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Lactobacillus acidophilus La-05 encapsulated by spray drying: Effect of mucilage and protein from flaxseed (Linum usitatissimum L.)
LWT - Food Science and Technology 62 (2015) 1162e1168
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Lactobacillus acidophilus La-05 encapsulated by spray drying: Effect of mucilage and protein from flaxseed (Linum usitatissimum L.) nica Rubilar a, Carolina Shene a, b Mariela Bustamante a, *, Mario Villarroel a, Mo a
Center of Food Biotechnology and Bioseparations, Scientific and Technological Bioresource Nucleus, BIOREN, and Department of Chemical Engineering, Universidad de La Frontera, Ave. Francisco Salazar 01145, Box 54-D, Temuco, Chile b Centre for Biotechnology and Bioengineering (CeBiB), Universidad de La Frontera, Temuco, Chile
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 August 2014 Received in revised form 9 February 2015 Accepted 14 February 2015 Available online 21 February 2015
Flaxseed mucilage (FM) and soluble protein (FSP) were used as wall materials for encapsulating Lactobacillus acidophilus La-05 by spray drying. The effects of the content of FM in the encapsulating solution and drying temperature on the survival of bacterium after drying and its viability during storage were evaluated. Optimal conditions for maximizing the survival of L. acidophilus (78%) predicted by the surface response methodology were 0.2% w/v of FM and 110 C. These encapsulating conditions permitted almost a 2-fold increase of viability compared to the product encapsulated without FM. A further increase in survival (90%) was obtained by supplementing the encapsulating solution with heat-treated FSP. Moreover, the encapsulation of L. acidophilus enhanced its viability during incubation in simulated gastric acid and bile solutions. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Microencapsulation Spray drying Lactobacillus acidophilus Flaxseed mucilage Flaxseed soluble protein
1. Introduction The consumers' interest in foods that confer health benefits has led, in recent decades, to the growth of the probiotic product market. Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit to the host” (FAO/WHO, 2001). The species most used in functional foods belong to the lactobacilli and bifidobacteria genera (Holzapfel, € rkroth, & Schillinger, 2001). In the developHaberer, Geisen, Bjo ment of fermented dairy products the final cell content is a function of fermentation conditions. However, the addition of probiotics in non fermented products such as chocolate, probiotic capsules, tablets, and chewables requires stages of fermentation, harvesting and stabilization. Stabilization stages, in which moisture is removed by hot air, are critical because process conditions determine cell survival. Several studies have established that microencapsulation can improve survival and viability of probiotic (Ann ~o Barrozo et al., 2007; de Lara Pedroso, Thomazini, Jorda Heinemann, & Favaro-Trindale, 2012; McMaster, Kokott, & Slatter, 2005). Spray drying is widely used in the food industry because it is easily scaled-up, offers low operating cost and high production
* Corresponding author. Tel.: þ56 45 2325491; fax: þ56 45 2732402. E-mail address: [email protected] (M. Bustamante). http://dx.doi.org/10.1016/j.lwt.2015.02.017 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
rates. Nevertheless, the elevated temperatures used in spray drying can be stressful to cells affecting their survival (Boza, Barbin, & Scamparini, 2004). The fast rate of water removal may induce irreversible changes in the functional integrity of bacterial membranes and proteins (Crowe, Crowe, Carpenter, & Aurell-Wistrom, 1987). Several studies have shown that the composition and concentration of the solids in the encapsulating solution can improve viability of probiotics (Ann et al., 2007; Corcoran, Ross, Fitzgerald, & ^ncio, Pinto, Mun ~ oz, & Amboni, Stanton, 2004; Fritzen-Freire, Prude 2013). Enhancement of survival and viability is explained because cell encapsulation into a solid matrix offers protection not only during drying but also during storage. Moreover, encapsulation of probiotics might also improve viability during gastric transit. It has been suggested that the use of prebiotics, as encapsulating material could improve the survival of probiotics during spray drying. Prebiotics that have been used as encapsulating materials include polydextrose (Corcoran et al., 2004), inulin and oligofructose (Fritzen-Freire et al., 2013), lactulose and raffinose (Ann et al., 2007). Today, research is directed to find alternatives having similar or better characteristics. Flaxseed (Linum usitatissimum L.), also known as linseed, is a source of bioactive phenolic compounds, polyunsaturated fatty acids, dietary fiber and proteins (Bozan & Temelli, 2008; Oomah,
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2001). Bioactivity of flaxseed proteins includes the control of blood glucose level stimulating insulin secretion (Nuttall, Mooradian, Gannon, Billington, & Krezowski, 1984). The flaxseed mucilage (FM) contains a mixture of rhamnogalacturonan I and arabinoxylan; prebiotic properties of arabinoxylan has been described (Naran, Chen, & Carpita, 2008; Pastell, Westermann, Meyer, Tuomainen, & Tenkanen, 2009; Sørensen, Pedersen, & Meyer, 2006). Besides, the intake of FM has been suggested for the reduction blood glucose and cholesterol in diabetics (Thakur, Mitra, Pal, & Rousseau, 2009), the increase of fecal fat excretion in human (Kristensen et al., 2012), and for suppressing postprandial lipemia and hunger in young men (Kristensen et al., 2013). Nevertheless, in spite the bioactive properties of the mucilage and protein of flaxseed, its use in the food industry is limited. The aim of this work is to evaluate FM and soluble protein from flaxseed (FSP) as wall materials for the spray drying encapsulation of Lactobacillus acidophilus La-05. Survival of the lactic acid bacterium (LAB) after spray drying and viability of the encapsulated bacterium during storage at 4 C were evaluated. The viability of encapsulated and free LAB in simulated gastric juice and bile solution was determined. Morphology of the encapsulated products was investigated. The impact of FM on the growth of the LAB was also evaluated. 2. Materials & methods 2.1. Strains, chemicals, and culture media nsholm, L. acidophilus La-05 (La-05®) was from Chr. Hansen, (Ho Denmark). Malt dextrin (MD) of food grade with a dextrose equivalent (DE) of 15 was purchased from PRINAL®. Flaxseeds were purchased in the local market. Pepsin (0.7 FIPeU mg1) was obtained from Merck (Germany). Bile bovine was obtained from Sigma (USA). Lactobacilli broth (MRS broth, de Man, Rogosa, & Sharpe, 1960) and agar were from Difco (USA). All the chemicals were of analytical grade. 2.2. Extraction of FM and FSP The FM was extracted according to the Esparza, Leyton, Rubilar, and Shene (2011). Briefly, seeds were mixed (30 min) with hot distilled water (90e95 C), pH 5.0 at a ratio 1:10 w/v; the extraction cycle was performed twice. The extract was spread on a tray and dried at 60 C in an air convection heat oven. Mucilage-free seeds were dried (60 C), milled and passed through a 0.5 mm mesh. The meal was defatted with hexane twice (ratio 1:10, w/v) in a shaker (160 rpm, 8 h at 10 C). The defatted meal was recovered by filtration (Whatman® N 1) and the solvent was evaporated. The FSP was extracted from the defatted meal with 0.10 M NaCl in 0.10 M Tris buffer at pH 8.6 (ratio 1:16 w/v) (Li-Chan & Ma, 2002). The extraction was carried out in a shaker at 160 rpm for 16 h at 4 C. The extract was passed through a double layer of cheesecloth and centrifuged (7000 g, 4 C, 20 min). The supernatant was dried (60 C), milled and stored at 20 C until use. For the drying experiments the FSP was dispersed in distilled water and heated at 90 C for 10 min to destroy microorganisms and to allow protein denaturation and aggregation (Kiokias, Dimakou, & Oreopoulou, 2007). The chemical composition of FM and FSP were determined following the AOAC methods (AOAC, 1995). 2.3. Bacterial culture preparation The LAB was sub-cultured twice with 5% v/v inoculum in 5 mL of MRS broth (37 C, 12 h). For the spray drying assays 200 mL of MRS broth were inoculated (5% v/v) with the grown culture and
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incubated under the same conditions. Cells in late-log phase were harvested by centrifuging (5000 g, 4 C, 5 min); the pellet was washed with sterile distilled water and re-suspended in sterile distilled water. 2.4. Effect of FM on the growth of the LAB The MRS broth (5 mL) without glucose was supplemented with FM to a final concentration of 0.2 and 1% w/v. After sterilization (121 C, 15 min) the medium was inoculated (5% v/v) with the LAB from a second sub-culture and incubated at 37 C for 24 h. The growth was followed at 600 nm after proper dilution. Titratable acidity (TA) was determined (AOAC, 1995) and expressed as percent of lactic acid. Acid production after 24 h of incubation was determined by HPLC. The assay was performed in triplicate. 2.5. Spray drying Drying runs were performed in a laboratory spray dryer unit (LabPlant SD-05 dryer, Huddersfield, England) equipped with a 1.5 mm diameter nozzle, a spray chamber (500 mm length, 215 mm height) and a peristaltic pump. Bacterial suspensions (total viable counts between 108 and 109 CFU mL1) fed to the spray dryer contained MD (15% w/v) and different concentrations (0, 0.1 and 0.2% w/v) of FM and FSP (2, 6, and 12% w/v). Feeding rate was 6 g min1. The dry powder was collected in sterile glass bottles attached to the bottom of the cyclone, cooled to room temperature and stored at 4 C until their characterization. The experimental design corresponded to the matrix of response surface methodology (RSM) for two variables at two levels (Table 1). Each drying run was carried out in duplicate except for the central point which was made in triplicate. Viability of the encapsulated LAB during the storage at 4 C was followed for 45 days. 2.6. Viability of the free and encapsulated LAB in simulated gastric juice and bile solution The methodology described by Rajam, Karthik, Parthasarathi, Joseph, and Anandharamakrishnan (2012) with modifications was used. The simulated gastric juice was prepared in MRS broth adjusting the pH to 2.0 with sterile 1 M HCl. Pepsin solution was filtered through a 0.2 mm sterile membrane and added to the acidic MRS broth to reach a final concentration of 0.3% v/v. Bile tolerance was evaluated in MRS broth containing bovine bile (2% v/v). In both assays 0.1 g of the encapsulated LAB or 0.1 mL of LAB suspension were added to 4.9 mL of sterile simulated gastric juice or bile solution. Viable cell count was determined in samples taken immediately after mixing and every 2 h during the incubation at 37 C. Results were expressed as relative viability given by the ratio between viable cells at time “t” and that at time zero. 2.7. Analysis Concentrations of butyric, lactic, acetic, pyruvic and formic acids in culture samples (filtered under 0.2 mm) were determined by HPLC, using a Shodex KC-811 (Showa Denko KK, Tokyo, Japan) column kept at 40 C in an Alliance Waters e2695 Separation Module (Waters Inc, Mass, USA), detection was made at 210 nm (Waters Inc, Mass, USA). The sample (10 mL) was eluted with phosphoric acid 0.1% (v/v) at a rate of 1 mL min1. Calibration curves were constructed using acetic acid, formic acid, propionic acid, sodium butyrate, and pyruvic acid sodium salt (Merck, Germany) and L-(þ)-lactic acid (Scharlau Chemie S.A.). Survival after drying and viability during storage of the LAB were determined by the standard plate count method. Briefly, the
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Table 1 Effect of the flaxseed mucilage (FM) content in the encapsulating solution and drying inlet air temperature (T) on the survival of L. acidophilus La-05, logarithmic reduction of viable cells (LRVC), and viscosity of encapsulating solution (m). Response given by the lineal model derived from the experimental results is also shown. Independent variables FM (% w/v)
T ( C)
0.0
110 125 140 110 125 140 110 125 140
0.1
0.2
a b
ma (mPa s)
Experimental survivalb (%)
3.61 ± 0.01
69.84 62.37 47.12 73.05 67.02 58.62 78.43 71.02 63.30
5.48 ± 0.11
7.87 ± 0.03
± ± ± ± ± ± ± ± ±
1.40 1.73 3.33 3.88 2.57 0.63 0.29 0.19 4.75
Predicted survival (%)
Student residual
LRVC Log10 (N0/N)
69.04 60.32 51.61 74.61 65.89 57.18 80.18 71.46 62.75
0.49 1.11 2.75 0.84 0.55 0.78 1.07 0.24 0.34
2.96 3.69 5.07 2.62 3.09 4.02 1.92 2.71 3.57
± ± ± ± ± ± ± ± ±
0.12 0.10 0.12 0.35 0.50 0.10 0.08 0.00 0.42
Mean values of four experiments. Mean values of two replicates, except in central point in which were three replicates.
sample (0.1 g) was diluted in sterile buffered peptone water 0.1% w/ v (4.9 mL); the suspension was kept at 4 C for 30 min to release the cells. The appropriate dilution of the suspension was seeded on MRS agar and incubated at 37 C for 48 h. Viability of LAB during storage was expressed as colony forming units per gram (CFU g1) of dry power. The results were expressed as Log10 CFU g1. The percent survival after drying was calculated from (Simpson, Stanton, Fitzgerald, & Ross, 2005):
Survivalð%Þ ¼
N N0
100
where, N is Log10 CFU per g of the spray-dried powder immediately after drying and N0 is Log10 CFU per g of dry matter in the suspension fed to the dryer. Reduction of viable cells with respect to counts after drying was calculated from (Boza et al., 2004):
Logarithmic reduction of viable cells ðLRVCÞ ¼ Log10
N0 N
where, N0 is Log10 CFU per g of dry matter in the suspension fed to the dryer and N is Log10 CFU per g of the spray-dried powder. The viscosity of feed solutions was determined at 20 C using an AND Vibro Viscometer SV-10 (A&D Company, Limited, Japan). 2.8. Physical properties of the spray dried product The outer structure of dried particles was observed with a scanning electron microscope (SEM). The dry particles containing L. acidophilus La-05 were stored at 4 C until microscopic analysis. Then, the samples were dispersed over the sample holder which was equipped with a double sided carbon tape. The dried particles were gold-coated with Edward S150 Sputter Coater. Morphology was observed with a microscope JEOL JSM-6380LV (Jeol, Tokyo, Japan) at 10 kV and filament current at 50 mA. The particle size of dried particles was measured by laser diffraction (Zetasizer model Nano-ZS90, Malvern, UK) at 25 C using isopropyl as the continuous phase. 2.9. Statistics Statistical analysis was carried out using the Design Expert 6.0 statistical software (Stat-Ease, Minneapolis, Mn, USA). Analysis of variance (ANOVA) was used to determine significance of the effects (P < 0.05). Differences between means were detected using Duncan test (SPSS version 15.0).
3. Results and discussion 3.1. Effect of FM on the growth of the LAB Changes in growth, pH, and TA during fermentation of MRS broth supplemented with FM (0.2 and 1% w/v) as the carbon source are shown in Fig. 1. Main component in FM was the non-nitrogen extract (67.37%); contents of other components were 14.89% protein, 0.75% lipids, 2.50% fiber and 11.36% ash. Even though the LAB was able to grow in medium containing FM as carbon source, a high cell concentration was obtained at 0.2% w/v FM; the slow growth of the LAB could be due to the increase of medium viscosity when FM was tested at 1% w/v. It has been shown that FM has prebiotic activity increasing the concentration of L. acidophilus and Bifidobacterium lactis when kefir formulations were prepared with 2 g of FM per liter of milk (HadiNezhad, Duc, Fong Han, & Hosseinian, 2013). The pH decrease was explained by the synthesis of lactic, formic, and propionic acids (2.09, 1.13, and 0.11 g L1, respectively). Production of organic acids is one of the properties of probiotic bacteria; these can act as antimicrobials displacing pathogenic bacteria (Gibson, Probert, Van Loo, Rastall, & Roberfroid, 2004). 3.2. Effect of FM on the survival of the LAB after spray drying and viability during storage The effect of FM as a component of the encapsulating solution on the survival of the LAB after spray drying was tested at different inlet air temperature. The RSM was used to determine the significance of the effects on the survival of the LAB after drying; the results obtained in the 11 drying runs are shown in Table 1. Because FM exerted an important increase on the viscosity of the encapsulating solution (3.61 mPa s and 7.87 mPa s for FM concentration of 0 and 0.2% w/v, respectively) this was tested in a small range (0.1e0.2% w/v). A high viscosity of the solution fed to spray drying is not desirable because the size of the droplets increases demanding higher air inlet temperatures. The highest LAB survival (78.43%) was obtained with FM at 0.2% w/v and the lowest inlet air temperature (110 C) (run 7, Table 1). Even though drying at 140 C significantly reduced the survival of the LAB, FM reduces this effect being this concentration dependent; compared with the control (without FM) addition of FM at 0.2% w/v increased 34% the survival of the LAB while FM at 0.1% w/v increased the survival by 24%. The lineal relationship for the survival in terms of FM concentration and inlet air temperature (T) that fits the experimental data is given by:
Sð%Þ ¼ 65:89 þ 5:57 FM 8:71 T
r2 ¼ 0:94
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Fig. 1. Effect of flaxseed mucilage (FM) added to MRS broth on growth of L. acidophilus La-05. (a) Cell concentration (Log10 CFU mL1), (b) pH, and (c) titratable acidity. Concentration of FM (B) 0.2% w/v and (C) 1% w/v.
The model has predictive capability deduced from the signal to noise ratio (higher than 4), the small residual value and Student residual (Table 1). The ANOVA (Table 2) showed that both factors significantly contributed (P < 0.05) to the predicted survival of the LAB after drying. The response surface plot (Fig. 2) shows that the survival of the LAB increased as the inlet air temperature decreased from 140 C to 110 C and the concentration of FM increased from 0.0% to 0.2%. Viability of the LAB encapsulated with or without FM decreased gradually during storage at 4 C (Fig. 3). However, after one day of drying, viability of the LAB encapsulated with 0.2% w/v of FM and dried at 110 C (Fig. 3c) was higher than the control (without FM Fig. 3a) and with 0.1% w/v of FM (Fig. 3b). After 45 days the count of viable LAB in the products encapsulated with 0.1 and 0.2% w/v of FM and dried at 110 C was 6.24 and 6.84 Log CFU g1, respectively. These values are higher than the levels recommended (6 Log CFU g1) for exerting health benefits to the consumer (Roy, 2005). The enhanced survival and viability of the LAB encapsulated with FM at 0.2% w/v suggests that this flaxseed component would act as thermoprotector of cells undergoing the drying process. The functions of seed mucilage are still matters of speculation. It has been proposed that seed mucilage facilitate seed hydration or improve resistance to desiccation during brief drought after
absorption (Naran et al., 2008). These properties could promote the survival of LAB during drying. 3.3. Effect of the FSP on the survival of the LAB after spray drying Main component of FSP was protein 44.98%; contents of other components were 2.33% lipid, 0.96% fiber, 14.04% ash and 36.77% non-nitrogen extract. The effect of FSP at concentrations of 2, 6, and 12% w/v in the encapsulating solution on the survival of LAB after spray drying was evaluated by replacing partially MD (Table 3); drying temperature and FM in the encapsulating solution, were 110 C and 0.2% w/v, respectively. Survival of the LAB was significantly (P < 0.05) affected by FSP. Relatively low survivals were obtained with 0% (78.51%) and 2% (79.00%) w/v of FSP in the
Table 2 ANOVA for the overall effects of flaxseed mucilage (FM) concentration in the encapsulating solution and drying inlet air temperature (T) on the survival of the L. acidophilus La-05 after spray drying. Factors
DFa
Mean square
Fexpb
P>F
FM T
1 1
186.15 455.53
40.35 98.75
0.0002 <0.0001
a b
DF ¼ Degree of freedom. Fexp ¼ Fisher ratio. Test for comparing model variance with residual variance.
Fig. 2. Response surface plot showing the effects of the flaxseed mucilage (FM) content in the encapsulating material and inlet air temperature (T) on the percent survival of L. acidophilus La-05 after spray drying.
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Fig. 3. Viability during storage at 4 C of L. acidophilus La-05 encapsulated by spray drying with different concentrations of flaxseed mucilage in the encapsulating solution, (a) control without the flaxseed mucilage (FM), (b) 0.1% w/v FM, and (c) 0.2% w/v FM. Inlet air temperature (C) 110 C, (:) 125 C, and (B) 140 C.
encapsulating solution whereas the highest survival was obtained with 12% w/v of FSP (90.35%). In these experiments FSP was heat denaturated before it was added to the encapsulated solution because preliminary tests showed better results (not shown). The major fraction of flaxseed protein is globulin (Li-Chan & Ma, 2002), which exhibits poor mechanical properties because most of the hydrophobic and SH groups are buried inside the molecule. Denaturation changes the native structure of proteins allowing protein-protein interactions, disulphide cross-linking and hydrogen-bonding. These interactions make the denatured proteins stiffer, stronger and stretchable. 3.4. Viability of the encapsulated LAB after incubation in simulated gastric juice and bile solution The ability to survive at the adverse environmental conditions characteristic of the digestive tract (low pH at the stomach and bile salts in the first portion of the intestinal tract) is a desirable property that probiotics should meet. Cell encapsulation could enhance survival due to the protection conferred by the wall material. Fig. 4a
Table 3 Effect of the content of flaxseed soluble protein (FSP) in the encapsulating solution on the survival L. acidophilus La-05 after spray drying at 110 C. Other components in the encapsulating solution were malt dextrin (15% w/v) and flaxseed mucilage (0.2% w/v). FSP (% w/v)
Survival (%)
0 2 6 12
78.51 79.00 81.86 90.35
± ± ± ±
1.31c 0.32c 0.66b 0.76a
Different superscript letters in the same column indicate significant differences (P < 0.05).
shows the relative viability of encapsulated and free LAB in MRS broth containing 2% of bile salts; viability of the LAB encapsulated with FM (0.2% w/v) decreased after the first 2 h, remained relatively stable the next 2 h and finally grew reaching a relative viability of 0.85. The LAB encapsulated with FM and FSP (12% w/v) grew for 4 h and then remained constant reaching a relative viability of almost 1.20 whereas the free LAB showed a stable viability during 4 h after which a sharp decline was recorded (0.27). Fig. 4b shows the relative viability of the encapsulated and free LAB in a simulated gastric juice. A sharp decline in the number of LAB in all samples was observed. After 4 h no survival was detected in the free LAB sample whereas the encapsulated LAB with FM showed a decreased in relative viability which was almost constant in the last 3 h of the test. The LAB encapsulated with FM and FSP reached a relative viability of 0.47 after 6 h of incubation. The results obtained in other studies have shown mixed results, depending on the methodology, composition of reagents and strains evaluated. Pan, Chen, Wu, Tang, and Zhao (2009) determined no growth of L. acidophilus NIT after 2 h exposure to pH 2 whereas Rajam et al. (2012) determined that Lactobacillus plantarum encapsulated with denatured whey protein showed better stability compared with that encapsulated with untreated whey protein after 4 h incubation in simulated acidic and bile conditions; this behavior was explained due to the stronger and insoluble film forming property of denatured protein, limiting cells release. Dolly, Anishaparvin, Joseph, and Anandharamakrishnan (2011) found that the relative viability of L. plantarum mtcc 5422 had a gradual decline reaching null viability after 4 h exposition to pH 2.0 and bile salts. 3.5. Physical properties of the dry powders Fig. 5 shows the SEM micrographs of dry particles containing the LAB encapsulated with solutions of different composition, stored 1
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Fig. 4. Effect of (a) bile salt (2% v/v), (b) acid (pH 2.0) and pepsine (0.3% v/v) on the relative viability of L. acidophilus La-05 incubated at 37 C: (C) encapsulated with flaxseed mucilage (FM) (0.2% w/v), (:) encapsulated with FM (0.2% w/v) and flaxseed soluble protein (12% w/v), and (B) control (free LAB).
Fig. 5. The SEM micrographs of dry particles containing L. acidophilus La-05 stored at 4 C. Particles were obtained with an encapsulating solution that contained 15% w/v of malt dextrin and 0.2% w/v of flaxseed mucilage and dried with inlet air temperature of 110 C. Dry particles after: (a) 1 day of storage, and (b) 30 days of storage. Dry particles after 1 day of dried with flaxseed soluble protein (c) 2% w/v and (d) 12% w/v.
and 30 days at 4 C. The particles showed a spherical shape, wrinkled surface and varied size. The external surface did not show evidence of fissures or cracks. Fig. 5(aeb) shows the “flat ball” effect which may be related to the heat penetration and water evaporanovas, Ortega-Rivas, tion from droplet during drying (Barbosa-Ca Juliano, & Yan, 2005). Morphology of the particles did not change during storage (Fig. 5aeb). The particles of the product encapsulated with 2 or 12% FSP (Fig. 5ced) did not reveal any difference in terms of microstructure; however, the presence of wrinkles is lower than in the particles of the product encapsulated without FSP (Fig. 5aeb). Sheu and Rosenberg (1998) determined that the incorporation of whey proteins into wall material consisting of MD with high DE (15) minimized the presence of surface wrinkles associated with wall materials consisting of only MD. Entrapped cells were not visible in powder. Average size of the particles was 3.4 mm, varying between 0.06 mm and 7.0 mm.
The high viscosity of aqueous solution containing FM restricts its concentration in encapsulating solutions. The positive effect of FM and heat-treated FSP on the survival of the LAB during spray drying and the enhanced survival against bile solution and simulated gastric juice suggest that flaxseed could be a source of fractions for the design of encapsulating wall. Conflict of interest statement The authors declare that there are not conflicts of interest. Acknowledgments This research was supported by funding CONICYT through FONDECYT project 3130561. References
4. Conclusions Survival of L. acidophilus La-05 encapsulated by spray drying was highly dependent on the inlet air temperature an effect that can be reduced when FM is added to the encapsulation solution.
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~o Barrozo Heinemann, R., & Favarode Lara Pedroso, D., Thomazini, M., Jorda Trindale, C. S. (2012). Protection of Bifidobacterium lactis and Lactobacillus acidophilus by microencapsulation using spray-chilling. International Dairy Journal, 26, 127e132. Li-Chan, E. C. Y., & Ma, C. Y. (2002). Thermal analysis of flaxseed (Linum usitatissimum) proteins by differential scanning calorimetry. Food Chemistry, 77, 495e502. de Man, J. C., Rogosa, M., & Sharpe, E. (1960). A medium for the cultivation of Lactobacilli. Journal of Applied Bacteriology, 60, 130e135. McMaster, L. D., Kokott, S. A., & Slatter, P. (2005). Micro-encapsulation of Bifidobacterium lactis for incorporation into soft food. World Journal of Microbiology and Biotechnology, 21, 723e728. Naran, R., Chen, G., & Carpita, N. C. (2008). Novel rhamnogalacturonan I and arabinoxylan polysaccharides of flax seed mucilage. Plant Physiology, 148, 132e141. Nuttall, F. Q., Mooradian, A. D., Gannon, M. C., Billington, C., & Krezowski, P. (1984). Effect of protein ingestion on the glucose and insulin response to a standardized oral glucose load. Diabetes Care, 7, 465e470. Oomah, B. D. (2001). Flaxseed as a functional food source. Journal of the Science of Food and Agriculture, 81, 889e894. Pan, X., Chen, F., Wu, T., Tang, H., & Zhao, Z. (2009). The acid, bile tolerance and antimicrobial property of Lactobacillus acidophilus NIT. Food Control, 20, 598e602. Pastell, H., Westermann, P., Meyer, A. S., Tuomainen, P., & Tenkanen, M. (2009). In vitro fermentation of arabinoxylan-derived carbohydrates by Bifidobacteria and mixed fecal microbiota. Journal of Agricultural and Food Chemistry, 57, 8598e8606. Rajam, R., Karthik, P., Parthasarathi, S., Joseph, G. S., & Anandharamakrishnan, C. (2012). Effect of whey protein e alginate wall systems on survival of microencapsulated Lactobacillus plantarum in simulated gastrointestinal conditions. Journal of Functional Foods, 4, 891e898. Roy, D. (2005). Technological aspects related to the use of bifidobacteria in dairy products. Lait, 85, 39e56. Sheu, T. Y., & Rosenberg, M. (1998). Microstructure of microcapsules consisting of whey proteins and carbohydrates. Journal of Food Science, 63, 491e494. Simpson, P. J., Stanton, C., Fitzgerald, G. F., & Ross, R. P. (2005). Intrinsic tolerance of Bifidobacterium species to heat and oxygen and survival following spray drying and storage. Journal of Applied Microbiology, 99, 493e501. Sørensen, H. R., Pedersen, S., & Meyer, A. S. (2006). Optimization of reaction conditions for enzymatic viscosity reduction and hydrolysis of wheat arabinoxylan in an industrial ethanol fermentation residue. Biotechnology Progress, 22, 505e513. Thakur, G., Mitra, A., Pal, K., & Rousseau, D. (2009). Effect of flaxseed gum on reduction of blood glucose and cholesterol in type 2 diabetic patients. International Journal of Food Sciences and Nutrition, 60, 126e136.
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Lactobacillus acidophilus La-05 encapsulated by spray drying: Effect of mucilage and protein from flaxseed (Linum usitatissimum L.) nica Rubilar a, Carolina Shene a, b Mariela Bustamante a, *, Mario Villarroel a, Mo a
Center of Food Biotechnology and Bioseparations, Scientific and Technological Bioresource Nucleus, BIOREN, and Department of Chemical Engineering, Universidad de La Frontera, Ave. Francisco Salazar 01145, Box 54-D, Temuco, Chile b Centre for Biotechnology and Bioengineering (CeBiB), Universidad de La Frontera, Temuco, Chile
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 August 2014 Received in revised form 9 February 2015 Accepted 14 February 2015 Available online 21 February 2015
Flaxseed mucilage (FM) and soluble protein (FSP) were used as wall materials for encapsulating Lactobacillus acidophilus La-05 by spray drying. The effects of the content of FM in the encapsulating solution and drying temperature on the survival of bacterium after drying and its viability during storage were evaluated. Optimal conditions for maximizing the survival of L. acidophilus (78%) predicted by the surface response methodology were 0.2% w/v of FM and 110 C. These encapsulating conditions permitted almost a 2-fold increase of viability compared to the product encapsulated without FM. A further increase in survival (90%) was obtained by supplementing the encapsulating solution with heat-treated FSP. Moreover, the encapsulation of L. acidophilus enhanced its viability during incubation in simulated gastric acid and bile solutions. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Microencapsulation Spray drying Lactobacillus acidophilus Flaxseed mucilage Flaxseed soluble protein
1. Introduction The consumers' interest in foods that confer health benefits has led, in recent decades, to the growth of the probiotic product market. Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit to the host” (FAO/WHO, 2001). The species most used in functional foods belong to the lactobacilli and bifidobacteria genera (Holzapfel, € rkroth, & Schillinger, 2001). In the developHaberer, Geisen, Bjo ment of fermented dairy products the final cell content is a function of fermentation conditions. However, the addition of probiotics in non fermented products such as chocolate, probiotic capsules, tablets, and chewables requires stages of fermentation, harvesting and stabilization. Stabilization stages, in which moisture is removed by hot air, are critical because process conditions determine cell survival. Several studies have established that microencapsulation can improve survival and viability of probiotic (Ann ~o Barrozo et al., 2007; de Lara Pedroso, Thomazini, Jorda Heinemann, & Favaro-Trindale, 2012; McMaster, Kokott, & Slatter, 2005). Spray drying is widely used in the food industry because it is easily scaled-up, offers low operating cost and high production
* Corresponding author. Tel.: þ56 45 2325491; fax: þ56 45 2732402. E-mail address: [email protected] (M. Bustamante). http://dx.doi.org/10.1016/j.lwt.2015.02.017 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
rates. Nevertheless, the elevated temperatures used in spray drying can be stressful to cells affecting their survival (Boza, Barbin, & Scamparini, 2004). The fast rate of water removal may induce irreversible changes in the functional integrity of bacterial membranes and proteins (Crowe, Crowe, Carpenter, & Aurell-Wistrom, 1987). Several studies have shown that the composition and concentration of the solids in the encapsulating solution can improve viability of probiotics (Ann et al., 2007; Corcoran, Ross, Fitzgerald, & ^ncio, Pinto, Mun ~ oz, & Amboni, Stanton, 2004; Fritzen-Freire, Prude 2013). Enhancement of survival and viability is explained because cell encapsulation into a solid matrix offers protection not only during drying but also during storage. Moreover, encapsulation of probiotics might also improve viability during gastric transit. It has been suggested that the use of prebiotics, as encapsulating material could improve the survival of probiotics during spray drying. Prebiotics that have been used as encapsulating materials include polydextrose (Corcoran et al., 2004), inulin and oligofructose (Fritzen-Freire et al., 2013), lactulose and raffinose (Ann et al., 2007). Today, research is directed to find alternatives having similar or better characteristics. Flaxseed (Linum usitatissimum L.), also known as linseed, is a source of bioactive phenolic compounds, polyunsaturated fatty acids, dietary fiber and proteins (Bozan & Temelli, 2008; Oomah,
M. Bustamante et al. / LWT - Food Science and Technology 62 (2015) 1162e1168
2001). Bioactivity of flaxseed proteins includes the control of blood glucose level stimulating insulin secretion (Nuttall, Mooradian, Gannon, Billington, & Krezowski, 1984). The flaxseed mucilage (FM) contains a mixture of rhamnogalacturonan I and arabinoxylan; prebiotic properties of arabinoxylan has been described (Naran, Chen, & Carpita, 2008; Pastell, Westermann, Meyer, Tuomainen, & Tenkanen, 2009; Sørensen, Pedersen, & Meyer, 2006). Besides, the intake of FM has been suggested for the reduction blood glucose and cholesterol in diabetics (Thakur, Mitra, Pal, & Rousseau, 2009), the increase of fecal fat excretion in human (Kristensen et al., 2012), and for suppressing postprandial lipemia and hunger in young men (Kristensen et al., 2013). Nevertheless, in spite the bioactive properties of the mucilage and protein of flaxseed, its use in the food industry is limited. The aim of this work is to evaluate FM and soluble protein from flaxseed (FSP) as wall materials for the spray drying encapsulation of Lactobacillus acidophilus La-05. Survival of the lactic acid bacterium (LAB) after spray drying and viability of the encapsulated bacterium during storage at 4 C were evaluated. The viability of encapsulated and free LAB in simulated gastric juice and bile solution was determined. Morphology of the encapsulated products was investigated. The impact of FM on the growth of the LAB was also evaluated. 2. Materials & methods 2.1. Strains, chemicals, and culture media nsholm, L. acidophilus La-05 (La-05®) was from Chr. Hansen, (Ho Denmark). Malt dextrin (MD) of food grade with a dextrose equivalent (DE) of 15 was purchased from PRINAL®. Flaxseeds were purchased in the local market. Pepsin (0.7 FIPeU mg1) was obtained from Merck (Germany). Bile bovine was obtained from Sigma (USA). Lactobacilli broth (MRS broth, de Man, Rogosa, & Sharpe, 1960) and agar were from Difco (USA). All the chemicals were of analytical grade. 2.2. Extraction of FM and FSP The FM was extracted according to the Esparza, Leyton, Rubilar, and Shene (2011). Briefly, seeds were mixed (30 min) with hot distilled water (90e95 C), pH 5.0 at a ratio 1:10 w/v; the extraction cycle was performed twice. The extract was spread on a tray and dried at 60 C in an air convection heat oven. Mucilage-free seeds were dried (60 C), milled and passed through a 0.5 mm mesh. The meal was defatted with hexane twice (ratio 1:10, w/v) in a shaker (160 rpm, 8 h at 10 C). The defatted meal was recovered by filtration (Whatman® N 1) and the solvent was evaporated. The FSP was extracted from the defatted meal with 0.10 M NaCl in 0.10 M Tris buffer at pH 8.6 (ratio 1:16 w/v) (Li-Chan & Ma, 2002). The extraction was carried out in a shaker at 160 rpm for 16 h at 4 C. The extract was passed through a double layer of cheesecloth and centrifuged (7000 g, 4 C, 20 min). The supernatant was dried (60 C), milled and stored at 20 C until use. For the drying experiments the FSP was dispersed in distilled water and heated at 90 C for 10 min to destroy microorganisms and to allow protein denaturation and aggregation (Kiokias, Dimakou, & Oreopoulou, 2007). The chemical composition of FM and FSP were determined following the AOAC methods (AOAC, 1995). 2.3. Bacterial culture preparation The LAB was sub-cultured twice with 5% v/v inoculum in 5 mL of MRS broth (37 C, 12 h). For the spray drying assays 200 mL of MRS broth were inoculated (5% v/v) with the grown culture and
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incubated under the same conditions. Cells in late-log phase were harvested by centrifuging (5000 g, 4 C, 5 min); the pellet was washed with sterile distilled water and re-suspended in sterile distilled water. 2.4. Effect of FM on the growth of the LAB The MRS broth (5 mL) without glucose was supplemented with FM to a final concentration of 0.2 and 1% w/v. After sterilization (121 C, 15 min) the medium was inoculated (5% v/v) with the LAB from a second sub-culture and incubated at 37 C for 24 h. The growth was followed at 600 nm after proper dilution. Titratable acidity (TA) was determined (AOAC, 1995) and expressed as percent of lactic acid. Acid production after 24 h of incubation was determined by HPLC. The assay was performed in triplicate. 2.5. Spray drying Drying runs were performed in a laboratory spray dryer unit (LabPlant SD-05 dryer, Huddersfield, England) equipped with a 1.5 mm diameter nozzle, a spray chamber (500 mm length, 215 mm height) and a peristaltic pump. Bacterial suspensions (total viable counts between 108 and 109 CFU mL1) fed to the spray dryer contained MD (15% w/v) and different concentrations (0, 0.1 and 0.2% w/v) of FM and FSP (2, 6, and 12% w/v). Feeding rate was 6 g min1. The dry powder was collected in sterile glass bottles attached to the bottom of the cyclone, cooled to room temperature and stored at 4 C until their characterization. The experimental design corresponded to the matrix of response surface methodology (RSM) for two variables at two levels (Table 1). Each drying run was carried out in duplicate except for the central point which was made in triplicate. Viability of the encapsulated LAB during the storage at 4 C was followed for 45 days. 2.6. Viability of the free and encapsulated LAB in simulated gastric juice and bile solution The methodology described by Rajam, Karthik, Parthasarathi, Joseph, and Anandharamakrishnan (2012) with modifications was used. The simulated gastric juice was prepared in MRS broth adjusting the pH to 2.0 with sterile 1 M HCl. Pepsin solution was filtered through a 0.2 mm sterile membrane and added to the acidic MRS broth to reach a final concentration of 0.3% v/v. Bile tolerance was evaluated in MRS broth containing bovine bile (2% v/v). In both assays 0.1 g of the encapsulated LAB or 0.1 mL of LAB suspension were added to 4.9 mL of sterile simulated gastric juice or bile solution. Viable cell count was determined in samples taken immediately after mixing and every 2 h during the incubation at 37 C. Results were expressed as relative viability given by the ratio between viable cells at time “t” and that at time zero. 2.7. Analysis Concentrations of butyric, lactic, acetic, pyruvic and formic acids in culture samples (filtered under 0.2 mm) were determined by HPLC, using a Shodex KC-811 (Showa Denko KK, Tokyo, Japan) column kept at 40 C in an Alliance Waters e2695 Separation Module (Waters Inc, Mass, USA), detection was made at 210 nm (Waters Inc, Mass, USA). The sample (10 mL) was eluted with phosphoric acid 0.1% (v/v) at a rate of 1 mL min1. Calibration curves were constructed using acetic acid, formic acid, propionic acid, sodium butyrate, and pyruvic acid sodium salt (Merck, Germany) and L-(þ)-lactic acid (Scharlau Chemie S.A.). Survival after drying and viability during storage of the LAB were determined by the standard plate count method. Briefly, the
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Table 1 Effect of the flaxseed mucilage (FM) content in the encapsulating solution and drying inlet air temperature (T) on the survival of L. acidophilus La-05, logarithmic reduction of viable cells (LRVC), and viscosity of encapsulating solution (m). Response given by the lineal model derived from the experimental results is also shown. Independent variables FM (% w/v)
T ( C)
0.0
110 125 140 110 125 140 110 125 140
0.1
0.2
a b
ma (mPa s)
Experimental survivalb (%)
3.61 ± 0.01
69.84 62.37 47.12 73.05 67.02 58.62 78.43 71.02 63.30
5.48 ± 0.11
7.87 ± 0.03
± ± ± ± ± ± ± ± ±
1.40 1.73 3.33 3.88 2.57 0.63 0.29 0.19 4.75
Predicted survival (%)
Student residual
LRVC Log10 (N0/N)
69.04 60.32 51.61 74.61 65.89 57.18 80.18 71.46 62.75
0.49 1.11 2.75 0.84 0.55 0.78 1.07 0.24 0.34
2.96 3.69 5.07 2.62 3.09 4.02 1.92 2.71 3.57
± ± ± ± ± ± ± ± ±
0.12 0.10 0.12 0.35 0.50 0.10 0.08 0.00 0.42
Mean values of four experiments. Mean values of two replicates, except in central point in which were three replicates.
sample (0.1 g) was diluted in sterile buffered peptone water 0.1% w/ v (4.9 mL); the suspension was kept at 4 C for 30 min to release the cells. The appropriate dilution of the suspension was seeded on MRS agar and incubated at 37 C for 48 h. Viability of LAB during storage was expressed as colony forming units per gram (CFU g1) of dry power. The results were expressed as Log10 CFU g1. The percent survival after drying was calculated from (Simpson, Stanton, Fitzgerald, & Ross, 2005):
Survivalð%Þ ¼
N N0
100
where, N is Log10 CFU per g of the spray-dried powder immediately after drying and N0 is Log10 CFU per g of dry matter in the suspension fed to the dryer. Reduction of viable cells with respect to counts after drying was calculated from (Boza et al., 2004):
Logarithmic reduction of viable cells ðLRVCÞ ¼ Log10
N0 N
where, N0 is Log10 CFU per g of dry matter in the suspension fed to the dryer and N is Log10 CFU per g of the spray-dried powder. The viscosity of feed solutions was determined at 20 C using an AND Vibro Viscometer SV-10 (A&D Company, Limited, Japan). 2.8. Physical properties of the spray dried product The outer structure of dried particles was observed with a scanning electron microscope (SEM). The dry particles containing L. acidophilus La-05 were stored at 4 C until microscopic analysis. Then, the samples were dispersed over the sample holder which was equipped with a double sided carbon tape. The dried particles were gold-coated with Edward S150 Sputter Coater. Morphology was observed with a microscope JEOL JSM-6380LV (Jeol, Tokyo, Japan) at 10 kV and filament current at 50 mA. The particle size of dried particles was measured by laser diffraction (Zetasizer model Nano-ZS90, Malvern, UK) at 25 C using isopropyl as the continuous phase. 2.9. Statistics Statistical analysis was carried out using the Design Expert 6.0 statistical software (Stat-Ease, Minneapolis, Mn, USA). Analysis of variance (ANOVA) was used to determine significance of the effects (P < 0.05). Differences between means were detected using Duncan test (SPSS version 15.0).
3. Results and discussion 3.1. Effect of FM on the growth of the LAB Changes in growth, pH, and TA during fermentation of MRS broth supplemented with FM (0.2 and 1% w/v) as the carbon source are shown in Fig. 1. Main component in FM was the non-nitrogen extract (67.37%); contents of other components were 14.89% protein, 0.75% lipids, 2.50% fiber and 11.36% ash. Even though the LAB was able to grow in medium containing FM as carbon source, a high cell concentration was obtained at 0.2% w/v FM; the slow growth of the LAB could be due to the increase of medium viscosity when FM was tested at 1% w/v. It has been shown that FM has prebiotic activity increasing the concentration of L. acidophilus and Bifidobacterium lactis when kefir formulations were prepared with 2 g of FM per liter of milk (HadiNezhad, Duc, Fong Han, & Hosseinian, 2013). The pH decrease was explained by the synthesis of lactic, formic, and propionic acids (2.09, 1.13, and 0.11 g L1, respectively). Production of organic acids is one of the properties of probiotic bacteria; these can act as antimicrobials displacing pathogenic bacteria (Gibson, Probert, Van Loo, Rastall, & Roberfroid, 2004). 3.2. Effect of FM on the survival of the LAB after spray drying and viability during storage The effect of FM as a component of the encapsulating solution on the survival of the LAB after spray drying was tested at different inlet air temperature. The RSM was used to determine the significance of the effects on the survival of the LAB after drying; the results obtained in the 11 drying runs are shown in Table 1. Because FM exerted an important increase on the viscosity of the encapsulating solution (3.61 mPa s and 7.87 mPa s for FM concentration of 0 and 0.2% w/v, respectively) this was tested in a small range (0.1e0.2% w/v). A high viscosity of the solution fed to spray drying is not desirable because the size of the droplets increases demanding higher air inlet temperatures. The highest LAB survival (78.43%) was obtained with FM at 0.2% w/v and the lowest inlet air temperature (110 C) (run 7, Table 1). Even though drying at 140 C significantly reduced the survival of the LAB, FM reduces this effect being this concentration dependent; compared with the control (without FM) addition of FM at 0.2% w/v increased 34% the survival of the LAB while FM at 0.1% w/v increased the survival by 24%. The lineal relationship for the survival in terms of FM concentration and inlet air temperature (T) that fits the experimental data is given by:
Sð%Þ ¼ 65:89 þ 5:57 FM 8:71 T
r2 ¼ 0:94
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Fig. 1. Effect of flaxseed mucilage (FM) added to MRS broth on growth of L. acidophilus La-05. (a) Cell concentration (Log10 CFU mL1), (b) pH, and (c) titratable acidity. Concentration of FM (B) 0.2% w/v and (C) 1% w/v.
The model has predictive capability deduced from the signal to noise ratio (higher than 4), the small residual value and Student residual (Table 1). The ANOVA (Table 2) showed that both factors significantly contributed (P < 0.05) to the predicted survival of the LAB after drying. The response surface plot (Fig. 2) shows that the survival of the LAB increased as the inlet air temperature decreased from 140 C to 110 C and the concentration of FM increased from 0.0% to 0.2%. Viability of the LAB encapsulated with or without FM decreased gradually during storage at 4 C (Fig. 3). However, after one day of drying, viability of the LAB encapsulated with 0.2% w/v of FM and dried at 110 C (Fig. 3c) was higher than the control (without FM Fig. 3a) and with 0.1% w/v of FM (Fig. 3b). After 45 days the count of viable LAB in the products encapsulated with 0.1 and 0.2% w/v of FM and dried at 110 C was 6.24 and 6.84 Log CFU g1, respectively. These values are higher than the levels recommended (6 Log CFU g1) for exerting health benefits to the consumer (Roy, 2005). The enhanced survival and viability of the LAB encapsulated with FM at 0.2% w/v suggests that this flaxseed component would act as thermoprotector of cells undergoing the drying process. The functions of seed mucilage are still matters of speculation. It has been proposed that seed mucilage facilitate seed hydration or improve resistance to desiccation during brief drought after
absorption (Naran et al., 2008). These properties could promote the survival of LAB during drying. 3.3. Effect of the FSP on the survival of the LAB after spray drying Main component of FSP was protein 44.98%; contents of other components were 2.33% lipid, 0.96% fiber, 14.04% ash and 36.77% non-nitrogen extract. The effect of FSP at concentrations of 2, 6, and 12% w/v in the encapsulating solution on the survival of LAB after spray drying was evaluated by replacing partially MD (Table 3); drying temperature and FM in the encapsulating solution, were 110 C and 0.2% w/v, respectively. Survival of the LAB was significantly (P < 0.05) affected by FSP. Relatively low survivals were obtained with 0% (78.51%) and 2% (79.00%) w/v of FSP in the
Table 2 ANOVA for the overall effects of flaxseed mucilage (FM) concentration in the encapsulating solution and drying inlet air temperature (T) on the survival of the L. acidophilus La-05 after spray drying. Factors
DFa
Mean square
Fexpb
P>F
FM T
1 1
186.15 455.53
40.35 98.75
0.0002 <0.0001
a b
DF ¼ Degree of freedom. Fexp ¼ Fisher ratio. Test for comparing model variance with residual variance.
Fig. 2. Response surface plot showing the effects of the flaxseed mucilage (FM) content in the encapsulating material and inlet air temperature (T) on the percent survival of L. acidophilus La-05 after spray drying.
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Fig. 3. Viability during storage at 4 C of L. acidophilus La-05 encapsulated by spray drying with different concentrations of flaxseed mucilage in the encapsulating solution, (a) control without the flaxseed mucilage (FM), (b) 0.1% w/v FM, and (c) 0.2% w/v FM. Inlet air temperature (C) 110 C, (:) 125 C, and (B) 140 C.
encapsulating solution whereas the highest survival was obtained with 12% w/v of FSP (90.35%). In these experiments FSP was heat denaturated before it was added to the encapsulated solution because preliminary tests showed better results (not shown). The major fraction of flaxseed protein is globulin (Li-Chan & Ma, 2002), which exhibits poor mechanical properties because most of the hydrophobic and SH groups are buried inside the molecule. Denaturation changes the native structure of proteins allowing protein-protein interactions, disulphide cross-linking and hydrogen-bonding. These interactions make the denatured proteins stiffer, stronger and stretchable. 3.4. Viability of the encapsulated LAB after incubation in simulated gastric juice and bile solution The ability to survive at the adverse environmental conditions characteristic of the digestive tract (low pH at the stomach and bile salts in the first portion of the intestinal tract) is a desirable property that probiotics should meet. Cell encapsulation could enhance survival due to the protection conferred by the wall material. Fig. 4a
Table 3 Effect of the content of flaxseed soluble protein (FSP) in the encapsulating solution on the survival L. acidophilus La-05 after spray drying at 110 C. Other components in the encapsulating solution were malt dextrin (15% w/v) and flaxseed mucilage (0.2% w/v). FSP (% w/v)
Survival (%)
0 2 6 12
78.51 79.00 81.86 90.35
± ± ± ±
1.31c 0.32c 0.66b 0.76a
Different superscript letters in the same column indicate significant differences (P < 0.05).
shows the relative viability of encapsulated and free LAB in MRS broth containing 2% of bile salts; viability of the LAB encapsulated with FM (0.2% w/v) decreased after the first 2 h, remained relatively stable the next 2 h and finally grew reaching a relative viability of 0.85. The LAB encapsulated with FM and FSP (12% w/v) grew for 4 h and then remained constant reaching a relative viability of almost 1.20 whereas the free LAB showed a stable viability during 4 h after which a sharp decline was recorded (0.27). Fig. 4b shows the relative viability of the encapsulated and free LAB in a simulated gastric juice. A sharp decline in the number of LAB in all samples was observed. After 4 h no survival was detected in the free LAB sample whereas the encapsulated LAB with FM showed a decreased in relative viability which was almost constant in the last 3 h of the test. The LAB encapsulated with FM and FSP reached a relative viability of 0.47 after 6 h of incubation. The results obtained in other studies have shown mixed results, depending on the methodology, composition of reagents and strains evaluated. Pan, Chen, Wu, Tang, and Zhao (2009) determined no growth of L. acidophilus NIT after 2 h exposure to pH 2 whereas Rajam et al. (2012) determined that Lactobacillus plantarum encapsulated with denatured whey protein showed better stability compared with that encapsulated with untreated whey protein after 4 h incubation in simulated acidic and bile conditions; this behavior was explained due to the stronger and insoluble film forming property of denatured protein, limiting cells release. Dolly, Anishaparvin, Joseph, and Anandharamakrishnan (2011) found that the relative viability of L. plantarum mtcc 5422 had a gradual decline reaching null viability after 4 h exposition to pH 2.0 and bile salts. 3.5. Physical properties of the dry powders Fig. 5 shows the SEM micrographs of dry particles containing the LAB encapsulated with solutions of different composition, stored 1
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Fig. 4. Effect of (a) bile salt (2% v/v), (b) acid (pH 2.0) and pepsine (0.3% v/v) on the relative viability of L. acidophilus La-05 incubated at 37 C: (C) encapsulated with flaxseed mucilage (FM) (0.2% w/v), (:) encapsulated with FM (0.2% w/v) and flaxseed soluble protein (12% w/v), and (B) control (free LAB).
Fig. 5. The SEM micrographs of dry particles containing L. acidophilus La-05 stored at 4 C. Particles were obtained with an encapsulating solution that contained 15% w/v of malt dextrin and 0.2% w/v of flaxseed mucilage and dried with inlet air temperature of 110 C. Dry particles after: (a) 1 day of storage, and (b) 30 days of storage. Dry particles after 1 day of dried with flaxseed soluble protein (c) 2% w/v and (d) 12% w/v.
and 30 days at 4 C. The particles showed a spherical shape, wrinkled surface and varied size. The external surface did not show evidence of fissures or cracks. Fig. 5(aeb) shows the “flat ball” effect which may be related to the heat penetration and water evaporanovas, Ortega-Rivas, tion from droplet during drying (Barbosa-Ca Juliano, & Yan, 2005). Morphology of the particles did not change during storage (Fig. 5aeb). The particles of the product encapsulated with 2 or 12% FSP (Fig. 5ced) did not reveal any difference in terms of microstructure; however, the presence of wrinkles is lower than in the particles of the product encapsulated without FSP (Fig. 5aeb). Sheu and Rosenberg (1998) determined that the incorporation of whey proteins into wall material consisting of MD with high DE (15) minimized the presence of surface wrinkles associated with wall materials consisting of only MD. Entrapped cells were not visible in powder. Average size of the particles was 3.4 mm, varying between 0.06 mm and 7.0 mm.
The high viscosity of aqueous solution containing FM restricts its concentration in encapsulating solutions. The positive effect of FM and heat-treated FSP on the survival of the LAB during spray drying and the enhanced survival against bile solution and simulated gastric juice suggest that flaxseed could be a source of fractions for the design of encapsulating wall. Conflict of interest statement The authors declare that there are not conflicts of interest. Acknowledgments This research was supported by funding CONICYT through FONDECYT project 3130561. References
4. Conclusions Survival of L. acidophilus La-05 encapsulated by spray drying was highly dependent on the inlet air temperature an effect that can be reduced when FM is added to the encapsulation solution.
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