Đồ án Kỹ thuật sấy probiotics

eptically to grow media Growing Centrifuging Centrifuging Washing media Prepare 29 1.2. Equipments and technological parameters: 1.2.1. Equipments: Figure 3.1: Structure of lyophilization equipment Freeze drying Suspending Adding cryoprotectant Storing Filling in glass vials or ampoules Loading onto trays Loading onto freeze dry chamber Ampoules, glass vials Sealing 30 1- Freeze dry chamber 2- Valve 3- Syphon 4- Hot water tank 5- Condenser 6- Liquid seperate tank 7- Amonium condenser 9- Compresser 10- Vacuum pump 11,12,13- Electric motors 14- Water pump 15- Filter 16- Heat exchanger 17- Vacuummeter 18- Control valve 19- Tray 20- Thermal net 21- Thermal controler Structure of freeze dryer: Drying chamber: The drying chamber of freeze dryer consist of a vacuum tight unit containing loading door and one or more inspector wimdow. Each chamber contains aset of shelves that has hydrolic stoppering mechanism and gas bleed system. Heat is applied directly through electrical resistantce coilsor by circulating hot water, silicon or glycol. Condenser: The condenser of freeze dryer is a vacuum tight unit separate from chamber by a vacuum Valve. They contain a set of cooling plate refrigerated by refrigeration system.Condenser is supplied with hot waterdefrosting system. It was a cold trap used to collect the moisture. Vacuum pump : Each freeze dryer is equipped with a two stage rotary gas ballast vacuum pump. Vacuum pump is an essential component of the freeze dryer and is required for evacuated emvironment around the product. Vacuum pump keeps the chamber and condenser sufficiently free from the residual gases, water vapor stream to be unable to flow from the drying meterial. Refridgeration system : The freeze dryer is provided with twin independent direct expansion, single or multi-component refrigeration plant. The commonly used refrigerant is Freon R- 502 gas. Control facilities : 31 The freeze dryer is instrumented to control and operate th various plant components like shelf staking mechanism. Hydraulic stoppering device and chamber condenser valve. Instrumentation is also available to mesure and record the various process variables like chamber pressure, shelf temperature, product temperature , and condenser temperature Figure 3.2: Freeze drier 1.2.2.Processing parameters: Growing: Temperature : 37C Time : until the late log phase or the early stationary phase of each strain (17 hours for L. paracasei ssp. paracasei NFBC 338 and L. rhamnosus under anaerobic conditions) Centrifuging : Purpose : to havest micro-organism 32 Temperature : 4C Time : 15 minutes Rotary velocity :9000 x g Washing : Purpose : to wash out fementation broth residues Wash 3 times with distilled water Suspend : Purpose : to prepare feed solution for freeze drying (the suspension of micro- organism with initial concentration from 108cfu/ml to 1011cfu/ml) 1% inoculum in MRS broth or 15% RSM (reconstituted skimmed milk; Golden Vale, Cork,Ireland) Often distilled water to make up to reach 20%(w/w) solid Votex to get a homogenous suspension Adding cryoprotectant : Purpose : to protect cells from damage during freezing step Figure 3.3: Filling suspension in glass vials 33 Freeze drying : Freezing step : at –80 C for 21 hours Freeze dried for 48 h ,P =0.08Pa ,(40 h of primary drying at -30ºC and 8 h of secondary drying at -10ºC). Moisture left:less than 1% Storage: Temperature :-18 C under vacuum, in dark place. 1.3. Factors influence freeze drying process 1.3.1. Depth of product in container: The greater the depth of product in the container, the longer the drying process will be 1.3.2. Vapor pressure diferential: The actual driving force for the process is the vapor pressure diferential between the vapor at the surface where drying of product is occuring and that at the surface of the ice on the condenser 1.3.3. Amount of solid in the product, their particle size and their thermal conductance : Amount of solid in the product, their particle size and their thermal conductance will affect the rate of drying. The more solid present, the more impedment will be provided to the escape of the water vapor. The smaller of the ice crystal size, the faster the drying rate is. The poor thermal conducting properties of the solid in the product, the slower of heat transfer rate through the frozen material to the drying boundary. Solid content must be appoximately between 5-25%, so mannitol or gelatin are used as bulk agent 1.4. The influence of processing factors on quality of product: The freeze drying medium, physiological state of the cells (Broadbent and Lin, 1999), freezing rate (Peter and Reichart, 2001), freeze drying parameters, 34 rehydration conditions, and initial cell concentration (Costa et al., 2000) are the different parameters that account for freeze drying tolerance. The percentage survival has been reported to increase with increasing initial cell concentration up to 1011 cfu/ml (Costa et al., 2000); a minimal concentration 107cfu/ml is generally recommended (Pocard et al., 1994). Bacterial cells in the stationary phase are more resistant, indicating that the age of the culture has a positiveeffect (Brashears and Gilliland, 1995). It has also been reported that the addition of Tween 80 increases the proportion of unsaturated fatty acids in the membrane (Beal et al., 2001), which in turn leads to modification of the membrane permeability favoring better survival. Intrinsic Tolerance of Cultures. Although variation in viability after drying among different lactic acid starter cultures is explicit, no clear explanation is available. Some studies found that viability is correlated to cell morphology, i.e., cell size and shape. Bozog lu et al. reported that small spherical streptococci cells are more resistant to freeze drying than long rod lactobacilli cells because of the lower surface area to volume ratio. It has been reported that the higher the surface area of the cell, the higher the membrane damage owing to extracellular ice crystal formation during freezing (Fonseca, Beal, & Corrieu, 2000). Consequently, cell size has a strong influence on survival of probiotics during freeze-drying, with small spherical cells such as enterococci being more resistant to freezing and freeze-drying than larger rod shaped lactobacilli (Fonseca et al., 2000). While a number of stress proteins induced by heat or oxidative stresses are known to vary among species or strains, the viability of different species or subspecies of Bifidobacterium sp. also varies with their heat or oxygen tolerance. The majority of species with high heat and moderate oxygen tolerance have relatively high viabilities (68- 102%) compared to species with a low tolerance to heat and a variety of tolerances to oxygen (19.4 -38%) . In a more specific instance, different strains of L. lactis can differ in their capability to accumulate glycine betaine as a compatible solute, which is due to some genotypical differences . This accumulation in lactic acid bacteria affects the ability to survive drying. Since the differences in sensitivity to dehydration seem to be an individual trait, it poses a difficulty in adopting drying conditions from one given strain to other strains. Table 3.1: Viabilities of different Strains of Lactic Acid Bacteria of the same species 35 Growth Media and Growth Conditions. Growth media Two recent studies have shown that the growth media can have a significant effect on the freeze dried survival of Enterococcus faecalis, Enterococcus durans and Lactobacillus bulgaricus (Carvalho et al., 2003, 2004). These studies also highlighted that the effects can be strain and protectant dependent. Lactobacillus bulgaricus showed the lowest decrease in viability after freeze- drying when grown in the presence of mannose, compared to fructose, lactose or glucose. Survival during room temperature storage was also found to be dependent on the drying protectant medium used. Carvalho and co-workers (2004) showed supplementation of the drying medium with any of the four carbohydrates tested enhanced the desiccation protection during storage. Other 36 sugar types, such as fructose and sorbitol also provided better protection than the standard growth media carbohydrate glucose (Carvalho et al., 2004). This is consistent with a study on the survival of Bradyrhizobium japonicum by Streeter and co-workers (2003) who showed presence of trehalose during growth was much more effective in protection against desiccation compared to adding trehalose to the culture at the time of desiccation. 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 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). 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 counter- act 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. Studies on the effects of different growth media on the viability of lactic acid starter cultures are rare. Most of Lactobacillus sp. are grown in the De Man Rogosa and Sharpe (MRS) medium, which is rather expensive for mass production. In a study by Linders et al., L.plantarum was grown in enriched MRS medium (55 g L-1 MRS medium supplemented with1gL 1 yeast extract and 10 g L-1 glucose) and diluted MRS medium (2.75 g L-1 MRS medium). It is well-known that some organisms confronted with a decreasing water activity accumulate compatible solutes such as amino acids, quaternary amines (e.g., glycine betaine, carnitine), and sugars. This adaptation can also prepare cells for the osmotic stress during the drying process. Lactic acid bacteria, like most bacteria, do not synthesize compatible solutes de novo and are dependent on the uptake of them or their precursors .It is supposed that the drying process duration is too short for the bacteria to accumulate compatible solutes, and the accumulation is an energy-dependent process where the energy source such as glucose must be available. Thus, compatible solutes or their precursors should be added to the growth medium and assimilated before drying. The presence of compatible solutes in the medium has been shown to increase viability of lactic acid starter cultures, especially under osmotic stress growth 37 conditions whereby compatible solute assimilation is promoted . On the other hand, results from a study by Linders et al. show lower glucose fermentation activities of dried L. plantarum grown under osmotic stress (1 mol L -1 NaCl) despite having a higher accumulation of compatible solutes betaine and carnitine compared with cells grown under standard conditions. The adverse influence of NaCl on the residual activity is even more prominent when cells are grown in batch mode (3% compared to 40% in chemostat mode). The influence of mode of cell cultivation per se has less effect on residual activity of cells grown in media without NaCl supplementation (76% in batch mode compared to 61% in chemostat mode) , although this effect was not very pronounced. These contradictory results, despite the same L. plantarum strain being used, are presumably due to the effect of initial cell concentration . Growth Conditions. The influence of pH on viability of cells after drying is still equivocal and seems to vary with the drying process applied. According to a study by Schweigart the pH optima before drying of lactic acid bacteria cultures are 4.6 -4.8 for freeze drying. In regard to alkaline solutions used to control pH during fermentation, calcium hydroxide was reported to favor viability of L. bulgarius after freezing and freeze drying. The mechanism of protection is not clearly known but is supposedly that calcium may reinforce cell integrity against damage from dehydration effects or ice crystals during freezing. There is no report about the effect of various neutralizing agents on the viability of cells dried without a freezing step. Stadhouders et al. state some advantages of calcium hydroxide that growth of lactic acid bacteria is less inhibited by calcium lactate than ammonium lactate or sodium lactate (which are the salts from neutralization of lactic acid produced during the growth of cells), and the dried powder obtained is less hygroscopic. Cell Harvesting Conditions. The optimal harvest time of cells for starter culture production depends on the specific organism. In general, lactic acid bacteria are harvested either in the late log phase or early stationary phase. Besides the maximal yield, cells harvested at the stationary phase render higher viability after drying.. In a study by Corcoran et al. ,three different growth phases of L. rhamnosus (lag, early exponential, and stationary phase) were investigated and similar results were found. Over 50% and 14% viability are obtained by the stationary phase and early exponential phase cells, while lag phase cells are most susceptible with only approximately 38 2% viability. The greater tolerance of stationary phase cells to dehydration is probably due to the depletion of nutrients. Glucose starvation is a known trigger for stress response leading to the resistance to many stresses, e.g., osmotic and heat stress.In the process of harvesting cells, centrifugation is considered to be efficient for concentrating cells . Initial cell concentration : There are few publications on the influence of initial cell concentration on viability after drying. Linders et al found that the initial cell density directly correlates to the activity of normally grown L. plantarum after drying. The ratios of the activity immediately after drying to the activity before drying ranging from 0.1 to 0.83 are achieved using initial cell densities between 0.025 and 0.23 g of cell/ g of sample, respectively. If cells are grown under osmotic stress, the residual activity is low (0.06) and not related to the initial cell density. Higher initial cell concentration can provide an additional benefit. When the method of freeze drying or lyophilization is mentioned in relation to the preservation of micro-organisms, it is nearly always with regards to long term storage of cell suspensions that contain greater than 108 cells ml–1 (Miyamoto-Shinohara et al., 2000; Costa et al., 2000). The reason for preserving high cell concentrations is on the premise that the majority of cells die during long term storage, but a sufficient number survive to enable the continuation of the strain. Bozoglu et al. (1987) suggest that survival of 0.1% of the original cell population is a “sufficient number” of survivors of freeze drying to allow continuation. Cryoprotectants : To and Etzel (1997) demonstrated that 60-70% of cells that survived the freezing step can live through the dehydration step. Addition of protective agents to starter cultures is a common means to protect cells during drying and storage. The excipients added may be simple components directly added to the cell concentrate or complex components/polymers used as suspending medium or carrier (such as skim milk, whey, gum acacia, gelatine). Protective agents are numerous. The effectiveness of a given protectant seems to vary largely with each type of culture. One of the most extensively investigated compounds is trehalose. This may be due to the attention being drawn by a phenomenon called anhydrobiosis, in which some organisms in nature survive long and extreme dehydration period by accumulating a large amount of disaccharides, especially trehalose . The protective ability is enhanced significantly by using it in concert 39 with borate ions, which cross-link the trehalose, thereby raising the glass transition temperature (Tg) of the dry matrix . It is thought that a high Tg of sugars correlates to their ability to stabilize lipid bilayer during dehydration . Trehalose-borate is found to be a better protectant than a control mixture which is currently used in industry for the stabilization of freeze-dried L. acidophilus, but the high price of trehalose may raise questions regarding its use in starter culture production at an industrial scale.According to the hypothesis of Crowe et al. (87), headgroups of lipid bilayer are brought closer together when water is removed during dehydration. The increased attraction forces the dry bilayer to the gel phase at room temperature. During rehydration the dry lipid then undergoes a phase transition from the gel back to liquid crystalline phase, leading to leakage of lipid membrane. Following the hypothesis, sugar can lower the Tm by replacing the water between lipid head groups, thus preventing phase transition and its accompanying leakage upon rehydration. However, the positive effect of sorbitol and maltose was not found to be due to their ability to lower Tm or to change the moisture distribution and water activities of the dried L. plantarum cells. Linders et al. therefore suggest that the effective sugars, i.e., sorbitol and maltose, act through their free radical scavenging activity.The synergistic effects may be acquired from the combination of different protectants. . Rate of freezing: It has been shown that cellular inactivation occurs mostly at the freezing step (Tsvetkov & Brankova, 1983). Indeed, To and Etzel (1997) demonstrated that 60-70% of cells that survived the freezing step can live through the dehydration step. During freezing, the formation of extracellular ice causes an increase in extra-cellular osmolality, so as soon as ice forms outside of the cell in solution, the cell begins to dehydrate. The intracellular and extra-cellular solution concentrations will increase as temperature drops until a eutectic point is reached. There are as such two kinds of freezing methods, i.e. slow freezing and fast freezing. During slow freezing, the process of gradually dehydrating the cell as ice is slowly formed outside the cell leads to extensive cellular damage, while fast freezing can avoid solute effects and excessive cellular shrinkage (Fowler & Toner, 2005). One of the main causes of cell viability loss is the need to freeze the sample in order for it to be dried. The process of freezing and especially the rate of freezing can be detrimental to the viability of a bacterial cell after drying (Uzunova-Doneva and Donev,2000). There are two ways to freeze a sample before drying, either within the freeze dryer chamber, through cooled shelves, or the product can be frozen prior to loading onto the freeze dryer. Freezing within 40 the freeze dryer can enable a slow uniform ice crystal structure to form and the freezing can be controlled to an extent that an annealing process can be used. An annealing process is a controlled rise and fall of temperature to increase ice crystal growth to form channels and pores to the surface for easier vapor escape during drying. Annealing and slow freezing form ice crystal structures that enhance the appearance of freeze dried products. In the case of freezing micro- organisms however, the viability of the cells after drying can be more important than the appearance of the product and so a faster rate of freezing is used. Large ice crystal formation can cause damage to fragile cell membranes, which cannot then repair after desiccation and thus reducing cell viability. To enable only small ice crystals to form the faster the rate of freezing, the better. Snap freezing cells in liquid nitrogen for example is a well known method for cryopreservation of biological cells. However, snap freezing means the product has to be frozen first and then loaded onto the freeze dryer. Maintaining the samples frozen during transportation and loading can be challenging and for this reason the simplicity of freezing the product within the freeze dryer is often preferred. Storage 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 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). Bruno and Shah (2003) demonstrated that a temperature maintained at -18 C was optimum for the long-term storage of freeze-dried probiotics to maximize viability of bifidobacteria, whereas a storage temperature of 20 C was unsuitable, resulting in significant reductions in viable counts. The moisture content of probiotic powders is a critical factor influencing shelf- life stability of the live bacteria. Work conducted in our laboratory has shown that viability of freeze-dried probiotics in skim milk is inversely related to relative vapour pressure (RVP), with 11.4% RVP yielding highest viability during storage at room temperature (unpublished). Zayed and Roos (2004) also demonstrated that the amount of water remaining after drying affects not only the viability of bacteria, as determined immedi-ately after the process, but also the rate of loss of viability during subsequent storage. Indeed, the optimum 41 moisture content for storage of freeze-dried L. salivarius subsp. sal-ivarius was reported to range from 2.8% to 5.6% (Zayed & Roos, 2004). The carrier used during freeze-drying of probiotics is known to have an influence on storage stability. Some studies have shown that the presence of disaccharides can stabilize the cell membrane during both freezing and storage (Carvalho et al., 2002; Conrad et al., 2000; Crowe et al., 1988).For example, it has been proposed that sorbitol prevents membrane damage by interaction with the membrane (Linders, de Jong, Meerdink, & Vantriet, 1997b), and stabilizes protein functionality and structure (Yoo & Lee, 1993). Control of the phase transition temperature in membranes of dry cells is an important factor determining desiccation tolerance of live probiotics, in addition to control of free radical activity (Linders et al., 1997a). It has been suggested that the decrease of viability during storage at high temperatures and/or relative humidity for sugar- containing products has been related to their glass transition temperature (Vega & Roos, 2006). The reason for this is that sugars are likely to form highly viscous glasses at room temperature when they are dehydrated, and the improved storage of anhydrobiotes and liposomes has been associated with the presence of a glassy state. Our data demonstrated that high viability of freeze- dried L. rhamnosus GG powders in trehalose, lactose/trehalose and lactose/maltose related to their high glass transition temperature (unpublished). However, Carvalho et al. (2002) and Linders et al. (1997a) demonstrated that sorbitol was the most effective protectant for L. plantarum and L. rhamnosus during storage, while in contrast, the superior glass former, trehalose was not an effective protectant. The impairment of viability during storage is related to oxidation of membrane lipids (Teixeira, Castro, & Kirby, 1996). Unsaturated acyl lipids such as oleic acid can not 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 D