The effect of black rice water extract on surface color, lipid oxidation, microbial growth, and antioxidant activity of beef patties during chilled storage

Journal Pre-proof The effect of black rice water extract on surface color, lipid oxidation, microbial growth, and antioxidant activity of beef patties during chilled storage

Ronnachai Prommachart, Thiago Sakomoto Belem, Suthipong Uriyapongson, Patricia Rayas-Duarte, Juntanee Uriyapongson, Ranjith Ramanathan PII:

S0309-1740(19)30778-8

DOI:

https://doi.org/10.1016/j.meatsci.2020.108091

Reference:

MESC 108091

To appear in:

Meat Science

Received date:

18 August 2019

Revised date:

30 December 2019

Accepted date:

12 February 2020

Please cite this article as: R. Prommachart, T.S. Belem, S. Uriyapongson, et al., The effect of black rice water extract on surface color, lipid oxidation, microbial growth, and antioxidant activity of beef patties during chilled storage, Meat Science (2019), https://doi.org/10.1016/j.meatsci.2020.108091

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© 2019 Published by Elsevier.

Journal Pre-proof The eff ect of black rice water extract on surface color, lipid oxidation, microbial growth, and antioxidant activity of beef patties during chilled storage

Ronnachai Prommachart1,2 , Thiago Sakomoto Belem1 , Suthipong Uriyapongson2 , Patricia

Department of Animal and Food Sciences, Oklahoma State University, Stillwater, OK 74078,

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Rayas-Duarte3 , Juntanee Uriyapongson4 , Ranjith Ramanathan1,*

Department of Animal Science, Faculty of Agriculture, Khon Kaen University, KhonKaen

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United States of America

40002, Thailand

Robert M. Kerr Food & Agricultural Products Center, Oklahoma State University, 123 FAPC,

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Faculty of Food Technology, Khon Kaen University, KhonKaen 40002, Thailand

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Stillwater, OK, 74078‐ 6055, United States of America

*Corresponding author: [email protected] (R. Ramanathan)

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Journal Pre-proof Abstract Black rice is rich in phenolic acids and anthocyanin; however, limited studies have determined its effect on ground beef quality. The objective was to determine the effects of 0, 0.4, 0.8, and 1.2% black rice water extract (BRWE) on ground beef patties quality when packaged in polyvinyl chloride (PVC). pH, surface color, lipid oxidation, total plate count, and antioxidant capacity were determined on 0, 3, and 6 days of storage under fluorescent light at 2 °C. Addition

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of BRWE had no effect (P = 0.98) on pH. Incorporating BRWE in ground beef improved (P <

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0.0001) redness compared with control. The addition of BRWE decreased (P < 0.0001) lipid

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oxidation than control during storage; while antioxidant capacity increased with the addition of

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extract. BRWE at 1.2% reduced (P = 0.007) aerobic microbial counts after 6 days of storage.

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lipid oxidation and discoloration.

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These results suggested that BRWE could be used as a natural antioxidant in ground beef to limit

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Keywords: black rice; beef color; lipid oxidation; discoloration; antioxidant activity.

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Journal Pre-proof 1. Introduction Ground meat is a popular meat product worldwide; however, ground meat has a shorter shelf-life than intact meat. More specifically, grinding can release various enzymes, prooxidants, and increase surface area, which can accelerate oxidative changes and microbial growth (Amiri, Aminzare, Azar, & Mehrasbi, 2019; Brodowska et al., 2019; Salami et al., 2019; Karre, Lopez, & Getty, 2013). Although quality deterioration in meat is a complicated and depends on the

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chemical compositions of meat, degree of unsaturation, enzymatic activity, pH, light, time of

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storage, pro-oxidant/antioxidant balance, oxygen access, and storage temperature (Ramanathan

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& Mancini, 2018; English, Mafi, VanOverbeke, & Ramanathan, 2016; Jiang & Xiong, 2016;

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Kanner, 1994); adoption of appropriate processing techniques have the potential enhance product quality (de Oliveira Ferreira, Rosset, Lima, Stuelp Campelo, & de Macedo, 2019; Wills et al.,

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2017; Ryu, Shim, & Shin, 2014; Ramanathan, Mancini, & Dady, 2011; Suman et al., 2010).

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Antioxidants are a group of compounds added in meat and products to extend shelf-life (Fruet, Nörnberg, Calkins, & De Mello, 2019; Jiang & Xiong, 2016; Cunha et al., 2018; Falowo,

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Fayemi, & Muchenje, 2014; Trindade, Mancini-Filho, & Villavicencio, 2010). The use of natural

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antioxidants has been increasing in the last few years as there are concerns about the safety of synthetic antioxidants (Aziz & Karboune, 2016; Firuzi et al., 2019; Maqsood, Abushelaibi, Manheem, Al Rashedi, & Kadim, 2015; Özünlü, Ergezer, & Gökçe, 2018). Several natural plant extracts with high potential antioxidant activity are incorporated in meat and meat product to improve shelf-life (Fruet, Nörnberg, Calkins, & De Mello, 2019; Lorenzo et al., 2018; Pateiro et al., 2018; Sadeghinejad, Amini Sarteshnizi, Ahmadi Gavlighi, & Barzegar, 2019). For example, addition of extracts derived from black currant (Jia, Kong, Liu, Diao, & Xia, 2012), pomegranate (Firuzi et al., 2019; Turgut, Işıkçı, & Soyer, 2017), blackberries (Ganhão, Estévez, Armenteros,

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Journal Pre-proof & Morcuende, 2013), and cranberry pomace (Tamkutė, Gil, Carballido, Pukalskienė, & Venskutonis, 2019) have been reported to have positive effects on preventing lipid and protein oxidation in meat and meat product. Black rice (Oryza sativa L.) is a natural source of food colorants and has high antioxidant activity (Laokuldilok, Shoemaker, Jongkaewwattana, & Tulyathan, 2011; Pang et al., 2018; Shao et al., 2018). Phenolic acids, anthocyanins (cyanidin-3-glucoside (C3G), and peonidin-3-

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glucoside (P3G)), and proanthocyanidins are the major bioactive compounds that contribute to

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antioxidant property in black rice (Min, McClung, & Chen, 2011). Moreover, anthocyanin and

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phenolic acids have demonstrated antimicrobial properties in vitro (Puu onen-Pimia et al.,

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2001; odr gue -Carpena, Morcuende, Andrade, Kylli, & Estévez, 2011; Zhao et al., 2009). Park et al. (2017) reported that black rice powder improved ground pork quality. Although black rice

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bran has antioxidant and antimicrobial properties, to the best of our knowledge, no published

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research has determined its effect on ground beef quality. Therefore, the objectives of this study were to analyze the effects of black rice water extract (BRWE) on surface color, lipid oxidation,

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microbial growth, and antioxidant capacity of beef patties during chilled storage.

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2. Materials and methods

2.1 Materials and chemicals

The black rice extract powder was purchased from Xi'an Rongsheng Biotechnology Co., Ltd, China. Folin-Ciocalteu reagent, gallic acid, DPPH (2,2-diphenyl-1-picrylhydrazyl), 2thiobarbituric acid (≥ 98%), and trichloroacetic acid (ACS reagent, ≥ 99%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2 Preparation of black rice water extract powder solution and determination of antioxidant activity 4

Journal Pre-proof 2.2.1. Preparation of black rick water extract (BRWE) Black rice extract powder was added in deionized distilled water at 0.4, 0.8, 1.2 g/100 mL to achieve 0.4, 0.8, and 1.2%. The mixture was vortexed for 5 min, and the solutions were centrifuged at 3,000 × g for 10 min at 4 ºC. The resultant black rice water extract (BRWE) supernatant was added in ground beef.

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2.2.2 Determination of total anthocyanin and total phenolic content Black rice bran powder (0.2 g) was mixed with 10 mL of acidified methanol dissolved in

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1.0 N HCl (85:15 v/v). The mixture was incubated at room temperature for 2 h with shaking. The

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extracts were centrifuged at 3,000 × g at 4 °C for 10 min, and the supernatants free of suspended

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particles were collected into a volumetric flask. In order to extract anthocyanin and phenolic

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compounds in the residue after centrifugation, 10 mL of acidified methanol was added to the residue and shook for 1 h. The total phenolic content in the supernatant was determined by

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measuring wavelength at 765 nm using the Folin-Ciocalteu method as described previously

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(Ainsworth & Gillespie, 2007). Gallic acid was used as the calibration standard, and the total phenolic content was expressed as mg gallic acid (GAE) equivalent per g dry weight.

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Total anthocyanin content in the extract was determined by the pH-differential method according to Lee et al. (2005). A 0.5 mL aliquot of the extract was diluted with buffer reagents (0.025 M otassium chloride at H1.0 and 0.4 M sodium acetate at H4.5 ). Total anthocyanin content was determined by measuring wavelengths at 520 and 700 nm respectively, using a UV– visible spectrophotometer (Shimadzu UV-Vis Spectrophotometer UV-2600 with TCCController, Columbia, MD, USA). The results were expressed in mg of cyanidin-3-O-glucoside equivalents per g dry weight using the equation: Anthocyanin content (cyaniding-3-glucoside equivalents, mg/L) = A×MW×DF×103 /(ε×1) 5

Journal Pre-proof where A =  H1.0 (A520 nm -A700 nm) – pH4.5 (A520 nm -A700 nm); MW = molecular weight of cyanidin-3-glucoside (449.2 g/mol); DF = dilution factor; 10 3 = factor for conversion from g to mg; ε = molar extinction coefficient of cyanidin-3-glucoside (26,900 M-1 ·cm-1 ); 1 = the path length (cm). 2.3 Ground beef patty preparation

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Fresh beef chuck muscles (Institutional Meat Purchase Specification number 116A; M. serratus ventralis; IMPS, 2014) were collected from six Angus cattle carcasses (Choice grade,

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market age cattle) harvested at a USDA approved slaughter facility (Robert M. Kerr Food &

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Agricultural Products Center, Oklahoma State University, Stillwater). Muscles from each animal

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served as replicates (n = 6 replications) and were vacuum-packaged separately and stored at 2 °C

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for 14 days. Meat samples from each animal were coarse ground through a meat grinder with mesh size 10 mm plates (LEM, #12 Big Bite Grinder - 0.75 HP, West Chester, OH, USA). After

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mincing, coarse ground beef samples were randomly divided into one of four treatments: control

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(CON; ground beef with deionized distilled water), 0.4% BRWE, 0.8% BRWE, and 1.2% BRWE. Each coarse ground beef treatment was hand mixed with deionized water (2 mL) or

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BRWE (2 mL from either 0.4, 0.8, or 1.2% solution) for 3 min and followed by fine grinding (through a 4.5-mm plate grinder). From each treatment, three beef patties were formed using a Kitchen Art Adjust-A-Burger (∼160 g/patty; average dimensions = 11 cm diameter and 2 cm thickness). The total number of patties prepared for each replication was 12 (3 days x 4 antioxidant treatment = 12; six replicates resulted in 12 x 6 = 72 patties). Patties from each replication were randomly assigned to 0, 3, and 6 days of storage. Patties were placed on foam tray, overwrapped with an oxygen-permeable polyvinyl chloride film (oxygen-permeable polyvinyl chloride fresh meat film; 15,500 to 16,275 cm3 O 2 /m2 /24 h at 23°C, E-Z Wrap Crystal 6

Journal Pre-proof Clear Polyvinyl Chloride Wrapping Film, Koch Supplies, Kansas City, MO) and displayed under fluorescent lights (800 lux; Sylvania, 95W, Fluorescent Lamp, F96T12-CW-HO-SS, Cool White Supersaver, Toronto, ON, Canada) at 2 °C. 2.4 Instrumental color evaluation Ground beef patty assigned to day 6 color analysis was repeatedly used to measure

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surface color on 0, 1, 2, 3, 4, 5, and 6 days of storage time at three locations using a HunterLab Miniscan XE Plus (2.54‐ cm‐ dia aperture, illuminant A, and 10° observer angle; HunterLab

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Associates, Reston, VA, USA). Lightness (L*), redness (a*) and yellowness (b*) were

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determined following CIE (Commission Internationals de I′Eclairage) color coordinates. Chroma

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(C*) and hue angle (hº) were calculated from a* and b* color coordinates (AMSA, 2012) using

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the following equations:

hº = arctan (b*/a*)

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C*= (a*2 +b*2 )1/2

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Chroma is correlated to the intensity of red color, while hue represents discoloration (AMSA,

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2012). In addition, total color change between respective day and day 0 is calculated as color difference (∆E*) as follows: ∆E* = (∆L*2 +∆a*2 +∆b*2)1/2 (AMSA, 2012). For each replication, 4 antioxidant levels x 7 days of storage x 3 technical readings on each patty = 84 observations were recorded and six replications (84 x 6 = 504) resulted in 504 observations. 2.5 Proximate composition and pH of beef patties The chemical composition of beef patties was conducted using an Association of Official Analytical Chemist (AOAC) approved (Official Method 2007.04) near-infrared spectrophotometer (FOSS Food Scan 78800; Dedicated Analytical Solutions, DK-3400 Hillerod,

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Journal Pre-proof Denmark), and data rocessing was conducted using ISIscan™ Software. The roximate composition values were reported on a percentage basis. The pH of meat patties was measured on 0, 3, and 6 days storage by using a calibrated pH meter specific for meat (HANNA, model HI 99163, Woonsocket, RI, USA) inserting a pH probe at three locations of each patty. 2.6 Microbiological analysis

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Aerobic plate count (APC) of beef patties were determined using Petrifilm™ (3M Microbiology Products, St. Paul, MN, USA) on days 0, 3, and 6 of storage. Ten grams of each

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patty were taken aseptically and stomached (Stomacher 400 Lab Blender, Seward Laboratory

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Systems Inc., London, UK) with 90 mL of sterile 0.1% peptone water (Bacto peptone, Difco

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Laboratories, Inc., Detroit, MI, USA) for 30 sec at 225 rpm (FILTRA-BAG, no.89085-570,

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Nobel Ste-Julie, QC, Canada). Each sample was serial ten-fold diluted in 0.1% peptone water. Duplicate plates were prepared from each dilution by plating on Petri-film plates. Plates for APC

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were incubated at 37 °C for 48 h. The APC was expressed as log10 CFU (colony forming units).

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For each replication, 4 antioxidant levels x 3 days of storage x 3 technical replicates = 36 observations were recorded, and six replications (36 x 6 = 216) resulted in 216 observations for

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statistical analysis.

2.7 2-Thiobarbituric acid reactive substances (TBARS) values of meat Lipid oxidation was evaluated as 2-thiobarbituric acid reactive substances (TBARS) values according to the procedure of Wills et al. (2017) on days 0, 3, and 6 of storage. To avoid pink color interfere from black rice extract, TBARS detections were performed with some minor modifications as described by Hodges, DeLong, Forney, and Prange (1999). Briefly, 3 g of the patty was mixed with 27 mL of 11% trichloroacetic acid solution. The mixture was homogenized using a Tissue Teaser (120 V with 7 mm Probe, BioSpec Products, Inc., Bartlesville, OK, USA) 8

Journal Pre-proof for 30 s. Following homogenization, solutions were filtered utilizing a Whatman #42 filter paper (GE Whatman; Sigma Aldrich, St. Louis, MO, USA). One mL of filtrate was added to a test tube with 1 mL of either (i) -TBA solution comprised of 11% (w/v) trichloroacetic acid or (ii) +TBA solution containing the above plus 20 mM TBA. The filtrate and solution were mixed, incubated at 95 °C in a water bath for 15 min, and absorbance was measured at 440, 532, and 600 nm using a Shimadzu UV-2600 PC spectrophotometer (Shimadzu Inc., Columbia, MD, USA). For each

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replication, 4 antioxidant levels x 3 days of storage x 3 technical replicates = 36 observations

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were recorded, and six replications (36 x 6 = 216) resulted in 216 observations for statistical

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analysis. The TBARS values were expressed as mg malondialdehyde/ kg meat. Malondialdehyde

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equivalents were calculated using following equations:

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1) [(Abs 532+T BA)-(Abs 600+T BA)-(Abs 532-T BA-Abs600-TBA) = A

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2) [(Abs 440+T BA-Abs 600+T BA) 0.0571] = B

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3) MDA equivalents (nmol∙ml-1 ) = (A-B/1,57,000) × 106 2.8 Radical scavenging activity assay

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The antioxidant activity of patties was determined as a radical scavenging activity of stable 2,2-diphenyl-1-picrylhydrazyl (DPPH; Fratianni et al., 2010) with slight modifications on days 0, 3, and 6 of storage. Briefly, 3 g of ground beef sample was mixed with 10 mL methanol, and homogenized for 30 min at room temperature. The homogenate was centrifuged at 3,000 × g and 4 ºC for 15 min (C 48-R, Awel International, Blain, Nantes, France) to collected clear supernatant. The analysis was performed in microplates by adding 200 μL of 0.5 mM DPPH solution (dissolved in methanol) to 100 μL of patty extracts and incubated in the dark for 20 min at room temperature. The absorbance was measured at 517 nm using a UV-Vis 9

Journal Pre-proof spectrophotometer (SpectraMax® M3 Multi-Mode Microplate Reader, Molecular Devices, San Jose, CA, USA). The absorbance of DPPH with methanol (control sample) was used for a baseline measurement. The DPPH scavenging activity was calculated by the following equation: DPPH scavenging activity (%) = where Ac is the absorbance of the control (DPPH solution with methanol) and As is the absorbance of the sample. For each replication, 4 antioxidant levels x 3 days of storage x 3

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technical replicates = 36 observations were recorded, and six replications (36 x 6 = 216) resulted

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in 216 observations for statistical analysis.

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2.9 Statistical analysis

The experimental design was a split-plot. Each muscle from each carcass served as a

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block and ground muscle from each animal was assigned randomly to 0 (control), 0.4, 0.8, and

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1.2% BRWE, resulting in a completely randomized block in the whole plot (n = 6 replications). Within the subplot, patties were assigned to 0, 3, or 6 days of storage (split-plot factor). The

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repeated option in PROC MIXED was used to assess the covariance-variance structure among

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the repeated measures for display color data. The most appropriate structure was determined using Akaike’s information criterion out ut. Type-3 fixed effects of antioxidant treatments, storage time, and their interactions were analyzed using the Mixed Procedure of SAS 9.4 (SAS Inst. Inc., Cary, NC, U.S.A.). The random terms included animal (block), animal*antioxidant (error A), and unspecified residual error B. Least squares means for protected F tests (P < 0.05) were separated using the probability of difference (pdiff) option and were considered significant at P < 0.05. A regression analysis was also performed to determine the effect of increasing dose of BRWE (linear or quadratic) on color, lipid oxidation, and antioxidant activity.

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Journal Pre-proof 3. Results 3.1 Total phenolic, anthocyanin, proximate composition, and pH value of beef patties The average total phenolic and anthocyanin content in black rice were 270.51 mg of gallic acid/g and 233.55 mg of cyanidin-3-O-glucoside equivalents per g dry weight, respectively. Based on the effective concentration of BRWE in 100 g patty, phenolic contents (with respect to gallic acid) were 216, 432, and 648 ppm in 0.4, 0.8, and 1.2%, respectively.

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Similarly, anthocyanin contents were 186, 373, and 559 ppm in 0.4, 0.8, and 1.2% treatments

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added in 100 g patty, respectively. Ground beef patties had 65.0 ± 0.2% moisture, 19.04 ± 0.1%

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protein, and 12.7 ± 1.2% fat. There was no main effect or interaction (P = 0.98) resulted for pH.

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3.2 Effect of BRWE on ground beef color

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Table 1 presents the changes in lightness (L*), redness (a*), yellowness (b*), chroma (C*) and hue angle (hº) value of beef patties with and without BRWE during chilled storage.

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Two-way significant interactions between BRWE treatment and day of the display were

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observed for a* (P < 0.0001), b* (P < 0.0001), C* (P < 0.0001), and hº (P < 0.0001). Addition of BRWE at 1.2% decreased lightness of ground beef patties compared to other

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treatments on day 0. However, from day 4, there were no differences (P > 0.05) between BRWE treatments on L* values. During storage, there were no differences in lightness between 0.4% BRWE and control (except on day 6). Incorporating 0.4% BRWE improved redness (a* values) of ground beef patties compared to control from day 1 to the end of storage. Chroma values also showed the same trend as a* values showing the color stabilizing effect of BRWE at a lower concentration. BWRE patties at 1.2% had greater bluish ting on day 0 of the display as indicated by lower b* values. By the end of the display, all BRWE had greater redness than control. BRWE at 1.2% had 11

Journal Pre-proof greater bluish tinge by the end of the display compared with other BREW and control treatments. However, changes in a*, chroma, and hue values were lower for 1.2% compared with control (∆E* = day 6 a* values – day 0 a* values; Table 2). Further, hue values demonstrated that 1.2% BREW had the least discoloration compared with other treatments. The regression analysis indicated that increasing levels of BRWE had a quadratic response to redness with storage time

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(R2 = 0.99; y = -0.1751x2 - 0.3415x + 25.481). 3.3 Effect of BRWE on microbiological of beef patties

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A significant BRWE treatment × day of the display interaction observed for APC (P =

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0.007; Figure 1). No clear trend was observed for the effect of BRWE on APC during storage.

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There were no differences (P = 0.85) in APC on day 0 among control, 0.8, and 1.2% BRWE.

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compared to control (4.80 CFU/g).

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However, on day 6, the lowest APC (4.25 CFU/g) was observed for BRWE 1.2% treated

3.4 Effect of BRWE on lipid oxidation

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A BRWE treatment × day of the display interaction (P < 0.0001) was observed for

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TBARS values (Figure 2). On day 0, lipid oxidation values showed no differences (P = 0.85) among all treatments. However, on 3 and 6 days of the display, control treatment showed a greater (P = 0.005) lipid oxidation (TBARS values) when compared with BRWE. On day 6 of display, 0.8 and 1.2% BRWE had lower (P = 0.01) lipid oxidation than 0.4% BRWE. The control had quadratic response to storage time, but 0.4 % BRWE had linear relationship and 1.2% BRWE had quadratic response to storage time (y = 0.0089x2 – 0.0144x + 0.298). 3.5 Effect of BRWE on antioxidant activity

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Journal Pre-proof A BRWE treatment × display time interaction resulted for antioxidant activity (P = 0.002; Figure 3). All the treatments demonstrated decrease (P = 0.01) in antioxidant activity with storage time as indicated by a lower percentage of DPPH. However, on all days, control treatment showed the lowest (P = 0.001) the percentage of DPPH compared with BRWE treatments. The antioxidant activity of control demonstrated a quadratic response to decrease in antioxidant activity with storage time (y = 1.32x2 – 7.54x + 23.23). Increase in BRWE

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concentration also showed quadratic response with storage time (y = 3.505x2 – 18.165x + 45.74;

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R2 = 1).

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4. Discussion

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In the current research, the addition of BRWE minimized discoloration compared to

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control samples. For example, 1.2 % BRWE resulted in 24.49% less discoloration than control patties, based on the hue angle. Previous research noted that polyphenols and anthocyanin are

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effective antioxidants that can limit oxidative changes in meat. In support, Jia et al. (2012) and

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Nowak, Czyzowska, Efenberger, and Krala, (2016) reported that black currant extract and black rice powder (Park et al., 2017) rich anthocyanin enhanced the red color stability of pork meat and

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meat product. In addition, Turgut et al. (2017) demonstrated that pomegranate extracts rich anthocyanin and phenolic content retarded discoloration of meat and pork patties during refrigerated storage. Firuzi et al. (2019) noted that 10 mg gallic acid equvilent in pomegrante rind limited frankfurte color change (ΔE) during chilled storage. Similarly, Ganh o, Est e , Kylli, Heinonen, and Morcuende (2010) reported that blackberry extract rich in anthocyanin enhanced color stability of pork burger patties, while the other fruits extract containing a small amount of anthocyanin did not the stabilize color. Therefore, improved color stability in BRWE might be due to the effects of higher antioxidant activity by anthocyanin and phenolic content. In 13

Journal Pre-proof support, BRWE extract had greater polyphenol and anthocyanin contents and radical scavenging capacity. Decreased lightness of BRWE patties at 0.8 and 1.2% can be attributed to color imparted by anthocyanin. However, 0.4% did not affect lightness compared to control. In support, b* values were lower for BRWE patties, indicating more bluish tinge for patties. Additionally, 0.4% of BRWE had greater red intensity (as indicated by chroma values) than control. Further,

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changes in a*, chroma, and hue values during storage (∆E*) were lower for BRWE treatments

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than control. There was a concentration-dependent effect on changes in chroma and hue values.

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Grinding of meat provides a favorable environment for myoglobin oxidation. Hence, in the

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current research greater concentration of antioxidants in meat had a positive effect on limiting discoloration.

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Rancidity can lead to unacceptable flavor and limits consumer acceptance of meat

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products (Falowo et al., 2014). Greene and Cumuze (1982) noted that oxidized flavor is detected in beef with TBARS values between 0.6 and 2.0 mg MDA/kg meat indicating a large variation in

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the threshold of the panelists. Moreover, Campo et al. (2006) reported that 2 mg MDA/kg meat

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could be considered as a threshold for the acceptability of oxidized beef. In this study, the highest TBARS value (0.78 mg MDA/kg meat) was observed in control patties, whereas BRWE had lower and stable values during 6 days of storage. BRWE showed strong potential to inhibit lipid oxidations on beef patties because of its antioxidant activity. Anthocyanin and phenolics can prevent the formation of fatty free radicals by inhibiting the free radical formation and blocking radical chain reaction in the oxidation process (Rice-Evans, Miller, & Paganga, 1997). Several studies demonstrated that natural extract rich in anthocyanin and phenolics can inhibit lipid oxidation in muscle food. Apple peel-based edible coating in ground beef (Shin, Chang,

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Journal Pre-proof Lacroix, & Han, 2017), artichoke extract rich phenolics in raw beef patties (Ergezer & Serdaroğlu, 2018), blackcurrant extract rich anthocyanins in raw pork patties (Jia et al., 2012), Mediterranean berries in pork burger patties (Ganhão et al., 2013), pistachio green hull extract in beef (Sadeghinejad et al., 2019) and pomegranate peel extract in beef meatballs (Turgut et al., 2017) found effectively inhibited lipid oxidation during chilled storage. Previous research reported 300 ppm of phenolic in pomegranate rind powder decreased ground goat lipid oxidation

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by 75% compared with control samples (Devatkal, Narsaiah, & Borah, 2010). Our results were

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consistent with these previous findings and suggested the potential application of BRWE as a

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natural antioxidant to protect against lipid oxidation during chilled storage for meat and meat

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products with improved quality traits.

In the current research, the aerobic plate counts of patties during storage were lower than

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those reported by Fruet et al. (2019); García-Lomillo, González-SanJosé, Del Pino-García,

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Rivero-Pérez, & Muñiz-Rodríguez, (2017) and lesser than 107 CFU/g, limit established by the International Commission for Microbial Specifications in Food in chilled meat (ICMSF, 2004).

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BRWE at 1.2% decreased bacterial count on day 6 of storage compared with other treatments.

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However, all treatments had all aerobic plate counts between 4 and 5 logs after 6 days of storage, indicating all samples were not microbiologically spoiled. The presence of bioactive compounds such as terpenes, phenolic compounds, tannins, carvacrol, and thymol essential oil can exert antimicrobial activity (Aziz & Karboune, 2016). For example, antimicrobial effect of cranberry pomace, red wine pomace, apple peel extract, pomegranate and red grape extract on mesophilic, lactic acid has been reported in meat products (Tamkutė et al., 2019; Shin et al., 2017; GarcíaLomillo et al., 2017; Andrés et al., 2017). Antimicrobial eff ect of anthocyanin in vitro was reported by various researchers (Casaburi, Di Martino, Ercolini, Parente, & Villani, 2015;

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Journal Pre-proof Cisowska, Wojnicz, & Hendrich, 2011; Zhao et al., 2009). Anthocyanin and phenolic compounds can act on the cytoplasmic membrane and change its structure and function; as a result bacteria losses structural integrity of the cell membrane, especially in Gram-positive bacteria (Holley & Patel, 2005; Lacombe, Wu, Tyler, & Edwards, 2010). 5. Conclusions

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The results of the present study showed that BRWE could be potentially used as natural additives in order to increase redness (greater a* values and lower hue angle) during simulated

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retail-display of beef patties. The addition of BRWE increased antioxidant capacity and

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decreased lipid oxidation. Greater concentration of BRWE had the least changes in chroma, hue,

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mechanisms of BRWE in muscle systems.

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and a* values during 6 days of storage. Further research is needed to elucidate the antioxidant

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Conflict of interest

Acknowledgments

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The authors declare that they have no conflict of interest.

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The authors would like to thank The Royal Golden Jubilee (RGJ) Ph.D. Programme for providing the grant to conduct research. This research was supported, in part, by Oklahoma State University and the USDA- National Institute of Food and Agriculture.

16

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Journal Pre-proof

Table 1. Instrumental color of beef patties treated with different levels of black rice water extract (BRWE) during 6 days of storage. Storage time (day) Parameter Treatments 0 1 2 3 4 5 6 Aa Aa Aa Aa Aa Aa Control 48.88 49.13 48.8 48.54 48.55 48.61 48.66Aa Ab Aa Aab Ab ABb ABb L* BRWE 0.4% 48.14 48.87 48.64 48.17 47.56 47.55 47.55Bb SE = 0.38 BRWE 0.8% 46.36Bb 47.35Ba 46.74Bb 47.19Bab 46.50Bb 46.50Bb 46.50Bb BRWE 1.2% 45.16Cb 46.69Ba 46.82Ba 46.46Ba 46.41Ba 46.48Ba 46.55Ba a* SE = 0.23

Control BRWE 0.4% BRWE 0.8% BRWE 1.2%

29.23Aa 28.45Ba 27.14Ca 24.83Da

25.70Bb 26.80Ab 26.16ABb 24.14Cb

22.02Cc 24.41Ac 24.73Ac 23.07Bc

b* SE = 0.22

Control BRWE 0.4% BRWE 0.8% BRWE 1.2%

24.08Aa 22.94Ba 21.20Ca 18.74Dab

23.17Ab 22.60Ba 21.55Ca 19.18Da

21.06Ac 20.84ABb 20.41Bb 18.39Cb

Chroma SE = 0.32

Control BRWE 0.4% BRWE 0.8% BRWE 1.2%

37.83Aa 36.54Ba 34.44Ca 31.08Da

34.60Ab 35.06Ab 33.89Ba 30.90Ca

Hue SE = 0.26

Control BRWE 0.4% BRWE 0.8% BRWE 1.2%

39.49Aa 38.89Ba 38.01Ca 37.06Da

rn

u o

J

l a

42.06Ab 40.15Bb 39.50Cb 38.50Db

o r p

f o

18.43Cd 22.28Ad 22.42Ad 21.58Bc

15.5Ce 19.67Ae 19.88Ae 19.06Be

14.08Cf 17.44Af 17.27ABf 16.84Bf

12.27Bg 15.12Ag 14.78Ag 14.77Ag

20.1Ad 20.57Ab 19.56Bc 18.20Cb

19.51Ae 19.73Ac 18.64Bd 17.10Cc

19.24Af 18.85Ad 17.52Be 16.17Cd

18.96Ag 17.97Be 16.41Cf 15.24De

30.47Bc 32.10Ac 32.07Ab 29.48Cb

27.28Cd 30.32Ad 29.75Ac 28.26Bc

24.93Ce 27.86Ae 27.25Ad 25.67Bd

23.73Cf 25.65Af 24.65Be 23.31De

22.59Bg 23.49Ag 22.09Bf 20.98Cf

43.75Ac 40.49Bc 39.55Cb 38.58Db

47.52Ad 42.73Bd 41.12Cc 40.15Dc

51.58Ae 45.11Be 43.16Cd 41.91Dd

54.20Af 47.32Bf 45.32Ce 43.69De

57.13Ag 49.95Bg 47.99Cf 45.89Df

r P

e

26

Journal Pre-proof A-D

Least square means with different letters within the same day of storage time are significantly

different (P < 0.001) a-g

Least square means with different letters within the same treatments are significantly different

(P < 0.001) L* represents lightness, a greater values means lighter meat; a* represents redness, a greater

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value means more red color; b* indicates blueness, a lower value indicates more blue color; a

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greater chroma indicates more intense red color; hue indicates discoloration and a greater hue

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angle represents more discoloration.

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Journal Pre-proof Table 2: Effects of black rice water extract (BRWE) on color change during 6 days of storage

Variable (∆E*) Control BRWE 0.4% BRWE 0.8% BRWE 1.2%

Storage time (day) 0 1 2 Af 3.65 7.82Ae Bf 1.83 4.58Be 1.43Bf 2.56Ce 1.73Bf 2.44Ce

3 11.5Ad 6.60Bd 5.06Bd 3.54Cd

4 14.74Ac 9.36Bc 7.69Cc 6.12Dc

5 15.90Ab 11.75Bb 10.53Cb 8.49Db

o r p

f o

6 17.71Aa 14.23Ba 13.25Ca 10.74Da

e

Total color change between respective day and day 0 is calculated as ∆E *= (∆L*2 +∆a*2 +∆b*2 )1/2 A-D

a-g

r P

Least square means with different letters within the same day of storage time are significantly different (P < 0.001).

l a

Least square means with different letters within the same treatments are significantly different (P < 0.001).

n r u

The standard error for ∆E* comparison is 0.42

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Journal Pre-proof Figure 1: Aerobic Plate Counts (log CFU/g) of beef patties with different levels of black rice water extract (BRWE) during 6 days of storage.

6

Ab

ABb ABa

Bc

ABb

Aa

Ab

Ba Bc ABa

3

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Log CFU

4

Aa

Aa

5

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2

0

0 day

3 day BRWE 0.4%

BRWE 0.8%

6 day

BRWE 1.2%

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Control

-p

1

A-B

lP

Least square means with different letters within the same day of storage time are significantly

Least square means with different letters within the same treatments are significantly different

(P < 0.05).

ur

a-c

na

different (P < 0.05).

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The standard error for antioxidant × storage time comparison is 0.45.

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Journal Pre-proof Figure 2: TBARS values (mg MDA/kg) of beef patties with different levels of black rice water extract (BRWE) during 6 days of storage.

Aa

0.8

Ab

0.6

Ba

0.5 c

b

a

0.3

Ca

Ba

Ca

Ca

a

of

0.4

Ba

ro

0.2

0.1 0.0 0 day

BRWE 0.4%

6 day

BRWE 0.8%

BRWE 1.2%

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Control

3 day

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TBARS value mgMDA/kg mea

0.7

A-C

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Least square means with different letters within the same day of storage time are significantly

Least square means with different letters within the same treatments are significantly different

(P < 0.05).

ur

a-c

na

different (P < 0.05).

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The standard error for antioxidant × storage time comparison is 0.04.

30

Journal Pre-proof Figure 3: Antioxidant activity (%DPPH) of beef patties with different levels of black rice water extract (BRWE) during 6 days of storage.

35 Aa

30 Ba

Ab Ca

20

Ba

Ab

Bb Bb

Da

Cb

15

Cb

Db

of

%DPPH

25

5

0 0 day

BRWE 0.4%

6 day

BRWE 0.8%

BRWE 1.2%

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Control

3 day

-p

ro

10

A-C

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Least square means with different letters within the same day of storage time are significantly

Least square means with different letters within the same treatments are significantly different

(P < 0.05).

ur

a-c

na

different (P < 0.05).

Jo

The standard error for antioxidant × storage time comparison is 0.96.

31

Journal Pre-proof Ronnachai Prommachart (conducted study, data analysis, prepared manuscript), Thiago Sakomoto Belem (conducted study), Suthipong Uriyapongson (conceptualization), Patricia Rayas-Duarte (conceptualization, proof reading, methodology), Juntanee Uriyapongson (conceptualization, methodology), Ranjith Ramanathan (conceptualization, proof reading,

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editing, data analysis, funding, project administration)

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Journal Pre-proof Highlights Black rice water extract (BRWE) had 270.51 mg of gallic acid/g



BRWE minimized color deterioration of beef patties during storage



BRWE decreased lipid oxidation of beef patties during storage



A concentration dependent effect of BRWE was observed for antioxidant activity

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

33

Figure 1

Figure 2

Figure 3