The Remnant photosynthetic pigments in tea dregs: identification, composition, and potential use as antibacterial photosensitizer
DOI:
https://doi.org/10.5219/1651Keywords:
antimicrobial photosensitization, chlorophyll, pigments, tea dregsAbstract
The production of tea dregs is continually increasing along with the growth of people's interest in ready-to-drink beverages. However, the recent development of research on the use of tea dregs is still very limited. The present study was aimed to identify the remnant photosynthetic pigments in tea dregs, determine their composition, and evaluate their potential use as natural antibacterial agents based on light-induced reaction (photosensitization). The tea dregs from six commercial teas, consisting of green and black teas, were analyzed using high-performance liquid chromatography (HPLC) with a photodiode array detector, and the spectroscopic data were analyzed from 350 to 700 nm. Pigment identification was performed based on spectral characteristics, and pigment composition in the extracts from the dregs was determined by a three-dimensional multi-chromatogram analysis method. The dominant pigment fractions in both tea types were pheophytin a and its isomers, as well as pheophytin b. Although the dregs of black teas generally contain fewer remnant pigments, they possess residual chlorophyll b, which is not found in the dregs of green teas. In thirty-minutes illumination under 50 W red light-emitting diode, the presence of pigments from tea dregs caused up to 0.87 and 0.35 log reduction of Staphylococcus aureus and Escherichia coli, respectively. The disparity of pigments composition among tea types does not strongly influence their photosensitization activity against both bacteria. Hence, upon further application, the amount of total remnant pigments in the dregs could be taken as substantial consideration instead of tea types.
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Acedo, P., Stockert, J. C., Cañete, M., Villanueva, A. 2014. Two combined photosensitizers: A goal for more effective photodynamic therapy of cancer. Cell Death and Disease, vol. 5, no. 3, p. 1-12. https://doi.org/10.1038/cddis.2014.77 DOI: https://doi.org/10.1038/cddis.2014.77
Alves, E., Faustino, M. A. F., Neves, M. G. P. M. S., Cunha, A., Nadais, H., Almedia, A. 2015. Potential applications of porphytins in photodynamic inactivation beyond the medical scope. Journal of Photochemistry and Photobiology C: Photochemistry Reviews, vol. 22, p. 34-57. https://doi.org/10.1016/j.jphotochemrev.2014.09.003 DOI: https://doi.org/10.1016/j.jphotochemrev.2014.09.003
Amaral, L. S., Azevedo, E. B., Perussi, J. R. 2018. The response surface methodology speeds up the search for optimal parameters in the photoinactivation of E. coli by photodynamic therapy. Photodiagnosis and Photodynamic Therapy, vol. 22, p. 26-33. https://doi.org/10.1016/j.pdpdt.2018.02.020 DOI: https://doi.org/10.1016/j.pdpdt.2018.02.020
Amaya, D. B. R. 2016. Natural food pigments and colorants. Current Opinion in Food Science, vol. 7, p. 20-26. https://doi.org/10.1016/j.cofs.2015.08.004 DOI: https://doi.org/10.1016/j.cofs.2015.08.004
Brotosudarmo, T. H. P., Heriyanto, Shioi, Y., Indriatmoko, Adhiwibawa, M. A. S., Indrawati, R., Limantara, L. 2018. Composition of the main dominant pigments from potential two edible seaweeds. Philippine Journal of Science, vol. 147, no. 1, p. 47-55.
Chaturvedula, V. S. P., Prakash, I. 2011. The aroma, taste, color and bioactive constituents of tea. Journal of Medicinal Plants Research, vol. 5, no. 11, p. 2110-2124. https://doi.org/10.5897/JMPR.9001187
Chen, M. 2014. Chlorophyll modifications and their spectral extension in oxygenic photosynthesis. Annual Review of Biochemistry, vol. 83, p. 26.1-26.24. DOI: https://doi.org/10.1146/annurev-biochem-072711-162943
https://doi.org/10.1146/annurev-biochem-072711-162943 DOI: https://doi.org/10.1146/annurev-biochem-072711-162943
Chowdhury, A., Sarkar, S., Chowdhury, A., Bardhan, S., Mandal, P., Chowdhury, M. 2016. Tea waste management: A case study from West Bengal, India. Indian Journal of Science and Technology, vol. 9, no. 42, p. 1-6. https://doi.org/10.17485/ijst/2016/v9i42/89790 DOI: https://doi.org/10.17485/ijst/2016/v9i42/89790
Cieplik, F., Deng, D., Crielaard, W., Buchalla, W., Hellwig, E., Al-Ahmad, A., Maisch, T. 2018. Antimicrobial photodynamic therapy – what we know and what we don’t. Critical Reviews in Microbiology, vol. 44, no. 5, p. 571-589. https://doi.org/10.1080/1040841X.2018.1467876 DOI: https://doi.org/10.1080/1040841X.2018.1467876
Croft, H., Chen, J. M. 2017. Leaf pigment content. Reference Module in Earth Systems and Environmental Sciences. Oxford : Elsevier Inc., p. 1-26. https://doi.org/10.1016/B978-0-12- 409548-9.10547-0
Deb, S., Pou, K. R. J. 2016. A review of withering in the processing of black tea. Journal of Biosystems Engineering, vol. 41, no. 4, p. 365-372. https://doi.org/10.5307/JBE.2016.41.4.365 DOI: https://doi.org/10.5307/JBE.2016.41.4.365
Diby, L., Kahia, J., Kouamé, C., Aynekulu, E. 2017. Tea, coffee, and cocoa. Encyclopedia of Applied Plant Sciences, 2nd ed, vol. 3, p. 420-425. https://doi.org/10.1016/B978-0-12-394807-6.00179-9 DOI: https://doi.org/10.1016/B978-0-12-394807-6.00179-9
Donlao, N., Ogawa, Y. 2019. The influence of processing conditions on catechin, caffeine and chlorophyll contents of green tea (Camelia sinensis) leaves and infusions. LWT – Food Science and Technology, vol. 116, p. 1-8. https://doi.org/10.1016/j.lwt.2019.108567 DOI: https://doi.org/10.1016/j.lwt.2019.108567
Dubey, K. K., Janve, M., Ray, A., Singhal, R. S. 2020. Ready-to-drink tea. Trends in Non-alcoholic Beverages, Oxford : Elsevier Inc., p. 101-140. https://doi.org/10.1016/B978-0-12-816938-4.00004-5 DOI: https://doi.org/10.1016/B978-0-12-816938-4.00004-5
Fu, W., Magnúsdóttir, M., Brynjólfson, S., Palsson, B. O., Paglia, G. 2012. UPLC-UV-MSE analysis for quantification and identification of major carotenoid and chlorophyll species in algae. Analytical and Bioanalytical Chemistry, vol. 404, p. 3145-3154. https://doi.org/10.1007/s00216-012-6434-4 DOI: https://doi.org/10.1007/s00216-012-6434-4
Fyrestam, J., Bjurshammar, N., Paulsson, E., Johannsen, A., Östman, C. 2015. Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry. Analytical and Bioanalytical Chemistry, vol. 407, no. 23, p. 7013-7023. https://doi.org/10.1007/s00216-015-8864-2 DOI: https://doi.org/10.1007/s00216-015-8864-2
Gerola, A. P., Santana, A., França, P. B., Tsubone, T. M., Oliveira, H. P. M., Caetano, W., Kimura, E., Hioka, N. 2011. Effects of metal and the phytil chain on chlorophyll derivatives: Physicochemical evaluation for photodynamic inactivation of microorganisms. Photochemistry and Photobiology, vol. 87, p. 884-894. https://doi.org/10.1111/j.1751-1097.2011.00935.x DOI: https://doi.org/10.1111/j.1751-1097.2011.00935.x
Ghate, V. S., Ng, K. S., Zhou, W., Yang, H., Khoo, G. H., Yoon, W. B., Yuk, H. G. 2013. Antibacterial effect of light emitting diodes of visible wavelengths on selected foodborne pathogens at different illumination temperatures. International Journal of Food Microbiology, vol. 166, p. 399-406. https://doi.org/10.1016/j.ijfoodmicro.2013.07.018 DOI: https://doi.org/10.1016/j.ijfoodmicro.2013.07.018
Ghate, V. S., Zhou, W., Yuk, H. G. 2019. Perspectives and trends in the application of photodynamic inactivation for microbiological food safety. Comprehensive Reviews in Food Science and Food Safety, vol. 18, p. 402-424. https://doi.org/10.1111/1541-4337.12418 DOI: https://doi.org/10.1111/1541-4337.12418
Hoenes, K., Wenzel, U., Spellerberg, B., Hessling, M. 2020. Photoinactivation sensitivity of Staphylococcus carnosus to visible-light irradiation as a function of wavelength. Photochemistry and Photobiology, vol. 96, no. 1, p. 156-169. https://doi.org/10.1111/php.13168 DOI: https://doi.org/10.1111/php.13168
Hong, J. E., Lim, J. H., Kim, T. Y., Jang, H. Y., Oh, H. B.,
Chung, B. G., Lee, S. Y. 2020. Photo-oxidative protection of chlorophyll a in C-phycocyanin aqueous medium. Antioxidants, vol. 9, no. 1235, p. 1-13. https://doi.org/10.3390/antiox9121235 DOI: https://doi.org/10.3390/antiox9121235
Hussain, S., Anjali, K. P., Hassan, S. T., Dwivedi, P. B. 2018. Waste tea as a novel adsorbent: a review. Applied Water Science, vol. 8, no. 165, p. 1-16. DOI: https://doi.org/10.1007/s13201-018-0824-5
https://doi.org/10.1007/s13201-018-0824-5 DOI: https://doi.org/10.1007/s13201-018-0824-5
Indrawati, R., Ozols, M., Indriatmoko, Heriyanto, Brotosudarmo, T. H. P., Limantara, L. 2012. Re-evaluation on multi-chromatogram approach of 3D-chromatograpic-data. Journal of Biomaterial Chemistry, vol. 1, p. 12-16.
Indrawati, R., Heriyanto, Brotosudarmo, T. H. P., Limantara,
L. 2019. Distribution of chlorophylls and carotenoids in the different parts of thallus structure from three Sargassum spp. as revealed by multi-chromatograms HPLC approach. In Proceedings of the Indonesian Chemical Society. Malang, Indonesia : Indonesian Chemical Society, p. 5-9. https://doi.org/10.34311/pics.2019.01.1.5
Indrawati, R., Lolita, A. M., Limantara, L. 2021. Terapi fotodinamik antimikroba: Prospek baru dalam penanganan pangan? (Antimicrobial photodynamic therapy: A new prospect in food handling?). Jurnal Sains dan Terapan Kimia, vol. 15, no. 1, p. 74-90. (In Indonesian) https://doi.org/10.20527/jstk.v15i1.8771 DOI: https://doi.org/10.20527/jstk.v15i1.8771
Jeffrey, S. W., Mantoura, R. F. C., Wright, S. W. 1997. Phytoplankton pigments in oceanography: Guidelines to modern methods. Paris, France : UNESCO Publishing, 661 p. ISBN 92-3-103275-5.
Kabir, M. M., Mouna, S. S. P., Akter, S., Khandaker, S., Didar-ul-Alam, Md., Bahadur, N. M., Mohinuzzaman, M., Islam, Md. A., Shenashen, M. A. 2021. Tea waste based natural adsorbent for toxic pollutant removal from waste samples. Journal of Molecular Liquids, vol. 322, p. 1-16. https://doi.org/10.1016/j.molliq.2020.115012 DOI: https://doi.org/10.1016/j.molliq.2020.115012
Koca, N., Karadeniz, F., Burdurlu, H. S. 2006. Effect of pH on chlorophyll degradation and colour loss in blanched green peas. Food Chemistry, vol. 100, p. 609-615. https://doi.org/10.1016/j.foodchem.2005.09.079 DOI: https://doi.org/10.1016/j.foodchem.2005.09.079
Küpper, H., Spiller, M., Küpper, F. C. 2000. Photometric method for the quantification of chlorophylls and their derivatives in complex mixtures: Fitting with Gauss- peak spectra. Analytical Biochemistry, vol. 286, no. 2, p. 247-256. https://doi.org/10.1006/abio.2000.4794 DOI: https://doi.org/10.1006/abio.2000.4794
Kustov, A. V., Belykh, D. V., Smirnova, N. L., Venediktov,
E. A., Kudayarova, T. V., Kruchin, S. O., Khudyaeva, I. S., Berezin, D. B. 2018. Synthesis and investigation of water- soluble chlorophyll pigments for antimicrobial photodynamic therapy. Dyes and Pigments, vol. 149, p. 553-559. https://doi.org/10.1016/j.dyepig.2017.09.073 DOI: https://doi.org/10.1016/j.dyepig.2017.09.073
Lefebvre, T., Talbi, A., Atwi-Ghaddar, S., Destandau, E., Lesellier, E. 2020. Development of an analytical method for chlorophyll pihments separation by reversed-phase supercritical fluid chromatography. Journal of Chromatography A, vol. 1612, p. 1-8. https://doi.org/10.1016/j.chroma.2019.460643 DOI: https://doi.org/10.1016/j.chroma.2019.460643
Lichtenthaler, H. K. 1987. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods in Enzymology, vol. 148, p. 350-382. https://doi.org/10.1016/0076-6879(87)48036-1 DOI: https://doi.org/10.1016/0076-6879(87)48036-1
Mesquita, M. Q., Dias, C. J., Neves, M. G. P. M. S., Almeida, A., Faustino, M. A. F. 2018. Revisiting current photoactive materials for antimicrobial photodynamic therapy. Molecules, vol. 23, no. 10, p. 1-47. https://doi.org/10.3390/molecules23102424 DOI: https://doi.org/10.3390/molecules23102424
Oktavia, L., Mulyani, I., Suendo, V. 2021. Investigation of chlorophyll-a derived compounds as photosensitizer for photodynamic inactivation. Bulletin of Chemical Reaction Engineering and Catalysis, vol. 16, no. 1, p. 161-169. https://doi.org/10.9767/bcrec.16.1.10314.161-169 DOI: https://doi.org/10.9767/bcrec.16.1.10314.161-169
Pareek, S., Sagar, N. A., Sharma, S., Kumar, V., Agarwal, T., Gonzales-Aguilar, G. A., Yahia, E. M. 2017. Chlorophylls: Chemistry and biological functions. In Yahia, E. M. Fruit and vegetable phytochemicals: Chemistry and human health. 2nd ed. Chichester, UK : John Wiley and Sons, Ltd., p. 269-284. ISBN 9781119158042. https://doi.org/10.1002/9781119158042.ch14 DOI: https://doi.org/10.1002/9781119158042.ch14
Pou, K. R. J., Paul, S. K., Malakar, S. 2019. Industrial processing of CTC black tea. In Grumezescu, A. M., Holban, A. M. Caffeinated and cocoa based beverages. Vol. 8. Duxford, UK : Woodhead Publishing, p. 131-162. ISBN 978- 0-12-815865-4. DOI: https://doi.org/10.1016/B978-0-12-815864-7.00004-0
Prasad, A., Du, L., Zubair, M., Subedi, S., Ullah, A., Roopesh, M. S. 2020. Applications of light-emitting diodes (LEDs) in food processing and water treatment. Food Engineering Reviews, vol. 12, p. 268-289. https://doi.org/10.1007/s12393-020-09221-4 DOI: https://doi.org/10.1007/s12393-020-09221-4
Purushothaman, T., Mol, K. I. 2021. A critical review on antimicrobial photodynamic inactivation using light emitting diode (LED). International Journal of Arts, Science and Humanities, vol. 8, no. 3, p. 124-130. https://doi.org/10.34293/sijash.v8i3.3476 DOI: https://doi.org/10.34293/sijash.v8i3.3476
Raish, M., Husain, S. Z. A., Bae, S. M., Han, S. J., Park, C. H., Shin, J. C. 2010. Photodynamic therapy in combination with green tea polyphenol EGCG enhances antitumor efficacy in human papillomavirus 16 (E6/E7) immortalized tumor cells. Journal of Applied Research, vol. 10, no. 2, p. 58-67.
Rodrigues, V. C., Silva, M. V., Santos, A. R., Zielinski, A.
A. F., Haminiuk, C. W. I. 2015. Evaluation of hot and cold extraction of bioactive compounds in teas. International Journal of Food Science and Technology, vol. 50, p. 2038- 2045. https://doi.org/10.1111/ijfs.12858 DOI: https://doi.org/10.1111/ijfs.12858
Roshanak, S., Rahimmalek, M., Goli, S. A. H. 2016. Evaluation of seven different drying treatments in respect to total flavonoid, phenolic, vitamin C content, chlorophyll, antioxidant activity and color of green tea (Camellia sinensis or C. assamica) leaves. Journal of Food Science and Technology, vol. 53, no. 1, p. 721-729. https://doi.org/10.1007/s13197-015-2030-x DOI: https://doi.org/10.1007/s13197-015-2030-x
Safdar, N., Sarfaraz, A., Kazmi, Z., Yasmin, A. 2016. Ten different brewing methods of green tea: comparative antioxidant study. Journal of Applied Biology and Biotechnology, vol. 4, no. 3, p. 33-40. https://doi.org/10.7324/JABB.2016.40306 DOI: https://doi.org/10.7324/JABB.2016.40306
Samide, A., Tutunaru, B. 2017. Thermal behavior of the chlorophyll extract from a mixture of plants and seaweed. Journal of Thermal Analysis and Calorimetry, vol. 127, p. 597- 604. https://doi.org/10.1007/s10973-016-5490-y DOI: https://doi.org/10.1007/s10973-016-5490-y
Sato, T., Shimoda, Y., Matsuda, K., Tanaka, A., Ito, H. 2018. Mg-dechelation of chlorophyll a by Stay-Green activates chlorophyll b degradation through expressing Non-Yellow Coloring 1 in Arabidopsis thaliana. Journal of Plant Physiology, vol. 222, p. 94-102. DOI: https://doi.org/10.1016/j.jplph.2018.01.010
https://doi.org/10.1016/j.jplph.2018.01.010 DOI: https://doi.org/10.1016/j.jplph.2018.01.010
Seifert, B., Pflanz, M., Zude, M. 2014. Spectral shift as advanced index for fruit chlorophyll breakdown. Food and Bioprocess Technology, vol. 7, p. 2050-2059. https://doi.org/10.1007/s11947-013-1218-1 DOI: https://doi.org/10.1007/s11947-013-1218-1
Senapathy, G. J., George, B. P., Abrahamse, H. 2020. Enhancement of phthalocyanine mediated photodynamic therapy by catechin on lung cancer cells. Molecules, vol. 25, p. 1-13. https://doi.org/10.3390/molecules25214874 DOI: https://doi.org/10.3390/molecules25214874
Son, M., Pinnola, A., Bassi, R., Schlau-Cohen, G. S. 2019. The electronic structure of Lutein 2 is optimized for light harvesting in plants. Chem., vol. 5, no. 3, p. 575-584. https://doi.org/10.1016/j.chempr.2018.12.016 DOI: https://doi.org/10.1016/j.chempr.2018.12.016
Stinco, C. M., Benítez-González, A. M., Meléndez-Martínez,
A. J., Hernanz, D., Vicario, I. M. 2019. Simultaneous determination of dietary isoprenoids (carotenoids, chlorophylls and tocopherols) in human faeces by Rapid Resolution Liquid Chromatography. Journal of Chromatography A, vol. 1583, p. 63-72. https://doi.org/10.1016/j.chroma.2018.11.010 DOI: https://doi.org/10.1016/j.chroma.2018.11.010
Suzuki, Y., Shioi, Y. 2003. Identification of chlorophylls and carotenoids in major teas by high-performance liquid chromatography with photodiode array detection. Journal of Agricultural and Food Chemistry, vol. 51, p. 5307-5314. https://doi.org/10.1021/jf030158d DOI: https://doi.org/10.1021/jf030158d
Suzuki, K., Kamimura, A., Hooker, S. B. 2015. Rapid and highly sensitive analysis of chlorophylls and carotenoids from marine phytoplankton using ultra-high performance liquid chromatography (UHPLC) with the first derivative spectrum chromatogram (FDSC) technique. Marine Chemistry, vol. 176, p. 96-109. https://doi.org/10.1016/j.marchem.2015.07.010 DOI: https://doi.org/10.1016/j.marchem.2015.07.010
Wei, Y., Fang, S., Jin, G., Ni, T., Hou, Z., Li, T., Deng, W. W., Ning, J. 2020. Effects of two yellowing process on colour, taste, and nonvolatile compounds of bud yellow tea. International Journal of Food Science and Technology, vol. 55, no. 8, p. 2931-2941. https://doi.org/10.1111/IJFS.14554 DOI: https://doi.org/10.1111/ijfs.14554
Wei, Y., Li, T., Xu, S., Ni, T., Deng, W. W., Ning, J. 2021.
The profile of dynamic changes in yellow tea quality and chemical composition during yellowing process. LWT – Food Science and Technology, vol. 139, p. 1-11. https://doi.org/10.1016/j.lwt.2020.110792 DOI: https://doi.org/10.1016/j.lwt.2020.110792
Wijaya, W., Heriyanto, Prasetyo, B., Limantara, L. 2010. Determination of chlorophylls and carotenoids content in three major teas based on peak area from HPLC chromatogram. In 38th Meeting of National Working Group on Indonesian Medicinal Plant and German Academic Exchange Service : Proceeding of International Conference on Medicinal Plants. Surabaya, Indonesia : Widya Mandala Catholic University, p. 103-111. ISBN 978-602-96839-1-2.
Wojdyło, A., Nowicka, P., Tkacz, K., Turkiewicz, I. P. 2021. Fruit tree leaves as unconventional and valuable source of chlorophyll and carotenoid compounds determined by liquid chromatography-photodiode-quadrupole/time of flight- electrospray ionization-mass spectrometry (LC-PDA-qTof- ESI-MS). Food Chemistry, vol. 349, p. 1-12. https://doi.org/10.1016/j.foodchem.2021.129156 DOI: https://doi.org/10.1016/j.foodchem.2021.129156
Yu, X., Hu, S., He, C., Zhou, J., Qu, F., Ai, Z., Chen, Y., Ni,
D. 2019. Chlorophyll metabolism in postharvest tea (Camellia sinensis L.) leaves: variations in color values, chlorophyll derivatives, and gene expression levels under different withering treatments. Journal of Agricultural and Food Chemistry, vol. 67, p. 10624-10636. https://doi.org/10.1021/acs.jafc.9b03477 DOI: https://doi.org/10.1021/acs.jafc.9b03477
Zapata, M., Rodriguez, F., Garrido, J. L. 2000. Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases. Marine Ecology Progress Series, vol. 195, p. 29-45. https://doi.org/10.3354/meps195029 DOI: https://doi.org/10.3354/meps195029
Zepka, L. Q., Jacob-Lopes, E., Roca, M. 2019. Catabolism and bioactive properties of chlorophylls. Current Opinion in Food Science, vol. 26, p. 94-100. https://doi.org/10.1016/j.cofs.2019.04.004 DOI: https://doi.org/10.1016/j.cofs.2019.04.004
Zhang, G., Yang, J., Hu, C., Zhang, X., Li, X., Gao, S., Ouyang, X., Ma, N., Wei, H. 2019. Green synthesis of Chlorin e6 and tests of its photosensitive bactericidal activities. Journal of Forestry Research, vol. 30, p. 2349-2356. https://doi.org/10.1007/s11676-018-0756-9 DOI: https://doi.org/10.1007/s11676-018-0756-9
Zhang, Y., Gao, W., Cui, C., Zhang, Z., He, L., Zheng, J., Hou, R. 2020. Development of a method to evaluate the tenderness of fresh tea leaves based on rapid, in-situ Raman spectroscopy scanning for carotenoids. Food Chemistry, vol. 308, p. 1-8. https://doi.org/10.1016/j.foodchem.2019.125648 DOI: https://doi.org/10.1016/j.foodchem.2019.125648
Zhou, R., Li, X., He, Y. 2017. Grading of green tea and quantitative determination of beta-carotene and lutein based on hyperspectral imaging. In 2017 ASABE Annual International Meeting. Washington, USA : American Society of Agricultural and Biological Engineers, p. 1-9. https://doi.org/10.13031/aim.201700625 DOI: https://doi.org/10.13031/aim.201700625
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