3.1 Characterization of microcapsules of Pediococcus pentosaceus P107
As for the moisture content for probiotic cultures must be constant and below 5%, in order to obtain stability in long-term storage [19, 20]. In the present study, the microcapsules showed humidity values of 0.86% (± 0.01), 1.93% (± 0.02), and 3.40% (± 0.01) for W, WX and WP respectively, being considered adequate according to the ideal value mentioned above.The different values can be attributed to differences in the wall materials of each microcapsule and to the total content of encapsulating material, since it facilitates the formation of more compact walls, which may restrict the diffusion of water vapor from the interior of the microcapsule to the surface during spray drying [21].
In literature, a value ratio above the recommended 5% or 5 g 100g− 1 was observed. Fávaro-Trindade & Grosso [22] found moisture content between 3.2 and 5.3 g 100g− 1 in the powders obtained from the microencapsulation of Lactobaillus acidophylus La-05 and Bifidobacterium lactis Bb-2 spray dried. Likewise, Rajam & Anandharamakrishnan [21], observed values that ranged from 5.52 to 9.47%, in microcapsules containing fructooligosaccharides or whey proteins [21]. Determining points such as equipment parameters and choice of wall material must be considered [23, 24].
The water activity values of the microcapsules obtained in this study, 0.233 ± 0.01, 0.244 ± 0.01 and 0.265 ± 0.02, respectively, are within the normal range (less than 0.6) for atomized product and the minimum required to maintain cell viability [25]. The literature reported that the water activity values of less than 0.3 increases the stability of the dried product, due to the lower amount of free water available for biochemical reactions, ie, longer life [11, 25, 26].
As for the color attribute, all microcapsules showed high values for L *, 95.65, 94.24 and 96.12 respectively for W, WX and WP, indicating light colors. Negative values for a *, -1.67, -0.56 and − 0.24 respectively for W, WX and WP, indicating shades of green and finally b * positive values, 5.68, 7.19 and 4.82 also respectively for W, WX and WP, indicating trend in yellow. These parameters can be assigned to the predominant use of whey for the three solutions produced microcapsules, since it has a light yellow color, explaining the tendency of microcapsules to yellow [11, 27]. The final color may impact the food matrix to be applied, white pattern microcapsules have convenient features for applications in different formulations [28].
Figure 1 shows Scanning Electron Microscopy images (SEM) of the microcapsules W, containing whey (ab), WX, containing xanthan and whey (bc), WP, containing whey and pectin (a). The microcapsules containing whey (W), and whey with xanthan gum (WX) are symmetrical and rounded with average sizes of 12.32 µm ± 0.80, 9.87 ± 0.76, respectively. In contrast, microcapsules with whey and pectin (WP) had an average size of 6.99 ± 0.67 µm and some deformations on it´s surface, which may be due to the interaction of the material with the high pressure exerted by spraying and to shrinkage during the process and cooling [29].
Considering the size of the microcapsules obtained, they are adequate (up to 80 µm) to be inserted in foods because small particles ensure a homogeneous and high quality product without affecting the sensory properties [9]. Rosolen et al. [15] observed midst values of 12.73 µm for L. lactis R7 microcapsules as whey and inulin. Otherwise, Nunes et al. [4] obtained microcapsules Lactobacillus acidophilus with inulin, trehalose, and Hi-maize by spraying, having sizes between 6.68 µm and 7.30 µm
3.1 Microcapsule rupture test, viability, encapsulation efficiency (EE) and process yield
Table 1 shows the plug effect of 100 mM PBS (pH 7.4) and the saline solution (0.5% pH. 2.5 with 3 pepsin mg.mL− 1), referred to from now on as PBS buffer and gastric solution, respectively, about the disruption of the microcapsules studied.
Table 1
Release of bacterial cells from microcapsules when immersed into the PBS buffer or simulated gastric fluids with various exposure times at 37 ° C.
Time
(min)
|
PBS buffer 100 mM pH 7.4
|
Gastric Solution (saline solution 0.5% pH 2.5 and pepsin 3 mg.mL− 1)
|
Log CFU g− 1
|
W WX WP W WX WP
|
30
60
120
|
12.85 ± 0.34a,A 7.09 ± 0.13c,B 0.00 ± 0.00a,B
12.69 ± 0.10a,A 8.15 ± 0.15b,B 0.00 ± 0.00a,B
11.87 ± 0.67a,A 9.15 ± 0.15a,B 0.00 ± 0.00a,B
|
8.92 ± 0.07a,B 12.99 ± 0.20a,A 13.63 ± 0.15a,A
8.76 ± 0.26a,B 11.58 ± 0.51b,A 9.15 ± 0.15b,A
8.44 ± 0.39a,B 11.15 ± 0.15b,A 9.00 ± 0.10b,A
|
W: microcapsule whey; |
WX: microcapsule whey and xanthan; |
WP: microcapsule whey and pectin; |
Results represent the mean (standard deviations), n = 3. |
a−c Different superscript lowercase letters in the same column represent statistical difference (p < 0.05). |
Means with different superscript capital letters represent a defined difference (p < 0.05) between the solutions tested for the same microcapsule. |
The PBS plug at a concentration of 100 mM has been described in the literature as a microcapsule tear solution [4, 11, 26, 30]. Conforming these results, the whey (W) microcapsule of the present study, when exposed to the PBS plug over 30 min time showed a viable cell concentration of 12.85 Log CFU g− 1. In contrast, the same plug promoted partial disruption of microcapsules in WX and was not able to rupture the microcapsules WP and cause total exposure of the microorganism.
When the gastric solution was analyzed, higher cell concentrations were obtained at 30 min for WX (12.99 Log CFU.g− 1) and WP (13.63 Log CFU.g− 1), reducing significantly (p < 0.05) the cell count depending on exposure time. It is noteworthy that both pectin as xanthan thickeners have properties that are strongly influenced by acidic pH and by the temperature of 37 ° C, compromising the structure of the microcapsule [16, 31].
Understanding the tearing process of microcapsules and delivery of its content is complex, as it involves several factors. There are few reports in the literature which explore the tearing properties of encapsulated bacteria, being some of the mechanisms described diffusion, erosion and fragmentation. In addition, factors such as changes in pH, activity of proteolytic enzymes, osmotic stress and time staying in a tearing solution, contribute to the process [32].
Whey proteins when bound to pectin polysaccharides form complexes through non-covalent interactions, as electrostatic forces in both solution and interfaces, hydrogen bonding, and hydrophobic interactions under various conditions. Xanthan, when in acid solution (pH 3.0) is negatively charged while whey proteins are positive, when this repulsive electrostatic force cancels out, the particles coated with both materials quickly separate, causing ruptures of the microcapsule and exposing the microorganism [33].
The initial viable cell count for W capsules, WX and WP was 13.50 Log CFU.g− 1, 13.67 Log CFU.g− 1 and 13.76 Log CFU.g− 1, respectively. After the drying process the microcapsules W and WX reduced 1.05 Log CFU.g− 1 and 1.28 Log CFU.g− 1, respectively, showing no significant difference (p > 0.05). The WP microcapsules obtained the lowest cell reduction (0.13 Log CFU.g− 1) showing the highest encapsulation efficiency of 99.05% in relation to the others, as shown in Table 2.
Table 2
Cell viability, encapsulation efficiency and yield of the process of Pediococcus pentosaceus P107 microencapsulated using different materials before and after spray drying.
|
Number of viable cells
(Log CFU.g− 1)
|
Reduction
(Log CFU.g− 1)
|
Encapsulation Efficiency (EE%)
|
Yield
(%)
|
|
Before spray drying
|
After spray drying
|
|
|
|
W
|
13.50 ± 0.20
|
12.45 ± 0.71
|
1.05 ± 0.21a
|
92.22 ± 0.50b
|
17.37 ± 1.05a
|
WX
|
13.67 ± 0.50
|
12.39 ± 0.80
|
1.28 ± 0.30a
|
90.63 ± 0.25c
|
9.58 ± 0.98b
|
WP
|
13.76 ± 0.22
|
13.63 ± 0.50
|
0.13 ± 0.28b
|
99.05 ± 0.20a
|
15.23 ± 0.67a
|
W: microcapsule whey; |
WX: microcapsule whey and xanthan; |
WP: microcapsule whey and pectin; |
Results represent the mean (standard deviations), n = 3. |
a−cMeans with different superscript lowercase letters in the same column represent statistical difference (p < 0.05). |
The combination of whey and pectin culminated in the best encapsulation efficiency, which can be explained in the fact that this polysaccharide a nano-porous polymer, (2–50 nm) allowing only water and smaller particles to diffuse into the produced microcapsules. P. pentosaceus P107 has an average size ranging from 2–8 µm, making it comparatively larger than the pores of the complex used, allowing the retention of a large number of bacteria inside, when compared to other encapsulating materials [34].
Whey proteins have the ability to interact with glycoproteins present on the bacterial surface, making them a biocompatible material due to the adhesive potential in the protection of the microorganism. When mixed solutions of whey proteins and xanthan polysaccharides are heated there is competition between the gelling and separation processes of phases. Once gelling occurs the basic structure of the gel is established and therefore the separation of phases is delayed, which contributes to the formation of a continuous and stable microcapsule to the drying process [35, 36].
Considering the yield of the process is influenced by drying parameters, added to environmental and physical-chemical properties of the encapsulating material, the yield obtained in the present study for W was 17.37%, 9.58% for WX and 15.23% for WP, with W and WP being significantly larger. Much of the work brings the yield factor as process yield, ie, the efficiency of the same. Being comparative studies only a few, Arslan-Tontul & Erbas [37] found that yields ranged from 39.33 g to 54.65 g to 100g when microencapsulating Saccharomyces cerevisiae var: Boulardii with different wall materials by spraying.
3.2 Feasibility of storage of microencapsulated cells of P. pentosaceus P107 at different temperature
In this study, the microcapsules were evaluated by observing their ability to maintain the viability of P. pentosaceus P107 during storage at different temperatures (-20, 4 and 25°C) (Table 3).
Table 3
Viability of Pediococcus pentosaceus P107 microencapsulated for 180 days.
*Temperature
|
Time -20°C
|
4°C
|
25°C
|
(days) W WX WP
|
W WX WP
|
W WX WP
|
0
7
14
21
30
45
60
75
110
140
180
|
12.45 ± 0.71a
10.95 ± 0.04b
9.15 ± 0.15c
8.52 ± 0.60c
8.00 ± 0.00c
7.15 ± 0.15b
7.77 ± 0.01b
7.44 ± 0.39a
7.84 ± 0.01a
7.84 ± 0.06a
7.58 ± 0.21a
|
12.39 ± 1.00a
10.40 ± 0.80b
10.58 ± 0.51b
10.00 ± 0.00b
9.88 ± 0.51b
10.00 ± 0.01a
9.58 ± 0.51a
8.12 ± 0.11b
7.85 ± 0.15a
7.54 ± 0.06b
7.27 ± 0.02a
|
13.63 ± 0.50a
13.54 ± 0.06a
13.00 ± 0.00a
12.69 ± 0.35a
12.58 ± 0.51a
8.88 ± 0.78a
8.15 ± 0.15b
7.95 ± 0.04a,b
7.77 ± 0.68a
7.74 ± 0.17a,b
7.59 ± 0.11a
|
12.45 ± 0.71a
10.00 ± 0.00b
8.80 ± 0.06b
8.58 ± 0.51c
8.58 ± 0.55b
7.47 ± 0.02c
7.54 ± 0.06b
7.44 ± 0.39a
7.59 ± 0.27a
7.46 ± 0.15a
7.15 ± 0.15a
|
12.39 ± 1.00a
9.95 ± 0.42b
9.58 ± 0.51b
9.99 ± 0.61b
9.89 ± 0.78b
9.00 ± 0.22b
8.72 ± 0.12a
8.00 ± 0.00a
7.58 ± 0.51a
7.52 ± 0.04a
7.27 ± 0.02a
|
13.63 ± 0.50a
13.54 ± 0.06a
13.51 ± 0.20a
12.00 ± 0.00a
12.00 ± 0.03a
11.59 ± 0.11a
7.69 ± 0.35b
7.88 ± 0.78a
7.85 ± 0.15a
7.52 ± 0.04a
7.15 ± 0.15a
|
12.45 ± 0.71a
11.25 ± 0.24b
7.90 ± 0.05b
7.62 ± 0.15b
7.60 ± 0.00b
7.56 ± 0.24b
7.54 ± 0.06a
7.47 ± 0.00a
7.47 ± 0.02a
7.18 ± 0.14a
6.13 ± 0.39a
|
12.39 ± 1.00a
8.16 ± 0.15c
7.16 ± 0.15c
VC
VC
VC
VC
VC
VC
VC
VC
|
13.63 ± 0.50a
13.39 ± 0.08a
13.25 ± 0.24a
13.15 ± 0.15a
13.00 ± 0.12a
10.69 ± 0.35a
6.44 ± 0.39b
VC
VC
VC
VC
|
*Storage at freezing: -20°C; refrigeration: 4°C, and ambient temperatures: 25°C. |
a−c Means ± standard deviation with different superscript capital letters indicate significant difference (p < 0.05) between microcapsules in the same period and storage temperature. |
VC = Viable cells < 6 log CFU.g− 1; |
W: microcapsule with only whey; |
WX: microcapsule with whey and xanthan; |
WP: microcapsule with whey and pectin; |
The Cell viability presented was > 6 log CFU.g− 1 in W microcapsules, at the end of 180 days for the three storage temperatures. Similar results were shown by the WX microcapsules (7.27, 7.27 log CFU.g− 1) and WP (7.59, 7.15 log CFU.g− 1) but at temperatures of -20 ° C and 4 ° C, respectively. Likewise, Liao et al. [38] evaluated the feasibility to storage at temperatures of -20°C, 4°C, 25°C and 37°C Lactobacillus casei K1, microencapsulated by spray drying. The authors reported a high viability when stored at -20°C over the other temperatures. According to De Castro- Cislaghi et al. [30] the stability of the microencapsulated probiotic is increased in low temperatures, such as refrigeration and freezing temperature.
During storage at 25 ° C, there was loss of cell viability from the 21st day for WX and 75th days for WP, showing that the temperature has negative effects on the wall of the microcapsule and thus, on the micro-organism protection. The whey used alone plays an important role as the encapsulating agent to maintain the viability of P. pentosaceus P107 during storage. Moreover, as many LAB are indigenous in milk, the whey is considered a suitable matrix, making the microcapsules an environment with chemical-physical and biological characteristics adequate to maintain these microorganisms [39].
Oliveira et al., [24] found higher viability losses at 37°C when compared to 7°C for microencapsulated Bifidobacterium lactis by coacervation with casein and pectin, followed by atomization. Just like Sagardia-Vega et al. [40] that encapsulated Lactobacillus fermentum UCO-979C with alginate and xanthan, added or not of oil and found high viability at 4°C when compared to 25°C. The adaptation to the environment, such as heat stress and moisture content are important conditions for probiotics to survive at high temperatures [41]. A greater reduction in the number of encapsulated microorganisms is observed in the stored at 25°C, because at this temperature the metabolic activity is higher and nutrients are consumed quickly. Similarly other studies have shown that microencapsulation using thermoprotective agents increases the survival of microorganisms during storage because mechanical, oxidative and osmotic stress is minimized [42].
3.3 Survival of microencapsulated Pediococcus pentosaceus P107 passage the gastrointestinal tract simulated
One of the main barriers for oral administration of probiotic bacteria is the low pH of the stomach and associated high concentration of hydrochloric acid, the encapsulation technique being a useful tool to increase the protection of such micro-organisms when exposed to these conditions.
In the present study, significant protection by the WX microcapsules, demonstrated by cell viability higher than 10 Log CFU.g− 1 of P. pentosaceus P107, 120 min after the passage in the simulated gastric tract at different pH (2.0, 2.5) (Fig. 2a, 2b) and greater than 9 Log CFU.g− 1 at pH 3.0 (Fig. 2c). It can be explained that the very low pH of electrostatic bonds between xanthan and whey begin to disappear once amine groups deionize, while carboxyl groups retain a negative charge. The network formed by the encapsulant material is capable of expanding and absorbing water to buffer the acid compounds present in the gastric fluid, as they penetrate the microcapsules [43].
In contrast, both W and WP microcapsules, after the same period (120 days) had significantly lower values (< 9 Log CFU.g− 1) demonstrating that the enzyme action time and acid concentration are crucial for the integrity of the microcapsule and protection of the micro-organism. It has been reported that the dense network of hydrogel formed by combining whey to xanthan or pectin reduces the diffusion rate from the microcapsule, thereby reducing the exposure of the microorganism to the acidic medium which it is exposed [39, 44].
The presence or absence of bile salts in the simulated intestinal juice did not affect the viability of P. pentosaceus P107 (Fig. 2d). In the absence of bile salts, the microcapsules showed no significant difference. However, the presence of bile salts promoted microcapsule rupture and exposure of the microorganism, showing a reduction of 4.16, 3.41, 5.94 Log CFU.g− 1 to W, WX and WP, respectively.
Chen et al. [43] demonstrated that xanthan microcapsules linked with chitosan, when exposed to 1% bile salts for 120 min presented a reduction in 2.06 Log CFU.g− 1. On the other hand, Rosolen et al. [15] found a high cell viability in cells of Lactococcus lactis subsp. lactis R7 microencapsulated, after 240 min exposure to simulated intestinal tract both in the presence (reduction of 2.3 Log CFU.g− 1) and in the absence of bile salts (2.88 Log CFU.g− 1).
However, results may be affected by the encapsulating material used or by the metabolic interaction of the microorganism or because the natural resistance of these bacteria to different pH and digestive enzymes [45].
3.4 Evaluation of heat resistance
The survival of P. pentosaceus P107 microencapsulated exposed at temperatures of 65 ° C for 30 min and 72 ° C for 15s, is shown in Table 4.
Table 4
Viability and percentage of survival of Pediococcus pentosaceus P107 microencapsulated and subjected to heat treatment at different temperatures
Microcapsule
|
Survival rate (%)
|
|
65°C by 30 min
|
72°C by 15 s
|
Whey (W)
|
84.37 ± 1.12c
|
90.75 ± 0.25b
|
Whey and Xanthan (WX)
|
87.91 ± 0.18b
|
99.18 ± 0.18a
|
Whey and Pectin (WP)
|
92.46 ± 0.78a
|
99.31 ± 0.69a
|
W: whey microcapsule; |
WX: whey and xanthan microcapsule; |
WP: m whey and pectin icrocapsule; |
Results represent the mean (standard deviations), n = 3. |
a−c Means with different superscript lowercase letters in the same column represent statistical difference (p < 0.05). |
The results showed that the microencapsulated cell with whey (W) was significantly more sensitive to heat treatment (p < 0.05), but still obtained counts that demonstrate probiotic potential (> 6 Log CFU.g− 1) in both of the tests. For heat treatment of 65°C for 30 min the best results were presented by WP microcapsules with a survival rate of 92.46%, followed by WX with 87.91% survival. It can be observed that there was no change in the survival of P. pentosaceus P107 (WP and WX) when subjected to 72°C for 15s.
In the study of Ilha et al. [19] the Lactobacillus paracasei FNU cells microencapsulated with skim milk and whey by spray drying were subjected to thermal testing at 65°C for 30 min and showed a percentage of survival of approximately 70%. Etchepare et al. [46] evaluated the viability of L. acidophillus microencapsulated in multiple layers by ion gelling using calcium alginate and whey. When the particle containing alginate is submitted to the thermal resistance test (72°C for 15 s) 77% survival was observed. When the particle receives a layer of whey the survival increases to 82%, demonstrating the effect of the coatings used in the protection of the microorganism. However the values are lower than those obtained in the present study
Few studies evaluate the protection the microcapsule can provide the microorganism when exposed to lethal temperatures. The high temperature and short time (72°C for 15 sec) is preferred for products containing encapsulated probiotics because the microcapsule is capable of protecting from the generated damage, such as protein denaturation and destruction of nucleic acids, which would lead to cellular apoptosis [47]. The viscoelastic properties of polysaccharides associated with the colloidal protein system improve electrostatic interactions between wall materials determining the level of thermotolerance of the same [46, 48].
The use of heat due to the microencapsulation process or heat treatment during food processing provides in matrices such as whey the release of sulfur amino acids, reducing redox potential, thus aiding probiotic survival [49]. There is a long way to go in research that relates wall materials to the thermal resistance of microencapsulated probiotics.