Currently in editing
Title: Turmeric and curcumin-based antioxidant treatment in growth medium improves critical quality attributes of cultivated meat
Author: Noorul H. Ali, MS (bioengineering), Tufts University, Medford, MA 02155, United States
Correspondence: noorul.ali@tufts.edu
Abstract
Introduction
Methods
Results
Discussion
References
Appendix
Meat oxidizes over time, changing its appearance, texture and flavor. An important aspect of cultivated meat is long-term storage and freshness. Antioxidants combat oxidation of lipids and other cellular material. Here, we treat bovine satellite muscle cells with different concentrations of two types of antioxidant treatments. Higher cell viability and lower oxidation is demonstrated under food-grade antioxidant treatment.
Oxidation of meat is a challenge to its long-term storage. Oxida>ve stress leads to the crea>on of free radical oxygen and reac>ve nitrogen species, which cause discolora>on, change in flavor and forma>on of toxic compounds. Lipid and protein oxida>on are the mechanis>c reasons for colorectal cancer’s link to red meat. Overall, it is desired for cul>vated meat to be resistant to oxida>ve stress. An>oxidants scavenge free radical species and slow down oxida>on. Endogenous produc>on of carotenoids phytoene, lycopene and β-carotene in mammalian cells through metabolic engineering has been demonstrated [1]. This was done to combat lipid oxida>on’s role in decreasing shelf-life, and impairing color stability and flavor. Here, we added an exogenous an>oxidant to growth medium to study its effect on viability and lipid oxida>on of bovine satellite muscle cells. We compare food-grade and lab-grade an>oxidant treatments at different concentra>ons and select one of each kind based on highest cell viability. We add op>mal lab-grade and food-grade an>oxidant concentra>ons to growth medium with cells. Lipid oxida>on levels of cells were tested with a thiobarbituric acid reac>ve substances (TBARS) assay using malondialdehyde (MDA), a byproduct of lipid oxida>on. We conclude food-grade an>oxidant treatment is beVer than lab-grade an>oxidant treatment due to higher cell viability and reduced oxida>on. Methods Cell thawing, seeding and viability tes/ng Bovine satellite muscle cells were thawed from cryopreserva>on at -80 ⁰C and incubated (37 ⁰C, 5% CO2). 10mL of food-grade an>oxidant treatment stock solu>on was prepared by mixing commercially available turmeric powder and water at a concentra>on of 10,100 ng/mL. 10mL of lab-grade an>oxidant treatment stock solu>on was prepared by mixing commercially available curcumin (carotenoid) powder and dimethyl sulfoxide (DMSO) at a concentra>on of 10,000 ng/mL. 2 control growth medium formula>ons and 10 growth medium formula>on with differing concentra>ons of food-grade and lab-grade an>oxidant treatments were made as described in Table 1. DF 10, DF100 and DF1000 growth medium formula>ons were prepared by serial dilu>on. Standard growth medium (Gibco DMEM 1X + GlutaMAX) for bovine satellite cells used. Cells were seeded at 910 cells/cm2. Each condi>on was triplicated and 200uL of respec>ve media formula>on was exchanged every 3 days for 1 week. The well-plate was incubated (37 ⁰C, 5% CO2). A6er 1 week, cell viability was measured for each of the 12 media formula>ons by adding 20uL of Prestoblue dye to each well and performing fluorescence plate reading
assay at 560/590nm. 1X growth medium with 10% Prestoblue dye was used as control for cell viability assay. Name Formula>on An>oxidant concentra>on Grade Control GM 1X standard growth medium None Control Control DMSO 1X growth medium and 0.1% DMSO None Control DF1 L 1X growth medium, 0.1% DMSO and 0.1% lab-grade stock solu>on
10 ng/mL curcumin Lab
DF10 L 1X growth medium, 0.1% DMSO and 10% DF 1 L solu>on 1 ng /mL curcumin Lab
DF100 L 1X growth medium, 0.1% DMSO and 10% DF 10 L solu>on 0.1 ng/mL curcumin Lab
DF1000 L 1X growth medium, 0.1% DMSO and 10% DF 100 L solu>on 0.01 ng/mL curcumin Lab
DF10000 L 1X growth medium, 0.1% DMSO and 10% DF 1000 L solu>on 0.001 ng/mL curcumin Lab
DF1 F 1X growth medium and 5% food-grade stock solu>on 500 ng/mL turmeric Food
DF10 F 1X growth medium and 10% DF 1 F solu>on 50 ng/mL turmeric Food DF100 F 1X growth medium and 10% DF 10 F solu>on 5 ng/mL turmeric Food DF1000 F 1X growth medium and 10% DF 100 F solu>on 0.5 ng/mL turmeric Food DF10000 F 1X growth medium and 10% DF 1000 F solu>on 0.05 ng/mL turmeric Food Table 1: Growth medium formula>ons varying in an>oxidant concentra>on and type Cell seeding for oxida/on test Three T-175 flasks were seeded at 17,000 cells/cm2 each, with 25 mL of standard growth medium in the first, 25mL of DF 1 F food-grade medium in the second and 25mL DF 10000 L lab-grade medium in the third. Flasks were incubated (37 ⁰C, 5% CO2) for 1 week with media exchanged every 3 days. Cells were counted using a hemocytometer a6er scraping. Cells in all flasks were normalized to have the same count by removing 214uL of cell suspension solu>on from lab-grade and 610uL from food-grade. Cell suspensions were then centrifuged (300g, 5 minutes) and resuspended in 2mL PBS each. 900uL of each cell suspension solu>on was stored in 6 Eppendorf tubes, 2 tubes for each condi>on (control, food-
grade, lab-grade). 1 tube of each condi>on was stored for 1 week at room temperature (22 ⁰C) and another of each was refrigerated for 1 week (4 °C) to introduce mul>ple oxida>ve challenges. Lipid oxida/on test with TBARS assay Cell pellet tubes were placed on a heat block (100 °C, 10 minutes) to simulate cooking. Cell pellets were mixed with 200uL water . Cells were lysed using the freeze-thaw method by placing tubes under refrigera>on (-80 °C, 5 minutes) and thawing, repea>ng the freeze-thaw process for 3 cycles. 5 standard solu>ons were prepared as described in Table 2 to plot MDA standard curve. To conduct the TBARS assay, 100uL of 10% trichloroace>c acid (TCA) reagent and 800uL of color reagent were added 100uL of standard solu>ons or 100uL of sample solu>ons from 6 Eppendorf tubes. The 11 vials were placed on the heat block (100 °C, 60 minutes). Vials were placed in the refrigerator (4 °C, 10 minutes). Vials were microcentrifuged (4 °C, 1,600g, 10 minutes). 200uL from each vial was triplicated on a 96-well plate. MDA and TCA form an adduct whose absorbance peaks at 535nm. This allowed us to conduct a TBARS assay that acts as a proxy for lipid oxida>on since MDA is a byproduct of lipid oxida>on. Absorbance was measured using a plate reader at 535nm. MDA standard equa>on was generated using Numpy, a Python library. MDA concentra>ons in each of the 6 condi>ons were extrapolated using the equa>on obtained for the MDA standard curve from absorbance data obtained for each vial. We use MDA absorbance measurements as a proxy for lipid oxida>on in cells.
Table 2: MDA standard solu>ons Results 500 ng/mL turmeric and 0.001 ng/mL curcumin treatments showed highest cell viability As seen in Figure 1, cell viability is highest under DF1 for food-grade an>oxidant treatments and DF10000 under lab-grade an>oxidant treatments. 500 ng/mL turmeric in growth medium shows highest cell viability amongst all food-grade an>oxidant treatments. 0.001 ng/mL curcumin in growth medium shows highest cell viability amongst all lab-grade an>oxidant treatments. While viability decreases as concentra>on of turmeric goes down in growth medium, it increases with decrease in curcumin concentra>on. Surprisingly, high levels of curcumin in growth medium significantly impacted growth as seen in DF1 under lab-grade an>oxidant treatments. Only DF1000 and DF10000 lab-grade treatments, and DF1 food-grade treatment show higher viability compared to growth medium control.
Figures 1-2: Cell viability measurements under food-grade and lab-grade an>oxidant treatments. Prestoblue absorbance is a measure of cell viability. DF: dilu>on factor, GM: growth medium control, DMSO: dimethyl sulfoxide control. Food-grade antioxidant treatment shows lower lipid oxidation levels Compared to control, food-grade antioxidant treatment containing 500 ng/mL turmeric in growth medium shows much lower oxidation level under refrigeration and slightly lower oxidation level at room temperature. Lab-grade antioxidant treatment shows lower oxidation levels at room temperature. Surprisingly, lab-grade antioxidant treatment shows much higher oxidation under refrigeration.
Figure 3: MDA standard curve obtained by plotting line of best fit to absorbance measurements of prepared MDA standards. MDA: malondialdehyde.
Figures 4-5: Oxidation level comparison between food-grade and lab-grade antioxidant treatments under room temperature and refrigeration. MDA molar concentrations are obtained from MDA standard curve using absorbance assay measurements to line of best fit in Figure 3. MDA concentrations to corresponding absorbance assay measurements from cells are noted. Discussion Increasing turmeric in growth medium leads to higher cell viability. Turmeric is known for its an>oxida>ve proper>es. High concentra>ons of turmeric in growth medium showed high cell viability, with the highest concentra>on demonstra>ng higher cell viability than standard growth medium. This result highlights turmeric’s poten>al in improving cell viability. In literature, turmeric has shown an>oxidant proper>es by inhibi>ng lipid peroxida>on [3]. Further research can be done to characterize turmeric’s effects on the growth of bovine cells. Curcumin, a component of turmeric, in the lab-grade an>oxidant treatment is essen>ally purified turmeric. High concentra>ons of curcumin in our growth medium showed reduced cell viability. At low temperatures, curcumin has reduced bioavailability [7]. This seems to be a plausible reason for the much higher oxida>on levels under lab-grade an>oxidant treatment and refrigera>on. Our lab-grade an>oxidant treatment contains the lowest concentra>on of curcumin. The low concentra>on of curcumin coupled with reduced bioavailability of curcumin at low temperature may have resulted in increased oxida>on. Further study should be done to confirm curcumin’s lower bioavailability under refrigera>on. Meat is generally refrigerated. Any detrimental effects of an an>oxidant treatment to cul>vated meat’s shelf-life can significantly impact adop>on and commercializa>on. In conclusion, higher cell viability and lower lipid oxida>on is shown in bovine satellite muscle cells under food-grade an>oxidant treatment with commercially available turmeric. Lab-grade an>oxidant treatment with curcumin demonstrates higher viability of cells under certain condi>ons, lower oxida>on at room temperature but much higher oxida>on under refrigera>on. Since refrigera>on is a major aspect of commercial meat produc>on, we conclude food-grade an>oxidant treatment is beVer than lab-grade an>oxidant treatment.
References [1]: Stout AJ, Mirliani AB, Soule-Albridge EL, Cohen JM, Kaplan DL. Engineering carotenoid produc>on in mammalian cells for nutri>onally enhanced cell-cultured foods. Metab Eng. 2020 Nov;62:126-137. doi: 10.1016/j.ymben.2020.07.011. Epub 2020 Sep 2. PMID: 32890703; PMCID: PMC7666109. [2]: Jakubczyk K, Drużga A, Katarzyna J, Skonieczna-Żydecka K. An>oxidant Poten>al of Curcumin-A Meta-Analysis of Randomized Clinical Trials. An>oxidants (Basel). 2020 Nov 6;9(11):1092. doi: 10.3390/an>ox9111092. PMID: 33172016; PMCID: PMC7694612. [3]: R. Selvam, Lalitha Subramanian, R. Gayathri, N. Angayarkanni. The an>-oxidant ac>vity of turmeric (Curcuma longa). 1995. Journal of Ethnopharmacology. hVps://doi.org/10.1016/0378-8741(95)01250-H. [4]: Marko J.A., Lew E., Trinidad K. Slides. BME 174: Cellular agriculture laboratory course. Spring 2024. Tu6s University. [5]: Marko J.A., Lew E., Trinidad K. Lab protocols weeks 10-13. BME 174: Cellular agriculture laboratory course. Tu6s University. [6]: Ali N. Lab notebook. BME 174: Cellular agriculture laboratory course. Tu6s University. [7]: Risa Indriani, Agus Muhamad HaVa, Ninik Irawa>, "Effect of the storage temperature on curcumin content in food supplement by spectrophotometry method," Proc. SPIE 11044, Third Interna>onal Seminar on Photonics, Op>cs, and Its Applica>ons (ISPhOA 2018), 110440M (11 April 2019); hVps://doi.org/10.1117/12.2504989
Turmeric and curcumin-based an/oxidant treatment in growth medium improves cri/cal quality a;ributes of cul/vated meat Noorul Hasan Ali, MS (bioengineering), Tu6s University Abstract Meat oxidizes over >me, changing its appearance, texture and flavor . An important aspect of cul>vated meat is long-term storage and freshness. An>oxidants combat oxida>on of lipids and other cellular material. Here, we treat bovine satellite muscle cells with different concentra>ons of two types of an>oxidant treatments. Higher cell viability and lower oxida>on is demonstrated under food-grade an>oxidant treatment. Introduc/on Oxida>on of meat is a challenge to its long-term storage. Oxida>ve stress leads to the crea>on of free radical oxygen and reac>ve nitrogen species, which cause discolora>on, change in flavor and forma>on of toxic compounds. Lipid and protein oxida>on are the mechanis>c reasons for colorectal cancer’s link to red meat. Overall, it is desired for cul>vated meat to be resistant to oxida>ve stress. An>oxidants scavenge free radical species and slow down oxida>on. Endogenous produc>on of carotenoids phytoene, lycopene and β-carotene in mammalian cells through metabolic engineering has been demonstrated [1]. This was done to combat lipid oxida>on’s role in decreasing shelf-life, and impairing color stability and flavor. Here, we added an exogenous an>oxidant to growth medium to study its effect on viability and lipid oxida>on of bovine satellite muscle cells. We compare food-grade and lab-grade an>oxidant treatments at different concentra>ons and select one of each kind based on highest cell viability. We add op>mal lab-grade and food-grade an>oxidant concentra>ons to growth medium with cells. Lipid oxida>on levels of cells were tested with a thiobarbituric acid reac>ve substances (TBARS) assay using malondialdehyde (MDA), a byproduct of lipid oxida>on. We conclude food-grade an>oxidant treatment is beVer than lab-grade an>oxidant treatment due to higher cell viability and reduced oxida>on. Methods Cell thawing, seeding and viability tes/ng Bovine satellite muscle cells were thawed from cryopreserva>on at -80 ⁰C and incubated (37 ⁰C, 5% CO2). 10mL of food-grade an>oxidant treatment stock solu>on was prepared by mixing commercially available turmeric powder and water at a concentra>on of 10,100 ng/mL. 10mL of lab-grade an>oxidant treatment stock solu>on was prepared by mixing commercially available curcumin (carotenoid) powder and dimethyl sulfoxide (DMSO) at a concentra>on of 10,000 ng/mL. 2 control growth medium formula>ons and 10 growth medium formula>on with differing concentra>ons of food-grade and lab-grade an>oxidant treatments were made as described in Table 1. DF 10, DF100 and DF1000 growth medium formula>ons were prepared by serial dilu>on. Standard growth medium (Gibco DMEM 1X + GlutaMAX) for bovine satellite cells used. Cells were seeded at 910 cells/cm2. Each condi>on was triplicated and 200uL of respec>ve media formula>on was exchanged every 3 days for 1 week. The well-plate was incubated (37 ⁰C, 5% CO2). A6er 1 week, cell viability was measured for each of the 12 media formula>ons by adding 20uL of Prestoblue dye to each well and performing fluorescence plate reading
assay at 560/590nm. 1X growth medium with 10% Prestoblue dye was used as control for cell viability assay. Name Formula>on An>oxidant concentra>on Grade Control GM 1X standard growth medium None Control Control DMSO 1X growth medium and 0.1% DMSO None Control DF1 L 1X growth medium, 0.1% DMSO and 0.1% lab-grade stock solu>on
10 ng/mL curcumin Lab
DF10 L 1X growth medium, 0.1% DMSO and 10% DF 1 L solu>on 1 ng /mL curcumin Lab
DF100 L 1X growth medium, 0.1% DMSO and 10% DF 10 L solu>on 0.1 ng/mL curcumin Lab
DF1000 L 1X growth medium, 0.1% DMSO and 10% DF 100 L solu>on 0.01 ng/mL curcumin Lab
DF10000 L 1X growth medium, 0.1% DMSO and 10% DF 1000 L solu>on 0.001 ng/mL curcumin Lab
DF1 F 1X growth medium and 5% food-grade stock solu>on 500 ng/mL turmeric Food
DF10 F 1X growth medium and 10% DF 1 F solu>on 50 ng/mL turmeric Food DF100 F 1X growth medium and 10% DF 10 F solu>on 5 ng/mL turmeric Food DF1000 F 1X growth medium and 10% DF 100 F solu>on 0.5 ng/mL turmeric Food DF10000 F 1X growth medium and 10% DF 1000 F solu>on 0.05 ng/mL turmeric Food Table 1: Growth medium formula>ons varying in an>oxidant concentra>on and type Cell seeding for oxida/on test Three T-175 flasks were seeded at 17,000 cells/cm2 each, with 25 mL of standard growth medium in the first, 25mL of DF 1 F food-grade medium in the second and 25mL DF 10000 L lab-grade medium in the third. Flasks were incubated (37 ⁰C, 5% CO2) for 1 week with media exchanged every 3 days. Cells were counted using a hemocytometer a6er scraping. Cells in all flasks were normalized to have the same count by removing 214uL of cell suspension solu>on from lab-grade and 610uL from food-grade. Cell suspensions were then centrifuged (300g, 5 minutes) and resuspended in 2mL PBS each. 900uL of each cell suspension solu>on was stored in 6 Eppendorf tubes, 2 tubes for each condi>on (control, food-
grade, lab-grade). 1 tube of each condi>on was stored for 1 week at room temperature (22 ⁰C) and another of each was refrigerated for 1 week (4 °C) to introduce mul>ple oxida>ve challenges. Lipid oxida/on test with TBARS assay Cell pellet tubes were placed on a heat block (100 °C, 10 minutes) to simulate cooking. Cell pellets were mixed with 200uL water . Cells were lysed using the freeze-thaw method by placing tubes under refrigera>on (-80 °C, 5 minutes) and thawing, repea>ng the freeze-thaw process for 3 cycles. 5 standard solu>ons were prepared as described in Table 2 to plot MDA standard curve. To conduct the TBARS assay, 100uL of 10% trichloroace>c acid (TCA) reagent and 800uL of color reagent were added 100uL of standard solu>ons or 100uL of sample solu>ons from 6 Eppendorf tubes. The 11 vials were placed on the heat block (100 °C, 60 minutes). Vials were placed in the refrigerator (4 °C, 10 minutes). Vials were microcentrifuged (4 °C, 1,600g, 10 minutes). 200uL from each vial was triplicated on a 96-well plate. MDA and TCA form an adduct whose absorbance peaks at 535nm. This allowed us to conduct a TBARS assay that acts as a proxy for lipid oxida>on since MDA is a byproduct of lipid oxida>on. Absorbance was measured using a plate reader at 535nm. MDA standard equa>on was generated using Numpy, a Python library. MDA concentra>ons in each of the 6 condi>ons were extrapolated using the equa>on obtained for the MDA standard curve from absorbance data obtained for each vial. We use MDA absorbance measurements as a proxy for lipid oxida>on in cells.
Table 2: MDA standard solu>ons Results 500 ng/mL turmeric and 0.001 ng/mL curcumin treatments showed highest cell viability As seen in Figure 1, cell viability is highest under DF1 for food-grade an>oxidant treatments and DF10000 under lab-grade an>oxidant treatments. 500 ng/mL turmeric in growth medium shows highest cell viability amongst all food-grade an>oxidant treatments. 0.001 ng/mL curcumin in growth medium shows highest cell viability amongst all lab-grade an>oxidant treatments. While viability decreases as concentra>on of turmeric goes down in growth medium, it increases with decrease in curcumin concentra>on. Surprisingly, high levels of curcumin in growth medium significantly impacted growth as seen in DF1 under lab-grade an>oxidant treatments. Only DF1000 and DF10000 lab-grade treatments, and DF1 food-grade treatment show higher viability compared to growth medium control.
Figures 1-2: Cell viability measurements under food-grade and lab-grade an>oxidant treatments. Prestoblue absorbance is a measure of cell viability. DF: dilu>on factor, GM: growth medium control, DMSO: dimethyl sulfoxide control. Food-grade antioxidant treatment shows lower lipid oxidation levels Compared to control, food-grade antioxidant treatment containing 500 ng/mL turmeric in growth medium shows much lower oxidation level under refrigeration and slightly lower oxidation level at room temperature. Lab-grade antioxidant treatment shows lower oxidation levels at room temperature. Surprisingly, lab-grade antioxidant treatment shows much higher oxidation under refrigeration.
Figure 3: MDA standard curve obtained by plotting line of best fit to absorbance measurements of prepared MDA standards. MDA: malondialdehyde.
Figures 4-5: Oxidation level comparison between food-grade and lab-grade antioxidant treatments under room temperature and refrigeration. MDA molar concentrations are obtained from MDA standard curve using absorbance assay measurements to line of best fit in Figure 3. MDA concentrations to corresponding absorbance assay measurements from cells are noted. Discussion Increasing turmeric in growth medium leads to higher cell viability. Turmeric is known for its an>oxida>ve proper>es. High concentra>ons of turmeric in growth medium showed high cell viability, with the highest concentra>on demonstra>ng higher cell viability than standard growth medium. This result highlights turmeric’s poten>al in improving cell viability. In literature, turmeric has shown an>oxidant proper>es by inhibi>ng lipid peroxida>on [3]. Further research can be done to characterize turmeric’s effects on the growth of bovine cells. Curcumin, a component of turmeric, in the lab-grade an>oxidant treatment is essen>ally purified turmeric. High concentra>ons of curcumin in our growth medium showed reduced cell viability. At low temperatures, curcumin has reduced bioavailability [7]. This seems to be a plausible reason for the much higher oxida>on levels under lab-grade an>oxidant treatment and refrigera>on. Our lab-grade an>oxidant treatment contains the lowest concentra>on of curcumin. The low concentra>on of curcumin coupled with reduced bioavailability of curcumin at low temperature may have resulted in increased oxida>on. Further study should be done to confirm curcumin’s lower bioavailability under refrigera>on. Meat is generally refrigerated. Any detrimental effects of an an>oxidant treatment to cul>vated meat’s shelf-life can significantly impact adop>on and commercializa>on. In conclusion, higher cell viability and lower lipid oxida>on is shown in bovine satellite muscle cells under food-grade an>oxidant treatment with commercially available turmeric. Lab-grade an>oxidant treatment with curcumin demonstrates higher viability of cells under certain condi>ons, lower oxida>on at room temperature but much higher oxida>on under refrigera>on. Since refrigera>on is a major aspect of commercial meat produc>on, we conclude food-grade an>oxidant treatment is beVer than lab-grade an>oxidant treatment.
References [1]: Stout AJ, Mirliani AB, Soule-Albridge EL, Cohen JM, Kaplan DL. Engineering carotenoid produc>on in mammalian cells for nutri>onally enhanced cell-cultured foods. Metab Eng. 2020 Nov;62:126-137. doi: 10.1016/j.ymben.2020.07.011. Epub 2020 Sep 2. PMID: 32890703; PMCID: PMC7666109. [2]: Jakubczyk K, Drużga A, Katarzyna J, Skonieczna-Żydecka K. An>oxidant Poten>al of Curcumin-A Meta-Analysis of Randomized Clinical Trials. An>oxidants (Basel). 2020 Nov 6;9(11):1092. doi: 10.3390/an>ox9111092. PMID: 33172016; PMCID: PMC7694612. [3]: R. Selvam, Lalitha Subramanian, R. Gayathri, N. Angayarkanni. The an>-oxidant ac>vity of turmeric (Curcuma longa). 1995. Journal of Ethnopharmacology. hVps://doi.org/10.1016/0378-8741(95)01250-H. [4]: Marko J.A., Lew E., Trinidad K. Slides. BME 174: Cellular agriculture laboratory course. Spring 2024. Tu6s University. [5]: Marko J.A., Lew E., Trinidad K. Lab protocols weeks 10-13. BME 174: Cellular agriculture laboratory course. Tu6s University. [6]: Ali N. Lab notebook. BME 174: Cellular agriculture laboratory course. Tu6s University. [7]: Risa Indriani, Agus Muhamad HaVa, Ninik Irawa>, "Effect of the storage temperature on curcumin content in food supplement by spectrophotometry method," Proc. SPIE 11044, Third Interna>onal Seminar on Photonics, Op>cs, and Its Applica>ons (ISPhOA 2018), 110440M (11 April 2019); hVps://doi.org/10.1117/12.2504989