http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, 2014; 31(8): 759–767 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.932026
Microencapsulation of Lactobacillus casei by spray drying Rebeka Cristiane Silva dos Santos1, Leandro Finkler2, and Christine Lamenha Luna Finkler1,2 1
Programa de Po´s-Graduac¸a˜o em Biotecnologia Industrial, Universidade Federal de Pernambuco, Cidade Universita´ria, Recife, PE, Brazil and Centro Acadeˆmico de Vito´ria, Universidade Federal de Pernambuco, Rua do Alto do Reservato´rio, Vito´ria de Santo Anta˜o, PE, Brazil
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
2
Abstract
Keywords
This study evaluates the use of spray drying to produce microparticles of Lactobacillus casei. Microorganism was cultivated in shaken flasks and the microencapsulation process was performed using a laboratory-scale spray dryer. A rotational central composite design was employed to optimise the drying conditions. High cell viability (1.1 1010 CFU/g) was achieved using an inlet air temperature of 70 C and 25% (w/v) of maltodextrin. Microparticles presented values of solubility, wettability, water activity, hygroscopicity and humidity corresponding to 97.03 ± 0.04%, 100% (in 1.16 min), 0.14 ± 0.0, 35.20 g H2O/100 g and 4.80 ± 0.43%, respectively. The microparticles were spherical with a smooth surface and thermally stable. Encapsulation improved the survival of L. casei during storage. After 60 days, the samples stored at 8 C showed viable cell concentrations of 1.0 109 CFU/g.
Experimental planning, maltodextrin, microparticles, probiotic
Introduction The spray drying technique is based on the formation of a powder from a liquid containing a dissolved or suspended solid. The process occurs by atomisation of the liquid suspension, producing droplets that are exposed to hot air in the drying chamber, resulting in evaporation of the liquid phase and formation of dry particles. The dried solid particles are then separated in a cyclone and collected as a powder. This method is used in the food industry to guarantee the stability of a product in soluble powder form (Paramita et al., 2010), avoid risks of chemical and biological degradation, reduce storage costs and obtain a product with specific desired properties, such as instant solubility (Gharsallaoui et al., 2007). The technique can be applied to heat sensitive materials (Lannes and Medeiros, 2003; Filkova´ et al., 2007), primarily involving biologically active compounds or cells (Doherty et al., 2011). One of the main drawbacks of the spray drying technique is a low cell survival rate (Ananta et al., 2005). However, spray drying is a viable way of producing micro-organisms such as lactic acid bacteria and probiotic cultures on an industrial scale. Bacteria belonging to the genus Lactobacillus are the most widely used probiotic supplements (Guarner and Malagelada, 2003). Lactobacillus casei, L. paracasei and L. rhamnosus are extensively employed in the food industry to produce fermented milks as starter cultures in fermentation processes. Lactobacillus casei has applications as a probiotic in the production of lactic acid and in the pharmaceutical industry. There is an extensive body of literature concerning its health-promoting properties, and
Address for correspondence: Christine Lamenha Luna Finkler, Centro Acadeˆmico de Vito´ria, Universidade Federal de Pernambuco, Rua do Alto do Reservato´rio, s/n, 55608-680, Bela Vista, Vito´ria de Santo Anta˜o, PE 55608680, Brazil. Tel: +55 81 35233351. E-mail: chrislluna@ yahoo.com.br
History Received 18 September 2013 Revised 7 May 2014 Accepted 3 June 2014 Published online 29 July 2014
it is one of the most important species for probiotic applications in processed foods. Due to exposure of the cells to high temperatures during the atomisation process, the greatest challenge in the use of spray drying is maintenance of cell viability. However, this difficulty can be mitigated by encapsulation of the active ingredient inside a polymeric matrix (Dziezak, 1998). Microencapsulation by spray drying has been widely used in the food industry for decades (Gouin, 2004). Applications include the encapsulation of flavour components (Baranauskiene et al., 2006), soybean oil (Hogan et al., 2001), fish oil (Drusch, 2006), egg (Banu et al., 2012) and probiotic micro-organisms (Cai and Corke, 2000; Gardiner et al., 2000; Peighambardoust et al., 2011; Silva et al., 2011; Fritzen-Freire et al., 2012; Ivanovska et al., 2012; Anekella and Orsat, 2013). The materials used as encapsulation agents during spray drying include gum Arabic (Ferrari et al., 2012), alginate, chitosan (Malmo et al., 2013), carrageenan (Krishnaiah et al., 2012), gelatin (Lian et al., 2002) and maltodextrin (Matsuno et al, 2002). Maltodextrin is a product obtained from the hydrolysis of starch consisting of units of D-glucose. It is inexpensive, highly soluble and of low hygroscopicity. In a spray drying process, addition of maltodextrin can increase the solid concentration in the feed and reduce the humidity of the powder produced (Anselmo et al., 2006; Phisut, 2012). The main factors that influence the efficiency of microencapsulation are the inlet and outlet air temperatures (Liu et al., 2004) and the type and concentration of the encapsulation agent. Studies designed to optimise a spray drying technique are often conducted empirically; such studies are seldom performed using statistical experimental design for planning experiments and interpreting data. The present study investigated the microencapsulation of L. casei by spray drying, using statistical experimental design to evaluate the influence of inlet air temperature and concentration of the encapsulation agent on the concentration of viable cells at
760
R. C. S. dos Santos et al.
the end of the drying process. The microparticles obtained were characterised according to their physicochemical characteristics, and cell viability was assessed after a storage time of 60 days.
Table 1. Codified levels and actual values of the variables studied in the first experimental design.
Methods
1 2 3 4 5 6 7 8 9 10 11
Materials The lyophilised culture of Lactobacillus casei UFPEDA used in this study was obtained from the culture collection of the Antibiotics Department of the Federal University of Pernambuco, Brazil. Whey was donated by the company Natural da VacaÕ , located in Gravata´ (Pernambuco, Brazil) and was kept frozen. Maltodextrin was supplied by NeoNutriÕ (Poc¸os de Caldas, MG, Brasil). Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
J Microencapsul, 2014; 31(8): 759–767
Fermentation A pre-inoculum was prepared by inoculating the micro-organism into a tube containing 5 ml of sterile saline solution (NaCl, 0.85% w/v) to obtain turbidity equivalent to tube number 0.5 on McFarland’s scale. The inoculum was subsequently prepared using 250 ml Erlenmeyer flasks containing 5 ml of pre-inoculum and 45 ml of De Man, Rogosa and Sharpe (MRS) medium supplemented with 2% (w/v) glucose, pH 6.5. Inoculum cultivation was conducted under stationary conditions, with incubation at 37 C for 12 h. For cell cultivation, experiments were conducted using 500 ml Erlenmeyer flasks containing 200 ml of growth medium based on whey supplemented with 20 g/l glucose. Cell growth was performed for 43 h under the same conditions, using an inoculum concentration of 5% (v/v). This time was established from previous experiments aimed at obtaining the maximum concentration of cells. Microencapsulation by spray drying The spray-drying of L. casei UFPEDA was performed using a laboratory-scale spray dryer (SD-BASIC). The equipment consisted of a spray system, blower, heater, 215 mm 500 mm main chamber and a cyclone. The feed solution (crude cell broth of L. casei with added maltodextrin) was atomised into a vertical drying chamber using a 1.5-mm nozzle at a feed flow rate of 12.5 ml/min. The dried powder was collected in a single cyclone separator. In order to define the conditions for L. casei microencapsulation and maximise the viable cell concentration, two independent variables (inlet air temperature and maltodextrin concentration) were evaluated using a full 22 factorial design, with three central points (level 0) and four axial points (levels ± a, where ¼ 1.4142), totalling 11 experiments (RCCD, rotational central composite design). The tests were performed randomly, and the data were analyzed using StatisticaÕ 8.0 software (Statsoft, Tulsa, OK), with a 95% confidence level. The experimental error was obtained from the mean and standard deviation of the central points. Tables 1 and 2 show the experimental conditions investigated for each experimental design. Microparticles characterisation The microparticles obtained using the selected drying conditions were characterised according to the methods described in the following sections. Humidity The humidity corresponds to the loss in weight suffered by the product when heated in conditions in which water is removed.
Assays
Inlet air temperature ( C) 77.3 112.7 77.3 112.7 70 120 95 95 95 95 95
(1) (+1) (1) (+1) (1.41) (+1.41) (0) (0) (0) (0) (0)
Concentration of maltodextrin (% w/v) 3.8 3.8 17.2 17.2 10.5 10.5 1 20 10.5 10.5 10.5
(1) (1) (+1) (+1) (0) (0) (1.41) (+1.41) (0) (0) (0)
Table 2. Codified levels and actual values of the variables studied in the second experimental design.
Assays 1 2 3 4 5 6 7 8 9 10 11
Inlet air temperature ( C) 52.9 67.1 52.9 67.1 50 70 60 60 60 60 60
(1) (+1) (1) (+1) (1.41) (+1.41) (0) (0) (0) (0) (0)
Concentration of maltodextrin (% w/v) 7.9 7.9 22.1 22.1 15 15 5 25 15 15 15
(1) (1) (+1) (+1) (0) (0) (1.41) (+1.41) (0) (0) (0)
This is an extensive property that depends on the amount of sample. A 5 g portion of the powder was heated for 3 h at 105 C. After cooling in a desiccator, the powder was weighed and the procedure was repeated until a constant weight (cw) was obtained. The humidity (Xp) was calculated using Equation 1. The tests were performed in quadruplicate. 5 1 ð1Þ Xp ¼ cw Solubility The microparticles solubility in water was determined by the maximum percentage (by weight) of the sample that will dissolve in a unit volume of water at certain (usually room) temperature. About 1 g of dry sample was diluted in 100 ml of distilled water. After homogenisation of the suspension at room temperature (28 C), the mixture was centrifuged at 30 000 g for 10 min. About 20 ml samples of the resulting solution were distributed into Petri dishes and dried in a vacuum drying oven at 75 C for 5 h. The percentage solubility was calculated by weight difference (Eastman and Moore, 1984). The tests were performed in quadruplicate. Hygroscopicity Hygroscopicity was determined in order to evaluate the ability of microparticles to absorb water from the surrounding environment. About 2 g portions of the sample were placed in aluminum capsules containing a saturated NaCl solution, under ambient conditions (75% relative humidity, 25 C), until equilibrium was achieved. The hygroscopicity was expressed as the amount of
Microencapsulation of L. casei by spray drying
DOI: 10.3109/02652048.2014.932026
water absorbed per 100 g of powder, on a dry basis (g/100 g). The tests were performed in quadruplicate. Water activity The water activity (aw) represents the ratio of the water vapour pressure of the product to the water vapour pressure of pure water under the same conditions. This is an intensive property that does not depend on the amount of sample and refers to this unbound water. Establishing the water activity of a product assists in predicting its tendency to spoil. The water activity was measured at 25 C using a Pawkit Water Activity Meter instrument (Decagon, WA). The tests were performed in quadruplicate.
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
Wettability The wettability of microparticles aiming to evaluate the ability of solid surface to be wetted when in contact with water. About 2 g portions of sample were placed in 600 ml beakers containing 400 ml of distilled water at 25 C. The time required for all particles to become wetted was determined visually (Magalha˜es Netto, 1997). The tests were performed in quadruplicate. Morphological analysis of microparticles The morphological characteristics of the microparticles were evaluated by using scanning electronic microscope (Aspex, model Express VP) at a magnification of 2500. Capsules size measurements were carried out as follows: dry microparticles were placed on graph paper under a microscope (Shimadzu) and photographed with a digital photo camera (Sony Cyber-shot DSC-WX80, Tokyo, Japan). Medium diameter of the particle size was measured with Image J 1.36b software (NIH, Bethesda, MD). From the cumulative particle distribution curve, we determined the values of d10, d50 and d90. Thermal analysis Glass transition as well as other thermal properties is a key to understand how drying conditions affect physical changes of materials. Thermal analysis was performed by differential scanning calorimetry (DSC) and differential thermal analysis (DTA) using a Shimadzu thermoanalyzer (Tokyo, Japan). All measurements employed a linear heating rate of 10 C/min, and an empty pan was used as a reference material. Portions of sample (10 mg) were heated linearly to 250 C. The Tg (glass transition temperature) was defined as the midpoint value of the change in specific heat, observed as an endothermic shift in the baseline of the DSC signal. Storage test Microparticles of L. casei were stored in penicillin tubes that were hermetically sealed and placed in a desiccator. Experiments were performed in triplicate, and probiotic survival was evaluated by performing viable cell counts after 7, 14, 28 and 60 days of storage at temperatures of 30 C, 4 C and 8 C. Viable cell counts Portions (0.1 g) of the encapsulated probiotic cells were solubilised in a solution of 0.1 M sodium citrate (pH 6.5) and serially diluted in tubes containing sterile saline solution (NaCl, 0.85% w/v). A 10 ml volume of the samples was plated on MRS agar supplemented with 2% (w/v) glucose, using the pour plate technique. The plates were incubated at 37 C for 48–72 h and results of viable and cultivable cell counts were expressed by CFU/g.
761
Results Figure 1(A and B) shows the response surface and the contour curve, respectively, for the concentration of viable cells during the first experimental design. The highest concentration of viable cells (1.5 108 CFU/g) was obtained for an inlet air temperature of 95 C and 20% (w/v) maltodextrin. The results indicated that the optimum ranges for maximisation of the concentration of viable cells covered the entire temperature range employed and maltodextrin concentrations above 18% (w/v). The Pareto graph (Figure 1C) revealed that the maltodextrin concentration was the most significant variable, with positive estimated effects of 34.9 and 30.4, demonstrating that the presence of a higher molecular weight carbohydrate (in this case maltodextrin) contributed to stability of the system (Ferrari et al., 2012). Temperature showed a statistically significant negative effect (8.097), indicating that higher viable cell counts were obtained at lower temperatures. The maximum viable cell concentration was not achieved using the first experimental design. A new experimental design was therefore performed using lower temperatures (50–70 C) and maltodextrin concentrations between 5% and 25% (w/v). Figure 2(A and B) shows the response surface and the contour curve, respectively, for the concentration of viable cells obtained using the second experimental design. In this case, it can be seen that a maximum concentration of viable cells was achieved. The critical values of the model for maximum response were a maltodextrin concentration of 8.75% (w/v) and a temperature of 59.3 C, giving a theoretical maximum viable cell concentration of 1.97 109 CFU/g. The predictive equation (Equation 2) contains the statistically significant variables, where X is the concentration of viable cells, T is the temperature and A is the adjuvant concentration. X ¼ 2:63 1010 7:86 106 T 2 þ 1:14 108 A 4:14 106 A
ð2Þ
The Pareto graph (Figure 2C) showed that the linear and quadratic terms of the maltodextrin concentration were significant, as well as the quadratic term of the temperature (p50.05). In all cases, the negative estimated effects indicated that lower temperatures and lower adjuvant concentrations resulted in higher concentrations of viable cells. Figure 3(A and B) shows typical DTA, TGA and DSC curves obtained for the thermal analysis of the microparticles and maltodextrin, respectively. In both cases, the DTA curves presented endothermic peaks at temperatures between 50 C and 120 C, which corresponded to water evaporation. The microparticles presented a shift in the heat capacity at around 160 C, compared to maltodextrin, and an additional endothermic peak just after 200 C. This was probably the temperature at which the formation of carbonaceous products (ash) from maltodextrin was initiated. The stability of the powder was evaluated over a storage period of 60 days. All the tests began at an initial count of 1.1 1010 CFU/g. The sample stored at 30 C only showed cell viability in the initial count. After 60 days, the samples stored at 8 C and 4 C showed viable cell concentrations of 1.0 109 and 3.0 108 CFU/g, respectively (Figure 4). The microparticles showed values of solubility, wettability, water activity, hygroscopicity and humidity corresponding to 97.03 ± 0.04%, 100% (in 1.16 min), 0.14 ± 0.0, 35.20 g H2O/100 g and 4.80 ± 0.43%, respectively. Figure 5 shows the morphological characteristics of the microparticles containing L. casei. Spray-dried particles were spherical with slightly roughened surfaces. The spraying system produced a very fine powder and particles presented a medium
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
762
R. C. S. dos Santos et al.
J Microencapsul, 2014; 31(8): 759–767
Figure 1. Response surface (A), contour curvature (B) and Pareto diagram (C) as a function of the inlet air temperature and concentration of maltodextrin for the first experimental design.
diameter of 14 mm. Values of d10, d50 and d90 were 1, 11 and 27.5 mm, respectively.
Discussion The inlet air temperature directly influences the outlet air temperature, which is a critical variable that affects microorganism survival (Teixeira et al., 1995). The experimental data indicated that the difference between the inlet and outlet air temperatures was around 20 ± 5 C. Previous work has demonstrated the negative effect of a high outlet air temperature on the viability of bacteria. For example, Gardiner et al. (2000) investigated the survival of L. paracasei NFBC 338 after spray drying, and obtained a survival rate of 97% when an outlet air temperature of 70 C was used. However, when a temperature of 120 C was employed, the survival rate was zero. Drying performed under the optimal conditions suggested by the second experimental design (maltodextrin concentration of 8.75% (w/v) and temperature of 59.3 C) resulted in a product with inadequate characteristics; the material formed was hygroscopic and showed a tendency to agglomerate and adhere to the walls of the equipment, which could have been due to the low maltodextrin concentration employed.
The quality of the final product obtained after spray drying is affected by the concentration of the adjuvant used during drying, and an increased quantity of adjuvant can help to produce a powder with satisfactory macroscopic characteristics. According to Roos (2010), less hygroscopic powders are obtained using higher maltodextrin concentrations. High hygroscopicity affects the efficiency of the process and compromises the microbiological stability of the final product, and maltodextrin acts to strengthen the glassy matrix during the drying process (Cha´vez and Ledeboer, 2007). Based on these results, drying was carried out using the extreme points of the second experimental design (70 C and 25% (w/v) of maltodextrin). The fine-grained powder obtained was easily collected from the walls of the cyclone and collection vessel, and presented a viable cell concentration of 1.1 1010 CFU/g. The high concentration of viable cells obtained could be explained by the incorporation of a greater amount of maltodextrin, as well as a lower inlet air temperature and a consequent decrease in the outlet air temperature. Higher concentrations of the encapsulation agent normally provide bacterial cells with greater protection against damage. In a previous study, it was shown that during drying of
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
DOI: 10.3109/02652048.2014.932026
Microencapsulation of L. casei by spray drying
763
Figure 2. Response surface (A), contour curvature (B) and Pareto diagram (C) as a function of the inlet air temperature and concentration of maltodextrin for the second experimental design.
L. fermentum, cell viability increased by about 10% when a higher adjuvant concentration was used (Cai et al., 2012). The resistance of probiotic bacteria is also determined by the outlet air temperature (Rokka and Rantamaki, 2010). Thermal stress directly affects the enzymatic functions required for maintenance of cell viability, and the greatest challenge in producing a probiotic powder is the high temperature used during the process (Jankovic et al., 2010). High temperatures can lead to the formation of pores in membranes and cause leakage of intracellular substances (Gardiner et al., 2000; Corcoran et al., 2004) as well as protein denaturation (Meng et al., 2008). Sunny-Roberts and Knorr (2009) investigated the survival of L. rhamnosus after a spray drying process and found that the survival rate was inversely proportional to the outlet air temperature. Use of a lower inlet air temperature and a higher maltodextrin concentration therefore favoured the cell viability of L. casei. The microparticles showed a solubility of 97.03 ± 0.04% and a high degree of wettability (100% in 1.16 min). Powders that have low wettability tend to form lumps during addition to water and mixing (Maia and Golgher, 1983). The high wettability was probably assisted by the low temperature used in the drying process, since high temperatures can result in the formation of a rigid coating on the microcapsule surface, preventing the diffusion of water molecules and reducing solubilisation of the
powder (Chegeni and Ghobadian, 2005). The high solubility was due to the influence of maltodextrin, which reduced the stickiness of the powder and increased the surface area of the particles in contact with water during the rehydration (Goula and Adamopoulos, 2005). Characterisation of a powdered product in terms of its water activity is important because this parameter affects the processing and handling properties, as well as the stability of the powder (Roos, 2010). In order to maintain the viability of a probiotic organism, the water activity should be as low as possible (Santivarangkna et al., 2008). The mortality of micro-organisms during storage can increase at higher aw values, due to the growth of other microbes as well as undesirable chemical reactions (Wang et al., 2004; Cha´vez and Ledeboer, 2007; Ying et al., 2010). Dried probiotic foods are therefore more stable because they have a lower aw, which enhances durability and reduces storage costs (Kearney et al., 2009). Low molecular mobility of water contributes to the stabilisation of biological systems during longterm storage (Buitink et al., 2000), and satisfactory survival of probiotics during storage requires an aw value below 0.3 (Cha´vez and Ledeboer, 2007). The aw should be between 0.11 and 0.23 for most species of Lactobacillus (Kumar and Mishra, 2004; Koc et al., 2010). The powder obtained in this study had a water activity of 0.14 ± 0.0,
764
R. C. S. dos Santos et al.
J Microencapsul, 2014; 31(8): 759–767
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
Figure 3. Values of TGA, DSC and DTA for microparticles of L. casei (A) and maltodextrin (B) (L. casei microparticles by spray drying at 70 C and maltodextrin concentration of 25% (w/v).
which was considered to be satisfactory for a powdered L. casei probiotic. A hygroscopicity value of 35.20 g H2O/100 g was obtained. The use of maltodextrin during drying can reduce hygroscopicity by around 50%, compared to samples without addition of this agent (Cai and Corke, 2000). The drying temperature also has a significant effect on the final properties of the powder, with lower temperatures resulting in less hygroscopic particles (Tonon et al., 2008). Another factor related to the hygroscopicity is humidity. Particles with low humidity content are more hygroscopic due to their greater ability to absorb moisture from the environment, which is related to the vapour pressure gradient between the material and the surrounding atmosphere. In this study, the microparticles showed a humidity of 4.80 ± 0.43%. This relatively low humidity could be explained by the concentration of maltodextrin employed. In other work, it was demonstrated that during the drying of L. kefir, bacterial survival was significantly higher under conditions of low humidity
(Golowczyc et al., 2010), while an outlet air temperature of around 60 C resulted in a powder with a moisture content of less than 10%, whose biological activity was maintained (Riveros et al., 2009). The thermogravimetric signal revealed a significant loss of mass from the microparticles, and from 160 C the sample was rapidly degraded during heating. In the case of maltodextrin, the mass loss occurred gradually, with onset of sample degradation after 225 C. Powdered food products containing amorphous carbohydrates undergo physical changes that are related to the glass transition temperature (Tg), such as stickiness and compaction during storage (Roos, 2010). The Tg serves as a benchmark to characterise the thermal stability of a product; below this temperature, the material is more stable. The DSC curve for the microparticles showed a Tg of around 56 C, revealed by a baseline shift; the biological activity of the probiotic powder should therefore be preserved below this temperature. The DSC curve showed two second-order
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
DOI: 10.3109/02652048.2014.932026
Figure 4. Viable cell counting of L. casei microparticles for 60 days.
Microencapsulation of L. casei by spray drying
765
agreement with the observation of sample degradation from 200 C. In relation to stability during storage (shelf life), for foods containing probiotics, the product should have an average shelf life of between 15 and 30 days, with 4106 CFU/mL remaining at the end of the period (Trabulsi and Sampaio, 2000). Teanpaisan et al. (2012) demonstrated that the maximum cell viability of L. paracasei SD1 during storage for 6 months occurred at 4 C, while survival rates were close to 0% when storage temperature 25 C was used. Hsiao et al. (2004) also reported that the storage of dried probiotic cultures in glass flasks at low temperature (4 C) increased the useful life of the product. The available evidence therefore indicates that storage at a low temperature favours maintenance of the viability of probiotic samples dried using spray drying. Another factor that assists the retention of cell viability during storage is the use of glass containers, which have low permeability and minimise the ingress of oxygen (Shah, 2000). Under favourable storage conditions, probiotics dried by spray drying should be stable for at least 30 days (Gardiner et al., 2000; Wang et al., 2004; Kearney et al., 2009). The morphological analysis demonstrated that some of the microparticles presented slight surface roughness, and there were a few structures that were not encapsulated. The size range was typical of particles produced by spray drying. Similar morphological characteristics were described by Tonon et al. (2011). The correlation between microcapsule size and stability is not completely understood, although the existence of an optimum size could be related to decreased stability when higher levels of imperfections are present.
Conclusions
Figure 5. Scanning electronic microscopy (Aspex, model Express VP) of L. casei microparticles (2500 magnification).
transformations. After 140 C, there was glass transition of the amorphous phase contained in the microparticles and fusion of the crystalline phase, shown by two consecutive drops of the curve, which could have been due to the presence of unencapsulated biological material. The endothermic peak at 140 C, corresponding to the melting heat of the microparticles during the phase change, showed that the microparticles had a good resistance to high temperatures. The onset of oxidative degradation was apparent above 200 C, as well as the presence of interferences (false peaks) due to expansion of the material. The results indicated that microcapsule decomposition began at around 160 C, in agreement with earlier findings (Haines, 1995). The DSC results for maltodextrin revealed an endothermic peak at 175 C, with onset of decomposition occurring at around 215 C, indicating that there was no formation of other material until this temperature was reached. As also seen for the microparticles, decomposition occurred at temperatures above 200 C. According to Elnaggar et al. (2010), heating of maltodextrin to 220 C results in carbonisation without fusion, in
Lactobacillus casei loaded microparticles were prepared by spraydrying technique. Experimental design was employed to determine the best operating ranges in terms of the inlet air temperature and the maltodextrin concentration. The drying condition selected was at the extreme point of the second experimental design (70 C and 25% w/v of maltodextrin), which resulted in formation of a powder with good macroscopic characteristics and a high concentration of viable cells (41010 CFU/g). The microparticles were homogeneous spherical particles, with slightly roughened surfaces, and were satisfactory in terms of their wettability, solubility, water activity, hygroscopicity and humidity. During storage for 60 days, the cells remained viable at temperatures of 8 and 4 C, with viable cell concentrations of 4108 CFU/g. Thermal analysis indicated that the glass transition temperature (Tg) was higher than any anticipated storage temperature. It could therefore be assumed that the bacteria were stored in the glassy state. The results demonstrated that microencapsulation of L. casei by spray drying, using maltodextrin as an encapsulation agent, offers a viable alternative technique for the production of probiotic powders used as food ingredients.
Acknowledgments Gratitude must go to Dr Luciano Bueno (Rural Federal University of Pernambuco, Brazil) for technical assistance during the thermal analysis, and Rafael Albuquerque for the assistance during microscopic analysis.
Declaration of interest This research was supported by Fundac¸a˜o de Amparo a` Cieˆncia e Tecnologia do Estado de Pernambuco – FACEPE (IBPG-0797-3.06/10).
766
R. C. S. dos Santos et al.
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
References Ananta E, Volkert M, Knorr, D. Cellular injuries and storage stability of spray-dried Lactobacillus rhamnosus GG. Int Dairy J, 2005;15: 399–409. Anekella K, Orsat V. Optimization of microencapsulation of probiotics in raspberry juice by spray drying. Lwt – Food Sci Technol, 2013;50: 17–25. Anselmo GCS, Eduardo MRM, Cavalcanti M, Arruda PC, Coelho M. Determination of hygroscopicity of caja powder through spray drying. J Biol Sci Earth, 2006;6:58–65. ¨ zlu¨han G, Melike S, Figen K, Gonca S, Neriman B. Banu K, Mehmet K, O Effects of formulation on stability of spray-dried egg. Drying Technol Int J, 2012;30:63–71. Baranauskiene R, Venskutonis PR, Dewettinck K, Verhe R. Properties of oregano (Origanumvulgare L.), citronella (Cymbopogonnardus G.) and marjoram (Majoranahortensis L.) flavors encapsulated into milk protein-based matrices. Food Res Int, 2006;39:413–25. Buitink J, Van Den Dries IJ, Hoekstra FA, Alberda M, Hemminga MA. High critical temperature above Tg may contribute to the stability of biological systems. Biophys J, 2000;79:1119–28. Cai CJ, Thaler B, Liu DW, Wang JJ, Qiao SY. Optimization of spray-drying workflow as a method for preparing concentrated cultures of Lactobacillus fermentum. J Anim Vet Adv, 2012;11: 2769–74. Cai YZ, Corke H. Production and properties of spray-dried Amaranthus betacyanin pigments. J Food Sci, 2000;65:1248–52. Cha´vez BE, Ledeboer AM. Drying of probiotics: Optimization of formulation and process to enhance storage survival. Drying Technol, 2007;25:1193–201. Chegeni GR, Ghobadian B. Effects of spray drying conditions on physical properties of orange juice powder. Drying Technol, 2005;23: 657–68. Corcoran BM, Ross RP, Fitzgerald GF, Stanton C. Comparative survival of probiotic Lactobacilli spray-dried in the presence of prebiotic substances. J Appl Microbiol, 2004;96:1024–39. Doherty SB, Gee RP, Stanton C, Fitzgerald GF, Brodkorb A. Development and characterisation of whey protein micro-beads as potential matrices for probiotic protection. Food Hydrocolloids, 2011; 25:1604–17. Drusch S. Sugar beet pectin: A novel emulsifying wall component for microencapsulation of lipophilic food ingredients by spray-drying. Food Hydrocolloids, 2006;21:1223–8. Dziezak JD. Microencapsulation and encapsulated ingredients. Food Technol, 1998;42:136–51. Eastman JE, Moore CO. 1984. Cold-water soluble granular starch for gelled food composition. Patent US 4.465.702 A. Elnaggar YS, El-Massik MA, Abdallah OY, Ebian AE. Maltodextrin: A novel excipient used in sugar-based orally disintegrating tablets and phase transition process. Am Association Pharm Scientists, 2010;11: 645–51. Ferrari CC, Germer SPM, Alvim ID, Vissotto FZ, Aguirre JM. Influence of carrier agents on the physicochemical properties of blackberry powder produced by spray drying. Int J Food Sci Technol, 2012;47: 1237–45. Filkova´ I, Huang LX, Mujumdar AS. 2007. Industrial spray drying systems. In: Mujumdar AS, ed. Handbook of industrial drying. London: CRC, pp. 215–56. Fritzen-Freire CB, Prudencio ES, Amboni RDMC, Pinto SS, Negra˜o-Murakami AN, Murakami FS. Microencapsulation of Bifidobacteria by spray drying in the presence of prebiotics. Food Res Int, 2012;45:306–12. Gardiner GE, O’Sullivan E, Kelly J, Auty MAE, Fitzgerald GF, Collins JK, Ross RP, Stanton C. Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray drying. Appl Environ Microbiol, 2000;66:2605–12. Gharsallaoui A, Roudaut G, Chambin O, Voilley A, Saurel R. Applications of spray-drying in microencapsulation of food ingredients: An overview. Food Res Int, 2007;40:1107–21. Golowczyc MA, Gerez CL, Silva J, Abraham AG, De Antoni, GL, Teixieira, P. Survival of spray-dried Lactobacillus kefir is affected by different protectants and storage conditions. Bioproc Biol Eng Storage Cond, 2010;33:681–6. Gouin S. Microencapsulation: Industrial appraisal of existing technologies and trends. Trends Food Sci Technol, 2004;15:330–47.
J Microencapsul, 2014; 31(8): 759–767
Goula AM, Adamopoulos KG. Spray drying of tomato pulp in dehumidified air: II. The effect on powder properties. J Food Eng, 2005;66:35–42. Guarner F, Malagelada JR. 2003. Gut flora in health and disease. London: The Lancet. Haines PJ. 1995. Thermal methods of analysis: Principles, applications and problems. London: Blackie Academic. Hogan SA, Mcnamee BF, O’Riordan ED, O’Sullivan M. Microencapsulation properties of sodium caseinate. J Agric Food Chem, 2001;49:1934–8. Hsiao HC, Lian WC, Chou CC. Effect of packaging conditions and temperature on viability of microencapsulated Bifidobacteria during storage. J Sci Food Agric, 2004;84:134–9. Ivanovska TP, Petrusˇevska-Tozi L, Kostoska MD, Geskovski N, Grozdanov A, Stain C, Stafilov T, Mladenovska K. Microencapsulation of Lactobacillus casei in chitosan-Ca-alginate microparticles using spray-drying method. Macedonian J Chem Chem Eng, 2012;31:115–23. Jankovic I, Sybesma W, Phothirath P, Ananta E, Mercenier A. Application of probiotics in food products – Challenges and new approaches. Curr Opinion Biotechnol, 2010;21:175–81. Kearney N, Meng XC, Stanton C, Kelly J, Fitzgerald GF, Ross RP. Development of a spray dried probiotic yoghurt containing Lactobacillus paracasei NFBC 338. Int Dairy J, 2009;19:684–9. Koc B, Yilmazer MS, Balkir P, Ertekin FK. Spray drying of yogurt: Optimization of process conditions for improving viability and other quality attributes. Drying Technol, 2010;28:495–507. Krishnaiah D, Sarbatly R, Nithyanandam R. Microencapsulation of Morinda citrifolia L. extract by spray-drying. Chem Eng Res Design, 2012;90:622–32. Kumar P, Mishra HN. Yoghurt powder – A review of process technology, storage and utilization. Food Bioproducts Process, 2004;82:133–42. Lannes SCS, Medeiros ML. Cupuassu chocolate drink powder processed by spray-dryer. Brazilian J Sci Pharm, 2003;39:115–23. Lian WC, Hsiao HC, Chou CC. Survival of Bifidobacteria after spraydrying. Int J Food Microbiol, 2002;74:79–86. Liu Z, Zhou J, Zeng Y, Ouyang X. The enhancement and encapsulation of Agaricus bisporus flavor. J Food Eng, 2004;65:391–6. Magalha˜es Netto F. 1997. Influence of water activity on the glass temperature. In: Germer DCP, Jardim SPM, eds. Water activity in foods. Campinas: ITAL, pp. 4–14. Maia ABR, Golgher M. Parameters for assessing the quality of reconstitution of dehydrated milk powder spray dryer (‘‘spray-drier’’). Boletim SBCTA, 1983;17:235–54. Malmo C, Storia AL, Mauriello G. Microencapsulation of Lactobacillus reuteri DSM 17938 cells coated in alginate beads with chitosan by spray drying to use as a probiotic cell in a chocolate souffle´. Food Bioprocess Technol, 2013;6:795–805. Matsuno R, Minemoto K, Hakamata S, Adachi S. Oxidation of linoleic acid encapsulated with gum Arabic or maltodextrin by spray-drying. J Microencapsulation, 2002;19:181–9. Meng XC, Stanton C, Fitzgerald GF, Daly C, Ross RP. Anhydrobiotics: The challenges of drying probiotic cultures. Food Chem, 2008;106: 1406–16. Paramita V, Iida K, Yoshii H, Furuta T. Effect of additives on the morphology of spray-dried powder. Drying Technol, 2010;28:323–9. Peighambardoust SH, GolshanTafti A, Hesari J. Application of spray drying for preservation of lactic acid starter cultures: A review. Trends Food Sci Technol, 2011;22:215–24. Phisut N. Spray drying technique of fruit juice powder: Some factors influencing the properties of product. Int Food Res J, 2012;19: 1297–306. Riveros B, Ferreira J, Borquez R. Spray drying of a vaginal probiotic strain of Lactobacillus acidophilus. Drying Technol, 2009;27:123–32. Rokka S, Rantamaki P. Protecting probiotic bacteria by microencapsulation: Challenges for industrial applications. Eur Food Res Technol, 2010;231:1–12. Roos YH. Glass transition temperature and its relevance in food processing. Ann Rev Food Sci Technol, 2010;1:469–96. Santivarangkna C, Kulozik U, Foerst P. Inactivation mechanisms of lactic acid starter cultures preserved by drying processes. J Appl Microbiol, 2008;105:1–13. Shah NP. Probiotic bacteria: Selective enumeration and survival in dairy foods. J Dairy Sci, 2000;83:894–907. Silva J, Freixo R, Gibbs P, Teixeira P. Spray-drying for the production of dried cultures. Int J Dairy Technol, 2011;64:321–35.
DOI: 10.3109/02652048.2014.932026
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
Sunny-Roberts EO, Knorr D. The protective effect of monosodium glutamate on survival of Lactobacillus rhamnosus GG and Lactobacillus rhamnosus E-97800 strains during spray-drying and storage in trehalose-containing powders. Int Dairy J, 2009;19:209–14. Teanpaisan R, Chooruk A, Wannun A, Wichienchot S, Piwat S. Survival rates of human-derived probiotic Lactobacillus paracasei SD1 in milk powder using spray drying. J Sci Technol, 2012;34:241–5. Teixeira P, Castro H, Kirby R. Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus. J Appl Microbiol, 1995;78:456–62. Tonon RV, Grosso CRF, Hubinger MD. Influence of emulsion composition and inlet air temperature on the microencapsulation of flaxseed oil by spray drying. Food Res Int, 2011;44:282–9.
Microencapsulation of L. casei by spray drying
767
Tonon RV, Brabet C, Hubinger MD. Influence of process conditions on the physicochemical properties of ac¸ai (Euterpe oleraceae mart.) powder produced by spray drying. J Food Eng, 2008;88:411–18. Trabulsi LR, Sampaio MMSC. 2000. Probio´ticos, prebio´ticos e simbio´ticos. In: Trabulsi LR, Carneiro-Sampaio MMS, eds. Os probio´ticos e a Sau´de Infantil. Sa˜o Paulo: Nestle´, pp. 15. Wang YC, Yu RC, Chou CC. Viability of lactic acid bacteria and Bifidobacteria in fermented soymilk after drying, subsequent rehydration and storage. Int J Food Microbiol, 2004;93:209–17. Ying DY, Phoon MC, Sanquansri L, Weerakkody R, Burgar I, Augustin MA. Microencapsulated Lactobacillus rhamnosus GG powders: Relationship of powder physical properties to probiotic survival during storage. J Food Sci, 2010;75:588–95.
Microencapsulation of Lactobacillus casei by spray drying Rebeka Cristiane Silva dos Santos1, Leandro Finkler2, and Christine Lamenha Luna Finkler1,2 1
Programa de Po´s-Graduac¸a˜o em Biotecnologia Industrial, Universidade Federal de Pernambuco, Cidade Universita´ria, Recife, PE, Brazil and Centro Acadeˆmico de Vito´ria, Universidade Federal de Pernambuco, Rua do Alto do Reservato´rio, Vito´ria de Santo Anta˜o, PE, Brazil
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
2
Abstract
Keywords
This study evaluates the use of spray drying to produce microparticles of Lactobacillus casei. Microorganism was cultivated in shaken flasks and the microencapsulation process was performed using a laboratory-scale spray dryer. A rotational central composite design was employed to optimise the drying conditions. High cell viability (1.1 1010 CFU/g) was achieved using an inlet air temperature of 70 C and 25% (w/v) of maltodextrin. Microparticles presented values of solubility, wettability, water activity, hygroscopicity and humidity corresponding to 97.03 ± 0.04%, 100% (in 1.16 min), 0.14 ± 0.0, 35.20 g H2O/100 g and 4.80 ± 0.43%, respectively. The microparticles were spherical with a smooth surface and thermally stable. Encapsulation improved the survival of L. casei during storage. After 60 days, the samples stored at 8 C showed viable cell concentrations of 1.0 109 CFU/g.
Experimental planning, maltodextrin, microparticles, probiotic
Introduction The spray drying technique is based on the formation of a powder from a liquid containing a dissolved or suspended solid. The process occurs by atomisation of the liquid suspension, producing droplets that are exposed to hot air in the drying chamber, resulting in evaporation of the liquid phase and formation of dry particles. The dried solid particles are then separated in a cyclone and collected as a powder. This method is used in the food industry to guarantee the stability of a product in soluble powder form (Paramita et al., 2010), avoid risks of chemical and biological degradation, reduce storage costs and obtain a product with specific desired properties, such as instant solubility (Gharsallaoui et al., 2007). The technique can be applied to heat sensitive materials (Lannes and Medeiros, 2003; Filkova´ et al., 2007), primarily involving biologically active compounds or cells (Doherty et al., 2011). One of the main drawbacks of the spray drying technique is a low cell survival rate (Ananta et al., 2005). However, spray drying is a viable way of producing micro-organisms such as lactic acid bacteria and probiotic cultures on an industrial scale. Bacteria belonging to the genus Lactobacillus are the most widely used probiotic supplements (Guarner and Malagelada, 2003). Lactobacillus casei, L. paracasei and L. rhamnosus are extensively employed in the food industry to produce fermented milks as starter cultures in fermentation processes. Lactobacillus casei has applications as a probiotic in the production of lactic acid and in the pharmaceutical industry. There is an extensive body of literature concerning its health-promoting properties, and
Address for correspondence: Christine Lamenha Luna Finkler, Centro Acadeˆmico de Vito´ria, Universidade Federal de Pernambuco, Rua do Alto do Reservato´rio, s/n, 55608-680, Bela Vista, Vito´ria de Santo Anta˜o, PE 55608680, Brazil. Tel: +55 81 35233351. E-mail: chrislluna@ yahoo.com.br
History Received 18 September 2013 Revised 7 May 2014 Accepted 3 June 2014 Published online 29 July 2014
it is one of the most important species for probiotic applications in processed foods. Due to exposure of the cells to high temperatures during the atomisation process, the greatest challenge in the use of spray drying is maintenance of cell viability. However, this difficulty can be mitigated by encapsulation of the active ingredient inside a polymeric matrix (Dziezak, 1998). Microencapsulation by spray drying has been widely used in the food industry for decades (Gouin, 2004). Applications include the encapsulation of flavour components (Baranauskiene et al., 2006), soybean oil (Hogan et al., 2001), fish oil (Drusch, 2006), egg (Banu et al., 2012) and probiotic micro-organisms (Cai and Corke, 2000; Gardiner et al., 2000; Peighambardoust et al., 2011; Silva et al., 2011; Fritzen-Freire et al., 2012; Ivanovska et al., 2012; Anekella and Orsat, 2013). The materials used as encapsulation agents during spray drying include gum Arabic (Ferrari et al., 2012), alginate, chitosan (Malmo et al., 2013), carrageenan (Krishnaiah et al., 2012), gelatin (Lian et al., 2002) and maltodextrin (Matsuno et al, 2002). Maltodextrin is a product obtained from the hydrolysis of starch consisting of units of D-glucose. It is inexpensive, highly soluble and of low hygroscopicity. In a spray drying process, addition of maltodextrin can increase the solid concentration in the feed and reduce the humidity of the powder produced (Anselmo et al., 2006; Phisut, 2012). The main factors that influence the efficiency of microencapsulation are the inlet and outlet air temperatures (Liu et al., 2004) and the type and concentration of the encapsulation agent. Studies designed to optimise a spray drying technique are often conducted empirically; such studies are seldom performed using statistical experimental design for planning experiments and interpreting data. The present study investigated the microencapsulation of L. casei by spray drying, using statistical experimental design to evaluate the influence of inlet air temperature and concentration of the encapsulation agent on the concentration of viable cells at
760
R. C. S. dos Santos et al.
the end of the drying process. The microparticles obtained were characterised according to their physicochemical characteristics, and cell viability was assessed after a storage time of 60 days.
Table 1. Codified levels and actual values of the variables studied in the first experimental design.
Methods
1 2 3 4 5 6 7 8 9 10 11
Materials The lyophilised culture of Lactobacillus casei UFPEDA used in this study was obtained from the culture collection of the Antibiotics Department of the Federal University of Pernambuco, Brazil. Whey was donated by the company Natural da VacaÕ , located in Gravata´ (Pernambuco, Brazil) and was kept frozen. Maltodextrin was supplied by NeoNutriÕ (Poc¸os de Caldas, MG, Brasil). Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
J Microencapsul, 2014; 31(8): 759–767
Fermentation A pre-inoculum was prepared by inoculating the micro-organism into a tube containing 5 ml of sterile saline solution (NaCl, 0.85% w/v) to obtain turbidity equivalent to tube number 0.5 on McFarland’s scale. The inoculum was subsequently prepared using 250 ml Erlenmeyer flasks containing 5 ml of pre-inoculum and 45 ml of De Man, Rogosa and Sharpe (MRS) medium supplemented with 2% (w/v) glucose, pH 6.5. Inoculum cultivation was conducted under stationary conditions, with incubation at 37 C for 12 h. For cell cultivation, experiments were conducted using 500 ml Erlenmeyer flasks containing 200 ml of growth medium based on whey supplemented with 20 g/l glucose. Cell growth was performed for 43 h under the same conditions, using an inoculum concentration of 5% (v/v). This time was established from previous experiments aimed at obtaining the maximum concentration of cells. Microencapsulation by spray drying The spray-drying of L. casei UFPEDA was performed using a laboratory-scale spray dryer (SD-BASIC). The equipment consisted of a spray system, blower, heater, 215 mm 500 mm main chamber and a cyclone. The feed solution (crude cell broth of L. casei with added maltodextrin) was atomised into a vertical drying chamber using a 1.5-mm nozzle at a feed flow rate of 12.5 ml/min. The dried powder was collected in a single cyclone separator. In order to define the conditions for L. casei microencapsulation and maximise the viable cell concentration, two independent variables (inlet air temperature and maltodextrin concentration) were evaluated using a full 22 factorial design, with three central points (level 0) and four axial points (levels ± a, where ¼ 1.4142), totalling 11 experiments (RCCD, rotational central composite design). The tests were performed randomly, and the data were analyzed using StatisticaÕ 8.0 software (Statsoft, Tulsa, OK), with a 95% confidence level. The experimental error was obtained from the mean and standard deviation of the central points. Tables 1 and 2 show the experimental conditions investigated for each experimental design. Microparticles characterisation The microparticles obtained using the selected drying conditions were characterised according to the methods described in the following sections. Humidity The humidity corresponds to the loss in weight suffered by the product when heated in conditions in which water is removed.
Assays
Inlet air temperature ( C) 77.3 112.7 77.3 112.7 70 120 95 95 95 95 95
(1) (+1) (1) (+1) (1.41) (+1.41) (0) (0) (0) (0) (0)
Concentration of maltodextrin (% w/v) 3.8 3.8 17.2 17.2 10.5 10.5 1 20 10.5 10.5 10.5
(1) (1) (+1) (+1) (0) (0) (1.41) (+1.41) (0) (0) (0)
Table 2. Codified levels and actual values of the variables studied in the second experimental design.
Assays 1 2 3 4 5 6 7 8 9 10 11
Inlet air temperature ( C) 52.9 67.1 52.9 67.1 50 70 60 60 60 60 60
(1) (+1) (1) (+1) (1.41) (+1.41) (0) (0) (0) (0) (0)
Concentration of maltodextrin (% w/v) 7.9 7.9 22.1 22.1 15 15 5 25 15 15 15
(1) (1) (+1) (+1) (0) (0) (1.41) (+1.41) (0) (0) (0)
This is an extensive property that depends on the amount of sample. A 5 g portion of the powder was heated for 3 h at 105 C. After cooling in a desiccator, the powder was weighed and the procedure was repeated until a constant weight (cw) was obtained. The humidity (Xp) was calculated using Equation 1. The tests were performed in quadruplicate. 5 1 ð1Þ Xp ¼ cw Solubility The microparticles solubility in water was determined by the maximum percentage (by weight) of the sample that will dissolve in a unit volume of water at certain (usually room) temperature. About 1 g of dry sample was diluted in 100 ml of distilled water. After homogenisation of the suspension at room temperature (28 C), the mixture was centrifuged at 30 000 g for 10 min. About 20 ml samples of the resulting solution were distributed into Petri dishes and dried in a vacuum drying oven at 75 C for 5 h. The percentage solubility was calculated by weight difference (Eastman and Moore, 1984). The tests were performed in quadruplicate. Hygroscopicity Hygroscopicity was determined in order to evaluate the ability of microparticles to absorb water from the surrounding environment. About 2 g portions of the sample were placed in aluminum capsules containing a saturated NaCl solution, under ambient conditions (75% relative humidity, 25 C), until equilibrium was achieved. The hygroscopicity was expressed as the amount of
Microencapsulation of L. casei by spray drying
DOI: 10.3109/02652048.2014.932026
water absorbed per 100 g of powder, on a dry basis (g/100 g). The tests were performed in quadruplicate. Water activity The water activity (aw) represents the ratio of the water vapour pressure of the product to the water vapour pressure of pure water under the same conditions. This is an intensive property that does not depend on the amount of sample and refers to this unbound water. Establishing the water activity of a product assists in predicting its tendency to spoil. The water activity was measured at 25 C using a Pawkit Water Activity Meter instrument (Decagon, WA). The tests were performed in quadruplicate.
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
Wettability The wettability of microparticles aiming to evaluate the ability of solid surface to be wetted when in contact with water. About 2 g portions of sample were placed in 600 ml beakers containing 400 ml of distilled water at 25 C. The time required for all particles to become wetted was determined visually (Magalha˜es Netto, 1997). The tests were performed in quadruplicate. Morphological analysis of microparticles The morphological characteristics of the microparticles were evaluated by using scanning electronic microscope (Aspex, model Express VP) at a magnification of 2500. Capsules size measurements were carried out as follows: dry microparticles were placed on graph paper under a microscope (Shimadzu) and photographed with a digital photo camera (Sony Cyber-shot DSC-WX80, Tokyo, Japan). Medium diameter of the particle size was measured with Image J 1.36b software (NIH, Bethesda, MD). From the cumulative particle distribution curve, we determined the values of d10, d50 and d90. Thermal analysis Glass transition as well as other thermal properties is a key to understand how drying conditions affect physical changes of materials. Thermal analysis was performed by differential scanning calorimetry (DSC) and differential thermal analysis (DTA) using a Shimadzu thermoanalyzer (Tokyo, Japan). All measurements employed a linear heating rate of 10 C/min, and an empty pan was used as a reference material. Portions of sample (10 mg) were heated linearly to 250 C. The Tg (glass transition temperature) was defined as the midpoint value of the change in specific heat, observed as an endothermic shift in the baseline of the DSC signal. Storage test Microparticles of L. casei were stored in penicillin tubes that were hermetically sealed and placed in a desiccator. Experiments were performed in triplicate, and probiotic survival was evaluated by performing viable cell counts after 7, 14, 28 and 60 days of storage at temperatures of 30 C, 4 C and 8 C. Viable cell counts Portions (0.1 g) of the encapsulated probiotic cells were solubilised in a solution of 0.1 M sodium citrate (pH 6.5) and serially diluted in tubes containing sterile saline solution (NaCl, 0.85% w/v). A 10 ml volume of the samples was plated on MRS agar supplemented with 2% (w/v) glucose, using the pour plate technique. The plates were incubated at 37 C for 48–72 h and results of viable and cultivable cell counts were expressed by CFU/g.
761
Results Figure 1(A and B) shows the response surface and the contour curve, respectively, for the concentration of viable cells during the first experimental design. The highest concentration of viable cells (1.5 108 CFU/g) was obtained for an inlet air temperature of 95 C and 20% (w/v) maltodextrin. The results indicated that the optimum ranges for maximisation of the concentration of viable cells covered the entire temperature range employed and maltodextrin concentrations above 18% (w/v). The Pareto graph (Figure 1C) revealed that the maltodextrin concentration was the most significant variable, with positive estimated effects of 34.9 and 30.4, demonstrating that the presence of a higher molecular weight carbohydrate (in this case maltodextrin) contributed to stability of the system (Ferrari et al., 2012). Temperature showed a statistically significant negative effect (8.097), indicating that higher viable cell counts were obtained at lower temperatures. The maximum viable cell concentration was not achieved using the first experimental design. A new experimental design was therefore performed using lower temperatures (50–70 C) and maltodextrin concentrations between 5% and 25% (w/v). Figure 2(A and B) shows the response surface and the contour curve, respectively, for the concentration of viable cells obtained using the second experimental design. In this case, it can be seen that a maximum concentration of viable cells was achieved. The critical values of the model for maximum response were a maltodextrin concentration of 8.75% (w/v) and a temperature of 59.3 C, giving a theoretical maximum viable cell concentration of 1.97 109 CFU/g. The predictive equation (Equation 2) contains the statistically significant variables, where X is the concentration of viable cells, T is the temperature and A is the adjuvant concentration. X ¼ 2:63 1010 7:86 106 T 2 þ 1:14 108 A 4:14 106 A
ð2Þ
The Pareto graph (Figure 2C) showed that the linear and quadratic terms of the maltodextrin concentration were significant, as well as the quadratic term of the temperature (p50.05). In all cases, the negative estimated effects indicated that lower temperatures and lower adjuvant concentrations resulted in higher concentrations of viable cells. Figure 3(A and B) shows typical DTA, TGA and DSC curves obtained for the thermal analysis of the microparticles and maltodextrin, respectively. In both cases, the DTA curves presented endothermic peaks at temperatures between 50 C and 120 C, which corresponded to water evaporation. The microparticles presented a shift in the heat capacity at around 160 C, compared to maltodextrin, and an additional endothermic peak just after 200 C. This was probably the temperature at which the formation of carbonaceous products (ash) from maltodextrin was initiated. The stability of the powder was evaluated over a storage period of 60 days. All the tests began at an initial count of 1.1 1010 CFU/g. The sample stored at 30 C only showed cell viability in the initial count. After 60 days, the samples stored at 8 C and 4 C showed viable cell concentrations of 1.0 109 and 3.0 108 CFU/g, respectively (Figure 4). The microparticles showed values of solubility, wettability, water activity, hygroscopicity and humidity corresponding to 97.03 ± 0.04%, 100% (in 1.16 min), 0.14 ± 0.0, 35.20 g H2O/100 g and 4.80 ± 0.43%, respectively. Figure 5 shows the morphological characteristics of the microparticles containing L. casei. Spray-dried particles were spherical with slightly roughened surfaces. The spraying system produced a very fine powder and particles presented a medium
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
762
R. C. S. dos Santos et al.
J Microencapsul, 2014; 31(8): 759–767
Figure 1. Response surface (A), contour curvature (B) and Pareto diagram (C) as a function of the inlet air temperature and concentration of maltodextrin for the first experimental design.
diameter of 14 mm. Values of d10, d50 and d90 were 1, 11 and 27.5 mm, respectively.
Discussion The inlet air temperature directly influences the outlet air temperature, which is a critical variable that affects microorganism survival (Teixeira et al., 1995). The experimental data indicated that the difference between the inlet and outlet air temperatures was around 20 ± 5 C. Previous work has demonstrated the negative effect of a high outlet air temperature on the viability of bacteria. For example, Gardiner et al. (2000) investigated the survival of L. paracasei NFBC 338 after spray drying, and obtained a survival rate of 97% when an outlet air temperature of 70 C was used. However, when a temperature of 120 C was employed, the survival rate was zero. Drying performed under the optimal conditions suggested by the second experimental design (maltodextrin concentration of 8.75% (w/v) and temperature of 59.3 C) resulted in a product with inadequate characteristics; the material formed was hygroscopic and showed a tendency to agglomerate and adhere to the walls of the equipment, which could have been due to the low maltodextrin concentration employed.
The quality of the final product obtained after spray drying is affected by the concentration of the adjuvant used during drying, and an increased quantity of adjuvant can help to produce a powder with satisfactory macroscopic characteristics. According to Roos (2010), less hygroscopic powders are obtained using higher maltodextrin concentrations. High hygroscopicity affects the efficiency of the process and compromises the microbiological stability of the final product, and maltodextrin acts to strengthen the glassy matrix during the drying process (Cha´vez and Ledeboer, 2007). Based on these results, drying was carried out using the extreme points of the second experimental design (70 C and 25% (w/v) of maltodextrin). The fine-grained powder obtained was easily collected from the walls of the cyclone and collection vessel, and presented a viable cell concentration of 1.1 1010 CFU/g. The high concentration of viable cells obtained could be explained by the incorporation of a greater amount of maltodextrin, as well as a lower inlet air temperature and a consequent decrease in the outlet air temperature. Higher concentrations of the encapsulation agent normally provide bacterial cells with greater protection against damage. In a previous study, it was shown that during drying of
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
DOI: 10.3109/02652048.2014.932026
Microencapsulation of L. casei by spray drying
763
Figure 2. Response surface (A), contour curvature (B) and Pareto diagram (C) as a function of the inlet air temperature and concentration of maltodextrin for the second experimental design.
L. fermentum, cell viability increased by about 10% when a higher adjuvant concentration was used (Cai et al., 2012). The resistance of probiotic bacteria is also determined by the outlet air temperature (Rokka and Rantamaki, 2010). Thermal stress directly affects the enzymatic functions required for maintenance of cell viability, and the greatest challenge in producing a probiotic powder is the high temperature used during the process (Jankovic et al., 2010). High temperatures can lead to the formation of pores in membranes and cause leakage of intracellular substances (Gardiner et al., 2000; Corcoran et al., 2004) as well as protein denaturation (Meng et al., 2008). Sunny-Roberts and Knorr (2009) investigated the survival of L. rhamnosus after a spray drying process and found that the survival rate was inversely proportional to the outlet air temperature. Use of a lower inlet air temperature and a higher maltodextrin concentration therefore favoured the cell viability of L. casei. The microparticles showed a solubility of 97.03 ± 0.04% and a high degree of wettability (100% in 1.16 min). Powders that have low wettability tend to form lumps during addition to water and mixing (Maia and Golgher, 1983). The high wettability was probably assisted by the low temperature used in the drying process, since high temperatures can result in the formation of a rigid coating on the microcapsule surface, preventing the diffusion of water molecules and reducing solubilisation of the
powder (Chegeni and Ghobadian, 2005). The high solubility was due to the influence of maltodextrin, which reduced the stickiness of the powder and increased the surface area of the particles in contact with water during the rehydration (Goula and Adamopoulos, 2005). Characterisation of a powdered product in terms of its water activity is important because this parameter affects the processing and handling properties, as well as the stability of the powder (Roos, 2010). In order to maintain the viability of a probiotic organism, the water activity should be as low as possible (Santivarangkna et al., 2008). The mortality of micro-organisms during storage can increase at higher aw values, due to the growth of other microbes as well as undesirable chemical reactions (Wang et al., 2004; Cha´vez and Ledeboer, 2007; Ying et al., 2010). Dried probiotic foods are therefore more stable because they have a lower aw, which enhances durability and reduces storage costs (Kearney et al., 2009). Low molecular mobility of water contributes to the stabilisation of biological systems during longterm storage (Buitink et al., 2000), and satisfactory survival of probiotics during storage requires an aw value below 0.3 (Cha´vez and Ledeboer, 2007). The aw should be between 0.11 and 0.23 for most species of Lactobacillus (Kumar and Mishra, 2004; Koc et al., 2010). The powder obtained in this study had a water activity of 0.14 ± 0.0,
764
R. C. S. dos Santos et al.
J Microencapsul, 2014; 31(8): 759–767
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
Figure 3. Values of TGA, DSC and DTA for microparticles of L. casei (A) and maltodextrin (B) (L. casei microparticles by spray drying at 70 C and maltodextrin concentration of 25% (w/v).
which was considered to be satisfactory for a powdered L. casei probiotic. A hygroscopicity value of 35.20 g H2O/100 g was obtained. The use of maltodextrin during drying can reduce hygroscopicity by around 50%, compared to samples without addition of this agent (Cai and Corke, 2000). The drying temperature also has a significant effect on the final properties of the powder, with lower temperatures resulting in less hygroscopic particles (Tonon et al., 2008). Another factor related to the hygroscopicity is humidity. Particles with low humidity content are more hygroscopic due to their greater ability to absorb moisture from the environment, which is related to the vapour pressure gradient between the material and the surrounding atmosphere. In this study, the microparticles showed a humidity of 4.80 ± 0.43%. This relatively low humidity could be explained by the concentration of maltodextrin employed. In other work, it was demonstrated that during the drying of L. kefir, bacterial survival was significantly higher under conditions of low humidity
(Golowczyc et al., 2010), while an outlet air temperature of around 60 C resulted in a powder with a moisture content of less than 10%, whose biological activity was maintained (Riveros et al., 2009). The thermogravimetric signal revealed a significant loss of mass from the microparticles, and from 160 C the sample was rapidly degraded during heating. In the case of maltodextrin, the mass loss occurred gradually, with onset of sample degradation after 225 C. Powdered food products containing amorphous carbohydrates undergo physical changes that are related to the glass transition temperature (Tg), such as stickiness and compaction during storage (Roos, 2010). The Tg serves as a benchmark to characterise the thermal stability of a product; below this temperature, the material is more stable. The DSC curve for the microparticles showed a Tg of around 56 C, revealed by a baseline shift; the biological activity of the probiotic powder should therefore be preserved below this temperature. The DSC curve showed two second-order
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
DOI: 10.3109/02652048.2014.932026
Figure 4. Viable cell counting of L. casei microparticles for 60 days.
Microencapsulation of L. casei by spray drying
765
agreement with the observation of sample degradation from 200 C. In relation to stability during storage (shelf life), for foods containing probiotics, the product should have an average shelf life of between 15 and 30 days, with 4106 CFU/mL remaining at the end of the period (Trabulsi and Sampaio, 2000). Teanpaisan et al. (2012) demonstrated that the maximum cell viability of L. paracasei SD1 during storage for 6 months occurred at 4 C, while survival rates were close to 0% when storage temperature 25 C was used. Hsiao et al. (2004) also reported that the storage of dried probiotic cultures in glass flasks at low temperature (4 C) increased the useful life of the product. The available evidence therefore indicates that storage at a low temperature favours maintenance of the viability of probiotic samples dried using spray drying. Another factor that assists the retention of cell viability during storage is the use of glass containers, which have low permeability and minimise the ingress of oxygen (Shah, 2000). Under favourable storage conditions, probiotics dried by spray drying should be stable for at least 30 days (Gardiner et al., 2000; Wang et al., 2004; Kearney et al., 2009). The morphological analysis demonstrated that some of the microparticles presented slight surface roughness, and there were a few structures that were not encapsulated. The size range was typical of particles produced by spray drying. Similar morphological characteristics were described by Tonon et al. (2011). The correlation between microcapsule size and stability is not completely understood, although the existence of an optimum size could be related to decreased stability when higher levels of imperfections are present.
Conclusions
Figure 5. Scanning electronic microscopy (Aspex, model Express VP) of L. casei microparticles (2500 magnification).
transformations. After 140 C, there was glass transition of the amorphous phase contained in the microparticles and fusion of the crystalline phase, shown by two consecutive drops of the curve, which could have been due to the presence of unencapsulated biological material. The endothermic peak at 140 C, corresponding to the melting heat of the microparticles during the phase change, showed that the microparticles had a good resistance to high temperatures. The onset of oxidative degradation was apparent above 200 C, as well as the presence of interferences (false peaks) due to expansion of the material. The results indicated that microcapsule decomposition began at around 160 C, in agreement with earlier findings (Haines, 1995). The DSC results for maltodextrin revealed an endothermic peak at 175 C, with onset of decomposition occurring at around 215 C, indicating that there was no formation of other material until this temperature was reached. As also seen for the microparticles, decomposition occurred at temperatures above 200 C. According to Elnaggar et al. (2010), heating of maltodextrin to 220 C results in carbonisation without fusion, in
Lactobacillus casei loaded microparticles were prepared by spraydrying technique. Experimental design was employed to determine the best operating ranges in terms of the inlet air temperature and the maltodextrin concentration. The drying condition selected was at the extreme point of the second experimental design (70 C and 25% w/v of maltodextrin), which resulted in formation of a powder with good macroscopic characteristics and a high concentration of viable cells (41010 CFU/g). The microparticles were homogeneous spherical particles, with slightly roughened surfaces, and were satisfactory in terms of their wettability, solubility, water activity, hygroscopicity and humidity. During storage for 60 days, the cells remained viable at temperatures of 8 and 4 C, with viable cell concentrations of 4108 CFU/g. Thermal analysis indicated that the glass transition temperature (Tg) was higher than any anticipated storage temperature. It could therefore be assumed that the bacteria were stored in the glassy state. The results demonstrated that microencapsulation of L. casei by spray drying, using maltodextrin as an encapsulation agent, offers a viable alternative technique for the production of probiotic powders used as food ingredients.
Acknowledgments Gratitude must go to Dr Luciano Bueno (Rural Federal University of Pernambuco, Brazil) for technical assistance during the thermal analysis, and Rafael Albuquerque for the assistance during microscopic analysis.
Declaration of interest This research was supported by Fundac¸a˜o de Amparo a` Cieˆncia e Tecnologia do Estado de Pernambuco – FACEPE (IBPG-0797-3.06/10).
766
R. C. S. dos Santos et al.
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
References Ananta E, Volkert M, Knorr, D. Cellular injuries and storage stability of spray-dried Lactobacillus rhamnosus GG. Int Dairy J, 2005;15: 399–409. Anekella K, Orsat V. Optimization of microencapsulation of probiotics in raspberry juice by spray drying. Lwt – Food Sci Technol, 2013;50: 17–25. Anselmo GCS, Eduardo MRM, Cavalcanti M, Arruda PC, Coelho M. Determination of hygroscopicity of caja powder through spray drying. J Biol Sci Earth, 2006;6:58–65. ¨ zlu¨han G, Melike S, Figen K, Gonca S, Neriman B. Banu K, Mehmet K, O Effects of formulation on stability of spray-dried egg. Drying Technol Int J, 2012;30:63–71. Baranauskiene R, Venskutonis PR, Dewettinck K, Verhe R. Properties of oregano (Origanumvulgare L.), citronella (Cymbopogonnardus G.) and marjoram (Majoranahortensis L.) flavors encapsulated into milk protein-based matrices. Food Res Int, 2006;39:413–25. Buitink J, Van Den Dries IJ, Hoekstra FA, Alberda M, Hemminga MA. High critical temperature above Tg may contribute to the stability of biological systems. Biophys J, 2000;79:1119–28. Cai CJ, Thaler B, Liu DW, Wang JJ, Qiao SY. Optimization of spray-drying workflow as a method for preparing concentrated cultures of Lactobacillus fermentum. J Anim Vet Adv, 2012;11: 2769–74. Cai YZ, Corke H. Production and properties of spray-dried Amaranthus betacyanin pigments. J Food Sci, 2000;65:1248–52. Cha´vez BE, Ledeboer AM. Drying of probiotics: Optimization of formulation and process to enhance storage survival. Drying Technol, 2007;25:1193–201. Chegeni GR, Ghobadian B. Effects of spray drying conditions on physical properties of orange juice powder. Drying Technol, 2005;23: 657–68. Corcoran BM, Ross RP, Fitzgerald GF, Stanton C. Comparative survival of probiotic Lactobacilli spray-dried in the presence of prebiotic substances. J Appl Microbiol, 2004;96:1024–39. Doherty SB, Gee RP, Stanton C, Fitzgerald GF, Brodkorb A. Development and characterisation of whey protein micro-beads as potential matrices for probiotic protection. Food Hydrocolloids, 2011; 25:1604–17. Drusch S. Sugar beet pectin: A novel emulsifying wall component for microencapsulation of lipophilic food ingredients by spray-drying. Food Hydrocolloids, 2006;21:1223–8. Dziezak JD. Microencapsulation and encapsulated ingredients. Food Technol, 1998;42:136–51. Eastman JE, Moore CO. 1984. Cold-water soluble granular starch for gelled food composition. Patent US 4.465.702 A. Elnaggar YS, El-Massik MA, Abdallah OY, Ebian AE. Maltodextrin: A novel excipient used in sugar-based orally disintegrating tablets and phase transition process. Am Association Pharm Scientists, 2010;11: 645–51. Ferrari CC, Germer SPM, Alvim ID, Vissotto FZ, Aguirre JM. Influence of carrier agents on the physicochemical properties of blackberry powder produced by spray drying. Int J Food Sci Technol, 2012;47: 1237–45. Filkova´ I, Huang LX, Mujumdar AS. 2007. Industrial spray drying systems. In: Mujumdar AS, ed. Handbook of industrial drying. London: CRC, pp. 215–56. Fritzen-Freire CB, Prudencio ES, Amboni RDMC, Pinto SS, Negra˜o-Murakami AN, Murakami FS. Microencapsulation of Bifidobacteria by spray drying in the presence of prebiotics. Food Res Int, 2012;45:306–12. Gardiner GE, O’Sullivan E, Kelly J, Auty MAE, Fitzgerald GF, Collins JK, Ross RP, Stanton C. Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray drying. Appl Environ Microbiol, 2000;66:2605–12. Gharsallaoui A, Roudaut G, Chambin O, Voilley A, Saurel R. Applications of spray-drying in microencapsulation of food ingredients: An overview. Food Res Int, 2007;40:1107–21. Golowczyc MA, Gerez CL, Silva J, Abraham AG, De Antoni, GL, Teixieira, P. Survival of spray-dried Lactobacillus kefir is affected by different protectants and storage conditions. Bioproc Biol Eng Storage Cond, 2010;33:681–6. Gouin S. Microencapsulation: Industrial appraisal of existing technologies and trends. Trends Food Sci Technol, 2004;15:330–47.
J Microencapsul, 2014; 31(8): 759–767
Goula AM, Adamopoulos KG. Spray drying of tomato pulp in dehumidified air: II. The effect on powder properties. J Food Eng, 2005;66:35–42. Guarner F, Malagelada JR. 2003. Gut flora in health and disease. London: The Lancet. Haines PJ. 1995. Thermal methods of analysis: Principles, applications and problems. London: Blackie Academic. Hogan SA, Mcnamee BF, O’Riordan ED, O’Sullivan M. Microencapsulation properties of sodium caseinate. J Agric Food Chem, 2001;49:1934–8. Hsiao HC, Lian WC, Chou CC. Effect of packaging conditions and temperature on viability of microencapsulated Bifidobacteria during storage. J Sci Food Agric, 2004;84:134–9. Ivanovska TP, Petrusˇevska-Tozi L, Kostoska MD, Geskovski N, Grozdanov A, Stain C, Stafilov T, Mladenovska K. Microencapsulation of Lactobacillus casei in chitosan-Ca-alginate microparticles using spray-drying method. Macedonian J Chem Chem Eng, 2012;31:115–23. Jankovic I, Sybesma W, Phothirath P, Ananta E, Mercenier A. Application of probiotics in food products – Challenges and new approaches. Curr Opinion Biotechnol, 2010;21:175–81. Kearney N, Meng XC, Stanton C, Kelly J, Fitzgerald GF, Ross RP. Development of a spray dried probiotic yoghurt containing Lactobacillus paracasei NFBC 338. Int Dairy J, 2009;19:684–9. Koc B, Yilmazer MS, Balkir P, Ertekin FK. Spray drying of yogurt: Optimization of process conditions for improving viability and other quality attributes. Drying Technol, 2010;28:495–507. Krishnaiah D, Sarbatly R, Nithyanandam R. Microencapsulation of Morinda citrifolia L. extract by spray-drying. Chem Eng Res Design, 2012;90:622–32. Kumar P, Mishra HN. Yoghurt powder – A review of process technology, storage and utilization. Food Bioproducts Process, 2004;82:133–42. Lannes SCS, Medeiros ML. Cupuassu chocolate drink powder processed by spray-dryer. Brazilian J Sci Pharm, 2003;39:115–23. Lian WC, Hsiao HC, Chou CC. Survival of Bifidobacteria after spraydrying. Int J Food Microbiol, 2002;74:79–86. Liu Z, Zhou J, Zeng Y, Ouyang X. The enhancement and encapsulation of Agaricus bisporus flavor. J Food Eng, 2004;65:391–6. Magalha˜es Netto F. 1997. Influence of water activity on the glass temperature. In: Germer DCP, Jardim SPM, eds. Water activity in foods. Campinas: ITAL, pp. 4–14. Maia ABR, Golgher M. Parameters for assessing the quality of reconstitution of dehydrated milk powder spray dryer (‘‘spray-drier’’). Boletim SBCTA, 1983;17:235–54. Malmo C, Storia AL, Mauriello G. Microencapsulation of Lactobacillus reuteri DSM 17938 cells coated in alginate beads with chitosan by spray drying to use as a probiotic cell in a chocolate souffle´. Food Bioprocess Technol, 2013;6:795–805. Matsuno R, Minemoto K, Hakamata S, Adachi S. Oxidation of linoleic acid encapsulated with gum Arabic or maltodextrin by spray-drying. J Microencapsulation, 2002;19:181–9. Meng XC, Stanton C, Fitzgerald GF, Daly C, Ross RP. Anhydrobiotics: The challenges of drying probiotic cultures. Food Chem, 2008;106: 1406–16. Paramita V, Iida K, Yoshii H, Furuta T. Effect of additives on the morphology of spray-dried powder. Drying Technol, 2010;28:323–9. Peighambardoust SH, GolshanTafti A, Hesari J. Application of spray drying for preservation of lactic acid starter cultures: A review. Trends Food Sci Technol, 2011;22:215–24. Phisut N. Spray drying technique of fruit juice powder: Some factors influencing the properties of product. Int Food Res J, 2012;19: 1297–306. Riveros B, Ferreira J, Borquez R. Spray drying of a vaginal probiotic strain of Lactobacillus acidophilus. Drying Technol, 2009;27:123–32. Rokka S, Rantamaki P. Protecting probiotic bacteria by microencapsulation: Challenges for industrial applications. Eur Food Res Technol, 2010;231:1–12. Roos YH. Glass transition temperature and its relevance in food processing. Ann Rev Food Sci Technol, 2010;1:469–96. Santivarangkna C, Kulozik U, Foerst P. Inactivation mechanisms of lactic acid starter cultures preserved by drying processes. J Appl Microbiol, 2008;105:1–13. Shah NP. Probiotic bacteria: Selective enumeration and survival in dairy foods. J Dairy Sci, 2000;83:894–907. Silva J, Freixo R, Gibbs P, Teixeira P. Spray-drying for the production of dried cultures. Int J Dairy Technol, 2011;64:321–35.
DOI: 10.3109/02652048.2014.932026
Journal of Microencapsulation Downloaded from informahealthcare.com by Laurentian University on 12/06/14 For personal use only.
Sunny-Roberts EO, Knorr D. The protective effect of monosodium glutamate on survival of Lactobacillus rhamnosus GG and Lactobacillus rhamnosus E-97800 strains during spray-drying and storage in trehalose-containing powders. Int Dairy J, 2009;19:209–14. Teanpaisan R, Chooruk A, Wannun A, Wichienchot S, Piwat S. Survival rates of human-derived probiotic Lactobacillus paracasei SD1 in milk powder using spray drying. J Sci Technol, 2012;34:241–5. Teixeira P, Castro H, Kirby R. Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus. J Appl Microbiol, 1995;78:456–62. Tonon RV, Grosso CRF, Hubinger MD. Influence of emulsion composition and inlet air temperature on the microencapsulation of flaxseed oil by spray drying. Food Res Int, 2011;44:282–9.
Microencapsulation of L. casei by spray drying
767
Tonon RV, Brabet C, Hubinger MD. Influence of process conditions on the physicochemical properties of ac¸ai (Euterpe oleraceae mart.) powder produced by spray drying. J Food Eng, 2008;88:411–18. Trabulsi LR, Sampaio MMSC. 2000. Probio´ticos, prebio´ticos e simbio´ticos. In: Trabulsi LR, Carneiro-Sampaio MMS, eds. Os probio´ticos e a Sau´de Infantil. Sa˜o Paulo: Nestle´, pp. 15. Wang YC, Yu RC, Chou CC. Viability of lactic acid bacteria and Bifidobacteria in fermented soymilk after drying, subsequent rehydration and storage. Int J Food Microbiol, 2004;93:209–17. Ying DY, Phoon MC, Sanquansri L, Weerakkody R, Burgar I, Augustin MA. Microencapsulated Lactobacillus rhamnosus GG powders: Relationship of powder physical properties to probiotic survival during storage. J Food Sci, 2010;75:588–95.
