Đồ án Kỹ thuật sấy probiotics (drying probiotics)

perhaps 50 m/s but quickly decelerate to thermal velocity (0.2 to 2 m/s). The drying chamber may be arranged either horizontally or vertically, with vertical orientation being the most common. Typically, the food droplets are sprayed at the top of the chamber and fall down to the bottom by gravity. In spray drying of foods, air is fed in the same direction as the food droplets in a concurrent operation. Thus, both air and food droplets enter the drying chamber at the top and gradually fall to the bottom of the chamber, where air is separated from dry power and the product is removed from the dryer. Co-current operation, with air and drying droplets moving in the same direction, is desired in most foods include probiotics due to the thermal sensitivity. Food droplets with the highest moisture content come into contact with air at the highest temperature. Since water being removed from the droplet provides an evaporative cooling effect, the product generally does not heat above the wet-bulb temperature for the air, typically below 50oC. When the product reaches its driest state, the air has also cooled, and chances of thermal degradation are reduced. Nevertheless, some products are heated with low temperature process to minimize protein destabilization. 50 Figure 4.11 Dryer chamber Fan In a spray drying process, high flow rates of drying air are generally obtained be the use of centrifugal fans. Usually a two-fan system is used, the main fan situated after the powder recovery equipment and the supply fan located in the inlet duct to the drying chamber. Two fans enable better control of the pressure in the chamber. With the single fan after the cyclone, the whole drying system operates under a high negative pressure. The operating pressure in the drying chamber determines the amount of power in the exhaust air and hence the capacity of the cyclones and their collection of efficiency. In special cases, more fans may be used in a drying process, for example, a centrifugal fan for blowing cool air to potential hot spots in the drying chamber and atomizer. The pressure developed by a fan depends upon the blade design. The most common type of fan in spray drying has backward-curving blades. Such blades are also used for the supply fan. If the powder-air ratio is high, the backward-curving blades may cause problems with deposit formation on the backside of the blades. In such cases, the use of the blade profile intermediate between the backward curving and the radial is recommended. 51 Figure 4.12 Typical centrifugal fan Powder Separation Once air and dried powder reach the bottom of the dryer, they are separated and the powdered product removed for either further processing or packaging. Primary separation of powder is accomplished by gravitational settling of the heavier powder particles. Separation of air and finer powder particles is usually accomplished in a cyclone device. The air containing fine powdered particles is circulated tangentially into the cyclone separator. Centrifugal force causes the particles to segregate from the air and settle to the bottom of the conical separator. Air flows back out the top of the cyclone, while powdered product is removed from the bottom, where it rejoins the main powdered product from the dryer. Since the cyclone does not remove all of the fine particles, in some cases an additional separation step is needed. This is often accomplished using a textile or bag filter. Here, air passes through a fabric filter before being exhausted into the atmosphere. Fine particles are trapped by the filter and eventually recovered by reverse air flow through the filters. This device is often used as the final separation prior to exhausting air at the atmosphere. 52 Figure 4.13 Drying cyclone designs with different types of air inlet. Spray Dryer Type Probiotics are heat-sensitive food products so they usually dried in co-current dryers. In a co-current dryer, the spray is directed into the hot air entering the dryer and both pass through the chamber in the same direction. Co-current dryers are the preferred design for heat-sensitive products because the hottest drying air contacts the droplets at their maximum moisture content. Spray evaporation is rapid, and the temperature of the drying air is quickly reduced by the vaporization of water. The product does not suffer from heat degradation because the droplet temperature is low during most of the evaporation time. Once the moisture content reaches the target level, the temperature of the particle does not increase greatly because the surrounding air is now much cooler. 53 Figure 4.14 Open Cycle Co-current Flow Layout 2.3. Factors influencing the spray drying 2.3.1. Kind of equipment and papameters Dispersion of the feed solution in small droplets The dispersion can be achieved with a pressure nozzle, a two fluid nozzle, a rotary disk atomizer or an ultrasonic nozzle. So different kinds of energy can be used to disperse the liquid body into fine particles. The selection upon the atomizer type depends upon the nature and amount of feed and the desired characteristics of the dried product. The higher the energy for the dispersion, the smaller are the generated droplets. 54 Figure 4.15 Nozzle and product Mixing of spray and drying medium (air) with heat and mass transfer The manner in which spray contacts the drying air is an important factor in spray dryer design, as this has great bearing on dried product properties by influencing droplet behavior during drying. This mixing is an important aspect and defines the method of spray drying: Co-Current flow Figure 4.16 Co-Current flow The material is sprayed in the same direction as the flow of hot air through the apparatus. The droplets come into contact with the hot drying air when they are the most moist. The product is treated with care due to the sudden vaporization. Counter-Current flow 55 Figure 4.17 Counter-Current flow The material is sprayed in the opposite direction of the flow of hot air. The hot air flows upwards and the product falls through increasingly hot air into the collection tray. The residual moisture is eliminated, and the product becomes very hot. This method is suitable only for thermally stabile products. Combined Figure 4.18 Combined The advantages of both spraying methods are combined. The product is sprayed upwards and only remains in the hot zone for a short time to eliminate the residual moisture. Gravity then pulls the product into the cooler zone. Due to the fact that the product is only in the hot zone for a short time, the product is treated with care. Disk atomizer (rotary wheel) 56 Figure 4.19 Disk atomizer The material to be sprayed flows onto a rapidly rotating atomizing disk and is converted to a fine mist. The drying air flows in the same direction. The product is treated with care, just as in the co-current flow method. 2.3.2. Dry matter content in the feed To obtain a high-quality powder, constant dry matter content in the concentrate produced in the evaporator preceding the drying process is necessary. The occurrence of changes in dry matter content in the feed to the dryer is one of the major sources of disturbance in the drying process. It is also advantageous to remove as much water as possible at the evaporation stage from an energy-saving point of view. In practice however, due to variations that occur in dry matter content of the concentrate as a consequence of variations in feed and process variables, the set point for this dry matter content is often lower than theoretically possible. This is in order to reduce the risk too high a viscosity of the concentrate. Less variation in dry matter content of the Concentrate enables a higher set-point and thus also improves the energy efficiency of the powder production process. When using conventional control technology, such as single-loop proportional-integral-derivative (PID) controllers, the long time delay from input (e.g. flow or dry matter content of food fed to the evaporator) to output (e.g. total solids content of concentrate by controlling the steam supply) will result in a relatively long period of off-spec concentrate. Modern design methods for multivariable control make it possible to design compensators that reduce or eliminate the off-spec period. For the design of such a multivariable control system one should determine the dynamic behavior of the evaporator involved. This can be done either by using a physical model simulating the dynamic behavior or by carrying out step-response measurements on the actual evaporator and using system identification techniques to draw up a black-box 57 model. The first approach is more flexible and robust for handling changes in the design and process operation. The advantage of the latter approach is that it requires less detailed knowledge about the design of the evaporator. Also in drying processes there is a trend to use more and more predictive models in the control strategy. The main issue for the automatic control of spray dryers is to achieve a reduced variation in the moisture content of the powder, enabling a higher set point for the moisture content, which strongly reduces the operating costs. 2.3.3. Air temperature Drying is enhanced by operation at elevated air temperature. Drying rate increases due to the grater rate of heat transfer, which results in higher vaporization rate. Also, for air at given moisture content, relative humidity goes down as temperature goes up. This results in a higher driving force for removing moisture from the product surface. In addition, higher temperatures mean faster internal diffusion process. That is, water molecules migrate more rapidly at higher temperatures, and internal drying is also enhanced. Thus, increased air temperature improves drying by effecting both internal (falling rate period) and external processes (constant rate period). However, extreme high temperatures may cause unwanted chemical or physical reactions to take place in the product. Barberan, 1976 explain, indeed, that a temperature of 180oC to 300oC, at inlet of a spraying device, is capable of killing all the live organisms. These observations are also confirmed in EP298605 (Unilever: page 2, lines 43-48), and EP63438 (Scottish Milk Marke: page 1, line 14-21). Some species of lactic acid bacteria are, however, naturally heat-resistant, that is to say they are capable of with-standing relatively high temperatures. Chopin et al. have thus shown that it is possible to spray-dry, at 215oC, a sporulating culture of Microbacterium lacticum andnto obtain slightly more than 10% survival after drying (Canadian J. Microb, 23, 755-762, 1977). Unfortunately, these species generally form part of the appearance of bad taste. These heat-resistant lactic acid bacteria are therefore not suitable for human consumption (“Fundamentals of Food Microbiology”, Marion L. Field, AVI Publishing Comp, Westport, 1979). Outlet air temperature is a major processing parameter affecting the number of survivors during spray-drying. Silva et al. (2002) found relatively small changes in the outlet temperature appear to have significant effects on the survival of Lactobacillus strains, indicating that spray drying temperatures need to be 58 optimized individually for every new application. Kim and Bhowmik (1990) reported that numbers of Streptococcus salivarius subsp. thermophilus and L. debrueckii subsp. Bulgaricus decreased with increasing outlet or inlet air temperatures and atomizing air pressure, while similar findings were reported by Gardiner et al., 2000 for both L. paracasei NFBC 338 and L. salivarius UCC 118. Consequently, improved viability can be achieved by reducing the outlet temperature during spray-drying (Fig. 4.20), but beyond probiotic viability, powder quality is also influenced by these parameters, with moisture content of ≈3.5% being preferred for shelf-stable products (Zayed & Roos, 2004). Figure 4.20. Survival of Lactobacillus paracasei NFBC 338 during spray drying in 20% (wt/vol) RSM supplemented with 0.5% (wt/vol) yeast extract at different air outlet temperatures (bar graph). The line shows the moisture contents of the resulting powders. The air inlet temperature was maintained at 170oC. The results are means based on data from duplicate spray-drying trials, and standard deviations are indicated by vertical bars (Gardiner et al., 2000). 2.3.4.Other factors Air Velocity The speed at which drying air blows across the product surface impacts the rate of moisture migration from the surface to the drying air. Evaporation from the 59 surface is enhanced by improved convective mass transfer rates as a result of increase air velocity. Thus, increasing air velocity (or wind speed) shortens the constant rate period. However, since drying in the falling rate period generally is not limited by external conditions, increasing air velocity normally has little effect on the falling rate period. Relative Humidity The amount of moisture in the air, as measured by the vapor pressure or relative humidity, affects the driving force for external mass transfer. The difference in moisture vapor pressure between the food surface and the drying air represents the driving force for external mass transfer. For a given food product (with a known surface vapor pressure or water activity of the food), increased relative humidity of air enhances drying in the constant rate period. However, relative humidity generally has little influence on drying in the falling rate period, where internal mass transfer limits the drying rate. 2.4. Products Certain characteristics of spray-dried powders are desirable. These include the ability to be wetted by water during reconstitution, dispersability of the powder into water and solubility in water. Changes in product attributes, particularly at the case-hardened surface of the droplets decreases the ability of a powder to be wetted and dispersed into water. Agglomeration of particles may also influence the amount of surface available for wetting. The rate of dissolution, and final solubility of the powder in water, also depends on the nature of the particle surface III. COMPARISON Freeze dry : - The most widespread technique for dehydration of probiotics,use for all strains - At low temperature, milder condition - Higher probiotic survival rate - High cost - Small scale Spray dry : - Apply to the hydration of limited number of probiotic culture. - High outlet temperature - Lower probiotic survival rate - Lower cost - Large scale 60 IV. ACHIEVEMENTS IMPROVE SPRAY DRYING AND QUALITY OF PRODUCT 1. Cell physiology The importance of cell physiology to the successful drying of probiotics has been demonstrated in a number of studies, and in this respect, several factors have been proposed which have an influence on the survival of probiotic bacteria during dehydration, e.g. stress treatment, growth phase of the probiotic culture prior to dehydration, growth media and genetic modification. 1.1. Application of mild stress prior to dehydration The application of sublethal stress to enhance the stress responses prior to dehydration has been demonstrated as one feasible approach, ensuring high viability of bacterial cultures and retention of physiological activity during dehydration (de Urraza & de Antoni, 1997; Desmond et al., 2002; Kim, Khunajakr, & Dunn, 1998; Lorca & de Valdez, 1998; Teixeira et al., 1995). It has been demonstrated that bacteria respond to changes in their immediate surroundings by a metabolic reprogramming which leads to a cellular state of enhanced resistance (Pichereau, Hartke, & Auffray, 2000). Resistance encoded by defence systems can be divided into two classes. The first comprises a specific system induced by a sublethal dose of a chemical or physical stress (e.g. heat shock), that permits survival against a challenge dose of the same agent (Desmond, Stanton, Fitzgerald, Collins, & Ross, 2001; Gouesbet, Jan, & Boyaval, 2001; Pichereau et al., 2000). The second class of resistance comprises more general systems which prepare cells to survive against very different environmental stresses, without the need for cultures to have had prior exposure to that stress (Desmond et al., 2001; Gouesbet et al., 2001; Pichereau et al., 2000). This mechanism is known as cross-protection (Kim, Perl, Park, Tandianus, & Dunn, 2001). Indeed, pre-adaptation with heat or salt led to improved heat tolerance of probiotics during spray-drying. For example, L. paracasei NFBC 338, pre-adapted by exposure to 0.3 M NaCl, was significantly more resistant to heat stress associated with spray-drying (outlet temperatures between 95oC and 100oC) than non-adapted control cells (33.46 ± 2.3% versus 8.27 ± 4.42% survival, 61 respectively) (Desmond et al., 2001). Although not as efficient as the homologous stress, the levels of cross-protection were in the order heat ≈ salt > hydrogen peroxide > bile (Stanton et al., 2002). In the case of the first type of resistance mentioned, mild heat treatments can lead to adaptation of the cell membrane by increasing the saturation and the length of the fatty acids in order to maintain optimal fluidity of the membrane and activity of intrinsic proteins (Russell & Fukanaga, 1990). The beneficial effect of heat stress in the conservation of probiotics can also be explained by the production of heat shock proteins (HSPs) which promote the correct folding of nascent polypeptides, assembly of protein complexes, degradation and translocation of proteins (Bukau & Horwich, 1998; De Angelis & Gobbetti, 2004). The two major groups of chaperone proteins are the 70-kDa DnaK family and the 60-kDa GroE family which function as chaperone machines (Georgopoulos & Welch, 1993). The components of the DnaK chaperone typically consist of DnaK, DnaJ and GrpE, while that of GroE is composed of GroEL and GroES (De Angelis & Gobbetti, 2004). Desmond, Fitzgerald, Stanton, and Ross (2004) reported that chaperone protein GroEL was among the most strongly expressed proteins in the cell under heat adaptation conditions. Viability of the heat-adapted L. paracasei NFBC 338 in RSM was enhanced 18-fold during spray-drying at outlet temperatures of 95–105oC (Desmond et al., 2001). Heat shock induction of the groESL chaperone in L. johnsonii also provided protection agains freezing (Walker, Girgis, & Klaenhammer, 1999). The increased cytoplasmic contentrations of GroES and GroEL favored cell viability and metabolic activity during and after starter preparation using freezing, lyophilisation or spray-drying (De Angelis & Gobbetti, 2004). Furthermore, pressure pre-treatment has also been shown to increase heat resistance of probiotics. For example, it has been demonstrated that incubation of L. rhamnosus GG at elevated pressure of 100 MPa for 5–10 min prior to exposure to lethal heat at 60oC led to increased heat resistance as compared to untreated controls (Ananta & Knorr, 2003). Jaya Prasad, Paul McJarrow, and Pramod Gopal, 2002 observed that when prestressed with either heat (50°C) or salt (0.6 M NaCl), Lactobacillus rhamnosus HN001 (also known as DR20) showed significant (P < 0.05) improvement in viability compared with the nonstressed control culture after storage at 30°C in the dried form. 62 1.2. Growth phase When grown in batch culture, the growth of bacterial cultures occurs during four distinct phases, i.e. log, lag, stationary and death phases. It is known that the stress responses of bacterial cultures vary depending on the growth phase. Indeed, bacteria that enter into stationary phase develop a general stress resistance and are thus more resistant to various types of stresses (including subsequent down-stream processing and storage) than bacteria in the log-phase, due to carbon starvation and exhaustion of available food sources that trigger stress responses to allow survival of the cell population (Brashears & Gilliland, 1995; Lorca & de Valdez, 1999; Morgan et al., 2006; van de Guchte et al., 2002). Therefore, the optimal growth phase for dehydration survival is the stationary phase. For example, it was reported that stationary phase cells of L. rhamnosus yielded the highest recovery rates after drying (31–50% survival), whereas early log-phase cells exhibited only 14% survival, and lag phase cells showed the highest susceptibility, with only a 2% cell survival under similar conditions of drying (Corcoran et al., 2004). 1.3. Growth media 1.3.1. Carbohydrates The composition of the growth media is a contributing factor to the survival rate of probiotic cultures during drying, and in this respect, the importance of the presence of carbohydrates has been demonstrated. The survival of L. sakei following spray-drying was enhanced when cells were grown in the presence of sucrose (Ferreira et al., 2005). Other sugars, such as fructose and sorbitol also provided better protection than glucose, the standard growth media carbohydrate (Carvalho et al., 2004). The mechanism for the protection of sugars in the growth media is likely that growth in the presence of various sugar substrates produces cells with distinct morphological and physiological traits, thus reflecting distinct resistances to the various stress treatments tested (Carvalho et al., 2004). Studies have shown that metabolites such as mannitol, sorbitol and glutamate which in most cases remain inside the cell may be responsible for the distinct survival behaviour during dehydration (Wisselink, Weusthuis, Eggink, Hugenholtz, & Grobben, 2002), and the formation of these metabolites depends on the carbon sources in the growth media (Kets, Galinski, de Wit, de Bont, & Heipieper, 1996). Adding trehalose to the growth media, enables cells to increase the amount of trehalose within the cytoplasm, which in turn stabilizes the cytoplasmic membrane during desiccation (Streeter, 2003). This accumulation of intracellular trehalose or 63 any carbohydrate is only possible however, if the microorganism cannot use the chosen carbohydrate as a carbon source (Streeter, 2003), or if the cell has the capacity to localize carbohydrate enzymes in vacuoles (Jules et al., 2004). 1.3.2. Compatible solutes: polyols, NaCl, amino acids and amino derivatives Another form of additives used in growth media are compatible solutes such as polyols, amino acids and amino derivatives. Micro-organisms undergoing drying are faced with an increasing osmotic stress as the water activity decreases. One way organisms counteract the osmotic stress is to accumulate compatible solutes to maintain the osmotic balance between the highly concentrated extracellular environment and the more dilute intracellular environment. These solutes can also help to stabilize proteins and the cell membrane during osmotic stress conditions brought on by low water activity during drying processes. One solute which is present in several groups of bacteria and has shown various halotolerant properties is the amino derivative betaine. Kets and de Bont (1994) found that Lactobacillus plantarum grew significantly better in the presence of betaine under osmotically stressful conditions (0.6 M sodium chloride). However, the increased growth rate and apparent salt tolerance did not produce high cell recoveries, with a maximum of 11% of viable cells surviving drying. This indicates that possible protectant (betaine) uptake by cells and stress induced tolerance from growth conditions, does not necessarily guarantee tolerance to desiccation. The presence of sodium chloride in the growth media and medium concentration also have effects on the survival of probiotic cultures following dehydration. Linders et al. (1997) demonstrated that the presence of 1 mol l-1 or 1.25 mol l-1 NaCl during growth of L. plantarum resulted in a decreased residual activity after drying in the fluidized bed. 1.3.3. pH The final pH of the growth media of the probiotic culture also influences the survival during desiccation. The cells obtained under non-controlled pH (4.5) conditions were more resistant to heat stress, spray-drying and storage in the dried state than those from culture under controlled pH (6.5) (Silva et al., 2005). This phenomenon may be related to acid shock/adaptation, which can alter the physiological state of bacterial cells leading to enhanced synthesis of heat shock 64 proteins, and hence improvement of resistance to drying. Silva et al. (2005) confirmed that the higher resistance of L. delbrueckii subsp. bulgaricus grown under non-controlled pH correlated with the enhanced production of heat shock proteins. 1.4. Genetic-modification of probiotic strains Advances in genomics and proteomics have led to the identification of genes involved in Lactobacillus stress responses, such as the molecular chaperone groESL and dnaK (heat stress) (Prasad, McJarrow, & Gopal, 2003; Schmidt, Hertel, & Hammes, 1999; Walker et al., 1999), and enhanced viability of probiotic cultures during dehydration has been obtained by over-expression of the genes encoding various stress inducible proteins. Walker et al. (1999) revealed that features of the groESL operon are shared between various lactic acid bacteria, notably other Lactobacillus species and Lactococcus lactis. Two-dimensional polyacrylamide gel electrophoresis also revealed that GroEL expression in probiotic L. paracasei NFBC 338 was increased under heat adaptation conditions (52oC for 15 min) (Desmond et al., 2004).. Furthermore, using transmission electron microscopy (TEM), it was apparent that the probiotic lactobacilli present in both spray-dried and freeze-dried powders were in close contact (Fig. 6.1) (Corcoran et al., 2006). Because an increased ability to accumulate betaine via BetL can improve the ability of an organism to prevail in diverse, stressful environments, the nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strain L. salivarius UCC118 (Sheehan, Sleator, Fitzgerald, & Hill, 2006).. 65 Figure 6.1. (a) TEM image of spray-dried log-phase L. paracasei NFBC 338, overproducing GroESL (60,000×). (b) TEM image of spray-dried log-phase L. paracasei NFBC 338, control (60,000×). (c) TEM image of freeze-dried log-phase L. paracasei NFBC 338, control (60,000×) (Corcoran et al., 2006). 2. Protective agents Protective agents can be added during growth of the micro-organism, or prior to freezing or drying. The type of protectant largely depends on the micro-organism; however, there are a few that appear to work well with many species. These include skim milk powder, whey protein, trehalose, glycerol, betaine, adonitol, sucrose, glucose, lactose and polymers such as dextran and polyethylene glycol (Hubalek, 2003; Morgan, Herman, White, & Vesey, 2006). Protective additives can be generally classed into two categories: amorphous glass forming, and eutectic crystallizing salts. 2.1. Amorphous glass forming The former matrix includes substances such as carbohydrates, proteins and polymers. A glass is a supersaturated thermodynamically unstable liquid with a very high viscosity. 2.2. Eutectic crystallizing salts A eutectic salt is a crystallizing salt that will leave solution and form crystal structures as the freezing point is reached. Each solute will crystallize at a different temperature. The formation of harmful crystals (either salt or ice) could potentially damage the cell membrane, thereby compromising the integrity of the cell and allowing valuable cell contents to leak out of the cell after thawing. As the freezing point for water is reached, ice crystals will form, and the remaining salts left in solution will be concentrated around the ice crystals. This highly 66 concentrated salt solution mixed with any substances released by the cells prior to freezing can also be detrimental to a cell by causing irreversible damage to the cell membrane (Orndorff and MacKenzie, 1973). Eutectic salts appear to only exert detrimental effects instead of protecting qualities. Used in spray drying In spray drying, amorphous glass forming is used. The use of gum acacia in the spray-drying medium resulted in enhanced probiotic survival of L. paracasei NFBC 338, which displayed 10-fold greater survival than control cells (20% RSM) when grown in a mixture of RSM (10% w/v) and gum acacia (10% w/v) prior to spray-drying at air outlet temperature of 100–105 oC (Desmond, Ross, O’Callaghan, Fitzgerald, & Stanton, 2002). RSM appears to be a very suitable media for efficacious spray-drying of probiotic cultures (Ananta, Volkert, & Knorr, 2005; Corcoran, Ross, Fitzgerald, & Stanton, 2004; Desmond et al., 2002) as skim milk protein can prevent cellular injury by stabilizing cell membrane constituents (Castro, Teixeira, & Kirby, 1995). 3. Rehydration Rehydration of probiotic powders is the final critical step for the revival of cells after dehydration. The reconstitution process in water can be divided into four steps: wetting, submersion, dispersion and dissolving (Freudig, Hogekamp, & Schubert, 1999). Among these steps, wetting of the particles is very often the reconstitution controlling step (Vega & Roos, 2006). The rehydration solution itself (in terms of osmolarity, pH and nutritional energy source), as well as the rehydration conditions (in terms of rehydration temperature and volume) may significantly affect the rate of recovery to the viable state, and thus influence survival rates (Carvalho et al., 2004). For optimum results, it is recommended to dry the cells at the stationary phase of growth and to use slow rehydration procedures (Teixeira et al., 1995). Studies have shown that the rehydration media also can influence probiotic recovery significantly. For example, complex media such as 10% (w/v) RSM and PTM media (1.5% (w/v) peptone), 1% (w/v) tryptone and 0.5% (w/v) meat extract as well as a 10% (w/v) sucrose solutions were found to produce significantly higher bacterial cell recovery 67 than media such as phosphate buffer, sodium glutamate and water (Costa, Usall, Teixido, Garcia, & Vinas, 2000). Some studies have indicated that the same rehydration solution as used for cryopreservation results in increased viability (Abadias, Teixido, Usall, Benabarre, & Vinas, 2001; Ray, Jezeski, & Busta, 1971). The reason for this is that such a solution provides a high osmotic pressure environment which could control the rate of hydration, and thus avoid osmotic shock. The temperature of rehydration of freeze dried and spray-dried probiotics also influences cell recovery. 4. Storage and packaging The method of storage and the packaging it is stored within will influence the shelf life of any dried product. Storage Following spray drying, Desmond et al. (2002) found 100% survival of Lactobacillus paracasei was achievable after one week storage at 4 °C and 15 °C. However, there was a 20 – 80% drop in cell viability at four weeks for both temperatures. This recovery relied on a high 7×107 cfu ml –1 initial bacterial load, and the enumeration of the high cell concentrations gave results with up to 20% variation. The variable survival rate indicates that cell injury can also occur during storage. To achieve long term storage of the spray dried powder, possible changes in the storage conditions should also be investigated. The storage conditions i.e. storage temperature, moisture content of powders, relative humidity, powder composition, oxygen content, exposure to light and storage materials, have significant influences on the survival of probiotics in dried powders, and the correct storage conditions are essential to maintain viable populations of freeze and spray-dried probiotic bacteria. Viability of probiotic bacteria during powder storage is inversely related to storage temperature (Gardiner et al., 2000; Mary, Moschetto, & Tailliez, 1993; Silva, Carvalho, Teixeira, & Gibbs, 2002; Teixeira et al., 1995b). Simpson et al. (2005) reported that there was a significant decline in viability of a number of bifidobacteria species when spray-dried in a skimmed milk-based carrier and stored at 15oC and 25oC. . 68 The carrier used during the spray-drying of probiotics is known to have an influence on storage stability. Ananta et al. (2005) evaluated the effect of a spray- dried carrier on protection of L. rhamnosus GG at 25oC and 37oC storage conditions, and found that the protection capacity decreased in the order RSM > RSM/Polydextrose > RSM/Raftilose ®P95. Moreover, stability of L. rhamnosus GG during long-term storage was impaired by partial substitution of skim milk with either of the prebiotic substances evaluated. Similar findings were previously reported by Corcoran et al. (2004) for spray-dried probiotic lactobacilli. These data justify the suitability of skim milk as a medium for the large-scale production of shelf-stable spray-dried probiotic bacteria. The impairment of viability during storage is related to oxidation of membrane lipids (Teixeira, Castro, & Kirby, 1996). Unsaturated acyl lipids such as oleic acid cannot be considered as stable food constituents during food storage, as the presence of one or more allyl groups within the fatty acid molecule are readily oxidized to hydroperoxides. Moreover, products of lipid peroxidation have been shown to induce DNA damage in a model system (Akasaka, 1986; Inouye, 1984) and in bacteria (Marnett et al., 1985). Therefore, to minimize oxidation and thereby optimize probiotic viability during storage, the presence of antioxidants (Teixeira et al., 1995b), in combination with storage under vacuum with controlled water activity should be effective. Packaging There are very few studies on packaging and viability, as the shelf life of products is generally assessed by private industry and therefore regarded as a commercial secret (Costa et al., 2002). Costa et al. (2002) compared glass vials and high and low barrier plastic bags for the storage of Pantoea agglomerans. The findings showed the high barrier bags provide a more consistent storage environment compared to the low barrier bags, which allowed higher levels of oxygen and moisture to ingress. Bozoglu et al. (1987) compared viability of Lactobacillus bulgaricus and Streptococcus thermophilus following storage in air compared to nitrogen and under vacuum. Storage within glass vials sealed under vacuum or nitrogen gas were found to be superior compared to storage in air. They concluded the poor cell recovery was due to oxygen diffusion into the dry cells through the 69 interfacial area of the cell, possibly because the cells remain permeable throughout storage. The accumulation of free radicals such as oxygen species within a cell that cannot metabolize them, or actively transport them out of the cell, can result in irreversible damaging processes occurring within the cell (Bozoglu et al., 1987). Teixeira et al. (1995) found evidence of damage to the cell wall, cell membrane and DNA during storage of L.delbrueckii ssp. bulgaricus. They attempted to use an antioxidant to prolong cell viability during storage. The results showed an increase in the death rate at –20 °C storage, but an increase in survival at 4 °C. They believed the antioxidant ascorbic acid, could have pro-oxidant properties as well and possibly produce hydroxyl radicals which can oxidize biological molecules. 5. Microencapsulation Microencapsulation helps to separate a core material from its environment until it is released. It protects the unstable core from its environment, thereby improving its stability, extends the core’s shelf life and provides a sustained and controlled release (Figure 6.2). The structure formed by the micro-encapsulation agent around the core substance is known as the wall. The properties of the wall system are designed to protect the core and to release it at controlled rates under specific conditions while allowing small molecules to pass in and out of the membrane (Franjione and Vasishtha, 1995; Gibbs et al., 1999). The capsules may range from submicron to several millimeters in size and can be of different shapes (Shahidi and Han, 1993; Franjione and Vasishtha, 1995). Compared to immobilisation/entrapment techniques, micro-encapsulation has many advantages. The microcapsule is composed of a semipermeable, spherical, thin and strong membranous wall. Therefore the bacterial cells are retained within the microcapsules (Jankowski et al., 1997). Moreover, compared to an entrapment matrix, here is no solid or gelled core in the microcapsule and its small diameter helps to reduce mass transfer limitations. The nutrients and metabolites can diffuse through the semipermeable membrane easily. The membrane serves as a barrier to cell release and minimises contamination. The encapsulated core material is released by several mechanisms such as mechanical rupture of the cell wall, dissolution of the wall, melting of the wall and diffusion through the wall (Franjione and Vasishtha, 1995). 70 Figure 6.2. Principle of Encapsulation: Membrane barrier isolates cells from the host immune system while allowing transport of metabolites and extracellular nutrients. Membrane with size selective pores (30-70 kDa). Source: INOTECH Encapsulation. Microencapsulation of various bacterial cultures including probiotics has also been used for extending their storage life (Krasaekoopt, Bhandari, & Deeth, 2003). Several methods of micro encapsulation of probiotic bacteria have been reported and include extrusion technique, emulsion technique, cross-linking with cationic polymers, coating with other polymers, mixing with starch and incorporation of additives (Krasaekoopt et al., 2003). Song, Cho, and Park (2003), Kim, Kamara, Good, and Enders (1988) Koo, Cho, Huh, Baek, and Park (2001) observed that both non-encapsulated and encapsulated cells stored at 4oC had comparable stability, while encapsulation provided a greater degree of protection against increased storage temperature. O’Riordan, Andrews, Buckle, and Conway (2001) prepared microencapsulated Bifidobacterium PL-1 with starch by spray-drying, however the starch-coated cells did not display any enhanced viability compared with free PL1 cells when exposed to acid conditions for 6 h or in two dry food preparations over 20 days storage at ambient temperature (19–24 oC). Hence, the efficiency of microencapsulation of probiotics depends on the encapsulating materials and techniques of micro-encapsulation. 71 V. PRODUCTS CONTAINED PROBIOTICS 1. Forms of probiotic powder Lyophilization (freeze drying) Figure 7.1: Forms of probiotic powder by freeze drying The concentrated of bacteria is first frozen at 40° C then, by sublimation, the ice is transformed into steam. One thus obtains a crust lasts ready to be crushed. Crushing Figure 7.2: Forms of probiotic powder - crushing The crust is crushed to obtain a powder with the concentration of bacteria is very high. It is the pure culture. Mixture Figure 7.3: Forms of probiotic powder by freeze drying - mixture Mix the pure culture is mixed with of the excipients (inert ingredients which serve as vehicle to the pure culture) to permit a standardized packaging. 72 VI. Storage the products contained probiotics Table 6.1: Stability of probiotics during the storehouse 73 74 Table 6.1 (continue) 75 Table 6.2: Ameloiration of the viable probiotics during the storehouse 76 Table 6.3: Effect of the probiotics addition in the milk products according to sensories 77 VII. Examples for product of probiotic powder Figure 7.4 : Mixture of probiotics in tablets Table 6.4: Informations of product Mixture for children 5 – 15 years old. Mixture for adults Strains: 8 Bacterial strains that is specific to children, appropriate dosage; formulated to reflect the healthy microflora of a child. 8 Bacterial strains specific to adults, appropriate dosage; formulated to reflect the healthy microflora of an adult. Quality 4 Billion of viable cells/active per capsule availability : Each vegetable capsule contains : a total of 4 billion of viable cells during the manufacturing (2,4 billion guaranteed until the expiry date) 12 Billion of viable cells/active per capsule availability: Each vegetable capsule contains : 12 billion of viable cells in the manufacture (8,3 billion guaranteed up to the expiry date). Availability : 60 vegetational capsules. 60 and 12 vegetational capsules Usage guide The intestinal flora sound of a child is similar to that of an Adults require the larger quantities of the same bacteria 78 adult. The major difference lies in the dosage because of their small size, children require smaller quantities of the same probiotics. The strains used for the mixture for children have been specifically selected by reason of their benefits for the health of children. If the child cannot swallow the capsules, if the child cannot swallow the capsules, the middle more easy to him to take this product of basic support is to mix the contents of the dish with its food. that children. The strains used for the mixture for adults have an appropriate dosage for adults in good health who wish to support their immune system. In the framework of food choices and of a lifestyle, the mixture for adults optimizes the digestion and intestinal health, which represent the foundations of the health of all the body systems. Medicinal ingredients: - Lactobacillus casei: 25% - Lactobacillus rhamnosus: 25% - Lactobacillus acidophilus: 20% - Lactobacillus plantarum - Streptococcus thermophilus: 7% - Bifidobacterium bifidum: 5% - Bifidobacterium breve: 5% - Lactobacillus bulgaricus: 3% - Lactobacillus casei: 25% - Lactobacillus rhamnosus: 25% - Lactobacillus acidophilus: 20% - Lactobacillus plantarum: 10% - Streptococcus thermophilus: 7% - Bifidobacterium bifidum: 5% - Bifidobacterium breve: 5% - Lactobacillus bulgaricus: 3% NON- MEDICINAL INGREDIENTS: microcrystalline cellulose, magnesium stearate, ascorbic acid. May contain traces of powdered milk, whey and soy lecithin Microcrystalline cellulose, magnesium stearate, ascorbic acid. May contain traces of powdered milk, whey and soy lecithin. Recommended Dosage : to restore the health of the intestinal flora, take three capsules per day after To restore the health of the intestinal flora, take three capsules per day after eating. To maintain the health of the intestinal flora, take a capsule per day after eating. 79 References [1] Chalat Santivarangkna, Alternative Drying Processes for the Industrial Preservation of Lactic Acid Starter Cultures, 2007, Biotechnol.Prog, 23, 302-315. [2] X.C.Meng, Anhydrobiotics:The challenges of drying probiotic cultures, 2008, Food chemistry, 106, 1406-1416. [3] Song Miao, Effect of disaccharides on survival during storage of freeze dried probiotics, 2008, Dairy Sci.Technol.,88, 19-30. [4] C.A.Morgan, Preservation of micro-organisms by drying; A review, 2006, Journal of Microbiological Methods , 66,183–193. [5] Kanchi Bhasker Praveen Kumar Reddy, Role of Cryoprotectants on the Viability and Functional Properties of Probiotic LacticAcid Bacteria during Freeze Drying, 2009, Food Biotechnology, 23,243–265. [6] C.A. Morgan, N. Herman, P.A. White, G. Vesey Preservation of micro-organisms by drying; A review Journal of Microbiological Methods 66 (2006) 183–193 [7] Colette Desmonda, Catherine Stantona, Gerald F. Fitzgeraldb, Kevin Collins, R. Paul Ross Environmental adaptation of probiotic lactobacilli towards improvement of performance during spray drying International Dairy Journal 12 (2002) 183–190 [8] Diamant Conference Centre Brussels, Belgium Powder Research to Promote Competitive Manufacture of Added-Value Food Ingredients 27th and 28th November 2002 [9] G. E. Gardiner, E. O’sullivan, J. Kelly, M. A. E. Auty Comparative Survival Rates of Human-Derived Probiotic Lactobacillus paracasei and L. salivarius Strains during Heat Treatment and Spray Drying Applied And Environmental Microbiology, June 2000, p. 2605–2612 [10] Gillian E. Gardiner, Paul Bouchier, Eilis O’Sullivanb, Jim Kellya, J. Kevin Collins, Gerald Fitzgeraldb, R. Paul Ross, Catherine Stantona, A spray-dried culture for probiotic Cheddar cheese manufacture International Dairy Journal 12 (2002) 749–756 [11] Guidelines for the Evaluation of Probiotics in Food Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food London Ontario, Canada April 30 and May 1, 2002 [12] Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria Report of a Joint FAO/WHO Expert Consultation on 80 Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria Amerian Córdoba Park Hotel, Córdoba, Argentina 1-4 October 2001 [13] Jaya Prasad,Paul McJarrow, and Pramod Gopal Heat and Osmotic Stress Responses of Probiotic Lactobacillus rhamnosus HN001 (DR20) in Relation to Viability after Drying Applied And Environmental Microbiology, Feb. 2003, p. 917–925 [14] Johan Palmfeldt , Barbel Hahn-Hagerdal Influence of culture pH on survival of Lactobacillus reuteri subjected to freeze-drying International Journal of Food Microbiology 55 (2000) 235–238 [15] J.P. 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Ross Anhydrobiotics: The Challenges of drying probiotic cultures Food Chemistry 106 (2008) 1406–1416 [20] Wen-Chian Lian, Hung-Chi Hsiao, Cheng-Chun Chou Survival of bifidobacteria after spray-drying International Journal of Food Microbiology 74 (2002) 79– 86 [21] RAPPORT Consultation mixte d’experts FAO/OMS sur l’évaluation des propriétés sanitaires et nutritionnelles des probiotiques dans les aliments, y compris le lait en poudre contenant des bactéries lactiques vivantes . Cordoba, Argentine . 1er - 4 octobre 2001 81 [22]Les probiotiques dans l’industrie alimentaire : que conseiller à nos patients ? G. CORTHIER (Jouy-en-Josas). Volume 37 - N° spécial CREGG - 2007 [23] 2185BF9DC143/16256/Rodier_Sylvain_ESP_H2008_corrige.pdf Dissertation critique Est-ce que cela vaut la peine du point de vue technologique, de rajouter des probiotiques dans les aliments autres que les laits fermentés ? Dans le cadre de l’épreuve synthèse de programme. Présentée à l’équipe programme TTA, Par Sylvain Rodier. Institut de technologie agroalimentaire, campus de Saint- Hyacinthe. Le 12 mai 2008 [24] otics_prebiotics_fr.pdf World Gastroenterology Organisation . Organisation mondiale de Gastroentérologie Recommandation Pratique: Probiotiques et Prébiotiques Mai 2008 [25] LES BACTÉRIES PROBIOTIQUES :INNOVATIONS ET TENDANCES DE DÉVELOPPEMENT TECHNOLOGIQUE. Membres du Réseau Bio-Innovation® : GRATUIT. Volume 6, Numéro 4 - Août 2007 [26] PROBIOTIQUES DOCUMENT DE TRAVAIL Rapport présenté à la Direction des produits de santé naturels, Santé Canada. Mars 2005. Nutritech Consulting Winnipeg, Manitoba Préparé par: K.C. Fitzpatrick 82 [27] Probiotiques - La sélection Udo® [28] Le concept de probiotique :DUCLUZEAU R. (1) ; (1) INRA, 46, rue Albert-Calmette, 78350 Jouy-en-Josas, FRANCE. Journal Title : Antibiotiques ISSN 1294-5501 Source / Source: 2002, vol. 4, no4, pp. 234-238 [5 page(s) (article)] (18 ref.) [29] Lactococcus lactis: nominated as the Wisconsin State Microbe © 2009 Kenneth Todar, PhD [30] LES PROBIOTIQUES, ALLIES D'UNE BONNE SANTÉ INTESTINALE PROFESSEUR L. DE VUYST .GROUPE DE RECHERCHE EN MICROBIOLOGIE INDUSTRIELLE ET BIOTECHNOLOGIE ALIMENTAIRE UNITE DE BIO-INGENIERIE – VRIJE UNIVERSITEIT BRUSSEL 83