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Plant material and chemicals
Apple pomace (Figure I.8) was obtained from Val-de-Vire Bioactives (Conde-sur-Vire, France) and kept in the dark until use. Standards of chlorogenic acid, (-) epicatechin and phloridzin, were purchased from Sigma Aldrich (St. Louis, USA). Other chemicals were of analytical grade and purchased from VWR International (Darmstadt, Germany).
In all extraction procedures, a 50 mM malate buffer in a pH 3.8 was used in order to mimetize fruit’s conditions. To determine the optimal extraction conditions, the solid/liquid ratio was evaluated in function of total polyphenols obtained by a conventional maceration method. The samples subjected to extraction ranged from 5 g to 35 g of dry material. The experiments were performed in flasks containing 100 mL of the buffer in a RT-10 magnetic stirrer plate (IKAMAG, Germany) over 8 hours in the dark. Samples were then pressed using a manual press and the liquid extract was filtered before analysis with a 0.45 μm mesh filter. The total polyphenols content (TPC) was measured using Folin–Ciocalteu’s reagent and results are expressed in mg of catechin equivalent per 100g of dry weight. All experiments were carried out in triplicates.
Ultrasound-assisted extractions (UAE) were performed in an ultrasonic extraction reactor PEX1 (R.E.U.S., Contes, France) with 14×10 cm internal dimensions and maximal capacity of 1 L, equipped with a transducer at the base of jug operating at a frequency of 25 kHz with maximum input power (output power of the generator) of 150 W (Figure I.9). The double-layered mantle (with water circulation) allowed the control of extraction temperature by cooling/heating systems. Considering the actual input power from the device is converted to heat which is dissipated in the medium, calorimetric measurements were performed to assess actual ultrasound power, calculated as shown in the equation 1 below (Toma et al., 2011).
Where Cp is the heat capacity of the solvent at constant pressure (J.g−1.°C−1), m is the mass of solvent (g) and dT/dt is temperature rise per second. Then, the applied ultrasonic intensity (UI) was calculated using the calculated power as shown in the equation 2 (Tiwari et al., 2008).
Where UI is the ultrasonic intensity (W.cm-2), P is the ultrasound power (W) as calculated by the equation 1, and D is the internal diameter (cm) of the ultrasound reactor. To the 500 mL of malate buffer (50 mM pH 3.8), 75 g of dried apple pomace were added and submitted to extraction and the obtained extracts were filtered with a 0.45 μm mesh filter before been lyophilized (for HPLC analysis) or analyzed for TPC. Conventional extraction was performed by agitation in the same conditions for comparison. All experiments were carried out in triplicates.
Isolated compounds study
In order to verify whether antioxidants present in the extracts undergo degradation during sonication, the following isolated compounds were submitted to ultrasound treatment: (-) epicatechin, phloridzin and chlorogenic acid. These compounds (in a final concentration of 0.5 mg/mL) were diluted in 2 mL of methanol and then introduced in the ultrasonic extraction reactor with 200mL of malate buffer (50 mM pH 3.8) followed by ultrasound treatment in the optimized conditions. The extractions were subsequently observed in the UV spectrophotometer (Spectronic Genesys 5, Thermo Fischer Scientific, France) at respective characteristic wavelengths for each molecule and then analyzed by HPLC-DAD for quantification purposes. All experiments were carried out in triplicates.
Total phenolics determination (TPC)
TPC was determined using Folin–Ciocalteu reagent (Singleton and Rossi, 1965). In a test tube, 50 μL of the filtered sample were mixed with 1 mL of a 10% Na2CO3 solution and 250 μL of Folin-Ciocalteu reagent. The absorbance was determined using a spectrophotometer (Spectronic Genesys 5, Thermo Fischer Scientific, France) after 1 hour at 765 nm against a calibration curve. The results were expressed in mg of catechin equivalent per 100 g of dry weight.
Identification and individual quantification of phenolic compounds by HPLC-DAD
Polyphenols were measured by HPLC after re-dissolution of the freeze-dried extracts in acidic methanol (1% acetic acid, v/v), or after thioacidolysis as described previously (Guyot et al., 2001), followed by filtration (PTFE, 0.45 μm). A Waters HPLC apparatus (Milford, MA, USA) was used, a system 717 plus autosampler equipped with a cooling module set at 4°C, a 600 E multisolvent system, a 996 photodiode array detector, and a Millenium 2010 Manager system. The column was a Purospher RP18 endcapped, 5μm (Merck, Darmstadt, Germany). The mobile phase was a gradient of solvent A (aqueous acetic acid, 25 mL/L) and solvent B (acetonitrile): initial, 3 % B; 0-5 min, 9 % B linear; 5-15 min, 16 % B linear; 15-45, 50 % B linear, followed by washing and reconditioning the column. HPLC peaks were identified on chromatograms according to their retention times and their UV-visible spectra by comparison with available standard compounds as described by Guyot et al. (2001). Quantification is performed by reporting the measured integration area in the calibration equation of the corresponding standard. Phloretin and phloretin xyloglucoside were calculated as phloridzi equivalent, all flavonols were quantified against quercetin (molar responses, then their respective contents of glycosides are used to calculate concentrations in g/L or g/kg). Total flavonols and total polyphenols were the sums of the corresponding compounds, quantified by HPLC. The average degree of polymerization of flavan-3-ols was calculated as the molar ratio of all the flavan-3-ols units (thioether adducts plus terminal units) to (-)-epicatechin and (+)-catechin corresponding to terminal units.
Antioxidant activity: inhibition of linoleic acid peroxidation
A freshly prepared 2.55 mM solution of linoleic acid (2 mL) in a pH 7.4 phosphate buffer with 100 mM of NaCl containing 10 mM SDS (sodium dodecyl sulfate) were placed at 37 °C in the spectrometer cell. At time zero, 25 μL of a freshly prepared 80 mM solution of AAPH (2,2′-azobis(2-amidinopropane)) in the same buffer was added (Roche et al., 2005). After 15 min, 25 μL of an antioxidant solution were added in MeOH. The experiments were repeated with different phenol concentrations (1 mM and lower). The initial level of hydroperoxides (molar absorption coefficient at 234 nm = 26 100 M-1.cm-1) were below 2% in all experiments. The uninhibited and inhibited peroxidation rates were calculated from the slope of the absorbance at 234 nm versus time before and after antioxidant addition using fixed time intervals. All experiments were carried out in triplicates. Standard deviations were lower than 10%.
Results of preliminary investigations showed the volume of solvent to be used in the extraction (thus, the solid/liquid ratio) affect the extraction of polyphenols due to an insufficient interaction between the solvent and the matrix. This parameter had an influence on the applied ultrasonic intensity, since a minimum of free liquid is necessary to the functioning of the apparatus. In addition, the temperature and sonication duration have an interaction in the experiment since the ultrasonic energy input tends to increase the temperature of the medium, and both parameters have a direct influence in the yield of extracted polyphenols. Therefore, results of preliminary studies showed polyphenols yield is mainly dependent on the ratio of solvent to sample, the extraction time, the temperature and the ultrasonic intensity.
In order to investigate the influence and relevance of the operating parameters required during extractions, a Central Composite Design (CCD) was used to analyze total polyphenol content (TPC) and extract main polyphenols. Three independent factors (namely temperature (T), sonication duration (t) and Ultrasonic intensity (UI)) were evaluated, as well as eventual interaction between these variables.
The full uniformly routable CCD presents the following characteristics (Bezerra et al., 2008): (1) total number of experiments (N) are given N= k2 +2k+cp, where k is the number of variables and cp is the number of replicates of the central point; (2) The star points are at a distance α from the center of the design and α-values are calculated by α = 2(k−p)/4; and (3) all factors are studied in five levels −α, −1, 0, +1, +α). Therefore, in the case of three variables, the number of experiments is 20, the number of replicates of the central point in 6 and the α-value is 1.68.
Preliminary experiments allowed us to distinguish the variables implied in the model at five separated coded levels: -α = -1.68), -1, 0, +1, +α = +1.68). The limit values of each variable range were chosen as function of limitations of ultrasonic apparatus (minimum and maximum power available in the device), temperature of extraction for polyphenols (which might degrade above 40°C) and time of sonication. Values are presented on Table I.2 and involved a total of 20 experiments; including six replications at the centre point to evaluate experimental error measurement, and randomized to avoid effects of extraneous variables. Variables were coded according to the following Equation (3), where Xi is the coded value, xi, the real value of a variable, ̅ , the real value of a variable at the center point, and Δxi, the step change: Experimental data for predicting TPC have then been represented using a second order polynomial Equation (4) as follows: Σ Σ ΣΣ (4).
Where: Y is the response variable TPC (mg of catechin equivalent per 100 g of dried apple pomace sample), β0 is the average response obtained during replicated experiments of the CCD, βi; βii; βij are the linear, quadratic and cross-product effects, respectively, Xi and Xj are the independent coded variables. The results were analyzed using the Statgraphics XV® software.
The extracts obtained were analyzed with a mathematical model derived from Fick’s second law (Herodez et al., 2003). The extraction of polyphenols from apple pomace follows first-order kinetics (Spiro and Jago, 1982), which can be represented as follows: Where Ct is the concentration of total polyphenols at time t, C∞ is the final concentration of total polyphenols and k is the apparent first-order rate constant of extraction.
When ln (C /[C -C]) is plotted against time, the points fall on two intersecting straight lines, the first with a relatively steep slope and the second with a relatively shallow one. The points of intersection of ln (C /[C -C]) vs. t plots for the fast and the slow stages are termed transition points.
Table of contents :
CHAPTER I : Applications of Ultrasound in Processing, Preservation and Extraction
1.2. ULTRASOUND PRINCIPLE
1.3. INFLUENCING PARAMETERS
1.3.1. Solvent type
1.3.3. Ultrasonic intensity
1.3.4. Ultrasonic power and frequency
1.3.5. Presence of dissolved gases
1.4.1. Laboratory scale
1.4.2. Industrial scale
1.5. ULTRASOUND IN FOOD PROCESSING
1.5.3. Filtration, Degassing/Deaeration
1.5.6. Freezind and Crystallization
1.5.8. Sterilization/ Pasteurization
1.5.10. Miscellaneous Effects
1.6. ULTRASOUND IN FOOD PRESERVATION
1.6.1. Microorganism inactivation
1.6.2. Spore inactivation
1.6.3. Enzyme inactivation
1.7. ULTRASOUND IN FOOD EXTRACTION
1.7.1. Fruits and vegetables
1.7.2. Herbs and spices
1.7.3. Oleaginous seeds
CHAPTER II : Ultrasound in The Extraction of Polyphenols From Apple Pomace
1.2. EXPERIMENTAL SECTION
1.2.1. Plant material and chemicals
1.2.2. Extraction Procedures
1.2.3. Isolated compounds study
1.2.4. Total phenolics determination (TPC)
1.2.5. Identification of phenolic compounds by HPLC-DAD
1.2.6. Antioxidant activity: inhibition of linoleic acid peroxidation
1.2.7. Experimental design
1.2.8. Kinetics studies
1.3. RESULTS AND DISCUSSION
1.3.1. Solid-liquid ratio
1.3.2. Experimental design studies
1.3.3. Results for TPC
1.3.4. Optimization of ultrasound-assisted extraction
1.3.5. Comparison and kinetic studies
1.3.6. Antioxidant Activity
1.3.7. Ultrasound effects on extracted molecules
1.3.8. Large scale ultrasound extraction
CHAPTER III : Ultrasound In Food Preparation
3.2. EXPERIMENTAL SECTION
3.2.1. Materials and equipments
3.2.2. Food preparation
3.2.3. Analysis Methodology
3.3. RESULTS AND DISCUSSION
3.3.1. Chocolate Genoise
3.3.2. Sponge cake
3.3.3. Chocolate mousse
CHAPTER I : Ultrasound Effects on Food Products
1.2. IMPACT OF ULTRASOUND ON FOOD PRODUCTS
1.2.1. Color modifications
1.2.2. Antioxidants modifications
1.2.3. Polysacharides modifications
1.3. IMPACT OF ULTRASOUND ON LIPID CONTAINING FOOD PRODUCTS
1.3.5. Microbial Inactivation
1.4. MECHANISMS AND FACTORS OF LIPID OXIDATION
1.5. LIPID DEGRADATION ASSESSMENT METHODS
1.5.1. Primary oxidation compounds
1.5.2. Secondary oxidation compounds
1.5.3. Other methods
CHAPTER II : Degradation of Edible Oils During Ultrasound Processing
2.2. MATERIALS AND METHODS
2.2.1. Materials and Reagents
2.2.2. Ultrasound treatment of samples
2.2.3. Physicochemical analysis
2.2.4. Electron paramagnetic resonance analysis
2.2.5. Fatty acid methyl esters derivatives analysis
2.2.6. Volatiles and off-flavors analysis
2.2.7. Sensory analysis
2.2.8. Statistical analysis
2.3. RESULTS AND DISCUSSION
2.3.1. Physicochemical analysis
2.3.2. Electron paramagnetic resonance analysis
2.3.3. Volatiles, sensory and off-flavors analysis
2.3.5. Comprehension of the mechanism of ultrasound lipid degradation