LIPID OXIDATION IN LOW MOISTURE FOODS: DEVELOPING TECHNOLOGIES FOR IMPROVING NUTRITIONAL COMPOSITION WITHOUT NEGATIVELY IMPACTING QUALITY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1007711
• Grant No.: 2016-67017-24611
• Cumulative Award Amt.: $469,775.00
• Proposal No.: 2015-05970
• Project Start Date: Jan 1, 2016
• Project End Date: Dec 31, 2019
• Grant Year: 2016
• Program Code: [A1361]- Improving Food Quality

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Food Science

Non Technical Summary
Dietary Guidelines for Americans (2010) and the new Scientific Report of the Dietary Guidelines committee (2015) both recommend that consumers increase consumption of polyunsaturated fats and decrease consumption of saturated fats to decrease risk for coronary heart disease. Low moisture foods are a large contributor of saturated fat in the American diet. For instance, grain-based desserts and snacks, including many low-moisture foods like crackers, cookies, and granola bars, are among the top three contributors of saturated fat to the American diet (National Cancer Institute, 2010). The association of high dietary saturated fat with consumption of low moisture foods suggests that improving the nutritional profiles of these products by substituting their saturated fatty acids with unsaturated fatty acids could have an important, positive impact on consumer health. However, such reformulations are challenging because fats high in saturated fatty acids are solid at room temperature and these solid fats play an important role in texture of low moisture foods. In addition, increasing polyunsaturated fatty acid composition in low moisture foods leads to an increase rancidity, which will negatively impact product quality, nutritional composition, and shelf-life.This project will determine how different ingredients in low moisture foods impact the development of rancidity. With this knowledge, technologies will be developed to help prevent rancidity which will allow for more unsaturated fats to be used in these projects thus making the foods healthier. In addition, these technologies will decrease food waste thus improving sustainability. This is important since over 40% of the foods produced on the farm become spoiled costing Americans over $180 billion in food/year.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	5010	1000	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
5010 - Food;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

lipid oxidation

antioxidants

low moisture foods

nutrition

polyunsaturated fatty acids

solid fat

Goals / Objectives
Dietary Guidelines for Americans (2010) and the new Scientific Report of the Dietary Guidelines committee (2015) both recommend that consumers increase consumption of polyunsaturated fats and decrease consumption of saturated fats to decrease risk for coronary heart disease. Low moisture foods are a large contributor of saturated fat in the American diet. For instance, grain-based desserts and snacks, including many low-moisture foods like crackers, cookies, and granola bars, are among the top three contributors of saturated fat to the American diet (National Cancer Institute, 2010). The association of high dietary saturated fat with consumption of low moisture foods suggests that improving the nutritional profiles of these products by substituting their saturated fatty acids with unsaturated fatty acids could have an important, positive impact on consumer health. However, such reformulations are challenging because fats high in saturated fatty acids are solid at room temperature and these solid fats play an important role in determining microstructure and physical properties of these products. In addition, increasing polyunsaturated fatty acid composition in low moisture foods leads to an increase in lipid oxidation, which will negatively impact product quality, nutritional composition, and shelf-life. Thus, improving the nutritional quality of the fats used in low moisture foods is extremely challenging as switching to more unsaturated fats impacts both their physical properties and chemical stability.The overall goal of this project is to identify the critical parameters involved in lipid oxidation pathways in low moisture foods so that a systematic approach can be utilized to develop effective antioxidant technologies. Our central hypothesis is that novel antioxidant technologies for low moisture foods could be developed if food manufacturers understood (i) the influence of lipid type and concentration on the structural organization and oxidation kinetics of lipids within model low moisture foods (crackers); (ii) the location of the lipids most prone to oxidation; and (iii) the location of antioxidants in relation to oxidizing lipids. Since the location and/or structural organization of lipids and antioxidants could also be influence by water it will be critical to determine the impact of water activity on lipid and antioxidant location.

Project Methods
The ingredients used to formulate the model crackers are shown in the Table below. Crackers will be prepared by mixing sifted flour, baking soda, and salt together followed by addition of interesterified soybean oil and mixing for 1 min (Speed 2) in a Kitchen Aid mixer until an agglomerated mass is formed. Water will then be added and further mixed with a dough hook for 1 min (Speed 2) to form a dough. Ten grams of additional flour will be kneaded into the dough by hand to form a non-sticky mass. The dough will be flattened by passing it through a Kitchen Aid pasta roller 2 times. The dough will be cut into 2.5 cm x 2.5 cm crackers and baked on an ungreased cookie sheet at 163 ºC for 21 min in an electric oven. After baking, crackers will be coarsely crushed and placed into acid-washed, 10-ml glass GC vials and stored at 55 ºC in the dark at relative humidities of 0, 11 and 33 and 43% by putting the samples in desiccator's containing silica gel and saturated LiCl, MgCl2, and K2CO3 solutions, respectively (Ponginebbi et al., 2000). Table 1. IngredientsMass (g)Percentage (w/w)Flour62.5050.6DI Water39.0031.6Interesterified Soybean Oil10.008.1Salt1.501.2Baking Soda0.580.5Flour for sprinkling on surface10.008.1Total123.58 g100 %The lipid soluble dye Nile Red (0.5%), the oxidatively sensitive dye, 5-(and-6)-carboxy-2′,7′-di?uorodihydro-?uorescein diacetate (carboxy-H2DFFDA) (0.1%) and fluorescent rosmarinic acid esters (varying concentrations) will be dissolved in ethanol, and then 1 mL of these solutions will be mixed with the fat phase or the final dough until even dispersal of colored dye is visually observed. Rhodamine B (protein specific dye; 0.3%) will be dissolved in the water and 1 mL will be added to the dry ingredients prior to formation of the dough. One mL ethanol will be added to control samples when necessary. All dye concentrations were determined by preliminary experiments which produced good confocal images (see earlier). The pH of the crackers will be determined using a standard pH meter by crushing crackers in deionized water (1-to-10 ratio) to form an aqueous slurry. The water activity of crushed crackers will be determined using a commercial device (Decagon Devices AquaLab Series 3). Water content will be determined in a vacuum oven and fat content will be determined using the Soxhlet method (Shahidi, 2005). A Nikon Confocal Microscope will be used to visualize the microstructure of the crackers. Nile Red will be excited at 488 nm and emission spectra will be collected at 515 nm. 5-(and-6)-carboxy-2′,7′-di?uorodihydro-?uorescein diacetate (carboxy-H2DFFDA) will be excited at 495 nm and emission spectra will be collected at 520 nm. Rhodamine B will be excited at 554 nm, and the emission spectra will be collected at 627 nm. Detector pinhole size will be 150 μm. While lipid hydroperoxides do not directly correlate to rancidity since they are not volatile aroma compounds, they can provide important information on lipid oxidation mechanisms such as the ability of transition metals to convert hydroperoxides into secondary lipid oxidation products. Lipid hydroperoxides will be measured using a modified version of the International Dairy Federation method as described by Shantha and Decker (1994). Cracker samples (0.1 g) will be pulverized using mortar and pestle, mixed with chloroform and methanol (2:1 v/v; 5 mL), vortexed briefly, and centrifuged (3400g) for 10 min to separate the solvent fraction. The hydroperoxide containing solvent fraction (200 μl) will be mixed with 17.0 μL of a 50/50 solution of ferrous sulfate dissolved in deionized water and barium chloride dissolved in 0.4 N HCl and 17 μl of ammonium thiocyanate dissolved in water. The final mixture will be vortexed, covered to prevent evaporation, and incubated at room temperature for 20 min to develop the color. Absorbance will be measured at 500 nm and the concentration of hydroperoxides will be calculated from a cumene hydroperoxide standard curve. Headspace hexanal will be used as a marker of omega-6 fatty acid oxidation. Sample vials will be closed with aluminum caps containing PTFE/silicone septa and headspace hexanal will be measured by a robotic solid phase microextraction (SPME) gas chromatograph (Panya et al. 2012). A 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/Carboxen/PDMS) stable flex SPME fiber will be inserted through the vial septum and exposed to the sample headspace for 10 min at 55°C. The volatiles on the SPME fiber will be desorbed at 250°C for 3 min in the GC injector at a split ratio of 1:7. The chromatographic separation of volatile aldehydes will be performed on a fused-silica capillary column (30 m × 0.32 mm i.d. × 1 μm) coated with 100% poly(dimethylsiloxane) (Equity-1, Supelco). The temperatures of the oven, injector, and flame ionization detector will be 65, 250, and 250° C respectively. Peak integration will be calculated using Shimadzu EZstart and concentrations will be calculated using a standard curve made from crushed crackers spiked with known hexanal concentrations. All experiments will be conducted in triplicate and repeated at least twice. Oxidation lag phases will be determined as the first data point that is significantly greater than the zero time value. Statistical analysis will be conducted using Minitab ® Statistical Software (Minitab, State College, PA). One-way analysis of variance (ANOVA) followed by Tukey's pairwise comparison will be conducted to determine differences of p<0.05.

Progress 01/01/16 to 12/31/19

Outputs
Target Audience:Target Audience Food Ingredient Suppliers DSM Monsanto Bunge Oils Kalsec Kemin Dupont Food Processing Companies Food Network International Frito Lay Kellogg Unilever Nestle Kraft Mondelez Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate student were trained on this grant. One is working for Dupont and the other for Ingredion. How have the results been disseminated to communities of interest?Talks have been given at the American Oil Chemist Society, Institute of Food Technologist, UMass Strategic Research Alliance, University of Massachusetts Chef's Conference, Clean Label Conference and NIFA Project Director Meeting. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Low moisture snacks, such as crackers, present a health concern for US consumers due to their high content of saturated fats, which increase a risk of coronary heart disease. This is because crackers are traditionally made from semi solid fats that are high in saturated fatty acids to provide structure. These semi-solid fats could be made healthier if they contained more unsaturated fatty acids. However, this increases lipid oxidation and thus decreases shelf-life. The objective of this study was to investigate the role of solid fat content (SFC) on the oxidative stability of a model cracker system. To determine how SFC effects lipid oxidation in crackers, different oils containing different SFCs were selected in 3 ways: 1) commercially available oils which had different SFCs, 2) blending fully hydrogenated soybean oil with liquid soybean oil to produce different SFCs but similar fatty acid and tocopherol homolog profile with interesterified soybean oil, 3) blending lipids to produce varied SFCs but fixed 18:2 percentage and tocopherol homolog concentrations. Oxidation stability was evaluated based on hydroperoxide and hexanal lag phases. Results from commercially available oils (1) show that increasing SFC resulted in increasing oxidative stability. Palm shortening (PS) had high SFC and were stable up to 42 days, the longest hexanal lag phase in this study. In contrast, liquid soybean oil (LSO) and canola/cotton blend (CB) had low SFCs and oxidized after 20 days. Interesterified soybean oil (ISO) which had a higher polyunsaturated fat content (40.6 % ) than CB (35.8 %) produced a more stable oxidatively cracker, presumably due to its higher SFC than CB (test conditions 1). In the second test, fully hydrogenated soybean oil was blended with liquid soybean oil to produce similar fatty acid profile as an interesterified soybean oil. Tocopherols isomers were adjusted to produce similar tocopherol profiles. Blended soybean oil had a longer lag phase than the interesterified soybean fat. In the third test, different fats, including fully hydrogenated soybean oil, interesterified soybean oil, palm oil, and sunflower oil were used to produce varied SFCs but fixed 18:2 at 20%. Tocopherols homologs were added to all mixtures to produce similar tocopherol profile. Unlike test conditions 1, the oxidative stability of blends of liquid and solid fats could not be predicted by SFC. A second project was completed on antioxidant strategies for low moisture foods. In this study, we examined the influence of water activity (aw), sugars (glucose, maltose, maltodextrin, and cyclodextrin), and proteins (casein and gluten) on the lipid hydroperoxide and hexanal lag phases of model crackers. Oxidative stability of crackers was in an order: aw 0.7 > aw 0.4 > aw 0.2 > aw 0.05. Higher water activities resulted in bigger differences between hydroperoxide lag phases and hexanal lag phases. Compared to non-reducing cyclodextrin and no added sugar controls, reducing sugars including glucose, maltose, and maltodextrin at the same dextrose equivalence increased both hydroperoxide and hexanal lag phases. At the same dextrose equivalence, oxidative stability was in the order of maltose > maltodextrin > glucose > control (no sugar added). The antioxidant effectiveness of maltose, a low sweetness profile sugar, increased with increasing concentrations from 1.1 to 13.8%. Increasing aw increased the antioxidant activity of maltose. For example, 1.1% maltose increased both hydroperoxides and hexanal lag phases by 9 days at an aw of 0.2, but increased hydroperoxide lag phase by 24 days and hexanal lag phase by 15 days at an aw of 0.7. Gluten was able to inhibit lipid oxidation with activity increasing with increasing aw while casein showed minimal antioxidant impact. Antioxidant activity of gluten decreased when its sulfhydryl groups were blocked by N-ethylmaleimide suggesting that cysteine was an important antioxidant component of gluten. Adjusting water activity and addition of reducing sugars and gluten could be strategies to increase oxidative stability of low moisture crackers.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Thanh Phuong Vu; He, L.; McClements, D.J.; Decker, E.A. Effects of water activity, sugars, and proteins on lipid oxidative stability of low moisture model crackers. Food Res. Int. 2020, 130, 108844.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Freund, M.A.; Chen, B.; Decker, E.A. The Inhibition of Advanced Glycation End Products by Carnosine and Other Natural Dipeptides to Reduce Diabetic and Age-Related Complications. Comprehensive Reviews in Food Science and Food Safety 2018, 17, 1367-1378.

Progress 01/01/18 to 12/31/18

Outputs
Target Audience:Food Ingredient Suppliers DSM Monsanto Bunge Oils Kalsec Kemin Dupont Food Processing Companies Frito Lay Kellogg Unilever Nestle Kraft Mondelez Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two PhD students were trained. How have the results been disseminated to communities of interest?Research has been presented at national conferences via presentations and posters. What do you plan to do during the next reporting period to accomplish the goals?We are looking at why fats with different solid fat contents impact oxidative stability differently. We will conduct triacylglycerol analysis, fatty acid compositions of liquid and solid fat fractions and analysis of oxidation products from n-9, n-6 and n-3 fatty acids. We are also investigating if surface fats on crackers oxidize faster and how antioxidants partition in liquid, solid and surface fats.

Impacts
What was accomplished under these goals? Low moisture snacks, such as crackers, present a health concern for US consumers due to their high content of saturated fats, which increase a risk of coronary heart disease. This is because crackers are traditionally made from semi solid fats that are high in saturated fatty acids to provide structure. These semi-solid fats could be made healthier if they contained more unsaturated fatty acids. However, this increases lipid oxidation and thus decreases shelf-life. The objective of this study was to investigate the role of solid fat content (SFC) on the oxidative stability of a model cracker system. To determine how SFC effects lipid oxidation in crackers, different oils containing different SFCs were selected in 3 ways: 1) commercially available oils which had different SFCs, 2) blending fully hydrogenated soybean oil with liquid soybean oil to produce different SFCs but similar fatty acid and tocopherol homolog profile with interesterified soybean oil, 3) blending lipids to produce varied SFCs but fixed 18:2 percentage and tocopherol homolog concentrations. Oxidation stability was evaluated based on hydroperoxide and hexanal lag phases. Results from commercially available oils (1) show that increasing SFC resulted in increasing oxidative stability. Palm shortening (PS) had high SFC and were stable up to 42 days, the longest hexanal lag phase in this study. In contrast, liquid soybean oil (LSO) and canola/cotton blend (CB) had low SFCs and oxidized after 20 days. Interesterified soybean oil (ISO) which had a higher polyunsaturated fat content (40.6 % ) than CB (35.8 %) produced a more stable oxidatively cracker, presumably due to its higher SFC than CB (test conditions 1). In the second test, fully hydrogenated soybean oil was blended with liquid soybean oil to produce similar fatty acid profile as an interesterified soybean oil. Tocopherols isomers were adjusted to produce similar tocopherol profiles. Blended soybean oil had a longer lag phase than the interesterified soybean fat. In the third test, different fats, including fully hydrogenated soybean oil, interesterified soybean oil, palm oil, and sunflower oil were used to produce varied SFCs but fixed 18:2 at 20%. Tocopherols homologs were added to all mixtures to produce similar tocopherol profile. Unlike test conditions 1, the oxidative stability of blends of liquid and solid fats could not be predicted by SFC. In a fourth experiment, the role of cracker ingredients on oxidative stability was determined. It was found that reducing sugars were strong antioxidants and could be used to extend shelf-life. This included maltose, a sugar with low sweetness that could be compatible with a cracker product. As little as 1.1% maltose was able to increase shelf life by 50%. Gluten and casein were also found to inhibit lipid oxidation with gluten being more effective. for example, 2.3% gluten increased shelf-life by 20%. The sulfhydryl groups of gluten were primarily responsible for its antioxidant activity.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Vu,T.P. and Decker, E.A. Oxidative stability of lipids in crackers in relations to water activity, reducing sugars, and solid fat content. Amer. Assoc. Cereal Chemist International Annual Meeting
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Vu,T.P.; McClements, D.J. and Decker, E.A. Role of solid fat on lipid oxidation in a model cracker system. Amer. Oil Chem. Society Annual Meeting

Progress 01/01/17 to 12/31/17

Outputs
Target Audience:Food Ingredient Suppliers DSM Monsanto Bunge Oils Kalsec Kemin Dupont Food Processing Companies Food Network International Frito Lay Kellogg Unilever Nestle Kraft Mondelez Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One PhD, 1 MS and 1 undergradature student have conducted research and learned laboratory skill on this project. How have the results been disseminated to communities of interest?The research was presented at the American Oil Chemist Society meeting in Orlando, FL. The research poster won first prize in the Lipid Oxidation and Quality Division. What do you plan to do during the next reporting period to accomplish the goals?We are looking at why fats with different solid fat contents impact oxidative stability differently. We will conduct triacylglycerol analysis, fatty acid compositions of liquid and solid fat fractions and analysis of oxidation products from n-9, n-6 and n-3 fatty acids. We are also investigating if surface fats on crackers oxidize faster and how antioxidants partition in liquid, solid and surface fats. Finally, we are studying how oxygen levels impact oxidation rates.

Impacts
What was accomplished under these goals? Crackers and other low moisture foods represent a potential health concern due to their high content of saturated fats, which increase a risk of coronary heart disease. Traditionally crackers are made from semi solid fats that are high in saturated fatty acids to provide structure. The objective of this study was to investigate the role of solid fat content (SFC) on the oxidative stability of a model cracker system. Oxidative stability was evaluated by lipid hydroperoxide and headspace hexanal measurements. Results from commercially available oils show that increasing SFC resulted in decreasing oxidative stability. Oxidative stabilities were in order: palm shortening (SFC at 55 °C = 6.3 - 8.7%) > interesterified soybean oil (SFC at 55 °C = 13.8 - 14.6%) > liquid soybean oil = canola/cotton blend (SFC at 55 °C = 0%). In second condition, different fats were used to produce varied SFCs but fixed 18:2 at 20%. Tocopherol homologs were adjusted to produce similar tocopherol profiles. Similar to the previous experiment, oxidative stabilities increased with increasing SFC: 55 °C = 31.1 - 32.7% > SFC at 55 °C = 24.4 - 25.4% > SFC at 55 °C = 12.9 - 13.5%. However, in the third test, fully hydrogenated soybean oil was blended with liquid soybean oil to produce similar fatty acid profile with interesterified soybean oil. Tocopherol homologs were also adjusted to produce similar tocopherol profiles. The oxidative stability in the third test could not be predicted by SFC.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Role of solid fat on lipid oxidation in a model cracker system

Progress 01/01/16 to 12/31/16

Outputs
Target Audience:Food Ingredient Suppliers DSM Monsanto Bunge Oils Kalsec Kemin Dupont Food Processing Companies Food Network International Frito Lay Kellogg Unilever Nestle Kraft Mondelez Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and 1 post doc have participated in the project. How have the results been disseminated to communities of interest?Research publication and presentations at national meetings. What do you plan to do during the next reporting period to accomplish the goals?We are looking at why fats with different solid fat contents impact oxidative stability differently. We will conduct triacylglycerol analysis, fatty acid compositions of liquid and solid fat fractions and analysis of oxidation products from n-9, n-6 and n-3 fatty acids. We are also investigating if surface fats on crackers oxidize faster and how antioxidants partition in liquid, solid and surface fats.

Impacts
What was accomplished under these goals? Low moisture snacks, such as crackers, present a health concern for US consumers due to their high content of saturated fats, which increase a risk of coronary heart disease. This is because crackers are traditionally made from semi solid fats that are high in saturated fatty acids to provide structure. These semi-solid fats could be made healthier if they contained more unsaturated fatty acids. However, this increases lipid oxidation and thus decreases shelf-life. The objective of this study was to investigate the role of solid fat content (SFC) on the oxidative stability of a model cracker system. To determine how SFC effects lipid oxidation in crackers, different oils containing different SFCs were selected in 3 ways: 1) commercially available oils which had different SFCs, 2) blending fully hydrogenated soybean oil with liquid soybean oil to produce different SFCs but similar fatty acid and tocopherol homolog profile with interesterified soybean oil, 3) blending lipids to produce varied SFCs but fixed 18:2 percentage and tocopherol homolog concentrations. Oxidation stability was evaluated based on hydroperoxide and hexanal lag phases. Results from commercially available oils (1) show that increasing SFC resulted in increasing oxidative stability. Palm shortening (PS) had high SFC and were stable up to 42 days, the longest hexanal lag phase in this study. In contrast, liquid soybean oil (LSO) and canola/cotton blend (CB) had low SFCs and oxidized after 20 days. Interesterified soybean oil (ISO) which had a higher polyunsaturated fat content (40.6 % ) than CB (35.8 %) produced a more stable oxidatively cracker, presumably due to its higher SFC than CB (test conditions 1). In the second test, fully hydrogenated soybean oil was blended with liquid soybean oil to produce similar fatty acid profile as an interesterified soybean oil. Tocopherols isomers were adjusted to produce similar tocopherol profiles. Blended soybean oil had a longer lag phase than the interesterified soybean fat. In the third test, different fats, including fully hydrogenated soybean oil, interesterified soybean oil, palm oil, and sunflower oil were used to produce varied SFCs but fixed 18:2 at 20%. Tocopherols homologs were added to all mixtures to produce similar tocopherol profile. Unlike test conditions 1, the oxidative stability of blends of liquid and solid fats could not be predicted by SFC.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Barden, L. & Decker, E. A. (2016). Lipid Oxidation in Low-moisture Food: A Review. Critical Reviews in Food Science and Nutrition 56(15), 2467-2482.