Hygroscopicity

Hygroscopicity assay was performed in the desiccators containing saturated potassium carbonate and potassium chloride solutions having RH of 43% and 85%, respectively, according to the method proposed by Cai & Corke (2000), with some modifications.

Each sample was weighed (up to 1 g) in the aluminum plate. Hygroscopicity was determined gravimetrically after no change in the mass of samples was observed. Each test was duplicated. Before samples were placed in desiccators weights of aluminum plates (Wplate) and total weights (W0 total) were measured. When no change of total weight was observed in measurements during storage, final total weight (Wfinal total) was measured. Amount of water absorbed per g of capsule or powder was determined by the following equation:

𝐻𝑦𝑔𝑟𝑜𝑠𝑐𝑜𝑝𝑖𝑐𝑖𝑡𝑦 =^𝑊𝑓𝑖𝑛𝑎𝑙 𝑡𝑜𝑡𝑎𝑙 −𝑊_{0 𝑡𝑜𝑡𝑎𝑙}

𝑊_{0 𝑡𝑜𝑡𝑎𝑙} −𝑊_{𝑝𝑙𝑎𝑡𝑒} (10)

31 2.9 Chemical analysis

2.9.1 Reducing sugar content

It was stated in section 2.6 that high reducing sugar content in the product may interfere with the total phenol reagent. Therefore, it was required to determine the reducing sugar content of the phenolic powders. The method described in the book of Cemeroğlu (2007) with some modifications was used. Fehling-I solution was prepared by dissolving 69.3 g CuSO₄.5H₂O in distilled water. Then, final volume was brought to 1 liter by addition of distilled water. Fehling-II solution was prepared by dissolving 346 g of potassium sodium tartrate tertrahydrate and 100 g of KOH in distilled water.

Distilled water was added until the final volume was 1 liter. Standardization was performed by using standard glucose solution, which was prepared by dissolving 0.5 g of glucose in 100 mL of distilled water. Firstly, 10 mL of standard glucose solution was mixed with 10 mL of Fehling solution (1:1 solution of Fehling-I and Fehling-II) and heated to its boiling point. After 2 min of boiling, few drops of methylene blue indicator solution were added. After 2 min of boiling, mixture was titrated with standard glucose solution. The amount of glucose required to titrate 10 mL of Fehling solution was determined in this step of standardization.

To prepare solutions of phenolic powders, 0.5 g of EPP and 0.5 g of PEPP were dissolved separately in 10 mL of distilled water. Then, 0.5 mL of each solution was added to 9.5 mL of distilled water in order to dilute the solutions. Each solution was gently mixed. Then, 10 mL of diluted solution was added to 10 mL of Fehling solution and the same procedure described in the standardization was repeated. The amount of reducing sugar in phenolic powders was determined by reducing sugar content of added standard solution, by multiplying with dilution factor and dividing by 10 mL. Results were reported as % (w/w).

32 2.9.2 Total phenolic content

Total phenolic content (TPC) of EPP, PEPP, capsules and cakes without sugar was determined by modified Folin-Ciocalteu method (Beretta et al., 2005). Two solutions were used in this assay. First solution contained 10% (v/v) of Folin-Ciocalteu’s phenol reagent and 90% of distilled water. Second solution was composed of 7.5 % (w/v) of sodium carbonate dissolved in distilled water. Extraction of polyphenols from the samples was performed with ethanol:water:acetic acid solvent (50:42:8) (Saenz et al.

2009). One hundred milligrams of EPP, PEPP or capsule was added to 1 mL of solvent and agitated by using vortex (ZX3; VELP Scientifica, Usmate, MB, Italy). For the analysis of TPC during storage, dispersions were ultrasonicated (80 W, 50% pulse) for 2 min. Extraction of phenolic compounds from the crumb of cake was performed with some modifications. Firstly, 20 mL of solvent were added to 10 g of crumb. Then, the crumb was crushed manually using glass rod to guarantee efficient ultrasonication.

Solution containing crumb particles was then ultrasonicated in two cycles each for 1 min and with 160 W adjusted power. After the first cycle, dispersion was manually agitated. After the ultrasonication, 7.5 mL of dispersion were centrifuged at 10,000 rpm for 2 min and liquid part was carefully collected. Polyphenolic extracts of EPP, PEPP, capsules and cakes were then filtered through 0.45-µm filter. The dilution rates varied for each sample and they were applied to ensure that phenolic content of the diluted sample fits the calibration curve. Calibration curve for this assay was prepared by using gallic acid as a standard and had a range of 0-100 mg GAE/mL (APPENDIX A1, Fig.A.1).

Samples for the spectrophotometric analysis were prepared in two steps. Firstly, 2.5 mL of 10% Folin-Ciocalteu’s phenol reagent was mixed with 0.5 mL of diluted sample.

Mixture was stored for 5 min in dark. Then, 2 mL of sodium carbonate solution were added to the mixture and stored for 1 h at room temperature in dark place. Finally, samples were analyzed spectrophotometrically at 760 nm. Phenolic content (TPCstd) of

33

the diluted samples was found from the standard curve. TPC of the EPP, PEPP, capsules and cakes was found from following equation:

𝑇𝑃𝐶 = 𝑇𝑃𝐶_𝑠𝑡𝑑𝑥_𝑊^{𝑉𝑠𝑜𝑙𝑣𝑒𝑛𝑡}

𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 𝐷𝐹 (11)

where, V_solvent is volume of solvent used in the extraction of phenolic compounds, DF is dilution factor and W_sample is weight of the sample.

Total phenolic content of EPP and PEPP was expressed in mg GAE/ g dry weight.

Total phenolic content loss during storage was expressed in % and was calculated by the following equation:

𝑇𝑃𝐶 𝑙𝑜𝑠𝑠 % =^{𝑇𝑃𝐶𝑡=0}_𝑇𝑃𝐶^{−𝑇𝑃𝐶𝑡=𝑡}

𝑡=0 𝑥100 (12) where, TPC_t=0 is the initial TPC and TPC_t=t is TPC at any time period during storage.

Similar equation was used for the calculation of the retention of polyphenols after baking. Retention of TPC (%) was found by subtracting TPC loss (%) from 100%. Due to the presence of antioxidants in margarine and a possibility that other ingredients may affect measurement of TPC of cakes, control cake without sugar was also determined.

This amount was then subtracted from the experimental value of TPC of cakes. In this calculation initial TPC was a theoretical value of polyphenols and TPCt=t was measured TPC of cakes. Weight loss factor which was not studied in this work was taken into consideration for the calculation of theoretical TPC of cakes and was equal to 1.1.

Correction factor due to the presence of the reducing sugars in the phenolic powders was not used. Reducing sugar content of EPP and PEPP was found to be 65.3% and 63.2%, respectively. However, in the analysis of TPC, all samples were diluted and final concentration of reducing sugars in any sample was ranging from 0.52% (w/v) to

34

0.44%. Waterhouse (2002) proposed to use the correction factor for the sweet and semisweet wines having reducing sugar content >2% (w/v) sugar.

2.9.3 Surface phenolic content and encapsulation efficiency of capsules

Surface phenolic content (SPC) of capsules is an important parameter for the evaluation of encapsulation efficiency (EE%). SPC was determined by modified Folin-Ciocalteu method (Beretta et al., 2005). One hundred milligrams of capsules were agitated for 1 min with 1 mL of methanol:ethanol solvent (1:1) and then filtered through 0.45-µm filter. Calibration curve was prepared with gallic acid and ethanol:methanol solvent (APPENDIX A.1, Fig.A.2). Measurement of SPC was performed at the same conditions described in section 2.9.2. SPC was expressed as mg GAE/g of capsules.

Encapsulation efficiency was calculated from the following equation:

𝐸𝐸% =^{𝑇𝑃𝐶 −𝑆𝑃𝐶}_𝑇𝑃𝐶 𝑥100 (13)

2.9.4 Total antioxidant activity of phenolic powders, capsules and cakes

Total antioxidant activity (TAA) of capsules, phenolic powders and cakes was measured by (1,1-diphenil-2-picrylhydrazyl) DPPH method (Yen & Duh, 1994) with some modifications. The same diluted samples, described in the section 2.9.2 were used for the TAA determination. One hundred microlitters of diluted samples were added to 25 ppm DPPH solution (DPPH dissolved in methanol) and left to stand in the dark place for 1 h. After that, samples were analyzed spectrophotometrically at 517 nm.

DPPH content corresponding to each sample was determined from the calibration curve (APPENDIXT A.1, Fig.A.3). TAA was calculated from the following formula:

35

𝑇𝐴𝐴 = 𝑇𝐴𝐴_𝑡=0− 𝑇𝐴𝐴_{𝑡=1 𝑕} (14)

where, TAAt=0 is initial DPPH concentration and TAAt=1 h is concentration of DPPH in the sample after 1 h.

Dilution rate, volume of solvent and weight of sample were used in the calculation of TAA of the samples. TAA was expressed as DPPH equivalent/g of sample.

During storage percentage of TAA loss was calculated from the following equation:

𝑇𝐴𝐴 𝑙𝑜𝑠𝑠 % = ^{𝑇𝐴𝐴𝑖𝑛𝑖𝑡𝑖𝑎𝑙 −𝑇𝐴𝐴𝑡 =𝑡}

𝑇𝐴𝐴_{𝑖𝑛𝑖𝑡𝑖𝑎𝑙} 𝑥100 (15)

where, TAA_initial is TAA of capsules before storage and TAA_t=t is TAA at any time period during storage.

Similarly with TPC analysis, TAA was also determined for the control cake. The correction factor due to weight loss was equal to 1.1. Retention of TAA after baking was also calculated by subtracting TAA loss (%) (Equation 15) from 100%. TAAinitial

was theoretical TAA of cake and TAAt=t was TAA of cake. TAA of cake was calculated by subtracting TAA of control cake from experimentally determined TAA of cake.

2.10 Surface morphology of phenolic powders and capsules

The micrographs of the surface of encapsulated phenolic powders, EPP and PEPP were obtained by using scanning electron microscopy (SEM). Dry samples were coated by

36

gold/palladium using Hummel VII sputter coating device (Anatech USA, Union City, CA, USA) and analyzed with JSM-6400 electron microscope (Jeol Ltd., Tokyo, Japan) operating at 20 kV. Images were taken at 100x and 500x magnification.

2.11 Sensory analysis of cakes

A 30-member panel consisting of students of Food Engineering Department of METU was used in the sensory evaluation of cake samples. Three types of cakes were analyzed by the panelists. One cake was the control cake (containing no encapsulated phenolic powder) and other two contained encapsulated phenolic powders prepared from a) 8% MD, 2% GA and EPP and b) 8% MD, 2% GA and PEPP. The panelists were asked to give scores for flavor, texture and color of cakes ranging from 1 to 5.

Each cake had a three digit code. Sensory analysis evaluation sheet and data of panelist scores are shown in the APPENDIX A.2.

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:

𝑉_𝑠𝑜 = ^{𝜌𝑆−𝜌}_18𝜇^{𝐿 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

38

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

40

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

43

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

EPP^A - - 34.5±0.24 20.1±0.09 11.7±0.03 69.5±0.57

PEPP^A - - 31.4±0.17 24.6±0.12 11.0±0.05 73.7±0.82

EPP^B 10 0 50.1±0.35b^C 17.4±0.05b 13.4±0.01a 54.5±0.34a

EPP^B 8 2 50.4±0.19b 18.3±0.08b 14.0±0.04a 54.7±0.19a

PEPP^B 10 0 56.0±1.54a 20.8±0.45a 11.0±0.10b 49.6±1.76b PEPP^B 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 ≤

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 ≤

Belgede EFFECT OF NANOENCAPSULATION OF PURIFIED POLYPHENOLIC POWDER ON ENCAPSULATION EFFICIENCY, STORAGE AND BAKING STABILITY (sayfa 47-0)