2. MATERIALS AND METHODS
2.12 Statistical analysis
All experiments if not implied in the method part were replicated twice. Data was analyzed by one-way or two-way analysis of variance (ANOVA) (P≤0.05) using MINITAB software 15 version (Minitab Inc., State College, PA, USA) and SAS software version 9.1 (SAS Institute Inc., NC, USA), respectively. Two-way ANOVA was used for storage experiment which had independent variables of powder type, composition of coating materials and RH. Dependent variables were, EE%, SPC, D[32], span, SSA, TAA, L*, a*, b*, ∆E* (of capsules, powders, crumb and crust), TAA loss (storage and baking), TPC loss (storage and baking), hygroscopicity, hardness, chewiness, gumminess, flavor, and texture.
37 CHAPTER 3
RESULTS AND DISCUSSION
3.1 Effect of degritting on physical and chemical properties of polyphenolic powders and capsules
3.1.1 Effect of degritting on particle size of extracted concentrates
In order to study the effect of centrifugation on particle size distribution of extracted polyphenolic concentrate, three different samples were analyzed (Fig 3.1). It was found that centrifugation had significant effect on particle size distribution of the samples.
Sample had larger particle size prior to centrifugation (P1). Sample spun at 10,000 rpm (P3) contained more particles with smaller size when compared to the sample spun at 5,000 rpm (P2). In other words, more particles with larger size were removed from the concentrate as centrifugation rotational speed was increased. This resulted in the shift of particle size distribution curve to the left (lower diameters) which can be explained by Stoke’s Law. General equation of Stoke’s Law (Leung, 2007) can be expressed as:
𝑉𝑠𝑜 = 𝜌𝑆−𝜌18𝜇𝐿 𝑔𝑑2 (16)
where, Vso is separation velocity, ρS density of solid, ρL density of liquid, g centrifugal acceleration, d diameter of the particle, and µ viscosity of liquid. Subscript “o” in
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separation velocity stands for separation of an individual particle with no interaction with other particles in an ideal dilute solution. In this analysis all parameters were the same for three samples except centrifugal acceleration. As angular velocity increased critical diameter of the particle to remain in the suspended form decreased.
Fig. 3.1 Effect of degritting on particle size distribution of concentrated polyphenolic extracts P1, P2 and P3.
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It can be seen from the Equation (16) that P3 had smaller particles in the suspended form when compared to P2 during centrifugation. Undoubtedly, P1 contained larger suspended particles when compared to P2 and P3, since no centrifugal acceleration was applied on it. Hence, sample spun at the higher angular velocity was comprised of particles with smaller Sauter mean diameter (D[32]) (Table 3.1; Table B.1). On the contrary, span of P3 had the highest value and it was significantly different (p≤0.001) from that of P1 and P2 samples (Table B.2). This can be explained by analyzing its particle size distribution curve (Fig 3.1). Since most of the particles of P3 are smaller this shifted the particle size distribution curve to the left (smaller size), therefore difference between d(v,90) and d(v,10) is high (Equation (4)). Definitely, this difference is lower for P2 and P1. In addition, d(v,50) is the lowest for P3. Removal of the large particles by centrifugation from P3 made it possible to detect smaller particles which could be shadowed during particle size analysis and thus, not present in the results of P1 and P2. Therefore, particle size distribution curve of P3 shifted to the left.
Table 3.1 Influence of angular velocity on purification of dispersion
* Columns having different letters (a, b & c) are significantly different (p ≤ 0.05).
Specific surface area was also significantly different (p≤0.001) for all samples (Table B.3). The reason for this is its indirect proportionality to D[32] (Table 3.1). The highest
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specific surface area was reported for P3. Consequently, it can be understood that filtration process was insufficient for the removal of particles with the diameters in the range of 1-100 µm from the polyphenolic extract, so extra treatment had to be applied.
The origin of these particles which were present in the extract can be organic or inorganic. Soluble and insoluble tissues of pomace with different size and geometry could be extracted during maceration. Drying of the pomace under the sun could lead to contamination with particles from the environment such as mineral crystals or dust suspended in the air. These particles with size under the critical diameter of suspension could remain in the extract.
3.1.2 Effect of degritting of concentrates on particle size distribution of emulsions Two different core materials (EPP and PEPP) were used to prepare emulsions. Core materials were entrapped in two different coating materials including 10% MD and combination of 8% MD and 2% GA. Results of the particle size analysis of the emulsions are given in the Table 3.2.
Table 3.2 Particle size analysis results of emulsions prepared with different coating materials and powder types
* Columns having different letters (a & b) are significantly different (p ≤ 0.05).
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Purification of polyphenolic extracted concentrates had significant (p≤0.001) influence on D[32] (Table B.4), span and specific surface area values of emulsions (Table B.5;
Table B.6). Emulsions prepared with PEPP contained smaller particles when compared to emulsions prepared with EPP. Most of these particles were in the nano range, resulting from the D[32] values of P3. Therefore, degritting was found to be a critical parameter in the preparation of nano-emulsions. Coating materials used in this study had no significant influence (p>0.05) on the particle size distribution of the emulsions.
Similarly, it can be seen on the Fig.3.2 that particle size distribution curves are very similar for emulsions prepared with different coating materials. On the contrary, they appear very different when emulsions containing PEPP were compared with emulsions containing EPP.
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Fig. 3.2 Particle size distribution of micro-emulsions (containing EPP) prepared with 10% MD (solid line) and 8% MD-2% GA (dotted line), nano-emulsions (containing PEPP) prepared with 10% MD (dashed line) and 8% MD-2% GA (dash dotted line)
Similar to extracted polyphenolic concentrates, degritting had significant (p≤0.05) effect on the span values of the emulsions. High span value of P3 resulted in the high span values of the emulsions prepared with PEPP. These emulsions contained large range of particles with different sizes. Most of these particles were in the nano range, and there was continuous and approximately equal percent volume range of large particles (Fig 3.2) which had remained in the concentrate after degritting, and were present in the PEPP. However, particle size distribution appeared more narrow (lower span values) for emulsions prepared with EPP, but still contained large range of
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particles with different diameters. Jafari et al. (2007a) reported that homogenization in blender and ultrasonication increased span values of the sub-micron emulsions. In addition, since PEPP contained lower amount of impurities, energy density of ultrasonication of total solids in the emulsions was higher than that of the emulsions prepared with EPP, leading to more disruption and formation of smaller particles.
Gordon and Pilosof (2010) reported that ultrasonication for 10 min caused the formation of many small particles. Specific surface areas of the emulsions prepared with PEPP were higher and significantly (p≤0.001) different when compared to emulsions prepared with EPP (Table 3.2), due to the indirect proportionality to Sauter mean diameter.
3.1.3 Effect of degritting of concentrates on particle size distribution of capsules For the capsules, particle size analysis was performed only for the ones prepared with 8% MD-2% GA-EPP (micro-emulsion) and 8% MD-2% GA-PEPP (nano-emulsion), since particle size distributions of the emulsions were not affected from the types of coating material. Most of the capsules were in the micro range (Fig 3.3). Capsules prepared from nano-emulsion were smaller in diameter when compared to capsules prepared from micro-emulsion.
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Fig. 3.3 Particle size distribution of capsules prepared from micro-emulsion (solid line) and nano-emulsion (dashed line)
Capsules prepared from nano-emulsion had approximately two times smaller D[32]
value (17.882±0.0230 µm) when compared to capsules prepared from micro-emulsion (39.331±0.0170 µm). However, span values, which were different in the analysis of emulsions, were approximately equal and were found to be 2.37±0.050 and 2.43±0.030 for capsules containing PEPP and EPP, respectively.
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3.1.4 Effect of degritting of concentrates on color of phenolic powder and capsules Lightness and redness of the polyphenolic powder increased as a result of degritting (Table 3.3). After centrifugation of the concentrates, the remaining wet sediment’s color was light brown (data not shown). Therefore, PEPP was lighter and more reddish than EPP. Correspondingly, purification significantly (p=0.0013) increased lightness of the capsules (Table B.7), which can be explained by the lower amount of phenolic compounds on the surface of capsules containing PEPP when compared to capsules containing EPP. Formulation of coating material type had no significant (p>0.05) effect on L*, a*, b* values and ∆E* (Table 3.3) (Table B.8; Table B.9; Table B.10). On the other hand, redness of capsules was significantly (p≤0.001) different for core material type and was higher for capsules containing PEPP, resulted from the higher redness value of uncoated PEPP. These results can also be observed in the pictures of phenolic powders and capsules (Appendix C, Fig C.1)
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Table 3.3 Color measurements of polyphenolic powders and capsules Powder
Type
Coating Type (%) L* a* b* ∆E*
MD GA
EPPA - - 34.5±0.24 20.1±0.09 11.7±0.03 69.5±0.57
PEPPA - - 31.4±0.17 24.6±0.12 11.0±0.05 73.7±0.82
EPPB 10 0 50.1±0.35bC 17.4±0.05b 13.4±0.01a 54.5±0.34a
EPPB 8 2 50.4±0.19b 18.3±0.08b 14.0±0.04a 54.7±0.19a
PEPPB 10 0 56.0±1.54a 20.8±0.45a 11.0±0.10b 49.6±1.76b PEPPB 8 2 56.2±1.08a 20.7±0.23a 10.8±0.18b 49.6±1.09b
A Uncoated phenolic powder
B Encapsulated phenolic powder
C Statistical analysis was performed only for capsules, without including data of EPP and PEPP. Columns having different letters (a & b) are significantly different (p≤0.05)
3.1.5 Effect of degritting of concentrates on total antioxidant activity of polyphenolic powders and capsules
TAA of polyphenolic powders was found to be 7.09 and 6.80 ppm DPPH/g dry weight for EPP and PEPP, respectively. Slight decrease in the TAA of PEPP could be caused by removal of some active compounds together with impurities after centrifugation.
However, results in the Table 3.4 show that, degritting of polyphenolic concentrates has no significant (p>0.05) effect on the TAA of capsules (Table B.11). Coating material type was also found to have no significant (p>0.05) influence on TAA of capsules.
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Table 3.4 Total antioxidant activity data for capsules prepared from EPP and PEPP with different MD and GA concentrations
Powder Type Coating type TAA
(ppm DPPH/g dry w)
MD (%) GA (%)
EPP 10 0 1.85±0.050a
EPP 8 2 1.87±0.020a
PEPP 10 0 2.01±0.005a
PEPP 8 2 1.91±0.005a
3.1.6 Effect of degritting of concentrates on encapsulation efficiency
Encapsulation efficiency is one of the most important parameters in the encapsulation technology, since it shows how much amount of core material was encapsulated or entrapped. Results in Fig 3.4 show that degritting had a significant (p≤0.001) effect on the encapsulation efficiency of polyphenolic powders (Table B.12). This can be explained by the reduction of the particle size of capsules, which caused reduction in the phenolic content on the surface of the capsules (Fig 3.3). This can be supported by a study performed by Jafari et al. (2007b). They reported that surface oil content decreased with decreasing particle size of the encapsulated powder. Coating material type had no significant (p>0.05) influence on the encapsulation efficiency.
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TPC of capsules were found to be 30.43 mg GAE/g dry weight and 37.10 mg GAE/g dry weight containing EPP and PEPP, respectively. Results of SPC are given in the Table 3.5.
Fig. 3.4 Encapsulation efficiency of capsules prepared with different coating materials and powder types. Bars with different letters (a & b) are significantly different (p ≤
0.05)
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Table 3.5 Surface phenolic content data for capsules prepared from EPP and PEPP with different MD and GA concentrations
Powder Type Coating type SPC
(mg GAE/g dry w)
MD (%) GA (%)
EPP 10 0 4.24±0.020a*
EPP 8 2 3.52±0.355a
PEPP 10 0 0.74±0.080b
PEPP 8 2 0.64±0.015b
* Columns having different letters (a & b) are significantly different (p ≤ 0.05).
There was a significant (p≤0.001) difference in SPC of capsules containing different core materials (Table B.13). On the other hand, coating material type had no significant (p>0.05) influence on SPC of capsules. Since emulsion prepared with PEPP had smaller particles when compared to emulsions prepared with EPP, less phenolic compounds were present on the surface of the capsules. Soottitantawat et al. (2003) showed in their study that there was greater loss of flavor during spray drying for emulsions with higher droplet size in the encapsulation of D-limonene. In the same study it was also found that surface oil content decreased for emulsions with smaller droplets.
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3.1.7 Effects of degritting of concentrates on surface morphology of polyphenolic powders and capsules
Surface morphology of phenolic powders and capsules was analyzed by SEM (Fig 3.5
& Fig 3.6). Surface of PEPP (Fig 3.5B) was smooth and did not contain any foreign particles, while the surface of EPP (Fig 3.5A) was wrinkled and particles had sharp edges. It can also be seen that there were many small particles on the surface of EPP some of which may be foreign substances. After degritting most of these particles were removed and therefore they could not be observed on the micrograph of PEPP.
Fig. 3.5 Scanning electron micrographs of polyphenolic powders: EPP (A) and PEPP (B)
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Fig. 3.6 Scanning electron micrographs of encapsulated phenolic powders with formulation of 10% MD-EPP (A), 10% MD-PEPP (B), 8% MD-2% GA-EPP (C) and
8% MD-2% GA-PEPP (D)
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Encapsulated EPP powders (Fig 3.6A & Fig 3.6C) were similar in structure although the coating composition was different, while entrapped in 10% MD PEPP capsules (Fig 3.6B) were larger than PEPP capsules (Fig 3.6D) entrapped in 8% MD-2% GA coating material. Among the capsules produced with 10% MD coating material more agglomeration was observed for sample containing PEPP, while the structure looked similar. On the other hand, among the samples prepared with 8% MD-2% GA coating material capsules containing PEPP were smaller than capsules prepared with EPP. The same result was obtained in the particle size analysis of capsules (Fig 3.3).
3.2 Effect of degritting of concentrates on hygroscopicity and storage stability of capsules and phenolic powders
3.2.1 Hygroscopicity of capsules and phenolic powders
Hygroscopicity was determined for EPP, PEPP and encapsulated EPP and PEPP at two different relative humidities of 43% and 85%. Coating material type used in this analysis was composed of 8% MD and 2% GA. Hygroscopicity of capsules and polyphenolic powders was lower at 43% RH when compared to 85% RH (Table 3.6).
An increase in the water sorption for microencapsulated cloudberry phenolics with increasing water activity was also reported by Laine et al. (2008). This resulted from higher vapor content at higher humidity which can be easily absorbed by capsules and powders. At 43% RH the highest and significantly different hygroscopicity value was reported for PEPP (p≤0.05) and the lowest for EPP (Table 3.6) (Table B.14).
Hygroscopicity of PEPP was also the highest and significantly (p≤0.05) different from hygroscopicity of EPP at 85% RH (Table B.15). This might be related to the degritting of concentrates. Most of the large particles which were removed from concentrate after degritting were absent in PEPP, therefore, particle size of PEPP was smaller. Since it
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contained smaller particles, the exposed surface area was higher and, consequently, water sorption from air was higher. Similar reason was reported by Tonon et al. (2009).
They stated that water adsorption was smaller for particles with large size. Another reason for the difference between hygroscopicity of EPP and PEPP can be water sorption characteristics of impurities. Inorganic and organic impurities present in the EPP can be less hydrophilic than polyphenolic compounds or sugars. Presence of these components could decrease water adsorption.
Table 3.6 Hygroscopicity of polyphenolic powders and capsules at 43% and 85% RH
Sample Hygroscopicity (%)
* Columns having different letters (a, b, c & d) are significantly different (p ≤ 0.05).
At 43% RH encapsulated PEPP had higher and significantly (p≤0.05) different hygroscopicity than encapsulated EPP. This can be explained by higher hygroscopicity of PEPP described earlier in this section, and by smaller particle size of capsules. At higher RH (85%) there was no significant (p>0.05) difference between encapsulated powders. However, their hygroscopicity was significantly (p≤0.05) smaller than hygroscopicity of polyphenolic powders at both RH’s. Similar results were reported by Martinelli et al. (2007) which showed that hygroscopicity of lemon powder decreased with the addition of coating materials. Encapsulated powders containing both maltodextrin and gum arabic had lower hygroscopicity resulting from nonhygroscopic
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properties of these compounds when they were in powder form (Silva et al., 2012).
Consequently, coated polyphenolic powders adsorb less water than uncoated phenolic powders, which is an advantage of encapsulation for storage of phenolic compounds or antioxidants.
3.2.2 Storage stability of capsules and phenolic powders
Storage stability of capsules and polyphenolic powders was expressed in the terms of loss of TPC and TAA. Four different samples a) EPP, b) PEPP, c) capsules prepared with 8% MD-2% GA-EPP, and d) capsules prepared with 8% MD-2% GA-PEPP were analyzed at two different RH of 43% and 85%. TPC loss of capsules and phenolic powders was less during storage at 43% RH (Fig 3.7) when compared to TPC loss at 85% RH (Fig 3.8). This can be resulted from the higher water activity of the samples which are in equilibrium with the ambient air at 85% RH, which speeded up degradation reactions. Similarly, Zheng et al. (2011) reported that degradation of microcapsules of bayberry polyphenols increased with the increase of moisture content in a short term study. At higher moisture content solubility of polyphenols increased and caused an increase in the oxidation rate. Encapsulated powders showed significantly (p≤0.05) smaller loss of TPC when compared to EPP and PEPP during storage at 43% RH (Table B.16). Tonon et al. (2009) observed a more pronounced reduction in total polyphenolics of freeze dried açai powder after 15 days of storage when compared to encapsulated powders, since polyphenolic compounds in the capsules were protected better by carrier agents. Similar to hygroscopicity results, PEPP showed higher TPC loss during storage at 43% RH (Fig 3.7). The main reason could be the absence of impurities in PEPP which can act as protecting materials. No significant (p>0.05) difference between TPC loss of encapsulated powders during storage at 43% RH were observed. Similar to storage results at 43% RH, PEPP had the highest and significantly (p≤0.05) different loss of TPC when compared to other
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samples (Fig 3.8). However, the loss of TPC of EPP and encapsulated PEPP during storage at 85% RH was not significantly (p>0.05) different (Table B.17). This may be due to higher loss of TPC and higher hygroscopicity of PEPP at 85% RH. The lowest loss of TPC was observed for encapsulated EPP. This may be related to smaller loss of EPP and higher particle size of encapsulated EPP. Since, surface area of capsules of encapsulated EPP was smaller, the rate of oxidation of phenolic compounds was less.
Fig. 3.7 TPC loss of phenolic powders and capsules during storage at 43% RH [(♦):
EPPb*, (■): PEPPa, (▲): encapsulated EPPc and (●): encapsulated PEPPc.* Samples having different letters (a, b & c) are significantly different (p ≤ 0.05).]
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Fig. 3.8 TPC loss of phenolic powders and capsules during storage at 85% RH [(♦):
EPPb*, (■): PEPPa, (▲): encapsulated EPPc and (●): encapsulated PEPPb. * Samples having different letters (a, b & c) are significantly different (p ≤ 0.05).]
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Table 3.7 shows final loss of TPC after storage for 91 and 45 days at 43% and 85%
RH, respectively. Results are similar to the results of TPC loss during storage. TPC loses of encapsulated powders were the smallest and significantly (p≤0.05) different than those of phenolic powders at both humidities (Table B.18; Table B.19).
Consequently, it should be emphasized that encapsulation had a positive effect on storage stability of polyphenols.
Table 3.7 Loss of TPC after storage for 91 and 45 days at 43% and 85% RH
Sample Loss of TPC (%)
91 days, 43% RH 45 days, 85% RH
EPP 37.6±0.27b* 41.2±0.09b
PEPP 43.2±0.27a 52.5±0.17a
Encapsulated EPP 11.0±1.67d 31.9±0.40d
Encapsulated PEPP 18.2±0.03c 34.0±0.04c
* Columns having different letters (a, b, c & d) are significantly different (p ≤ 0.05).
Loss of TAA during storage at 43% and 85% RH for the same samples is shown in the Fig.3.9 and Fig.3.10, respectively. Similar to the loss of TPC, loss of TAA was higher at 85% RH for the same reasons as explained before. There are limited studies on the loss of TAA during storage in the literature. During storage at 43% RH, the highest and significantly (p≤0.05) different loss of TAA was reported for PEPP (Fig 3.9) (Table B.20). The significantly lower TAA loss was observed for encapsulated EPP during storage at 43% RH.
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Fig. 3.9 TAA loss of phenolic powders and capsules during storage at 43% RH [(♦):
EPPb*, (■): PEPPa, (▲): encapsulated EPPd and (●): encapsulated PEPPc. * Samples having different letters (a, b, c & d) are significantly different (p ≤ 0.05).]
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During storage at 85% RH the highest and significantly (p≤0.05) different loss of TAA was reported for PEPP (Table B.21), while encapsulated EPP had the smallest loss of TAA (Fig 3.10). In addition there was no significant (p>0.05) difference between TAA losses of EPP and encapsulated PEPP for the same reasons as explained in the case of loss of TPC.
Fig. 3.10 TAA loss of phenolic powders and capsules during storage at 85% RH [(♦): EPPb*, (■): PEPPa, (▲): encapsulated EPPc and (●): encapsulated PEPPb. * Samples having different letters (a, b & c) are significantly different (p ≤ 0.05).]
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Final values of the losses of TAA of the samples after 91 and 45 days of storage at 43%
and 85% RH, respectively, are given in the Table 3.8.
Table 3.8 Loss of TAA after storage for 91 and 45 days at 43% and 85% RH
Sample Loss of TAA (%)
91 days, 43% RH 45 days, 85% RH
EPP 40.1±2.84a* 49.2±0.40a
PEPP 45.3±0.61a 51.7±0.85a
Encapsulated EPP 7.6±0.41c 33.3±0.83b
Encapsulated PEPP 18.4±0.22b 38.1±1.77b
* Columns having different letters (a, b & c) are significantly different (p ≤ 0.05).
After storage for 91 days at 43% RH there was no significant (p>0.05) difference
After storage for 91 days at 43% RH there was no significant (p>0.05) difference