This contribution proposes an enzyme-assisted eco-friendly process for the extraction of non-extractable polyphenols (NEPPs) from black tea leftover (BTLO), an underutilized tea waste. BTLO hydrolyzed with various enzyme formulations was extracted using supercritical carbon dioxide and ethanol as co-solvent (SC-CO2 + EtOH). A conventional solvent extraction (CSE) was performed using EtOH + H2O (80:20, v/v) for comparison purposes. The results revealed that hydrolysis of BTLO with 2.9% (w/w) kemzyme at 45 °C and pH 5.4 for 98 min improved the liberation of NEPPs offering 5-fold higher extract yield (g/100 g) as compared with non-treated BTLO. In vitro antioxidant evaluation and LC-MS characterization of extracts revealed the presence of phenolic acids (mainly caffeic and para-coumaric acid) of high antioxidant value. Scanning electron micrograph of the hydrolyzed BTLO samples indicated noteworthy changes in the ultrastructure of BTLO. Moreover, polyphenol extracts obtained by SC-CO2 + EtOH extraction were found to be cleaner and richer in polyphenols as compared to CSE. The devised enzyme-assisted SC-CO2 + EtOH extraction process in the present work can be explored as an effective biotechnological mean for the optimal recovery of antioxidant polyphenols.
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Cheng A, Chen X, Wang W, Gong Z, Liu L. Contents of extractable and non-extractable polyphenols in the leaves of blueberry. Czech J Food Sci. 2013;31(3):275–82.
Dai J, Mumper RJ. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules. 2010;15(10):7313–52.
Bertsch W. The use of tea bricks as currency among the Tibetans. Tibet J. 2009;34(2):35–81.
Kim Y, Goodner KL, Park J-D, Choi J, Talcott ST. Changes in antioxidant phytochemicals and volatile composition of Camellia sinensis by oxidation during tea fermentation. Food Chem. 2011;129(4):1331–42.
Spiro M, Jaganyi D. Kinetics and equilibria of tea infusion. Part 11—the kinetics of the formation of tea scum. Food Chem. 1994;49(4):359–65.
Kinugasa H, Takeo T. Deterioration mechanism for tea infusion aroma by retort pasteurization. Agric Biol Chem. 1990;54(10):2537–42.
Traoré D. Cocoa and coffee value chains in West and Central Africa: constraints and options for revenue-raising diversification. Food Agric Organ. 2009;3:1–78.
Acosta-Estrada BA, Gutiérrez-Uribe JA, Serna-Saldívar SO. Bound phenolics in foods, a review. Food Chem. 2014;152:46–55.
Soto ML, Moure A, Domínguez H, Parajó JC. Recovery, concentration and purification of phenolic compounds by adsorption: a review. J Food Eng. 2011;105(1):1–27.
Martins S, Mussatto SI, Martinez-Avila G, Montanez-Saenz J, Aguilar CN, Teixeira JA. Bioactive phenolic compounds: production and extraction by solid-state fermentation. A review Biotechnol Adv. 2011;29(3):365–73.
Bimakr M, Rahman RA, Taip FS, Adzahan NM, Sarker MZ, Ganjloo A. Optimization of ultrasound-assisted extraction of crude oil from winter melon (Benincasa hispida) seed using response surface methodology and evaluation of its antioxidant activity, total phenolic content and fatty acid composition. Molecules. 2012;17(10):11748–62.
Carrera C, Ruiz-Rodriguez A, Palma M, Barroso CG. Ultrasound assisted extraction of phenolic compounds from grapes. Anal Chim Acta. 2012;732:100–4.
Beejmohun V, Fliniaux O, Grand E, Lamblin F, Bensaddek L, Christen P, et al. Microwave-assisted extraction of the main phenolic compounds in flaxseed. Phytochem Anal. 2007;18(4):275–82.
Pataro G, Donsì G, Ferrari G. Aseptic processing of apricots in syrup by means of a continuous pilot scale ohmic unit. LWT-Food Sci Technol. 2011;44(6):1546–54.
Yaqoob S, Sultana B, Mushtaq M. In vitro antioxidant activities of Trianthema portulacastrum L. hydrolysates. Prev Nutri Food Sci. 2014;19(1):1–27.
Bener M, Shen Y, Apak R, Finley JW, Xu Z. Release and degradation of anthocyanins and phenolics from blueberry pomace during thermal acid hydrolysis and dry heating. J Agric Food Chemi. 2013;61(27):6643–9.
Komes D, Belscak-Cvitanovic A, Horzic D, Rusak G, Likic S, Berendika M. Phenolic composition and antioxidant properties of some traditionally used medicinal plants affected by the extraction time and hydrolysis. Phytochem Anal. 2011;22(2):172–80.
Wai CM, Laintz K inventors. Idaho Research Foundation, Inc., assignee. Supercritical fluid extraction. United States patent US 5,356,538. 1994.
Herrero M, Cifuentes A, Ibanez E. Sub-and supercritical fluid extraction of functional ingredients from different natural sources: plants, food-by-products, algae and microalgae: a review. Food Chem. 2006;98(1):136–48.
Lang Q, Wai CM. Supercritical fluid extraction in herbal and natural product studies—a practical review. Talanta. 2001;53(4):771–82.
Kong Y, Fu Y-J, Zu Y-G, Liu W, Wang W, Hua X, et al. Ethanol modified supercritical fluid extraction and antioxidant activity of cajaninstilbene acid and pinostrobin from pigeonpea [Cajanus cajan (L.) Millsp.] leaves. Food Chem. 2009;117(1):152–9.
Fathordoobady F, Mirhosseini H, Selamat J, Manap MYA. Effect of solvent type and ratio on betacyanins and antioxidant activity of extracts from Hylocereus polyrhizus flesh and peel by supercritical fluid extraction and solvent extraction. Food Chem. 2016;202:70–80.
Tow WW, Premier R, Jing H, Ajlouni S. Antioxidant and antiproliferation effects of extractable and nonextractable polyphenols isolated from apple waste using different extraction methods. J Food Sci. 2011;76(7):T163–72.
Saura-Calixto F, Serrano J, Goñi I. Intake and bioaccessibility of total polyphenols in a whole diet. Food Chem. 2007;101(2):492–501.
Chaovanalikit A, Wrolstad R. Total anthocyanins and total phenolics of fresh and processed cherries and their antioxidant properties. J Food Sci. 2004;69(1):67–72.
Chen Z, Bertin R, Froldi G. EC50 estimation of antioxidant activity in DPPH assay using several statistical programs. Food Chem. 2013;138(1):414–20.
Arts MJ, Haenen GR, Voss HP, Bast A. Antioxidant capacity of reaction products limits the applicability of the Trolox Equivalent Antioxidant Capacity (TEAC) assay. Food Chem Toxicol. 2004;42(1):45–9.
Gülçin Ì, Güngör Şat İ, Beydemir Ş, Elmastaş M, İrfan KÖ. Comparison of antioxidant activity of clove (Eugenia caryophylata Thunb) buds and lavender (Lavandula stoechas L.). Food Chem. 2004;87(3):393–400.
Beker BY, Bakir T, Sonmezoglu I, Imer F, Apak R. Antioxidant protective effect of flavonoids on linoleic acid peroxidation induced by copper(II)/ascorbic acid system. Chem Phys Lipids. 2011;164(8):732–9.
Agostini F, Bertussi RA, Agostini G, Atti Dos Santos AC, Rossato M, Vanderlinde R. Supercritical extraction from vinification residues: fatty acids, alpha-tocopherol, and phenolic compounds in the oil seeds from different varieties of grape. Scientific World J. 2012;2012:1–9.
Li B, Smith B, Hossain MM. Extraction of phenolics from citrus peels: II. Enzyme-assisted extraction method. Sep Purif Technol. 2006;48(2):189–96.
Zou Y, Chang SK, Gu Y, Qian SY. Antioxidant activity and phenolic compositions of lentil (Lens culinaris var. Morton) extract and its fractions. J Agric Food Chem. 2011;59(6):2268–76.
Bonoli M, Marconi E, Caboni MF. Free and bound phenolic compounds in barley (Hordeum vulgare L.) flours. Evaluation of the extraction capability of different solvent mixtures and pressurized liquid methods by micellar electrokinetic chromatography and spectrophotometry. J Chromatogr A. 2004;1057(1–2):1–12.
Soylu S. Accumulation of cell-wall bound phenolic compounds and phytoalexin in Arabidopsis thaliana leaves following inoculation with pathovars of Pseudomonas syringae. Plant Sci. 2006;170(5):942–52.
Walch SG, Tinzoh LN, Zimmermann BF, Stuhlinger W, Lachenmeier DW. Antioxidant capacity and polyphenolic composition as quality indicators for aqueous infusions of Salvia officinalis L. (sage tea). Front Pharmacol. 2011;2:79.
Ercisli S, Tosun M, Karlidag H, Dzubur A, Hadziabulic S, Aliman Y. Color and antioxidant characteristics of some fresh fig (Ficus carica L.) genotypes from northeastern Turkey. Plant Food Hum Nutr. 2012;67(3):271–6.
Zhu F, Cai YZ, Sun M, Ke J, Lu D, Corke H. Comparison of major phenolic constituents and in vitro antioxidant activity of diverse Kudingcha genotypes from Ilex kudingcha, Ilex cornuta, and Ligustrum robustum. J Agric Food Chem. 2009;57(14):6082–9.
Zhang Y, Seeram NP, Lee R, Feng L, Heber D. Isolation and identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties. J Agric Food Chem. 2008;56(3):670–5.
Pohanka M, Bandouchova H, Sobotka J, Sedlackova J, Soukupova I, Pikula J. Ferric reducing antioxidant power and square wave voltammetry for assay of low molecular weight antioxidants in blood plasma: performance and comparison of methods. Sensors. 2009;9(11):9094–103.
Wang Y, Yang M, Lee SG, Davis CG, Kenny A, Koo SI, et al. Plasma total antioxidant capacity is associated with dietary intake and plasma level of antioxidants in postmenopausal women. J Nutri Biochem. 2012;23(12):1725–31.
Zulueta A, Esteve MJ, Frígola A. ORAC and TEAC assays comparison to measure the antioxidant capacity of food products. Food Chem. 2009;114(1):310–6.
Fu Y-J, Liu W, Zu Y-G, Tong M-H, Li S-M, Yan M-M, et al. Enzyme assisted extraction of luteolin and apigenin from pigeonpea [Cajanus cajan (L.) Millsp.] leaves. Food Chem. 2008;111(2):508–12.
Wu X, Yu X, Jing H. Optimization of phenolic antioxidant extraction from Wuweizi (Schisandra chinensis) pulp using random-centroid optimization methodology. Int J Mol Sci. 2011;12(9):6255–66.
Rene A, Abasq ML, Hauchard D, Hapiot P. How do phenolic compounds react toward superoxide ion? A simple electrochemical method for evaluating antioxidant capacity. Anal Chem. 2010;82(20):8703–10.
Palmer DM, Kitchin JS. A double-blind, randomized, controlled clinical trial evaluating the efficacy and tolerance of a novel phenolic antioxidant skin care system containing Coffea arabica and concentrated fruit and vegetable extracts. J Drugs Dermatol. 2010;9(12):1480–7.
The work presentment in this paper was financially sponsored by Higher Education Commission (HEC), Pakistan, under the International Research Support Initiative Program (IRSIP); PIN: IRSIP 24 PS 17.
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The authors declare that they have no conflicts of interest.
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Mushtaq, M., Sultana, B., Akram, S. et al. Enzyme-assisted supercritical fluid extraction: an alternative and green technology for non-extractable polyphenols. Anal Bioanal Chem 409, 3645–3655 (2017). https://doi.org/10.1007/s00216-017-0309-7