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Encapsulation Technology to Protect Probiotic BacteriaMaria Chavarri, Izaskun Maranon and Maria Carmen Villaran[1] Bioprocesses & Preservation Area, Health Division, Tecnalia,Parque Tecnologico de Alava, Minano (Alava),, Spain1. Probiotics are bacteria that help maintain the natural balance of organisms (microflora) in the intestines. I have officially switched to inner-eco and find it very reasonably priced for 30 servings coupled with great results. I’m sharing some of my favorite recipes using protein powder that are perfect for taking on a trail run or stowing away in your gym bag.
Plant Protein Complex (Creamy Vanilla) – Great for vegetarians and vegans (and even meat-eaters who want a change of pace), the NOW Sports Plant Protein Complex is a versatile choice made with a pea, brown rice, hemp and quinoa protein blend.
Sprouted Brown Rice Protein (Natural Vanilla) – Everyone needs protein to thrive and survive, however those with certain food allergies (such as whey, egg, soy, etc) are often left with few choices when it comes to protein powder. Additionally, I recently tried these two other NOW Foods products – thumbs up to both, especially for convenience.
Lemonade Slender Sticks – These single-serve drink mix packets are so convenient for summer travel or to stash in a gym bag. Probiotic-10 + Inulin: More single-serve packets, so you can get probiotics anytime, anywhere! Increases helpful Lactobacilli, restrains growth of harmful bacteria, and help with bowel movement.
You may have noticed that probiotics are now featuring in articles relating to all sorts of health problems, including obesity, diabetes, depression and heart disease. Schematic diagram of a spray-dry encapsulation process and image of a Mini Spray Dryer B-290 (BUCHI), available at TECNALIA.Table 1. Flow-Focusing technology to make droplets and Cellena® equipment from Ingeniatrics Tecnologias. Image of Inotech Encapsulator IE-50R and schematic diagram of jet destabilization and breakage for single and concentric nozzles. Schematic diagram of the JetCutter technology and representation of fluid losses due to the cutting wire impact. Examples of two bioencapsulation process carried out at TECNALIA changing the nozzle diameter, cutting tool and inclination angle to obtain different bead size necessaries for several applications.Table 3. Gelation of an alginate bead when the Ca2+ gelling ions diffuse into the alginate-containing system. IntroductionProbiotic bacteria are used in production of functional foods and pharmaceutical products.
The normal human digestive tract contains about 400 types of probiotic bacteria that reduce the growth of harmful bacteria and promote a healthy digestive system.
For me, they have kept inflammation down in my body, my guts regulated, and definitely help me during allergy season.
I do all the proteins – beef, chicken, fish, and so on.  But often, I’m in such a rush for breakfast in the mornings or after a workout, that I’m not able to cook up a big juicy steak. Each scoop provides 22 grams of proteins and 3,900 mg of branched chain amino acids and 1,800 of L-Arginine. NOW® Sprouted Brown is a great solution and can be used with the confidence that only natural and pure ingredients are being used – no soy or dairy and is suitable for vegans and vegetarians.
What I like about them, other than the sweet-tart taste, is peace of mind knowing I’m using a product with made natural sweeteners and non-GMO ingredients, no artificial flavors or preservatives, and contains 50% of nine daily vitamins. This convenient supplement helps to maintain a healthy microbial balance in the gut – thanks to 10 probiotic strains and 10 billion organisms per little packet! The largest group of probiotic bacteria in the intestine is lactic acid bacteria, of which Lactobacillusacidophilus, found in yogurt with live cultures, is the best known.
Chez l'homme, lactobacilli sont couramment utilise comme les probiotiques, soit d ’ espece unique ou en culture melange avec d'autres bacteries. In order, to produce health benefits probiotic strains should be present in a viable form at a suitable level during the product is shelf life until consumption and maintain high viability throughout the gastrointestinal tract. I came down with a slight head cold and noticed this probiotic was helpful in reducing symptoms and duration. Using protein powder is a go-to on these days when I have a light appetite, but realize my body needs adequate protein for muscle repair and maintenance. Many reports indicated that there is poor survival of probiotic bacteria in products containing free probiotic cells [1].
Providing probiotic living cells with a physical barrier to resist adverse environmental conditions is therefore an approach currently receiving considerable interest [2].The encapsulation techniques for protection of bacterial cells have resulted in greatly enhanced viability of these microorganisms in food products as well as in the gastrointestinal tract.
Encapsulation is a process to entrap active agents within a carrier material and it is a useful tool to improve living cells into foods, to protect [3, 4, 5, 6, 7], to extend their storage life and to convert them into a powder form for convenient use [8, 9, 10, 11]. In addition, encapsulation can promote controlled release and optimize delivery to the site of action, thereby potentiating the efficacy of the respective probiotic strain. This process can also prevent these microorganisms from multiplying in food that would otherwise change their sensory characteristics. Otherwise, materials used for design of protective shell of encapsulates must be food-grade, biodegradable and able to form a barrier between the internal phase and its surroundings.2. DefinitionProbiotics are defined as live microorganisms which, when administered in adequate amounts, confer health benefits to the host [12], including inhibition of pathogenic growth, maintenance of health promoting gut microflora, stimulation of immune system, relieving constipation, absorption of calcium, synthesis of vitamins and antimicrobial agents, and predigestion of proteins [13]. Several health benefits have been proved for specific probiotic bacteria, and recommendations for probiotic use to promote health have been published [14].The term ‘‘probiotic’’ includes a large range of microorganisms, mainly bacteria but also yeasts.
Because they can stay alive until the intestine and provide beneficial effects on the host health, lactic acid bacteria (LAB), non-lactic acid bacteria and yeasts can be considered as probiotics.
LAB are the most important probiotic known to have beneficial effects on the human gastro-intestinal (GI) tract [15].The effects of probiotics are strain-specific [16, 17, 18] and that is the reason why it is important to specify the genus and the species of probiotic bacteria when proclaiming health benefits.
Health benefitsThere is evidence that probiotics have the potential to be beneficial for our health [22]. Multiple reports have described their health benefits on gastrointestinal infections, antimicrobial activity, improvement in lactose metabolism, reduction in serum cholesterol, immune system stimulation, antimutagenic properties, anti-carcinogenic properties, anti-diarrheal properties, improvement in inflammatory bowel disease and suppression of Helicobacter pylori infection by addition of selected strains to food products [23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33].The beneficial effects of probiotic microorganisms appear when they arrive in the intestinal medium, viable and in high enough number, after surviving the above mentioned harsh conditions [34]. Apart from the MBV index, daily intake (DI) of each food product is also determinable for their probiotic effectiveness. This microbiota plays an important role in human health, not only due to its participation in the digestion process, but also for the function it plays in the development of the gut and the immune system [42].
The mechanisms of action of probiotic bacteria are thought to result from modification of the composition of the endogenous intestinal microbiota and its metabolic activity, prevention of overgrowth and colonization of pathogens and stimulation of the immune system [43]. With regard to pathogen exclusion, probiotic bacteria can produce antibacterial substances (such as bacteriocins and hydrogen peroxide), acids (that reduce the pH of the intestine), block adhesion sites and be competitive for nutrients [44].Recent studies have shown differences in the composition of the gut microbiota of healthy subjects [45], underlining the difficulties in defining the normal microbiota at microbial species level. Moreover, studies suggest that some specific changes in gut microbiota composition are associated with different diseases [46, 47].
This was confirmed by the comparison of the microbiome from healthy individuals with those of diseased individuals, allowing the identification of microbiota imbalance in human diseases such as inflammatory bowel disease or obesity [48, 49].3. The benefits of encapsulation to protect probiotics against low gastric pH have been shown in numerous reports [50] and similarly for liquid- based products such as dairy products [21, 52].Encapsulation refers to a physicochemical or mechanical process to entrap a substance in a material in order to produce particles with diameters of a few nanometres to a few millimetres. So, the capsules are small particles that contain an active agent or core material surrounded by a coating or shell.
Encapsulation shell materials include a variety of polymers, carbohydrates, fats and waxes, depending of the core material to be protected, and this aspect will be discussed below in the this section.The protection of bioactive compounds, as vitamins, antioxidants, proteins, and lipids may be achieved using several encapsulation technologies for the production of functional foods with enhanced functionality and stability. Encapsulation technologies can be used in many applications in food industry such as controlling oxidative reaction, masking flavours, colours and odours, providing sustained and controlled release, extending shelf life, etc.
In the probiotic particular case, these need to be protected during the time from processing to consumption of a food product.

The coating or shell of sealed capsules needs to be semipermeable, thin but strong to support the environmental conditions maintaining cells alive, but it can be designed to release the probiotic cell in a specific area of the human body. The scientific references related with probiotic encapsulation stress the degradation in the gastrointestinal tract, more than the processing conditions and the coating material usually employed can withstand acidic conditions in the stomach and bile salts form the pancreas after consumption. In this way, the protection of the biological integrity of probiotic bacteria is achieved during gastro-duodenal transit, achieving a high concentration of viable cells to the jejunum and the ileum.The selection of the best encapsulation technology for probiotics needs to consider numerous aspects in order to guarantee the survival of bacteria during the encapsulation process, in storage conditions and consumption, as well as the controlled release in the specific desired area of gut.
Spray-dryingSpray-drying is a commonly used technique for food ingredients production because it is a well-established technique suitable for large-scale, industrial applications. The first spray dryer was constructed in 1878 and, thus, it is a relatively old technique compared with competing technologies [53].
This technique is probably the most economic and effective drying method in industry, used for the first time to encapsulate a flavour in the 1930s. However, it is not so useful for the industrial production of encapsulated probiotics for food use, because of low survival rate during drying of the bacteria and low stability upon storage.Drying is an encapsulation technique which is used when the active ingredient is dissolve in the encapsulating agent, forming an emulsion or a suspension. The solvent is commonly a hydrocolloid such as gelatine, vegetable gum, modified starch, dextrin, or non-gelling protein. The solution that is obtained is dried, providing a barrier to oxygen and aggressive agents [54].In the spray-drying process a liquid mixture is atomized in a vessel with a single-fluid nozzle, a two-fluid nozzle or spinning wheel (depending of the type of spray dryer in use) and the solvent is then evaporated by contacting with hot air or other gas. Inlet temperatures of above 60 °C resulted in poor drying and the sticky product often accumulated in the cyclone. The logarithmic number of probiotics decreases linearly with outlet air temperature of the spray-drier (in the range of 50 ?C - 80 ?C) [56]. So, the optimal outlet air temperature might be as low as possible, enough to assure the drying of the product and to avoid the sticky effect. Alternatively, a second draying step might be applied, using a fluid bed or a vacuum oven, for example, due to the optimal survival of probiotics is achieved with low water activity.The successful spray drying of Lactobacillus and Bifidobacterium have previously reported for a number of different strains, including L. The atomization process and encapsulant agent cellulose acetate phthalate were effective in protecting these micro-organisms in acidic medium (hydrochloric acid solutions pH 1 and 2) during incubation for up to 2 h.
Bifidobacteria in the encapsulated form showed a small reduction in their populations when exposed to acidic media and bile solutions when compared with those exposed in the free form. Among the encapsulants tested, gelatin and soluble starch were the most effective in providing protection to the micro-organisms in acidic medium and milk was the least effective [9]. They used the spray drying process, in which entry and exit temperatures of 170 ?C and 90–85 ?C respectively, and observed a reduction of 2 log cycles in the microbial population.
The presence of some prebiotics in the encapsulating material show higher count after spray drying for Bifidobacterium, depending of the physical properties of the prebiotic compound selected (thermoprotector effect, crystalinity, etc.) [62, 63] and a similar effect occurs for Lactobacillus bacteria [61, 64]. Some researchers have proposed the addition of thermo-protectants as inputs before drying with the intention of improving the resistance to the process and stability during storage [65].
In the case of Rodriguez-Huezo and collaborators [63] used a prebiotic as encapsulant (‘aguamiel’) and a mixture of polymers composed of concentrated whey protein, ‘goma mesquista’ and maltodextrin. In fact, Ross and collaborators [66] reported that neither inulin nor polydextrose enhanced probiotic viability of spray-dried probiotics. In another study, it was also observed that when quercetin was added together with probiotics, the microencapsulation yields and survival rates were lower than for the micro-organism without quercetin [67]. However, most probiotic strains do not survive well the high temperatures and dehydratation during the spray-drying process. Loss of viability is principally caused by cytoplasmatic membrane damage although the cell wall, ribosomes and DNA are also affected at higher temperatures [74]. It was reported that the stationary phase cultures are more resistant to heat compare to cells in exponential growth phase [61].One approach used by a number of researchers to improve probiotic survival is the addition of protectants to the media prior to drying. Nevertheless, this economical and effective technology for protecting materials is rarely considered for cell immobilization because of the high mortality resulting from simultaneous dehydration and thermal inactivation of microorganisms. Spray-coolingThis process is similar to spray-drying described before in relation with the production of small droplets.
The principal difference in the spray-cooling process is the carrier material and the working conditions related with him. In the case, a molten matrix with low melting point is used to encapsulate the bacteria and the mixture is injected in a cold air current to enable the solidification of the carrier material.
It is interesting because the capsules produced in this way are generally not soluble in water. However, due the thermal conditions of the process, the spray-cooling is used rarely for probiotics encapsulation. As example of successful development, the patent US 5,292,657 [81] present the spray-cooling of probiotics in molten lipid atomized by a rotary disk in a cooling chamber. Fluid-bed agglomeration and coatingThe fluid-bed technology evolved from a series of inventions patented by Dr.
Wurster and colleagues at the University of Wisconsin Alumni Research Foundation (WARF) between 1957 and 1966 [82, 83, 84, 85].
These patents are based on the use of fluidising air to provide a uniform circulation of particles past an atomising nozzle.
This nozzle is used to atomize a selected coating material (a melt product or an aqueous solution) which solidifies in a low temperature or by solvent evaporation.
A proper circulation of the particles is recognised as the key to assure that all particles in the fluid-bed achieve a uniform coating.
As particles flow is spray direction countercurrent, collisions involving wet particles are more probable and these collisions agglomerate particles. Bur the particles agglomerate become heavier and have less fluidization, so this phenomenon selectively agglomerates smaller particles and promotes agglomerate uniformity.Placement of the nozzle at the bottom of a fluid bed provides the most uniform film on small particles and minimises agglomeration of such particles in the coating process compared with any other coating technique. This uniform coating is achieved because particles move further apart as they pass through the atomised spray from the nozzle and into an expansion region of the apparatus. This configuration allows the fluidising air to solidify or evaporate coating materials onto particles prior to contact between particles. This technique is among all, probably the most applicable technique for the coating of probiotics in industrial productions since it is possible to achieve large batch volumes and high throughputs. As example, Lallemand commercialize ProbiocapTM, and these particles are made in a fluid bed coating of freeze-dried probiotics with low melting lipids [87].Specifically, Koo and collaborators [88], reported that L. Later, Lee and researchers [69] showed that the microencapsulation in alginate microparticules coating with chitosan offers an effective way of delivering viable bacterial cells to the colon and maintaining their survival during refrigerated storage.Fluidized-bed drying was recently investigated by Stummer and collaborators [89] as method for dehydration of Enterococus faecium.
This study concludes to use fluidized-bed technology as a feasible alternative for the dehydration of probiotic bacteria by layering the cells on spherical pellets testing different protective agents as glucose, maltodextrin, skim milk, trehalose or sucrose, preferably skim milk or sucrose. This drying technique is a dehydration process which works by freezing the product and then reducing the surrounding pressure to allow the frozen water to sublimate directly from the solid phase to the gas phase.
The process is performed by freezing probiotics in the presence of carrier material at low temperatures, followed by sublimation of the water under vacuum.
One of the most important advantages is the water phase transition and oxidation are avoided. In order to improve the probiotic activity upon freeze-drying and also stabilize them during storage, it is frequent the addition of cryoprotectans.One of the most important aspects to decide is the choice of the optimal ending water content.
This decision have to be a compromise between the highest survival rate after drying (higher survival rate with higher water content) and the lowest inactivation upon storage (better at low water activity, but not necessarily 0% of water content).
According with King and collaborators [90], the loses in survival rates of freeze-dried probiotic bacteria under vacuum may be explained with a first-order kinetic and the rate constants can be described by an Arrhenius equation. But this equation might be affected by other factors as phase transition, atmosphere and water content.In any case, the lyophilisation or freeze-drying is a very expensive technology, significantly more than spray-drying [56], even if it is probably most often used to dry probiotics. However, most of freeze-drying process only provide stability upon storage and not or limited during consumption. The freeze-drying is useful to dry probiotics previously encapsulated by other different techniques, as emulsion [91] or entrapment in gel microspheres [92].

In this way it is possible to improve the stability in the gastrointestinal tract and optimize the beneficial effect of probiotic consumption.The Vacuum-drying is a similar process as freeze-drying, but it takes place at 0 - 40 ?C for 30 min to a few hours. The advantages of this process are that the product is not frozen, so the energy consumption and the related economic impact are reduced. Emulsion-based techniquesAn emulsion is the dispersion of two immiscible liquids in the presence of a stabilizing compound or emulsifier.
Emulsions are simply produced by the addition of the core phase to a vigorously stirred excess of the second phase that contains, if it is necessary, the emulsifier (Figure 4). Nevertheless, even if the technique readily scalable, it produce capsules with an extremely large size distributions. The technique is a modification of the basic technique in which an emulsion is made in of an aqueous solution in a hydrophobic wall polymer. This emulsion is the poured with vigorous agitation, into an aqueous solution containing stabilizer. The loading capacity of the hydrophobic core is limited by the solubility and diffusion to the stabilizer solution. The principal application of this technology is in pharmaceutical formulations.Entrapment of probiotic bacteria in emulsion droplets has been suggested as a means of enhancing the viability of microorganism cells under the harsh conditions of the stomach and intestine. For example, Hou and collaborators [93] reported that entrapment of cells of lactic bacteria (Lactobacillus delbrueckii ssp.
The study was performed with Lactobacillus paracasei and their entrapment in the oil phase of protein-stabilized emulsions protected the cells when exposed to GI tract enzymes, provided that the emulsions were freshly prepared. Following, however, treatment of aged for up to 4 weeks emulsions under conditions simulating those of the human GI environment, the microorganism did not survive in satisfactory numbers. Moreover, even if the emulsion techniques described before are easily scalable, these techniques have an important disadvantage to be applied in an industrial process because are batch processes. The static mixers are small devices placed in a tube consisting in static obstacles or diversions where the two immiscible fluids are pumped [118, 119]. This system improves the size distribution, reduce shear and allows keeping the aseptic conditions because it might be a closed system (Figure 5). CoacervationThis process involves la precipitation of a polymer or several polymers by phase separation: simple or complex coacervation, respectively. Simple coacervation is based on “salting out” of one polymer by addition of agents as salts, that have higher affinity to water than the polymer.
It is essentially a dehydration process whereby separation of the liquid phase results in the solid particles or oil droplets (starting in an emulsion process) becoming coated and eventually hardened into microcapsules. With regard to complex coacervation, it is a process whereby a polyelectrolyte complex is formed. This process requires the mixing of two colloids at a pH at which both polymers are oppositely charged (i.e.
But it is not the only use of this technique and the complex coacervation is also suitable for probiotic bacteria microencapsulation.
And the most frequent medium used might be a water-in-oil emulsion [120].Oliveira and collaborators [121] encapsulated B.
The process used and the wall material were efficient in protecting the microorganisms under study against the spray drying process and simulated gastric juice; however, microencapsulated B.
However, the scale-up of coacervation is difficult, since it is a batch process that yields coacervate in an aqueous solution. Extrusion techniques to encapsulate in microspheresThe methods of bioencapsulation in microspheres include two principal steps: (1) the internal phase containing the probiotic bacteria is dispersed in small drops a then (2) these drops will solidify by gelation or formation of a membrane in their surface.
Before this section, there are described emulsion systems and coacervation as different methods to obtain these drops and even the membrane formation, but also extrusion technology is useful in order to produce probiotic encapsulation in microspheres. There are different technologies available for this purpose and the selection of the best one is related with different aspects as desired size, acceptable dispersion size, production scale and the maximum shear that the probiotic cells can support.When a liquid is pumped to go through a nozzle, first this is extruded as individual drops.
Increasing enough the flow rate, the drop is transformed in a continuous jet and this continuous jet has to be broken in small droplets. So, the extrusion methods could be divided in two groups, dropwise and jet breakage (Figure 6), and the limit between them is established according to the minimum jet speed according to this equation (eq. Moreover, the flow is around several millilitres by hour and the method is not interesting for an industrial application. For example, in the Figure 7 is showed a cell immobilization process carried out at TECNALIA using the method of dripping by gravity. Drop generation by gravity using a 160 µm nozzle.? Air o liquid coaxial flow and submerged nozzlesApplying a coaxial air flow around the extrusion nozzle it is possible to reduce the microsphere diameter between a few micrometres and 1 mm. The air flow might be replaced for a liquid one: with a suitable selection of the liquid flow the control of the surface tension is improved. Drops produced in air are generated as aerosols, while the drops produced, for example, in water are made as emulsions. An example of the former consist of a static cup immersed in a water-immiscible oil such as mineral oil or vegetal oil and a concentric nozzle as is schematically showed in the Figure 9.
Each droplet consist of core material being encapsulatd totally surrounded by a finite film of aqueous polymer solution, as gelatine, for example.
Ltd in Japan These capsules are composed of three layers: a core freeze-dried probiotic bacteria in solid fat, with an intermediate hard fat layer and a gelatin-pectin outer layer [122]. The droplet generation improves replacing the dragging forces by a high electrostatic potential between the capillary nozzle and the harvester solution. Even if the capsules size is appropriated and the size distribution is narrow enough, this technique is more expensive than other extrusion ones and it is not fast enough to be scaled.? Vibration technology for jet break-upApplying a vibration on a laminar jet for controlled break-up into monodisperse microcapsules is one among different extrusion technologies for encapsulation of probiotic bacteria. The vibration technology is based on the principle that a laminar liquid jet breaks up into equally sized droplets by a superimposed vibration (Figure 10).
He showed that the frequency for maximum instability is related to the velocity of the jet and the nozzle diameter (eq. The possibility of working with a wide range of materials (hot melt products, hydrogels, etc.) is also an important aspect to be considered, as well as the design with also concentric nozzles in the lab scale devices and with this kind of nozzles it is possible to produce capsules with a defined core region (solid or liquid) surrounded by a continuous shell layer. On the other side, the principal disadvantage of this technology is the limit in the viscosity for the liquid to be extruded.But may be one of the most important advantage of the vibration devices commercialized is that the scale up of this technology is relatively “simple” and it consist in the multiplication of the number of nozzles, developing multinozzle devices.
The only challenge is that each nozzle of a multinozzle plant must operate in similar production conditions: equal frequency and amplitude, and equal flow rate. The droplet generation is based on a mechanical impact of the cutting wire on the liquid jet.
Some techniques as emulsion, simple dropping, electrostatic-enhanced dropping, vibration technique or rotating disc and nozzle techniques have in common that the fluids have to be low in viscosity, and not all of them may be used for large-scale applications. Directly underneath the nozzle the jet is cut into cylindrical segments by a rotating cutting tool made of small wires fixed in a holder (Figure 11).
Driven by the surface tension the cylindrical segments form spherical beads while falling further down, where they finally can be harvested. The size of beads can be adjusted within a range between approximately 200 µm up to several millimetres, adjusting parameters as nozzle diameter, flow rate, number of cutting wires and the rotating speed of cutting tool.Bead generation by a JetCutter device is achieved by the cutting wires, which cut the liquid jet coming out of the nozzle. The device is designed to recover these losses, but it is important to minimize de lost volume selecting a smaller diameter of the cutting wire and angle of inclination of the cutting tool with regard to the jet (Figure 11). According with Pruesse and Vorlop [124], a suitable model of the cutting process might help to operator in the parameters selection. One of the most important parameters is the ratio of the velocities of the fluid and cutting wire, necessary to determinate the proper inclination angle (eq.

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