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Encapsulation of Lactobacillus acidophilus in moist-heat-resistant multilayered microcapsules
Journal of Food Engineering 192 (2017) 11e18
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Encapsulation of Lactobacillus acidophilus in moist-heat-resistant multilayered microcapsules Panithan Pitigraisorn a, Kantaphong Srichaisupakit a, Nutnicha Wongpadungkiat a, Saowakon Wongsasulak b, * a
Department of Food Engineering, Faculty of Engineering, King Mongkut's University of Technology Thonburi, 126 Pracha Uthit Road, Tungkru, Bangkok, 10140, Thailand Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, 126 Pracha Uthit Road, Tungkru, Bangkok, 10140, Thailand
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2015 Received in revised form 24 July 2016 Accepted 31 July 2016 Available online 2 August 2016
The development of state-of-the-art moist-heat-resistant microcapsules for a probiotic is described in this report. The survival of the heat-sensitive probiotic Lactobacillus acidophilus upon exposure to moistheat treatment was improved by encapsulating the bacterial cells in multilayered microcapsules, which were formed via a technique that combined electrospraying and fluidized bed coating. A composite composed of egg albumen (EA) and stearic acid (SA) was coated onto the L. acidophilus-encapsulating alginate microcapsules to produce the first shell layer. These microcapsules were subsequently coated with cassava starch granules while they were dried in a fluidized bed dryer using cassava pearls as the drying aid. The effect of the EA:SA ratio on the morphology, encapsulation efficiency (EE) and degree of moist-heat resistance (at 70 C and 100% relative humidity for 30 min) of the microcapsules was investigated. The results showed that the EE of the microcapsules was greater than 90%. When the relative proportion of SA was increased, the survival rate of the cells encapsulated in the moist-heattreated microcapsules significantly improved. Compared with earlier reports, the current multilayered microcapsules conferred an extremely high degree of protection of the encapsulated cells upon moistheat exposure, with the cells suffering a loss of vitality of only 0.6 log CFU/g. These newly developed microcapsules can serve as a prototype encapsulation structure for the protection of other thermosensitive microorganisms and compounds used to fortify foods and feeds. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Cassava starch Egg albumen Encapsulation Probiotics Solid lipid Thermo-tolerance
1. Introduction Probiotics are live microorganisms that provide health benefits, such as preventing diarrhea, treating Helicobacter pylori, ameliorating respiratory tract infections, reducing serum cholesterol levels and improving the host's lactose tolerance levels, when they are consumed in adequate quantities (Lee and Salminen, 2009; Xu et al., 2016). The two most popular genera that are normally added to feed and food products are Bifidobacterium and Lactobacillus (Corona-Hernandez et al., 2013). Bacteria of the latter genus are more tolerant of acidic conditions than bacteria of the former genus. Nonetheless, bacteria of the former genus are much more sensitive to oxygen and the environmental temperature. The
* Corresponding author. E-mail address: [email protected] (S. Wongsasulak). http://dx.doi.org/10.1016/j.jfoodeng.2016.07.022 0260-8774/© 2016 Elsevier Ltd. All rights reserved.
optimum temperature for the growth of Lactobacillus acidophilus is 40e42 C, and a major loss of viability occurs when the temperature is higher than 45 C (Tripathi and Giri, 2014). Therefore, fortification of food and feed products by probiotics is normally performed during a downstream manufacturing process. Nevertheless, the temperature to which most thermally processed foods are exposed during the downstream steps and the temperature used for drying feed products is greater than 60e70 C. The recommended minimum number of live probiotic cells in foods to provide health benefits to the host is 106e107 CFU/mL or g of food (Tripathi and Giri, 2014). Thus, encapsulation is employed to provide an effective physical barrier to the environmental factors that are harmful to the probiotics (Corona-Hernandez et al., 2013; Tripathi and Giri, 2014; Vandenplas et al., 2015; Zhang et al., 2014). Three common encapsulation techniques are used for probiotics: extrusion, emulsification and polymer cross-linking and spray drying. The first two methods can leave solvent residues in
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fortified feeds and foods. Spray drying is a simple encapsulation technique in which microcapsules are formed and dried in one step (Ray et al., 2016; Vemmer and Patel, 2013) without leaving any solvent residue. However, this technique adversely affects the encapsulation efficiency, which can be attributed to the high temperature conditions required to evaporate the solvent (Laelorspoen et al., 2014; Martín et al., 2015). For encapsulation via electrospraying/electrospinning, electrical charges are applied to a polymer solution to initiate an electrospraying/electrospinning jet from a Taylor cone (Wongsasulak et al., 2010; Weiss et al., 2012). The jet is projected towards a grounded collector. While moving through the electric field, the solvent rapidly evaporates from the travelling jet and solidifies to form electrosprayed/electrospun particles (beads, beaded fibers or fibers) on the collector. The important advantage of this technique over the previously mentioned probiotic encapsulation techniques is that microcapsules can be produced without the application of heat, thereby resulting in a relatively high encapsulation efficiency (Dong et al., 2013; GomezMascaraque et al., 2016). Moreover, the particle size distribution of electrospun and electrosprayed products is narrow and controllable (Laelorspoen et al., 2014). Additionally, this technique can be applied to produce microcapsules with a core-shell structure by using either coaxial spinneret tips (Sakuldao et al., 2011; Kiatyongchai et al., 2014) or an electrostatic interaction technique (Laelorspoen et al., 2014). The viability of probiotics encapsulated in microcapsules following exposure to a thermal process depends on many crucial factors, e.g., the use of a proper encapsulation technique, the architecture of the produced microcapsules and the selection of suitable encapsulating polymers. The structure of the microcapsule can be divided into one of three major categories as follows: conventional, bi/multilayered and multi-compartmental (Vemmer and Patel, 2013; Leong et al., 2016). The latter two structures are generally much more effective for encapsulation. To date, there are a limited number of reports of successfully encapsulating probiotics in thermal-tolerant microcapsules. Sodium alginate, which is an edible natural polysaccharide, is one of the most widely used capsule materials for probiotic microencapsulation due to its nontoxicity, its egg-box gel-forming capacity and its gelation under mild conditions. However, an alginate component alone may be ineffective for improving probiotic cell viability due to the high porosity of the formed structure (Mokarram et al., 2009; Laelorspoen et al., 2014). Zein was recently used to produce a shell coating of alginate-based microcapsules encapsulating L. acidophilus to maximize their viability during exposure of the microcapsules to gastrointestinal fluid (Laelorspoen et al., 2014). Due to the highly hydrophobic property of zein, the viability of the encapsulated cells in the simulated gastric fluid (pH 1.2) containing pepsin was improved five-fold compared with cells encapsulated in non-coated alginate-based microcapsules. However, a zein solution might not be appropriate for use because it might affect the viability of the cells during microcapsule formation. The main constituent of egg albumen is amphoteric proteins, which can be acidified to allow them to interact with negatively charged alginate molecules. As noted by Wongsasulak et al. (2006, 2007), the presence of cassava starch (CS) granules in a cold-set EA gel matrix substantially improved the elastic modulus of this matrix during exposure to moisture and heat. This composite system exhibited an excellent protective capacity for heat-sensitive compounds, even at high relative humidity values (Wongsasulak et al., 2006). Acidified egg albumen (EA) was chosen as the reagent to coat the shell of the alginate-based microcapsules in this study. The high level of hydrophobicity and low level of thermal diffusivity of the obtained microcapsules was maximized by dispersing a solid lipid (i.e., stearic acid or SA) into the EA matrix. Electrospraying was
employed to produce alginate-based microcapsules encapsulating L. acidophilus. Multilayered microcapsules were obtained by manipulating CS granules to produce a top shell layer on the surface of the microcapsules via fluidized bed drying. The effect of the EASA composite shell layer on the moist-heat resistance of the encapsulated cells was studied. The hypothesis underlying this investigation was that the survival of L. acidophilus upon exposure to moist heat could be improved by encapsulating these cells in alginate-based microcapsules coated with a material with a low level of thermal diffusivity and a high level of hydrophobicity. Alginate with these properties would hinder the penetration of moist heat into the microcapsules, thereby improving the survival rate of the encapsulated cells upon exposure of the microcapsules to moist heat. 2. Experimental procedures 2.1. Materials The sodium alginate (food grade) used in this study was purchased from the Qingdao Bright Moon Seaweed Group Co., Ltd, (Shangdong, China). Egg albumen powder (86.25% db. protein content determined using the Kjeldahl analytical method (Methods 925.31, AOAC, 2012), with an N factor of 6.25) was obtained from the Winner Group Enterprise Co. (Bangkok, Thailand). Calcium chloride and citric acid were obtained from Italmar Co., Ltd. (AR grade, Carlo-Erba Reagents SAS, France). Lactobacilli de Man, Rogosa and Sharpe (MRS) broth and agar were obtained from Difco (MI, USA). The MRS media were used without supplementation. Tween® 40 (AR grade) was purchased from Fluka (Germany). 2.2. Methods 2.2.1. Preparation of the L. acidophilus suspension A culture of L. acidophilus TISTR 1338 grown on MRS agar with an average viable cell count of 107 colony forming units (CFU)/mL was provided by the Thailand Institute of Scientific and Technology Research (TISTR; Pathum Thani, Thailand). The cells were transferred to 5 mL of sterilized MRS broth, and the cultures were incubated at 37 C for 24 h under anaerobic conditions. Cells from late-log growth phase cultures with an average cell count of 109 CFU/mL were collected after centrifugation (Hitachi, Model CR22N, Japan) of the culture at 6000g for 10 min at 4 C. Four milliliters of the supernatant was discarded, and only 1 mL containing the pelleted cells was collected. The pelleted cells were then suspended in MRS broth. 2.2.2. Preparation of the core shell solutions To prepare the core solution, sodium alginate was added to distilled water to a final concentration of 2.5% w/v, and the solution was constantly stirred for 2 h. Then, glycerol (8% w/v) was added. The solution was mixed with the cell suspension to produce the core solution. The ratio of the alginate solution to the cell suspension was set at 20:2 (v/v). To prepare the shell solution, 8% w/v of an EA solution was prepared by dissolving the EA powder in distilled water and gently stirring the mixture for 2 h using a magnetic stirrer (IKA, Model C-MAGHS7, China). The EA solution was heated to 50 C for 10 min. Stearic acid (SA) was completely melted at 80 C and then mixed with Tween® 40 at a weight ratio of 1:1.25 using a high shear speed homogenizer (IKA, Model T25, Ultra-Turrax, Germany) operated at 12,600 rpm for 5 min and then at 3000 rpm for 1 min. The homogenization temperature was controlled at 76 ± 1 C using a water bath. The SA solution was allowed to cool to 53 ± 1 C prior to mixing with the EA solution. Calcium chloride was added to the mixture at a 1.5% w/v ratio.
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Fig. 1. SEM images taken at 35 magnification of multilayered capsules containing L. acidophilus: (A) alginate capsules without a shell, (B) with an EA shell, (C) with an EA-SA composite shell (EA:SA ¼ 1:0.05), and (D) with an EA-SA composite shell (EA:SA ¼ 1:0.25). The scale bar is 500 mm.
Finally, the pH of the EA emulsion was adjusted to 3.65 using citric acid. This pH was chosen based on the results of our previous study (Laelorspoen et al., 2014).
2.2.3. Electrospraying and fluidized bed drying of the microcapsules Electrospraying was conducted as described in our previous report (Laelorspoen et al., 2014). First, 20 mL of the core solution was loaded into a glass syringe with a stainless steel needle with a 0.69 mm inner diameter (Hamilton #91021, Reno, NV, USA). The syringe was mounted onto a syringe pump (NE100, New Era Pump System, Farmingdale, NY, USA), and the needle tip was connected to a high-voltage generator (Model ES30Pe5W, Gamma High Voltage, Ormond Beach, FL, USA) set to 8 kV. The feed rate of the core solution was fixed at 10 mL/h. The shell solution, which was contained in a completely grounded stainless steel bowl, was constantly stirred using a magnetic stirrer. The temperature of the shell solution was controlled at 50 ± 0.5 C using a water bath. The distance from the needle tip to the surface of the shell solution was set to 6 cm. The core solution was electrosprayed into the shell solution, and the resulting mixture was left for 30 min. The, the microcapsules were separated from the mixture by filtration. The multilayered microcapsules were finally obtained upon fluidized bed drying; cassava pearls (Fish brand, E.T.C. Eiab Tong
Encapsulation efficiency ð%Þ ¼
Chan Co., Ltd., Bangkok, Thailand) were used as the drying aid. The weight ratio of the microcapsules to cassava pearls was 1:5. The air inlet temperature was controlled at 37 ± 1 C. The minimum fluidization air velocity (Umf) was set at 2.7 m/s for the first 15 min and then reduced to 1 m/s; this velocity was maintained for 105 min. These drying conditions were determined in a preliminary study in which they were shown to provide the most rapid drying rate without significantly adversely affecting the morphology of the dried microcapsules (data not shown). The final moisture content of the microcapsules was ~13.6 ± 1% (dry basis or d.b.), and the water activity (aw) was 0.85 ± 0.01.
2.2.4. Microcapsule encapsulation efficiency and payload The encapsulation efficiency (EE) of the microcapsules was defined as the number of viable cells encapsulated in the microcapsules compared with the initial number of viable cells used to form the microcapsules (Annan et al., 2008; Ayama et al., 2014). The cell payload of the microcapsules was defined as the number of viable cells encapsulated in the microcapsules compared with the microcapsule weight (Lakkis, 2007; Wongsasulak et al., 2014). The EE and payload were calculated using Equations (1e2), respectively, as follows:
Number of viable cells encapsulated in capsules 100 Number of viable cells taken to form capsules
(1)
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Cell payload ð%Þ ¼
P. Pitigraisorn et al. / Journal of Food Engineering 192 (2017) 11e18
Number of viable cells encapsulated in capsules 100 Weight of capsules
2.2.5. Examination of the microcapsule morphology and microstructure The general morphology of the microcapsules before and after moist-heat treatment was determined using scanning electron microscopy (SEM) (JEOL-JSM-5410LV, Tokyo, Japan). The microcapsules were mounted onto a metal stub using double-sided tape
(2)
and sputter-coated with gold prior to the evaluation. The diameters of the microcapsules were determined using ImageJ software (NIH, MD, USA). The average diameter was determined by measuring 100 microcapsules.
Fig. 2. SEM image taken at 200 magnification of multilayered capsules containing L. acidophilus: (A) alginate capsules without a shell, (B) with an EA shell, (C) with an EA-SA composite shell (EA:SA ¼ 1:0.05), and (D) with an EA-SA composite shell (EA:SA ¼ 1:0.25). The scale bar is 500 mm. (E) is an SEM image taken at 100 of a cross-sectioned alginate capsule that was coated with EA. The image in the inset was taken at a magnification of 4500 .
P. Pitigraisorn et al. / Journal of Food Engineering 192 (2017) 11e18
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Fig. 3. Images taken under UV light of multilayered capsules containing L. acidophilus: (A) alginate capsules without a shell, (B) with an EA shell, (C) with an EA-SA composite shell (EA:SA ¼ 1:0.05), and (D) with an EA-SA composite shell (EA:SA ¼ 1:0.25).
2.2.6. Determination of the viable L. acidophilus cell count encapsulated in the microcapsules The viable cell count was determined as described by Laelorspoen et al. (2014). To liberate the encapsulated cells, microcapsules containing L. acidophilus were dissociated by dissolving 1 g of microcapsules in 75 mL of 1% w/v sodium citrate and vigorously stirring the mixture for 30 min using a magnetic stirrer. To determine the number of encapsulated cells, the liberated cells were incubated in MRS broth at 37 C for 4 h prior to spreading them on MRS agar and incubating the plates at 37 C under anaerobic condition for 48 h. The incubation was performed to recover cells that were injured during the process used to dry the microcapsules and the exposure of the microcapsules to moist heat. The incubation period (4 h) was shown to not affect the cell count numbers in a preliminary experiment (data not shown). The numbers of colonies formed by the viable cells were counted and reported as CFU/g of dried microcapsules. The analyses were performed in triplicate. 2.2.7. Determination of the survival rate of microencapsulated L. acidophilus exposed to moist heat Free cell and encapsulated cell samples were spread as a 1-mmthick layer on petri dishes. Then, the dishes were incubated in a moist heat chamber (70 ± 0.5 C and 100% relative humidity) for 30 min to evaluate the moist-heat resistance of the samples. The viable cell counts of the treated samples were determined following the method described in Section 2.2.6. 2.2.8. Statistical analysis Significant differences among the experimental data were determined using a one-way analysis of variance (ANOVA) for completely randomized designs using the SPSS computational software (SPSS 17.0). The differences among the mean values were evaluated using Duncan's multiple range test at a 95% confidence level (p < 0.05). All experiments were performed at least in duplicate.
3. Results and discussion 3.1. Morphology and microstructure of the multilayered microcapsules As shown in the SEM images presented in Fig. 1AeD, the microcapsules obtained using all of the tested formulations were moderately spherical and had the same average diameter of approximately 450 ± 50 mm (p 0.05). Structural collapse of the alginate-based microcapsules lacking shell coatings that were dried using the fluidized bed process (Fig. 1A) was observed more often than collapse of the similarly treated microcapsules with shell coatings (Fig. 1BeD). During drying in the fluidized bed, water evaporated from the uncoated microcapsules, leading to their structural collapse. In contrast, when the alginate-based microcapsules were incubated in a shell-coating solution containing an EA or EA-SA composite, the EA molecules might have diffused into the egg-box structure of the microcapsules. This diffusion may have resulted in a positive interaction between the alginate and EA molecules that improved the mechanical strength of the composite matrix network, thereby allowing these microcapsules to maintain their structures during the drying process (Devi et al., 2016; Wongsasulak et al., 2007; Zhang et al., 2016). Magnified images of the microcapsule surfaces (Fig. 2AeD) showed that they were covered or embedded with CS granules in all cases. During drying,
Table 1 Encapsulation efficiency and cell payload of the alginate based microcapsules. Microcapsules
Encapsulation efficiency (%)
Payload (log CFU/gdry)
Without shell 95.3 ± 5.4 12.6 ± With shell coating (EA/SA ratio) 1/0 85.0 ± 6.5 11.0 ± 1/0.05 88.6 ± 2.6 11.7 ± 1/0.25 92.7 ± 4.4 12.3 ± 1/0.5 Unable to form core-shell microcapsules
0.7 1.3 0.4 1.0
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P. Pitigraisorn et al. / Journal of Food Engineering 192 (2017) 11e18 Table 2 Viability of free cells and encapsulated cells in microcapsules with and without shell coating before and after exposure to moist-heat treatments. Sample
Free Cells Encapsulated cells without shell Encapsulated cells with shell EA to SA ratio 1/0 1/0.05 1/0.25
Viability (log CFU/gdry)
Reduction (log CFU/gdry)
Before
After
12.2 ± 0.1 12.6 ± 0.7
N/D 10.2 ± 9.3
12.2d ± 0.1 3.3c±0.6
11.0 ± 1.3 11.7 ± 0.4 12.3 ± 1.0
9.7 ± 0.3 10.5 ± 0.7 11.6 ± 0.0
1.3b ± 0.6 1.2b ± 0.3 0.6a±0.1
the CS granules that disintegrated from the cassava pearls became embedded into or adhered to the surface of the microcapsules. Fig. 2E is the SEM image of the cross-sectioned capsules, which revealed that the bacterial cells were completely embedded in the core matrix of the multilayered microcapsules. Fig. 3 shows images of microcapsules without and with the EA shell coating (Fig. 3A,B) and with the EA-SA composite shell coating (Fig. 3CeD) taken under UV light. As shown in the figures, the microcapsules coated with an EA or EA-SA composite shell layer (Fig. 3BeD) were much less transparent than the non-coated microcapsules (Fig. 3A). Under UV light, the microcapsules with a multilayered shell coating or a thicker shell layer were opaque (Xu et al., 2013).
3.2. Microcapsule encapsulation efficiency and cell payload As shown in Table 1, the encapsulation efficiency (EE) of the non-coated microcapsules was 95.3 ± 5.4, whereas the EE of the microcapsules coated with EA was 83.2 ± 9.8%. When SA was added to the coating layer at EA:SA ratios of 1:0.05 and 1:0.25, the EEs were 88.6 ± 2.6 and 92.7 ± 0.3%, respectively. The alginate-based microcapsules that had shell coatings had lower encapsulation efficiencies than the non-coated microcapsules. This phenomenon
could be attributed to the acidity of the acidified EA solution (pH ~3.65). Although L. acidophilus tolerates acidic conditions relatively well compared with other probiotic genera, the viability of this microorganism is significantly reduced at pH values lower than 3.3e3.4 (Lee and Salminen, 2009). Nonetheless, the encapsulation efficiency of the microcapsules was slightly improved when SA was present in the EA matrix. This result could be due to the dispersed SA particles retarding the diffusion of oxygen into the microcapsules and thereby protecting the microorganisms from oxygen exposure (Salminen et al., 2016) during fluidized bed drying. Compared with the results of other probiotic encapsulation studies, the encapsulation technique proposed here resulted in a significantly higher encapsulation efficiency (93%); moreover, encapsulation was performed in this study under mild conditions via a combination of electrospraying and layer coating. The EE of probiotics is generally in the range of 60e95% (Ayama et al., 2014; brard et al., 2009; Liu et al., 2016; Shi et al., 2013). However, an He EE value greater than 90% has rarely been reported. These results can be explained by the use of an encapsulation technique involving spray drying, which normally causes major stress to probiotic cells, leading to a lower EE. Lopez-Rubio and Lagaron (2012) also reported that the EE of probiotics could be
Fig. 4. SEM images taken at 200 magnification of multilayered capsules containing L. acidophilus that had been exposed to moist heat at 70 C (100% RH) for 30 min: (A) alginate capsules without a shell, (B) with an EA shell, (C) with an EA-SA composite shell (EA:SA ¼ 1:0.05), and (D) with an EA-SA composite shell (EA:SA ¼ 1:0.25). The scale bar is 100 mm.
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significantly improved by using electrospraying as the encapsulation technique. 3.3. Effects of moist heat on the viability of free and encapsulated L. acidophilus cells As shown in Table 2, free or non-encapsulated L. acidophilus cells were destroyed by exposure to moist heat at 70 C for 30 min. In contrast, the viability rates of L. acidophilus cells encapsulated in the EA-SA-coated microcapsules, which exhibited a very high capacity to protect the encapsulated cells during exposure to moist heat, were reduced by only 1.3 to 0.6 log CFU/g. The EA molecules gelled upon exposure to moist heat, thereby increasing the density of the matrix network (Wongsasulak et al., 2006, 2007). As shown in the SEM images (Fig. 4AeD), the CS granules coating the surfaces of the microcapsule samples partially gelatinized. The rate of gelatinization in the microcapsules lacking SA (Fig. 4AeB) was much greater than the rate of gelatinization of the microcapsules containing SA (Fig. 4CeD). The presence of CS granules in the EA matrix has been shown to improve the gel strength of the EA matrix, resulting in a superior barrier to moist-heat penetration of the microcapsules (Wongsasulak et al., 2006). The results of this study indicated that the starch granules absorbed moisture from the EA matrix upon exposure of the EA-starch composite matrix to moist heat, leading to an increase in the apparent EA concentration and inducing protein-starch interactions. Thus, the gel strength of the composite matrix is substantially increased, leading to an increased level of moist-heat tolerance. This explanation was supported by the results of the study of Corona-Hernandez et al. (2013), which showed that the viscoelastic properties of an alginate-protein coacervation complex were an important factor in determining the level of thermotolerance of composite microcapsules. Additionally, the SA molecules incorporated into the EA matrix played an important role in inhibiting the diffusion of hot moisture into the microcapsules (Fabra et al., 2009; Sabikhi et al., 2010). As strongly indicated by the higher-magnification SEM images (Fig. 3CeD), the SA molecules melted and migrated to the exterior of the microcapsules after the moist-heat treatment. The amount of melted lipid that migrated to the surface of the microcapsules would have increased when the proportion of SA present increased, thereby enhancing the barrier to hot moisture diffusion into the capsules. This phenomenon might be the major mechanism underlying the excellent moist-heat-resistant property of the multilayered microcapsules. Furthermore, after the microcapsules were removed from the moist-heat environment and cooled to room temperature, the melted lipid molecules crystalized and formed the outermost surface of the microcapsule shells, which may have maximized the resistance of these microcapsules to digestion by gastrointestinal fluids. Studies of the resistance of these microcapsules to gastrointestinal fluid digestion and the release characteristics of the probiotics encapsulated in these structures in the gastrointestinal tract are currently underway, and the results will be reported in a future publication. 4. Conclusions This study focused on the architecture of multilayered microcapsules that provided moist-heat protection for a probiotic strain that underwent the thermal processes necessary for the production of foods and feeds. The goal of protecting the probiotic by encapsulating it in an appropriately formulated microcapsule was achieved. The viability rate of the encapsulated probiotic after the exposure of the microcapsules to moist heat was reduced by less than 1 log unit. Coating the alginate-based microcapsules with an EA-SA composite significantly improved the resistance of the
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encapsulated cells to moist heat exposure. SA played a significant role in the improved protection of the encapsulated probiotic. The encapsulation efficiency of the new microcapsules was greater than 90%, which was attributed to the use of a combination of electrospraying and a fluidized bed coating to produce the microcapsule and the use of mild encapsulation conditions. The newly developed multilayered microcapsules might serve as prototype encapsulation structures for the protection of other heat-sensitive microorganisms, compounds and nutrients used to fortify foods and feeds. Acknowledgements This study was granted by the Asahi Glass Foundation (Year 2014) and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission. Additionally, this project was partially funded by the National Research Council of Thailand (Year 2015). The authors especially thank Assoc. Prof. Parichat Hongsprabhas for her constructive discussion of this study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jfoodeng.2016.07.022. References Annan, N.T., Borza, A.D., Truelstrup Hansen, L., 2008. Encapsulation in alginatecoated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions. Food Res. Int. 41, 184e193. Association of official analytical chemists (AOAC), 2012. Official Methods of Analysis of AOAC International Methods 925.31, 19th ed. AOAC Int, Gaithersburg, MD. Ayama, H., Sumpavapol, P., Chanthachum, S., 2014. Effect of encapsulation of selected probiotics cell on survival in simulated gastrointestinal tract condition. Songklanakarin J. Sci. Technol. 36, 291e299. Corona-Hernandez, R.I., Alvarez-Parrilla, E., Lizardi-Mendoza, J., Islas-Rubio, A.R., de la Rosa, Laura A., Wall-Medrano, A., 2013. Structural stability and viability of microencapsulated probiotic bacteria: a review. Compr. Rev. Food Sci. Food Saf. 12, 614e628. Devi, N., Sarmah, M., Khatun, B., Maji, T.K., 2016. Encapsulation of active ingredients in polysaccharides-protein complex coacervates. Adv. Colloid interface Sci. http://dx.doi.org/10.1016/j.cis.2016.05.009. Dong, Q.Y., Chen, M.Y., Xin, Y., Qin, X.Y., Cheng, Z., Shi, L.E., Tang, Z.X., 2013. Alginatebased and protein-based materials for probiotics encapsulation: a review. Int. J. Food Sci. Technol. 48, 1339e1351. Fabra, M.J., Hambleton, A., Talens, P., Debeaufort, F., Chiralt, A., Voilley, A., 2009. Influence of films used for flavour encapsulation. Carbohydr. Polym. 325e332. rez-Masia , R., Sanchez, G., Lopez-Rubio, A., Gomez-Mascaraque, L.G., Morfin, R.C., Pe 2016. Optimization of electrospraying condition for the microencapsulation of probiotics and evaluation of their resistance during storage and in-vitro digestion. LWT Food Sci. Technol. 69, 438e446. brard, G., Hoffart, V., Beyssac, E., Cardot, J.M., Alric, M., Subirade, M., 2009. Coated He whey protein/alginate microparticles as oral controlled delivery systems for probiotic yeast. J. Microencapsul. 27, 292e302. Kiatyongchai, T., Wongsasulak, S., Yoovidhya, T., 2014. Coaxial electrospinning and release characteristics of cellulose acetate-gelatin blend encapsulating a model drug. J. Appl. Polym. Sci. 131, 40167e40175. Laelorspoen, N., Wongsasulak, S., Yoovidhya, T., Devahastin, S., 2014. Microencapsulation of Lactobacillus acidophilus in zeinealginate coreeshell microcapsules via electrospraying. J. Funct. Foods 7, 342e349. Lakkis, J.M., 2007. Encapsulation and Controlled Release Technologies in Food Systems. Patent, USA, pp. 1e3. Lee, Y.K., Salminen, S., 2009. Handbook of Probiotics and Prebiotic, second ed. John Wiley & Sons, Inc, New Jersey. Leong, J., Lam, W., Ho, K., Voo, W., Lee, M.F., Lim, H., Lim, S., Tey, B., Poncelet, D., Chan, E., 2016. Advances in fabricating spherical alginate hydrogels with controlled particle designs by ionotropic gelation as encapsulation systems. Particuology 24, 44e60. Lopez-Rubio, A., Lagaron, J.M., 2012. Whey protein capsules obtained though electrospraying for the encapsulation of bioactive. Innov. Food Sci. Emerg. Technol. 13, 200e206. Liu, Y., Sun, Y., Sun, L., Rizwan-ur-Rehman, Wang, Y., 2016. In vitro and in vivo study of sodium polyacrylate grafted alginate as microcapsule matrix for live probiotic delivery. J. Funct. Foods 24, 429e437. Martín, M.J., Lara-Villoslada, F., Ruiz, M.A., Morales, M.E., 2015. Microencapsulation
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of bacteria: a review of different technologies and their impact on the probiotics effects. Innov. Food Sci. Emerg. Technol. 27, 15e25. Mokarram, R.R., Mortazavi, S.A., Habibi, N., Shahidi, F., 2009. The influence of multistage alginate coating on survivability of potential probiotic bacteria in simulated gastric and intestinal juice. Food Res. Int. 42, 1040e1045. Ray, S., Raychaudhuri, U., Chakraborty, R., 2016. An overview of encapsulation of active compounds used in food products by drying technology. Food Biosci. 13, 76e83. Sabikhi, L., Babu, R., Thompkinson, D.K., Kapila, S., 2010. Resistance of microencapsulated Lactobacillus acidophilus LA1 to processing treatments and simulated gut conditions. Food Bioprocess Technol. 3, 586e593. Sakuldao, S., Yoovidhya, T., Wongsasulak, S., 2011. Coaxial electrospinning and sustained release properties of gelatin-cellulose acetate core-shell ultrafine fibres. ScienceAsia 37, 335e343. € Salminen, H., GOmmel, C., Leuenberger, B.H., Weiss, J., 2016. Influence of encapsulated functional lipids on crystal structure and chemical stability in solid lipid nanoparticles: towards bioactive-based design of delivery systems. Food Chem. 190, 928e937. Shi, L., Li, Z., Zhang, Z., Zhang, T., Yu, W., Zhou, M., Tang, Z., 2013. Encapsulation of Lactobacillus bulgaricus in carrageenan-locust bean gum coated milk microspheres with double layer structure. LWT Food Sci. Technol. 54, 147e151. Tripathi, M.K., Giri, S.K., 2014. Probiotic functional foods: survival of probiotics during processing and storage. J. Funct. Foods 9, 225e241. Vandenplas, Y., Huys, G., Daube, G., 2015. Probiotics: an update. J. de pediatria 91, 6e21. Vemmer, M., Patel, A.V., 2013. Review of encapsulation methods suitable for microbial biological control agents. Biol. Control 67, 380e389. Wongsasulak, S., Yoovidhya, T., Bhumiratana, S., Hongsprabhas, P., McClements, D.J.,
Weiss, J., 2006. Thermo-mechanical properties of egg albumen-cassava starch composite films containing sunflower-oil droplets as influenced by moisture content. Food Res. Int. 39, 277e284. Wongsasulak, S., Yoovidhya, T., Bhumiratana, S., Hongsprabhas, P., 2007. Physical properties of composite network of egg albumen and cassava starch formed by salt-induced gelation method. Food Res. Int. 40, 249e256. Wongsasulak, S., Patapeejumruswong, M., Weiss, J., Supaphol, P., Yoovidhya, T., 2010. Electrospinning of food-grade nanofibers from cellulose acetate and egg albumen blends. J. Food Eng. 98, 370e376. Wongsasulak, S., Pathumban, S., Yooovidhya, T., 2014. Effect of entrapped ɑtocopherol on mucoadhesivity and evaluation of the release, degradation, and swelling characteristics of zeinechitosan composite electrospun fibers. J. Food Eng. 120, 110e117. Weiss, J., Kanjanapongkul, K., Wongsasulak, S., Yoovidhya, T., 2012. Electrospun fibers: fabrication, functionalities and potential food industry applications. In: Huang, Q. (Ed.), Nanotechnology in the Food, Beverage and Nutraceutical Industries. Woodhead Publishing Limited, Cambridge, UK. -Bourque, F., Dumont, M.J., Jabaji, S., 2016. Encapsulation of LactobaXu, M., Gagne cillus casei ATCC 393 cells and evaluation of their survival after freeze-drying, storage and under gastrointestinal conditions. J. Food Eng. 168, 52e59. Xu, Q., Qin, H., Yin, Z., Hua, J., Pack, D.W., Wang, C., 2013. Coaxial electrohydrodynamic atomization process for production of polymeric composite microsphere. Chem. Eng. Sci. 104, 330e334. Zhang, L., Huang, S., Ananingsih, V.K., Zhou, W., Chen, X.D., 2014. A study on Bifidobacterium lactis Bb12 viability in bread during baking. J. Food Eng. 122, 33e37. Zhang, Z., Zhang, R., Zou, L., McClements, D.J., 2016. Protein encapsulation in alginate hydrogel beads: effect of pH on microgel stability, protein retention and protein release. Food Hydrocoll. 58, 308e315.
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Encapsulation of Lactobacillus acidophilus in moist-heat-resistant multilayered microcapsules Panithan Pitigraisorn a, Kantaphong Srichaisupakit a, Nutnicha Wongpadungkiat a, Saowakon Wongsasulak b, * a
Department of Food Engineering, Faculty of Engineering, King Mongkut's University of Technology Thonburi, 126 Pracha Uthit Road, Tungkru, Bangkok, 10140, Thailand Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, 126 Pracha Uthit Road, Tungkru, Bangkok, 10140, Thailand
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2015 Received in revised form 24 July 2016 Accepted 31 July 2016 Available online 2 August 2016
The development of state-of-the-art moist-heat-resistant microcapsules for a probiotic is described in this report. The survival of the heat-sensitive probiotic Lactobacillus acidophilus upon exposure to moistheat treatment was improved by encapsulating the bacterial cells in multilayered microcapsules, which were formed via a technique that combined electrospraying and fluidized bed coating. A composite composed of egg albumen (EA) and stearic acid (SA) was coated onto the L. acidophilus-encapsulating alginate microcapsules to produce the first shell layer. These microcapsules were subsequently coated with cassava starch granules while they were dried in a fluidized bed dryer using cassava pearls as the drying aid. The effect of the EA:SA ratio on the morphology, encapsulation efficiency (EE) and degree of moist-heat resistance (at 70 C and 100% relative humidity for 30 min) of the microcapsules was investigated. The results showed that the EE of the microcapsules was greater than 90%. When the relative proportion of SA was increased, the survival rate of the cells encapsulated in the moist-heattreated microcapsules significantly improved. Compared with earlier reports, the current multilayered microcapsules conferred an extremely high degree of protection of the encapsulated cells upon moistheat exposure, with the cells suffering a loss of vitality of only 0.6 log CFU/g. These newly developed microcapsules can serve as a prototype encapsulation structure for the protection of other thermosensitive microorganisms and compounds used to fortify foods and feeds. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Cassava starch Egg albumen Encapsulation Probiotics Solid lipid Thermo-tolerance
1. Introduction Probiotics are live microorganisms that provide health benefits, such as preventing diarrhea, treating Helicobacter pylori, ameliorating respiratory tract infections, reducing serum cholesterol levels and improving the host's lactose tolerance levels, when they are consumed in adequate quantities (Lee and Salminen, 2009; Xu et al., 2016). The two most popular genera that are normally added to feed and food products are Bifidobacterium and Lactobacillus (Corona-Hernandez et al., 2013). Bacteria of the latter genus are more tolerant of acidic conditions than bacteria of the former genus. Nonetheless, bacteria of the former genus are much more sensitive to oxygen and the environmental temperature. The
* Corresponding author. E-mail address: [email protected] (S. Wongsasulak). http://dx.doi.org/10.1016/j.jfoodeng.2016.07.022 0260-8774/© 2016 Elsevier Ltd. All rights reserved.
optimum temperature for the growth of Lactobacillus acidophilus is 40e42 C, and a major loss of viability occurs when the temperature is higher than 45 C (Tripathi and Giri, 2014). Therefore, fortification of food and feed products by probiotics is normally performed during a downstream manufacturing process. Nevertheless, the temperature to which most thermally processed foods are exposed during the downstream steps and the temperature used for drying feed products is greater than 60e70 C. The recommended minimum number of live probiotic cells in foods to provide health benefits to the host is 106e107 CFU/mL or g of food (Tripathi and Giri, 2014). Thus, encapsulation is employed to provide an effective physical barrier to the environmental factors that are harmful to the probiotics (Corona-Hernandez et al., 2013; Tripathi and Giri, 2014; Vandenplas et al., 2015; Zhang et al., 2014). Three common encapsulation techniques are used for probiotics: extrusion, emulsification and polymer cross-linking and spray drying. The first two methods can leave solvent residues in
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fortified feeds and foods. Spray drying is a simple encapsulation technique in which microcapsules are formed and dried in one step (Ray et al., 2016; Vemmer and Patel, 2013) without leaving any solvent residue. However, this technique adversely affects the encapsulation efficiency, which can be attributed to the high temperature conditions required to evaporate the solvent (Laelorspoen et al., 2014; Martín et al., 2015). For encapsulation via electrospraying/electrospinning, electrical charges are applied to a polymer solution to initiate an electrospraying/electrospinning jet from a Taylor cone (Wongsasulak et al., 2010; Weiss et al., 2012). The jet is projected towards a grounded collector. While moving through the electric field, the solvent rapidly evaporates from the travelling jet and solidifies to form electrosprayed/electrospun particles (beads, beaded fibers or fibers) on the collector. The important advantage of this technique over the previously mentioned probiotic encapsulation techniques is that microcapsules can be produced without the application of heat, thereby resulting in a relatively high encapsulation efficiency (Dong et al., 2013; GomezMascaraque et al., 2016). Moreover, the particle size distribution of electrospun and electrosprayed products is narrow and controllable (Laelorspoen et al., 2014). Additionally, this technique can be applied to produce microcapsules with a core-shell structure by using either coaxial spinneret tips (Sakuldao et al., 2011; Kiatyongchai et al., 2014) or an electrostatic interaction technique (Laelorspoen et al., 2014). The viability of probiotics encapsulated in microcapsules following exposure to a thermal process depends on many crucial factors, e.g., the use of a proper encapsulation technique, the architecture of the produced microcapsules and the selection of suitable encapsulating polymers. The structure of the microcapsule can be divided into one of three major categories as follows: conventional, bi/multilayered and multi-compartmental (Vemmer and Patel, 2013; Leong et al., 2016). The latter two structures are generally much more effective for encapsulation. To date, there are a limited number of reports of successfully encapsulating probiotics in thermal-tolerant microcapsules. Sodium alginate, which is an edible natural polysaccharide, is one of the most widely used capsule materials for probiotic microencapsulation due to its nontoxicity, its egg-box gel-forming capacity and its gelation under mild conditions. However, an alginate component alone may be ineffective for improving probiotic cell viability due to the high porosity of the formed structure (Mokarram et al., 2009; Laelorspoen et al., 2014). Zein was recently used to produce a shell coating of alginate-based microcapsules encapsulating L. acidophilus to maximize their viability during exposure of the microcapsules to gastrointestinal fluid (Laelorspoen et al., 2014). Due to the highly hydrophobic property of zein, the viability of the encapsulated cells in the simulated gastric fluid (pH 1.2) containing pepsin was improved five-fold compared with cells encapsulated in non-coated alginate-based microcapsules. However, a zein solution might not be appropriate for use because it might affect the viability of the cells during microcapsule formation. The main constituent of egg albumen is amphoteric proteins, which can be acidified to allow them to interact with negatively charged alginate molecules. As noted by Wongsasulak et al. (2006, 2007), the presence of cassava starch (CS) granules in a cold-set EA gel matrix substantially improved the elastic modulus of this matrix during exposure to moisture and heat. This composite system exhibited an excellent protective capacity for heat-sensitive compounds, even at high relative humidity values (Wongsasulak et al., 2006). Acidified egg albumen (EA) was chosen as the reagent to coat the shell of the alginate-based microcapsules in this study. The high level of hydrophobicity and low level of thermal diffusivity of the obtained microcapsules was maximized by dispersing a solid lipid (i.e., stearic acid or SA) into the EA matrix. Electrospraying was
employed to produce alginate-based microcapsules encapsulating L. acidophilus. Multilayered microcapsules were obtained by manipulating CS granules to produce a top shell layer on the surface of the microcapsules via fluidized bed drying. The effect of the EASA composite shell layer on the moist-heat resistance of the encapsulated cells was studied. The hypothesis underlying this investigation was that the survival of L. acidophilus upon exposure to moist heat could be improved by encapsulating these cells in alginate-based microcapsules coated with a material with a low level of thermal diffusivity and a high level of hydrophobicity. Alginate with these properties would hinder the penetration of moist heat into the microcapsules, thereby improving the survival rate of the encapsulated cells upon exposure of the microcapsules to moist heat. 2. Experimental procedures 2.1. Materials The sodium alginate (food grade) used in this study was purchased from the Qingdao Bright Moon Seaweed Group Co., Ltd, (Shangdong, China). Egg albumen powder (86.25% db. protein content determined using the Kjeldahl analytical method (Methods 925.31, AOAC, 2012), with an N factor of 6.25) was obtained from the Winner Group Enterprise Co. (Bangkok, Thailand). Calcium chloride and citric acid were obtained from Italmar Co., Ltd. (AR grade, Carlo-Erba Reagents SAS, France). Lactobacilli de Man, Rogosa and Sharpe (MRS) broth and agar were obtained from Difco (MI, USA). The MRS media were used without supplementation. Tween® 40 (AR grade) was purchased from Fluka (Germany). 2.2. Methods 2.2.1. Preparation of the L. acidophilus suspension A culture of L. acidophilus TISTR 1338 grown on MRS agar with an average viable cell count of 107 colony forming units (CFU)/mL was provided by the Thailand Institute of Scientific and Technology Research (TISTR; Pathum Thani, Thailand). The cells were transferred to 5 mL of sterilized MRS broth, and the cultures were incubated at 37 C for 24 h under anaerobic conditions. Cells from late-log growth phase cultures with an average cell count of 109 CFU/mL were collected after centrifugation (Hitachi, Model CR22N, Japan) of the culture at 6000g for 10 min at 4 C. Four milliliters of the supernatant was discarded, and only 1 mL containing the pelleted cells was collected. The pelleted cells were then suspended in MRS broth. 2.2.2. Preparation of the core shell solutions To prepare the core solution, sodium alginate was added to distilled water to a final concentration of 2.5% w/v, and the solution was constantly stirred for 2 h. Then, glycerol (8% w/v) was added. The solution was mixed with the cell suspension to produce the core solution. The ratio of the alginate solution to the cell suspension was set at 20:2 (v/v). To prepare the shell solution, 8% w/v of an EA solution was prepared by dissolving the EA powder in distilled water and gently stirring the mixture for 2 h using a magnetic stirrer (IKA, Model C-MAGHS7, China). The EA solution was heated to 50 C for 10 min. Stearic acid (SA) was completely melted at 80 C and then mixed with Tween® 40 at a weight ratio of 1:1.25 using a high shear speed homogenizer (IKA, Model T25, Ultra-Turrax, Germany) operated at 12,600 rpm for 5 min and then at 3000 rpm for 1 min. The homogenization temperature was controlled at 76 ± 1 C using a water bath. The SA solution was allowed to cool to 53 ± 1 C prior to mixing with the EA solution. Calcium chloride was added to the mixture at a 1.5% w/v ratio.
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Fig. 1. SEM images taken at 35 magnification of multilayered capsules containing L. acidophilus: (A) alginate capsules without a shell, (B) with an EA shell, (C) with an EA-SA composite shell (EA:SA ¼ 1:0.05), and (D) with an EA-SA composite shell (EA:SA ¼ 1:0.25). The scale bar is 500 mm.
Finally, the pH of the EA emulsion was adjusted to 3.65 using citric acid. This pH was chosen based on the results of our previous study (Laelorspoen et al., 2014).
2.2.3. Electrospraying and fluidized bed drying of the microcapsules Electrospraying was conducted as described in our previous report (Laelorspoen et al., 2014). First, 20 mL of the core solution was loaded into a glass syringe with a stainless steel needle with a 0.69 mm inner diameter (Hamilton #91021, Reno, NV, USA). The syringe was mounted onto a syringe pump (NE100, New Era Pump System, Farmingdale, NY, USA), and the needle tip was connected to a high-voltage generator (Model ES30Pe5W, Gamma High Voltage, Ormond Beach, FL, USA) set to 8 kV. The feed rate of the core solution was fixed at 10 mL/h. The shell solution, which was contained in a completely grounded stainless steel bowl, was constantly stirred using a magnetic stirrer. The temperature of the shell solution was controlled at 50 ± 0.5 C using a water bath. The distance from the needle tip to the surface of the shell solution was set to 6 cm. The core solution was electrosprayed into the shell solution, and the resulting mixture was left for 30 min. The, the microcapsules were separated from the mixture by filtration. The multilayered microcapsules were finally obtained upon fluidized bed drying; cassava pearls (Fish brand, E.T.C. Eiab Tong
Encapsulation efficiency ð%Þ ¼
Chan Co., Ltd., Bangkok, Thailand) were used as the drying aid. The weight ratio of the microcapsules to cassava pearls was 1:5. The air inlet temperature was controlled at 37 ± 1 C. The minimum fluidization air velocity (Umf) was set at 2.7 m/s for the first 15 min and then reduced to 1 m/s; this velocity was maintained for 105 min. These drying conditions were determined in a preliminary study in which they were shown to provide the most rapid drying rate without significantly adversely affecting the morphology of the dried microcapsules (data not shown). The final moisture content of the microcapsules was ~13.6 ± 1% (dry basis or d.b.), and the water activity (aw) was 0.85 ± 0.01.
2.2.4. Microcapsule encapsulation efficiency and payload The encapsulation efficiency (EE) of the microcapsules was defined as the number of viable cells encapsulated in the microcapsules compared with the initial number of viable cells used to form the microcapsules (Annan et al., 2008; Ayama et al., 2014). The cell payload of the microcapsules was defined as the number of viable cells encapsulated in the microcapsules compared with the microcapsule weight (Lakkis, 2007; Wongsasulak et al., 2014). The EE and payload were calculated using Equations (1e2), respectively, as follows:
Number of viable cells encapsulated in capsules 100 Number of viable cells taken to form capsules
(1)
14
Cell payload ð%Þ ¼
P. Pitigraisorn et al. / Journal of Food Engineering 192 (2017) 11e18
Number of viable cells encapsulated in capsules 100 Weight of capsules
2.2.5. Examination of the microcapsule morphology and microstructure The general morphology of the microcapsules before and after moist-heat treatment was determined using scanning electron microscopy (SEM) (JEOL-JSM-5410LV, Tokyo, Japan). The microcapsules were mounted onto a metal stub using double-sided tape
(2)
and sputter-coated with gold prior to the evaluation. The diameters of the microcapsules were determined using ImageJ software (NIH, MD, USA). The average diameter was determined by measuring 100 microcapsules.
Fig. 2. SEM image taken at 200 magnification of multilayered capsules containing L. acidophilus: (A) alginate capsules without a shell, (B) with an EA shell, (C) with an EA-SA composite shell (EA:SA ¼ 1:0.05), and (D) with an EA-SA composite shell (EA:SA ¼ 1:0.25). The scale bar is 500 mm. (E) is an SEM image taken at 100 of a cross-sectioned alginate capsule that was coated with EA. The image in the inset was taken at a magnification of 4500 .
P. Pitigraisorn et al. / Journal of Food Engineering 192 (2017) 11e18
15
Fig. 3. Images taken under UV light of multilayered capsules containing L. acidophilus: (A) alginate capsules without a shell, (B) with an EA shell, (C) with an EA-SA composite shell (EA:SA ¼ 1:0.05), and (D) with an EA-SA composite shell (EA:SA ¼ 1:0.25).
2.2.6. Determination of the viable L. acidophilus cell count encapsulated in the microcapsules The viable cell count was determined as described by Laelorspoen et al. (2014). To liberate the encapsulated cells, microcapsules containing L. acidophilus were dissociated by dissolving 1 g of microcapsules in 75 mL of 1% w/v sodium citrate and vigorously stirring the mixture for 30 min using a magnetic stirrer. To determine the number of encapsulated cells, the liberated cells were incubated in MRS broth at 37 C for 4 h prior to spreading them on MRS agar and incubating the plates at 37 C under anaerobic condition for 48 h. The incubation was performed to recover cells that were injured during the process used to dry the microcapsules and the exposure of the microcapsules to moist heat. The incubation period (4 h) was shown to not affect the cell count numbers in a preliminary experiment (data not shown). The numbers of colonies formed by the viable cells were counted and reported as CFU/g of dried microcapsules. The analyses were performed in triplicate. 2.2.7. Determination of the survival rate of microencapsulated L. acidophilus exposed to moist heat Free cell and encapsulated cell samples were spread as a 1-mmthick layer on petri dishes. Then, the dishes were incubated in a moist heat chamber (70 ± 0.5 C and 100% relative humidity) for 30 min to evaluate the moist-heat resistance of the samples. The viable cell counts of the treated samples were determined following the method described in Section 2.2.6. 2.2.8. Statistical analysis Significant differences among the experimental data were determined using a one-way analysis of variance (ANOVA) for completely randomized designs using the SPSS computational software (SPSS 17.0). The differences among the mean values were evaluated using Duncan's multiple range test at a 95% confidence level (p < 0.05). All experiments were performed at least in duplicate.
3. Results and discussion 3.1. Morphology and microstructure of the multilayered microcapsules As shown in the SEM images presented in Fig. 1AeD, the microcapsules obtained using all of the tested formulations were moderately spherical and had the same average diameter of approximately 450 ± 50 mm (p 0.05). Structural collapse of the alginate-based microcapsules lacking shell coatings that were dried using the fluidized bed process (Fig. 1A) was observed more often than collapse of the similarly treated microcapsules with shell coatings (Fig. 1BeD). During drying in the fluidized bed, water evaporated from the uncoated microcapsules, leading to their structural collapse. In contrast, when the alginate-based microcapsules were incubated in a shell-coating solution containing an EA or EA-SA composite, the EA molecules might have diffused into the egg-box structure of the microcapsules. This diffusion may have resulted in a positive interaction between the alginate and EA molecules that improved the mechanical strength of the composite matrix network, thereby allowing these microcapsules to maintain their structures during the drying process (Devi et al., 2016; Wongsasulak et al., 2007; Zhang et al., 2016). Magnified images of the microcapsule surfaces (Fig. 2AeD) showed that they were covered or embedded with CS granules in all cases. During drying,
Table 1 Encapsulation efficiency and cell payload of the alginate based microcapsules. Microcapsules
Encapsulation efficiency (%)
Payload (log CFU/gdry)
Without shell 95.3 ± 5.4 12.6 ± With shell coating (EA/SA ratio) 1/0 85.0 ± 6.5 11.0 ± 1/0.05 88.6 ± 2.6 11.7 ± 1/0.25 92.7 ± 4.4 12.3 ± 1/0.5 Unable to form core-shell microcapsules
0.7 1.3 0.4 1.0
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P. Pitigraisorn et al. / Journal of Food Engineering 192 (2017) 11e18 Table 2 Viability of free cells and encapsulated cells in microcapsules with and without shell coating before and after exposure to moist-heat treatments. Sample
Free Cells Encapsulated cells without shell Encapsulated cells with shell EA to SA ratio 1/0 1/0.05 1/0.25
Viability (log CFU/gdry)
Reduction (log CFU/gdry)
Before
After
12.2 ± 0.1 12.6 ± 0.7
N/D 10.2 ± 9.3
12.2d ± 0.1 3.3c±0.6
11.0 ± 1.3 11.7 ± 0.4 12.3 ± 1.0
9.7 ± 0.3 10.5 ± 0.7 11.6 ± 0.0
1.3b ± 0.6 1.2b ± 0.3 0.6a±0.1
the CS granules that disintegrated from the cassava pearls became embedded into or adhered to the surface of the microcapsules. Fig. 2E is the SEM image of the cross-sectioned capsules, which revealed that the bacterial cells were completely embedded in the core matrix of the multilayered microcapsules. Fig. 3 shows images of microcapsules without and with the EA shell coating (Fig. 3A,B) and with the EA-SA composite shell coating (Fig. 3CeD) taken under UV light. As shown in the figures, the microcapsules coated with an EA or EA-SA composite shell layer (Fig. 3BeD) were much less transparent than the non-coated microcapsules (Fig. 3A). Under UV light, the microcapsules with a multilayered shell coating or a thicker shell layer were opaque (Xu et al., 2013).
3.2. Microcapsule encapsulation efficiency and cell payload As shown in Table 1, the encapsulation efficiency (EE) of the non-coated microcapsules was 95.3 ± 5.4, whereas the EE of the microcapsules coated with EA was 83.2 ± 9.8%. When SA was added to the coating layer at EA:SA ratios of 1:0.05 and 1:0.25, the EEs were 88.6 ± 2.6 and 92.7 ± 0.3%, respectively. The alginate-based microcapsules that had shell coatings had lower encapsulation efficiencies than the non-coated microcapsules. This phenomenon
could be attributed to the acidity of the acidified EA solution (pH ~3.65). Although L. acidophilus tolerates acidic conditions relatively well compared with other probiotic genera, the viability of this microorganism is significantly reduced at pH values lower than 3.3e3.4 (Lee and Salminen, 2009). Nonetheless, the encapsulation efficiency of the microcapsules was slightly improved when SA was present in the EA matrix. This result could be due to the dispersed SA particles retarding the diffusion of oxygen into the microcapsules and thereby protecting the microorganisms from oxygen exposure (Salminen et al., 2016) during fluidized bed drying. Compared with the results of other probiotic encapsulation studies, the encapsulation technique proposed here resulted in a significantly higher encapsulation efficiency (93%); moreover, encapsulation was performed in this study under mild conditions via a combination of electrospraying and layer coating. The EE of probiotics is generally in the range of 60e95% (Ayama et al., 2014; brard et al., 2009; Liu et al., 2016; Shi et al., 2013). However, an He EE value greater than 90% has rarely been reported. These results can be explained by the use of an encapsulation technique involving spray drying, which normally causes major stress to probiotic cells, leading to a lower EE. Lopez-Rubio and Lagaron (2012) also reported that the EE of probiotics could be
Fig. 4. SEM images taken at 200 magnification of multilayered capsules containing L. acidophilus that had been exposed to moist heat at 70 C (100% RH) for 30 min: (A) alginate capsules without a shell, (B) with an EA shell, (C) with an EA-SA composite shell (EA:SA ¼ 1:0.05), and (D) with an EA-SA composite shell (EA:SA ¼ 1:0.25). The scale bar is 100 mm.
P. Pitigraisorn et al. / Journal of Food Engineering 192 (2017) 11e18
significantly improved by using electrospraying as the encapsulation technique. 3.3. Effects of moist heat on the viability of free and encapsulated L. acidophilus cells As shown in Table 2, free or non-encapsulated L. acidophilus cells were destroyed by exposure to moist heat at 70 C for 30 min. In contrast, the viability rates of L. acidophilus cells encapsulated in the EA-SA-coated microcapsules, which exhibited a very high capacity to protect the encapsulated cells during exposure to moist heat, were reduced by only 1.3 to 0.6 log CFU/g. The EA molecules gelled upon exposure to moist heat, thereby increasing the density of the matrix network (Wongsasulak et al., 2006, 2007). As shown in the SEM images (Fig. 4AeD), the CS granules coating the surfaces of the microcapsule samples partially gelatinized. The rate of gelatinization in the microcapsules lacking SA (Fig. 4AeB) was much greater than the rate of gelatinization of the microcapsules containing SA (Fig. 4CeD). The presence of CS granules in the EA matrix has been shown to improve the gel strength of the EA matrix, resulting in a superior barrier to moist-heat penetration of the microcapsules (Wongsasulak et al., 2006). The results of this study indicated that the starch granules absorbed moisture from the EA matrix upon exposure of the EA-starch composite matrix to moist heat, leading to an increase in the apparent EA concentration and inducing protein-starch interactions. Thus, the gel strength of the composite matrix is substantially increased, leading to an increased level of moist-heat tolerance. This explanation was supported by the results of the study of Corona-Hernandez et al. (2013), which showed that the viscoelastic properties of an alginate-protein coacervation complex were an important factor in determining the level of thermotolerance of composite microcapsules. Additionally, the SA molecules incorporated into the EA matrix played an important role in inhibiting the diffusion of hot moisture into the microcapsules (Fabra et al., 2009; Sabikhi et al., 2010). As strongly indicated by the higher-magnification SEM images (Fig. 3CeD), the SA molecules melted and migrated to the exterior of the microcapsules after the moist-heat treatment. The amount of melted lipid that migrated to the surface of the microcapsules would have increased when the proportion of SA present increased, thereby enhancing the barrier to hot moisture diffusion into the capsules. This phenomenon might be the major mechanism underlying the excellent moist-heat-resistant property of the multilayered microcapsules. Furthermore, after the microcapsules were removed from the moist-heat environment and cooled to room temperature, the melted lipid molecules crystalized and formed the outermost surface of the microcapsule shells, which may have maximized the resistance of these microcapsules to digestion by gastrointestinal fluids. Studies of the resistance of these microcapsules to gastrointestinal fluid digestion and the release characteristics of the probiotics encapsulated in these structures in the gastrointestinal tract are currently underway, and the results will be reported in a future publication. 4. Conclusions This study focused on the architecture of multilayered microcapsules that provided moist-heat protection for a probiotic strain that underwent the thermal processes necessary for the production of foods and feeds. The goal of protecting the probiotic by encapsulating it in an appropriately formulated microcapsule was achieved. The viability rate of the encapsulated probiotic after the exposure of the microcapsules to moist heat was reduced by less than 1 log unit. Coating the alginate-based microcapsules with an EA-SA composite significantly improved the resistance of the
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encapsulated cells to moist heat exposure. SA played a significant role in the improved protection of the encapsulated probiotic. The encapsulation efficiency of the new microcapsules was greater than 90%, which was attributed to the use of a combination of electrospraying and a fluidized bed coating to produce the microcapsule and the use of mild encapsulation conditions. The newly developed multilayered microcapsules might serve as prototype encapsulation structures for the protection of other heat-sensitive microorganisms, compounds and nutrients used to fortify foods and feeds. Acknowledgements This study was granted by the Asahi Glass Foundation (Year 2014) and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission. Additionally, this project was partially funded by the National Research Council of Thailand (Year 2015). The authors especially thank Assoc. Prof. Parichat Hongsprabhas for her constructive discussion of this study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jfoodeng.2016.07.022. References Annan, N.T., Borza, A.D., Truelstrup Hansen, L., 2008. Encapsulation in alginatecoated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions. Food Res. Int. 41, 184e193. Association of official analytical chemists (AOAC), 2012. Official Methods of Analysis of AOAC International Methods 925.31, 19th ed. AOAC Int, Gaithersburg, MD. Ayama, H., Sumpavapol, P., Chanthachum, S., 2014. Effect of encapsulation of selected probiotics cell on survival in simulated gastrointestinal tract condition. Songklanakarin J. Sci. Technol. 36, 291e299. Corona-Hernandez, R.I., Alvarez-Parrilla, E., Lizardi-Mendoza, J., Islas-Rubio, A.R., de la Rosa, Laura A., Wall-Medrano, A., 2013. Structural stability and viability of microencapsulated probiotic bacteria: a review. Compr. Rev. Food Sci. Food Saf. 12, 614e628. Devi, N., Sarmah, M., Khatun, B., Maji, T.K., 2016. Encapsulation of active ingredients in polysaccharides-protein complex coacervates. Adv. Colloid interface Sci. http://dx.doi.org/10.1016/j.cis.2016.05.009. Dong, Q.Y., Chen, M.Y., Xin, Y., Qin, X.Y., Cheng, Z., Shi, L.E., Tang, Z.X., 2013. Alginatebased and protein-based materials for probiotics encapsulation: a review. Int. J. Food Sci. Technol. 48, 1339e1351. Fabra, M.J., Hambleton, A., Talens, P., Debeaufort, F., Chiralt, A., Voilley, A., 2009. Influence of films used for flavour encapsulation. Carbohydr. Polym. 325e332. rez-Masia , R., Sanchez, G., Lopez-Rubio, A., Gomez-Mascaraque, L.G., Morfin, R.C., Pe 2016. Optimization of electrospraying condition for the microencapsulation of probiotics and evaluation of their resistance during storage and in-vitro digestion. LWT Food Sci. Technol. 69, 438e446. brard, G., Hoffart, V., Beyssac, E., Cardot, J.M., Alric, M., Subirade, M., 2009. Coated He whey protein/alginate microparticles as oral controlled delivery systems for probiotic yeast. J. Microencapsul. 27, 292e302. Kiatyongchai, T., Wongsasulak, S., Yoovidhya, T., 2014. Coaxial electrospinning and release characteristics of cellulose acetate-gelatin blend encapsulating a model drug. J. Appl. Polym. Sci. 131, 40167e40175. Laelorspoen, N., Wongsasulak, S., Yoovidhya, T., Devahastin, S., 2014. Microencapsulation of Lactobacillus acidophilus in zeinealginate coreeshell microcapsules via electrospraying. J. Funct. Foods 7, 342e349. Lakkis, J.M., 2007. Encapsulation and Controlled Release Technologies in Food Systems. Patent, USA, pp. 1e3. Lee, Y.K., Salminen, S., 2009. Handbook of Probiotics and Prebiotic, second ed. John Wiley & Sons, Inc, New Jersey. Leong, J., Lam, W., Ho, K., Voo, W., Lee, M.F., Lim, H., Lim, S., Tey, B., Poncelet, D., Chan, E., 2016. Advances in fabricating spherical alginate hydrogels with controlled particle designs by ionotropic gelation as encapsulation systems. Particuology 24, 44e60. Lopez-Rubio, A., Lagaron, J.M., 2012. Whey protein capsules obtained though electrospraying for the encapsulation of bioactive. Innov. Food Sci. Emerg. Technol. 13, 200e206. Liu, Y., Sun, Y., Sun, L., Rizwan-ur-Rehman, Wang, Y., 2016. In vitro and in vivo study of sodium polyacrylate grafted alginate as microcapsule matrix for live probiotic delivery. J. Funct. Foods 24, 429e437. Martín, M.J., Lara-Villoslada, F., Ruiz, M.A., Morales, M.E., 2015. Microencapsulation
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