Encapsulated bacteria là gì

3.1.1. Spray-drying

Spray-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. Most of spray dryers used in food industry are concurrent in design, i.e. product enters the dryer flowing in the same direction as the drying air. The objective is to obtain a very rapid drying and to avoid that the temperature of the material dried exceeds the exit air temperature of the dryer [Figure 1].

Figure 1.

Schematic diagram of a spray-dry encapsulation process and image of a Mini Spray Dryer B-290 [BÜCHI], available at TECNALIA.

But also in a concurrent design, the conventional procedure requires to expose cells to high temperature and osmotic stresses due to dehydration witch results in relatively high viability and activity losses immediately after spraying and most likely also affects storage stability. However, some strains survive better than others. And parameters as drying temperature and time and shell material have also an important effect.

Using gelatinised modified starch as a carrier material, ORiordan obtained good results in Bifidobacteriumcells encapsulation with an inlet temperature of 100 ºC and oulet temperature of 45 ºC. Inlet temperatures of above 60 °C resulted in poor drying and the sticky product often accumulated in the cyclone. Higher inlet temperatures [>120 °C] resulted in higher outlet temperatures [>60 °C] and significantly reduced the viability of encapsulated [55]. The logarithmic number of probiotics decreases linearly with outlet air temperature of the spray-drier [in the range of 50 ºC - 80 ºC] [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 Lactobacillusand Bifidobacteriumhave previously reported for a number of different strains, including L. paracasei[57, 58], Lactobacillus curvatus[59], L. acidophilus[60], L. rhamnosus[61] and Bifdobacterium ruminantium[8]. Specifically, Favaro-Trindade and Grosso [6] used spray drying to encapsulate B. lactisand L. acidophilusin the enteric polymer cellulose acetate phthalate enriched with the fructooligosaccharide Raftilose1 [a prebiotic]. In this work, the process was also appropriate, especially for B. lactis[Bb-12], since for entry temperature of 130 ºC and exit of 75 ºC, the counts in the powder and dispersion [feed] were similar; however, the L. acidophiluspopulation showed a reduction of two log cycles. 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. In another study, B. longumB6 and B. infantiswere encapsulated by spray drying, with gelatin, soluble starch, milk and gum arabic as encapsulating agents. 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]. Desmond and collaborators [57] encapsulated L. acidophilusin β-cyclodextrin and gum arabic. They used the spray drying process, in which entry and exit temperatures of 170 ºC and 9085 ºC respectively, and observed a reduction of 2 log cycles in the microbial population. However, the microencapsulation process extended the shelf-life of the culture.

On the other hand, the most typical materials used as carrier in probiotic bacteria encapsulation are proteins and/or carbohidrates, which may be in the glassy state at storage temperatures to minimize molecular mobility and thus degradation. 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 Lactobacillusbacteria [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 Rodríguez-Huezo and collaborators [63] used a prebiotic as encapsulant [aguamiel] and a mixture of polymers composed of concentrated whey protein, goma mesquista and maltodextrin. It is important to mention that not all the compound employees were efficient protectors. 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]. A lot of other studies have employed of spray-drying technology to encapsulate probiotic cells, as noted in the table 1.

In summary, spray-drying technology offers high production rates at relatively low operating costs and resulting powders are stable and easily applicable [73]. 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. For example, the incorporation of thermoprotectants, such as trehalose [75], non-fat milk solids and/ or adnitol [76], growth promoting factors including various probiotic/prebiotic combinations [77] and granular starch [78] have been shown to improve culture viability during drying and storage [79, 80].

Microencapsulation by spray-drying is a well-established process that can produce large amounts of material. 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.

3.1.2. Spray-cooling

This 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. In any case, the contact time of the probiotics with the melt carrier material should remain very sort.

3.1.3. Fluid-bed agglomeration and coating

The 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. The most commonly used techniques are referred to as the bottom-spray [Wurster] fluid-bed process and the top-spray fluid-bed process [Figure 2]; however, variations such as tangential-spray are also practised.

Figure 2.

Schematic Diagrams of two types of the most commonly used fluid-bed coaters.

The top-spray fluid-bed coater is characterized by placement of nozzle above a fluidising bed and spraying down ware into the circulating flow of particles. This technique is useful for agglomeration or granulation. 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. A partition [centre tube] is used in Wurster fluid-bed coating to control the cyclic flow of particles in the process better than with de air distribution plate alone [Figure 3].

Figure 3.

Expansion chamber for a bottom-spray [Wurster] fluid-bed process and detail of air distribution plate [from Glatt available at TECNALIA].

The most common coating material used for probiotics is lipid based, but proteins or carbohydrates can also be used [86]. 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. bulgaricusloaded in chitosan-coated alginate microparticles showed higher storage stability than free cell culture. 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. According with the described procedure, it is possible to combine two manufacturing steps: [1] cell-dehydration preserving the optima cell properties and [2] the processing into suitable solid formulation with appropriate physical properties [the spherical pellets improve the flowability for filling capsules or dosing in different formulations.]

3.1.4. Freeze and vacuum-drying

Freeze-drying is also named lyophilisation. 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. Because of that, this technique is used as a second step of encapsulation process. 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 ºC 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. In the product point of view, the freezing damage is avoided.

3.1.5. Emulsion-based techniques

An emulsion is the dispersion of two immiscible liquids in the presence of a stabilizing compound or emulsifier. When the core phase is aqueous this is termed a water-in-oil emulsion [w/o] while a hydrophobic core phase is termed an oil-in-water emulsion [o/w]. 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. Because of this limitation, there are several industrial efforts to achieve a narrow particle size distribution controlling the stirring and homogenization of the mixture.

There are also double emulsions, such water-in-oil-in-water [w/o/w]. 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 delbrueckiissp. bulgaricus] in the droplets of reconstituted sesame oil body emulsions increased approximately 104 times their survival rate compared to free cells when subjected to simulated GI tract conditions.

Figure 4.

Probiotic cell encapsulation by water-in-oil and water-in-oil-in-water emulsions.

Nevertheless, Mantzouridou and collaborators [94] have presented an study investigating the effect of cell entrapment inside the oil droplets on viable cell count over storage and under GI simulating conditions, according to the type of emulsifier used: egg yolk, gum arabic/xanthan mixture or whey protein isolate. The study was performed with Lactobacillus paracaseiand 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. The probiotic cells survived in larger numbers in aged emulsions when the cells were initially dispersed in the aqueous phase of a yolk-stabilized dressing-type emulsion and their ability to survive enzymatic attack was further enhanced by inulin incorporation.

Lactobacillus rhamnosushas been encapsulated in a w/o/w emulsion. According to Pimentel-González and collaborators [95] the survival of the entrapped L.rhamnosusin the inner water phase of the double emulsion increased significantly under low pH and bile salt conditions in an in vitrotrial, meanwhile the viability and survival of control cells decrease significantly under the same conditions.

In the table 2 details probiotic strains and carrier materials that have employed some researchers in the emulsification technology.

The emulsion methods produce capsules sized from a few micrometres to 1mm, approximately, but with a high dispersion compared to other techniques, as extrusion ones. 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. Nevertheless, it exist another promising technique different to the turbine used. 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]. For example, nowadays this technology is used in dairy industry for viscous products, as admixing fruit pieces or cultures to yoghurt or to process ice cream or curds.

Figure 5.

Schematic diagram of a static mixer system to make emulsions.

3.1.6. Coacervation

This 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. gelatine [+] and arabic gum [-]], leading to phase separation and formation of enclosed solid particles or liquid droplets.

The complex coacervation is one of the most important techniques used for flavour microencapsulation. 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. lactis[BI 01] and L. acidophilus[LAC 4] through complex coacervation using a casein/pectin complex as the wall material. To ensure higher stability, the coacervated material was atomized. 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. lactislost its viability before the end of the storage time. Specifically, microencapsulated L. acidophilusmaintained its viability for a longer storage [120 days] at 7 and 37 ºC, B. lactislost viability quickly.

Advantages of coacervation, compared with other methods for the encapsulation of probiotics, are a relatively simple low-cost process [which does not necessarily use high temperatures or organic solvents] and allow the incorporation of a large amount of micro-organisms in relation to the encapsulant. However, the scale-up of coacervation is difficult, since it is a batch process that yields coacervate in an aqueous solution. Therefore, to extend its shelf-life, an additional drying process should be applied, which can be harmful to cells.

3.1.7. Extrusion techniques to encapsulate in microspheres

The 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. 1]:

vj,min=2[σdj]0,5         vj,min=minimumjetspeed[m/s]                          σ=surfacetension[N/m]                      ρ=liquidflowingdensity[kg/m3] dj=jetdiameter[m]                                          E1

Regardless of the selected technique, the liquid obtained drops have to be solidifying by gelation or external membrane formation [Figure 6]. The resulting hydrogel beads are very porous and a polymeric coating is usually applied in order to assure a better retention of the encapsulated probiotic bacteria.

Figure 6.

Classification of methods to make and solidify drops

Dripping by gravity

This method is the simplest dripping method to make individual drops, but the size of the droplet will be determined by his weight and surface tension, as well as the nozzle perimeter. The typical diameter of a drop made by this technique is higher than 2 mm. 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. The nozzle diameter is 160 µm and the final size of hydrogel bead [after solidification in a Calcium Chloride solution] is 2,4±0,15 mm.

Figure 7.

Cell encapsulation in an alginate matrix. Drop generation by gravity using a 160 µm nozzle.

Air o liquid coaxial flow and submerged nozzles

Applying a coaxial air flow around the extrusion nozzle it is possible to reduce the microsphere diameter between a few micrometres and 1 mm. However, the flow rate is limited, less than 30 mL/h to avoid a continuous jet formation. 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. The aerosol beads could be solidified using ionic gelation or hot air. The beads recovered as emulsion are usually extracted or the water is evaporated.

The Spanish enterprise Ingeniatrics Tecnologías has patent an owner Flow Focusing® technology, valid to work with air and liquid flow, and also an user-friendly bioencapsulation device for biotechnological research and clinical microbiology able to encapsulate high molecular weight compounds, microorganisms and cells in homogeneous particles of predictable and controllable size based on Flow Focusing® technology named Cellena® distributed by Biomedal [Figure 8].

Nevertheless, despite all the advantages, due to the mentioned low flow rate, this technique is not used in an industrial scale and also in a laboratory scale it is being replaced for the jet breakage techniques stated below.

Figure 8.

Flow-Focusing technology to make droplets and Cellena® equipment from Ingeniatrics Tecnologías.

The submerged nozzles usually are static, but they can be also rotating or vibrating to improve the droplet generation, but are always immersed in a carrier fluid. 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. The carrier fluid, a warm oil phase that cools after droplet formation, gels this polymer solution thereby forming gel beads with a continuous core/shell structure. The smaller diameter using this technique is typically around 1 mm.

Figure 9.

Schematic diagram of a submerged two-fluid static nozzle.

An example of this technology is provided by Morishita Jintan Co. 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]. However, the size of the capsules produced is quite large to be applied in food products [1.8-6.5 mm] and the technique is quite expensive for use in many food applications.

Electrostatic potential

This technique is the last one of drop generation techniques. The droplet generation improves replacing the dragging forces by a high electrostatic potential between the capillary nozzle and the harvester solution. The electric forces help the gravity force in front of the surface tension.

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-up

Applying 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]. The instability of liquid jets was theoretically analysed for Lord Rayleigh [123]. He showed that the frequency for maximum instability is related to the velocity of the jet and the nozzle diameter [eq. 2 and eq. 3].

fopt=vJopt                         fopt=optimalfrequency[Hz]  vJ=jetvelocity[m/s]               λopt=optimalwavelength[m]E2

opt=π2dN1+3ρσdN              dN=nozzlediameter[m]                       η=dynamicviscosity[kg/ms]ρ=density[kg/m3]                           σ=Surfacetension[N/m]         E3

Using this technology, it is possible to obtain monodisperse droplets which size can be freely chosen in a certain range depending on the nozzle diameter and the frequency of the sinusoidal force applied [eq. 4]. The droplets made are harvested in an accurate hardening bath. To avoid large size distributions due to coalescence effects during the flight and the hitting phase at the surface of hardening solution the use of a dispersion unit with an electrostatic dispersion unit is essential [Figure 10].

dD=1.5dN2opt3             dD=dropletdiameter[m]       dN=nozzlediameter[m]         λopt=optimalwavelength[m]E4

Figure 10.

Image of Inotech Encapsulator IE-50R and schematic diagram of jet destabilization and breakage for single and concentric nozzles.

The Encapsulator BIOTECH [the updated version of IE-50R] from EncapBioSystems and Spherisator form BRACE GmbH are two different devices labels to produce microencapsuled probiotic bacteria using the vibration technology for jet breakage. The principal advantages of this technology are the low size dispersion [5-10%], a high flow rate [0.1-2 L/h] and is able to work in sterile conditions. 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. In this way, the scale up is direct from the lab to a pilot or industrial scale.

JetCutter technology

The bead production by JetCutter [from geniaLab] is achieved cutting a jet into cylindrical segments by a rotating micrometric cutting tool. 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. On the contrary, the JetCutter technique is especially capable of processing medium and highly viscous fluids up to viscosities of several thousand mPas.

For bead production by the JetCutter the fluid is pressed with a high velocity out of a nozzle as a solid jet. 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. But in each cut the wire produce a cutting loss. 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. 5], but the fluid velocity is also related with the bead size [eq. 6], while the diameter of the nozzle and wire determine the volume of cutting loses [eq. 7].

=arcsin[ufluiduwire]         α=inclinationangle                    ufluid=velocityofthefluid                     uwire=velocityofthecuttingwire E5

dbead=32·D2·[ufluidn·zdwire]3        dbead=beaddiameter                  D=nozzlediameter        dwire=cuttingwirediameterE6

Vloss=π·D24·dwire                 n=numberofrotations          z=numberofcuttingwiresVloss=VolumeoftheoveralllossE7

Regarding the advantages of the JetCutter technology, besides the capacity for work with medium and highly viscous fluids, there are the narrow bead size dispersion and the wide range of possible sizes, as well as the high flow rate [approx. 0.1-5 L/h].

To scale up the JetCutter technology there are two ways. First, a multi-nozzle device can be used, in which nozzles are strategically distributed in the perimeter of the cutting tool. The second way is the increase of the cutting frequency, but this approach needs also a higher velocity of the jet and a too high speed of the beads might cause problems, as coalescence or deformation in the collection bath entrance. In order to overcome this problem, the droplets can be pre-gelled prior entering the collection bath using, for example, a tunnel equipped with nozzles spraying the hardening solution or refrigerating the falling beads.

The extrusion technique is the most popular microencapsulation or immobilization technique for micro-organisms that uses a gentle operation which causes no damage to probiotic cells and gives a high probiotic viability [21]. This technology does not involve deleterious solvents and can be done under aerobic and anaerobic conditions. The most important disadvantage of this method is that it is difficult to use in large scale productions due to the slow formation of the microbeads [15]. Various polymers can be used to obtain capsules by this method, but the most used agents are alginate, κ-carrageenan and whey proteins [125].

Figure 11.

Schematic diagram of the JetCutter technology and representation of fluid losses due to the cutting wire impact.

Figure 12.

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.

There are many studies with the extrusion techniques for probiotic protection and stabilization. In 2002, Shah and Ravula [126] encapsulated Bifidobacteriumand Lactobacillus acidophilusin calcium alginate in frozen fermented milk-based dessert, and, in general, the survival of bacteria cells was improved by encapsulation. Some studies employed to encapsulate Bifidobacteria alginate alone and a mixture with other compounds and observed more resistant to the acidic medium than the free cells [5, 112, 127]. A similar result was observed by Chávarri and collaborators [67], where chitosan was used as coating material to improve the stability of alginate beads with probiotics. In this study, with extrusion technique, they showed an effective means of maintaining survival under simulated human gastrointestinal conditions. In the table 3 details probiotic strains and carrier materials that have employed some researchers in the extrusion technology.

3.1.8. Adhesion to starch granules

Starch is unique among carbohydrates because it occurs naturally as discrete particles called granules. Their size depends on the starch origin ranging from 1 to 100 µm. They are rather dense and insoluble, and hydrate only slightly in water at room temperature. The granular structure is irreversibly lost when the granules are heated in water about 80 ºC, and heat and mechanical energy are necessaries to totally dissolve the granules.

Usually starches are partially or totally dissolved before they are used in food application, for example to be used as texturizing. Starch hydrolysates or chemically modified starches are used as microencapsulation matrices for lipophilic flavours [152, 153]. Partially hydrolysed and crosslinked starch granules were suggested to be suitable carriers for various functional food components [154]. To hydrolyse the starch granules, the use of amylases is the preferred way and corn starch seems to be the most suitable starch for his purpose.

Some probiotic bacteria were shown to be able to adhere to starch and a few investigations about the utilisation of starch granules to protect these bacteria were reported.

3.1.9. Compression coating

This technique involves compressing dried bacteria powder into a core tablet or pellet and the compressing coating material around the core to form the final compact [Figure 13]. The compression coating has received a renewed interest for probiotic bacteria encapsulation used with gel-forming polymers in order to improve the stabilization of lyophilized bacteria during storage [155]. The viability in process of the bacteria is affected by the compression pressure and to improve the storage survival the coating material has a significant effect.

Due of the size of the final product obtained by compression coating, this technique is used for pharmaceutical and nutraceutical compounds development, but not for food ingredients obtention.

Figure 13.

Schematic diagram of compression coating of probiotics.