In the last decade many studies have been performed on the possible sources of natural antioxidants, since there is a demand in different industries towards the replacement of synthetic antioxidants by natural ones. However, the factors such as loss of quality during storage and processing, unpleasant taste, and in some cases, off-color decrease
20
the effectiveness of the utilization of natural antioxidants in food products.
Encapsulation of these functional food ingredients can improve their quality attributes and mask unwanted features. In addition, food ingredients which are encapsulated in particles or dispersed in emulsions with particle size less than 200 nm can be released under control depending on the pH or temperature of the environment and bioavailability of the coating material.
There is a lack of study on the encapsulation of phenolic materials from the residual sources. In addition, there are limited researches on nano-emulsion containing food ingredients other than oils. The primary aim of this study was to develop a method to obtain nano-emulsion containing dry polyphenolic powder extracted from sour cherry pomace. It was also aimed to investigate the effect of degritting of the extract of sour cherry pomace on the encapsulation efficiency and particle size distribution of the emulsions. It was important to obtain capsules of both unpurified and degritted phenolic powders. In addition, surface morphology, color and particle size of capsules obtained at the optimum conditions were studied.
Another objective of this research was to compare storage stability of capsules prepared from micro- and nano-emulsions. There are limited studies on the baking or cooking stability of capsules of phenolic powders. Effect of encapsulation on retention of phenolic compounds and total antioxidant capacity of phenolic powders and capsules during processing at high temperatures was investigated by incorporating them into cakes. Quality of cakes containing capsules and degritted phenolic powders were also compared.
21 CHAPTER 2
MATERIALS AND METHODS
2.1 Materials
2.1.1 Supplying of samples
Sour cherry pomace was supplied by Karmey Fruit Juice Factory (Karaman, Turkey).
Seeds, stems and other foreign materials were removed from pomace by screening.
Fine pomace was stored in a refrigerator (D 8340 SM; Beko, Istanbul, Turkey) at 4 °C until the extraction.
2.1.2 Reagents
Maltodextrin (MD) (DE=4–7), gum arabic (GA), potassium carbonate, potassium chloride, Folin-Ciocalteu’s phenol reagent, sodium carbonate, 1,1-diphenyl-2-picrylhydrazyl (DPPH), ethanol (absolute), potassium sodium tartrate tetrahydrate, methanol G CHROMASOLV®, CuSO4.5H2O, gallic acid, glucose, potassium hydroxide, acetic acid (100%) were purchased from Sigma-Aldrich Chemical Co. (St.
Louis, MO, USA). Ingredients used in the production of the cake were obtained from the local commercial markets.
22 2.2 Extraction of polyphenolic compounds
Extraction of phenolic compounds (Fig.2.1) from sour cherry pomace was performed by conventional maceration method in shaking water bath (GFL, GFL Gesellschaft für Labortechnik mbH, Burgwedel, Germany) at 30 °C and 70 rpm for 24 h. In maceration 20 g of pomace and 400 ml ethanol:water (1:1, v/v) solvent were used (Simsek, 2010).
At the end of maceration process large particles of pomace were removed by filter cloth followed by vacuum filtration. Clear extract was then concentrated by a vacuum rotary evaporator (Heidolph Laborota 4000 efficient; Heidolph Instruments GmbH & Co, Schwabach, Germany) at 40 °C in order to remove ethanol and sufficient amount of water.
Fig. 2.1 Flow chart of the processes in the preparation of extracted phenolic powders
23 2.3 Degritting of extracted phenolic concentrate
Firstly, concentrates were purified in SIGMA 2–16PK centrifuge (Sigma Laborzentrifugen GmbH; Osterode am Harz, Germany) at different angular velocities in order to define appropriate operating conditions (Fig.2.1). For this purpose, 7.5 ml of concentrate was centrifuged at 5000 and 10000 rpm angular velocities for 2 min.
Supernatant was accurately collected and used for particle size analysis. As shown in the Fig.2.1 three samples analyzed were P1 (unpurified), P2 (purified at 5000 rpm) and P3 (purified at 10000 rpm).
According to the results of particle size analysis degritting of 7.5 ml of concentrate at 10000 rpm for 2 min was selected as operating parameter and was used in the production of purified extracted phenolic powder (PEPP).
2.4 Production of phenolic powders
In order to investigate the effects of degritting, control powder was produced from unpurified concentrate which was frozen at –20 °C in a deep freeze (D 8340 SM; Beko, Istanbul, Turkey) and then dried (Christ Alpha 1–2 LD plus; Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) at –52 °C and 0.075 mbar for 48 h (Fig.2.1). At the end of freeze drying, samples were manually ground to obtain homogeneous extracted phenolic powder (EPP). PEPP was prepared from purified concentrate by freezing, freeze drying and then grinding as in the case of EPP (Fig.2.1). Both powders were stored in a deep freeze at –20 °C and kept until utilization.
24 2.5 Preparation of emulsions and capsules
Maltodextrin (MD) solution was prepared one day prior to emulsification by dissolving MD in distilled water and kept in shaking water bath at 27 °C for 24 h. Gum arabic (GA) was dissolved in distilled water and stirred for 30 min at 50 °C using magnetic stirrer (MR 3001K; Heidolph Instruments GmbH & Co, Schwabach, Germany) 2 h prior to encapsulation. In addition, GA solution which was used in the preparation of emulsions containing PEPP was centrifuged at 10000 rpm for 2 min and filtered through 0.45–µm filter (Gema Medical S. L.; Barcelona, Spain). Total soluble solid content of coating material solution was 10% (w/w) and it was prepared with two different formulations composed of: a) 10% (w/w) MD and 90% distilled water, b) 8%
MD, 2% GA and 90% distilled water.
All emulsions were prepared in two stages: a) pre-emulsions were obtained by mixing dry phenolic powder with coating material solution at the ratio of 1:20 (core-to-coating), and by stirring at 4000 rpm for 5 min using high-speed blender (IKA Works Co, Rawang, Selangor, Malaysia). b) Pre-emulsions (21 g) were further homogenized by ultrasonication (Sonic Ruptor 400; OMNI International, Kennesaw, GA, USA) for 20 min (160 W, 50% pulse, 20 kHz). Ultrasonic homogenizer was equipped with titanium 5/32" stepped micro tip with diameter of 3.8 mm and length of 255.8 mm. In order to prevent overheating of the emulsions during ultrasonication, they were placed in a water bath at 4 °C.
Emulsions were frozen in deep freezer at –20 °C and then dried under vacuum at –52
°C and 0.075 mbar for 48 h. Dry matrices of encapsulated phenolics were manually crushed using a glass rod and stored at –20 °C until analysis or utilization.
25 2.6 Preparation of cake
Two types of cake batters were used for the preparation of cakes for different analyses.
Cakes were formulated with sugar for quality analysis. Sugar content has a negative effect on the analysis of total phenolic content (Waterhouse, 2002). Therefore, cakes containing encapsulated phenolic powders used in the measurement of baking stability of phenolic compounds were prepared from batters with no sugar content. The compositions of cake mixes are given in the Table 2.1.
Table 2.1 Composition of cake batters batter for cake without sugar this step was omitted). Then, margarine melt was added and mixing continued at the same speed for 1 min. All remaining dry ingredients and water were added simultaneously and mixed at 85 rpm for 1 min followed by mixing at 140 rpm for 1 min and one more mixing at 85 rpm for 2 min until the smooth cake
26
batter was obtained. 500 mg of encapsulated phenolic powder or phenolic powder (EPP, PEPP) was added to 100 g of batter. Then batter was further mixed at 85 rpm for 1 min. Encapsulated phenolic powders added to batter were of two types: a) prepared with 8% MD, 2% GA and EPP, b) prepared with 8% MD, 2% GA and PEPP.
Cake batters of 100 g were placed in glass pans lined with wax paper and baked at 175
°C for 22 min in a preheated conventional electrical oven (Arçelik 9411 FT; Arçelik, Istanbul, Turkey). Six cakes were baked at a time. After baking, cakes were removed from the pans and cooled for 1 h at room temperature. Control cake was prepared under the same conditions and contained neither phenolic powder nor encapsulated phenolic powder. Each experiment was duplicated.
2.7 Storage of phenolic powder and encapsulated phenolic powder
Phenolic powders (EPP and PEPP) and encapsulated phenolic powders (prepared with 8% MD, 2% GA and EPP; 8% MD, 2% GA and PEPP) were used in the evaluation of storage stability. Samples were stored in two desiccators with different relative humidities (RH) at room temperature (21±2.0 °C). Saturated aqueous solutions of potassium carbonate and potassium chloride were used to obtain 43% and 85% RH, respectively (Greenspan, 1977). Before placing the sample, each desiccator was kept closed overnight to achieve equilibrium. Storage stability analyses were performed once in 5 days for samples stored at 85% RH and once in 10 days for samples stored at 43% RH. The parameters analyzed were total phenolic content, total antioxidant activity and hygroscopicity.
27 2.8 Physical analysis
2.8.1 Particle size analysis
Particle size analysis was performed for extracted concentrates, emulsions and encapsulated phenolic powders by the laser light scattering method using Mastersizer 2000 (Malvern Instruments, Worcestershire, UK). The mean diameter of the particles was expressed as Sauter mean diameter (D[32]) and calculated with the following formula:
𝐷[32] = 𝑛𝑖𝑑𝑖3 𝑛𝑖𝑑𝑖2 (3)
where, ni stands for the frequency of occurrence of particles in size class i, and di for mean diameter (µm) of these particles.
Span of the particle size distributions was calculated with the following formula:
𝑆𝑝𝑎𝑛 = 𝑑 𝑣,90 −𝑑(𝑣,10)
𝑑(𝑣,50) (4)
where, d(v,10), d(v,50), and d(v,90) are the diameters at 10%, 50%, and 90%
cumulative volume, respectively (Elversson et al., 2003). The instrument also reported the specific surface area (m2/g) of the particles.
2.8.2 Color analysis
Color analysis was performed for phenolic powders, capsules, crumb and crust of cakes.
28
2.8.2.1 Color measurement of phenolic powder and encapsulated samples
Color determination was performed in CIE (L*, a*, b*) color space by using a UV-2450 UV-Vis spectrophotometer (Shimadzu Co., Kyoto, Japan). It was measured as reflected color from the surface of capsules or powders. Illuminant type C (2° standard observer) was used in this analysis. Mean of 3 determinations was calculated for L*
(darkness/whiteness), a* (greenness/redness) and b* (blueness/yellowness) parameters of each sample. The difference in the color (∆E*) with the reference sample (barium sulfate) was calculated for each sample using the following formula:
∆𝐸∗= ∆𝐿∗2+ ∆𝑎∗2+ ∆𝑏∗2 (5)
where, ∆L*, ∆a* and ∆b* are the differences between the color values of reference and sample.
2.8.2.2 Determination of color of crumb and crust of cakes
Color reflected from the crumb and crust of the cake was measured separately in CIE (L*, a*, b*) color space by using Minolta color reader (CR-10; Japan). The instrument was standardized each time by a white sample. Duplicate readings were performed from different position and mean value of ∆E* calculated by the instrument was recorded. Color measurement was performed only for cakes which contained sugar.
29 2.8.3 Specific volume of cake
Specific volume of cakes which contained sugar was determined by the rape seed displacement method (AACC, 1988). Density of rape seed was determined by filling a glass container with known volume (Vcontainer) and weight (Wcontainer) with seeds through tapping and smoothing the surface with ruler until the constant weight was reached.
Bulk density (ρseeds) of rape seeds was found to be 693.2 g/cm3. Then, weights of cakes (Wcake) were measured. Finally, cakes were placed in the container and the remaining part was filled with the seed. The container was taped and the surface was smoothed with a ruler. Total weight (Wtotal) was recorded when no change in consecutive weight measurements was observed. Weight of seeds (Wseeds) required for filling of the container was calculated from the following equation:
𝑊𝑠𝑒𝑒𝑑𝑠 = 𝑊𝑡𝑜𝑡𝑎𝑙 − 𝑊𝑐𝑎𝑘𝑒 − 𝑊𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑒𝑟 (6)
Volume of rape seeds (Vseeds) was calculated from the following equation:
𝑉𝑠𝑒𝑒𝑑𝑠 = 𝑊𝜌𝑠𝑒𝑒𝑑𝑠
𝑠𝑒𝑒𝑑𝑠 (7) Finally, volume of cakes was determined using equation (8):
𝑉𝑐𝑎𝑘𝑒 = 𝑉𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑒𝑟 − 𝑉𝑠𝑒𝑒𝑑𝑠 (8)
Specific volume (SVcake) was calculated from the following equation:
𝑆𝑉𝑐𝑎𝑘𝑒 =𝑊𝑉𝑐𝑎𝑘𝑒
𝑐𝑎𝑘𝑒 (9) All parameters were in the SI units.
30 2.8.4 Crumb texture
Crumb hardness (N), chewiness (N) and gumminess (N) of cakes with sugar samples were measured by using texture analyzer (TAPlus; Lloyd Instruments, Bognor Regis, UK). Fresh cakes (1 h after baking) with cubic shape (25 mm each side) without crust were compressed for 25% of original thickness by 50 N load cell equipped with cylindrical probe (10 mm in diameter) at a speed of 55 mm/min. Each measurement was duplicated and mean of a selected parameter was calculated.
2.8.5 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 −𝑊0 𝑡𝑜𝑡𝑎𝑙
𝑊0 𝑡𝑜𝑡𝑎𝑙 −𝑊𝑝𝑙𝑎𝑡𝑒 (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 = 𝑇𝑃𝐶𝑠𝑡𝑑𝑥𝑊𝑉𝑠𝑜𝑙𝑣𝑒𝑛𝑡
𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 𝐷𝐹 (11)
where, Vsolvent is volume of solvent used in the extraction of phenolic compounds, DF is dilution factor and Wsample 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=0𝑇𝑃𝐶−𝑇𝑃𝐶𝑡=𝑡
𝑡=0 𝑥100 (12) where, TPCt=0 is the initial TPC and TPCt=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 −𝑆𝑃𝐶𝑇𝑃𝐶 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 = 𝑇𝐴𝐴𝑡=0− 𝑇𝐴𝐴𝑡=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
𝑇𝐴𝐴𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑥100 (15)
where, TAAinitial is TAA of capsules before storage and TAAt=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
The micrographs of the surface of encapsulated phenolic powders, EPP and PEPP were