Journal of Society of Preventive Korean Medicine (대한예방한의학회지)

Society of Preventive Korean Medicine (대한예방한의학회)
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In vitro anti-skin-aging effects of dried pomegranate concentrated powder

  • Lee, Dae-Geon (The Medical Research Center for Globalization of Herbal Medicine, Daegu Haany University) ;
  • Choi, Beom-Rak (Research Institute, HL Science Co., Ltd.) ;
  • Ku, Sae-Kwang (The Medical Research Center for Globalization of Herbal Medicine, Daegu Haany University) ;
  • Kang, Su-Jin (The Medical Research Center for Globalization of Herbal Medicine, Daegu Haany University) ;
  • Park, Hye-Rim (Research Institute, HL Science Co., Ltd.) ;
  • Sung, Mi-Sun (Research Institute, HL Science Co., Ltd.) ;
  • Lee, Young-Joon (The Medical Research Center for Globalization of Herbal Medicine, Daegu Haany University) ;
  • Park, Ki-Moon (Department of Foodscience and biotechnology, Sungkyunkwan University)
  • Received : 2018.07.09
  • Accepted : 2018.07.22
  • Published : 2018.08.31
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Abstract

Purpose : In this study, we intended to observe the anti-wrinkle and moisturizing effects of dried pomegranate juice concentration powder (PCP) using in vitro test. Materials and methods : Antioxidant effects of PCP were determined by free radical scavenging capacity (DPPH assay) and the cytotoxicity of PCP was examined in human keratinocyte (HaCaT) and human primary dermal fibroblast-neonatal (HDF) cells. To investigate the moisturizing effect of PCP, hyaluronan synthesis was examined in HaCaT cells. Activity of procollagen production were assessed in HDF cells and elastase inhibition properties of PCP were evaluated in cell free condition, to determine their anti-wrinkle effects. Metalloproteinase 1 (MMP-1) activity was also assessed following UVB irradiation, in the current in vitro experiment. Results : No PCP treatment related significant cytotoxic effects were demonstrated against to the both HDF and HaCaT cells. PCP showed favorable free radical scavenging activities in dose-dependent manner. In PCP-treated HaCaT cells, hyaluronan synthesis was non-significantly but markedly increased, and pro-collagen productions were significantly increased in HDF cells, at all three different concentrations (0.25, 0.75 and 1 mg/ml), and elastase inhibitory activities were observed by PCP treatment. A significant decrease in UVB-induced MMP-1 activity was also observed in 1 mg/ml PCP-treated HDF cells as compared to those of UVB-exposed cells. Conclusions : Taken together, these results suggest that PCP has favorable antioxidant, anti-wrinkle and moisturizing effects without meaningful cytotoxicity on HDF and HaCaT cell lines.

Keywords

References

  1. Gilchrest BA. Skin aging and photoaging. Dermatology nursing. 1990;2(2):79-82.
  2. Cevenini E, Invidia L, Lescai F, Salvioli S, Tieri P, Castellani G, Franceschi C. Human models of aging and longevity. Expert opinion on biological therapy. 2008;8(9):1393-1405. https://doi.org/10.1517/14712598.8.9.1393
  3. Jurkiewicz BA, Buettner GR. Ultraviolet light-induced free radical formation in skin: an electron paramagnetic resonance study. Photochemistry and photobiology. 1994;59(1):1-4. https://doi.org/10.1111/j.1751-1097.1994.tb04993.x
  4. Messarah M, Boumendjel A, Chouabia A, Klibet F, Abdennour C, Boulakoud MS, Feki AE. Influence of thyroid dysfunction on liver lipid peroxidation and antioxidant status in experimental rats. Experimental and toxicologic pathology : official journal of the Gesellschaft fur Toxikologische Pathologie. 2010;62(3):301-310. https://doi.org/10.1016/j.etp.2009.04.009
  5. Rodriguez KJ, Wong HK, Oddos T, Southall M, Frei B, Kaur S. A purified feverfew extract protects from oxidative damage by inducing DNA repair in skin cells via a PI3-kinase-dependent Nrf2/ARE pathway. Journal of dermatological science. 2013;72(3):304-310. https://doi.org/10.1016/j.jdermsci.2013.08.004
  6. Campanini MZ, Pinho-Ribeiro FA, Ivan AL, Ferreira VS, Vilela FM, Vicentini FT, Martinez RM, Zarpelon AC, Fonseca MJ, Faria TJ, Baracat MM, Verri WA, Jr., Georgetti SR, Casagrande R. Efficacy of topical formulations containing Pimenta pseudocaryophyllus extract against UVB-induced oxidative stress and inflammation in hairless mice. Journal of photochemistry and photobiology B, Biology. 2013;127:153-160. https://doi.org/10.1016/j.jphotobiol.2013.08.007
  7. Kim SR, Jung YR, An HJ, Kim DH, Jang EJ, Choi YJ, Moon KM, Park MH, Park CH, Chung KW, Bae HR, Choi YW, Kim ND, Chung HY. Anti-wrinkle and anti-inflammatory effects of active garlic components and the inhibition of MMPs via NF-kB signaling. PloS one. 2013;8(9):e73877. https://doi.org/10.1371/journal.pone.0073877
  8. Oh J, Kim JH, Park JG, Yi YS, Park KW, Rho HS, Lee MS, Yoo JW, Kang SH, Hong YD, Shin SS, Cho JY. Radical scavenging activity-based and AP-1-targeted anti-inflammatory effects of lutein in macrophage-like and skin keratinocytic cells. Mediators of inflammation. 2013;2013:787042.
  9. Poswig A, Wenk J, Brenneisen P, Wlaschek M, Hommel C, Quel G, Faisst K, Dissemond J, Briviba K, Krieg T, Scharffetter-Kochanek K. Adaptive antioxidant response of manganese-superoxide dismutase following repetitive UVA irradiation. The Journal of investigative dermatology. 1999;112(1):13-18. https://doi.org/10.1046/j.1523-1747.1999.00465.x
  10. Fuchs J, Kern H. Modulation of UV-light-induced skin inflammation by D-alpha-tocopherol and L-ascorbic acid: a clinical study using solar simulated radiation. Free radical biology & medicine. 1998;25(9):1006-1012. https://doi.org/10.1016/S0891-5849(98)00132-4
  11. Kim YH, Chung CB, Kim JG, Ko KI, Park SH, Kim JH, Eom SY, Kim YS, Hwang YI, Kim KH. Anti-wrinkle activity of ziyuglycoside I isolated from a Sanguisorba officinalis root extract and its application as a cosmeceutical ingredient. Bioscience, biotechnology, and biochemistry. 2008;72(2):303-311. https://doi.org/10.1271/bbb.70268
  12. Hunt G, Kyne S, Ito S, Wakamatsu K, Todd C, Thody A. Eumelanin and phaeomelanin contents of human epidermis and cultured melanocytes. Pigment cell research. 1995;8(4):202-208. https://doi.org/10.1111/j.1600-0749.1995.tb00664.x
  13. Vincensi MR, d'Ischia M, Napolitano A, Procaccini EM, Riccio G, Monfrecola G, Santoianni P, Prota G. Phaeomelanin versus eumelanin as a chemical indicator of ultraviolet sensitivity in fair-skinned subjects at high risk for melanoma: a pilot study. Melanoma research. 1998;8(1):53-58. https://doi.org/10.1097/00008390-199802000-00009
  14. Kvam E, Dahle J. Pigmented melanocytes are protected against ultraviolet-A-induced membrane damage. The Journal of investigative dermatology. 2003;121(3):564-569. https://doi.org/10.1046/j.1523-1747.2003.12418.x
  15. Yamazaki F, Okamoto H, Miyauchi-Hashimoto H, Matsumura Y, Itoh T, Tanaka K, Kunisada T, Horio T. XPA gene-deficient, SCF-transgenic mice with epidermal melanin are resistant to UV-induced carcinogenesis. The Journal of investigative dermatology. 2004;123(1):220-228. https://doi.org/10.1111/j.0022-202X.2004.22710.x
  16. Wagner JK, Parra EJ, H LN, Jovel C, Shriver MD. Skin responses to ultraviolet radiation: effects of constitutive pigmentation, sex, and ancestry. Pigment cell research. 2002;15(5):385-390. https://doi.org/10.1034/j.1600-0749.2002.02046.x
  17. Ortonne JP. Photoprotective properties of skin melanin. The British journal of dermatology. 2002;146 Suppl 61:7-10.
  18. Yamaguchi Y, Takahashi K, Zmudzka BZ, Kornhauser A, Miller SA, Tadokoro T, Berens W, Beer JZ, Hearing VJ. Human skin responses to UV radiation: pigment in the upper epidermis protects against DNA damage in the lower epidermis and facilitates apoptosis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2006;20(9):1486-1488. https://doi.org/10.1096/fj.06-5725fje
  19. Tada M, Kohno M, Niwano Y. Scavenging or quenching effect of melanin on superoxide anion and singlet oxygen. Journal of clinical biochemistry and nutrition. 2010;46(3):224-228. https://doi.org/10.3164/jcbn.09-84
  20. Ito S, Wakamatsu K. Chemistry of mixed melanogenesis--pivotal roles of dopaquinone. Photochemistry and photobiology. 2008;84(3):582-592. https://doi.org/10.1111/j.1751-1097.2007.00238.x
  21. Khazaeli P, Goldoozian R, Sharififar F. An evaluation of extracts of five traditional medicinal plants from Iran on the inhibition of mushroom tyrosinase activity and scavenging of free radicals. International journal of cosmetic science. 2009;31(5):375-381. https://doi.org/10.1111/j.1468-2494.2009.00503.x
  22. Momtaz S, Mapunya BM, Houghton PJ, Edgerly C, Hussein A, Naidoo S, Lall N. Tyrosinase inhibition by extracts and constituents of Sideroxylon inerme L. stem bark, used in South Africa for skin lightening. Journal of ethnopharmacology. 2008;119(3):507-512. https://doi.org/10.1016/j.jep.2008.06.006
  23. Ng LT, Ko HH, Lu TM. Potential antioxidants and tyrosinase inhibitors from synthetic polyphenolic deoxybenzoins. Bioorganic & medicinal chemistry. 2009;17(13):4360-4366. https://doi.org/10.1016/j.bmc.2009.05.019
  24. Nugroho A, Choi JK, Park JH, Lee KT, Cha BC, Park HJ. Two new flavonol glycosides from Lamium amplexicaule L. and their in vitro free radical scavenging and tyrosinase inhibitory activities. Planta medica. 2009;75(4):364-366. https://doi.org/10.1055/s-0028-1112216
  25. Wang KH, Lin RD, Hsu FL, Huang YH, Chang HC, Huang CY, Lee MH. Cosmetic applications of selected traditional Chinese herbal medicines. Journal of ethnopharmacology. 2006;106(3):353-359. https://doi.org/10.1016/j.jep.2006.01.010
  26. Solomon EI, Chen P, Metz M, Lee SK, Palmer AE. Oxygen Binding, Activation, and Reduction to Water by Copper Proteins. Angewandte Chemie (International ed in English). 2001;40(24):4570-4590. https://doi.org/10.1002/1521-3773(20011217)40:24<4570::AID-ANIE4570>3.0.CO;2-4
  27. Chen P, Solomon EI. $O_2$ activation by binuclear Cu sites: noncoupled versus exchange coupled reaction mechanisms. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(36):13105-13110. https://doi.org/10.1073/pnas.0402114101
  28. Inoue T, Shiota Y, Yoshizawa K. Quantum chemical approach to the mechanism for the biological conversion of tyrosine to dopaquinone. Journal of the American Chemical Society. 2008;130(50):16890-16897. https://doi.org/10.1021/ja802618s
  29. Rob D. Tyrosinase. In: Lontie R, editor. Copper Proteins and Copper Enzymes. IIth ed. Florida: CRC Press; 1984.p.207-240.
  30. Cho EA, Cho EH, Choi SJ, Park KH, Kim SY, Jeong YJ, Ku CS, Ha BJ, Jang DI, Chae HJ. Screening of anti-wrinkle resource from herbal medicinal extracts and stability test of its cosmetic products. Korean J Medicinal Crop Sci. 2011;19(2):126-135. https://doi.org/10.7783/KJMCS.2011.19.2.126
  31. Kwak YJ, Lee DH, Kim NM, Lee JS. Screening and extraction condition of anti-skin aging elastase inhibitor from medicinal plants. Korean J Medicinal Crop Sci. 2005;13(6):213-216.
  32. Wiedow O, Schroder JM, Gregory H, Young JA, Christophers E. Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence. The Journal of biological chemistry. 1990;265(25):14791-14795.
  33. Gil MI, Tomas-Barberan FA, Hess-Pierce B, Holcroft DM, Kader AA. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. Journal of agricultural and food chemistry. 2000;48(10):4581-4589. https://doi.org/10.1021/jf000404a
  34. Noda Y, Kaneyuki T, Mori A, Packer L. Antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin. Journal of agricultural and food chemistry. 2002;50(1):166-171. https://doi.org/10.1021/jf0108765
  35. Singh RP, Chidambara Murthy KN, Jayaprakasha GK. Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. Journal of agricultural and food chemistry. 2002;50(1):81-86. https://doi.org/10.1021/jf010865b
  36. Kim ND, Mehta R, Yu W, Neeman I, Livney T, Amichay A, Poirier D, Nicholls P, Kirby A, Jiang W, Mansel R, Ramachandran C, Rabi T, Kaplan B, Lansky E. Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer. Breast cancer research and treatment. 2002;71(3):203-217. https://doi.org/10.1023/A:1014405730585
  37. Maity N, Nema NK, Abedy MK, Sarkar BK, Mukherjee PK. Exploring Tagetes erecta Linn flower for the elastase, hyaluronidase and MMP-1 inhibitory activity. Journal of ethnopharmacology. 2011;137(3):1300-1305. https://doi.org/10.1016/j.jep.2011.07.064
  38. Chan YY, Kim KH, Cheah SH. Inhibitory effects of Sargassum polycystum on tyrosinase activity and melanin formation in B16F10 murine melanoma cells. Journal of ethnopharmacology. 2011;137(3):1183-1188. https://doi.org/10.1016/j.jep.2011.07.050
  39. Imokawa G. Recent advances in characterizing biological mechanisms underlying UV-induced wrinkles: a pivotal role of fibrobrast-derived elastase. Arch dermatol Res. 2008;300(1):7-20. https://doi.org/10.1007/s00403-007-0798-x
  40. Kim EJ, Kim MK, Jin XJ, Oh JH, Kim JE, Chung JH. Skin aging and photoaging alter fatty acids composition, including 11,14,17-eicosatrienoic acid, in the epidermis of human skin. Journal of Korean medical science. 2010;25(6):980-983. https://doi.org/10.3346/jkms.2010.25.6.980
  41. Kang SJ, Choi BR, Kim SH, Yi HY, Park HR, Park SJ, Song CH, Park JH, Lee YJ, Kwang S. Inhibitory effects of pomegranate concentrated solution on the activities of hyaluronidase, tyrosinase, and metalloproteinase. Journal of cosmetic science. 2015;66(3):145-159.
  42. Kang SJ, Choi BR, Kim SH, Yi HY, Park HR, Song CH, Park SJ, Ku SK, Lee YJ. Effect of pomegranate concentration solution on photoaging. J Soc Prev Korean Med. 2015;19:109-116.
  43. Kang SJ, Choi BR, Lee EK, Kim SH, Yi HY, Park HR, Song CH, Lee YJ, Ku SK. Inhibitory effect of dried pomegranate concentration powder on melanogenesis in B16F10 melanoma cells; involvement of p38 and PKA signaling pathways. International journal of molecular sciences. 2015;16(10):24219-24242. https://doi.org/10.3390/ijms161024219
  44. Kang SJ, Choi BR, Kim SH, Yi HY, Park HR, Song CH, Ku SK, Lee YJ. Beneficial effects of dried pomegranate juice concentrated powder on ultraviolet B-induced skin photoaging in hairless mice. Experimental and therapeutic medicine. 2017;14(2):1023-1036. https://doi.org/10.3892/etm.2017.4626
  45. Hu JR, Choi BR, Park HR, Sung MS, Yi HY, Kang SJ, Ku SK, Lee YJ. Skin tissue homogenate analysis for ceramide and TGF-1 contents with TGF-1 mRNA expressions after treatment of pomegranate concentrated solution and dried pomegranate concentrate powder in mice. J Korean Med. 2016;37(4):1-9. https://doi.org/10.13048/jkm.16042
  46. Kang SJ, Choi BR, Kim SH, Yi HY, Park HR, Sung MS, Song CH, Cho IJ, Lee YJ, Ku SK. Evaluation of the in vivo skin moisturizing effects and underlying mechanisms of pomegranate concentrate solution and dried pomegranate concentrate powder. J Korean Med. 2016;37(2):12-22. https://doi.org/10.13048/jkm.16017
  47. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature. 1958;181(4617):1199. https://doi.org/10.1038/1811199a0
  48. Jiang P, Li X, Thompson CB, Huang Z, Araiza F, Osgood R, Wei G, Feldmann M, Frost GI, Shepard HM. Effective targeting of the tumor microenvironment for cancer therapy. Anticancer research. 2012;32(4):1203-1212.
  49. Heo JC, Park JY, An SM, Lee JM, Yun CY, Shin HM, Kwon TK, Lee SH. Anti-oxidant and anti-tumor activities of crude extracts by Gastrodia elata Blume. Korean J Food Preserv. 2006;13(1):83-87.
  50. Losso JN, Munene CN, Bansode RR, Bawadi HA. Inhibition of matrix metalloproteinase-1 activity by the soybean Bowman-Birk inhibitor. Biotechnology letters. 2004;26(11):901-905. https://doi.org/10.1023/B:bile.0000025900.33812.7c
  51. Chen AY, Lu JM, Yao Q, Chen C. Entacapone is an Antioxidant More Potent than Vitamin C and Vitamin E for Scavenging of Hypochlorous Acid and Peroxynitrite, and the Inhibition of Oxidative Stress-Induced Cell Death. Medical science monitor : international medical journal of experimental and clinical research. 2016;22:687-696.
  52. Lijnen P, Petrov V. Transforming growth factor-beta 1-induced collagen production in cultures of cardiac fibroblasts is the result of the appearance of myofibroblasts. Methods and findings in experimental and clinical pharmacology. 2002;24(6):333-344. https://doi.org/10.1358/mf.2002.24.6.693065
  53. Morihara K, Tsuzuki H. Phosphoramidon as an inhibitor of elastase from Pseudomonas aeruginosa. The Japanese journal of experimental medicine. 1978;48(1):81-84.
  54. Kim DW, Kim HJ, Chae HS, Park NG, Kim YB, Jang A. Anti-oxidation and Anti-wrinkling Effects of Jeju Horse Leg Bone Hydrolysates. Korean journal for food science of animal resources. 2014;34(6):844-851. https://doi.org/10.5851/kosfa.2014.34.6.844
  55. Kikuchi K, Matahira Y. Oral N-acetylglucosamine supplementation improves skin conditions of female volunteers: Clinical evaluation by a microscopic three-dimensional skin surface analyzer. Journal of applied cosmetology. 2002;20(2):143-152.
  56. Uitterlinden EJ, Koevoet JL, Verkoelen CF, Bierma-Zeinstra SM, Jahr H, Weinans H, Verhaar JA, van Osch GJ. Glucosamine increases hyaluronic acid production in human osteoarthritic synovium explants. BMC musculoskeletal disorders. 2008;9:120. https://doi.org/10.1186/1471-2474-9-120
  57. Lateef H, Stevens MJ, Varani J. All-transretinoic acid suppresses matrix metalloproteinase activity and increases collagen synthesis in diabetic human skin in organ culture. The American journal of pathology. 2004;165(1):167-174. https://doi.org/10.1016/S0002-9440(10)63285-3
  58. Jurzak M, Latocha M, Gojniczek K, Kapral M, Garncarczyk A, Pierzchala E. Influence of retinoids on skin fibroblasts metabolism in vitro. Acta poloniae pharmaceutica. 2008;65(1):85-91.
  59. Afaq F, Saleem M, Krueger CG, Reed JD, Mukhtar H. Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-kB pathways and inhibits skin tumorigenesis in CD-1 mice. International journal of cancer. 2005;113(3):423-433. https://doi.org/10.1002/ijc.20587
  60. Wang RF, Xie WD, Zhang Z, Xing DM, Ding Y, Wang W, Ma C, Du LJ. Bioactive compounds from the seeds of Punica granatum (pomegranate). Journal of natural products. 2004;67(12):2096-2098. https://doi.org/10.1021/np0498051
  61. Yoshimura M, Watanabe Y, Kasai K, Yamakoshi J, Koga T. Inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-induced pigmentation. Bioscience, biotechnology, and biochemistry. 2005;69(12):2368-2373. https://doi.org/10.1271/bbb.69.2368
  62. Ross JA, Kasum CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annual review of nutrition. 2002;22:19-34. https://doi.org/10.1146/annurev.nutr.22.111401.144957
  63. Soobrattee MA, Bahorun T, Aruoma OI. Chemopreventive actions of polyphenolic compounds in cancer. BioFactors (Oxford, England). 2006;27(1-4):19-35. https://doi.org/10.1002/biof.5520270103
  64. Maas JL, Galletta GJ, Stoner GD. Ellagic acid, an anticarcinogen in fruits, especially in strawberries: a review. HortScience. 1991;26(1):10-14.
  65. Thresiamma KC, Kuttan R. Inhibition of liver fibrosis by ellagic acid. Indian journal of physiology and pharmacology. 1996;40(4):363-366.
  66. Osawa T, Ide A, Su JD, Namiki M. Inhibition of in vitro lipid peroxidation by ellagic acid. Journal of agricultural and food chemistry. 1987;35(5):808-812. https://doi.org/10.1021/jf00077a042
  67. Shimogaki H, Tanaka Y, Tamai H, Masuda M. In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibition. International journal of cosmetic science. 2000;22(4):291-303. https://doi.org/10.1046/j.1467-2494.2000.00023.x
  68. Yamane T, Nakagami G, Yoshino S, Muramatsu A, Matsui S, Oishi Y, Kanazawa T, Minematsu T, Sanada H. Hydrocellular foam dressing promotes wound healing along with increases in hyaluronan synthase 3 and PPARa gene expression in epidermis. PloS one. 2013;8(8):e73988. https://doi.org/10.1371/journal.pone.0073988
  69. Frenkel JS. The role of hyaluronan in wound healing. International wound journal. 2014;11(2):159-163. https://doi.org/10.1111/j.1742-481X.2012.01057.x
  70. Liao PL, Li CH, Chang CY, Lu SR, Lin CH, Tse LS, Cheng YW. Anti-ageing effects of alpha-naphthoflavone on normal and UVB-irradiated human skin fibroblasts. Experimental dermatology. 2012;21(7):546-548. https://doi.org/10.1111/j.1600-0625.2012.01517.x
  71. Kim HD, Cho HR, Moon SB, Shin HD, Yang KJ, Park BR, Jang HJ, Kim LS, Lee HS, Ku SK. Effects of beta-glucan from Aureobasidium pullulans on acute inflammation in mice. Archives of pharmacal research. 2007;30(3):323-328. https://doi.org/10.1007/BF02977613
  72. Adams LS, Seeram NP, Aggarwal BB, Takada Y, Sand D, Heber D. Pomegranate juice, total pomegranate ellagitannins, and punicalagin suppress inflammatory cell signaling in colon cancer cells. Journal of agricultural and food chemistry. 2006;54(3):980-985. https://doi.org/10.1021/jf052005r
  73. Orgil O, Schwartz E, Baruch L, Matityahu I, Mahajna J, Amir R. The antioxidative and anti-proliferative potential of non-edible organs of the pomegranate fruit and tree. LWT-Food Science and Technology. 2014;58(2):571-577. https://doi.org/10.1016/j.lwt.2014.03.030