Research in Experimental

Click or tap here to enter text. NHM 454 Research in Experimental & Functional Foods Worksheet Instructions: Answer the following questions, based on the article, “Total antioxidant content of alternatives to refined sugar”. 1. What is the hypothesis for this research? Explain. What other research term can be used and is commonly used in a research article to “call out” the hypothesis? Explain. Click or tap here to enter text. 2. How were “potential” confounding variables controlled for in this study? Explain. Click or tap here to enter text. 3. What outcome measure was used to test the hypothesis? Explain. Click or tap here to enter text. 4. What characteristics were most notable about the sugars with the greatest antioxidant content? Explain. Click or tap here to enter text. Instructions: Answer the following questions, based on the article, “Acute intake of phenolic-rich juice improves antioxidant status in health subjects”. 1. Among the exclusion criteria of this study, why were participants with inflammatory disease excluded? Explain. Click or tap here to enter text. 2. The basic design for this study follows that of a Click or tap here to enter text. RCT to determine causal relationship between phenolic-rich juice intake and antioxidant status. 3. What were the controlled variables in this study? Explain. Why is it important to control for these? Explain. Click or tap here to enter text. 4. What two outcomes were conflicting from this study based on the body of data suggesting that diets rich in antioxidant-dense foods decrease risk of oxidative stress-related diseases? Explain. Click or tap here to enter text. 1|Page RESEARCH Current Research Total Antioxidant Content of Alternatives to Refined Sugar KATHERINE M. PHILLIPS, PhD; MONICA H. CARLSEN, MSc; RUNE BLOMHOFF, PhD ABSTRACT Background Oxidative damage is implicated in the etiology of cancer, cardiovascular disease, and other degenerative disorders. Recent nutritional research has focused on the antioxidant potential of foods, while current dietary recommendations are to increase the intake of antioxidantrich foods rather than supplement specific nutrients. Many alternatives to refined sugar are available, including raw cane sugar, plant saps/syrups (eg, maple syrup, agave nectar), molasses, honey, and fruit sugars (eg, date sugar). Unrefined sweeteners were hypothesized to contain higher levels of antioxidants, similar to the contrast between whole and refined grain products. Objective To compare the total antioxidant content of natural sweeteners as alternatives to refined sugar. Design The ferric-reducing ability of plasma (FRAP) assay was used to estimate total antioxidant capacity. Major brands of 12 types of sweeteners as well as refined white sugar and corn syrup were sampled from retail outlets in the United States. Results Substantial differences in total antioxidant content of different sweeteners were found. Refined sugar, corn syrup, and agave nectar contained minimal antioxidant activity (⬍0.01 mmol FRAP/100 g); raw cane sugar had a higher FRAP (0.1 mmol/100 g). Dark and blackstrap molasses had the highest FRAP (4.6 to 4.9 mmol/ 100 g), while maple syrup, brown sugar, and honey showed intermediate antioxidant capacity (0.2 to 0.7 mmol FRAP/100 g). Based on an average intake of 130 g/day refined sugars and the antioxidant activity measured in typical diets, substituting alternative sweeteners could increase antioxidant intake an average of 2.6 mmol/day, similar to the amount found in a serving of berries or nuts. K. M. Phillips is a research scientist and director of the Food Analysis Laboratory Control Center, Biochemistry Department, Virginia Tech, Blacksburg. M. H. Carlsen is a doctoral student and R. Blomhoff is professor and head, Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Norway. Address correspondence to: Katherine M. Phillips, PhD, Biochemistry Department (0308), 304 Engel Hall, Virginia Tech, Blacksburg, VA 24061. E-mail: kmpvpi@ vt.edu Manuscript accepted: June 13, 2008. Copyright © 2009 by the American Dietetic Association. 0002-8223/09/10901-0005$36.00/0 doi: 10.1016/j.jada.2008.10.014 64 Journal of the AMERICAN DIETETIC ASSOCIATION Conclusion Many readily available alternatives to refined sugar offer the potential benefit of antioxidant activity. J Am Diet Assoc. 2009;109:64-71. O xidative damage has been implicated in the etiology of cancer, cardiovascular disease, and other degenerative disorders (1-3). Antioxidants are compounds with a reductive-oxidative potential and, therefore, have the ability to scavenge free radicals and other reactive oxygen species. Naturally occurring antioxidants in foods include vitamin E (tocopherols), vitamin C (ascorbic acid), flavonoids, lycopene, phenolic acids, and polyphenols, as well as some food additives (eg, butylated hydroxyanisole and butylated hydroxytoluene). Antioxidants prevent oxidative damage induced by free radicals and reactive oxygen species generated in vivo as byproducts of metabolism or inflammatory processes by suppressing their formation, acting as scavengers, or acting as their substrate. The total antioxidant capacity (TAC) of diets has been correlated with increased concentration of specific antioxidants (eg, carotenoids, tocopherols, vitamin C) and foods (eg, coffee, wine, fruits) (4). Serafini and colleagues (5) observed an inverse relationship between dietary TAC and incidence of gastric cancer. Current dietary recommendations are to increase the intake of antioxidant-rich foods rather than supplement specific nutrients (6). While the interaction of specific antioxidants and other food nutrients as related to physiological effects remain to be completely determined, TAC is generally considered a valuable parameter for identifying potentially rich food sources of biologically active antioxidants that might have beneficial health effects. TAC is assayed by several methods, including ferric-reducing ability of plasma (FRAP) (7), oxygen radical absorbance capacity (ORAC) (8), Trolox-equivalent antioxidant capacity (9), 2,2=-azinobis (3-ethylbenzothiazoline 6-sulfonate) (10), and 2,2-diphenyl-1-picrylhydrazyl (11) radical scavenging assays. The FRAP assay is a simple, fast, and inexpensive method for quantitative determination of the amounts of antioxidants in samples. The assay has little selectivity and measures most reductants above a certain reduction potential. The FRAP assay does not detect glutathione or protein thiols. This is an advantage over the ORAC and Trolox-equivalent antioxidant capacity assays because these thiols, which are present in high concentrations in animal and plant cells, are mainly degraded in the intestine and poorly absorbed. The original FRAP assay has a limited ability to measure fat-soluble antioxidants (7). Therefore, a modified FRAP assay was developed and reported previously (12) that also measures fat-soluble © 2009 by the American Dietetic Association antioxidants. On the basis of these and other considerations (13), the FRAP assay was chosen for assessing TAC. Some recommended dietary changes involve adding or increasing the intake of antioxidant-rich foods, such as berries, dark chocolate, nuts, green tea, and red wine (14-17). Refined sugar and corn syrup are the predominate sweeteners in Western diets. The estimated annual intake of added sugars in the United States (predominately from refined cane and beet sugar, corn syrup, glucose, and dextrose) is 47.5 kg per capita (31 tsp or 130 g per person per day) (18), yet sugar and refined corn syrup are virtually devoid of vitamins, minerals, and phytochemicals. Substitution of whole grains for refined flours and baked goods is recommended because whole grains are richer in antioxidants and nutrients lost in the refining process (19,20). It might be similarly expected that unrefined sweeteners derived from plants would also be richer in antioxidants, but little data exist on the composition of these products. Current attention to reducing refined sugar intake largely translates into replacement by artificial sweeteners (sucralose, aspartame, etc) with the purpose of reducing energy and carbohydrate intake, whereas natural whole-food alternatives represent a way to increase antioxidant and nutrient consumption. Many alternatives to refined sugar are available, though not widely used. These include plant saps/syrups (eg, maple syrup, agave nectar), syrups made from raw sugar and grains (eg, molasses, barley malt, and brown rice syrup), honey, and fruit sugars (eg, date sugar), as well as raw cane sugar. It was hypothesized that some of these alternatives contain higher levels of antioxidants compared to refined white sugar. Blomhoff and colleagues (12,21,22) recently published values for the TAC of foods using the FRAP method. Results of the analysis of approximately 200 fruits, vegetables, spices and herbs, cereals, supplements, juices and drinks sampled mainly from European countries have been reported (21,22), and a table of the FRAP content of 1,113 US food samples was published recently (12). In the present study, additional results are reported for sweeteners, along with estimates of the impact on total antioxidant intake they could make if used as alternatives to refined sugar. METHODS Samples Samples (Table 1) were procured locally and also through the United States Department of Agriculture’s (USDA) National Food and Nutrient Analysis Program (NFNAP) between 2002 and 2006 for the analysis of other nutrients (23). NFNAP is designed to update and improve the food composition data in the USDA’s National Nutrient Database for Standard Reference (24). Data for selected artificial sweeteners (eg, aspartame and sucralose) were reported previously (12) and found to contain FRAP ⬍0.05 mmol/100 g; these products were not further considered in this study because the focus was on natural alternatives to refined white sugar that might be utilized by consumers. The sampling design for NFNAP has been described previously (25). Local samples were procured from major retail outlets and/or health food stores or online distributors and represented major brands available in the US marketplace. Because the purpose of this study was to screen antioxidant content, a full statistical sampling plan was not implemented for all foods, although multiple samples of most products were obtained (see Table 1). Samples were handled according to standardized, thoroughly documented procedures (26). When composites were prepared, each sample unit was mixed, if necessary, and a representative subsample of no less than 1 cup (240 mL) of liquids and 4 oz (113 g) solids was taken, then combined and stirred thoroughly. Subsamples were dispensed among 30-mL glass jars with Teflon-lined lids (Qorpak, Bridgeville, PA), sealed under nitrogen, and stored at ⫺60⫾5°C in darkness before analysis. Samples were shipped on dry ice via express air delivery from Blacksburg, VA to Oslo, Norway, received in frozen condition, and stored at ⫺80°C prior to analysis. The range of storage time in Oslo was from 0 to 25 weeks prior to analysis. Reagents TPTZ (2,4,6-tri-pyridyl-s-triazine) was obtained from Fluka (Sigma-Aldrich, Deisenhofen, Switzerland), sodium acetate trihydrate and FeSO4⫻7 H2O from Riedel-deHaën (SigmaAldrich, Germany), acetic acid and hydrochloric acid from Merck (Merck, Darmstadt, Germany), FeCl3⫻6 H2O from BDH Laboratory Supplies (Poole, Dorset, UK). MilliQ water (Millipore, Bedford, MA) was used to ensure proper water quality. Methanol of high-performance liquid chromatography⫺grade was obtained from Merck. FRAP Analysis The antioxidant assay of Benzie and Strain (7) was used with minor modifications that allowed quantitation of most water- and fat-soluble antioxidants, as described previously (12). A Technicon RA 1000 system (Technicon Instruments Corporation, Tarrytown, NY) was used for the measurement of absorption changes that appear when the Fe3⫹-TPTZ2 complex is reduced to the Fe2⫹TPTZ2 form in the presence of antioxidants. An intense blue color with absorption maximum at 593 nm develops. Measurements were performed at 600 nm after 4 minutes incubation. An aqueous solution of 500 ␮mol/L FeSO4⫻7 H2O was used for calibration of the instrument. Three analytical portions of each sample were extracted, each extract was analyzed in triplicate, and results are given as reduced TPTZ-Fe2⫹-complexes in mmol/100 g. Quality Control Stability of samples during storage was established in a previous study (12), where it was determined that different homogenized foods could be stored at ⫺80°C for 65 weeks with only negligible changes in antioxidant content. The assay was also fully validated as described in a previous report (12). The within-day repeatability measured as relative standard deviation ranged from 0.4% to 6%. The variation in the values for replicate items obtained from the same source was typically between 3% and 10% relative standard deviation. January 2009 ● Journal of the AMERICAN DIETETIC ASSOCIATION 65 66 January 2009 Volume 109 Number 1 Table 1. Antioxidant capacity [ferric-reducing ability of plasma (FRAP)] of sweeteners Product NDB numbera Honey 19296 Corn syrup, light 19350 Molasses, blackstrap NA Molasses, dark NA Maple syrup, 100% pure 19353 Agave nectar, light NA Agave nectar, amber Agave nectar, raw Blue agave nectar NA NA NA Brown rice syrup NA Brown rice syrup, powdered Brown rice malt syrup Barley malt syrup Sugar, granulated white NA NA NA 19335 Sugar, light brown 19334g Brandb Composite typec No. of sample units per composite Sue Bee Sue Bee Store brand Store brand Multiple brandsf Dutch Gold Golden Blossom FMV Karo Clement Foods Co Clement Foods Co Clement Foods Co Slow as Molasses Plantation Brer Rabbit Grandma’s Golding Farms Brer Rabbit Private Selection Cary’s Spring Tree Madhava Madhava Madhava Madhava Molino Real Live Superfoods Lundberg Family Farms NOW Foods Emperor’s Kitchen N N N N N L L L N P P P L L L L L L L L L L L L L L L L L L Sweet Cloud Eden Organic Sweet Cloud Domino, C&H Domino, C&H Store Brand Store Brand Kroger Domino, C&H, Dixie Crystals Store brand FRAP mean mmol/100 g Standard errord Serving sizee 10 10 10 10 10 1 1 1 12 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.165 0.139 0.154 0.159 0.135 0.138* 0.193* 0.161* 0.008* 0.005 0.019 30 mL (1 Tbsp⫽21 g) L L L R R R R L N 1 1 1 3 3 1 2 1 10 0.717* 1.008 2.121* 0.009 0.017 0.004 0.009 0.004* 0.385 N 7 0.337 4.394z 4.776y 5.513x 4.251 4.533 4.900 0.412 0.371 0.454* 0.032 0.005 0.031 0.010 0.034z 0.143y 0.394z 0.006y 1.041* 0.003 30 mL (1 Tbsp⫽20 g) 0.001 0.018 0.064 0.048 0.090 0.142 30 mL (1 Tbsp⫽20 g) 30 mL (1 Tbsp⫽20 g) 0.026 0.014 30 mL (1 Tbsp⫽20 g) 0.024 0.003 0.003 0.002 0.003 0.010 0.042 0.003 0.295 30 mL (1 Tbsp⫽21 g) 0.166 30 mL (1 Tbsp⫽21 g) 30 mL (1 Tbsp⫽21 g) 30 mL (1 Tbsp⫽21 g) 30 mL (1 Tbsp⫽20 g) 1 oz (28.35 g) 30 mL (1 Tbsp⫽20 g) 30 mL (1 Tbsp⫽20 g) 0.002 0.009 0.001 0.002 1 oz (28.35 g) 0.018 1 oz (28.35 g) 0.015 FRAP mmol/ serving 0.035 0.029 0.032 0.033 0.028 0.029 0.041 0.034 0.002 0.001 0.879 0.955 1.103 0.850 0.907 0.980 0.082 0.074 0.091 0.007 0.001 0.006 0.002 0.007 0.030 0.079 0.001 0.295 0.143 0.202 0.424 0.002 0.005 0.001 0.003 0.001 0.109 Product mean FRAP mmol/100 g Product mean FRAP mmol/ serving 0.156 0.033 0.006 0.0012 4.894 0.979 4.562 0.912 0.412 0.082 0.019 0.004 0.031 0.010 0.089 0.0065 0.0021 0.019 0.200 0.040 1.041 0.295 0.717 1.565 0.143 0.313 0.009 0.003 0.361 0.102 0.096 (continued) Table 1. Antioxidant capacity [ferric-reducing ability of plasma (FRAP)] of sweeteners (continued) Product NDB numbera Sugar, dark brown Sugar, turbinado 19334g NA Sugar, raw cane NA Date sugar NA a Brandb Composite typec No. of sample units per composite FRAP mean mmol/100 g Standard errord Serving sizee Store brand Sugar in the Raw Sugar in the Raw Hain Pure Foods Florida Crystals Wholesome Sweeteners Sweet Cloud Bob’s Red Mill Barry Farm NOW Foods N R L R L L L L L L 6 3 1 3 1 1 1 1 1 1 0.689 0.079 0.210* 0.090 0.165 0.120 0.327* 4.586z 2.996y 2.237x 0.039 0.019 1 oz (28.35 g) 1 oz (28.35 g) 0.013 0.017 0.004 1 oz (28.35 g) 0.020 0.053 0.034 1 oz (28.35 g) FRAP mmol/ serving 0.195 0.022 0.059 0.026 0.047 0.034 0.093 1.300 0.849 0.634 Product mean FRAP mmol/100 g Product mean FRAP mmol/ serving 0.689 0.126 0.195 0.036 0.204 0.058 3.273 0.928 January 2009 ● Journal of the AMERICAN DIETETIC ASSOCIATION Entry reference number from US Department of Agriculture (USDA) Nutrient Database for Standard Reference (24). NA⫽not applicable (food not in database). Supplier information: Barry Farm (Wapakoneta, OH), Bob’s Red Mill (Bob’s Red Mill Natural Foods, Milwaukie, OR), Brer Rabbit (B&G Foods, Inc, Roseland, NJ), C&H (C&H Sugar Company, Inc, Crockett, CA), Cary’s (Specialty Brands of America, Inc, Westbury, NY), Clements Foods Co (Oklahoma City, OK), Dixie Crystals (Imperial Sugar Company, Sugar Land, TX), Domino (Domino Foods, Inc, Yonkers, NY), Dutch Gold (Dutch Gold Honey, Inc, Lancaster, PA), Eden Organic (Eden Foods, Inc, Clinton, MI), Emperor’s Kitchen (Great Eastern Sun, Asheville, NC), Florida Crystals (Florida Crystals Food Corp, West Palm Beach, FL), FMV (Inter-American Products, Inc, Cincinnati, OH), Golden Blossom (John Paton, Inc, Doylestown, PA), Golding Farms (Golding Farms Foods, Inc, Winston-Salem, NC), Grandma’s (Mott’s, Inc, Stamford, CT), Hain Pure Foods (The Hain Celestial Group, Inc, Boulder, CO), Karo (ACH Food Companies, Inc, Memphis, TN), Kroger (The Kroger Co, Cincinnati, OH), Live Superfoods (Bend, OR), Lundberg Family Farms (Richvale, CA), Madhava (Madhava Honey, Lyons, CO), Molino Real (Dictor S.A. de C.V., Guadalajara, Jalisco, Mexico), NOW Foods (Bloomingdale, IL), Plantation (Allied Old English, Inc., Port Reading, NJ), Private Selection (Inter-American Products, Inc, Cincinnati, OH), Slow as Molasses (Honeytree, Inc, Onsted, MI), Spring Tree (Spring Tree Maple Products, Westbury, NY), Sue Bee (Sue Bee Honey, Sioux City, IA), Sugar in the Raw (Cumberland Packing Corp, Brooklyn, NY), Sweet Cloud (Great Eastern Sun, Asheville, NC), Wholesome Sweeteners (Wholesome Sweeteners, Inc, Sugar Land, TX). c N⫽national composite of samples; R⫽regional composite of samples (25); L⫽sample(s) from a single outlet; P⫽commodity product provided by directly by producer. d Standard error, based on values from analysis of replicate subsamples. e Based on product label and/or US Department of Agriculture Nutrient Database for Standard Reference (24). f Composite of seven brands (ie, Madhava Mountain, Deep South, Barkmans Busy Bee, Billy Bee, Stollers, Winnie the Pooh, and Beemaid). g “Sugars, brown.” xyz Means with different superscript letter (x,y,z), within data for that product, were significantly different for n⫽3 analytical replicates (␣⫽.05); samples assayed with n⫽1 are marked with *. b 67 Serving Sizes and Sweetening Equivalents Weight and serving size of a typically consumed portion of each food was determined from the USDA National Nutrient Database for Standard Reference (24) and/or the product label, generally based on the US Food and Drug Association Nutrition Labeling and Education Act guidelines (27), or actual measurement of average portion weights taken during sample preparation. Statistical Methods Means, standard deviations, and standard errors were calculated using Microsoft Excel 2000 (version 9.0, Microsoft Corp, Redmond, WA). Data were subjected to an analysis of variance and Tukey test for multiple comparisons, with ␣⫽0.05, using SAS (version 8.2 [TS2M0], 2001, SAS Institute, Cary, NC). RESULTS Antioxidant Content of Sweeteners Table 1 summarizes the FRAP content of the individua ..