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Characteristics of the emulsion stabilized by polysaccharide conjugates alkali-extracted from green tea residue and its protective effect on catechins
Industrial Crops & Products 140 (2019) 111611
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Characteristics of the emulsion stabilized by polysaccharide conjugates alkali-extracted from green tea residue and its protective effect on catechins
T
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Xiaoqiang Chena,b,c, ,1, Yu Hana,1, Hong Mengb, Wei Lia, Qian Lia, Yayuan Luoa, Chunpeng Wanga, Jianchun Xiec, Long Wua, Xinyi Zhanga, Zhengqi Wua, Yinjun Zhangd National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, China b Key Laboratory of Cosmetic(Beijing Technology and Business University), China National Light Industry, Beijing 100048, China c Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU) Beijing 100048, China d College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China a
A R T I C LE I N FO
A B S T R A C T
Keywords: Tea polysaccharide conjugates Emulsion Emulsify Stability Natural emulsifier
Alkali-extracted tea polysaccharide conjugates, termed TPC-A, are obtained from the residue derived from tea after water extraction. Oil-in-water emulsions stabilized by different concentrations of TPC-A (0.5 wt%, 1.0 wt %, 1.5 wt %, 2.0 wt %, and 3.0 wt%) were prepared using medium chain triglycerides (MCT). The optimal concentrations of TPC-A were 2.0 wt % and 3.0 wt %. We investigated the effects of the storage time of 10 days, pH 2.0–10.0, and metal ions (Na+ and Ca2+) on the emulsion stabilized using 2.0 wt % TPC-A at 25 ℃. During the 10-day storage period, the mean particle diameter (MPD) (d32) of the emulsion stabilized using 2.0 wt % TPC-A slightly increased from 0.31 μm to 0.37 μm, and the absolute value of the zeta potential increased from 44.9 mV to 41.4 mV. Under neutral or alkaline conditions, the emulsion stabilized using TPC-A had the MPD (d32) of less than 0.30 μm and the absolute value of the zeta potential ranging from 40.52 to 41.53 mV. The MPD (d32) of the emulsion in different Na+ concentrations from 0 mmol/L to 500 mmol/L, increased from 0.30 μm to 2.80 μm, and their absolute value of zeta potential decreased from 41.0 mV to 37.7 mV. The emulsion stabilized using TPC-A had a favorable protective effect on EGCG and EGC, and the retention rates of EGCG and EGC during the 10 days were 7 and 6 times higher than those without TPC-A emulsion protection, respectively. TPC-A extracted from tea residue, which is abundantly available and cheap, can be used as a natural emulsifier, in order to develop a milky food or beverage products with excellent health benefits.
1. Introduction Due to the oversupply of traditional tea, especially low-grade tea, the development of deep-processed tea products, such as tea beverages, is necessary. Tea residue, a by-product of tea beverage processing, is underutilized and discarded, resulting in wastage of resources, and environmental pollution (He et al., 2015). Our previous studies have shown that alkali-extracted tea polysaccharide conjugates (TPC-A) obtained from tea residue have significant hypoglycemic effects (Chen et al., 2010). Many studies on TPC still focus on the biological activity (Nie and Xie, 2011). The functional activity and function-based applications of TPC are more often overlooked when compared to those of many plant polysaccharides, such as ganoderma lucidum
polysaccharide, dendrobium polysaccharide, lentinan polysaccharide, and pumpkin polysaccharide. In order to expand the use of TPC, it is necessary to explore their unique and significant functions. Oil-in-water emulsions are widely utilized in food processing, including the processing of dairy products, flavorings, and beverages. Since oil-in-water systems are thermodynamically unstable systems, additional emulsifiers are often required to reduce interfacial tension and create repulsive interactions (space or static) in order to stabilize the entire system (Mezdour et al., 2017). At present, emulsifiers used in the food industry such as fatty acid monoglycerides, sucrose esters etc. are mainly synthetic (Krog et al., 2004, Wang et al. 2016). With the growing awareness to consume healthy foods, the development of natural emulsifiers has received much attention(Mcclements et al.,
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Corresponding author at: College of Bioengineering and Food, Hubei University of Technology, NO. 28, Nanli Road, Hongshan District, Wuhan City, Hubei Prov. 430068, China. E-mail address: [email protected] (X. Chen). 1 Equal contribution. https://doi.org/10.1016/j.indcrop.2019.111611 Received 12 February 2019; Received in revised form 22 July 2019; Accepted 25 July 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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2017). Current studies on natural emulsifiers mainly focus on either proteins or their complexes with exogenous polysaccharides (Krstonosic et al., 2015). Tea polysaccharide conjugate (TPC) is macromolecules containing small amounts of covalently bound protein (Chen et al., 2017a, 2018; Chen et al., 2019a, b). We initially found that these have emulsification activity (Chen et al., 2017b, 2018), and do not need extra protein in order to form a protein-polysaccharide complex. The purpose of this study was to quantify the emulsification properties and the emulsion stability of TPC-A, and investigate the effect of TPC-A emulsion on the storage stability of catechins (EGCG and EGC). In order to achieve this objective, emulsions were prepared using different concentrations of TPC-A and high speed blenders and microfluidizers, following which the droplet sizes, zeta potential, microstructure and interfacial properties of the oil in water emulsions were measured. In order to maintain systemicity, tea source was consistency with those used in our previous research studies (Chen et al., 2009, 2010; Chen et al., 2014a, b). The study results provide a theoretical basis for the application of tea polysaccharides as emulsifiers and for the expansion of the function of tea polysaccharides in order to further promote their wide application.
Fig. 1. Effect of TPC-A concentration on the interfacial tension tested at the MCT oil-water interface at 25℃. The test time for each sample was 1 h, and the experiment was conducted at 25℃.
2. Materials and methods 2.1. Materials Low-grade green tea was purchased from the Xihu District, Hangzhou. D-glucuronic acid (D-GluA), D-galacturonic acid (D-GalA), L-fucose (L-Fuc), D-arabinose (D-Ara), D-mannose (D-Man), D-xylose (D-Xyl), D-fructose (D-Fru), L-rhamnose (L-Rha), D-galactose(D-Gal), Dribose (D-Rib), D-glucose (D-Glu), gum arabic (GA), and Nile red were purchased from Sigma-Aldrich (St. Louis, MO, USA). Medium chain triglycerides (MCT) were obtained from a commercial food supplier (Mazola, ACH Food Companies, Memphis, TN, USA). Double-distilled water was used throughout the study, to prepare all solutions and emulsions. 2.2. Preparation of TPC-A Low-grade green tea (1000 g) was pulverized, mixed with distilled water at a ratio of 1:15, heated at 90 ℃ for 2.0 h and centrifuged at 9190 g for 10 min in order to collect the precipitate. The precipitate was mixed with 4.0 L of 1.0 wt% aqueous sodium hydroxide at 50 ℃. The mixture was heated at 50 ℃ for 2.0 h, and the supernatant was collected by centrifugation at 8000 r/min. The supernatant was then concentrated, adjusted to pH 9.0 using HCl, decolorized using H2O2, precipitated, dialyzed, and finally lyophilized as previously described in order to obtain alkali-extracted tea polysaccharide conjugates (Chen et al., 2009). 2.3. Chemical properties of TPC-A The monosaccharide composition, amino acid composition, and relative molecular weight of tea polysaccharide conjugates were determined, as per a previous study. The sample was derivatized using acetic anhydride, and the monosaccharide fraction was determined using gas chromatography (Wang et al., 2017). After the sample was hydrolyzed using HCl, the amino acid composition was determined using the external standard method and the Hitachi L-8900 Amino Acid Auto Analyzer (Chen et al., 2017b). Gel Permeation ChromatographyMulti angle light scattering (GPC- MALS) was used to analyze the molecular weight of the TPC-A (Chen et al., 2019a).
Fig. 2. Mean particle diameter (d32) distribution (A), and particle size distribution (B) of emulsions stabilized by different concentrations of TPC-A at 25℃ using a Mastersizer 2000 laser particle diameter analyzer with the refractive index of the disperse phase of 1.475 and the continuous phase of 1.30.
2.4. Determination of TPC-A interfacial tension Axisymmetric dynamic drop formation was used to determine the interfacial tension (γ) in TPC-A samples adsorbed on the oil-water 2
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Fig. 3. zeta potential of TPC-A emulsions at different concentration at 25℃ using a Zetasizer Nano-ZS particle diameter and potentiometric analyzer (light scattering detection angle of 17°, laser wavelength 633 nm, He/Ne gas laser).
Fig. 5. The effect of different concentrations of calcium ions on zeta potential and mean particle diameter (d32) (A), and particle size distribution (B) of the emulsions stabilized by TPC-A at 25℃.
interface during the adsorption time (t). An appropriate amount of the polysaccharide aqueous solution was weighed using the sample tank, and a U-pin with the MCT was immersed in the sample tank containing the polysaccharide solution. The sample was pushed into the capillary using an electric motor in order to form a droplet of 10.0 μL volume on the needle tip, and a video camera was used to continuously collect the droplet shape image and detect the change in the interfacial tension γ with time t. The test time for each sample was 1.0 h, and the experiment was conducted at 25 °C. 2.5. Emulsion stabilized using different concentrations of TPC-A 2.5.1. Emulsion preparation MCT, forming the oil phase, was blended with distilled water containing the TPC-A prepared emulsion (8.0 wt % oil phase and 92.0 wt % aqueous phase) prepared using a high-speed PT-MR2100 Polytron-type mixer (Kinematica Co., Switzerland) at 26,000 rpm for 3 min, followed by a single passage through a high-pressure homogenizer (Microfluidics M-110 L, USA) at 75 MPa (Li et al., 2012). The entire process was performed in an ice bath in order to minimize lipid oxidation. Sodium azide (0.02 w/v %) was added to the final emulsion in order to prevent microbial growth. The concentration gradient of TPC-A was set to 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, and 3.0 wt%.
Fig. 4. Mean particle diameter (d32) and zeta potential(A) and particle size distribution (B) of emulsion stabilized by 2% TPC-A during storage at 25℃. The assay was performed by using a Zetasizer Nano-ZS particle diameter and potentiometric analyzer.
2.5.2. Measurement of mean particle diameter (MPD) (d32) and zeta potential The particle diameter distribution and MPD (d32) of the emulsion were determined using a Mastersizer 2000 laser particle diameter 3
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2.6.3. Effect of metal ions on the stability of TPC-A emulsion The emulsions containing either 0.10, 0.20, 0.30, 0.40, or 0.50 mol/ L NaCl and either 0.01, 0.02, 0.03, 0.04, or 0.05 mol/L CaCl2 (pH 7.0) were prepared. MPD (d32) and zeta potential were determined as previously described. A laser scanning confocal microscope (LSCM) was used to observe the microscopic appearance of the emulsion supplemented with Ca2+ and Na+ ions. The MCT in these emulsions was stained with Nile red dye. The excitation and emission wavelengths of Nile Red were 488 nm and 520 nm, respectively. All microstructure images were analyzed using Olympus image processing software for the observation of morphology. 2.7. Protective effect of TPC-A emulsion on EGCG and EGC TPC-A emulsion containing EGCG (3.0, 5.0, and 8.0 mg/mL) and EGC (3.0, 5.0, and 8.0 mg/mL) were prepared according to previously described methods. These samples were termed as either TPC-A emulsion EGCG-3, TPC-A emulsion EGCG-5, TPC-A emulsion EGCG-8, TPC-A emulsion EGC-3, and TPC-A emulsion EGC-5, or TPC-A emulsion EGC8. EGCG (5.0 mg/mL) and EGC (5.0 mg/mL) in PBS at pH 7.0, were prepared as controlled and termed EGCG-5 and EGC-5, respectively. These emulsion samples were stored at 25 °C for 10 days. Zeta potential and MPD (d32) of these emulsions were determined on the 1st and 10th day. The content of EGCG and EGC in the emulsion samples and control was determined using high performance liquid chromatography (HPLC). 2.8. Statistical analysis Three parallel samples were analyzed, and statistical analysis was performed using the statistical analysis software system (SPASS 13.0). Differences between the samples were calculated using analysis of variance (ANOVA) and Duncan test with a confidence interval of 95%.
Fig. 6. The effect of different concentrations of calcium ions on zeta potential and mean particle diameter (d32) (A), and particle size distribution (B) of the emulsions stabilized by TPC-A at 25℃.
3. Results and discussion 3.1. Chemical properties of TPC-A
analyzer. Phosphate buffer solution (PBS) with a pH 7.0 was used as the dispersion medium. Before the sample was added, the emulsion was slightly shaken and added dropwise to the dispersion medium until the signal met the test requirements. The refractive index of the dispersion phase and the continuous phase were 1.48 and 1.30, respectively. Each sample was measured parallelly three times, and an average of the three values was obtained. The emulsion (25.0 μL) was added to 4975.0 μL of 200-fold diluted PBS and the mixture vortexed for 1 min on a shaker. The zeta potential of the emulsion was measured at 25 °C using a Zetasizer Nano-ZS particle diameter and potentiometric analyzer (light scattering detection angle of 17°, laser wavelength 633 nm, and He/Ne gas laser).
GPC results indicated that TPC-A possessed one component with an average molecular weight of 117,800 Da. The polysaccharide chain of TPC-A was composed of Rha, Fuc, Ara, Xyl, Man, Glc, and Gla, with molar ratios of 13.54, 1.38, 34.22, 4.98, 5.17, 7.13, and 33.58 respectively. The free amino acids (Cf.a.a) were not detected in TPC-A. The amino acid composition of TPC-A was as follows: Asp, Glu, Ser, Gly, Thr, Arg, Ile, Val, Cys2, Tyr, Pro, Ala, Leu, Lys, Phe, and Leu, with mass contents (mg/g) of 11.06, 10.00, 5.99, 7.97, 6.28, 4.77, 7.17, 6.08, 7.34, 3.33, 3.39, 6.29, 8.14, 5.83, 5.66, and 8.14, respectively. The total amount of these amino acids (Ca.a.) were 10.74%. The content of protein moiety of TPC-A was calculated using the formula (Chen et al., 2017a, 2018): (Ca.a.－Cf.a.a.) × 110/128 = 9.23% (w/w).
2.6. Factors influencing emulsion stability
3.2. Interfacial tension measurement
2.6.1. Effect of storage time on the stability of TPC-A emulsion Freshly prepared emulsions (pH 7.0) were distributed into different beakers, while samples of these emulsions were placed in glass test tubes and incubated at 25 °C for 10 days. The appearance of the emulsion was observed daily, and the zeta potential and the MPD (d32) of the emulsion were determined as previously described.
Surfactants have lipophilic and hydrophilic groups. When they are mixed with water, the hydrophilic groups are attracted by the water molecules; the attractive force pulls a short non-polar hydrocarbon chain into water, whereas the lipophilic groups are repelled by the water molecules (Dickinsion, 2003). In order to overcome this instability, only the surface of the solution is occupied, with the lipophilic groups extending into the gas phase and the hydrophilic groups extending into the aqueous phase (Jiang et al., 2018). The adsorption of a targeted single-molecule reduces the tension at the gas-water and oilwater interfaces (Brocca et al., 2018). In order to investigate the emulsifying activity of TPC-A, we measured the interfacial tension of TPC-A at different concentrations. As the concentration of TPC-A
2.6.2. Effect of pH on the stability of TPC-A emulsion Freshly prepared emulsions (pH 7.0) were distributed into different beakers, and a sample of each emulsion was adjusted to a different pH value ranging between 2.0–8.0 using HCl or NaOH solutions. These samples were then placed in a test tube. MPD (d32) and zeta potential were determined as previously described. 4
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Fig. 7. Microscopic appearance of TPC-A emulsions stained with nile red dye and observed by using LSCM under different metal ion concentrations. The ionic strength of the solution was prepared by CaCl2 (Ca2+: 10–50 mmol/L) and NaCl (Na+:100-500 mmol/L).
colloidal dispersions (zeta potential) were used to evaluate the emulsifying ability of TPC-A (McClements and Gumus, 2016). With the concentration of TPC-A increasing from 1.0 wt% to 3.0 wt%, the MPD (d32) distribution of the TPC-A emulsion reduced from 2.82 to 0.27 μm, and the absolute value of the zeta potential slightly slightly decreased from 42.3 to 39.3 mV (Fig. 2A). As shown in Fig. 2B, the particle size distribution (PSD) of the emulsions stabilized by 0.5 wt% and 1.0 wt% TPC-A was bimodal distribution, while those of the emulsions stabilized by 1.5 wt%, 2.0 wt%, and 3.0 wt% TPC-A shifted to small particle size with their MPDs (d32) were 0.43 μm, 0.28 μm, and 0.27 μm, respectively. On the tenth day of storage, the emulsion containing 0.5 wt% and 1.0 wt% have demulsification with the absolute value of the zeta potential declining from 50.0 to 15.9 mV, and 46.9 to 17.8 mV respectively. The emulsion stabilized using 1.5 wt%, 2.0 wt% and 3.0 wt%
increased, the interfacial tension decreased rapidly. The interfacial tension of the 0.5 wt% TPC-A system dropped from 26.98 mN/m to 10.48 mN/m (Fig. 1). This process was due to the adsorption of TPC on the oil-water interface, which reduced the interfacial tension of oil and water and the reduction in interfacial tension was useful in tentatively determining the properties of TPC-A as a surfactant. Therefore, followup experiments using TPC-A as an emulsifier were performed in order to explore its emulsifying properties. 3.3. Effect of TPC-A concentration on emulsion formation The stability of the emulsion is usually characterized by its MPD (d32) distribution and ion-carrying capacity (Xu et al., 2016). The distribution uniformity of MPD (d32) and the electro-kinetic potential in 5
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3.4. Effect of storage time on emulsion stability Emulsion storage stability plays an important role in the shelf life of commercial food and beverage products. Based on the above effect of TPC-A concentration on emulsion formation described above, the storage of emulsions stabilized using 2.0 wt% TPC-A at 25 ℃ for 10 days was further investigated. Day 0 indicated the initial time of emulsion preparation. The MPD (d32) distribution of TPC-A emulsion and the change in MPD (d32) with time at 25 °C are shown in Fig. 4. During the storage time of 10 days, MPD (d32) slightly increased from 0.31 μm to 0.37 μm, and the absolute value of the zeta potential varied from 44.9 mV to 41.4 mV. The bimodal distribution of the two sizes was observed until the 10th day, and demulsification was observed on the 23rd day. During the 10 days, the particle size and zeta potentials showed minor changes, indicating that the emulsions stabilized using TPC-A have good stability. 3.5. Effect of metal ions on the stability of TPC-A emulsions The emulsion will be practically put to use in different solutions having a wide range of ionic strengths, and in different types of food products with various pH values. Therefore, it is necessary to investigate the stability of the emulsion when exposed to high ionic strength and/or acidic or alkaline environments. The high concentration of metal ions is inversely related to the stability of the emulsion. When the concentration of NaCl in these emulsions changed from 0 mol/L to 0.50 mol/L, the MPD (d32) increased from 0.33 to 2.77 μm and the absolute value of the zeta potential varied from 41.0 to 37.7 mV (Shown in Fig. 5A). When the sodium ion concentration is higher than 0.2, the shoulder-shape peak appeared in the particle size distribution of the emulsion(Shown in Fig. 5B).With the CaCl2 concentration increasing from 0 mol/L to 0.05 mol/L, the emulsion MPD (d32) increased from 0.33 μm to 5.21 μm as well as the absolute value of the zeta potential changing from 41.0 to 37.0 mV (Fig. 6A), and the peaks shifted to the large particle size distribution. In addition, the particle size distributions of TPC-A emulsions containing above 0.02 mol/L CaCl2 showed bimodal distribution (Fig. 6B). The metal ions destroys the lyophilic protective layer, resulting in emulsion droplet polymerization and demulsification (Goldstein et al., 2007). In addition, confocal microscopy results confirmed that the microstructure of the emulsion exposed to the highest metal ion concentration changed significantly during the 10-day storage period (Fig. 7).
Fig. 8. Mean particle diameter (d32) and zeta potential(A) and particle size distribution (B) of emulsion stabilized by 2% TPC-A during storage at 25℃. The assay was performed by using a Zetasizer Nano-ZS particle diameter and potentiometric analyzer.
TPC-A maintained stability with the variation range of zeta potential absolute value from 42.2 to 39.3 mV, 44.8 to 41.3 mV, and 42.5 to 40.2 mV, respectively (Fig. 3).The stability of the emulsion is positively correlated with its absolute value of zeta potential (Ichikawa et al., 2006; Wang et al., 2001). A complex in which a polysaccharide is either covalently or noncovalently bound to a protein is usually used as an emulsion stabilizer in order to synergistically exert the emulsification activity of the protein and enhance the stability of the polysaccharide over a wider range of pH and ionic strength (Krstonosic et al., 2015). TPC-A contained covalently bound protein. It is thought that the binding protein in TPC is coated by the carbohydrate chain and forms a hydrophobic "core"(Chen et al., 2017b, 2018). Polysaccharides and proteins confer hydrophilic and hydrophobic amphiphilic properties of TPC, respectively. This polysaccharide and protein conjugate can endue an emulsification function to TPC.
3.6. Effect of pH on the stability of TPC-A emulsions The initial pH value of the emulsion was 7.0, and different ratios of acid (HCl) and alkali (NaOH) were added to obtain a range of different pH values from 2.0 to 10.0. When the pH value changed from 2.0 to 6.0, MPD decreased from 9.17 μm to 0.31 μm, while zeta potential varied from 2.3 mV to −40.50 mV(Fig. 8). With pH varying from 7.0 to 10.0, MPD (d32) of these emulsions was less than 0.33 μm, and their absolute value of the zeta potential ranged from 40.52 to 41.53 mV. Thus, the
Table 1 Content and retention rate of EGCG and EGC in the emulsion stabilized by 2.0 wt% TPC-A under storage for 10 days at 25 ℃ (n = 3).
1 day 10 day Retention Rate
TPC-A emulsion EGCG3 (μg/mL)
TPC-A emulsion EGCG-5 (μg/mL)
TPC-A emulsion EGCG-8 (μg/mL)
EGCG-5 (μg/mL)
TPC-A emulsion EGC-3 (μg/mL)
TPC-A emulsion EGC-5 (μg/mL)
TPC-A emulsion EGC-8 (μg/mL)
EGC-5 (μg/mL)
2768.02 ± 26.18 505.70 ± 14.37 18.27%
4429.02 ± 36.37 3271.04 ± 14.37 73.85%
7648.10 ± 19.04 5014.18 ± 19.37 65.42%
4099.31 ± 14.40 351.47 ± 18.35 8.57%
2124.11 ± 20.01 1219.08 ± 8.37 57.41%
4264.53 ± 23.78 1534.96 ± 16.47 35.99%
6964.55 ± 79.85 5018.53 ± 12.76 72.06%
3749.03 ± 12.55 399.53 ± 13.50 10.66%
6
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emulsion stabilized using TPC-A was more stable in an alkaline solution. The polysaccharide moiety of TPC-A provided pH adaptability of these emulsions.
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3.7. Stability of EGCG and EGC in TPC-A emulsions The emulsions stabilized using 2.0 wt % TPC-A could protect EGCG and EGC from oxidation at 25 ℃ (Table 1). As shown in Fig. 9, all the emulsions containing EGCG or EGC were in a stable state during the 10day storage time, while the EGCG and EGC in PBS solution oxidized and appeared brown. The stable emulsification of TPC-A significantly increased the retention of EGCG and EGC. The retention rate of EGCG increased 2–10-fold, and the retention rate of EGC increased by 4–7fold. The emulsion containing TPC-A exhibited long-term stability and a protective effect on EGCG and EGC. The structural mechanism of TPC-A emulsion loading and protection of catechins remains to be further studied. The results of our research provide a theoretical basis for the wide application of TPC-A emulsion. 4. Conclusions The aim of this study was to investigate the emulsification ability of TPC-A, the stability of the emulsion, and the effect of the emulsion on the storage of major catechins (EGCG and EGC). Our results showed that TPC-A had significant surface activity and could effectively form a relatively stable emulsion, even at low TPC-A concentrations. This emulsion had good long-term stability, remained stable even after storage for 10 days at 25 °C, and prevented droplet aggregation and phase separation. The emulsion stabilized using low concentrations of TPC-A could effectively prolong the storage time of catechins and reduce the loss of catechins. The result indicates that TPC-A has the potential to be used as a novel emulsifier in the food, cosmetic, and pharmaceutical industry. In summary, this work has discovered a new application area for TPC-A. Acknowledgements This research was funded by the National Natural Science Foundation of China (grant number31871813), the Open Research Fund Program of the Key Laboratory of Cosmetic(Beijing Technology and Business University), China National Light Industry (grant number KLC-2018-YB6), and Beijing Advanced Innovation Center for Food Nutrition and Human Health (grant number 20161012). References Brocca, P., Rondelli, V., Corti, M., Del, F.E., Deleu, M., Cantù, L., 2018. Interferometric investigation of the gas-state monolayer of mono-rhamnolipid adsorbing at an oil/ water interface. J. Mol. Liq. 266, 687–691. Chen, X., Lin, Z., Ye, Y., Zhang, R., Yin, J., Jiang, Y., Wan, H., 2010. Suppression of diabetes in non-obese diabetic (nod) mice by oral administration of water-soluble and alkali-soluble polysaccharide conjugates prepared from green tea. Carbohydr. Polym. 82 (1), 28–33.
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Characteristics of the emulsion stabilized by polysaccharide conjugates alkali-extracted from green tea residue and its protective effect on catechins
T
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Xiaoqiang Chena,b,c, ,1, Yu Hana,1, Hong Mengb, Wei Lia, Qian Lia, Yayuan Luoa, Chunpeng Wanga, Jianchun Xiec, Long Wua, Xinyi Zhanga, Zhengqi Wua, Yinjun Zhangd National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, China b Key Laboratory of Cosmetic(Beijing Technology and Business University), China National Light Industry, Beijing 100048, China c Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU) Beijing 100048, China d College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China a
A R T I C LE I N FO
A B S T R A C T
Keywords: Tea polysaccharide conjugates Emulsion Emulsify Stability Natural emulsifier
Alkali-extracted tea polysaccharide conjugates, termed TPC-A, are obtained from the residue derived from tea after water extraction. Oil-in-water emulsions stabilized by different concentrations of TPC-A (0.5 wt%, 1.0 wt %, 1.5 wt %, 2.0 wt %, and 3.0 wt%) were prepared using medium chain triglycerides (MCT). The optimal concentrations of TPC-A were 2.0 wt % and 3.0 wt %. We investigated the effects of the storage time of 10 days, pH 2.0–10.0, and metal ions (Na+ and Ca2+) on the emulsion stabilized using 2.0 wt % TPC-A at 25 ℃. During the 10-day storage period, the mean particle diameter (MPD) (d32) of the emulsion stabilized using 2.0 wt % TPC-A slightly increased from 0.31 μm to 0.37 μm, and the absolute value of the zeta potential increased from 44.9 mV to 41.4 mV. Under neutral or alkaline conditions, the emulsion stabilized using TPC-A had the MPD (d32) of less than 0.30 μm and the absolute value of the zeta potential ranging from 40.52 to 41.53 mV. The MPD (d32) of the emulsion in different Na+ concentrations from 0 mmol/L to 500 mmol/L, increased from 0.30 μm to 2.80 μm, and their absolute value of zeta potential decreased from 41.0 mV to 37.7 mV. The emulsion stabilized using TPC-A had a favorable protective effect on EGCG and EGC, and the retention rates of EGCG and EGC during the 10 days were 7 and 6 times higher than those without TPC-A emulsion protection, respectively. TPC-A extracted from tea residue, which is abundantly available and cheap, can be used as a natural emulsifier, in order to develop a milky food or beverage products with excellent health benefits.
1. Introduction Due to the oversupply of traditional tea, especially low-grade tea, the development of deep-processed tea products, such as tea beverages, is necessary. Tea residue, a by-product of tea beverage processing, is underutilized and discarded, resulting in wastage of resources, and environmental pollution (He et al., 2015). Our previous studies have shown that alkali-extracted tea polysaccharide conjugates (TPC-A) obtained from tea residue have significant hypoglycemic effects (Chen et al., 2010). Many studies on TPC still focus on the biological activity (Nie and Xie, 2011). The functional activity and function-based applications of TPC are more often overlooked when compared to those of many plant polysaccharides, such as ganoderma lucidum
polysaccharide, dendrobium polysaccharide, lentinan polysaccharide, and pumpkin polysaccharide. In order to expand the use of TPC, it is necessary to explore their unique and significant functions. Oil-in-water emulsions are widely utilized in food processing, including the processing of dairy products, flavorings, and beverages. Since oil-in-water systems are thermodynamically unstable systems, additional emulsifiers are often required to reduce interfacial tension and create repulsive interactions (space or static) in order to stabilize the entire system (Mezdour et al., 2017). At present, emulsifiers used in the food industry such as fatty acid monoglycerides, sucrose esters etc. are mainly synthetic (Krog et al., 2004, Wang et al. 2016). With the growing awareness to consume healthy foods, the development of natural emulsifiers has received much attention(Mcclements et al.,
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Corresponding author at: College of Bioengineering and Food, Hubei University of Technology, NO. 28, Nanli Road, Hongshan District, Wuhan City, Hubei Prov. 430068, China. E-mail address: [email protected] (X. Chen). 1 Equal contribution. https://doi.org/10.1016/j.indcrop.2019.111611 Received 12 February 2019; Received in revised form 22 July 2019; Accepted 25 July 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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2017). Current studies on natural emulsifiers mainly focus on either proteins or their complexes with exogenous polysaccharides (Krstonosic et al., 2015). Tea polysaccharide conjugate (TPC) is macromolecules containing small amounts of covalently bound protein (Chen et al., 2017a, 2018; Chen et al., 2019a, b). We initially found that these have emulsification activity (Chen et al., 2017b, 2018), and do not need extra protein in order to form a protein-polysaccharide complex. The purpose of this study was to quantify the emulsification properties and the emulsion stability of TPC-A, and investigate the effect of TPC-A emulsion on the storage stability of catechins (EGCG and EGC). In order to achieve this objective, emulsions were prepared using different concentrations of TPC-A and high speed blenders and microfluidizers, following which the droplet sizes, zeta potential, microstructure and interfacial properties of the oil in water emulsions were measured. In order to maintain systemicity, tea source was consistency with those used in our previous research studies (Chen et al., 2009, 2010; Chen et al., 2014a, b). The study results provide a theoretical basis for the application of tea polysaccharides as emulsifiers and for the expansion of the function of tea polysaccharides in order to further promote their wide application.
Fig. 1. Effect of TPC-A concentration on the interfacial tension tested at the MCT oil-water interface at 25℃. The test time for each sample was 1 h, and the experiment was conducted at 25℃.
2. Materials and methods 2.1. Materials Low-grade green tea was purchased from the Xihu District, Hangzhou. D-glucuronic acid (D-GluA), D-galacturonic acid (D-GalA), L-fucose (L-Fuc), D-arabinose (D-Ara), D-mannose (D-Man), D-xylose (D-Xyl), D-fructose (D-Fru), L-rhamnose (L-Rha), D-galactose(D-Gal), Dribose (D-Rib), D-glucose (D-Glu), gum arabic (GA), and Nile red were purchased from Sigma-Aldrich (St. Louis, MO, USA). Medium chain triglycerides (MCT) were obtained from a commercial food supplier (Mazola, ACH Food Companies, Memphis, TN, USA). Double-distilled water was used throughout the study, to prepare all solutions and emulsions. 2.2. Preparation of TPC-A Low-grade green tea (1000 g) was pulverized, mixed with distilled water at a ratio of 1:15, heated at 90 ℃ for 2.0 h and centrifuged at 9190 g for 10 min in order to collect the precipitate. The precipitate was mixed with 4.0 L of 1.0 wt% aqueous sodium hydroxide at 50 ℃. The mixture was heated at 50 ℃ for 2.0 h, and the supernatant was collected by centrifugation at 8000 r/min. The supernatant was then concentrated, adjusted to pH 9.0 using HCl, decolorized using H2O2, precipitated, dialyzed, and finally lyophilized as previously described in order to obtain alkali-extracted tea polysaccharide conjugates (Chen et al., 2009). 2.3. Chemical properties of TPC-A The monosaccharide composition, amino acid composition, and relative molecular weight of tea polysaccharide conjugates were determined, as per a previous study. The sample was derivatized using acetic anhydride, and the monosaccharide fraction was determined using gas chromatography (Wang et al., 2017). After the sample was hydrolyzed using HCl, the amino acid composition was determined using the external standard method and the Hitachi L-8900 Amino Acid Auto Analyzer (Chen et al., 2017b). Gel Permeation ChromatographyMulti angle light scattering (GPC- MALS) was used to analyze the molecular weight of the TPC-A (Chen et al., 2019a).
Fig. 2. Mean particle diameter (d32) distribution (A), and particle size distribution (B) of emulsions stabilized by different concentrations of TPC-A at 25℃ using a Mastersizer 2000 laser particle diameter analyzer with the refractive index of the disperse phase of 1.475 and the continuous phase of 1.30.
2.4. Determination of TPC-A interfacial tension Axisymmetric dynamic drop formation was used to determine the interfacial tension (γ) in TPC-A samples adsorbed on the oil-water 2
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Fig. 3. zeta potential of TPC-A emulsions at different concentration at 25℃ using a Zetasizer Nano-ZS particle diameter and potentiometric analyzer (light scattering detection angle of 17°, laser wavelength 633 nm, He/Ne gas laser).
Fig. 5. The effect of different concentrations of calcium ions on zeta potential and mean particle diameter (d32) (A), and particle size distribution (B) of the emulsions stabilized by TPC-A at 25℃.
interface during the adsorption time (t). An appropriate amount of the polysaccharide aqueous solution was weighed using the sample tank, and a U-pin with the MCT was immersed in the sample tank containing the polysaccharide solution. The sample was pushed into the capillary using an electric motor in order to form a droplet of 10.0 μL volume on the needle tip, and a video camera was used to continuously collect the droplet shape image and detect the change in the interfacial tension γ with time t. The test time for each sample was 1.0 h, and the experiment was conducted at 25 °C. 2.5. Emulsion stabilized using different concentrations of TPC-A 2.5.1. Emulsion preparation MCT, forming the oil phase, was blended with distilled water containing the TPC-A prepared emulsion (8.0 wt % oil phase and 92.0 wt % aqueous phase) prepared using a high-speed PT-MR2100 Polytron-type mixer (Kinematica Co., Switzerland) at 26,000 rpm for 3 min, followed by a single passage through a high-pressure homogenizer (Microfluidics M-110 L, USA) at 75 MPa (Li et al., 2012). The entire process was performed in an ice bath in order to minimize lipid oxidation. Sodium azide (0.02 w/v %) was added to the final emulsion in order to prevent microbial growth. The concentration gradient of TPC-A was set to 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, and 3.0 wt%.
Fig. 4. Mean particle diameter (d32) and zeta potential(A) and particle size distribution (B) of emulsion stabilized by 2% TPC-A during storage at 25℃. The assay was performed by using a Zetasizer Nano-ZS particle diameter and potentiometric analyzer.
2.5.2. Measurement of mean particle diameter (MPD) (d32) and zeta potential The particle diameter distribution and MPD (d32) of the emulsion were determined using a Mastersizer 2000 laser particle diameter 3
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2.6.3. Effect of metal ions on the stability of TPC-A emulsion The emulsions containing either 0.10, 0.20, 0.30, 0.40, or 0.50 mol/ L NaCl and either 0.01, 0.02, 0.03, 0.04, or 0.05 mol/L CaCl2 (pH 7.0) were prepared. MPD (d32) and zeta potential were determined as previously described. A laser scanning confocal microscope (LSCM) was used to observe the microscopic appearance of the emulsion supplemented with Ca2+ and Na+ ions. The MCT in these emulsions was stained with Nile red dye. The excitation and emission wavelengths of Nile Red were 488 nm and 520 nm, respectively. All microstructure images were analyzed using Olympus image processing software for the observation of morphology. 2.7. Protective effect of TPC-A emulsion on EGCG and EGC TPC-A emulsion containing EGCG (3.0, 5.0, and 8.0 mg/mL) and EGC (3.0, 5.0, and 8.0 mg/mL) were prepared according to previously described methods. These samples were termed as either TPC-A emulsion EGCG-3, TPC-A emulsion EGCG-5, TPC-A emulsion EGCG-8, TPC-A emulsion EGC-3, and TPC-A emulsion EGC-5, or TPC-A emulsion EGC8. EGCG (5.0 mg/mL) and EGC (5.0 mg/mL) in PBS at pH 7.0, were prepared as controlled and termed EGCG-5 and EGC-5, respectively. These emulsion samples were stored at 25 °C for 10 days. Zeta potential and MPD (d32) of these emulsions were determined on the 1st and 10th day. The content of EGCG and EGC in the emulsion samples and control was determined using high performance liquid chromatography (HPLC). 2.8. Statistical analysis Three parallel samples were analyzed, and statistical analysis was performed using the statistical analysis software system (SPASS 13.0). Differences between the samples were calculated using analysis of variance (ANOVA) and Duncan test with a confidence interval of 95%.
Fig. 6. The effect of different concentrations of calcium ions on zeta potential and mean particle diameter (d32) (A), and particle size distribution (B) of the emulsions stabilized by TPC-A at 25℃.
3. Results and discussion 3.1. Chemical properties of TPC-A
analyzer. Phosphate buffer solution (PBS) with a pH 7.0 was used as the dispersion medium. Before the sample was added, the emulsion was slightly shaken and added dropwise to the dispersion medium until the signal met the test requirements. The refractive index of the dispersion phase and the continuous phase were 1.48 and 1.30, respectively. Each sample was measured parallelly three times, and an average of the three values was obtained. The emulsion (25.0 μL) was added to 4975.0 μL of 200-fold diluted PBS and the mixture vortexed for 1 min on a shaker. The zeta potential of the emulsion was measured at 25 °C using a Zetasizer Nano-ZS particle diameter and potentiometric analyzer (light scattering detection angle of 17°, laser wavelength 633 nm, and He/Ne gas laser).
GPC results indicated that TPC-A possessed one component with an average molecular weight of 117,800 Da. The polysaccharide chain of TPC-A was composed of Rha, Fuc, Ara, Xyl, Man, Glc, and Gla, with molar ratios of 13.54, 1.38, 34.22, 4.98, 5.17, 7.13, and 33.58 respectively. The free amino acids (Cf.a.a) were not detected in TPC-A. The amino acid composition of TPC-A was as follows: Asp, Glu, Ser, Gly, Thr, Arg, Ile, Val, Cys2, Tyr, Pro, Ala, Leu, Lys, Phe, and Leu, with mass contents (mg/g) of 11.06, 10.00, 5.99, 7.97, 6.28, 4.77, 7.17, 6.08, 7.34, 3.33, 3.39, 6.29, 8.14, 5.83, 5.66, and 8.14, respectively. The total amount of these amino acids (Ca.a.) were 10.74%. The content of protein moiety of TPC-A was calculated using the formula (Chen et al., 2017a, 2018): (Ca.a.－Cf.a.a.) × 110/128 = 9.23% (w/w).
2.6. Factors influencing emulsion stability
3.2. Interfacial tension measurement
2.6.1. Effect of storage time on the stability of TPC-A emulsion Freshly prepared emulsions (pH 7.0) were distributed into different beakers, while samples of these emulsions were placed in glass test tubes and incubated at 25 °C for 10 days. The appearance of the emulsion was observed daily, and the zeta potential and the MPD (d32) of the emulsion were determined as previously described.
Surfactants have lipophilic and hydrophilic groups. When they are mixed with water, the hydrophilic groups are attracted by the water molecules; the attractive force pulls a short non-polar hydrocarbon chain into water, whereas the lipophilic groups are repelled by the water molecules (Dickinsion, 2003). In order to overcome this instability, only the surface of the solution is occupied, with the lipophilic groups extending into the gas phase and the hydrophilic groups extending into the aqueous phase (Jiang et al., 2018). The adsorption of a targeted single-molecule reduces the tension at the gas-water and oilwater interfaces (Brocca et al., 2018). In order to investigate the emulsifying activity of TPC-A, we measured the interfacial tension of TPC-A at different concentrations. As the concentration of TPC-A
2.6.2. Effect of pH on the stability of TPC-A emulsion Freshly prepared emulsions (pH 7.0) were distributed into different beakers, and a sample of each emulsion was adjusted to a different pH value ranging between 2.0–8.0 using HCl or NaOH solutions. These samples were then placed in a test tube. MPD (d32) and zeta potential were determined as previously described. 4
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Fig. 7. Microscopic appearance of TPC-A emulsions stained with nile red dye and observed by using LSCM under different metal ion concentrations. The ionic strength of the solution was prepared by CaCl2 (Ca2+: 10–50 mmol/L) and NaCl (Na+:100-500 mmol/L).
colloidal dispersions (zeta potential) were used to evaluate the emulsifying ability of TPC-A (McClements and Gumus, 2016). With the concentration of TPC-A increasing from 1.0 wt% to 3.0 wt%, the MPD (d32) distribution of the TPC-A emulsion reduced from 2.82 to 0.27 μm, and the absolute value of the zeta potential slightly slightly decreased from 42.3 to 39.3 mV (Fig. 2A). As shown in Fig. 2B, the particle size distribution (PSD) of the emulsions stabilized by 0.5 wt% and 1.0 wt% TPC-A was bimodal distribution, while those of the emulsions stabilized by 1.5 wt%, 2.0 wt%, and 3.0 wt% TPC-A shifted to small particle size with their MPDs (d32) were 0.43 μm, 0.28 μm, and 0.27 μm, respectively. On the tenth day of storage, the emulsion containing 0.5 wt% and 1.0 wt% have demulsification with the absolute value of the zeta potential declining from 50.0 to 15.9 mV, and 46.9 to 17.8 mV respectively. The emulsion stabilized using 1.5 wt%, 2.0 wt% and 3.0 wt%
increased, the interfacial tension decreased rapidly. The interfacial tension of the 0.5 wt% TPC-A system dropped from 26.98 mN/m to 10.48 mN/m (Fig. 1). This process was due to the adsorption of TPC on the oil-water interface, which reduced the interfacial tension of oil and water and the reduction in interfacial tension was useful in tentatively determining the properties of TPC-A as a surfactant. Therefore, followup experiments using TPC-A as an emulsifier were performed in order to explore its emulsifying properties. 3.3. Effect of TPC-A concentration on emulsion formation The stability of the emulsion is usually characterized by its MPD (d32) distribution and ion-carrying capacity (Xu et al., 2016). The distribution uniformity of MPD (d32) and the electro-kinetic potential in 5
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3.4. Effect of storage time on emulsion stability Emulsion storage stability plays an important role in the shelf life of commercial food and beverage products. Based on the above effect of TPC-A concentration on emulsion formation described above, the storage of emulsions stabilized using 2.0 wt% TPC-A at 25 ℃ for 10 days was further investigated. Day 0 indicated the initial time of emulsion preparation. The MPD (d32) distribution of TPC-A emulsion and the change in MPD (d32) with time at 25 °C are shown in Fig. 4. During the storage time of 10 days, MPD (d32) slightly increased from 0.31 μm to 0.37 μm, and the absolute value of the zeta potential varied from 44.9 mV to 41.4 mV. The bimodal distribution of the two sizes was observed until the 10th day, and demulsification was observed on the 23rd day. During the 10 days, the particle size and zeta potentials showed minor changes, indicating that the emulsions stabilized using TPC-A have good stability. 3.5. Effect of metal ions on the stability of TPC-A emulsions The emulsion will be practically put to use in different solutions having a wide range of ionic strengths, and in different types of food products with various pH values. Therefore, it is necessary to investigate the stability of the emulsion when exposed to high ionic strength and/or acidic or alkaline environments. The high concentration of metal ions is inversely related to the stability of the emulsion. When the concentration of NaCl in these emulsions changed from 0 mol/L to 0.50 mol/L, the MPD (d32) increased from 0.33 to 2.77 μm and the absolute value of the zeta potential varied from 41.0 to 37.7 mV (Shown in Fig. 5A). When the sodium ion concentration is higher than 0.2, the shoulder-shape peak appeared in the particle size distribution of the emulsion(Shown in Fig. 5B).With the CaCl2 concentration increasing from 0 mol/L to 0.05 mol/L, the emulsion MPD (d32) increased from 0.33 μm to 5.21 μm as well as the absolute value of the zeta potential changing from 41.0 to 37.0 mV (Fig. 6A), and the peaks shifted to the large particle size distribution. In addition, the particle size distributions of TPC-A emulsions containing above 0.02 mol/L CaCl2 showed bimodal distribution (Fig. 6B). The metal ions destroys the lyophilic protective layer, resulting in emulsion droplet polymerization and demulsification (Goldstein et al., 2007). In addition, confocal microscopy results confirmed that the microstructure of the emulsion exposed to the highest metal ion concentration changed significantly during the 10-day storage period (Fig. 7).
Fig. 8. Mean particle diameter (d32) and zeta potential(A) and particle size distribution (B) of emulsion stabilized by 2% TPC-A during storage at 25℃. The assay was performed by using a Zetasizer Nano-ZS particle diameter and potentiometric analyzer.
TPC-A maintained stability with the variation range of zeta potential absolute value from 42.2 to 39.3 mV, 44.8 to 41.3 mV, and 42.5 to 40.2 mV, respectively (Fig. 3).The stability of the emulsion is positively correlated with its absolute value of zeta potential (Ichikawa et al., 2006; Wang et al., 2001). A complex in which a polysaccharide is either covalently or noncovalently bound to a protein is usually used as an emulsion stabilizer in order to synergistically exert the emulsification activity of the protein and enhance the stability of the polysaccharide over a wider range of pH and ionic strength (Krstonosic et al., 2015). TPC-A contained covalently bound protein. It is thought that the binding protein in TPC is coated by the carbohydrate chain and forms a hydrophobic "core"(Chen et al., 2017b, 2018). Polysaccharides and proteins confer hydrophilic and hydrophobic amphiphilic properties of TPC, respectively. This polysaccharide and protein conjugate can endue an emulsification function to TPC.
3.6. Effect of pH on the stability of TPC-A emulsions The initial pH value of the emulsion was 7.0, and different ratios of acid (HCl) and alkali (NaOH) were added to obtain a range of different pH values from 2.0 to 10.0. When the pH value changed from 2.0 to 6.0, MPD decreased from 9.17 μm to 0.31 μm, while zeta potential varied from 2.3 mV to −40.50 mV(Fig. 8). With pH varying from 7.0 to 10.0, MPD (d32) of these emulsions was less than 0.33 μm, and their absolute value of the zeta potential ranged from 40.52 to 41.53 mV. Thus, the
Table 1 Content and retention rate of EGCG and EGC in the emulsion stabilized by 2.0 wt% TPC-A under storage for 10 days at 25 ℃ (n = 3).
1 day 10 day Retention Rate
TPC-A emulsion EGCG3 (μg/mL)
TPC-A emulsion EGCG-5 (μg/mL)
TPC-A emulsion EGCG-8 (μg/mL)
EGCG-5 (μg/mL)
TPC-A emulsion EGC-3 (μg/mL)
TPC-A emulsion EGC-5 (μg/mL)
TPC-A emulsion EGC-8 (μg/mL)
EGC-5 (μg/mL)
2768.02 ± 26.18 505.70 ± 14.37 18.27%
4429.02 ± 36.37 3271.04 ± 14.37 73.85%
7648.10 ± 19.04 5014.18 ± 19.37 65.42%
4099.31 ± 14.40 351.47 ± 18.35 8.57%
2124.11 ± 20.01 1219.08 ± 8.37 57.41%
4264.53 ± 23.78 1534.96 ± 16.47 35.99%
6964.55 ± 79.85 5018.53 ± 12.76 72.06%
3749.03 ± 12.55 399.53 ± 13.50 10.66%
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emulsion stabilized using TPC-A was more stable in an alkaline solution. The polysaccharide moiety of TPC-A provided pH adaptability of these emulsions.
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3.7. Stability of EGCG and EGC in TPC-A emulsions The emulsions stabilized using 2.0 wt % TPC-A could protect EGCG and EGC from oxidation at 25 ℃ (Table 1). As shown in Fig. 9, all the emulsions containing EGCG or EGC were in a stable state during the 10day storage time, while the EGCG and EGC in PBS solution oxidized and appeared brown. The stable emulsification of TPC-A significantly increased the retention of EGCG and EGC. The retention rate of EGCG increased 2–10-fold, and the retention rate of EGC increased by 4–7fold. The emulsion containing TPC-A exhibited long-term stability and a protective effect on EGCG and EGC. The structural mechanism of TPC-A emulsion loading and protection of catechins remains to be further studied. The results of our research provide a theoretical basis for the wide application of TPC-A emulsion. 4. Conclusions The aim of this study was to investigate the emulsification ability of TPC-A, the stability of the emulsion, and the effect of the emulsion on the storage of major catechins (EGCG and EGC). Our results showed that TPC-A had significant surface activity and could effectively form a relatively stable emulsion, even at low TPC-A concentrations. This emulsion had good long-term stability, remained stable even after storage for 10 days at 25 °C, and prevented droplet aggregation and phase separation. The emulsion stabilized using low concentrations of TPC-A could effectively prolong the storage time of catechins and reduce the loss of catechins. The result indicates that TPC-A has the potential to be used as a novel emulsifier in the food, cosmetic, and pharmaceutical industry. In summary, this work has discovered a new application area for TPC-A. Acknowledgements This research was funded by the National Natural Science Foundation of China (grant number31871813), the Open Research Fund Program of the Key Laboratory of Cosmetic(Beijing Technology and Business University), China National Light Industry (grant number KLC-2018-YB6), and Beijing Advanced Innovation Center for Food Nutrition and Human Health (grant number 20161012). References Brocca, P., Rondelli, V., Corti, M., Del, F.E., Deleu, M., Cantù, L., 2018. Interferometric investigation of the gas-state monolayer of mono-rhamnolipid adsorbing at an oil/ water interface. J. Mol. Liq. 266, 687–691. Chen, X., Lin, Z., Ye, Y., Zhang, R., Yin, J., Jiang, Y., Wan, H., 2010. Suppression of diabetes in non-obese diabetic (nod) mice by oral administration of water-soluble and alkali-soluble polysaccharide conjugates prepared from green tea. Carbohydr. Polym. 82 (1), 28–33.
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