in the following section.

Capsule Features Capsule Size The desired size of capsules for ICT-applications is dependent on the required cell growth, the mechanical strength, and the separation characteristics of the capsule (Lacroix et al., 2005). It was found that beads with diameters below 1 mm can result in separation problems in continuous inoculation processes, such as clogging of the filter when the capsules are removed from the substrate after the fermentation (Champagne et al., 1994). Furthermore, reducing the size of the gel beads below 1 mm may result in mechanical instability during longterm continuous fermentation (Audet et al., 1992). Therefore, in ICT applications, spheres with size ranges between 1 and 3 mm are preferably used.

Gel Network Density The production of cell biomass within spherical gel-matrices is mainly controlled by diffusion and mass-transfer phenomena. Therefore, non-uniform cell growth in the colonized microcapsules results in the formation of a high cell density region near the capsule surface, leading to a 20- to 30-fold higher cell concentration than in the center of the gel (Champagne et al., 1994). A sometimes undesired leakage of cells into the surrounding medium occurs once the matrix space in the gels has been fully occupied and the gel then breaks due to mechanical stresses resulting from cell growth (Champagne et al., 1992; Klinkenberg et al., 2001). Therefore, a low density gel network, which provides sufficient space for the production of concentrated biomass within the polymer-gel, is a possible way to circumvent the problem of cell leakage (Park and Chang, 2000). Biopolymers, such as alginate, gellan-gum, xanthan, carrageenan, locust-bean-gum, or mixtures thereof are suitable and most commonly used in ICT for dairy applications, because of their ability to easily build hydrogels at low concentrations of about 1% in aqueous solutions (Burey et al., 2008; Champagne et al., 1994). Since the gel beads are removed from the dairy product after incubation, the non-dairy origin of such polymers is not a major issue.

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ICT-Capsules in Food Applications If microencapsulated probiotic cells are to be employed in final food applications, capsule-features that are important for a successful ICT-application can have an adverse impact. A low matrix density, required for concentrated biomass production within the capsules, is in contrast to the proposed protective mechanism of microencapsulation, that is, the creation of a physical barrier against unfavorable external conditions. Moreover, it must be considered that, in contrast to ICTapplications, probiotic microcapsules for food applications are clearly intended to remain in the food product until consumption. As a consequence, the size of the capsules must be considered with respect to the sensorial impact on the food. The matrix properties and release characteristics of microcapsules from ICT were not designed and optimized with respect to requirements of capsules intended to pass through the human gastro-intestinal tract. Alginate, which is most commonly used for probiotic encapsulation, can be extracted from the cell walls of marine algae. While it serves as a carbohydrate source for various marine molluscs (Gacesa, 1992), it is indigestible for humans, and behaves much like a dietary fiber (Brownlee et al., 2005). To effectively use probiotic microcapsules in food products, it must be ensured that the hydrocolloids used as matrix-material not only provide the desired barrier effect under acidic pH conditions, but are also digestible and, therefore, release the probiotic cells into the human gut. It can therefore be concluded that most of the important characteristics of microcapsules based on ICT do not meet the requirements of probiotic microcapsules, that are to be added directly to a food product. Encapsulation Techniques for Probiotic Cells The vast majority of microcapsules produced for ICTapplications in dairy systems are generated by two methods, the extrusion technique and the emulsion technique. Biopolymers such as alginate, gellan gum, xanthan, carrageenan, locust bean gum or mixtures thereof are commonly used as gelation material, since low concentrated (0.75–4%) aqueous solutions of these polymers can undergo mild ionotrophic and/or thermal gelation. Consequently, in most of the studies on probiotic cell encapsulation for food applications these methods are applied, mostly using alginate or gellan-xanthan mixtures as gelling agent (Champagne et al., 1994; Doleyres and Lacroix, 2005; Krasaekoopt et al., 2003). In recent years, spray drying was also utilized to encapsulate probiotic cells as an alternative to the encapsulation methods based on ICT, as outlined in the section titled “Spray Drying.” Extrusion Technique The extrusion technique involves preparing an aqueous hydrocolloid solution, adding concentrated microorganisms to

Figure 1

Microencapsulation by means of the extension technique.

it, and extruding the hydrocolloid-cell-mixture through a nozzle that forms droplets that fall into a hardening solution (see Fig. 1). In case of the most commonly used sodium-alginate, gelation can be achieved by dropping the droplets into a CaCl2-solution (Krasaekoopt et al., 2003). The size of the resulting capsules depends on the diameter of the orifice, the distance between the outlet, and the hardening-solution, and the viscosity of the hydrocolloid-cell mixture (Anal and Singh, 2007). Since the extrusion method was readily available from ICT, it was used by many researchers for the microencapsulation of probiotic cells, despite the large bead size ranges of 0.5–3 mm (Krasaekoopt et al., 2003).

Emulsion Technique For probiotic cell encapsulation, the most suitable method concerning control and flexible adjustment of the resulting capsule size is the emulsion technique. In this method a small volume of the aqueous hydrocolloidcell-mixture (discontinuous phase) is emulsified into a larger volume of vegetable oil (continuous phase). Once a water-inoil emulsion has been formed, the dispersed hydrocolloid-cellmixture must be insolubilized to form small beads within the oil phase (Krasaekoopt et al., 2003). When alginate capsules are produced, the microcapsules are hardened by slowly adding CaCl2-solution to the emulsion while stirring (see Fig. 2). When the calcium solution gets into contact with the dispersed alginate phase, instantaneous gelling occurs. Thus, the gelation kinetic is inhomogeneous, which sometimes leads to capsules having irregular shape (Sheu and Marshall, 1993). The technique was first developed by Nilsson et al. (1983) as a general method for immobilization of sensitive living cells. The authors stated that by adjusting the speed of a magnetic stirrer

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Figure 2

Microencapsulation by means of the emulsifying technique.

during the emulsifying process capsules with average diameters between 0.1 and 5 mm could be produced at that time. The major parameters to control the size of the capsules are similar to those that influence particle size formation in common emulsifying processes, that is, the energy input during emulsification, the addition of emulsifiers, and the viscosity ratio between the dispersed and the continuous phase. For probiotic cell encapsulation, the emulsifying step is often accomplished by means of a magnetic stir bar or a directly driven mechanical stirrer (Ding and Shah, 2009a). In these cases the shear forces and resulting particle size reduction is rather undefined. However, with the emulsion method capsule sizes below 100 µm can be achieved when sufficiently high.

Spray Drying Probiotic cell concentrates often need to be stored over longer periods prior to food manufacture and ingestion (De Giulio et al., 2005; Su et al., 2007). Hence, probiotic microcapsules are sometimes usually dried after production. In case of hydrogel-based microcapsules generated by extrusion or emulsification processes, freeze drying is frequently used to dry the capsules in a subsequent step after production (Godward and Kailasapathy, 2003; Heidebach et al., 2010, Kim et al., 2008; Lahtinen et al., 2007, Lee et al., 2004; Reid et al., 2007). An alternative method to achieve capsule-building and drying in a single step is spray-drying. Spray drying is a routine process in the food industry to convert liquids into dry powders. In recent years, spray drying has been utilized to encapsulate probiotic cells with the intention of not just simply drying, but as an alternative to the encapsulation methods based on ICT. In this context mixtures of probiotic cell concentrates were spray dried with aqueous solutions of various polymers, such as modified starch (O’Riordan et al., 2001), gum arabic (Desmond et al., 2002), gelatin (Lian et al., 2003), whey protein isolate (Picot and Lacroix, 2004), maltodextrin mixed with gum arabic (Su et al., 2007), and ß-cyclodextrin mixed with gum arabic (Zhao et al., 2008), and their ability to protect the probiotic cells against adverse conditions was investigated.

The advantage of spray drying is its wide availability in the food industry and that often favored small capsules with average diameters below 100 µm are usually generated at comparably low costs. However, in contrast to microcapsules generated from freeze-dried hydrogels, microcapsules prepared by this method are water soluble in most cases. Therefore, the cells are early released and are no longer protected from adverse conditions during product storage in non-dried products and during gastrointestinal transit (Krasaekoopt et al., 2003). The comparably high temperatures and rapid dehydration during spray drying generally lead to a deterioration of the cells, resulting in significant losses of living cells and a diminished resistance against unfavorable environmental conditions (Meng et al., 2008). It was shown that the survival of probiotic cells during spray drying increases with decreasing outlet air temperature (Ananta et al., 2005; Gardiner et al., 2000; Lian et al., 2002; To and Etzel, 1997). The chosen outlet air temperature is therefore often a compromise between the required residual water content and the probiotic cell survival, which is often as low as 1–10% in sufficiently dried powders (Desmond et al., 2002; Gardiner et al., 2000; Lian et al., 2002; Picot and Lacroix, 2004; Wang et al., 2004; Zhao et al., 2008).

ASSESSMENT OF CURRENT METHODS FOR PROBIOTIC ENCAPSULATION Encapsulation Yield One of the main reasons for the application of biopolymers within ICT is the mild ionotrophic gelation suitable for a virtually loss-free entrapment of living microbial cells (Kailasapathy, 2002; Poncelet et al., 1992). This is in contrast to the abovementioned rather low probiotic survival generally found during probiotic encapsulation by spray-drying. By using the original extrusion method known from ICT, encapsulation yields (EY) of 100% were achieved for the encapsulation of various probiotic cells in large microcapsules



(Krasaekoopt et al., 2004; 2006; Kushal et al., 2006; Leverrier et al., 2005; Sun and Griffiths, 2000; Talwalkar and Kailasapathy, 2003; Urbanska et al., 2007). The EY is usually calculated by comparing the probiotic colony forming units (CFU) per gram dry matter of the initial polymer-cell-solution versus the generated microcapsules. The EY is therefore a combined parameter that describes the survival of viable cells and the efficacy of entrapment during the encapsulation procedure. The main reason for an EY below 100% is mainly probiotic cell damage due to detrimental conditions caused by the encapsulation process itself, such as heating, shear stress, or the application of concentrated solutes. Furthermore, a physical loss of cells into the hardening solution during the encapsulation process can appear in significant numbers. It should also be noted that a disintegration process is required to measure the concentration of living cells in the microcapsules. In the case of alginate-based capsules, dissolution of the capsules can be easily achieved by gently shaking them in a phosphate-buffer solution (Sheu and Marshall, 1993). In contrast, for capsules based on irreversible gelation, mechanical disintegration methods are often required. An incomplete disintegration as well as detrimental influences of the disintegration process can shift the found EY towards lower levels (Annan et al., 2008).

Therapeutic Minimum and Core Load In contrast to capsules used for ICT, probiotic microcapsules for food applications are generally not intended to be propagated via a fermentation process that is accompanied by cell growth within the capsules. Entrapped cell growth preferentially takes place near the capsule surface because of better nutrient availability (Audet et al., 1992). This is undesirable in view of the physical protective effect of microencapsulation, being most effective within the core of the capsule. In probiotic foods a concentration of 106–107 CFU probiotic cells per gram or mL of the resulting product has been suggested as an effective or therapeutic minimum (Agrawal, 2005; Champagne et al., 2005). Therefore, a high initial EY, that is, a high core load with living probiotic cells after the encapsulation, is required to match the therapeutic minimum in the food, especially at a preferably low capsule addition ratio. However, when ICT-methods were modified to produce capsules with physical characteristics suitable to meet the requirements of probiotic encapsulation in foods, that is, small capsules sizes or the use of more suitable matrix materials such as proteins, several problems that are associated with having high EY values may arise.

Technological Challenges Arising from Probiotic Encapsulation Hydrocolloid-Based Microcapsules Besides spray-drying, the creation of microcapsules with diameters below 100 µm can also be achieved by modification

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of ICT methods. In most studies, the impact on the resulting EY was not assessed. However, some authors found that the required modifications led to undesirably low EY, as outlined below. Capela et al. (2007) encapsulated various probiotic strains in a 3% alginate solution by means of the emulsification technique using a magnetic stirrer system, resulting in microcapsules with an average diameter of 381 µm. Aqueous solutions of hydrocolloids used as precursors for encapsulation in ICT, such as alginate, carrageenan, gellan, or xanthan can have very high viscosities even at low concentrations. Hence, due to a high viscosity ratio between the dispersed and the continuous phase (mostly vegetable oil), a high energy input is necessary to produce sufficiently small microcapsules. An additional high-shear step during the emulsifying process was applied by means of an Ultra-Turrax, a Silverson mixer, or a high-pressure homogenizer in the study of Capela et al. (2007), to reduce the capsule size below 100 µm. The resulting EY differed significantly between strains and the method of homogenization. For Lactobacillus casei, Lactobacillus acidophilus, and Bifidobacterium longum, EY of 30% and less than 5% were found, respectively. Lactobacillus rhamnosus had an all over higher EY of 5% and 65%. The authors stated that individual probiotic strains may vary in their sensitivities to mechanical or thermal stresses caused by homogenization processes leading to a low survival of cells during the encapsulation process. In a study by Ding and Shah (2009a) a high shear process was applied using an Ultra-Turrax or microfluidizer to reduce the capsule-size of alginate-based capsules generated by the emulsifcation technique. For both devices it was found that the content of living cells from each of the eight different, individually encapsulated strains within the capsules gradually decreased from approximately 0.5 up to 3.5 log cycles CFU with increasing energy input during the emulsion process. Relatively low EY were also found by Cui et al. (2000), when small alginate microcapsules with sizes between 5 and 200 µm were prepared by spraying an aqueous mixture of alginate and Bifidobacteria into a CaCl2 solution using an air-driven atomization device. The authors found an EY of only 12.3%. The low EY was explained by the loss of cells in the surrounding aqueous CaCl2-solution during the gelation step. Apparently this was caused by the high surface-to-volume ratio, compared to large capsules that were produced by the standard dropping method.

Protein-Based Microcapsules An alternative strategy of using different matrix-materials compared to ICT-technology involves the application of protein solutions as precursor material for the capsule matrix. On the one hand, small microcapsules can be produced by the emulsion technique with less effort due to the good emulsifying properties and the rather low viscosity of food protein solutions, leading to a lower shear stress and resulting in a higher EY (Heidebach et al., 2009a). Furthermore, in contrast to the commonly used


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hydrocolloids, even highly concentrated aqueous solutions of most proteins have a relatively low viscosity. This facilitates the formation of microcapsules with dense gel network that provide a substantial buffering capacity, thereby supporting the idea of a protective barrier between the sensitive core material and the surrounding environment. However, the application of food protein based matrix materials as alternatives to commonly used polysaccharide-based matrices sometimes requires the modification or even establishment of novel encapsulation techniques, involving different gelation mechanisms during encapsulation. Annan et al. (2008) encapsulated Bifidobacterium adolescentis in small alginatecoated gelatine microcapsules, with average diameters of 50 µm. The capsules were produced by covalently cross-linking the gelatine-cell mixture with genipin, a non-toxic cross-linker from plants, during the emulsification process. An EY of only 41–43% was achieved. The authors stated that the strong physical stability of covalently cross-linked gels could have prevented a complete release of the cells leading to a lower EY. Hence, in this case it is not clear as to what extent a decrease of the EY is caused by the encapsulation process itself. Picot and Lacroix (2004) mixed various strains of Bifidobacteria separately with heat treated, denatured whey protein solutions at 40◦ C and then spray dried these mixtures to generate water-insoluble microcapsules with sizes of 3–75 µm. The authors found EY between 0.71 and 25.7%, depending on the heat tolerance of the strain. Yet, the authors concluded that cell damage caused by the relatively high shear levels and the thermal inactivation during the spray-drying process was a major drawback of the process. Reid et al. (2005) reported an EY of 22% during encapsulation by Ca2+-induced cold gelation via extrusion of a pre-heated whey protein solution that contained Lactobacillus ssp. in a concentrated CaCl2-solution. The authors stated that the exposure to concentrated CaCl2-solution during the gelation process may be responsible for the high mortality rates of the entrapped microorganisms during the encapsulation process. In a study of Weinbreck et al. (2010) water-insoluble microcapsules were created by spraying of pre-denaturated whey protein solution mixed with probiotic Lactobacillus rhamnosus onto core-particles in a fluidized bed coater. In this case a 103fold decline in cell viability during encapsulation was found, which was attributed to cell damage during drying. An emulsion process based on enzymatic gelation of caseinate solutions to produce probiotic microcapsules was used by Heidebach et al. (2009b). With this method an EY of 70% and 93% was achieved for Lactobacillus paracasei and Bifidobacterium lactis, respectively. The high physical stability of covalently cross-linked gels could prevent a complete release of cells, and therefore be responsible for an EY of slightly less than 100%. Encapsulation of these strains into another protein based gel matrix, produced by enzymatic rennet gelation of a 35% skim-milk concentrate, led to a complete recovery of viable cells after the encapsulation process (Heidebach et al., 2009a).

Optimization of ICT methods to match the specific requirements associated with the protection of probiotic cells in food applications is common practice (Kailasapathy, 2002). However, from the above-mentioned studies it can be seen that one of the most important features of ICT-capsules, namely the high initial EY can be lost during the above-mentioned modifications. The use of protein based hydrogels for the encapsulation of probiotic cells instead of polysaccharide biopolymers appears to be more promising to obtain small microcapsules if mild gelling mechanisms are applied, such as cold-induced gelation of whey concentrates or enzymatic gelation (Chen et al., 2006; 2003; Heidebach et al., 2009a; 2009b).

Lipid-Based Microcapsules Aside from carbohydrates and proteins, the use of lipid-based encapsulation systems for the encapsulation of probiotics has not yet been well explored. Matrix-encapsulation can be achieved by mixing probiotic cells with molten fat and subsequent cooling. However, dispersion of probiotic cell concentrates in oil was reported to be a difficult technological task (Modler and Villa-Garcia, 1993; Picot and Lacroix, 2004). Premature melting of the capsules at elevated temperatures, like the ones in the human body must be considered. Because of possible separation problems it is likely that applications are limited to solid foods (Lahtinen et al., 2007). Studies revealed that encapsulation of probiotics in butterfat afforded no protective effect during storage in frozen yogurt (Modler and Villa-Garcia, 1993). Lahtinen et al. (2007) found only a slight protective effect when probiotics were encapsulated in cocoa butter during storage in fermented and non-fermented oat-drinks. Hence, fat-based microcapsules currently seem less suitable for probiotic encapsulation compared with polysaccharide- or protein-based microcapsules.

IMPACT OF MICROCAPSULES ON PROBIOTIC SURVIVAL AND FOOD CHARACTERISTICS For food applications, creating microcapsules with sufficiently small average diameter is one of the most significant bottlenecks. On the one side, larger capsule diameters, and hence a higher volume-to-surface-ratio increases the likeliness of a protective effect (Anal and Singh, 2007). On the other side, the capsules must be small enough to not negatively impact the sensory properties of the food-product (Champagne and Fustier, 2007). This conflict of targets can lead to difficulties when it comes to application of probiotic microcapsules in food. From sensory studies dealing with the mouth feel sensation of particles in foods it can be concluded that large, hard, or sharp particles added in a high concentration to a low viscous medium produce a more rough, gritty, and unpleasant sensation, compared to small, soft, and spherical particles that are added at a lower concentration to a high viscous medium or gel (Engelen et al., 2005; Imai et al., 1995).

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MICROENCAPSULATION OF PROBIOTIC CELLS Table 1

Applications of encapsulated probiotics in yogurt

Encapsulated strain1


Bifidobacterium bifidum and Bifidobacterium infantis Bifidobacterium infantis Two strains of Bifidobacterium longum Lactobacillus acidophilus and Bifidobacterium infantis Two strains of Bifidobacterium longum Two strains of Lactobacillus acidophilus Lactobacillus acidophilus, Bifidobacterium bifidum, and Lactobacillus casei Bifidobacterium lactis and Lactobacillus acidophilus 1If

Matrix material and encapsulation technique Alginate; emulsion

Average size of capsules (µm)

Storage time

Increased survival due to microen-capsulation

Reference

not given

1 week

1 log cycle for each strain

(Hussein and Kebary, 1999)

3000 235

6 weeks 30 days

(Sun and Griffiths, 2000) (Adhikari et al., 2000).

Alginate, co-encapsulation with 2% resistant starch; emulsion κ-carrageenan; emulsion

500–1000

8 weeks

235

30 days

1 log cycle 0.5 log cycles for each strain 0.5 log cycles for each strain 1 log cycle for each strain

Alginate, co-encapsulation with 1% resistant starch, coated with chitosan; extrusion Alginate, coated with chitosan; extrusion

450–500

6 weeks

3 log cycles for each strain

(Iyer and Kailasapathy, 2005)

1900

4 weeks

1 log cycle for each strain

(Krasaekoopt et al., 2006)

Alginate, co-encapsulation with 1% resistant starch; emulsion

500–1000

7 weeks

Bifidobacterium lactis: 1 log cycle Lactobacillus acidophilus: 2 log cycles

(Kailasapathy, 2006)

Gellan-xanthan; extrusion κ-carrageenan; emulsion

(Sultana et al., 2000) (Adhikari et al., 2003)

not stated otherwise, probiotic strains were separately microencapsulated

While sugar crystals, that is, hard and irregular particles, can already be detected in various foods at sizes ranging from about 10 to 20 µm (Imai et al., 1999), soft spherical hydrogel microcapsules have a higher threshold level regarding a graininess detection. Hansen et al. (2002) reported a size below 100 µm as desirable to avoid having negative sensorial impacts of microcapsules in food.

Application of Probiotic Microcapsules in Yogurt Various fermented food products have already been supplemented with probiotic microcapsules. A major focus of most of these studies is an evaluation of the protective effect due to microencapsulation during product storage. Yogurt is the most extensively supplemented product so far, due to the fact that the probiotic activity found in yogurt is often rather low (Kailasapathy and Rybka, 1997; Shah, 2000). Table 1 gives an overview of the application of probiotic microcapsules in yogurt including the most important experimental conditions. From the studies shown in Table 1 it can be concluded that encapsulation of various probiotic strains in hydrocolloid gels enhances their survival during storage in yogurt at about 0.5 to 3 log cycles CFU. The protective effect is generally explained by limited diffusion of inhibitory substances such as metabolic products from the starter cultures, H2O2, lactic acid, and bacteriocin into the capsules (Krasaekoopt et al., 2006; Sun and Griffiths, 2000). Taken together with the rather low pH of 4.5 or below (Lourens-Hattingh and Viljoen, 2001), cell death from presence of oxygen has been discussed as one of the major factors for the low survival rates of probiotics in yogurt. Talwalkar and Kailasapathy (2003) showed that encapsulation in alginate hydrogels offers substantial protection for probiotics under aer-

obic conditions for several probiotic strains and could therefore be responsible for higher survival rates of encapsulated cells during storage in yogurt. In all cases, a protective effect could only be achieved by using capsules with sizes of 0.2–3 mm. In this case, a negative sensorial impact of the capsules on the food product is most likely. This was confirmed by sensory evaluations of such products which was investigated in some studies mentioned in Table 1. In the study of Adhikari et al. (2000), sensory analyses showed that consumers preferred the yogurt containing free probiotic cells over the one containing probiotic microcapsules by ranking with a higher “overall liking.” It is not quite clear if the inferior sensory characteristics were due to off-flavor or grittiness. While the survival tests were performed with plain yogurt in the study of Adhikari et al. (2003), yogurt used for sensorial evaluation additionally contained 13% blackberry jam. Panellists detected a “grainy structure” for the yogurts containing probiotic microcapsules and a worse “overall acceptability” compared to yogurts containing free cells. Similarly, Kailasapathy (2006) reported that sensorial analyses revealed a slight grittiness for the yogurts containing microcapsules compared to the yogurts containing free cells. Based on the literature available so far, it appears that microencapsulation by using capsules based on ICT-methods results in increased probiotic survival during storage in yogurt. However, the addition of probiotic microcapsules also leads to inferior sensory attributes, compared to yogurt containing the respective probiotic cells in free form. While there is a direct relationship between large capsules sizes and graininess in yogurt, further changes in flavor due to more complex reactions between the hydrocolloid from the capsules and the yogurt matrix, as well as altered metabolic profiles of microorganisms caused by encapsulation, should be considered in future investigations.

298


Table 2

T. HEIDEBACH ET AL. Applications of encapsulated probiotics in cheese


Matrix material and encapsulation technique


Bifidobacterium bifidum

κ-carrageenan; extrusion

Not given

6 months in cheddar cheese

Bifidobacterium bifidum, Bifidobacterium infantis, and Bifidobacterium longum, encapsulated together Two different strains of Lactobacillus acidophilus, Bifidobacterium lactis, and Bifidobacterium infantis Lactobacillus acidophilus and Bifidobacterium lactis

Alginate; extrusion

250

2 weeks in fresh cheese


500–1000


Bifidobacterium bifidum and Lactobacillus acidophilus

Alginate; extrusion (a) κ-carrageenan; emulsion (b)

1If

Storage time and food matrix

Increased survival due to microencapsulation

Reference

Higher cell survival in samples containing non-encapsulated cells No protective effect

(Dinakar and Mistry, 1994)

6 months in cheddar cheese

Higher cell survival in samples containing non-encapsulated cells

(Godward and Kailasapathy, 2003)

Not given

7 weeks in feta cheese

(Kailasapathy and Masondole, 2005)

200–300 (a) 300–400 (b)

90 days in white-brined cheese

Higher cell survival in samples containing non-encapsulated cells 2 log cycles for each strain for (a) and (b)

(Gobbetti et al., 1998)

(Ozer et al., 2009)


Application of Probiotic Microcapsules in Cheese Next to yogurt, cheese is often used as a target of supplementation with probiotic microcapsules (Table 2). Table 2 shows that in the case of rennet cheese, encapsulation is not always useful, since physiological conditions in a hydrocolloid matrix of carrageenan or alginate were less favorable for probiotic cells compared to a milk protein based rennet-gel-matrix. This was expressed in higher cell counts of cheeses containing free cells, compared to those with encapsulated cells at the end of storage (Dinakar and Mistry, 1994; Godward and Kailasapathy, 2003; Kailasapathy and Masondole, 2005). Conflicting results found by Ozer et al. (2009) could be possibly explained by the high concentration of salt (12%) in the brine, rendering the cheese-matrix inappropriate for probiotic survival in this case. The sensory impact of microcapsules supplementation on the cheese was evaluated by Dinakar and Mistry (1994). Capsule sizes were not stated, but since the authors used the extrusion method, relatively large capsule diameters are likely to have been present. However, about 3% capsules within the cheese matrix did not alter the cheeses’ sensory. Accordingly, Ozer et al. (2009) and Gobbetti et al. (1998) report that cheeses containing encapsulated probiotics did not differ from cheeses containing free cells in terms of their sensory properties. In contrast to this, Godward and Kailasapathy (2003) found no difference in flavor between cheeses containing free or encapsulated probiotics. However, in this case, grittiness for cheeses containing capsules was detected (Table 2). Other Foods Containing Encapsulated Probiotics Table 3 shows results from studies with other foods, supplemented with probiotic microcapsules. Food matrices generally

differ in their suitability as a carrier for probiotic cells. As an example, probiotic cells show a higher stability in frozen foods, such as ice-cream, compared to refrigerated foods, such as yogurt. From Table 3 it is apparent that in frozen products even very small microcapsules have the ability to protect probiotic cells during storage. It was reported by Sheu et al. (1993) and Homayouni et al. (2008) that the use of such small capsules avoids a negative impact on sensory impression. Also, Hansen et al. (2002) stated that such capsules were small enough to avoid a grainy structure in milk. Nevertheless, in contrast to milk containing free probiotic cells, a “bitter, sharp” off-flavor was detected by the panellists in samples containing encapsulated cells (Table 3). Hence, there is evidence that also small capsules with an average diameter of about 30 µm that are not detectable by sensory tests can protect probiotic cells in foods. However, it has not been proven yet, whether the application of such small microcapsules can lead to an improved survival during storage in yogurt without simultaneously inducing a negative sensorial effect. In some of the other studies presented in Table 3, sensory evaluation was performed as well. In the study of McMaster et al. (2005), sensory evaluation revealed no off-flavor or grittiness due to incorporated microcapsules. This can be explained by the low level of addition of only 0.04% capsules to the beverages. It should be noted though that the mouthfeel of Mahewu has been described as “grainy” per se and Amasi has a “thick, smooth” structure as stated by the authors. Muthukumarasamy and Holley (2006) added probiotic microcapsules to a sausage batter before fermentation, at a level of 1%. A sensory evaluation revealed that no difference concerning texture, flavor and overall acceptability was found between sausages containing free or encapsulated probiotics. In the study of Khalil and Mansour (1998), improved sensory properties were reported for mayonnaise containing encapsulated cells, with

299

MICROENCAPSULATION OF PROBIOTIC CELLS Table 3

Applications of encapsulated probiotics in other foods



Lactobacillus bulgaricus

Matrix material and encapsulation technique Alginate; emulsion


Storage time and food matrix

15 (a) 30 (b)

2 weeks in frozen ice-milk at −18◦ C

Bifidobacterium bifidum Alginate; emulsion and Bifidobacterium infantis Lactobacillus acidophilus Alginate; emulsion and Bifidobacterium ssp

Not given

8 weeks in mayonnaise with a pH of 4.4.

Not given

Bifidobacterium longum Bifidobacterium lactis

20 640

12 weeks in fermented frozen dairy dessert with a pH of 4.5 at −18◦ C 2 weeks in non-acidified milk (a): 3 weeks in African beverage based on fermented maize with a pH of 3.5 (Mahewu) (b): 3 weeks in African beverage based on fermented milk with a pH of 4.5 (Amasi) 27 days in dry fermented sausages 180 days in ice-cream at −20◦ C

Alginate; emulsion Gellan-xanthan; extrusion

Lactobacillus reuteri

Alginate; emulsion (a) Alginate; extrusion (b) Bifidobacterium lactis and Alginate, co-encapsulation Lactobacillus casei with 2% resistant starch; emulsion 1If

40 (a) 2000– 3000 (b) 18

Increased survival due to microencapsulation

Reference

No protective effect for (a); (Sheu et al., 1993) doubled survival rate for (b) About 5 log cycles for each (Khalil and Mansour, strain 1998) 2–3 log cycles for each strain

(Shah and Ravula, 2000)

0.5 log cycles (a): 2 log cycles (b): No protective effect

(Hansen et al., 2002) (McMaster et al., 2005a)

2 log cycles for (a) and (b)

(Muthukumarasamy and Holley, 2006) (Homayouni et al., 2008)

2 log cycles for each strain


respect to flavor and texture. However, since no information about the addition level and the capsule size was given, hardly any conclusion can be drawn (Table 3). These studies show that large hydrogel-capsules can be successfully applied in some types of foods without altering the sensory properties. Compatibility mainly depends on the physical characteristics of the surrounding food matrix. While for yogurt capsules with average sizes above 200 µm were shown to adversely affect the mouthfeel, gelled foods such as cheese and foods with a structure that is naturally associated with coarseness seem to be more suitable for the application of large microcapsules.

SURVIVAL DURING GASTRIC TRANSIT To obtain a notable health effect from the ingestion and colonization of probiotic cells in the gut, microorganisms must survive transit through the low pH gastric environment, which is an even tougher challenge compared to surviving processing and product conditions. The strong acidic conditions in the human stomach as a natural barrier of the host considerably reduce the number of living probiotic cells (Naidu et al., 1999; Ross et al., 2005). This makes the gastric transit the most crucial hurdle with respect to probiotic survival in food applications (Agrawal, 2005). In some cases enteric polymers, originally developed for controlled release of drugs in medical applications, were used to microencapsulate probiotic cells. By using cellulose-acetatephthalate (Favaro-Trindade and Grosso, 2002; Rao et al., 1989),

R R R , and AcrylEze (Liserre et al., 2007) or Eudragit Sureteric (Graff et al., 2008) impressive protective effects under simulated gastric conditions were found. However, these substances, suitable for medical applications, are not permitted in food products (O’Riordan et al., 2001). All common encapsulation techniques for probiotics in food applications result in matrix capsules, where the living cells as core material are embedded and immobilized in a continuous hydrogel matrix. On this account, it can be presumed that the protective effect of encapsulated probiotics depend on the physical characteristics of the capsule matrix and the capsule size. Many other factors influence the survival rates of encapsulated probiotic cells during simulated gastric transit, especially the sensitivity of the microbial strain as such, and the composition of the gastric fluid. The survival of a certain strain of bacteria under acidic conditions generally depends on the ability to control the activity of the membrane-bound ion transport system that generates the proton motive force (Booth, 1985). The regulation capacity of the cytoplasmic pH under stongly acidic external conditions is believed to strongly depend on the activity of the H+-ATPase, which can widely differ from strain to strain (Matsumoto et al., 2004). In the majority of studies a low concentrated (0.2–0.5%) NaCl-solution, adjusted with HCl to the target pH was used to simulate gastric juice without pepsin, according to the USPharmacopeia (USP, 2008). The rather simple composition of the simulated gastric juice commonly used can be explained by the fact that the gastric bactericidal barrier in vivo is primarily pH-hydrochloric acid dependent, with other constituents of gastric juice contributing little, if any, detectable effect on the killing of microorganisms (Giannellra et al., 1972).

300

T. HEIDEBACH ET AL.

However, aside from the application of various pH-values, sometimes additional substances have been added, leading to a wide variation of simulated gastric conditions as outlined in the following sections.

In Vitro Investigations


Alginate-Based Microcapsules Many researchers have investigated whether a protective effect of an alginate gel-matrix, generated by ionotrophic gelation of alginate solutions at various concentrations of 1%–3% during gastric transit can be achieved. An important prerequisite is structural integrity of the microcapsules during gastric transit. Several in vitro studies have shown that alginate microcapsules remain physically stable at low pH-values in gastric fluid (AllanWojtas et al., 2008; Annan et al., 2008; Cui et al., 2000; Hansen et al., 2002; Iyer et al., 2004; Martoni et al., 2007). However, with respect to the achievable protective effect, published data available so far are inconsistent, as outlined in Table 4. In some of the studies mentioned in Table 4, prebiotic resistant starch granules were co-encapsulated together with the microorganisms, a concept referred to as synbiotics. Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and activity of one or a limited number of bacteria in the colon. Synbiotics are created by a combined application of pro- and prebiotics (Fooks et al., 1999; Rastall and Maitin, 2002). It is generally thought that the resistance of probiotics against the harsh pH-conditions in the human gastro-intestinal-tract can be enhanced by coupling it with a selective growth promoter (Gibson, 2004). Iyer and Kailasapathy (2005), found an increase in the protective effect, due to co-encapsulation with resistant starch, compared to probiotic cells that were encapsulated in alginate alone. The authors assumed that the water-insoluble starch corns could block the pores of the capsules and therefore abate diffusion of acid into the network. An important conclusion that can be drawn from the results presented in Table 4 is that no direct relationship between capsule size or alginate concentration and the respective protective effect can be found. Some authors further studied the influence of capsule size or alginate concentration on the protective effect under equal conditions (Table 5). From the results displayed in Table 4 and Table 5 it becomes clear that a general statement about the suitability of an alginate gel matrix as a protective barrier towards a low pH environment is not possible. Table 5 shows that in direct comparison larger capsule sizes as well as higher alginate concentrations tend to provide a better protective effect. In contrast, as illustrated in Table 4, no such relationship can be found. A possible explanation could be that the variation of experimental simulated gastric conditions and the strain dependent acid-sensitivity dominates over the general improvement of alginate encapsulated cells in comparison to free cells.

From Table 4 it can be further seen that in general the highest increase in survival is achieved with capsules that were freezedried before the simulated gastric experiments. A possible explanation is that the addition of dried powders to simulated gastric juice instead of hydrated capsules requires certain time for rehydration of the capsules and could therefore lead to a delayed penetration of the capsules with acid. Furthermore, the addition of capsules to simulated gastric juice can generally lead to an increase in pH of the resulting mixture. Despite the fact that this effect can greatly affect survival rates, it is generally not considered in most studies on simulated gastric survival of encapsulated probiotics. This was extensively discussed by Saarela et al. (2006) as generally aflaw in experimental design, when probiotic cells were tested against acid stress. In case of freeze-dried capsules, the addition of the same amount of dry powder instead of hydrated capsules can result in a higher buffering capacity. This, in turn, leads to a higher resulting pH which may explain the outstanding results that have in some cases been obtained with respect to freezedried capsules. Standardized simulated gastric conditions would therefore allow a more meaningful comparison of encapsulation systems for probiotics. For the failure of a protective effect of alginate during incubation of hydrated microcapsules under simulated gastric conditions different explanations can be found. The porosity of the alginate gel allows the diffusion of H+-ions into the gel, thus affecting the cells (Trindade and Grosso, 2000). Hansen et al. (2002) suggested that the porosity of the alginate matrix is increased due to the presence of the bacteria during the gelation process. Le-Tien et al. (2004) encapsulated a pH-sensitive color indicator in 3 mm alginate beads from 1.5 or 2.5% alginate solutions. The authors found that after incubation in simulated gastric juice at pH 1.5 the internal capsule pH went below 2 after approximately 8 min incubation time, independent of the alginate concentration used. It was concluded that alginate gels have a limited buffering capacity, and the alginate gel structure should be seen as a highly porous hydrogel, that provides virtually no barrier effect against the diffusion of H+-ions into the gel.

Coating of the Capsule-Matrix Coating deposits an additional membrane-layer on the capsule surface. This leads to an increase in mechanical strength and a more pronounced barrier function. This is typically achieved by immersing the hydrogel capsules into a solution of coating polymer. Coating of biopolymer capsules has been a well known technique from the field of ICT, with its original goal to slow and reduce the release of cells into the surrounding medium (Champagne et al., 1992). It is also widely applied in the field of artificial cell therapy (Prakash and Martoni, 2006). The highly porous alginate network found in bacteria-loaded microcapsules may explain the limited protective effect under simulated gastric conditions (Allan-Wojtas et al., 2008). Thus, it was investigated whether coating can increase the protective effect under such conditions. Poly-l-lysine and chitosan are

301

500–1000

1% alginate 2% alginate together with 2% resistant starch 1.8% alginate

Lactobacillus acidophilus and Bifidobacterium Lactis Lactobacillus acidophilus and Bifidobacterium infantis Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium breve Lactobacillus casei

2If

1If

Not given

3% alginate

Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei, and two strains of Bifidobacterium lactis Bifidobacterium lactis Lactobacillus acidophilus

No protective effect

About 4 log cycles

Increased survival due to microencapsulation, compared with free cells

4 log cycles

3 log cycles

pH 1.5 for 2 h MRS-medium adjusted to pH 1.5, for 3 h

No protective effect 4 log cycles

Bifidobacterium bifidum no protective effect; Lactobacillus acidophilus: about 2.5 log cycles Lactobacillus casei: about 1 log cycle NGYC-medium adjusted to pH About 2 log cycles for each strain 2, for 3 h Lactate broth, adjusted to pH No protective effect 2.0, for 1 h MRS-medium adjusted to pH 2–3 log cycles for each of the strains 2.0, for 2 h

pH 1.55 for 2 h

pH 2.0 for 90 min

pH 1.2 for 3 h

NGYC-medium adjusted to pH No protective effect 2.0 or pH 3.0, for 3 h pH 2.0 and 3.0 for 2 h No protective effect for (a) and (b)

pH 1.5, for 2 h (solution contains 0.5% yeast extract and 0.05% L-cysteine) pH 1.0 or pH 2.0 for 2 h

Simulated gastric conditions2

not stated otherwise, probiotic strains were separately microencapsulated only the pH is given, a low concentrated (0.2–0.5%) NaCl-solution, adjusted with HCl to the target pH, was used.

2% alginate 60 2% alginate and 0.15% xanthan gum, 75 capsules were freeze-dried

3000

450–500

1.8% Alginate together with 1% resistant starch 2% alginate

1% alginate, capsules were 50 freeze-dried 2% alginate and 0.26% xanthan gum, 60 capsules were freeze-dried 2% alginate 1600

20 (a) and 70 (b)

two different strains of Lactobacillus acidophilus Propionibacterium freudenreichii

Lactobacillus acidophilus, Bifidobacterium bifidum, and Lactobacillus casei


2000–4000

1.5% alginate, capsules were freeze-dried


100

Matrix precursor (aqueous solution)


Effect of encapsulation in alginate-based microcapsules on probiotic survival after simulated gastric passage


Table 4


(Liserre et al., 2007) (Kim et al., 2008)

(Ding and Shah, 2007)

(Iyer and Kailasapathy, 2005) (Leverrier et al., 2005)


(Lee et al., 2004)

(Song et al., 2003)

(Hansen et al., 2002)

(Trindade and Grosso, 2000) (Sultana et al., 2000)

(Cui et al., 2000)

Reference

302 Table 5

T. HEIDEBACH ET AL. Influence of capsule size or alginate concentration on survival after simulated gastric passage



Matrix precursor (aqueous solutions)

Increased survival due to microencapsulation, compared Average size of with free cells capsules (µm) Simulated gastric conditions2

Two different strains of 2% alginate Bifidobacterium longum

1000, 1750, and 2600

pH 1.55 for 2 h

Two different strains of 2, 3, and 4% alginate Bifidobacterium longum Lactobacillus acidophilus 1.5% alginate

2600

pH 1.55 for 3 h

200, 450 and 1000

NGYC-medium, adjusted to pH 2.0, for 2 h

Lactobacillus rhamnosus

1.5 and 2.5% alginate

3000

Lactobacillus acidophilus

0.75, 1 and 2% alginate

450

Lactobacillus casei

2, 3 and 4% alginate

Not given

pH 1.5, containing pepsin, for 30 min NGYC-medium, adjusted to pH 2.0, for 2 h pH 1.5 for 3 h

Lactobacillus reuteri

3% alginate

40 and 2400

pH 1.5 for 2 h

1If 2If

1.0 mm: no survival, regardless of treatment; 1.75 mm: about 3 log cycles 2.6 mm: about 6.5 log cycles 2%: 4.5 log cycles, 3%: 5 log cycles 4%: 5.5 log cycles 0.2 mm: about 1 log cycle; 0.45 and 1.0 mm: about 1.5 log cycles About 7 log cycles for both alginate concentrations 0.75 and 1%: no protective effect; 2%: 1 log cycle 2%: 1 log cycle, 3%: 1 log cycle 4%: 2 log cycles 40 µm: 6 log cycle; 2.4 mm: 6.5 log cycles

Reference (Lee and Heo, 2000)

(Lee and Heo, 2000) (Chandramouli et al., 2004) (Le-Tien et al., 2004) (Chandramouli et al., 2004) (Mandal et al., 2006) (Muthukumarasamy et al., 2006)


commonly used as coating materials, because they form strong complexes with alginates (Krasaekoopt et al., 2004). Table 6 shows the protective effect that can be achieved with coated probiotic microcapsules. Table 6 shows that chitosan-coated microcapsules in comparison to non-coated capsules had an increased protective effect under simulated gastric conditions. In contrast, no enhancement of the protective effect was found in the case of coating with poly-l-lysine. However, only a few countries, for example, Japan, have to date permitted the use of chitosan in food so far (Agullo et al., 2003). In the US it is not allowed in foods (Park et al., 2002).

Ding and Shah (2009b) encapsulated ten different probiotic strains each separately in 3% solutions from different hydrocolloid precursors by means of the emulsion method. The authors found that xanthan gum and carageenan gum had slow protective effects comparable to those of alginate, while guar gum and locust-bean gum had poor protective capacities after 2 h of incubation in MRS-broth previously adjusted to pH 2.0. The usage of other polysaccharides besides alginate is only sporadic, but from the available results it seems rather unlikely that superior protective characteristics are achievable.

Protein Based Microcapsules Other Polysaccharide-Based Matrix-Materials Alginate is by far the most frequently used polymer to encapsulate probiotic cells. However, some studies were carried out with gellan-xanthan gum mixtures (see Table 7). Gellanxanthan-based microcapsules have a higher mechanical stability compared to alginate-based capsules (Sun and Griffiths, 2000) and could thus be advantageous in protecting probiotic cells during gastric transit. However, compared to the results of alginatebased capsules, the results are also inconsistent. Muthukumarasamy et al. (2006) directly compared capsules generated from different precursors. They encapsulated different strains of Lactobacillus reuteri in microcapsules from 0.5% gellan with 1% xanthan solutions, 1.75%.-carrageenan with 0.75% locust bean gum solutions or solely 3% alginate solutions. The authors found that alginate capsules generally provided an enhanced protective effect during incubation at pH 1.5 for 2 h, compared to the other polymers used. This result was found for small capsules (40–300 µm) generated by means of the emulsion technique, as well as for large capsules (2.5 mm) generated by the extrusion technique.

The application of polysaccharide-protein mixtures or solely protein-based matrix materials is a relatively new strategy and can be seen as a promising alternative approach directly developed for probiotic encapsulation and not originating from ICT. However, in this case, often alternative gelling-mechanisms are required (See section titled “Protein Based Microcapsules”). Table 8 shows approaches containing the application of proteins for probiotic encapsulation. In the studies mentioned in Table 8, the higher survival of encapsulated cells compared to free cells after incubation under simulated gastric conditions was explained by the buffering capacity of the protein-containing gel-matrix. This was confirmed by Gbassi et al. (2009), who found 5–7 log cycles CFU higher survival in alginate-based capsules that were soaked in wheyprotein solution, compared to capsules solely based on alginate, after simulated gastric treatment (see Table 8). Hebrard et al. (2006), who encapsulated a recombinant yeast in whey protein-based microcapsules, came to a similar conclusion, that is, that a protein matrix could exert a buffering effect at low pH. The limited protection, found in the studies of Reid

303

1.5% alginate, capsules were freeze-dried

2If

1If

(a): Chitosan; (b): 2% alginate gel mixed non-coated with 0.26% Xanthan, capsules were freeze-dried 1.8% Alginate together (a): Poly-l-lysine (b): with 1% resistant starch Chitosan (c): non-coated

(a): Poly-l-lysine (b): Chitosan (c): non-coated

(a): Poly-l-lysine (b) non-coated

Coating

450–500

60

1600

100


Bifidobacterium bifidum no protective effect for (a), (b), and (c); Lactobacillus acidophilus: about 3 log cycles for (b), about 2.5 log cycles for (a) and (c) Lactobacillus casei: about 2 log cycles for (b), about 1 log cycle for (a), and (c) About 4.5 log cycles for (a), 4 log cycles for (b)

4 log cycles for (a) and (b)


(Lee et al., 2004)


(Cui et al., 2000)

Reference

NGYC-medium adjusted to pH About 2.5 log cycles, for each strain for (b), (Iyer and 2, for 3 h about 2 log cycles for each strain for (a) and Kailasapathy, (c) 2005)

pH 2.0 for 90 min

pH 1.5 for 2 h (solution contains 0.5% yeast extract and 0.05% L-cysteine) pH 1.55 for 2 h



Two different strains of Lactobacillus acidophilus


Lactobacillus acidophilus, 2% alginate Bifidobacterium bifidum, and Lactobacillus casei



Effect of encapsulation and subsequent coating on probiotic survival after simulated gastric passage


Table 6


304 Table 7

T. HEIDEBACH ET AL. Effect of encapsulation in gellan-xanthan-based microcapsules on probiotic survival after simulated gastric passage



Bifidobacterium infantis

0.75% gellan gum mixed with 1% xanthan gum

3000

Bifidobacterium lactis Propionibacterium freudenreichii

0.75% gellan gum mixed with 1% xanthan gum 0.75% gellan gum mixed with 1% xanthan gum

600

1If 2If



3000

Simulated gastric conditions2 pH 2.5 for 2 h (a); 2 h incubation at pH 2.0 or 1.5 (b) pH 1.5 for 4 h lactate broth, adjusted to pH 2.0 for 1 h


Reference

(a): about 6 log cycles; (b) no (Sun and Griffiths, 2000) survival found, regardless of treatment. 2 log cycles (McMaster et al., 2005a) No protective effect (Leverrier et al., 2005)


et al. (2005) and Picot and Lacroix (2004) was related to the detrimental influence of the encapsulation process itself, probably leading to sub-lethal damage of the encapsulated cells prior to the addition to the simulated gastric model. (See section titled “Protein Based Microcapsules.”) It can be concluded that the application of protein-supported or protein-based capsule-matrices seems promising and could provide substantial protection for probiotic cells during gastric transit if gentle encapsulation techniques are applied. Furthermore, most of the probiotic foods available today are dairy products. A higher consumer acceptance for dairy based microcapsules compared to those of non-dairy origin in dairy products seems likely.

In Vivo Investigations There are only a few published in vivo studies, and these are dealing with the supplementation of probiotic microcapsules to animals. Kushal et al. (2006) encapsulated Lactobacillus acidophilus and Bifidobacterium bifidum in alginate microcapsules with an average diameter of 85 µm. The capsules, as well as the free cells were fed to mice with a skim-milk-carrier solution for a period of 10 days. The authors found higher levels of probiotic cells with a longer persistence period after application and post-withdrawal in mouse feces that were fed with encapsulated cells, compared to mice fed with free cells. Furthermore, a more pronounced displacement of coliform cells was reported in case of a treatment with encapsulated cells. However, the authors did not provide any quantitative information about the obtained living cell numbers of probiotics. Similar results were obtained in an in vivo study by Graff et al. (2008), who encapsulated Saccharomyces boulardii in capsules of alR with averginate, mixed with the enteric polymer Eudragit age diameters of about 350 µm. After a single oral dose together with the standard diet, significantly higher amounts of yeast were found in feces from mice, fed with encapsulated probiotics. However, both authors did not report on cell release in the host or probiotic health effects on the host.

INTESTINAL SURVIVAL AND CELL RELEASE Protective Effect of Microencapsulation under Simulated Intestinal Conditions After the transit through the stomach, probiotic cells have to withstand the conditions in the intestine. Incubation in simulated bile solutions was shown to be generally less detrimental to the probiotic cells, compared to simulated gastric conditions at low pH-values. In some cases even an increase in CFU was found during incubation under simulated intestinal conditions of encapsulated and free probiotic cells (Annan et al., 2008; Guerin et al., 2003; Picot and Lacroix, 2004). In other cases, no decrease of living cell numbers induced by incubation in simulated bile solution was found, regardless of whether free or encapsulated cells were used (Favaro-Trindade and Grosso, 2002; Muthukumarasamy et al., 2006; Reid et al., 2005; Trindade and Grosso, 2000). In cases where a cell-count reduction was observed, most authors found a protective effect due to microencapsulation (Chandramouli et al., 2004; Ding and Shah, 2007; Iyer and Kailasapathy, 2005; Kim et al., 2008; Krasaekoopt et al., 2004; Lee and Heo, 2000; Mandal et al., 2006; McMaster et al., 2005; Song et al., 2003). Only a few authors reported no protective effect of encapsulation (Leverrier et al., 2005; Reid et al., 2005; Sultana et al., 2000). Release of Microencapsulated Cells in the Intestine In Vitro Investigations To effectively use probiotic microcapsules in food products, it must be ensured that the hydrocolloids used as matrix material not only provide the desired barrier effect during storage in the food and under low pH-conditions, but that capsules must also be digestible in order for the probiotic cells to be released in the human gut. This release is an essential step for an effective interaction of the probiotic cells with the human intestinal ecosystem, and therefore a mandatory prerequisite for a successful application of probiotics in functional foods. Since the pore size of alginate gels is small enough to retain microorganisms within the gel network (Allan-Wojtas et al., 2008), a

305

2If

1If

2% alginate, capsules were soaked in a 2% whey-protein solution and subsequently freeze-dried 35% skim-milk-solids

Gelatine (13%), capsules coated with alginate 15% sodium-caseinate

2800

Ionotrophic gelation with Ca2+

Enzymatic gelation by means of rennet

70

Not given

170

50

50

Dehydration by means of spray-drying

Non-toxic cross-linker from plants: genipin Enzymatic gelation by means of transglutaminase Ionotrophic gelation with Ca2+

2000–3000


Ionotrophic gelation with Ca2+

Gelling-mechanism

pH 2.5 for 90 min

pH 1.8, containing pepsin, for 2h

dynamic simulated gastric system, with decreasing pH from 4 to 2 within 90 min pH 2.0, containing pepsin, for 2h pH 2.5 for 90 min

pH 1.5 (a) or 2.5 (b), containing pepsin, solution buffered with KCL, for 2 h pH 1.9, containing pepsin, for 30 min



Lactobacillus paracasei and Bifidobacterium lactis

Bifidobacterium adolescentis Lactobacillus paracasei and Bifidobacterium lactis 3 different strains of Lactobacillus plantarum

Alginate (2%) pectin (3.5%) whey protein (6.25%) mixture Bifidobacterium breve and Pre-heated whey proteins (10%) Bifidobacterium longum Lactobacillus rhamnosus Pre-heated whey proteins (12%)



Lactobacillus paracasei: 0.8 log cycles; Bifidobacterium lactis: 2.8 log cycles

5–7 log cycles, depending on strain

Lactobacillus paracasei: 2 log cycles; Bifidobacterium lactis: 2.8 log cycles

2 log cycles

Bifidobacterium longum: no survival, regardless of treatment; Bifidobacterium breve: 1 log cycle Slight increase

(a): no survival, regardless of treatment; (b): 5 log cycles


Effect of encapsulation polysaccharide-protein mixtures or solely protein-based microcapsules on probiotic survival after simulated gastric passage


Table 8


(Heidebach et al., 2009a)

(Gbassi et al., 2009)

(Heidebach et al., 2009b)

(Annan et al., 2008)

(Reid et al., 2005)

(Picot and Lacroix, 2004)

(Guerin et al., 2003)

Reference


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quantitative release is only achieved upon complete degradation of the gel-matrix during digestion. Some authors studied the cell release under various in vitro simulated intestinal conditions. Shah and Ravula (2000), as well as Mandal et al. (2006) reported a complete release of probiotic cells from alginate capsules in simulated intestinal juice and concluded that the organisms would be released in the human intestine. Iyer et al. (2004) investigated the release of an E. coli strain from alginate microcapsules using natural gastric secrets from different sections of the gastro-intestinal tract of hogs ex vivo. The authors found that cells were released from the capsules into the ex vivo porcine small-intestinal content within 1 hour. A slower release of probiotic cells into ex vivo porcine intestinal juice was reported for chitosan-coated alginate microcapsules (Iyer et al., 2005). In contrast to this, Urbanska et al. (2007) found that chitosan-coated alginate capsules containing Lactobacillus acidophilus, did not dissolve after incubation in simulated intestinal fluid for 24 h. Cui et al. (2000) reported that poly-l-lysine coated alginate microcapsules were completely dissolved within 12 h in simulated intestinal juice as well, and therefore did release all cells. Contrary to that Martoni et al. (2007) found poly-l-lysine coated alginate capsules containing Lactobacillus plantarum to be physically stable after incubation in simulated intestinal fluid for 10 h. In the case of gellan-xanthan-based microcapsules it was reported that no dissolution of the capsules in intestinal juice occurs and that a release of probiotics in the colon could only be achieved by mechanical breakdown due to the peristaltic motion (McMaster et al., 2005; Sun and Griffiths, 2000). However, it seems rather unlikely that a quantitative release of cells into the human gut is achievable by mechanical breakdown of capsules only and therefore digestibility of the gel matrix appears to be necessary. In contrast to this, by using natural food-matrices, such as highly concentrated protein hydrogels for probiotic encapsulation, a digestion accompanied by a probiotic cell release in the gut can be ensured. Picot and Lacroix (2004), for example, showed that whey protein-based microcapsules did not dissolve during incubation in simulated gastric juice, even in the presence of pepsin, but could be completely hydrolyzed in simulated intestinal juice within 3 h. Similarly, Reid et al. (2005) concluded that whey protein gel capsules might be preferable to alginate capsules, since their proteinaceous structure is hydrolyzed during ingestion. With proteins hydrogels of diverse mechanical and micro-structural properties can be generated, that have different release characteristics in the human intestinal tract (Chen et al., 2006, Livney, 2010). In Vivo Investigations Published data from in vivo trials regarding the digestibility of alginate capsules are scarce. However, the available studies seem to indicate that results differ from those reported in the

in vitro studies mentioned in the previous section. Simulated gastric experiments that focus on the reduction of living cell numbers predominantly caused by H+-concentration, allow for an accurate correlation with in vivo experiments (Berrada et al., 1991). However, this is not the case for a simulated digestion of microcapsules in the intestine, since the disintegration of a microcapsule in vivo as a part of the metabolic pathway of the host is driven by many complex enzymatic and chemical reactions that can not be easily mimicked in vitro. Hoad et al. (2008) investigated the fate of alginate microcapsules, generated from a 1.5% alginate solution, in the human intestinal tract after ingestion in vivo by means of magnetic resonance imaging. Physically intact capsules were still visible in the human intestine after 4 h of ingestion, while they were dissolved in a parallel in vitro trial (Rayment et al., 2009). The authors therefore concluded that in vitro trials did not sufficiently reflect the conditions in the human intestinal tract. Similarly, when chitosan- or poly-l-lysine coated alginate microcapsules containing an E.coli strain were administered to mice by Lin et al. (2008), the authors reported that both types of capsules remained intact in the mouse intestine after 6 h of ingestion, with chitosan coated capsules having a higher mechanical strength. Van Venrooy (2004) encapsulated Lactobacillus acidophilus in microcapsules using 1.5% alginate solutions. Six different alginate preparations were investigated that varied in their molecular weight and viscosity. After feeding the probiotic microcapsules to pigs, capsules were apparently excreted undigested, independent of the type of alginate used. Alginate-based polymer gel microcapsules are the most commonly used vehicles in the adjacent field of artificial cell therapy. Artificial cell therapy is a technique used to encapsulate biologically active materials in semi-permeable polymer membranes. One of the main characteristics of such microcapsules is that they must remain intact during the passage through the intestinal tract for safety reasons and are then excreted intact in the stool (Prakash and Jones, 2005). Future investigations will have to clarify the influence of physical characteristics of alginate-systems on the human intestinal tract, since the dissolution of probiotic microcapsules in the gut is a mandatory prerequisite for an effective application.

CONCLUSIONS AND FURTHER DIRECTIONS Conclusions Immobilized cell technology originally provided the technical background for the development of microencapsulation techniques for probiotic cells. However, other technical or physiological requirements play a decisive role in the microencapsulation of living probiotic cells. These may differ significantly from ICT. In particular, probiotic microcapsules

MICROENCAPSULATION OF PROBIOTIC CELLS • •

•

•


•

should be generated by encapsulation processes that do not decrease the living cell count or induce sub-lethal damages, should not alter the sensory properties of the food system, a fact that may be caused by having large, detectable capsules or changes in flavor profiles, should provide protection against adverse conditions that may be caused by food processing and the environment of the foodmatrix, should stabilize the probiotic cells against stress induced by the high acidic gastric-conditions, and should be digestible in the intestine and release the cells at a high level of activity.

Most of these features are not delivered by ICT-based probiotic microcapsules. Since for a successful implementation of probiotic microcapsules in foods all the above mentioned demands must be met, the development of enhanced encapsulation systems for probiotics is of utmost importance. To date most of the probiotic microcapsules available so far fail to fulfil all the necessary requirements.

Development of Novel Encapsulation Methods Efforts for novel approaches point in two directions, the development of advanced encapsulation techniques and the use of alternative matrix materials. Emulsion technology is prevalent and has been widely studied in the field of food science. Nevertheless, in the case of probiotic microencapsulation the emulsion process is mostly accomplished by simple mechanical or magnetic stirring, leading to a rather undefined particle size reduction. Since particle size has been identified as one of the key-factors for a successful application in foods, advanced emulsifying systems should be introduced in the emulsificationbased microencapsulation-technique. For instance, membrane emulsification might serve as an alternative technique since it was already successfully applied to the microencapsulation of Lactobacillus casei by Song et al. (2003), and suggested in a recent review by Charcosset (2009). To avoid the laborious separation of capsules from oil, it might be better if the emulsion technique would be translated to water-in-water emulsions, a system that is generated by thermodynamic incompatibility between proteins and polysaccharides inmixtures. Syrbe et al. (1998) already demonstrated the feasibility of creating whey-protein-based microgel particles by means of this method. Another promising strategy could be the application of waterinsoluble microcapsules from spray-drying. Some innovative studies already demonstrated that it is feasible to produce such microcapsules by spray drying, using pre-denatured whey protein solutions (Picot and Lacroix, 2004), protein-sugar-mixtures (Crittenden et al., 2006), or even alginate and carrageenan as precursors, as shown by Burey et al. (2009). With respect to the negative influence of the drying process on cell survival, the selection and identification of more heat stable strains (Meng et al., 2008), as well as the application of protective substances

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(Santivarangkna et al., 2008), co-encapsulated together with the probiotic cells appear to be most promising. The technique could thus be advantageous over the classical encapsulation techniques known from ICT, particularly with respect to cost effectiveness. In search of alternative matrix materials for probiotic encapsulation, proteins have begun to be more widely explored in the last 5 years. Generally, the use of engineered food structures, based on gels with high dry-matter content could be a suitable approach as matrix-materials for encapsulation (Aguilera, 2005; Parada and Aguilera, 2007). This is because they may provide a more pronounced physical barrier to protect the core while ensuring the release of the cells in the gut. It appears as if proteins would be the preferred source, because they are compatible with most food systems, provide flexibility with respect to various possible mechanisms for capsule formation, and they are digestible, which is important for the release characteristics of probiotic cells. However, when protein-solutions are used as precursors for probiotic encapsulation, the gelation mechanism seems crucial, since proteins are usually gelled by heating. This is not applicable during encapsulation of sensitive core materials, such as living microorganisms. A useful gelling mechanism that could serve as a processing step for the microencapsulation of probiotics could be cold-set gelation of whey-protein solutions (Hebrard et al., 2006; Reid et al., 2007). The use of enzymes as natural biocatalysts (Heidebach et al., 2009a; 2009b), or the use of non-toxic cross-linking agents from plants, such as genipin (Annan et al., 2008), could allow the formation of protein gels under mild conditions. The use of fat-based microcapsules currently seems rather unsuitable for probiotic encapsulation (see section titled “Lipid Based Microcapsules”). However, a related alternative could be hydrophobic coating. Recently, coating of 100–300 µm sized alginate capsules with hydrophobic stearic acid, was performed by Sabikhi et al. (2008), to delay penetration of gastric juice. In this case, the authors found remarkably increased survival rates of Lactobacillus acidophilus during simulated gastric- and bile conditions, compared to free cells.

The Demand for In Vivo Studies The overall task of probiotic encapsulation, from the first addition of cells to the capsule matrix-solution until the desired colonization in the human gut comprises different phases, such as the encapsulation process, product storage, and gastric transit. These steps are often investigated separately and in an isolated manner. Therefore, approaches would be useful that reflect all influences coming along with an actual implementation. In this context in vivo trials are of utmost importance. However, only a few in vivo studies are available (Graff et al., 2008; Kushal et al., 2006). Even without offering insights about cell release in the host or probiotic health effects of the host, these results are promising with respect to possible benefits for future food applications of microencapsulated probiotics. An important task for future applications is to provide clinical evidence

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that ingestion of microencapsulated probiotics is a more effective way to achieve health benefits from probiotic cells. Finally, it must be considered that above a certain population density microbial cells might undergo complex cell-to-cell interactions (quorum-sensing) that could lead to a different expression of genes compared to free cells (Lacroix and Yidirim, 2007). Some Lactobacillus strains at least have been shown to have undergone morphological changes due to entrapment in alginate gels (Lamboley et al., 2003). However, possible consequences of these phenomena on probiotic bioactivity have not yet been investigated.


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