Spontaneously Assembled Nano-aggregates in Clear Green Tea

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Spontaneously-assembled Nano-aggregates in Clear Green Tea Infusions from Camellia ptilophylla and Camellia sinensis Xiaorong Lin, Xiong Gao, Zhongzheng Chen, Yuanyuan Zhang, Wei Luo, Xiaofei Li, and Bin Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00068 • Publication Date (Web): 15 Apr 2017 Downloaded from http://pubs.acs.org on April 16, 2017

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Journal of Agricultural and Food Chemistry

Spontaneously-assembled Nano-aggregates in Clear Green Tea Infusions from Camellia ptilophylla and Camellia sinensis

Xiaorong Lin, Xiong Gao, Zhongzheng Chen, Yuanyuan Zhang, Wei Luo, Xiaofei Li, Bin Li *

College of Food Science, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou 510642, People’s Republic of China *Corresponding Author: Phone: +86 20 8528 1029. Fax: +86 20 8528 1029. Email: [email protected]

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ABSTRACT

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Tea nano-aggregates spontaneously assembled in clear tea infusions are considered as the precursors

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of tea cream, though their molecular basis remains obscure. Here, we characterized nano-aggregates

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in green tea infusions from Camellia ptilophylla, a peculiar tea variety with 6.0% of theobromine,

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and Camellia sinensis as the control for comparative purpose. Numerous negatively-charged

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spherical colloidal particles of 50-100 nm in diameter were primarily found in both green tea

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infusions. Catechins, proteins and carbohydrates were confirmed as the dominant components in

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green tea nano-aggregates. In addition, iron, copper, nickel, proteins and gallated catechins exhibited

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higher aggregating affinity than other components, while methylxanthines and calcium contributed to

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the transformation of nano-aggregates into tea cream. Green tea nano-aggregates were partly

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destroyed by simulated gastrointestinal digestion, and removing theses peculiar particles

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dramatically attenuated the bioaccessibility of methylxanthines, theanine, and some catechin

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monomers in green tea infusions. This study enhanced our knowledge of molecular interactions in

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the formation of green tea cream and provided insight into physicochemical profiles, phytochemical

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nature and functional effects of green tea nano-aggregates.

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Keywords: green tea, Camellia ptilophylla, Camellia sinensis, tea cream, nano-aggregates


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INTRODUCTION When a hot and strong tea infusion is cooled, intermolecular interactions among tea polyphenols,

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proteins and methylxanthines are enhanced, resulting in visible hazes and precipitates, which is

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so-called tea cream 1. In the industrial production of clear ready-to-drink tea beverages, tea cream is

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usually discarded via filtration. However, during this process numerous bioactive components like

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catechins and caffeine are removed simultaneously, leading to dramatic decrease in the in vitro

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antioxidant and anti-inflammatory activities of green teas 2-4. Accordingly, many attempts are

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devoted to prevent formation of tea cream, including lowering the extraction temperature 5, removing

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calcium (Ca) 6, regulating the pH of brewing solution 7, and adjusting the solid content of tea

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infusions 8, etc. However, such efforts are not entirely successful because the underlying molecular

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interactions of tea components behind the transformation of tea infusions from clear appearance to

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cloudness are not completely elucidated.

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Dated back to 1971, Rutter observed that a great number of spherical nano colloidal particles

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naturally suspended in clear black tea infusions, and these particles spontaneously transformed into

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tea cream via coacervation with an increase in tea concentration, or a decrease in temperature 9.

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These peculiar nano-aggregates in pre-creaming tea infusions are considered as the precursors of tea

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cream particles. Further characterization using dynamic light scattering (DLS), Cryo-transmission

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electron microscopy (TEM) and scanning electron microscopy (SEM) indicates that about 1015

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spherical nano-aggregates of 200 nm in diameter presented in a 150 mL black tea infusion of

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drinkable concentration at 20 °C 10. Moreover, the addition of caffeine (1,3,7-trimethylxanthine)

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results in an increase in the diameter of nano-aggregates in a decaffeinated black tea infusion. While

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insignificant differences are observed when caffeine is replaced by its analogue theophylline



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(1,3-dimethylxanthine) 11. It implies that caffeine may play a vital role in formation of black tea

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nano-aggregates and the involvement of methylxanthines in nano-aggregation may be related to the

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number or the location of methyl groups in their molecules. These findings suggest that the

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spontaneously assembled tea nano-aggregates provide a new perspective to explore molecular

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interactions in clear black tea infusions. Is it also a common property of unfermented teas?

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To present colloidal chemical evidences linking spontaneous molecular interactions of tea

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components in clear green tea infusions to the formation of tea cream, nano-aggregates in clear green

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tea infusions from Camellia ptilophylla Chang (C. ptilophylla) which naturally contains 6.0% of

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theobromine (3,7-dimethylxanthine) instead of caffeine and Camellia sinensis (C. sinensis) as the

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control were profiled for comparative purpose. Initially, physicochemical features of tea

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nano-aggregates were characterized using DLS, TEM and laser Doppler Velocimetry (LDV).

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Subsequently, nano-aggregates were separated via ultrafiltration of 100 kDa in molecular weight

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cut-off (MWCO) for further chemical analysis with high performance liquid chromatography

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(HPLC), flame atomic absorption spectrometry (FAAS) and inductively coupled plasm-optical

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emission spectrometer (ICP-OES). In addition, affinities of major chemical components for

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aggregation were compared by calculating their distribution coefficients between nano-aggregates

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and supernatants. Roles of various tea components in the transformation of nano-aggregates to tea

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cream particles were also evaluated by comparing their percentages distributed in nano-aggregates

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and in tea cream. Furthermore, the concentrations of major chemical components and

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physicochemical parameters of green tea infusions were traced in the dynamical extraction process to

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summarize the possible relationship between chemical composition of original tea infusions and the

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formation of tea nano-aggregates. Finally, the simulated gastrointestinal digestion of green tea


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nano-aggregates was performed to determine their fate under digestion to elucidate the impact of

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nano-aggregation on the in vitro bioaccessibility of major bioactive components in green tea.

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MATERIALS AND METHODS

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Materials

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Shoots with 2-3 leaves and a bud of C. ptilophylla plucked in spring were fixed with microwave

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(800 W, 2 min) following with microwave drying (800 W, 4 min) and hot air drying (60 °C, 12-18 h).

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Steamed green tea leaves of C. sinensis were purchased from Huahai Sugar Development Co., Ltd.

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(Guangdong, China). Dried tea leaves ground with a mortar and pestle were sieved for small

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particles ranging from 0.60 to 0.85 millimeters in size. Milli-Q water (18.2 MΩ·cm) was produced

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by Milli-Q Integral 3 (Molsheim, France).

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Following chemicals were used: -(-)catechin (C, ≥98%), epicatechin (EC, ≥98%), gallocatechin

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(GC, ≥98%), epigallocatechin (EGC, ≥98%), catechin gallate (CG, ≥98%), epicatechin gallate (ECG,

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≥98%), gallocatechin gallate (GCG, ≥98%), epigallocatechin gallate (EGCG, ≥95%), gallic acid (GA,

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97.5-102.5% by titration), theophylline (≥99.0%), theobromine (≥99.0%), theanine (BR grade),

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formic acid (~98%), pepsin from porcine gastric mucosa (≥400 units/mg protein), lipase from

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porcine pancreas (Type II, 100-400 units/mg protein), pancreatin from porcine pancreas (reagent

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grade) and bile salts (approximately 50% sodium cholate and 50% sodium deoxycholate) were

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purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China); Caffeine (99.9%) was

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purchased from Sangon Biotech Co., Ltd. (Shanghai, China). HPLC grade methanol and acetonitrile

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was obtained from Spectrum China Ltd (Shanghai, China). Phosphotungstic acid solution (2%, pH

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6.5) was purchased from Beijing Zhongjingkeyi Technology Co., Ltd. (Beijing, China).



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Extraction of green tea Green tea leaves (9.0 g) were extracted with 45 mL of boiled Milli-Q water in a boiling water

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bath (100 °C) for 30 min. Tea slurry was subsequently filtrated with quantitative filter paper and the

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filtrate was diluted to 50 mL to make up for the loss of water due to the evaporation in extracting

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process. Diluted infusions were used for further physicochemical characterization. To explore the

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formation of nano-aggregates, green tea leaves were extracted for different time duration (2, 4, 6, 8,

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10, 15, 20, 30 and 40 min) and their infusions were used for physicochemical and chemical analysis.

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Physicochemical characterization of nano-aggregates

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Total intensity (derived count rates), hydrodynamic diameter (DH) and polydispersity index

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(PDI) of nano-aggregates were tested by DLS in a disposable sizing cuvette (DTS 0012), and their

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Zeta potentials and conductivities were analyzed by LDV in a disposable folded capillary cell (DTS

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1070) on Zetasizer Nano ZS 90 with Malvern DTS 6.20 software (Malvern Instruments Ltd., UK) at

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25 °C.

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Morphological features of tea nano-aggregates were observed by TEM (TECNAI G2 12, FEI

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Company, Netherland) at 100 kV with the magnification of 4800 folds. Tea nano-aggregates were

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negatively stained by 2% phosphotungstic acid solution for 30 s on a carbon film coated copper grids

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(230 mesh, Beijing Zhongjingkeyi Technology Co., Ltd., China) using Milli-Q water as the control.

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Separation of nano-aggregates

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An aliquot (4 mL) of tea infusion was centrifuged for 20 min at 4 °C by 4,000 g (Centrifuge

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5840R with a A4-444 rator, Eppendof A G, Hamburg, Germany) in an Amicon centrifugal filter Unit

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(polyethersulfone membrane, MWCO of 100 kDa, EMD Millipore Corporation, Massachusetts,

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USA). Supernatants, re-dissolved pellets, and 4 mL of original infusion were diluted to 5 mL for


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physicochemical and phytochemical analysis and the in vitro digestive simulation.

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Analysis of chemical components

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Concentrations of tea polyphenols, proteins, carbohydrates, and free amino acids in original

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infusions, supernatants, and pellets of green tea were detected on a ultraviolet-visible spectrometry

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(UV-Vis) (UV-2102 C, Unico Instruments Co., Ltd., Shanghai, China), and their catechins (EGCG,

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GCG, ECG, EGC, CG, GC, EC, and C), caffeine, theobromine, theophylline and GA were tested on

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Agilent 1200 series HPLC system (G1322A vacuum degasser, G1311A quaternary pump, G1329A

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standard autosampler, G1315D diode-array detector, Agilent Technologies, Inc., Santa Clara, USA)

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with Eclipse XDB-C18 column (4.6 × 250 mm, 5 µm, Agilent) as we previously described 2.

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Theanine was determined as described by Bindal and Gupta with a slight modification on

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Agilent 1200 series HPLC system 12. In detail, 2 µL of tea solution filtered with a 0.22 µm

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polyvinylidene fluoride filter (Shanghai Anpel Scientific Instrument Co., Ltd., China) was separated

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on a Poroshell 120 Bonus-RP column (4.6 × 50 mm, 2.7 µm, Agilent) at a flow rate of 0.5 mL/min

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with the column temperature of 30 °C, using acetonitrile (A) and 0.05% (v/v) trifluoroacetic acid

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aqueous solution (B) as mobile phases. Tea solutions were eluted as following: 0-3 min, a isocratic

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elution with 100% phase A; 3-5 min, a gradient elution with phase A of 100% decreased to 50%;

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8-11 min, a isocratic elution with 50% phase A; 11-13 min, a gradient elution with phase A of 50%

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increased to 100%; 13-20 min, a isocratic elution with 100% phase A. Calibration plots were

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constructed with authentic standards by plotting peak areas from the absorbance signal at 199 nm

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versus standard concentration using the Agilent Chemstation B.04.02 software.

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Ca, magnesium (Mg), potassium (K), sodium (Na), ion (Fe), manganese (Mn), copper (Cu),

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zinc (Zn), and nickel (Ni) were detected by FAAS (HITACHI Z-2300, Hitachi Koki Co., Ltd., Japan)



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at 240 V 3. Aluminium (Al) was detected by ICP-OES (710-ES, Varian Inc., USA). The power was

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1.2 P and the nebulizer gas pressure was 230 kPa.

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Distribution coefficient of each chemical component between tea nano-aggregates and

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supernatants was calculated according to Penders et al 13 as follow. KA＞1 implied that component A

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might tend to aggregate. K ⁄ =

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⁄ ⁄

Simulated gastrointestinal digestion of nano-aggregates

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The in vitro gastrointestinal digestion of tea nano-aggregates were conducted on a mimic

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digestive system using saline (0.9%) as the control as we described previously 2. In detail, tea

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nano-aggregates, original infusions and supernatants (4 mL) were firstly diluted with saline (0.9%, 2

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mL) and then mixed with gastric liquid (40 mg mL-1 porcine pepsin in 0.1 M HCl, 0.6 mL) in an

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amber vial. HCl (1.0 M) was subsequently added to adjust the pH of mixtures to 2.0 ± 0.1. To

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replace oxygen, nitrogen was filled the headspace of vial for 5 min. The mixtures were then

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incubated at 37 °C with shaking (150 rpm, 1 h), and their pH was adjusted to 5.3 ± 0.1 using 0.1 M

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NaHCO3 and 1.0 M NaOH. Secondly, the gastro digesta was mixed with intestinal solution (1 mg

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mL-1 porcine lipase, 2 mg mL-1 pancreatin and 12 mg mL-1 bile salts in 0.1 M NaHCO3, 1.8 mL) and

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the pH was adjusted to 7.2 ± 0.1 via NaOH (1.0 M). These mixtures were subsequently diluted with

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saline (0.9%) to 10 mL and filled with nitrogen for 5 min. After incubating at 37 °C (150 rpm, 2 h),

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the intestinal digesta was collected for further analysis. The distribution of DH and total intensity of

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tea nano-aggregates with or without simulated digestion was analyzed by DLS. Digestive recoveries

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of catechins, methylxanthines, and theanine in the supernatants and their original infusions were


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determined by HPLC.

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Statistical analysis

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Data obtained in duplicate were analyzed by ANOVA using SAS Software 9.0 for Windows,

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and mean separation was determined by Least Significant Difference test. Data in figures and tables

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marked with different superscript letters were significant different (P

Spontaneously Assembled Nano-aggregates in Clear Green Tea

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