Bibi S, Kowalski JR, Zhang S, Ganjyal MG and Zhu JM*
School of Food Science, Washington State University, Pullman, WA 99164, USA
Received Date: June 08, 2017; Accepted Date: June 26, 2017; Published Date: July 03, 2017
Citation: Bibi S, Kowalski JR, Zhang S, Ganjyal MG, Zhu JM (2017) Stability and Functionality of Grape Pomace used as a Nutritive Additive During Extrusion Process. J Food Process Technol 8: 680. doi: 10.4172/2157-7110.1000680
Copyright: © 2017 Bibi S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Grape pomace (GP) is a major byproduct of wine and juice industry, rich in polyphenolics with demonstrated health benefits. Extrusion processing for development of healthy and quality GP supplemented corn starch snack foods was evaluated using response surface methodology. The retainability of polyphenolic content and antioxidant activity after extrusion processing were further assessed. The processing variables were feed moisture (16, 20, and 24 ± 0.2% w.b.), screw speed (150, 200, and 250 rpm), and the level of GP supplementation (0, 5, and 10% w/w). Extrudates with 5% GP and 16 ± 0.2% feed moisture had a high overall expansion ratio (ER) of 3.83 ± 0.14, and overall low density (0.11 ± 0.00 g/cm3). Total polyphenolic content (TPC) of the extrudates (5% GP, and 16% feed moisture) extruded at 150 and 250 rpm retained up to 74.1% and 78.57% respectively, while TPC was retained at 95% when extruded under 200 rpm with 10% GP and 16% feed moisture. Additionally, the total antioxidant activity and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of the 5% GP extrudates retained 98% after extrusion processing. Moreover, polyphenolic extract of 5% GP extrudates suppressed reactive oxygen species (ROS) in CaCO2 cells induced by hydrogen peroxide. In conclusion, GP incorporation in cornstarch extrudates improved both the physicochemical quality as well as nutritional value of products. Our study indicates that GP can be effectively incorporated into extruded foods by providing enhanced nutritional value without losing the expansion characteristics.
Grape pomace extrudates; Expansion; Polyphenolics; Antioxidant activity; Reactive oxidative species; CaCO2 cells
Grapes is the 4th largest fruit crop in the world with a production of 67 million tons per year . Grapes are generally cultivated for the wine production with about 80% of the global grape production being used in the wine industry . After the juicing process of grapes, huge amounts of the grape pomace (GP), consisting of seeds, skin and stems is obtained as the byproduct. GP is currently used as animal feed because of its protein value, as a fertilizer, and as a source for extraction of bioactive compounds [3,4]. GP is a rich source of polyphenolic compounds including gallic acid, catechin, epicatechin, and proanthocyanins among others . GP also contains substantial amount of non-digestible fiber (60% to 70%), essential fatty acids (13% to 19%), proteins (11%), as well as non-phenolic tocopherols, beta-carotene, and minerals [6,7]. The polyphenolics from GP are known for their antioxidant and anti-inflammatory effects [8,9], which demonstrate preventive effect on cancer , cardiovascular diseases , and inflammatory bowel disease . The GP polyphenolics have been used in food technology as antioxidants to inhibit the oxidation of fish lipids, frozen fish muscle, and fish oil . GP is used as a source of dietary fiber and antioxidant to enhance functionality and shelf life of yogurt and salad dressing , while grape seed flour can be used as an ingredient in baking products .
As another alternative usage, GP has been used in extrusion cooking , by mixing with barley flour at levels of 2% to 10% (w/w). However, using response surface methodology, product responses including bulk density, expansion, texture, and color were greatly affected by the proportion of GP and temperature of the extruder . The GP barley flour extrudates with 2% GP (at 160°C, 200 rpm) had higher preferences for overall product quality, while enhancing GP proportion above 6% reduced expansion . Extrusion processing also reduced the total phenolic content (TPC) of the GP barley flour extrudates compared to the raw material . However, both of these studies lack a reasonable explanation of the positive interactions between GP and barley flour, which can greatly affect the textural properties and is evident from our recent findings on carrot and cherry pomace incorporation in cornstarch extrudates [18,19]. Further feed moisture can also affect the textural as well as nutritional quality of the extruded products. Extrusion of GP with white sorghum (at ratio of 30:70 w/w, barrel temperature of 170°C and screw speed of 200 rpm) with a moisture content of 45% resulted in 120% increase in monomer contents of the polyphenolics in GP extrudates . All these suggest that extruded products can serve as a great vehicle for the delivery of beneficial nutrients of the GP, as extrusion is a high shear, high pressure, and short time processing technology , proven to help improve digestibility, nutrient bioavailability [20,22], and to have distinct textural properties . However, both the nutritional and textural quality of the extrudates is highly affected by the operational process including screw speed, feed moisture and raw material composition used , which remain unclear for GP cornstarch extrudates.
Expansion, an important quality parameter of the extruded puffed products, is greatly affected by the feed moisture content and feed material composition [18,19,25]. Cornstarch, widely used in extrusion cooking, undergoes gelatinization and mechanical degradation during extrusion, and expands well, while other raw ingredients such as proteins, added sugars, lipids, fiber and moisture generally tend to reduce expansion [24,26]. The cornstarch expanded products are usually energy dense, with high glycemic index and low nutrients (vitamins, minerals, proteins and fiber). Nutritional value of these products can be improved via incorporation of bioactive compounds and fiber from legumes, fruits, and vegetables . Studies on the incorporation of pomace into extruded products showed that increased pomace levels beyond a certain level had negative effect on the expansion quality [16,17,20,27,28]. However, recently our other colleagues found that carrot and cherry pomace incorporation into cornstarch extrudates resulted in better expansion and nutritional quality [18,19]. The objective of the current study was to examine the impact of GP incorporation on the quality of the cornstarch extrudates during extrusion processing to produce a nutrient enhanced extruded product with good quality attributes.
Cornstarch (native dent cornstarch with 23% amylose) was obtained from Tate & Lyle (Decatur, IL, USA). White GP was received from Woodward Canyon Winery (Lowden, WA, USA) that mainly contained Chardonnay grape skins, seeds, as well as some stems. The GP was freeze-dried in VirTis freeze drier (Vertis Comp. Gardiner, NY, USA) and ground using a cyclone mill (Model# 3010-060, UDY Corp. Fort Collins, CO, USA). Particle size of the white GP was determined through the USA standard test sieves of 75, 125, 150, 212 and 250 μm (Model# 78-700, Fieldmaster, Science First, Yulee, FL, USA). The GP bulk density was determined by measuring weight and volume of the GP content using standard graduated cylinder, and expressed in grams per cubic centimeter . The moisture (oven drying), ash, protein content (Kjeldahl, protein factor: 6.25), and total sugar (using Fehling’s reagents) were determined by the official methods (number 968.21, 945.18, and 974.06, respectively) of AOAC . Total dietary fiber content was determined using Sigma total dietary assay kit (TDF-A100 Sigma, St. Louis, MO, USA).
Feed mixture preparation
The cornstarch and GP (0%, 5% and 10% w/w) were mixed in a Hobart mixer (Model #A-200, Hobart, OH, USA) and equilibrated at three moisture levels: 16, 20, and 24 ± 0.2 (% w.b.) by adding the calculated amount of water. The feed mixtures were then stored in airtight plastic containers at 4°C overnight.
Processing conditions of extruder and process response
A 20 mm co-rotating twin-screw extruder (Model# TSE 20/40, CW Brabender, S. Hackensack, NJ, USA) with a length of 400 mm and the length to diameter (L/D) ratio of 20:1, was used for all the extrusion experiments. The temperature profile of the four independent zones of the extruder were kept constant at 50°C, 100°C, 140°C, and 140°C. A cylindrical die with a diameter of 3 mm was used. The feed rate of the material was fixed at 3.25 kg/h using a calibrated twin-screw volumetric feeder (Model# 15-37-000, CW Brabender Instrument, NJ, USA). The screw speeds were varied from 150 rpm to 250 rpm. These extrusion parameters were selected based on the literature data available on cornstarch extrusions, and our previous extrusion research experiments .
The premixed cornstarch and GP combinations were extruded as per the experimental design. After 5 min, when the extruder attained the stable conditions of pressure, torque and output flow, extrudates were collected. The collected extrudates were dried in a convection oven (Model# 414004-568, VWR International, LLC, PA, USA) at 45°C for 18 h. The extrudates had final average moisture of 4% to 6% (w.b.) and were stored in airtight plastic bags at 4°C until analysis. During processing, continuous data from the extruder, including die pressure, motor torque, and zone temperature, were recorded using Data Acquisition System for ATR and Intelli-Torque (CW Brabender, S. Hackensack, NJ, USA). The average of 10 data points, being taken at 20 second intervals during stable conditions, was used to calculate the specific mechanical energy (SME), motor torque, and die pressure according to previously reported method .
Expansion ratio (ER): Radial expansion was determined by measuring the diameter of 10 randomly chosen extrudates from one processing condition with a calipers (Mitutoyo America Corp., Aurora, IL, US) according to the method reported previously . For each experimental trial, two data points per single extrudate, with a total of 10 random extrudates, were taken and the mean diameter was calculated. ER was determined by dividing the mean diameter of the extrudates by the die diameter (3 mm).
Unit density: Unit density of the extrudates was determined in triplicate through displacement of 1.0 mm diameter glass beads (General Laboratory Supply, Pasadena, TX, USA) according to the method reported previously . Glass beads (30 ml) were filled in a 50 ml graduated cylinder, and the displacement of the beads by the extruded sample (2.0 g) was recorded. The density was equal to the sample mass divided by the sample volume.
Water absorption and water solubility index: Water absorption index (WAI) and water solubility index (WSI) were determined by the previously described procedure . Briefly, a portion of milled extrudates (2.5 g) was taken in 50 ml vial containing 30 ml of 30°C distilled water. Samples were mixed for 30 min in a 30°C water bath and then centrifuged at 3000 g speed for 10 min. The supernatant was removed and dried overnight. The weight of wet precipitate was recorded after 10 min for WAI. WAI was calculated as the ratio of the mass of the wet precipitate to the mass of the original dry weight. WSI was expressed as percentage of the overnight dried solid weight of the collected supernatant to the original sample weight.
Hardness: Hardness of the extrudates was determined by the previously reported method . Briefly, texture analyzer TA.XT2i (Texture Technologies, Scarsdale, NY, USA) with a single-blade and a 25 kg load cell with a test speed of 1 mm/s was used. The maximum force required to break an extrudate was recorded. Ten extrudates were analyzed per treatment and the mean peak force was reported.
Color: Color measurements were performed on milled extruded samples from all the trials, using a CM-5 spectrophotometer (Konica Minolta, NJ, USA) according to the method reported previously . The color was recorded using a Hunter color scale as the mean of three L*, a*, and b* readings, where L* indicates lightness, a* indicates redness, and b* indicates yellowness. A standard white and black calibration plate was used to equilibrate the spectrophotometer prior to color measurements.
Total polyphenolic compounds extraction and analysis: Total polyphenolics were extracted from raw material before processing and from extrudates after processing as previously reported . Briefly, samples were defatted twice with n-hexane at 70°C for 20 min. Total polyphenols were extracted from 1 g of GP using 10 ml of 80% ethanol that contained 1% formic acid (v/v) for 12 h, and then centrifuged at 10,000 × g for 15 min. The supernatants were collected and residue samples were re-extracted once under the same conditions. The total polyphenolic content (TPC) was determined by modified Folin–Ciocalteu assay in 96-well plate format . Briefly, 200 μl of the extracts were mixed with 12.5 μl of Folin-Ciocalteu reagent and then 37.5 μl 20% Na2CO3 in a 96-well plate. The plate was incubated at room temperature for 2 h when the absorbance was read at 760 nm on Synergy™ H1 microplate reader (BioTek, Winooski, VT, US). Gallic acid was used to generate the standard curve. The TPC was expressed as milligrams of gallic acid equivalents per gram of dried weight (mg GAE/ g DW). TPC data was subjected to General Linear Model of Statistical Analysis System-2000 and was expressed as mean ± standard error of mean. A significant difference was considered as P ≤ 0.05.
Total anti-oxidant activity and 2,2 Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging analysis: Assay was performed in a 96-well plate using previously published method with some modifications . Briefly, 200 μl of DPPH solution (60 μM) (Sigma, St. Louis, MO, USA) was added to wells of 96-well plate containing 50 μl of diluted extracts of the raw mix and GP extrudates or gallic acid standard solution. After incubation for 90 min at room temperature, the DPPH scavenging activity was measured at 517 nm absorbance on Synergy™ H1 microplate reader (BioTek, Winooski, VT, US). The total antioxidant activity was expressed as μg GAE/g DW based on the gallic acid standard calibration curve. The DPPH radical scavenging activity expressed as percentage of inhibition was calculated using the formula:
A is the absorbances
Acontrol is reading without samples.
Effects of GP extrudates extracts on intracellular reactive oxygen species (ROS) production in CaCO2 human colonic epithelial cells: CaCO2 cell line was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were grown in in Dulbecco’s Modified Eagle’s medium complete media: DMEM (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin–streptomycin (Sigma) at 37°C with 5% CO2 in a humidified incubator. Intracellular ROS levels were measured using a cell-permeable fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFH-DA) (Millipore, MA, USA) as previously described with some modifications . Briefly, 100 l of CaCO2 cells (5 × 105 cell/ml) were seeded in 96-well plate, and cultured in DMEM media with 10% FBS at 37°C with 5% CO2 for 12 h. Then, cells were pre-treated with 10 l of the raw mix and GP extrudates extracts in DMEM media for 12 h. After washing with PBS once, the cells were incubated with 100 μl of 10 M fresh DCFH-DA in PBS for 30 min. Then the cells were washed with PBS once, and incubated with 100 μl of PBS or 0.5 mm H2O2 for 30 min. Fluorescence of each well was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm on Synergy™ H1 microplate reader (BioTek, Winooski, VT, US). All the values were normalized with the control (no extract no H2O2).
Experimental design and statistical analysis
Response surface methodology was used to investigate the effects of extrusion processing conditions on the process responses (SME, motor torque, and die pressure) and product responses (ER, unit density, hardness, WAI, WSI, and color) of the extrudates. The independent variables were feed moisture (16, 20, and 24 ± 0.2% w.b.), screw speed (150 rpm, 200 rpm, and 250 rpm), and the GP level (0%, 5%, and 10%). A Box-Behnken design was used to determine the experimental conditions (Table 1). Data was subjected to regression analysis and a second-order polynomial regression model:
|Corn Starch (%)||GP
|Screw Speed (rpm)|
Table 1: Extrusion parameters and experimental design.
was used to fit the data for each response. Terms βo, βi, βij where I = j, and βij where I ≠ j are the coefficients for intercept, linear, quadratic, and interactive effects respectively. The term Y is the response, and Xi and Xj are the independent variables of moisture, screw speed, and GP content. The responses were plotted as a function of two independent variables, keeping the other one constant at the middle level using Origin software (version 9.0, Origin Lab, Northhampton, MA, US). A significant difference for a coefficient was considered as P ≤ 0.05.
GP composition is a potential nutritive adjunct ingredient for extrudates
The GP had a moisture content of 13.96 ± 0.14 g/100 g, ash content of 3.79 ± 0.36 g/100 g, protein content of 7.15 ± 0.12 g/100 g, total sugar content of 29.31 ± 0.63 g/100 g, total dietary fiber content of 39.24 ± 1.40 g/100 g, and crude fats content of 6.55 ± 0.5 g/100 g. The bulk density of the GP was 0.48 ± 0.01 g/cm3. The particle size distribution in GP was 44.65% >250 μm, 19.15% = 250-212 μm, 26.34% = 212-150 μm, 2.27% = 150-125 μm, 2.27% = 125-75 μm, and 0.85% <75 μm. GP is rich in dietary fiber and other nutrients, and its combination in cornstarch expanded products can enhance the nutritional quality and market value of GP incorporated products. The percentage of fiber, sugar, protein and fat in the GP can interact with cornstarch forming complexes that can greatly affect the textural properties of the extrudates . Though fiber was previously considered as inert material that reduces expansion [38,39], recently we have found that carrot and cherry pomace in cornstarch can play an active role in the expansion of extrudates that is potentially due to the interaction between pomace fiber particles and starch [18,19].
The process response parameters (SME, motor torque, and die pressure) indicate energy input to the materials in the extrusion process, which are affected by the extrusion processing inputs such as material composition (%GP, moisture), and screw speed . Regression results obtained for SME, motor torque, and pressure are shown in Table 2. Feed moisture had a negative linear effect (P≤0.01) on the die pressure. Screw speed had a positive linear effect (P≤0.01) on SME but no effect on motor torque or die pressure was observed. Feed moisture, GP, and screw speed had negative quadratic effects on the SME. The interactive effects of moisture and GP were significant for SME (P≤0.01), motor torque (P≤0.01), and die pressure (P≤0.05). The calculated SME ranged from 160.88 ± 4.79 to 511.21 ± 11.08 kJ/kg, and measured torque values ranged from 8.29 ± 0.19 to 21.71 ± 0.52 Nm, while the mean calculated values for pressure were 396.10 ± 3.33 to 859.70 ± 12.20 psi (Table 3).
|Level||SME||Motor Torque||Die Pressure||Expansion||Density||WSI||WAI||Hardness||Color|
|M × M||-0.996*||-0.041||9.440**||0.004||0.003||0.171||-0.041**||0.070||0.002|
|G × G||-1.151**||-0.051**||0.485||-0.001||0.002**||-0.050||-0.024**||0.140*||0.021**|
|SS × SS||-0.010**||0.000*||-0.004||0.000||0.000||0.001||0.000||0.001||0.000*|
|M × G||1.312**||0.057**||-1.173*||-0.012**||0.004**||-0.152||0.009||0.124||-0.012**|
|M × SS||-0.071||0.003||0.151**||0.000||0.000**||-0.014||0.000||-0.003||0.000|
|G × SS||-0.020||-0.001||0.060||0.000||0.000*||-0.008||0.001||0.002||0.000|
SME:Specific mechanical energy; WAI:Water absorption index; WSI:Water solubility index; C:Model constant; M, G and SS:Linear effects of moisture content, grape pomace, and screw speed, respectively; M × G:Interaction of moisture content and grape pomace; M × SS:Interaction of moisture content and screw speed; M × M, G × G; SS × SS:Quadratic effects of moisture content, grape pomace, and screw speed, respectively.
*and** indicate significant at P≤0.05and 0.01 respectively.
Table 2: Results of regression analysis for cornstarch grape pomace extrudates.
|Screw Speed (rpm)||SME
|16||0||200||404.6 ± 4.51||17.56 ± 0.20||814.00 ± 4.57|
|441.00 ± 6.79||19.14 ± 0.29||816.10 ± 5.28|
|5||150||374.47 ± 10.5||21.67 ± 0.16||843.50 ± 17.39|
|375.16 ± 8.92||21.71 ± 0.52||859.70 ± 12.2|
|250||511.21 ± 11.08||17.75 ± 0.38||730.90 ± 16.86|
|500.56 ± 9.05||17.38 ± 0.31||700.90 ± 12.12|
|10||200||409.20 ± 4.14||17.76 ± 0.18||852.50 ± 5.74|
|382.47 ± 7.08||16.6 ± 0.31||858.00 ± 7.71|
|20||0||150||251.26 ± 2.8||14.54 ± 0.16||462.00 ± 2.61|
|233.80 ± 2.42||13.53 ± 0.14||460.50 ± 2.56|
|250||376.14 ± 13.59||13.06 ± 0.47||402.80 ± 3.34|
|346.47 ± 2.98||12.03 ± 0.10||400.40 ± 3.99|
|5||200||365.19 ± 4.38||15.85 ± 0.20||464.90 ± 6.22|
|352.29 ± 6.02||15.29 ± 0.26||464.90 ± 5.50|
|359.66 ± 4.34||15.61 ± 0.19||470.20 ± 4.94|
|402.98 ± 8.18||17.49 ± 0.36||461.90 ± 6.84|
|358.51 ± 10.75||15.56 ± 0.47||471.60 ± 6.06|
|357.59 ± 6.10||15.52 ± 0.26||468.00 ± 3.74|
|10||150||284.78 ± 5.42||16.48 ± 0.31||502.60 ± 2.74|
|262.66 ± 3.47||15.2 ± 0.20||507.30 ± 3.27|
|250||397.16 ± 11.60||13.79 ± 0.40||504.10 ± 2.54|
|347.34 ± 7.29||12.06 ± 0.25||507.3 ± 4.63|
|24||0||200||197.46 ± 3.86||8.57 ± 0.17||453.00 ± 2.70|
|191.24 ± 2.4||8.3 ± 0.10||450.70 ± 2.68|
|5||150||160.88 ± 4.79||9.31 ± 0.28||437.40 ± 2.19|
|185.07 ± 3.40||10.71 ± 0.20||439.20 ± 3.19|
|250||238.76 ± 5.47||8.29 ± 0.19||423.40 ± 3.60|
|256.04 ± 5.11||8.89 ± 0.18||423.30 ± 2.96|
|10||200||250.45 ± 8.00||10.87 ± 0.35||400.30 ± 3.42|
|294.23 ± 5.06||12.77 ± 0.22||396.10 ± 3.33|
Table 3: Results values of process response (SME, motor torque and pressure).
The feed moisture and GP levels affected all the process response parameters (Table 2). The values of SME, motor torque, and pressure were lower at higher feed moisture and GP levels (Table 3). The feed moisture and pomace affect the melt viscosity of the material being processed [18,19]. The main reason for lower values of the process parameters at elevated feed moisture levels is reduction in the viscosity of the melt inside the extruder. Low viscosity melt faces less resistance in the screws and thus will be easily pushed out through the extruder with minimum energy input. It is likely that the inclusion of the GP disrupted the starch melt and contributed to the decrease in viscosity by acting as lubricant inside the extruder, which is seen in other studies [18,19]. A lower melt viscosity due to GP could account for the lower SME, motor torque and pressure observed in our study. This trend was also in line with reduced expansion of the extrudates at a higher level of GP (10%) and moisture content (24%). Further, the SME integrates torque, screw speed, and pressure. The SME, motor torque, and pressure, values were low at high moisture content via regression analysis (Table 2). These results were coincided with the correlation results, which showed that SME, motor torque and pressure positively correlated to each other (Table 4). Increase in screw speed increased SME while the motor torque decreased (Table 2). At GP level of 5%, the SME increased with the increasing screw speed and the decreasing feed moisture level (Figure 1). Generally, with increased screw speed, the SME increases. It is because increasing screw speed applies increased shear to the material being processed, resulting in greater SME input and decrease in motor torque. This is associated with decreased viscosity due to degradation of the starch in the material being processed [18,19,30].
|Level||SME||Motor Torque||Pressure||Expansion||Density||Hardness||WSI||WAI||Color (L)|
SME:Specific mechanical energy; WAI:Water absorption index; WSI:Water solubility index; L:Lightness.
*and** indicate significant at P ≤0.05, and 0.01, respectively.
Table 4: Cross-correlation values for cornstarch grape pomace extrudates.
Expansion ratio: The ER of cornstarch extrudates with GP ranged from 2.06 ± 0.12 to 3.83 ± 0.14. ER was found to be most dependent on GP and feed moisture levels, as shown by the regression analysis (Table 2). GP had a positive linear effect (P≤0.05) on the ER, along with a negative interactive effect (P≤0.01) in combination with moisture. It was observed in the regression response surface plot (Figure 2A) that increased feed moisture led to significant reduction in ER. As explained earlier, the high moisture level in the feed led to a reduction in the SME that could cause reduced physicochemical transformation of starch, and hence reduced the ER. Moreover, the post expansion collapse of cells at the die exit due to decrease in temperature, and migration of additional moisture from inside to the outside in expanding starch reduced the ER. Previous researchers also found an inverse relation between the ER and feed moisture [25,39], which are in line with our observations.
Addition of GP (5%) at low feed moisture (16%) resulted in an increase in the ER compared to the control with 0% GP, while the expansion ratio decreased with the addition of GP (10%) at a high feed moisture (24%) (Figure 2A,2B). The expansion phenomenon of the extruded products is mainly based on the flashing off of the steam that expands the starch matrix, forming a porous product, along with the die swell property of the materials [24,25,40]. Traditionally, it was thought that addition of fiber reduces the ER of the final product . However, the results suggest that instead of the GP being inert at low moistures, it is playing an active role either through the fiber, or the other components of the GP. It is possible that the fiber could have played an active role at 5% GP by being uniformly distributed in the starch matrix, which would allow maximum expansion, with fiber not disrupting the expanded starch structure. This trend is prominent in the carrot pomace and cherry pomace incorporated cornstarch expended products [18,19]. Other possibilities could be that sugar in addition to fiber in GP might have more specific effects. Sugar crystals act as filler at lower feed moisture and enhances expansion of the extrudates . Fiber in GP might have acted as a nucleating agent allowing cornstarch to trap more air cells by surrounding them firmly, while sugars in GP at low feed moisture might have enhanced the film forming effect by allowing bubble walls to be stabilized, resulting in larger porous structure. On the other hand, high GP and high moisture level has the tendency to disturb the starch matrix and the GP distribution during extrusion. This could have affected the expansion phenomenon such as collapsing of the cells due to aggregated GP in different spots of the starch  and plasticizing effect of sugars on starch  resulting in the reduction of the ER. Similarly, cherry pomace at 15% in cornstarch significantly reduces the ER compared to control . Our findings are opposite to that of Altan and colleagues who found a decrease in the expansion with up to 6% GP of barley flour extrudates . This difference could be due to difference in the raw material ingredients, as well as processing parameters, which is also evident from our previous studies on carrot and cherry pomace incorporation in cornstarch [18,19]. Overall, this suggests that there was positive active interaction between the GP constituents (sugars and fiber) and starch during extrusion that enhanced the expansion.
Unit density: Unit density is inversely proportional to expansion. Unit density values ranged from 0.11 ± 0.00 to 0.58 ± 0.13 g/cm3 for all extrudates. The regression analysis (Table 2) indicated that the linear and quadratic effect of GP significantly affected the unit density of the extrudates. The individual two-way interactive effects of all the variables: moisture, GP, and screw speed positively affected the unit density of the extrudates (P≤0.01). Increase in GP resulted in low unit density of the extrudates at a constant screw speed, while unit density of the extrudates decreased with decrease in moisture content (Figure 2C). This trend of the unit density can be attributed to the same factors that increased the ER at low feed moisture. Also with the inclusion of GP, the starting flour mix became less dense as GP unit density (0.48 ± 0.01 g/cm3) was significantly lower than that of cornstarch (0.78 ± 0.02 g/cm3). Further, the ER was negatively correlated with unit density (Table 4). Lower density and higher expansion are considered as favorable characteristics of extruded products. The GP levels effect on the unit density, are opposite to those found for extruded product of barley flour with GP , and corn flour with pineapple pomace , which could be due to the unique physical properties of GP and cornstarch.
Hardness: Hardness is a measure of the amount of force applied to break the sample. Lower the hardness the crispier and crunchier the extruded product is typically. GP had a significant negative linear, and significant positive quadratic effect on hardness (Table 2). The response surface plot for the GP and moisture content at fixed screw speed (200 rpm) is shown in Figure 3A. For low feed moisture (16%), increase in the GP led to lower hardness than the control samples. This low hardness, is anticipated from the fact that low density and high expansion correlates with low hardness [32,42]. These results are in line with unit density, which had a positive correlation with hardness (Table 1). Similar results are found in barley flour extrudates with GP  and the chick pea flour extruded snack foods . The decrease in feed moisture resulted in an increased melt viscosity and SME, while the GP incorporation would result in uniform distribution of GP in cornstarch, forming more air cells with a final porous structure, which could decrease hardness. The lower hardness with GP inclusion (at 5%) can provide a crispier texture than only cornstarch extrudates when extruded under low moisture condition, in addition to health beneficial attributes.
Water absorption index (WAI) and water solubility index (WSI)
WAI measures the volume occupied by starch after swelling in excess water, which can indicate the starch integrity in water after the extrusion processing . Feed moisture had positive linear, and negative quadratic effect while GP had positive quadratic effect on the WAI (Table 2). According to the response surface plots (Figure 3B) with increased feed moisture and GP, the WAI was lowered (screw speed 200 rpm). A similar trend was observed for GP (5% and 10%) levels that with an increase in moisture and screw speed heightened the WAI. At each feed moisture level (16, 20 or 24 ± 0.2%), the increase in screw speed resulted in lower WAI values, while the inclusion of GP resulted in higher WAI values. As increased screw speed results in an increased SME, that would ultimately result in more breakdown of the starch. This suggests that high screw speed renders more soluble products . GP contains fiber, carbohydrates, and proteins components that can provide more hydrophilic forces to compete for water than the starch , which could be another explanation to the higher WAI found in extrudates with GP. WAI is an indicator of the hydrophilic groups and their gel-forming capability within the starch matrix . High WAI values indicate that the extrudates can hold water with lesser solubility. This is a valuable property in processing breakfast cereal products, which helps to increase bowl life.
WSI measures soluble polysaccharides liberated from the starch after extrusion process, indicating molecular components degradation during extrusion . WSI values ranged from 14.23 ± 3.85 to 72.99 ± 2.57% for all the extrudates. GP exhibited positive linear effect on WSI (Table 2). At screw speed of 200 rpm, WSI had inverse relation with GP and feed moisture as shown by the response surface plot (Figure 3C). At 16% moisture level, WSI exhibited a direct trend with increased screw speed, and an inverse trend with 5% GP inclusion. These results are in line with the previously reported WSI range . The lower WSI of GP extrudates was consistent with their higher WAI, which was further proved by the negative correlation (-0.76) between WSI and WAI (Table 1). Lower values of WSI are often associated with the less dextrinization of starch during extrusion. Addition of GP provides insoluble fiber and other hydrophilic groups that can interact with starch to reduce the overall gelatinization, and hold water instead of being solubilized [27,43]. In fact cherry pomace in swelled starch granules trapes water and lowers WSI . However, soluble sugars within the GP could also lead to an increase in WSI since they are more dextrinized components. The overall WSI values are likely a balance between the WSI from the starch components and from the GP components. This is likely why WSI is seen to increase with GP inclusion at low moistures, but decrease at high moistures. The lower moistures favor more mechanical breakdown in the starch leading to dextrinization as opposed to at higher moisture. It is also relevant from the high SME as it enhanced the starch breakdown and hence increased WSI. The low WSI value is favorable for the extruded product to maintain the structure; indicating GP can be utilized in the development of direct expanded breakfast cereals with crispier textures and longer bowl life.
The regression analysis for the lightness (L) is shown in Table 2. GP level had a prominent effect on color parameters (L, a, b) with positive linear effect and quadratic effects on a, and b values (P≤0.01). Screw speed had a linear and quadratic effect on L and b values (P≤0.01). In addition, the L value was also affected by interactive effect of feed moisture and GP level (P≤0.01). Reduction in lightness with increasing GP level was observed at screw speed of 200 rpm. These findings are in agreement with those of Altan and colleagues who also found a reduction in lightness with GP inclusion . The low L value with increased GP could be due to the browning Maillard reaction and caramelization due to the presence of more simple sugars and proteins in the GP compared to cornstarch. Increased a and b values could also be due to the yellowish pigments in the GP and its heat degraded products.
Total polyphenolic content of GP extrudates extracts
Based on the quality analysis of cornstarch extrudates with GP, the extrudates at low feed moisture (16%) conditions were further chosen for TPC quantification. TPC of the dried GP was 58.15 ± 5.21 mg GAE/g DW. TPC of the raw material mixes at 16% feed moisture with 0%, 5% and 10% GP were beyond detection level, 1.12 ± 0.03, and 1.89 ± 0.06 mg GAE/g DW, respectively. The loss of polyphenolics depended on the extrusion process combinations. TPC retention in the extrudates with 5% GP was 74.10% (150 rpm), and 78.57% (250 rpm), while TPC in products with 10% GP under 200 rpm had no significant loss (Figure 4A). Results indicated that most of the polyphenolics retained in extrusion processing and thus could contribute to the nutritional quality of the extrudates. It has been reported that heat (baking) above 180°C negatively affects the TPC of the grape seed flour . The difference may be attributed to the short residence time usually less than 45 S in extrusion processing  as opposed to significantly longer baking times. In support, recently, we found that extrusion technology has a very minimal effect on the loss of water soluble TPC in cherry pomace incorporated in cornstarch, with no significant decrease of TPC in the extruded product .
Figure 4: Total phenolic content, antioxidant activity, and effect on ROS production of raw material mixes and cornstarch extrudates with GP processed at 16 % feed moisture content and 150-250 rpm screw speed. Total phenolic content (A), Antioxidant actvity (B), Percent DPPH inhibition (C), and intracellular reactive oxygen species (ROS) scavanging activity in CaCO2 cells.
Effect of extrusion on the total antioxidant activities of GP extrudates
Based on the percent retention of TPC in extrudates with 5% GP, we further accessed the possible influence of extrusion processing on GP functionality. We evaluated the antioxidant activity of the 5% GP level before and after extrusion process. The antioxidant activity of the 5% GP at 16% feed moisture before extrusion was 22.15 ± 0.03 μg GAE/g DW, after extrusion at 150 rpm was 21.80 ± 0.21 μg GAE/g DW, and at 250 rpm was 21.31 ± 0.38 μg GAE/g DW. The antioxidant activity and DPPH free radical scavenging activity of extrudates with 5% GP at 150 rpm maintain the same as the raw material before extrusion with a 98% retention (Figure 4B,4C). Our findings showed that functionality of the GP bioactives could be maintained if the extrusion processing was conducted at an optimal condition, and hence GP incorporation enhances the nutritional value of extrudates. Previously, the baking process of grape seed flour significantly reduced its antioxidant activity . The difference in time duration of exposure to heat which is less than 45s in extrusion processing  can explain the retention of antioxidant activity of the GP extrudates. Based on the physicochemical quality of the extruded products with GP, the addition of 5% GP level in cornstarch processed at 16% moisture level and 150 rpm screw speed is promising to produce healthy extrudates.
Effect of GP extrudates extracts on intracellular ROS production in Caco-2 human colonic epithelial cells
Furthermore, we examined the protective effect of GP extrudates against ROS production in human colonic epithelial cells using poplyphenolic extract prepared from GP raw mixes and extrudates. Hydrogen peroxide (H2O2) treatment induced enhanced ROS production in Caco-2 cells, which was mitigated by the GP polyphenolic extract treatment (Figure 4D). In fact, GP polyphenolics quenched ROS production in Caco-2 cells bringing it lower than the control with no detrimental effect of extrusion cooking. GP polyphenolics possess antioxidant as well as anti-infla mmatory effects with a well-established health beneficial effect on intestine [9,12,45,46]. These finding suggest that GP can be used as adjunctive ingredient in the production of healthy extrusion based cornstarch expended products.
Grape pomace incorporation in combination with feed moisture greatly affected the expansion quality of cornstarch extrudates. The 5% grape pomace level at the 16% feed moisture and 150 rpm resulted in enhanced expansion with substantial retention of TPC, total antioxidant activity in the cornstarch extrudates. This research can be applied in the making of corn starch expanded products with good nutritional value, which will enhance usage of this byproduct maximizing revenue of expending grape and wine industry. Further research on the sensory characteristics and physical and chemical interactions of GP with cornstarch need to be investigated.
This work was financially supported by Washington State University new faculty seed grant (10A-3057-9906) to Dr. Mei-Jun Zhu. We thank the Woodward Canyon Winery (Lowden, WA) for providing the grape pomace and the Tate & Lyle (Decatur, IL, US) for providing cornstarch for the experiments. We also thank to Frank Younce and Bhim Thapa at Washington State University for their help during the project.