Studies on the extraction and characterization of pectin and bitter principles from New Zealand grapefruit and Philippine calamansi : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University

A study was conducted to determine the presence of bitter components in NZ grapefruit and Philippine calamansi; describe the effect of maturity on the bitter components and other chemical constituents of grapefruit; reduce the bitterness of grapefruit juice by adsorption on polyvinylpyrrolidone; and to extract and characterize pectin from grapefruit peel. Naringin (995 PPm), narirutin (187 ppm), and limonoids (7.9 ppm) were detected in NZ grapefruit juice concentrate (27° Brix). Naringin was not detected in the calamansi juice, and limonin was detected at the level of 10.5 ppm in juice containing 5% crushed seeds. Maturation of the grapefruit caused an increase in pH from 3.00 to 3.50, an increase in total soluble solids from 10.8 to 14.4 with a decline to 13.5° Brix later in the season, a steady fall in acidity from 2.50 to 1.31 g citric acid/100 mL, and a continuous rise in the Brix/acid ratio from 4.2 to 10.3. Juice yield fluctuated throughout the season. Ascorbic acid remained fairly steady in the early and mid-season fruit but decreased in the late-season fruit. Naringin content was highest at the beginning of the season and fluctuated throughout the season. Naringin content in the grapefruit peel remained constant as the fruit matured. Narirutin was detected in the early-season fruit but disappeared later in the season. Limonoid content in both unpasteurized and pasteurized juices decreased with ripening. The use of polyvinylpyrrolidone significantly reduced naringin in grapefruit juice by up to 78.1% and limonin by up to 17.5% depending on the amount and reaction time of the adsorbent. A loss of 23.1% in ascorbic acid occurred with 5% PVP with a reaction time of 1 h. Pectin extraction at 85°C and the use of acidified isopropyl alcohol yielded a product with the following characteristics: 8.9% yield; 1.3% moisture content; 1.9% ash; 759 equivalent weight; 9.2% methoxyl content; 82.2% anhydrogalacturonic acid; 63.2% degree of esterification; 4.2 intrinsic viscosity; 89,362 molecular weight and setting time of 0.55 minute

Optimizing culture medium for debittering constitutive enzyme naringinase production by Aspergillus oryzae JMU316

The objective of this study was to investigate nutrient requirements for extracellular constitutive naringinase production by Aspergillus oryzae JMU316. The one-factor-at-a-time method was used to determine the impact of different carbon and nitrogen sources on naringinase production. Naringin exhibited the highest naringinase activity compared to all other carbon sources and pomelo pericarp powder produced comparable naringinase activity. Pomelo pericarp powder was selected as carbon source because it is a waste of fruit process, which means that it is a cheap resource and has additional environmental benefits. Peptone proved to be the most suitable nitrogen source for naringinase production. Subsequently, the orthogonal matrix method was used to further optimize the concentration of pomelo pericarp powder, peptone, and minerals. The optimal concentration of the components were15 g pomelo pericarp powder, 12 g peptone, 0.2 g CaCl2, 0.4 g NaCl, 2 g MgSO4·7H2O and 1 g K2HPO4 in 1 L distilled water for producing 408.28 IU/mL naringinase activity. The effects of medium components on naringinase were in the order of pomelo pericarp powder, K2HPO4, NaCl, peptone, CaCl2, MgSO4·7H2O. This two-step optimization strategy used in this study can be widely applied to other microbial fermentation processes.Key words: Pomelo pericarp powder, orthogonal matrix method, naringinase, culture medium optimization, Aspergillus oryzae JMU316

Covalent Immobilization of Naringinase over Two-Dimensional 2D Zeolites and its Applications in a Continuous Process to Produce Citrus Flavonoids and for Debittering of Juices

This is the peer reviewed version of the following article: J. M. Carceller, J. P. Martínez Galán, R. Monti, J. C. Bassan, M. Filice, J. Yu, M. J. Climent, S. Iborra, A. Corma, ChemCatChem 2020, 12, 4502, which has been published in final form at https://doi.org/10.1002/cctc.202000320. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] The crude naringinase from Penicillium decumbens and a purified naringinase with high a-L-rhamnosidase activity could be covalently immobilized on two-dimensional zeolite ITQ-2 after surface modification with glutaraldehyde. The influence of pH and temp. on the enzyme activity (in free and immobilized forms) as well as the thermal stability were detd. using the specific substrate: p-nitrophenyl-alpha-L-rhamnopyranoside (Rha-pNP). The crude and purified naringinase supported on ITQ-2 were applied in the hydrolysis of naringin, giving the flavonoids naringenin and prunin resp. with a conversion >90% and excellent selectivity. The supported enzymes showed long term stability, being possible to perform up to 25 consecutive cycles without loss of activity, showing its high potential to produce the valuable citrus flavonoids prunin and naringenin. We have also succeeded in the application of the immobilized crude naringinase on ITQ-2 for debittering grapefruit juices in a continuous process that was maintained operating for 300 h, with excellent results.The authors acknowledge financial support from PGC2018-097277-B-100 (MCIU/AEI/FEDER,UE) project and Program Severo Ochoa (SEV-2016-0683). Jilin agreement 111 Project (Grant No. B17020). JMC thanks to Universitat Politecnica de Valencia for predoctoral fellowships.Carceller-Carceller, JM.; Martínez Galán, JP.; Monti, R.; Bassan, JC.; Filice, M.; Yu, J.; Climent Olmedo, MJ.... (2020). Covalent Immobilization of Naringinase over Two-Dimensional 2D Zeolites and its Applications in a Continuous Process to Produce Citrus Flavonoids and for Debittering of Juices. ChemCatChem. 12(18):4502-4511. https://doi.org/10.1002/cctc.202000320S450245111218Puri, M., & Banerjee, U. C. (2000). Production, purification, and characterization of the debittering enzyme naringinase. Biotechnology Advances, 18(3), 207-217. doi:10.1016/s0734-9750(00)00034-3Vila-Real, H., Alfaia, A. J., Rosa, M. E., Calado, A. R., & Ribeiro, M. H. L. (2010). An innovative sol–gel naringinase bioencapsulation process for glycosides hydrolysis. Process Biochemistry, 45(6), 841-850. doi:10.1016/j.procbio.2010.02.004RoitNer, M., Schalkhammer, T., & Pittner, F. (1984). Preparation of prunin with the help of immobilized naringinase pretreated with alkaline buffer. Applied Biochemistry and Biotechnology, 9(5-6), 483-488. doi:10.1007/bf02798402Ribeiro, I. A., Rocha, J., Sepodes, B., Mota-Filipe, H., & Ribeiro, M. H. (2008). Effect of naringin enzymatic hydrolysis towards naringenin on the anti-inflammatory activity of both compounds. Journal of Molecular Catalysis B: Enzymatic, 52-53, 13-18. doi:10.1016/j.molcatb.2007.10.011Puri, M., Marwaha, S. S., Kothari, R. M., & Kennedy, J. F. (1996). Biochemical Basis of Bitterness in Citrus Fruit Juices and Biotech Approaches for Debittering. Critical Reviews in Biotechnology, 16(2), 145-155. doi:10.3109/07388559609147419Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Rodrigues, R. C., & Fernandez-Lafuente, R. (2015). Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts. Biotechnology Advances, 33(5), 435-456. doi:10.1016/j.biotechadv.2015.03.006Garcia-Galan, C., Berenguer-Murcia, Á., Fernandez-Lafuente, R., & Rodrigues, R. C. (2011). Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance. Advanced Synthesis & Catalysis, 353(16), 2885-2904. doi:10.1002/adsc.201100534ONO, M., TOSA, T., & CHIBATA, I. (1978). Preparation and properties of immobilized naringinase using tannin-aminohexyl cellulose. Agricultural and Biological Chemistry, 42(10), 1847-1853. doi:10.1271/bbb1961.42.1847Tsen, H.-Y., & Tsai, S.-Y. (1988). Comparison of the kinetics and factors affecting the stabilities of chitin-immobilized naringinases from two fungal sources. Journal of Fermentation Technology, 66(2), 193-198. doi:10.1016/0385-6380(88)90047-7SOARES, N. F. F., & HOTCHKISS, J. H. (1998). Naringinase Immobilization in Packaging Films for Reducing Naringin Concentration in Grapefruit Juice. Journal of Food Science, 63(1), 61-65. doi:10.1111/j.1365-2621.1998.tb15676.xPuri, M., Kaur, H., & Kennedy, J. F. (2005). Covalent immobilization of naringinase for the transformation of a flavonoid. Journal of Chemical Technology & Biotechnology, 80(10), 1160-1165. doi:10.1002/jctb.1303Lei, S., Xu, Y., Fan, G., Xiao, M., & Pan, S. (2011). Immobilization of naringinase on mesoporous molecular sieve MCM-41 and its application to debittering of white grapefruit. Applied Surface Science, 257(9), 4096-4099. doi:10.1016/j.apsusc.2010.12.003Luo, J., Li, Q., Sun, X., Tian, J., Fei, X., Shi, F., … Liu, X. (2019). The study of the characteristics and hydrolysis properties of naringinase immobilized by porous silica material. RSC Advances, 9(8), 4514-4520. doi:10.1039/c9ra00075eNunes, M. A. P., Vila-Real, H., Fernandes, P. C. B., & Ribeiro, M. H. L. (2009). Immobilization of Naringinase in PVA–Alginate Matrix Using an Innovative Technique. Applied Biochemistry and Biotechnology, 160(7), 2129-2147. doi:10.1007/s12010-009-8733-6Busto, M. D., Meza, V., Ortega, N., & Perez-Mateos, M. (2007). Immobilization of naringinase from Aspergillus niger CECT 2088 in poly(vinyl alcohol) cryogels for the debittering of juices. Food Chemistry, 104(3), 1177-1182. doi:10.1016/j.foodchem.2007.01.033Huang, W., Zhan, Y., Shi, X., Chen, J., Deng, H., & Du, Y. (2017). Controllable immobilization of naringinase on electrospun cellulose acetate nanofibers and their application to juice debittering. International Journal of Biological Macromolecules, 98, 630-636. doi:10.1016/j.ijbiomac.2017.02.018Gong, X., Xie, W., Wang, B., Gu, L., Wang, F., Ren, X., … Yang, L. (2017). Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage. Scientific Reports, 7(1). doi:10.1038/s41598-017-17172-wCarceller, J. M., Martínez Galán, J. P., Monti, R., Bassan, J. C., Filice, M., Iborra, S., … Corma, A. (2019). Selective synthesis of citrus flavonoids prunin and naringenin using heterogeneized biocatalyst on graphene oxide. Green Chemistry, 21(4), 839-849. doi:10.1039/c8gc03661fPuri, M., Marwaha, S. S., & Kothari, R. M. (1996). Studies on the applicability of alginate-entrapped naringiase for the debittering of kinnow juice. Enzyme and Microbial Technology, 18(4), 281-285. doi:10.1016/0141-0229(95)00100-xNorouzian, D., Hosseinzadeh, A., Inanlou, D. N., & Moazami, N. (1999). World Journal of Microbiology and Biotechnology, 15(4), 501-502. doi:10.1023/a:1008980018481Saallah, S., Naim, M. N., Lenggoro, I. W., Mokhtar, M. N., Abu Bakar, N. F., & Gen, M. (2016). Immobilisation of cyclodextrin glucanotransferase into polyvinyl alcohol (PVA) nanofibres via electrospinning. Biotechnology Reports, 10, 44-48. doi:10.1016/j.btre.2016.03.003Cipolatti, E. P., Valério, A., Henriques, R. O., Moritz, D. E., Ninow, J. L., Freire, D. M. G., … de Oliveira, D. (2016). Nanomaterials for biocatalyst immobilization – state of the art and future trends. RSC Advances, 6(106), 104675-104692. doi:10.1039/c6ra22047aCorma, A., Fornes, V., & Rey, F. (2002). Delaminated Zeolites: An Efficient Support for Enzymes. Advanced Materials, 14(1), 71-74. doi:10.1002/1521-4095(20020104)14:13.0.co;2-wGallego, E. M., Portilla, M. T., Paris, C., León-Escamilla, A., Boronat, M., Moliner, M., & Corma, A. (2017). «Ab initio» synthesis of zeolites for preestablished catalytic reactions. Science, 355(6329), 1051-1054. doi:10.1126/science.aal0121Margarit, V. J., Díaz-Rey, M. R., Navarro, M. T., Martínez, C., & Corma, A. (2018). Direct Synthesis of Nano-Ferrierite along the 10-Ring-Channel Direction Boosts Their Catalytic Behavior. Angewandte Chemie, 130(13), 3517-3521. doi:10.1002/ange.201711418Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Rodrigues, R. C., & Fernandez-Lafuente, R. (2014). Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization. RSC Adv., 4(4), 1583-1600. doi:10.1039/c3ra45991hSmith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., … Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150(1), 76-85. doi:10.1016/0003-2697(85)90442-7Marolewski, A. (1996). Fundamentals of Enzyme Kinetics. Revised Edition By Athel Cornish-Bowden. Portland Press, London. 1995. xiii + 343 pp. 17.5 cm × 24.5 cm. ISBN 1-85578-072-0. $29.00. Journal of Medicinal Chemistry, 39(4), 1010-1011. doi:10.1021/jm9508447Miller, G. L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31(3), 426-428. doi:10.1021/ac60147a030Cheong, M. W., Liu, S. Q., Zhou, W., Curran, P., & Yu, B. (2012). Chemical composition and sensory profile of pomelo (Citrus grandis (L.) Osbeck) juice. 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Procjena utjecaja visokoga tlaka na hidrolizu naringina u soku grejpfruta pomoću naringinaze imobilizirane u kapsulama kalcijeva alginata

The reduction of bitterness in citrus juices would increase their acceptance by the consumer. This reduction in grapefruit juices can be achieved as a result of an enzymatic process, with improved commercial value and maintenance of health properties. The use of a cheap, simple and effective immobilisation method combined with high pressure can be a key asset in the debittering of citrus juices. The aim of this study is the debittering of grapefruit juice under high pressure, with naringinase immobilized in calcium alginate beads. Naringinase, an α-rhamnopyranosidase, hydrolyzes naringin (a flavanone glycoside and primary bitter component in grapefruit juice) to naringenin, which is tasteless. High pressure can activate or inhibit enzymatic activities depending on the proteins and conditions. The hydrolysis of naringin was first evaluated in model solution (acetate buffer 0.02 M, pH=4.0) and then in grapefruit juice. In model solution, at 160 MPa and 37 °C, a 50 % increase in the concentration of reducing sugars was obtained when compared to the reaction at atmospheric pressure. The higher naringenin concentration (33 mg/L) was obtained at 54 °C under high pressure of 200 MPa, which corresponds to a naringin reduction of 72 % in model solution, while at atmospheric pressure (0.1 MPa), the naringin reduction was only 35 %. The decrease in naringin content can be directly correlated with the reduction in bitterness. From the concentration of residual naringin, the percentage of reduction in bitterness was evaluated. In grapefruit juice, a debittering of 75 % occurred with a pressure of 160 MPa at 37 °C for 20 minutes.Smanjenjem gorčine sokova citrusa povećala bi se njihova prihvatljivost za potrošače. Gorčina soka grejpfruta može se smanjiti enzimskim procesom koji poboljšava komercijalnu vrijednost soka, a ne smanjuje njegov pozitivan utjecaj na zdravlje. Uporaba jeftinih, jednostavnih i djelotvornih metoda imobilizacije, kombiniranih uz primjenu visokoga tlaka, ključni su za odgorčavanje sokova citrusa. Svrha je ovog istraživanja smanjiti gorčinu soka grejpfruta primjenom visokoga tlaka i naringinaze imobilizirane u kapsulama kalcijeva alginata. Naringinaza, tj. α-ramnopiranozidaza, hidrolizira naringin (flavanon glikozida što je osnovna gorka komponenta soka grejpfruta) u naringenin koji nema okus. Visoki tlak može aktivirati ili inhibirati aktivnost enzima ovisno o sastavu proteina i uvjetima hidrolize. U radu je najprije istražena hidroliza naringina u modelnoj otopini (acetatni pufer 0,02 M; pH=4,0), a zatim u soku grejpfruta. U modelnoj je otopini pri 160 MPa i 37 °C postignuto 50 %-tno povećanje koncentracije reducirajućih šećera, u usporedbi s reakcijom pri atmosferskom tlaku. Veća koncentracija naringenina (33 mg/L) postignuta je pri 54 °C i visokom tlaku od 200 MPa, što odgovara 72 %-tnoj redukciji narinigina u modelnoj otopini, dok je pri atmosferskom tlaku (0,1 MPa) redukcija naringina iznosila samo 35 %. Smanjenje koncentracije naringina može se povezati sa smanjenjem gorčine soka, tako da je postotak smanjenja procijenjen prema koncentraciji preostalog naringina. U soku grejpfruta smanjenje gorčine od 75 % postignuto je pri tlaku od 160 MPa, na 37 °C, tijekom 20 minuta

New fungal sources for α-L-Rhamnosidase: an important enzyme used in the synthesis of drugs and drug precursors

Two fungal strains were isolated and tentatively identified as Penicillium VY and Aspergillus VY. All the isolated species show the maximum production on third day in a liquid culture media. The pH optimum was found to be 10.0 for Penicillium VY and 11.0 for Aspergillus VY. The temperature optima were 50ºC in both the cases. The enzyme produced by Penicillium VY was found to be stable in the pH range 3.0-7.0 and 3.0–6.0 in case of Aspergillus VY. The enzyme does not loose activity up to 40º C in case of Penicillium VY and 40ºC in case of Aspergillus VY if exposed for 1 h.
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Purification and characterization of Aspergillus niger α-L-rhamnosidase for the biotransformation of naringin to prunin

This study was conducted to increase the bioactivity of litchi pericarp polysaccharides (LPPs) biotransformed by Aspergillus awamori. Comparedtothenon-A. awamori-fermented LPP, the growth effects of A. awamori-fermented LPP on Lactobacillus bulgaricus and Streptococcus thermophilus were four and two times higher after 3 days of fermentation, respectively. Increased 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and DNA protection activity of litchi pericarp polysaccharides were also achieved after A. awamori fermentation. Moreover, the relative content of glucose and arabinose in LPP after fermentation decreased from 58.82% to 22.60% and from 18.82% to 10.09%, respectively, with a concomitant increase in the relative contents of galactose, rhamnose, xylose, and mannose. Furthermore, lower molecular weight polysaccharides were obtained after A. awamori fermentation. It can be concluded that A. awamori was effective in biotransforming LPP into a bioactive mixture with lower molecular weight polysaccharides and higher antioxidant activity and relative galactose content

Comparative studies on inducers in the production of naringinase from Aspergillus niger MTCC 1344

This research provides detailed systematic study of the effect of different inducers (hesperidin, naringenin, naringin, rhamnose and rutin) in naringinase production by Aspergillus niger MTCC 1344. Cultures were carried out in shake flasks and they produce extracellular naringinase in a complex (molasses, peptone and salts) medium. The optimized concentration (%) of naringin, rhamnose, naringenin, rutin and hesperidin for   maximized naringinase production are 0.1, 0.375, 0.01, 0.2 and 0.2, respectively. Compared with control,  inducers increased the naringinase production by many folds in the order of naringin (6.63) > rhamnose (4.87) > naringenin (3.26) > rutin (2.84) > hesperidin (2.35). Under optimum conditions, about 9.68 units of enzyme per ml complex medium containing naringin were obtained on the 7th day. The activity to inducer (A/I) ratio was 968 Ug-1 naringin, and the cultivation time was shorter in submerged production. The results indicate that naringinase activity used naringin as an inducer which was significantly higher than the other four inducers. Therefore naringin is recommended for naringinase production.Key words: Naringin, naringenin, rutin, hesperidin, rhamnose, naringinase, Aspergillus, inducer, molasses

Immobilized enzyme technology for debittering citrus fruit juices

There has been increased interest in the use of immobilized enzymes in fruit juice industry for debittering of citrus fruit juices due to their high efficiency to remove bitter flavonoids. The structure of naringin, responsible for immediate bitterness, and of limonin, responsible for &quot;delayed bitterness&quot; has been discussed. This chapter also discusses various attempts that have been made to immobilize enzymes on an appropriate support so as to enable their use in debittering of citrus fruit juices. These include physicochemical and enzyme biotechnological approaches which makes the fruit juice more acceptable and cost effective to the consumer. Despite of high volume of production of citrus fruits and fruit juices, suitable processes to produce non-bitter citrus juice by immobilized enzymes technology has not yet commercialized globally.<br /

Changes in Bitterness, Antioxidant Activity and Total Phenolic Content of Grapefruit Juice Fermented by Lactobacillus and Bifidobacterium Strains

Four strains of Lactobacillus and Bifidobacterium including L. plantarum 01, L. fermentum D13, L. rhamnosus B01725, and B. bifidum B7.5 exhibiting naringinase production were applied in grapefruit juice fermentation. All investigated strains grew well in grapefruit juice without nutrition supplementation. In all cases, cell counts were 108–109 CFU ml−1 after 24 hours of fermentation. The highest lactic acid and acetic acid productions were observed in the case of strain L. plantarum 01. The L. plantarum 01 and L. fermentum D13 strains prefer glucose over fructose and sucrose, whereas fructose was the most favoured sugar for L. rhamnosus B01725 and B. bifidum B7.5. At the end of the fermentation process, antioxidant activity and total polyphenol content of grapefruit juice decreased in all cases, but the changes were not significant. Significant decrease of naringin was observed in the case of L. plantarum 01, 28% naringin in grapefruit juice was removed after fermentation. This result is promising for development of technology for production of probiotic grapefruit juice

Selective synthesis of citrus flavonoids prunin and naringenin using heterogeneized biocatalyst on graphene oxide

[EN] Production of citrus flavonoids prunin and naringenin was performed selectively through the enzyme hydrolysis of naringin, a flavonoid glycoside abundant in grapefruit wastes. To produce the monoglycoside flavonoid, prunin, crude naringinase from Penicillium decumbens was purified by a single purification step resulting in an enzyme with high -rhamnosidase activity. Both crude and purified enzymes were covalently immobilized on graphene oxide. The activity of the immobilized enzymes at different pH levels and temperatures, and the thermal stability were determined and compared with those exhibited by the free naringinases using specific substrates: p-nitrophenyl--d-glucoside (Glc-pNP) and p-nitrophenyl-alpha-l-rhamnopyranoside (Rha-pNP). The crude and purified naringinase supported on GO were tested in the hydrolysis of naringin, giving naringenin and prunin, respectively, in excellent yields. The supported enzymes can be reused many times without loss of activity. The naringinase stabilized on GO has high potential to produce the valuable citrus flavonoids prunin and naringenin.Authors acknowledge the financial support from MICINN Project CTQ-2015-67592-P and Program Severo Ochoa (SEV-2016-0683). JVC thanks Universitat Politecnica de Valencia for predoctoral fellowships. JY and AC thank the support from the National Natural Science Foundation of China (Grant No. 21320102001) and the 111 Project (Grant No. B17020).Carceller-Carceller, JM.; Martínez Galán, JP.; Monti, R.; Bassan, JC.; Filice, M.; Iborra Chornet, S.; Yu, J.... (2019). Selective synthesis of citrus flavonoids prunin and naringenin using heterogeneized biocatalyst on graphene oxide. Green Chemistry. 21(4):839-849. https://doi.org/10.1039/c8gc03661fS839849214Puri, M., & Banerjee, U. C. (2000). Production, purification, and characterization of the debittering enzyme naringinase. Biotechnology Advances, 18(3), 207-217. doi:10.1016/s0734-9750(00)00034-3Vila-Real, H., Alfaia, A. 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