Microencapsulation of Lactobacillus paracasei by spray freeze drying

Food Research International 43 (2010) 193–202

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Microencapsulation of Lactobacillus paracasei by spray freeze drying David Semyonov, Ory Ramon, Zoya Kaplun, Luba Levin-Brener, Nadya Gurevich, Eyal Shimoni * Faculty of Biotechnology and Food Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel

a r t i c l e

i n f o

Article history: Received 2 June 2009 Accepted 22 September 2009

Keywords: Probiotics Microencapsulation Spray freeze drying Trehalose Maltodextrin

a b s t r a c t This study evaluates the implementation of a new process: spray freeze drying (SFD), to produce dry micro-capsules of Lactobacillus paracasei with high viability. The study concentrated on determining the survival of the cells, encapsulated in a matrix of maltodextrin and trehalose. SFD was compared with the conventional bulk freeze drying (BFD). Overall it was shown that SFD is a successful method to generate dry micro-capsules of probiotic cells with high viability (>60%). The spraying stage did not affect the viability of the bacteria. In the freezing stage, high osmotic pressures originated by elevated trehalose concentrations, helped preserving the cells viability. It was also found that the lower the maltodextrin molecular weight, the larger the beads volume and solids concentration, the higher is the bacteria survival during the freezing and drying stages. In the drying stage, trehalose concentration was also the critical factor that increased final probiotic viability. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction A food is regarded as functional if it is satisfactorily demonstrated to beneficially affect one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either improved state of health and well-being and/or reduction of risk of disease (Stanton et al., 2001). Such foods are the result of major research efforts where the developments include new technologies, and innovative marketing strategies. Novel aspects of functional foods include probiotics, nutraceuticals and phytochemicals (Sanders, 1998). Probiotics represent probably the archetypal functional food, and are defined as alive microbial supplement which beneficially affect the host by improving its intestinal microbial balance (Guarner & Schaafsma, 1998). There is a growing scientific evidence to support the concept that maintenance of healthy gut micro-flora may provide protection against gastrointestinal disorders including gastrointestinal infections and inflammatory colon diseases (Gibson & Roberfroid, 1995). Presently the industrial marketing of functional foods is dominated by gut health products, in particular probiotics. Among those, dairy products are the key product sector (Berner & O’Donnell, 1998) showing an impressive growth during the recent years. Due to their sensitivity to environmental factors such as heat, oxygen and humidity, probiotic bacteria should be protected from deterioration processes. Supplementation of functional foods with probiotic bacteria raises considerable technological challenges because in order to provide health benefits the level of viable probi* Corresponding author. Tel.: +972 4 8292484; fax: +972 4 8293399. E-mail address: [email protected] (E. Shimoni). 0963-9969/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2009.09.028

otic bacteria should be >107 cfu ml1 or g1/product at time of consumption (Adhikari, Mustapha, Grun, & Fernando, 2000; Doleyres & Lacroix, 2005). Before a probiotic can benefit human health it must fulfill several criteria. It must have good technological properties so that it can be manufactured and incorporated into food products without losing viability and functionality or creating unpleasant flavors or textures. It must survive passage through the upper gastrointestinal (GI) tract and arrive alive and bioactive at its site of action, preserving its ability to function in the gut environment. Other requirements include their application in uniform distribution in the food formula, as well as their stability during product processing, distribution, and storage. The commonly used solutions to these problems often provide inefficient protection to probiotic bacteria thus limit their incorporation in foods. A promising solution to this problem is microencapsulation. Encapsulation of probiotics is employed in order to increase the bacteria resistance to freezing and freeze drying of the food (Champagne, Gardner, Brochu, & Beaulieu, 1991; Kearney, Upton, & Mcloughlin, 1990; Maitrot, Paquin, Lacroix, & Champagne, 1997; Shah & Ravula, 2000; Sheu, Marshall, & Heymann, 1993). In most of the studies the probiotic bacteria were entrapped in a gel matrix of biological nature materials such as alginate, k-carrageenan, and gellan/xanthan. The core and wall solution was turned into drops of desired size by an extrusion method, employing an emulsion, or by transfer from organic solvents. One problem in the probiotic entrapment approach is that the gel beads technologies stabilize the bacteria mostly in liquid products, and are difficult to scale up. To extend their storage shelf-life it is convenient to convert the micro-capsules into a dry powder by employing techniques such as

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spray drying, freeze drying, and/or fluidized bed drying. The spraydrying is an economic and effective technology, however, it causes high mortality as a result of simultaneous dehydration, thermal, and oxygen stresses imposed to bacteria during the drying process (Anal & Singh, 2007). Freeze drying is considered one of the most adequate methods for drying biological materials and sensitive foods. However, when this method was employed for drying probiotic bacteria and other cells, undesirable effects such leakage of the cell membrane due to changes in the physical state of membrane lipids or changes in the structure of sensitive proteins in the bacteria cell occur (Leslie, Israeli, Lighthart, Crowe, & Crowe, 1995; Teixeira, Castro, & Kirby, 1996; Teixeira, Castro, Malcata, & Kirby, 1995). Protective solutes such as cryoprotectants (saccharides and polyols) and other compatible solutes like adonitol, betaine, glycerol and skim milk were used to increase bacteria’s viability and increase their survival during freeze-drying and subsequent storage (de Valdez, Degiori, Holgado, & Oliver, 1983; Hamoudi, Goulet, & Ratti, 2007; Selmer-Olsen, Birkeland, & Sorhaug, 1999). These studies lead to the conclusion that the effect of each protective agent on the viability of a specific LAB strain during or following the freeze-drying process have to be determined on a case-bycase basis (Carvalho et al., 2004). As mentioned above, dried probiotic micro-capsules can be coated by an additional layer (shell) in order to protect the bacterial core from the acidic environment of the stomach and to avoid the deleterious effect of bile salts on the cell’s membrane. This additional shell can help to release the bacterial core at a desired site in the GIT. In order to be further coated, bulk freezed powders are micronized to a narrow particle distribution. This process is complex, requires intensive energy, and decrease the viability of the dried cells (Picot & Lacroix, 2003). The pharmaceutical industry utilized recently the spray freeze drying (SFD) for pharmaceutical powders preparation (Costantino et al., 2000, 2002; Maa, Nguyen, Sweeney, Shire, & Hsu, 1999; Maa & Prestrelski, 2000; Webb, Cleland, Carpenter, & Randolph, 2002). This method combines the narrow particle size distribution of an extrusion device and the freeze-drying process to prepare a dry powder of desired particle size and of narrow distribution. SFD basic principle is to spray a solution containing dissolved/suspended material (e.g. protein) by an atomization nozzle into a cold vapor phase of a cryogenic liquid, such as liquid nitrogen, so the droplets may start freezing during their passage through the cold vapor phase, and completely freeze upon contact with the cryogenic liquid phase (Costantino et al., 2000, 2002; Maa and Prestrelski, 2000; Maa et al., 1999; Webb et al., 2002; Yu, Johnston, & Williams, 2006). The frozen droplets are then dried by lyophilization. SFD powders have a controlled size, larger specific surface area and a better porous character than spray-dried powders. The particles retain their spherical and porous morphology and can be further coated with an enteric food grade biological polymer which is designed to disintegrate at specific loci in the GIT. Recently this method was further developed and the solution is sprayed under adequate pressure via a needle directly in liquid nitrogen (Yu et al., 2006). The cooling rates in the spray freezing section are dependent on many factors and thus are also very difficult to estimate. However, it was claimed (Franks, 1982; Siuta-Cruce & Goulet, 2001) that maximum cooling rates by freezing in liquid nitrogen are at the order of 300 K/s, considered as upper boundary for the cooling rate (Heller, Carpenter, & Randolph, 1999). To the best of our knowledge the SFD method was not used yet to produce dry powder of probiotic cells. The main objective of this study is to explore and evaluate the application of SFD method to produce dry micro-capsules of highly viable probiotics from LAB strain, Lactobacillus paracasei.

The wall matrix of the micro-capsules in the present study is maltodextrin, a polysaccharide that shows a decreased tendency to bind with the cell membrane, and its potency to penetrate the cell membrane is largely dependent on its molecular weight (Oldenhof, Wolkers, Fonseca, Passot, & Marin, 2005; Taylor & Zografi, 1998). Another matrix component is a disaccharide, trehalose, that act as a protective excipient, known to improve cell viability during freezing (cryoprotectant), freeze-drying (lyophilization), as well as during the storage of the dried bacteria (Crowe, Crowe, Rudolph, Womersley, & Appel, 1985; Leslie et al., 1995; Patist & Zoerb, 2005). 2. Materials and methods 2.1. Materials 2.1.1. Bacterial culture The bacterial strain used in this study was pure freeze-dried culture of L. paracasei LMG P-21380 provided by Probiotical s.r.l, Novara, Italy. Encapsulation aids were trehalose (Cargill, Minneapolis, USA) and maltodextrins (Galam, Kibbutz Maanit, Israel). All other reagents were of analytical grade. 2.2. Methods 2.2.1. Preparation of the probiotic solutions Solutions of maltodextrin and trehalose formulation of various ratios were prepared as follows: distilled water was heated to >90 °C, maltodextrin and trehalose were added, the solution stirred to complete dissolution, and then cooled to room temperature. The dry probiotic cultures were suspended in the formulation solution for at least 1 h, for both bulk freeze drying and spray freeze drying possesses. Different solutions with L. paracasei (0.25% w/v) were prepared for the examination: saline (0.85% NaCl in distilled water); maltodextrin–trehalose solutions with different ratios (1:0, 2:1, 1:1, 1:2 and 0:1, respectively) with total of 20% and 30% w/v solids. In addition, in order to evaluate effect of L. paracasei concentration on its survival, solutions of maltodextrin–trehalose (1:1) 30% w/v with 0.75%, 2%, 5% and 10% w/w bacteria concentration were also prepared. 2.2.2. Freezing methods Samples were frozen by a variety of methods: Freezing: samples (5 ml) were frozen at (A) 18 °C and (B) 80 °C freezers to reach thermal equilibrium (120 min). After freezing, samples were transferred (in liquid nitrogen to prevent melting) to the freeze dryer. (C) Quench frozen: quench freezing involved dipping samples vials (5 ml) in liquid nitrogen (196 °C) long enough to reach thermal equilibrium (3 min). By using this protocol the sample temperature rapidly drops below the expected glass-transition temperature after ice is formed. After the freezing stage the samples were transferred (in liquid nitrogen) to the freeze dryers. (D) Spray freezing: samples were sprayed as fine drops directly into liquid nitrogen (as described below) to provide the fastest cooling rates in this study. While cooling rates in this spray-freezing system are dependent on many factors and are thus difficult to estimate accurately, claims have been made (Franks, 1982) that the maximum cooling rates achievable with liquid nitrogen are of the order of 300 K/s, which could be considered an upper boundary for the cooling rate. To evaluate the survival rate of L. paracasei after freezing at 18 °C and 80 °C, quench freezing in liquid nitrogen, and spray freezing into liquid nitrogen, the frozen samples were collected, thawed at room temperature for an hour, followed by determination of viable bacteria concentration by the methods described below.

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2.2.3. Lyophilization methods Samples were lyophilized using two different freeze dryers: (I) Low temperature drying: the frozen samples were freeze dried using a SECFROID RIN-1362 lyophilizer (Lausanne, Suisse) at a constant controlled shelf temperature of 30 °C and 0.1 mbar for 48 h. (II) Room temperature drying: the frozen samples were freeze dried using a CHRIST, Alpha 1–4, Lo1-m (Martin Crist Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), at constant controlled shelf temperature of 20 °C and 0.05 mbar for 24 h. To evaluate the survival rate of L. paracasei after lyophilization, the dried samples were dissolved in saline solution followed by determination of viable bacteria concentration according to the methods described below. 2.2.4. Spray freeze drying (SFD) In this investigation, spray freeze drying involves spraying drops of the L. paracasei solution directly into liquid nitrogen. The droplet sizes and large temperature cooling gradient DT allow for a rapid cooling and freezing. Solutions were fed by a syringe pump model 351 (Sage instruments, Cambridge, MA, USA), and sprayed through a pneumatic nozzle (diameter 0.41 mm ± 0.02 mm) into liquid nitrogen. The bench top encapsulation unit setup (Nisco Encapsulation Unit, Var J1, SPA – 00336, Zurich, Switzerland) is represented in Fig. 1. The operating parameters that were tested: solution feeding 0.15, 0.3 and 0.8 ml/min; air pressure 1.01 bar; air flow 2.12, 3.08 and 4.52 l/min. After the probiotic solution was sprayed, the drops fell free (constant height of 10 cm) into a liquid nitrogen containing vessel (keeping constant amount of liquid nitrogen), and the instantly freeze. Frozen droplets were collected and transferred in liquid nitrogen to the freeze dryers. In order to evaluate the spraying (the air–liquid interface) effect and to examine possible damage caused by the spraying though the pneumatic nozzle, probiotic solutions with different compositions were sprayed (feed supply 0.3 ml/min) into a test tube using two air supplies 3.08 and 4.53 l/min. Sprayed probiotic solutions were collected and their viability was determined by the methods described below. 2.2.5. Viability of L. paracasei 2.2.5.1. Viability in the probiotic solutions. The viability of the probiotic cells in solutions, feed solution and after spraying or freezing stages, was determined as follows: probiotic samples were spread plated on MRS agar plates (DifcoTM Lactobacilli MRS agar, BD, Sparks, MD, USA), after appropriate 10-fold serial dilutions in saline solution. Viable cells counts were determined after 48 h incubation under anaerobic conditions at 37 °C. Anaerobic jars and gas generating kits (Oxoid Ltd.) were used for anaerobic conditions.

Fig. 1. Schematic description of the Nisco encapsulation unit.

195

Plates containing 20–350 colonies were measured and recorded as colony forming units (CFU) per gram of the product or ml of solution. The percent survival at each of the samples tested was calculated as follows:

Viability ¼ ð100  N=N  Þ;

ð1Þ

where N is the number of bacteria per ml of solution before the process and N is the number of bacteria per ml solution after spraying or freezing stages. 2.2.5.2. Viability in dry samples. Dry samples in four replicates (100–300 mg) were rehydrated at ambient temperature and dissolved in 4.5 ml saline (0.85% NaCl). Dissolved samples were spread plated on MRS agar as described above. The percent survival at each of the samples tested was calculated as follows:

Viability ¼ ð100  N  =N  Þ;

ð2Þ

where N is the number of bacteria per gram of dry matter before drying, and N is the number of bacteria per gram of dry matter in the capsules. 2.2.6. Water activity determination Water activity of micro-capsules at the end of the freeze-drying process was measured by ‘‘Hygropalm Aw1” water activity indicator (Rotronic Instrument Corp., Basserdorf, Germany). 2.2.7. Capsules size Capsules size measurements were carried out as follows: dry capsules produced by SFD process, were placed on graph paper under a microscope (SMZ-168, Motic, China) and photographed with a digital photo camera (Coolpix 995, NIKON, Japan). Capsule diameter was measured with Image J 1.36b software. 2.2.8. Data and statistical analysis All experiments were performed with at least in four replicates, and results hereto are expressed as their means ± standard deviation (SD). Where necessary, the number of repetition is noted in the text. The significance of the differences between groups was tested using t-test analysis. A probability level (p value) of <0.05 was considered to be statistically significant unless stated otherwise. Statistical analysis was performed by the data analysis tool pack of Microsoft Excel 2003 software. 3. Results and discussion When one wish to obtain viable probiotics in a dry state, considerations have to refer to both the freezing stage where damage can occur due the freezing stresses (Leslie et al., 1995), as well to the drying stage that also effect the cells. Spray freeze drying is a relatively new method employed in the pharmaceutical industry, for powder preparation. The idea is to minimize irreversible damage to proteins such as denaturation and aggregation, which occur due to freeze concentration that induce phase separation (Heller, Carpenter, & Randolph, 1997). It was proposed (Heller et al., 1999) to use kinetic strategies to avoid this protein destructive damage. They suggested to increase the cooling rate in the region of 3 °C to 23 °C in order to minimize the residence time of the sensitive formulation in that range. In this temperature range phase separation is both thermodynamically favorable and kinetically realizable. A basic difference between SFD of proteins and probiotics is that probiotic survival can be related mainly to leakage and fusion of their membrane, while protein aggregation and destabilization are of secondary importance (Yu et al., 2006).

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The preliminary experiments in the present work were performed in order to determine the influence of the operational parameters (solution and air flow rate) and solution formulation (Maltodextrins DE5 and DE19 20% w/v and 30% w/v) on the micro-capsule size, and the results are described in Fig. 2. SFD yield spherical particles of controllable size, that maintain their spherical shape and size upon the fast freezing process, and the drying process did not affect their uniform spherical nature. The microcapsule size was distributed between 400 lm and 1800 lm. The solution and air flow rate during spraying have an opposing effect on the final micro-capsules size as can be observed in Fig. 2. High solution feed rate resulted in bigger micro-capsules while higher airflow in smaller micro-capsules size. It was also found that the airflow affected the size of the micro-capsules more than the flow rate of the solution. The smallest size 400 lm was produced with solution and air flow rates of 0.15–0.3 ml/min and 4.5 l/min, respectively.

100

3.2. The spraying stage The effects of atomization and freezing stages were examined independently of the drying step. The first stage in SFD was spraying the solution to form droplets that were collected in aqueous media in order to evaluate the effect of the spraying stage, namely the effect of the air–liquid interface, on the probiotic viability. The viability of L. paracasei in relation to solution composition and various air flow rates is presented in Fig. 3. The spraying stage did not affect the probiotic viability. Thus regardless of formulation or air flow, the viability was practically 100%. This high viability indicates that the shear forces applied to the interface by the flow in the nozzle remains moderate at the air flow rates employed, and that the solution composition as well did not affect the probiotic viability. Low cell concentration in the solution on one hand, and matrix components that can absorb the shear stress on the other hand, prevented any damage during the spraying. 3.3. The freezing stage Samples were frozen by four methods: freezing at 18 °C and 80 °C, quench freezing in liquid nitrogen (196 °C) and spray freezing into liquid nitrogen. In the freezing stage the spray freezing process applies a double stress to the probiotic cells: thermal

Survival [%]

3.1. Optimization of micro-capsule production

80 60 40 20

M

D

D

DE 6

Sa lin

:T

e

(0 .8 5%

Na Cl )

M D re DE DE ha 6 l 6 os :T M e D re [2 :1 DE ha ] lo 6 se :T re [1 :1 ha ] lo se [1 :2 Tr ] eh al os 3. e 08 lit /m in 4. 53 lit /m in

0

M

196

Fig. 3. Effect of solution composition (at air flow 3.08 l/min) and flow rate (MD DE6–trehalose 1:1, 30% w/v) on the survival of L. paracasei during spraying. The error bars represent standard deviation of means (n = 4).

stress and osmotic stress which act simultaneously during the cooling stage (Dumont, Marechal, & Gervais, 2003; Morris, Coulson, & Clarke, 1988; Muldrew & McGann, 1990). In order to prevent the fusion of the membrane of L. paracasei and denaturation of their proteins, in both freezing and drying stages, we used stabilizing additives such as trehalose, and the polysaccharide maltodextrin of various dextrose equivalents (DE) (see the Section 2.2). In the present step of the study the frozen beads were thawed to enumerate the viable bacteria. In the spray freezing process the freezing rate can be controlled by controlling the drops diameter. The freezing rate is mostly affected by the area available for heat transfer. The effect of the spray frozen beads diameter on L. paracasei survival is described in Table 1. It is obvious that the cooling rate of the smaller beads (400 lm) is faster because the surface area of the larger beads (1000– 1400 lm) is 6.25–12.25 times higher than those of 400 lm diameter. However, spray freezing probiotic bacteria in smaller beads (400 lm) did not provide higher survival than in large beads (1000–1400 lm).

Fig. 2. Optimization of SFD process parameters in terms of capsules’ size. (A) Solution feed 0.15 ml/min; (B) solution feed 0.3 ml/min. d Maltodextrin DE19 20% w/v, j maltodextrin DE19 30% w/v, maltodextrin DE5 20% w/v, maltodextrin DE5 30% w/v. The error bars represent standard deviation of means (n P 25).

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D. Semyonov et al. / Food Research International 43 (2010) 193–202 Table 1 Influence of the particle size and the dextrose equivalent on L. paracasei survival after spray freezing and drying stages. Particle size (lm)

1000–1400 lm Spray freezing

Formulation MD MD MD MD a

DE6–trehalose (2:1) DE6–trehalose (1:1) DE19–trehalose (2:1) DE19–trehalose (1:1)

64 ± 8a 89 ± 7 75 ± 11 75 ± 13

400 lm Freeze-drying temperature 20 °C

30 °C

8±1 22 ± 3 36 ± 7 29 ± 5

14 ± 3 40 ± 4 40 ± 9 49 ± 8

Spray freezing

67 ± 9 74 ± 10 70 ± 5

Freeze-drying temperature 20 °C

30 °C

18 ± 3 10 ± 2 17 ± 2

20 ± 3 15 ± 2 25 ± 2

The error represent standard deviation of means (n = 4).

In addition, in order to determine the effect of the freezing rate on the viability of L. paracasei we employed the bulk freeze drying (BFD) method. Probiotic solutions were poured in Petri glass plates preserving the same area and thickness and were frozen at 18 and 80 °C and quench frozen in liquid nitrogen 196 °C. The bulk freezing experiments were performed with the scope to serve as a baseline for comparison with the SFD process. In addition to the freezing rate, effect of solution compositions (maltodextrin and trehalose ratio) and solids concentrations on probiotic survival were evaluated (Fig. 4). A significant increase in probiotic survival is apparent with increasing the trehalose fraction in the matrix. The effect of solids concentration on the probiotic survival was even more prominent. In BFD experiments, at low trehalose concentrations the survival of L. paracasei was the highest at the lowest freezing rate (18 °C). However, it can be seen in Fig. 4 that by increasing trehalose concentration the effect of the freezing rate was reduced. At 30% w/v maltodextrin–trehalose (1:2), the freezing rate did not affect the survival of the probiotics and their survival percentages were the highest. The freezing rate controls the nucleation and growth of ice crystals that are necessary to initiate the freezing process (Maa and Prestrelski, 2000). Slow freezing creates conditions where the ice nuclei grow in larger crystals. Rapid freezing affects mainly the number of the nuclei and not their size, however, fast freezing creates smaller ice crystals than slow freezing (Maa and Prestrelski, 2000). These findings are associated with changes of proteins state, as well as of the cells phospholipid membrane, during the freezedrying process. The combined effect of the freezing rate and additives on the bacterialsurvival needs to be explained. Several hypotheses were

suggested to elucidate the stabilizing mechanism of disaccharides and/or polysaccharides on bacterial survival during freeze drying (Colaco, Sen, Thangavelu, Pinder, & Roser, 1992; Crowe, Crowe, & Carpenter, 1993a, 1993b; Leslie et al., 1995). The vitrification theory is based on the fact that disaccharides as well as polysaccharides form glasses of very high viscosity. The bacteria as well as water are immobilized in the viscous glass preventing any deteriorative reactions to occur due the low mobility. The deteriorative reactions are: damages created by large crystals to the cells membrane and freezing induced unfolding of proteins. Protection of low molecular weight sugars such as trehalose, minimizes water crystal size in the inter-membrane space, thus preventing changes in the physical state of the membrane lipids. This in turn reduce the mechanical stresses in membranes (Koster, Lei, Anderson, Martin, & Bryant, 2000). In the present study, this task is performed by trehalose, a low molecular weight disaccharide (Mw = 342). Maltodextrin, a polysaccharide with a higher molecular weight, the other matrix component, is probably excluded from the inter-lamellar region during drying (Koster, Maddocks, & Bryant, 2003). Thus, the effect of the high molecular weight maltodextrins is mostly due their external glass formation. In the case of vitrification, the glass-transition temperature (Tg) of an additive or a mixture (as maltodextrin and trehalose) is significant. When the Tg is exceeded the glassy material becomes a highly viscous rubbery material and may collapse. The Tg of the excipients mixture can be calculated by using the Gordon–Taylor or Fox equations (Gordon & Taylor, 1952; Schneider, 1997), knowing the weight of each fraction in the mixture and the Tg dependency on moisture content of the excipient. Indeed, it can be seen that the freezing rate effect was prominent

Fig. 4. The combined effect of freezing processes and formulation composition on L. paracasei survival. (A) Various maltodextrin–trehalose ratios and (B) various solids concentration. j Freezing in bulk at 18 °C; N freezing in bulk at 80 °C; d quench freezing in liquid nitrogen 196 °C; and spray freezing in liquid nitrogen. The error bars represent standard deviation of means (n = 4). Increase of trehalose fraction as well as solids concentration accompanied by an increase of the freezing survival (P < 0.05). At low trehalose fraction or low solids concentrations the survival was significantly higher after freezing at low temperature (P < 0.05).

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D. Semyonov et al. / Food Research International 43 (2010) 193–202

when the formulation had a low trehalose fraction (Fig. 4). The results also indicate that the trehalose fraction in the matrix as well as the solids concentration are more significant than the bead size. At highest trehalose concentration, the lowering of the glass transition (Tg) become more significant, and the effect of vitrification on viability decreased. This situation is expressed as a decrease in the survival (Fig. 4A). The water replacement theory suggests that sugars reduce the transition temperature of membranes via replacement of the water between the phospholipid head groups, thus preventing phase transitions and leakage (Patist and Zoerb, 2005). It is also assumed that the sugars bind to the proteins, and serve as water substitute when the hydration shell is disrupted (Allison, Chang, Randolph, & Carpenter, 1999; Carpenter, Arakawa, & Crowe, 1991). In addition, sugars facilitate the formation of a glassy state in the cytoplasm upon dehydration (Oldenhof et al., 2005). Moreover, according to hydration forces explanations, disaccharides in the inter-membrane, space limit an increase in the fluid–gel transition temperature, acting as osmotic and volumetric spacers preventing close approach of the membranes. Indeed, in our experiments the probiotics and the added saccharides and polysaccharide were incubated for an hour before the spray-freezing. This could be accompanied by water removal from the probiotic cells, an event that increase cell viability (Dumont et al., 2003) the intracellular quantity of water before freezing determine the cell resistance to cooling or osmotic stress shock). The results of the present study fit well the conclusion of Dumont et al. (2003) that identified four distinct ranges of cooling rates based on studies on yeasts. The first range corresponds to very slow cooling rates (<5 °C/min) and results in low viability. The second range corresponds to low cooling rates (5–100 °C/ min), that does not damage the cells. The third range corresponds to rapid cooling ratio 100–2000 °C/min. In this case a considerable water outflow causes lethality due to high extracellular osmotic pressure and membrane lipid phase transitions. The fourth range correspond to ultra high cooling rates (>5000 °C), where the cell viability is preserved by high heat flow. In the present experiments the freezing rate was determined by the medium temperature (18, 80 and 196 °C), and solution volume. Since it is very difficult to calculate the exact freezing rate by performing heat transfer balances as function of time, it will be reasonable to explain the obtained viability results by assuming that the freezing rate when the temperature gradient is 18 °C is in the second group proposed by Dumont et al. (2003) while the rates created when the probiotics and their matrix at 80 °C and 196 °C is closer to the third group of Dumont et al. (2003) in a range of decreased viability. It is true that the four groups proposed by Dumont et al. (2003) were related to yeast behavior during cooling, however we observe that the approach can be useful in explaining the effect of cooling rate on LBA survival. As noted, during freezing water crystallizes and the size of the crystals is influenced by the cooling rate. The polysaccharide– disaccharide (maltodextrin–trehalose) solution containing the probiotics is located between these crystals and its concentration increase considerably as the freezing progress (the freeze concentration phenomenon). This concentrated solution exerts an

osmotic pressure that can be represented by the following expression:

ln aw ¼ 

PV w

ð3Þ

RT

where P – the osmotic pressure (MPa); Vw – the partial volume of the water (18  106 m3 mol1); T – the temperature (°K); R – the gas constant (8.314  106 m3 MPa K1 mol1). One can estimate the osmotic pressure P from the water sorption isotherm of trehalose (Iglesias, Chirife, & Buera, 1997). This water sorption isotherm presents data of moisture content m on dry basis (gr water/gr dry solids) vs. water activity (Table 2). Since, the moisture content is the reciprocal of the concentration:

m¼

1 C

ð4Þ

where C is gr solids/gr water, by knowing the specific volume V (cm3/gr) of the mixture solution it is possible to evaluate the solution concentration C (gr solids/ml solution) from the following expression (Mizrahi, Ramon, SilberbergBouhnik, Eichler, & Cohen, 1997):

C ¼

C 1 þ VC

ð5Þ

From this expression dividing by C and replacing 1/C by m (Eq. (4)) the relation between C and m can be obtained:

C ¼

1 mþV

ð6Þ

The value of the specific volume of the trehalose at 25 °C is assumed to be 0.67 (cm3/gr) (Simperler et al., 2007), while the specific volume of water can be assumed to be 1 (cm3/gr). From the adsorption isotherm of trehalose at 25 °C (57), we calculated the osmotic pressure (P) exerted by the solution (C) (Table 2). At aw = 0.8 the trehalose exerts high osmotic pressures of 30 MPa. According to Dumont et al. (2003), at 30 MPa, for cooling rates between 50 and 200 °C/min, the viability was >55%, while slow cooling rates such as 5–7 °C/min can result in very high viability for yeasts (100%) in a water glycerol solution. Thus, the high viability of L. paracasei during the freezing stage at low cooling rates can be related mainly to the high osmotic pressure; the higher the trehalose percentage in the matrix the higher the osmotic pressure exerted. The increase in the survival % of the cells with raising the solid concentration is shown in Fig. 4B. This increase occurs since the probiotic population is immobilized in a larger protective layer of trehalose and maltodextrin at higher solid concentrations. However, the effect of the freezing rate is observed to be similar to what was presented in Fig. 4A, very likely from the same reasons. As one increase the solid concentration, the freezing rate will be lower due to a lower heat transfer and a viscosity increase. Thus at high solid concentration the effect of the freezing rate tend to be less significant, as shown already for proteins during freeze drying (Maa and Prestrelski, 2000). The effect of the Maltodextrins DE6 and DE19 (of different molecular weights) on the probiotic survival is shown in Table 1.

Table 2 The effect of moisture content on the osmotic pressure (MPa) in maltodextrin–trehalose (1:1) formulation. Moisture content – m (gr water/gr dry solids) Water activity – aw Concentration – C (gr solids/ml solution)a Osmotic pressure – P (MPa)a a

0.09 0.33 1.32 152

0.11 0.44 1.29 113

0.12 0.6 1.26 70

0.14 0.7 1.24 49

The osmotic pressure values and concentration C data at 25 °C were calculated from the trehalose sorption isotherm (Iglesias et al., 1997).

0.15 0.8 1.22 30

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The larger bead sizes at the formulation of 1:1 trehalose:maltodextrin DE6 yielded the highest survival percentage, while in other formulations the survival rate was lower by 20% in both bead sizes (400 and 1400 lm) (Table 1). At lower trehalose fraction and the two maltodextrins, DE19 (Mw  9000) (Avaltroni, Bouquerand, & Normand, 2004) and DE6 (Mw  20,000) (Setser & Racette, 1992) the smaller Mw maltodextrin it exerts a greater osmotic pressure. But it cannot be concluded that the lower the Mw of the maltodextrin component the higher is its effect on the survival % of the probiotic cells.

3.4. The drying stage during the freeze-drying process In the primary drying stage, the rate of moisture removal can be enhanced by increasing the temperature of the lyophilizer shelves. It was shown that ice nucleation temperature determines the primary drying rate in lyophilization on samples frozen on a temperature controlled shelf (Searles, Carpenter, & Randolph, 2001). In the present experiments the shelf was kept at a constant temperature along the drying process. The effect of the freeze dryer shelf temperature (20 and 30 °C) during the drying stage on the survival of L. paracasei is shown in Fig. 5.

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As could be expected, when the shelf temperature was 30 °C the survival was higher. Surprisingly, freezing rate (determined by the freezing temperature) affected also the survival during the drying stage (Fig. 6). Probiotics that were bulk-frozen at a higher temperature (18 °C) survived the drying stage better than probiotics that were frozen at liquid nitrogen. As mentioned before, high freezing rate causes more damage to the cells than lower freezing rate. Thus, at the beginning of the drying stage, samples that were quench frozen in liquid nitrogen (fast freezing) had more injured cells causing lower survival percentage in comparison to samples that were frozen at higher temperature (slow freezing). Maltodextrin–trehalose matrix significantly improved freeze drying survival of L. paracasei, and increased viability by increased solids concentration in the probiotic solution. Increase of trehalose concentration (from 0% to 50%) was accompanied by increase in probiotic survival during drying until a maximal viability at the range of 50–100% trehalose (Fig. 5). As expected, the increase in trehalose fraction has a prominent effect on survival. This effect of trehalose during drying is a known phenomenon in anhydrobiosis (Crowe & Crowe, 1986). Trehalose, as well as maltodextrin, has the ability to form glasses, thus increasing the stability of the probiotics due to low mobility in the cells in the glassy media (Aldous, Auffret, & Franks, 1995; Levine & Slade, 1992). In

Fig. 5. The combined effect of shelves temperature (20 and 30 °C) and formulation composition on L. paracasei survival. (A) Lyophilization at 20 °C shelf (effect of trehalose fraction in the matrix); (B) lyophilization at 30 °C shelf (effect of trehalose fraction in the matrix); (C) lyophilization at 20 °C shelf (effect of solids concentration); (D) lyophilization at 30 °C shelf (effect of solids concentration). j Freezing in bulk at 18 °C; N freezing in bulk at 80 °C; d quench freezing in liquid nitrogen 196 °C; and spray freezing in liquid nitrogen. The error bars represent standard deviation of means (n = 4). Increase of trehalose fraction as well as solids concentration accompanied by an increase of the freezing survival (P < 0.01). Spray frozen bacteria survived better after drying at low shelf temperature (30 °C) than drying at higher shelf temperature (20 °C) (P < 0.05).

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Fig. 6. The effect of the freezing temperature on L. paracasei survival during drying stage. (A) Freeze drying at 20 °C shelf. (B) Freeze drying at 30 °C shelf. j Freezing in bulk at 18 °C; and d quench freezing in liquid nitrogen 196 °C. The error bars represent standard deviation of means (n = 4). Samples that were frozen at higher temperature survived the drying stage better than samples that were frozen by quenching in liquid nitrogen (P < 0.05).

addition, below Tg the stability of the cells is preserved, while above Tg the difference between the transient temperature T and Tg, (T–Tg) controls the physical and biological changes. Maltodextrins (DE6 and DE19) that have a higher Tg than trehalose are less effective in protecting the probiotic cells in both freezing and lyophilization stages. It was previously discussed in the discussion section that the creation of an extracellular glass alone is probably not enough to prevent membrane fusion and that direct interaction between the disaccharide, membrane phospholipids, and proteins, is essential for preserving cell viability not only during freezing, but also during dehydration processes (Crowe, Carpenter, Crowe, & Anchordoguy, 1990). Apparently the glass transition and the glassy state cannot provide full protection and stability to probiotic cells during both freezing and drying stages. The explanation for the stability of the cells under freezing and dehydration can be related to the trehalose presence and to additional protection mechanisms which prevail during the freezing as well in the drying stages as presented and demonstrated (Carpenter & Crowe, 1989; Crowe, Carpenter, & Crowe, 1998; Lambruschini, Relini, Ridi, Cordone, & Gliozzi, 2000).

Fig. 7. Effect of probiotic concentration on SFD and BFD encapsulation processes, maltodextrin DE6–trehalose 1:1, solids concentration 30% w/v.d Quench freezing in liquid nitrogen; and spray freezing in liquid nitrogen The error bars represent standard deviation of means (n = 4).

3.5. Probiotic concentration During SFD and BFD the effect of the probiotic cells concentration on the viability was minor at low probiotic concentration (Fig. 7). At higher concentrations the viability was higher for BFD than for SFD. This may explained by the fact that beads formed by SFD have a fix volume, and the partition of the cells into that volume is random. Hence, part of the cells can be located at the bead surface; therefore, the matrix formulation cannot protect them during the freezing and drying stages. At low probiotic concentrations, fewer cells are at the beads surface and the survival in both methods is similar. At higher cells concentration due to the large volume and effects of the protectants that embedded them, BFD provided higher protection than SFD.

4. Conclusions The present study demonstrate that spray freeze drying (SFD) is an appropriate process to generate dried micro-capsules of defined dimensions containing probiotic bacteria, L. paracasei, that retain high viability during the spraying, freezing, and drying stages. While BFD process resulted in slightly higher survival, in order to be further coated, the BFD dried mass requires a secondary process of particle size reduction that reduces significantly the viability of the dried probiotic cells. Thus, the SFD process is advantageous for creating matrix type micro-capsules that can be further coated by employing the fluidized bed industrial method. Coating with additional layers can enhance the protection of the dry probiotic cells during storage and in the GIT. The high viability in both SFD and BFD can be related to the adequate protection of the maltodextrin–trehalose additives combination during the freezing and drying stages. The important effect of the additives hints that the cooling rate, in the present study, is of less significance in both SFD and BFD processes. The shelves temperature during the drying stage affect too the probiotic survival, the lower the temperature (30 °C vs. 20 °C) the higher was their survival. In the freezing stage the concentrated solutions that exert high osmotic pressures on the cells (30–150 MPa) contribute to the high probiotic survival even at low and moderate cooling rates. In the drying stage, the major role in preserving the cells viability is attributed to the low molecular weight disaccharide trehalose that is known to create hydrogen bonds with proteins and the polar head groups of the lipid membrane of the cells preventing struc-

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tural damage during dehydration. Trehalose and maltodextrin mixtures also vitrify and create a glassy state. The polysaccharide maltodextrin of a much higher Mw than trehalose and contribute mainly due to its external vitrification that reduce the mobility of the cells in the glassy state.

Acknowledgment The research was supported by the Israeli Ministry of Industry Commerce and Trade.

References Adhikari, K., Mustapha, A., Grun, I. U., & Fernando, L. (2000). Viability of microencapsulated bifidobacteria in set yogurt during refrigerated storage. Journal of Dairy Science, 83(9), 1946–1951. Aldous, B. J., Auffret, A. D., & Franks, F. (1995). The crystallisation of hydrates from amorphous carbohydrates. Cryo Letters, 16, 181. Allison, S. D., Chang, B., Randolph, T. W., & Carpenter, J. F. (1999). Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Archives of Biochemistry and Biophysics, 365(2), 289–298. Anal, A. K., & Singh, H. (2007). Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends in Food Science and Technology, 18(5), 240–251. Avaltroni, F., Bouquerand, P. E., & Normand, V. (2004). Maltodextrin molecular weight distribution influence on the glass transition temperature and viscosity in aqueous solutions. Carbohydrate Polymers, 58(3), 323–334. Berner, L. A., & O’Donnell, J. A. (1998). Functional foods and health claims legislation: Applications to dairy foods. International Dairy Journal, 8(5–6), 355–362. Carpenter, J., Arakawa, T., & Crowe, J. (1991). Interactions of stabilizing additives with proteins during freeze-drying and freeze-thawing. Development of Biology Standard, 74, 225. Carpenter, J. F., & Crowe, J. H. (1989). An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry, 28(9), 3916–3922. Carvalho, A. S., Silva, J., Ho, P., Teixeira, P., Malcata, F. X., & Gibbs, P. (2004). Relevant factors for the preparation of freeze-dried lactic acid bacteria. International Dairy Journal, 14(10), 835–847. Champagne, C. P., Gardner, N., Brochu, E., & Beaulieu, Y. (1991). The freeze-drying of lactic acid bacterial. A review. Canadian Institute of Food Science and Technology Journal, 24, 118–128. Colaco, C., Sen, S., Thangavelu, M., Pinder, S., & Roser, B. (1992). Extraordinary stability of enzymes dried in trehalose – Simplified molecular-biology. Nature Biotechnology, 10(9), 1007–1011. Costantino, H. R., Firouzabadian, L., Hogeland, K., Wu, C., Beganski, C., Carrasquillo, K. G., Córdova, M., Griebenow, K., Zale, S. E., & Tracy, M. A. (2000). Protein sprayfreeze drying, effect of atomization conditions on particle size and stability. Pharmaceutical Research, 17(11), 1374–1382. Costantino, H. R., Firouzabadian, L., Wu, C. C., Carrasquillo, K. G., Griebenow, K., Zale, S. E., & Tracy, M. A. (2002). Protein spray freeze drying. 2. Effect of formulation variables on particle size and stability. Journal of Pharmaceutical Sciences, 91(2), 388–395. Crowe, J. H., Carpenter, J. F., & Crowe, L. M. (1998). The role of vitrification in anhydrobiosis. Annual Review of Physiology, 60, 73–103. Crowe, J. H., Carpenter, J. F., Crowe, L. M., & Anchordoguy, T. J. (1990). Are freezing and dehydration similar stress vectors – A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology, 27(3), 219–231. Crowe, J. H., & Crowe, L. M. (1986). Stabilization of membranes in anhydrobiotic animals. In C. A. Leopold (Ed.), Membranes, metabolism and dry organisms (pp. 188–209). Ithaca: Cornell University Press. Crowe, J. H., Crowe, L. M., & Carpenter, J. F. (1993a). Preserving dry biomaterials: The water replacement hypothesis, Part 1. Biopharm, 6, 28–32. Crowe, J. H., Crowe, L. M., & Carpenter, J. F. (1993b). Preserving dry biomaterials: The water replacement hypothesis, Part 2. Biopharm, 6, 40–43. Crowe, L. M., Crowe, J. H., Rudolph, A., Womersley, C., & Appel, L. (1985). Preservation of freeze-dried liposomes by trehalose. Archives of Biochemistry and Biophysics, 242(1), 240–247. de Valdez, G. F., Degiori, G. S., Holgado, A. A. P. D., & Oliver, G. (1983). Protective effect of adonitol on lactic-acid bacteria subjected to freeze-drying. Applied and Environmental Microbiology, 45(1), 302–304. Doleyres, Y., & Lacroix, C. (2005). Technologies with free and immobilised cells for probiotic bifidobacteria production and protection. International Dairy Journal, 15(10), 973–988. Dumont, F., Marechal, P. A., & Gervais, P. (2003). Influence of cooling rate on Saccharomyces cerevisiae destruction during freezing: Unexpected viability at ultra-rapid cooling rates. Cryobiology, 46(1), 33–42. Franks, F. (1982). The properties of aqueous solutions at subzero temperatures. In F. Franks (Ed.). Water: A comprehensive treatise (Vol. 7, pp. 15–338). New York: Plenum.

201

Gibson, G. R., & Roberfroid, M. B. (1995). Dietary modulation of the human colonic microbiota – Introducing the concept of prebiotics. Journal of Nutrition, 125(6), 1401–1412. Gordon, M., & Taylor, J. S. (1952). Ideal copolymers and the second order transitions of synthetic rubbers. I: Non-crystalline copolymers. Journal of Applied Chemistry, 2, 493–500. Guarner, F., & Schaafsma, G. J. (1998). Probiotics. International Journal of Food Microbiology, 39(3), 237–238. Hamoudi, L., Goulet, J., & Ratti, C. (2007). Effect of protective agents on the viability of Geotrichum candidum during freeze-drying and storage. Journal of Food Science, 72(2), M45–M49. Heller, M. C., Carpenter, J. F., & Randolph, T. W. (1997). Manipulation of lyophilization-induced phase separation: Implications for pharmaceutical proteins. Biotechnology Progress, 13(5), 590–596. Heller, M. C., Carpenter, J. F., & Randolph, T. W. (1999). Protein formulation and lyophilization cycle design: Prevention of damage due to freeze-concentration induced phase separation. Biotechnology and Bioengineering, 63(2), 166–174. Iglesias, H. A., Chirife, J., & Buera, M. P. (1997). Adsorption isotherm of amorphous trehalose. Journal of the Science of Food and Agriculture, 75(2), 183–186. Kearney, L., Upton, M., & Mcloughlin, A. (1990). Enhancing the viability of Lactobacillus plantarum inoculum by immobilizing the cells in calcium– alginate beads incorporating cryoprotectants. Applied and Environmental Microbiology, 56(10), 3112–3116. Koster, K. L., Lei, Y. P., Anderson, M., Martin, S., & Bryant, G. (2000). Effects of vitrified and nonvitrified sugars on phosphatidylcholine fluid-to-gel phase transitions. Biophysical Journal, 78(4), 1932–1946. Koster, K. L., Maddocks, K. J., & Bryant, G. (2003). Exclusion of maltodextrins from phosphatidylcholine multilayers during dehydration: Effects on membrane phase behaviour. European Biophysics Journal with Biophysics Letters, 32(2), 96–105. Lambruschini, C., Relini, N., Ridi, A., Cordone, L., & Gliozzi, A. (2000). Trehalose interacts with phospholipid polar heads in Langmuir monolayers. Langmuir, 16(12), 5467–5470. Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H., & Crowe, L. M. (1995). Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied and Environmental Microbiology, 61(10), 3592–3597. Levine, H., & Slade, L. (1992). Another view of trehalose for drying and stabilizing biological materials. BioPharm, 5, 36. Maa, Y.-F., & Prestrelski, S. J. (2000). Biopharmaceutical powders particle formation and formulation considerations. Current Pharmaceutical Biotechnology, 1(3), 283–302. Maa, Y. F., Nguyen, P. A., Sweeney, T., Shire, S. J., & Hsu, C. C. (1999). Protein inhalation powders: Spray drying vs spray freeze drying. Pharmaceutical Research, 16(2), 249–254. Maitrot, H., Paquin, C., Lacroix, C., & Champagne, C. P. (1997). Production of concentrated freeze-dried cultures of Bifidobacterium longum in kappacarrageenan locust bean gum gel. Biotechnology Techniques, 11(7), 527–531. Mizrahi, S., Ramon, O., SilberbergBouhnik, M., Eichler, S., & Cohen, Y. (1997). Scaling approach to water sorption isotherms of hydrogels and foods. International Journal of Food Science and Technology, 32(2), 95–105. Morris, G. J., Coulson, G. E., & Clarke, K. J. (1988). Freezing-injury in Saccharomyces cerevisiae – The effect of growth-conditions. Cryobiology, 25(5), 471–482. Muldrew, K., & McGann, L. E. (1990). Mechanisms of intracellular ice formation. Biophysical Journal, 57(3), 525–532. Oldenhof, H., Wolkers, W. F., Fonseca, F., Passot, S. P., & Marin, M. (2005). Effect of sucrose and maltodextrin on the physical properties and survival of air-dried Lactobacillus bulgaricus: An in situ Fourier transform infrared spectroscopy study. Biotechnology Progress, 21(3), 885–892. Patist, A., & Zoerb, H. (2005). Preservation mechanisms of trehalose in food and biosystems. Colloids and Surfaces B: Biointerfaces, 40(2), 107–113. Picot, A., & Lacroix, C. (2003). Effects of micronization on viability and thermotolerance of probiotic freeze-dried cultures. International Dairy Journal, 13(6), 455–462. Sanders, M. E. (1998). Overview of functional foods: Emphasis on probiotic bacteria. International Dairy Journal, 8(5–6), 341–347. Schneider, H. A. (1997). Conformational entropy contributions to the glass temperature of blends of miscible polymers. Journal of Research of the National Institute of Standards and Technology, 102(2), 229–248. Searles, J. A., Carpenter, J. F., & Randolph, T. W. (2001). The ice nucleation temperature determines the primary drying rate of lyophilization for samples frozen on a temperature-controlled shelf. Journal of Pharmaceutical Sciences, 90(7), 860–871. Selmer-Olsen, E., Birkeland, S. E., & Sorhaug, T. (1999). Effect of protective solutes on leakage from and survival of immobilized Lactobacillus subjected to drying, storage and rehydration. Journal of Applied Microbiology, 87(3), 429–437. Setser, C. S., & Racette, W. L. (1992). Macromolecule replacers in food-products. Critical Reviews in Food Science and Nutrition, 32(3), 275–297. Shah, N. P., & Ravula, R. R. (2000). Microencapsulation of probiotic bacteria and their survival in frozen fermented dairy desserts. Australian Journal of Dairy Technology, 55(3), 139–144. Sheu, T. Y., Marshall, R. T., & Heymann, H. (1993). Improving survival of culture bacteria in frozen desserts by microentrapment. Journal of Dairy Science, 76(7), 1902–1907. Simperler, A., Kornherr, A., Chopra, R., Jones, W., Motherwell, W. D. S., & Zifferer, G. (2007). The glass transition temperatures of amorphous trehalose–water mixtures and the mobility of water: An experimental and in silico study. Carbohydrate Research, 342(11), 1470–1479.

202

D. Semyonov et al. / Food Research International 43 (2010) 193–202

Siuta-Cruce, P., & Goulet, J. (2001). Improving probiotic survival rates: Microencapsulation preserves the potency of probiotic microorganisms in food systems. Food Technology, 55, 37–39. Stanton, C., Gardiner, G., Meehan, H., Collins, K., Fitzgerald, G., Lynch, P. B., & Ross, R. P. (2001). Market potential for probiotics. American Journal of Clinical Nutrition, 73(2), 476S–483S. Taylor, L. S., & Zografi, G. (1998). Sugar–polymer hydrogen bond interactions in lyophilized amorphous mixtures. Journal of Pharmaceutical Sciences, 87(12), 1615–1621. Teixeira, P., Castro, H., & Kirby, R. (1996). Evidence of membrane lipid oxidation of spray-dried Lactobacillus bulgaricus during storage. Letters in Applied Microbiology, 22(1), 34–38.

Teixeira, P. C., Castro, M. H., Malcata, F. X., & Kirby, R. M. (1995). Survival of Lactobacillus delbrueckii ssp. bulgaricus following spray–drying. Journal of Dairy Science, 78(5), 1025–1031. Webb, S. D., Cleland, J. L., Carpenter, J. F., & Randolph, T. W. (2002). A new mechanism for decreasing aggregation of recombinant human interferongamma by a surfactant: Slowed dissolution of lyophilized formulations in a solution containing 0.03% polysorbate 20. Journal of Pharmaceutical Sciences, 91(2), 543–558. Yu, Z. S., Johnston, K. P., & Williams, R. O. (2006). Spray freezing into liquid versus spray-freeze drying: Influence of atomization on protein aggregation and biological activity. European Journal of Pharmaceutical Sciences, 27(1), 9–18.