SSRN Id4375852

1 Extraction optimization of tea saponins from Camellia oleifera seed meal with
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2 deep eutectic solvents: composition identification and properties evaluation
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3 Xinjin Yu, Zhimei Zhao, Xiaoli Yan, Jianhua Xie, Qiang Yu, Yi Chen*
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5 State Key Laboratory of Food Science and Technology, Nanchang University,
6 Nanchang 330047, People’s Republic of China
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9 *To whom correspondence should be addressed.
10 Professor Yi Chen, Ph.D.; E-mail address: chenyi-417@163.com

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11 Tel.: 0791-88304449. Fax: 0791-88304449.

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4375852
13 Abstract
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14 Camellia oleifera seed meal is an important source of tea saponins. This study established an
15 environment-friendly and efficient technology based on deep eutectic solvents (DESs) to extract
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16 tea saponins from Camellia oleifera seed meal. The solvent consisting of choline chloride and
17 methylurea was screened as optimal DES. Under the optimal extraction conditions obtained by
18 response surface methodology, the extraction yield of tea saponins reached 94.36 mg/g, which
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19 increased by 27% compared with that of ethanol extraction, while the extraction time reduced by
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20 50%. Analysis through UV, FT-IR and UPLC-Q/TOF-MS indicated tea saponins did not alter
21 during DES extraction. Surface activity and emulsification evaluation indicated that extracted tea
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22 saponins could reduce interfacial tension at the oil-water interface with excellent foamability and
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23 foam stability, furthermore, they could form nano-emulsions (d32 < 200 nm) with excellent
24 stability. This study provides a theoretical basis for the efficient extraction and in-depth
25 development of tea saponins.

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26 Keywords: tea saponins, Camellia oleifera seed meal, DESs, response surface methodology,
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27 UPLC-Q/TOF-MS, properties evaluation.
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30 1. Introduction
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31 Tea saponins are mixtures of oleanane-type pentacyclic triterpenoid saponins with similar
32 structures (Yu et al., 2022). As natural glycosides, tea saponins are widespread in the leaves,
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33 flowers and fruits of Camellia species (Yang et al., 2020). Tea saponins contain both hydrophobic
34 and hydrophilic groups, which are natural nonionic surfactants with excellent foaming,
35 emulsifying, dispersing and wetting properties (Zhao et al., 2020). In recent years, the medicinal
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36 and health-related benefits of tea saponins, such as their antioxidant (Li et al., 2014), anticancer
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37 (Zhao et al., 2015), antibacterial (Yu et al., 2022), hypoglycemic (Di et al., 2017), and
38 anti-inflammatory effects (Yang et al., 2014), have increasingly drawn the attention of researchers.
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39 Because of these advantages, tea saponins are widely employed in daily chemical, food, and
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40 pharmaceutical fields.
41 Camellia oleifera (C. oleifera) stands as one of the four major woody oil crops worldwide,
42 which has a history of cultivation in China for more than 2,000 years (Tang et al., 2021; Zhao et
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43 al., 2020). C. oleifera seed meal is generated as a residue after the extraction of oil from C.
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44 oleifera seed, which contains abundant saponins (accounting for > 10%) (Guo et al., 2018).
45 According to the incomplete statistics of China, the production of C. oleifera seed meal is more
46 than 690,000 tons each year (Zhao et al., 2020). However, during past production, C. oleifera seed
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47 meal was usually discarded or used as low-value fertilizer, which was a great waste of C. oleifera
48 resources. It is essential for the efficient extraction of tea saponins out of C. oleifera seed meal to
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49 improve the commercial value of C. oleifera seed. Currently, tea saponins are usually extracted
50 using organic solvents. However, organic solvents are toxic, costly to the treatment of waste
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51 liquids and harmful to the environment (Lin et al., 2022). With the increasing awareness of
52 environmental protection, "green extraction" has gradually become the focus of research.
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53 Therefore, it is still necessary to explore a preferred method to extract tea saponins in a green way.
54 Deep eutectic solvents (DESs) are fluid systems that consist of eutectic mixtures of several
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55 natural components (Tang et al., 2021). DESs are considered to be new green solvents because
56 they have the advantages of easy synthesis, inexpensive, degradable, environment-friendly, and
57 toxic-free (Chen et al., 2022). In recent years, DESs have been widely used to extract biologically
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58 active ingredients from plants, including polysaccharides (Cai et al., 2020), phenolics (Cvjetko
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59 Bubalo et al., 2016), flavonoids (Shang et al., 2018), and alkaloids (Jiang et al., 2019).
60 Unfortunately, there are fewer about the use of DESs for tea saponin extraction (Tang et al.,
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61 2021).
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62 Based on the above, this study optimized the conditions for the tea saponins extraction by
63 response surface methodology (RSM) and established an environmentally friendly and efficient
64 DES-based technology to extract tea saponins from C. oleifera seed meal. The structure and
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65 composition of DES-extracted tea saponins were analyzed by ultraviolet (UV), Fourier transform
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66 infrared (FT-IR) spectrum and ultra-performance liquid chromatography-quadrupole-time of
67 flight-mass spectrometry (UPLC-Q/TOF-MS). Furthermore, the surface activity and
68 emulsification of the tea saponins that were extracted using DES from C. oleifera seed meal were
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69 investigated for the first time. The research results will contribute to the efficient extraction and
70 in-depth development of tea saponins, as well as to the high-value utilization of C. oleifera seed
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71 meal.
72 2. Materials and methods

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73 2.1. Chemicals and reagents
74 Vanillin, tea saponin standard (S9981, ≥98%), AB-8 macroporous resin, Tween 20, and
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75 Tween 80 were provided by Shanghai Maclean Co. (Shanghai, China). Choline chloride, urea,
76 methylurea, N, N'-dimethyl urea, acetamide, hydrochloric acid, Nile red, and potassium bromide
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77 were provided by Shanghai Aladdin Biochemical Technology Co.(Shanghai, China). Methanol
78 and acetonitrile of chromatographic grade were purchased from Sigma-Aldrich (St. Louis, MO,
79 USA). Petroleum ether, concentrated sulfuric acid, anhydrous ethanol, sodium hydroxide, and
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80 sodium chloride were purchased from Xilong Science Co. (Guangdong, China). Unless
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81 specifically stated, all chemicals were of analytical grade.
82 2.2. Samples er
83 C. oleifera seed was produced in Jiangxi, China. After being dried at 60 ℃ for 12 h in a hot
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84 air drying oven, C. oleifera seed was fully crushed in a high-speed grinder. After passing through
85 a 60-mesh sieve, the fat in the powder was removed with petroleum ether. The defatted C. oleifera
86 seed meal was stored in a closed desiccator after drying.

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87 2.3. Extraction and isolation of tea saponins

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88 2.3.1. Preparation and screening of DESs
89 The preparation of DESs was referred to the previous method with slight modifications
90 (Duan et al., 2016). Four DESs (abbreviated as Chcl-Ur, Chcl-Met, Chcl-Dme, Chcl-Ace) were
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91 respectively prepared with choline chloride (Chcl) as the hydrogen bond acceptor (HBA) and
92 various amides, including urea (Ur), methylurea (Met), N, N'-dimethyl urea (Dme), and acetamide
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93 （Ace）, as the hydrogen bond donors (HBDs) at a molar ratio of 1:1, and 20% water was added
94 to reduce the viscosity of the system after magnetic stirring at 80 °C for 1 h.

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95 1 g of sample and 20 ml of DESs were mixed and extracted by stirring in the water bath at 60
96 ºC for 1 h. After extraction, the extracts were centrifuged at 4800 rpm for 15 min and the
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97 supernatant was collected to determine the tea saponins content. The DES with the highest tea
98 saponins extraction yield was selected for subsequent experiments.
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99 2.3.2. Extraction procedure
100 According to the above extraction method, the following five factors were each individually
101 examined for their impact on the yield of tea saponins extraction: DES molar ratio, water content,
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102 liquid-material ratio, extraction temperature, and extraction time.
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103 2.3.3. Experimental design
104 The Box-Behnken design-based RSM was employed to optimize the extraction conditions to
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105 obtain the highest yield of tea saponins from C. oleifera seed meal. According to the single-factor
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106 experimental results, extraction temperature (A), water content (B), and liquid-material ratio (C)
107 that had greater impact on the tea saponin yield were chosen to conduct a three-factor and
108 three-level response surface design, and the tea saponin yield (Y) was utilized for the response
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109 value.
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110 2.3.4. Conventional extraction
111 1 g of sample and 20 ml of 60% ethanol were mixed and extracted by stirring in the water
112 bath at 70 ºC for 2 h. After extraction, the extracts were centrifuged at 4800 rpm for 15 min and
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113 the supernatant was collected to determine the tea saponins content.
114 2.3.5 Isolation and purification

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115 Isolation and purification of the extract were carried out by modified macroporous resin
116 column chromatography. The extract was dialyzed, concentrated and lyophilized to obtain the tea
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117 saponins crude extract. After being dissolved in distilled water and adsorbed on AB-8
118 macroporous resin, the crude extract was successively eluted with distilled water, 0.2% sodium
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119 hydroxide, and distilled water. Subsequently, the tea saponins part was separated by eluting with
120 90% ethanol, and the tea saponins purified were finally produced by concentration and
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121 lyophilization.
122 2.4. Determination of total tea saponins content
123 The total saponin content of tea was determined by referring to the method from Li et al. with
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124 a slight modification (Li et al., 2014). 0.5 ml of 8% vanillin-anhydrous ethanol solution and 5 ml
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125 of 77% sulfuric acid solution were added sequentially to 0.6 ml of the extract in an ice-water bath.
126 Then the mixture was retained in the 60 °C water bath for 15 min. After cooling for 10 min, the
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127 absorbance was measured at 550 nm. The results were compared with the standard curves (r2 =
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128 0.9994) of tea saponin standards (0.2, 0.4, 0.6, 0.8, 1.0 mg/mL) to calculate the total tea saponins
129 content.
130 2.5. Identification of the extracted tea saponins

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131 2.5.1. UV analysis

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132 The sample was solubilized in methanol and scanned on a UV-vis spectrophotometer
133 (TU-1900, PGENERAL, Beijing, China) in the range of 190-600 nm.
134 2.5.2. FT-IR analysis

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135 The sample was mixed with potassium bromide crystals after drying. After being ground and
136 pressed, the mixture was scanned on an FT-IR spectrometer (Nicolet iS50, Thermo Fisher
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137 Scientific, USA) between 400 cm-1 and 4000 cm-1.
138 2.5.3. Identification of tea saponins by UPLC-Q/TOF-MS

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139 Agilent 1290 UPLC system coupled to Agilent 6538 Q/TOF mass spectrometer (Agilent,
140 Santa Clara, CA, USA) was used in the identification of tea saponins. An Agilent Eclipse
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141 XDB-C18 column (2.1 mm × 100 mm, 1.8 μm, Agilent Technologies, USA) was used for
142 separation at 35 °C. The mobile phase was composed of water with 0.1% formic acid (A) and
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143 acetonitrile containing 0.1% formic acid (B) with a flow rate of 0.25 mL/min. The injection
144 volume was 5 μL. The elution gradient was as follows: 0-3 min, 95%-82% (A); 3-8 min,
145 82%-65% (A); 8-11 min, 65%-63% (A); 11-15 min, 63%-59% (A); 15-25 min, 59%-0% (A);
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146 25-35 min, 0%-59% (A). The mass spectra were obtained under negative ion mode employing
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147 electrospray ionization over the m/z 100–1700 range. The following operating parameters were
148 used: spray voltage of 2.5 kV, sheath gas of 45 arb, auxiliary gas of 15 arb, the capillary
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149 temperature at 320 °C, scan modes with full MS (resolution 70,000) and MS/MS (resolution
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150 17,500).
151 2.6. Determination of the surface activity of the extracted tea saponins
152 2.6.1. Measurement of interfacial tension

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153 Following the method of Zhu et al. (Zhu et al., 2017) a slight modification, the oil-water
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154 interfacial tension of tea saponins was determined at 25 ℃ using the contact angle meter (OCA25,
155 Data Physics Instruments GmbH, Germany). A single drop (15 μL) of the aqueous tea saponins
156 solution was added to a quartz cuvette containing corn germ oil using a syringe. A high-speed
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157 camera was used to record the droplet morphology change process, and the interfacial tension was
158 calculated by fitting the Laplace-Young equation.

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159 2.6.2. Determination of foamability and foam stability
160 The hand-cranking method was used to determine the foamability and foam stability (Yang et
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161 al., 2020). Solutions of extracted tea saponins with different mass fractions and Tween 20, Tween
162 80 with mass fractions of 1% were formulated and emptied into a stoppered measuring cylinder.
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163 Afterward, the stopper was closed tightly and the cylinder was turned upside down 30 times. The
164 foam heights at 0 min and 15 min of standing were recorded as the results of foamability and foam
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165 stability measurements, respectively.
166 2.7. Determination of emulsifying properties of the extracted tea saponins
167 2.7.1. Emulsion preparation
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168 Different concentrations of surfactants were dissolved in pure water and mixed continuously
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169 for two hours at room temperature. The solutions were kept at 4 °C for 12 hours as aqueous phase
170 to ensure full dissolution and hydration. After that, the crude emulsions were created by adding
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171 corn germ oil to the aqueous phase in a specific ratio (oil phase: aqueous phase = 1:9) and
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172 homogenizing the mixture in a high-speed dispersion emulsifier (T18 Ultra-Turrax, IKA,
173 Germany) at 13000 rpm for 5 min. The desired nanoemulsions were produced after the crude
174 emulsions were homogenized with a high-pressure homogenizer (NCJJ0.007/200, Langfang

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175 General Machinery Manufacturing Co., China).

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176 2.7.2. Particle diameter analysis
177 The particle diameter of emulsions was measured at 25 °C using a zeta potential particle sizer
178 (Zetasizer Nano ZS90, Malvern Instruments, UK).

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179 2.7.3. Microstructure analysis
180 An inverted fluorescence microscope (CKX41, Olympus Corporation, Japan) was used to
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181 characterize the microstructure of emulsions. 1% Nile red was dissolved in isopropanol,
182 thoroughly mixed, and then stored in darkness at 4°C to prepare a lipid stain. To emphasize the
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183 position of the lipid phase, 1 mL of emulsions were colored with 40 μL of the lipid stain before
184 analysis. The fluorescence signal of the Nile red was detected at a wavelength of 488 nm.
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185 2.7.4. Stability testing of emulsion
186 The effect of environmental stresses that might be encountered during industrial production
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187 and processing on the stability of the prepared emulsions was studied.
188 PH: The emulsions were adjusted to various pH values (3, 5, 7, 9, 11) using HCl or NaOH
189 solution, after that they were kept at room temperature for 24 hours before analysis.
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190 Thermal processing: The emulsions were put into test tubes and maintained in water baths
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191 that ranged in temperatures from 40 to 80 °C for 30 min. After that, they were restored to room
192 temperature and kept for 24 hours before being analyzed.

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193 Storage stability: The fresh emulsions were moved to sealed test tubes. Afterward, they were
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194 stored at room temperature for 30 days while being regularly sampled for analysis.
195 2.8. Statistical analysis
196 The extraction conditions were optimized by Design Expert software. All assays were
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197 performed in triplicate except for UPLC-Q/TOF-MS analysis. The data were presented as mean ±
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198 standard deviation. Statistical analysis was conducted by Duncan’s test and ANOVA with SPSS
199 19.0 software.
200 3. Results and discussion

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201 3.1. DESs Extraction
202 3.1.1. Preparation and screening of DESs

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203 In this study, it was discovered that all four DESs prepared were stable and clear viscous
204 liquids. In general, DESs' physicochemical characteristics, such as hydrogen bonding interactions,
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205 viscosity, polarity, and pH, influence their capacity to extract bioactive compounds (Duan et al.,
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206 2016). Due to the easy handling, some water was inserted to decrease the viscosity of DESs. The
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207 four DESs tested in this study were blended with water at 8:2 (w/w). Fig. S1A showed the
208 selection results of the four DESs using a highly polar extractant (60% ethanol) as control. The
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209 comparatively high extraction yield of the control indicated that tea saponins were polar
210 molecules. Among the four DESs, Chcl-Met had the highest yield of 81.51 mg/g, followed by
211 Chcl-Ace of 75.94 mg/g. In addition, Chcl-Dme (72.02 mg/g) and Chcl-Ur (63.47 mg/g) had
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212 lower yields compared to 60% ethanol (74.12 mg/g). The results above suggested that the
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213 capability of DESs to extract tea saponins varied depending on the type of HBD, which was
214 probably due to the fact that DESs with different HBD can form intermolecular hydrogen bonds of
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215 different strengths with tea saponins, resulting in different extraction capabilities (Duan et al.,
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216 2016). Therefore, Chcl-Met was selected as the optimal DES for subsequent experiments.
217 3.1.2. Analysis of single-factor experimental
218 The properties of DESs are mainly dependent on the types and molar ratios of HBA and
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219 HBD. As shown in Fig. S1B, the maximum extraction yield was reached at a molar ratio of 1:5
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220 and then the extraction yield decreased. To a certain extent, it is advantageous to the extraction of
221 tea saponins to increase the amount of Met because it can reduce the viscosity and surface tension
222 of DESs, enhance diffusion and improve the mass transfer effect. However, the excessive increase
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223 of Met will compete with tea saponins for the chloride anion, causing a significant steric
224 hindrance, which is detrimental to the interaction between tea saponins and Chcl, and thus leads to
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225 a decrease in extraction yield (Dong et al., 2021). Therefore, the subsequent experiments were
226 performed with Chcl-Met at a molar ratio of 1:5.

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227 The viscosity of DESs can impede mass-transfer extraction from plant matrix. The viscosity
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228 and polarity of DESs can be effectively adjusted by changing the water content (Liu et al., 2022a).
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229 As presented in Fig. S1C, the maximum extraction yield was achieved when the water content of
230 Chcl-Met was 20%, which was similar to the results of Lei et al. (Lei et al., 2022). The results
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231 were caused by the fact that a small increase in water content can decrease the viscosity of DESs
232 and enhance the solubility of tea saponins. However, excessive water content (>20%) can restrict
233 or destroy the interaction between DESs and tea saponins, reducing the extraction yield (Liu et al.,
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234 2022b). So Chcl-Met with 20% water content was used for the subsequent experiments.
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235 It is well known that the amount of extraction solvent is an important factor affecting the
236 extraction yield. The extraction yield of tea saponins increased with the increase of extraction
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237 solvent and reached a peak at the liquid-material ratio of 50 mL/g, after that it decreased
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238 significantly (Fig. S1D). The trend was in agreement with the results of Shirsath et al. (Shirsath et
239 al., 2017). It was likely due to the excess of solvent promoting the dissolution of tea saponins
240 along with that of impurities as well. Impurities will prevent tea saponins from dissolving,
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241 resulting in a decrease in extraction yield (Dong et al., 2021). As a consequence, the
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242 liquid-material ratio for subsequent experiments was selected at 50 mL/g.
243 Temperature is also an important factor affecting extraction yield. As shown in Fig. S1E,
244 with the increase in temperature, the extraction yield of tea saponins followed a trend of initially
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245 increasing and then declining, reaching its maximum at 60 °C. The results were due to the fact that
246 raising the temperature can decrease the viscosity of DESs and promote solute diffusion (Chen et
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247 al., 2022). However, the structure of tea saponins can be destroyed by excessive heat, which will
248 cause a decrease in the yield of extraction (Yang et al., 2020). Therefore, the subsequent
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249 experiments were performed with an extraction temperature of 60 °C.
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250 In general, the content of the extracts improves as the extraction time increases, and enough
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251 time can ensure that the extracts dissolve completely (Yue et al., 2022). As shown in Fig. S1F, the
252 lower extraction yield for a shorter extraction time was attributed to the incomplete dissolution of
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253 tea saponins in the solvent. There was an obvious increase in the extraction yield when the
254 extraction time was increased to 60 min, after which there was no longer a significant difference
255 because tea saponins had completely dissolved. In consideration of energy consumption, 60 min
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256 was considered as the optimal extraction time.
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257 3.1.3. Analysis of response surface model
258 Based on the results of single-factor experiments, the Box-Behnken experimental design was
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259 employed for investigating the effects of the three factors on tea saponins extraction yield. The
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260 experimental factors variables and levels used for optimization were shown in Table S1. The
261 results of the extraction of tea saponins according to the designed experimental protocol were
262 shown in Table S2. The experimental data were analyzed using RSM, and Table 1 provided a
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263 summary of the model's applicability. The second-order polynomial equation in below showed a
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264 correlation between the yield of tea saponins and each factor.
Y = 93.22 ― 2.57A + 7.27B ― 1.00C ― 6.43𝐴2 ― 9.08𝐵2 ― 5.75𝐶2 + 0.34𝐴𝐵 ― 1.32𝐴𝐶

265 + 2.34𝐵𝐶
266 The R2 value of the equation was 0.9711, which indicated that the equation fitted well and it
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267 was a significant correlation between predicted and actual values. According to Table 1, the
268 F-value and P-value of the model were 26.17 and 0.0001 respectively, indicating the model was
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269 significant. The model suggested that A, B, A2, B2 and C2 exhibited significant effects on the
270 extraction yield of tea saponins (P< 0.05). Generally, the lack of fit test was used to measure the
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271 validity of the goodness of fit (Jang et al., 2017). The F-value and P-value of the lack of fit were
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272 3.59 and 0.1243 respectively, indicating the non-experimental factors did not affect the
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273 experimental results. The insignificance of the lack of fit implied that the model fitted data well.
274 The interactions of different experimental variables on the effect of tea saponins extraction yield
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275 were visualized in the three-dimensional and two-dimensional response surface plots shown in
276 Fig. 1. Combined with the ANOVA of the model, it was found that the interaction between the
277 water content and the liquid-material ratio had the strongest effect on the tea saponins extraction
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278 yield. Therefore, the correlation between the experimental factors variables and tea saponin
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279 extraction yield could be adequately represented by this model.
280 3.1.4. Validation of optimal extraction conditions

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281 The optimal conditions to extract tea saponins from C. oleifera seed meal using Chcl-Met
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282 were determined by the regression equation of RSM. The optimal conditions obtained were as
283 follows: 27.97% water content, 50.16 mL/g liquid-material ratio, and 58.09 °C extraction
284 temperature. Under these conditions, the extraction yield of tea saponins was expected to be 94.91
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285 mg/g. The extraction conditions used in the practical experiment were: 28% water content, 50
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286 mL/g liquid-material ratio, and an extraction temperature of 60 °C. Under these conditions, the
287 experimental value of tea saponins extraction yield was 94.36 ± 1.23 mg/g. It was remarkably near
288 between the experimental values and the predicted values, confirming the validity of the model.
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289 3.1.5. Comparison of DES extraction and traditional solvent extraction
290 The extraction yield of tea saponins by using traditional ethanol extraction was 74.30 mg/g.
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291 Compared with traditional ethanol extraction, DES extraction not only increased the extraction
292 yield of tea saponin by 27% but also reduced the extraction time by 50%. In response to the
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293 problems of traditional organic reagent extraction, such as time-consuming, high cost, complicated
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294 operation, volatile reagents and high toxicity, the optimized DES extraction method has the
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295 advantages of short time, low cost, high efficiency, green and non-polluting.
296 3.2. Identification of the extracted tea saponins
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297 After purification, the structure and molecular formulas of the extracted tea saponins were
298 determined by UV, FT-IR and UPLC-Q/TOF-MS analysis. The DES extract showed a
299 characterized peak at 215 nm on the UV spectra (Fig. 2A), indicating the main component of the
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300 extract was saponins, which was in agreement with the findings of Yuan et al. (Yuan et al., 2018).
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301 The absorption peak was attributed to α and β conjugated double bonds in angelic acid.
302 Furthermore, the inconspicuous peak of the extract at 280 nm corresponded to cinnamic acid or
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303 implied flavonoid impurities (Tang et al., 2021). These substances were non-toxic and did not
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304 affect the usage of tea saponin.
305 FT-IR spectra provide information on molecular functional groups, which can be used to
306 identify some chemical compounds (He et al., 2013). The FT-IR spectra of tea saponins crude
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307 extract, tea saponins purified and tea saponin standard was shown in Fig. 2B. Through comparison
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308 and analysis, the characteristic absorption peak near 3420 cm-1 owed to the stretching vibration of
309 O-H, and the stretching vibration band of crude extract was significantly broader here. The
310 stretching vibrational peak of methyl (-CH3) and methylene (-CH2-) was observed near 2930 cm-1.
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311 The absence of characteristic absorption peak at wavenumbers from 2500 cm-1 to 1900 cm-1
312 indicated that there was no accumulation of triple or double bonds (Yuan et al., 2018). The
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313 absorption peaks from 1718 cm-1 to 1600 cm-1 suggested the existence of a carbonyl group (C=O).
314 The characteristic absorption peaks near 1420 cm-1 and 1386 cm-1 were owed to the symmetric
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315 and antisymmetric bending vibrations of the methyl group (-CH3). A clear absorption peak of
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316 bending vibrations in the C-O-H plane was observed near 1260 cm-1. The peaks observed near
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317 1070 cm-1 were due to the C-O-C stretching vibration. The tea saponins purified had the same
318 infrared characteristic peaks as the standard and were very similar to the infrared spectra of tea
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319 saponins reported in previous studies (He et al., 2013; Tang et al., 2021). As a result, it was
320 certainly confirmed that the Chcl-Met extract in this study was tea saponins, which belonged to
321 the mixtures of oleanane pentacyclic triterpene saponins.
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322 According to the previous study (Wu et al., 2019), a stronger signal intensity of saponin was
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323 achieved in the negative ionization mode than in the positive ionization mode. Therefore, the
324 composition of extracted tea saponins was further analyzed using UPLC-Q/TOF-MS with
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325 negative ionization mode. The total ion chromatogram (TIC) of extracted tea saponins was
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326 presented in Fig. 2C. Table 2 listed the retention time, molecular ion, fragments, molecular
327 formula and proposed compounds. A total of 20 saponins, including Teaseedsaponins,
328 Theasaponins, Oleiferasaponins, Assamsaponins, Floratheasaponins, and an unknown saponin

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329 were tentatively identified by comparing accurate masses and MS/MS spectroscopy data with
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330 previous reports and saponin mass spectrometry database（http://47.92.73.208:8082）(Guo et al.,
331 2018; Wu et al., 2019).
332 Tea saponins are oleanane pentacyclic triterpene saponins. Depending on the level and type
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333 of glycosylation, their primary structures are aglycones or glycosides with various sugars (Tang et
334 al., 2021). To begin with, 10 peaks possessing uniquely molecular ions [M-H]- were tentatively
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335 identified. Peak 1 showed molecular ion [M-H]- at m/z 1189.5702 and fragment ions at m/z 1057
336 (-132 Da), 895 (-162-132 Da) (Fig. S2A). These information were in agreement with that of
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337 theasaponin A1 (21-O-angeloyltheasapogenol A 3-O-β-D-galactopyranosyl
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338 (1→2)-[β-D-xy-lopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→3)]-β-D-glucopyranosiduronic
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339 acid), whose molecular formula was C57H90O26. The counterpart fragments were
340 [M-H-arabinopyranosyl]-, [M-H-galactopyranosyl-arabinopyranosyl]-. Peak 5 showed molecular
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341 ion [M-H]- at m/z 1219.5820 and fragment ions at m/z 1057 (-162 Da), 1039 (-162-18 Da), 925
342 (-162-132 Da) and 587 (-162-132-162-176 Da) (Fig. S2B). These information were in agreement
343 with that of theasaponin A4 （ 21-O-angeloyltheasapogenol A
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344 3-O-β-D-galactopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→3)]-β-
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345 D-glucuronopyranoside ） , whose molecular formula was C58H92O27. The counterpart fragments
346 were [M-H-glucopyranosyl]-,

er [M-H-glucopyranosyl-H2O]-,
347 [M-H-glucopyranosyl-arabinopyranosyl]- and

pe
348 [M-H-glucopyranosyl-arabinopyranosyl-galactopyranosyl-glucuronopyranosyl]-. Likewise, peaks
349 15, 17, 18, 19, and 20 were tentatively identified as floratheasaponin A, teaseedsaponin I,
350 theasaponin A7, teaseedsaponin D, and teaseedsaponin E, respectively. In addition, there were
ot
351 multiple saponins with structure and mass spectral data consistent with the information of peaks 6
tn
352 and 7, so peak 6 was probably theasaponin G1/G2 or assamsaponin A/E, and peak 7 was probably
353 theasaponin C1/B5. Peaks 3 and 8 had similar molecular ions and the same fragment ions at m/z
354 1099 (-162 Da), 1081 (-162-18 Da), and 949 (-162-18-132 Da). These information were in
rin
355 agreement with that of theasaponin A5/A6, whose molecular formula was C60H94O28. According to
356 the results of Wu et al. (Wu et al., 2019), theasaponin A6 had a smaller Log P value than that of
ep
357 theasaponinA5, indicating that theasaponin A6 was more hydrophilic and supposed to be eluted
358 earlier. Therefore, peak 3 and peak 8 were tentatively identified to be theasaponin A6 and
Pr
359 theasaponin A5, respectively. The molecular ions of peaks 4, 14 and 15 were similar. Peaks 14 and
17
360 15 possessed the same fragment ions at m/z 1055 (-132 Da), 1025 (-162 Da), 1007 (-162-18 Da),
ed
361 and 875 (-132-162-18 Da), however peak 4 lacked any at m/z 1007 and 875. The mass spectral
362 data of peaks 14 and 15 were consistent with those of camelliasaponin A1/A2, and the mass
iew
363 spectral data of peak 4 were consistent with oleiferasaponin D1, with the molecular formula
364 C58H92O25. Therefore, peaks 14 and 15 were preliminarily identified to be camelliasaponin A1/A2
365 and peak 4 was preliminarily identified to be oleiferasaponin D1. The molecular ions [M-H]- of
v
366 peaks 9, 11, 12 and 16 were at m/z 1201.5707-1201.5760 and they possessed the same fragment
re
367 ions at m/z 1069 (-132 Da), 1039 (-162 Da), 1021 (-162-18 Da), 907 (- 132-162 Da). It was found
368 that camelliasaponin B1/B2, theasaponin H1, and oleiferasaponin D2/D3 possessed corresponding
er
369 molecular and fragment ions by comparison with literature and database, but the number of these
pe
370 saponins were greater than the number of peaks, so it was impossible to determine the saponin
371 corresponding to each peak. Peak 2 showed molecular ion [M-H]- at m/z 1217.5644 and fragment
372 ions at m/z 1037（-162-18 Da）、887（-162-132-18-18 Da）、761（-162-132-162 Da）. The only

ot
373 known saponin in C. oleifera seed that possessed the corresponding molecular weight was
tn
374 theasaponin F1, but its structure was inconsistent with the above information. So it was supposed
375 to be a new saponin, and its oligosaccharide moiety was presumed to be
376 -β-D-galactopyranosyl-(1→2)-[β-d-glucopyranosyl-(1→2)-α-l-arabinopyranosyl-(1→3)]-β-d-gluc
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377 uronopyranosyl based on the fragment ions. Its specific structure is required to be further
378 characterized through NMR. It was reported that more than 70 saponins have been isolated from
ep
379 different species of Camellia sinensis seeds (Guo et al., 2018). In this study, 20 saponins were
380 tentatively identified by UPLC-Q/TOF-MS in the extracted tea saponins, which have also been
Pr
381 reported in previous studies on Camellia sinensis seeds (Wu et al., 2019). Furthermore, a greater
18
382 number of saponins were identified in this study compared to the results of tea saponins extracted
ed
383 with DES by Tang et al. (Tang et al., 2021).
384 3.3. Surface activity of the extracted tea saponins
iew
385 3.3.1. Interfacial tension
386 Tea saponins contain a hydrophobic aglycone fraction as well as a hydrophilic carbohydrate
387 fraction, which are natural surfactants (Zhu et al., 2019). The capacity of surfactants to prepare
v
388 and stabilize emulsions depends significantly on their interfacial characteristics. It is well known
re
389 that a lower interfacial tension value is beneficial for emulsification because less energy is
390 required to produce smaller droplets, which increases the stability of the emulsions (Zhu et al.,
er
391 2017). Therefore, the interfacial tension of extracted tea saponins at the oil-water interface was
pe
392 determined to better understand the interfacial properties of the extracted tea saponins. As shown
393 in Fig. 3A, the interfacial tension gradually decreased with the increase of the extracted tea
394 saponins concentration. Finally, the surface tension tended to stabilize due to the saturation of the
ot
395 interface. The interfacial tension of the 0.05% extracted tea saponins solution was only 6.5 mN/m,
tn
396 which was similar to the results of Zhu et al. (Zhu et al., 2019). The above results indicated that
397 the extracted tea saponins could effectively reduce the interfacial tension at the oil-water interface.
398 3.3.2. Foamability and foam stability

rin
399 Foamability and foam stability are important properties of surfactants. The interfacial tension
400 of the solution is highly dynamically reduced due to the presence of surfactant, which is favorable
ep
401 for the formation of foam. As shown in Fig. 3B, the foamability of extracted tea saponins was
402 positively correlated with its concentration. The change in its foamability tended to level off after
Pr
403 the concentrations were higher than 0.05%, which was similar to the results of Yang et al. (Yang
19
404 et al., 2020). In addition, Compared to the same concentration of Tween 20 and Tween 80, the
ed
405 initial foam height of the extracted tea saponins was the highest (7.8 cm), and the foam height
406 decreased the least (0.2 cm) after 15 min, indicating that the extracted tea saponins possessed
iew
407 better foamability and foam stability (Fig. 3C). In summary, the extracted tea saponins were
408 surfactants with strong foamability and foam stability.
409 3.4. Emulsifying properties of the extracted tea saponins
v
410 3.4.1. Effect of surfactant type and concentration on emulsions formation
re
411 As shown in Fig. 4A, the mean particle diameter (d32) of three emulsions significantly
412 decreased in the surfactant concentrations range of 0.5-1.5 wt%, especially for emulsions
er
413 containing Tween 80. This was attributed to the fact that are insufficient surfactants to protect all
pe
414 the oil droplets at low surfactant concentrations resulting in small oil droplets colliding and
415 aggregating together, increasing the mean particle diameter. In surfactant-limited regimes, the
416 mean particle diameter of emulsions was mainly dependent on the type and concentration of the
ot
417 surfactant (Zhu et al., 2019). The mean particle diameter tended to stabilize at surfactant
tn
418 concentrations higher than 1.5 wt%. The mean particle diameter of emulsions was limited by the
419 strength of the homogenizer's destructive force rather than by the characteristics of the surfactant
420 because there was sufficient surfactant protecting all the small droplets of oil at high surfactant
rin
421 concentrations (Yang et al., 2013). All three surfactants were capable of forming emulsions with
422 nano-sized droplets (d32 < 200 nm). The extracted tea saponins could produce smaller droplets at
ep
423 low surfactant concentrations due to their ability to promote the formation of droplets at the
424 water-oil interface more rapidly (Gao et al., 2020). The smallest droplets (d32 = 162 nm) were
Pr
425 created by Tween 80 at high concentrations, which was in concordance with the results of Yang et
20
426 al. (Yang et al., 2013). In addition, the microstructure of emulsions was investigated by inverted
ed
427 fluorescence microscopy (Fig. S3). The droplet distribution was observed to be sparse, and there
428 was no significant droplet aggregation. The droplets produced by Tween 80 and Tween 20 were
iew
429 smaller than that of extracted tea saponins, which was the same as the results of the particle
430 diameter analysis. The above results indicated that all three surfactants could produce small
431 droplets (d32 < 200 nm) during homogenization, and the effectiveness of Tween 80 was slightly
v
432 higher.
re
433 3.4.2. Effect of environmental stresses on emulsions stability
434 3.4.2.1 Effect of pH er

435 The pH value of the emulsions affects the surface charge, which is essential to stabilize the
pe
436 emulsions under electrostatic repulsive force (Zhao et al., 2021). The effect of pH 3-11 on the
437 mean particle diameter of emulsions stabilized by two surfactants was investigated (Fig. 4B). The
438 emulsions containing Tween 80 were stable overall pH ranges investigated, as they consistently
ot
439 maintained a small mean particle diameter despite slight variations. Additionally, the research of
tn
440 Zhu et al. also demonstrated that Tween 80-containing emulsions are stable in the pH ranges of 2
441 to 9 (Zhu et al., 2019). This was due to the fact that Tween 80 is a non-ionic surfactant that
442 stabilizes emulsions mainly through steric repulsion because of its large polymeric head groups
rin
443 (McClements et al., 2016). The emulsions containing extracted tea saponins were also stable in all
444 pH ranges investigated, with no significant change in particle diameter and no evidence of droplet
ep
445 aggregation, which was in agreement with the findings of Gao et al. (Gao et al., 2020). According
446 to the study of Zhu et al. (Zhu et al., 2019), the surface charge of emulsions containing tea
Pr
447 saponins changes significantly with pH, which affects the stability of emulsions under electrostatic
21
448 repulsion. Therefore, it was hypothesized that the droplets of emulsions containing tea saponins
ed
449 seemed stabilized by the combined effects of electrostatic and steric repulsion (Gao et al., 2020).
450 The above results indicated that the emulsions containing extracted tea saponins and Tween 80
iew
451 were stable in most of the pH range (pH 3-11).
452 3.4.2.2 Effect of thermal processing
453 The thermodynamic stability of emulsions is related to the ambient temperature (Zhao et al.,
v
454 2021). Emulsions stabilized by nonionic surfactant may become unstable when heated to the phase
re
455 inversion temperature (PIT) (Yang et al., 2013). As shown in Fig. 4C, the emulsions containing
456 either Tween 80 or extracted tea saponins were stable over the examined temperature range, with
er
457 no significant change in the mean particle diameter, which was possibly because the maximum
pe
458 temperature used in this study is lower than the PIT. According to the study of Zhu et al. (Zhu et
459 al., 2019), emulsions stabilized by tea saponins maintain relatively high Zeta potential over the
460 temperature range of 30-90 °C, which results in strong electrostatic repulsion among the droplets
ot
461 thus keeping the emulsions stable, but emulsions stabilized by Tween 80 becomes unstable at
tn
462 90 °C as it approaches its PIT. In summary, emulsions stabilized by extracted tea saponins were
463 stable for thermal processing.
464 3.4.2.3 Effect of long-term storage

rin
465 In commercial food applications, the long-term storage stability of emulsions is critical to
466 their shelf life (Yang et al., 2013). The effect of storage time on the mean particle diameter of
ep
467 emulsions at room temperature was investigated to evaluate the storage stability of emulsions (Fig.
468 4D). The emulsions stabilized by 3% extracted tea saponins remained stable during 30 days of
Pr
469 storage, with only slight changes in their mean particle diameter. However, the mean particle
22
470 diameter of the emulsions with lower concentrations of extracted tea saponins increased
ed
471 significantly with storage time, and the lower the concentration, the less stable the emulsions
472 were. Similar results were also observed for the storage stability of emulsions prepared with
iew
473 rhamnolipid (Bai et al., 2016). These results were probably because the storage stability of
474 emulsions is determined by droplet size and Brownian motion, so the smaller the particle
475 diameter, the better the storage stability (Kumar et al., 2018). In summary, emulsions stabilized by
v
476 3% extracted tea saponins possessed excellent storage stability, and increasing the use of tea
re
477 saponins at low concentrations was effective in improving the storage stability.
478 4. Conclusions er
479 In this study, an environmentally friendly and efficient DES-based technology was
pe
480 established to extract tea saponins from C. oleifera seed meal. By comparison of four different
481 DESs, the DES composed of choline chloride and methylurea obtained the highest extraction yield
482 of tea saponins. The extraction yield of tea saponins reached 94.36 mg/g under the optimal
ot
483 extraction conditions obtained by response surface methodology. Compared to conventional

tn
484 ethanol extraction, DES extraction exhibited higher extraction efficiency, which increased the
485 extraction yield by 27% while reducing the extraction time by 50%. UV and FT-IR analysis
486 indicated that DES extraction did not affect the structure of tea saponins, and 20 saponins were
rin
487 tentatively identified by UPLC-Q/TOF-MS in the extracted tea saponins. The comprehensive
488 properties of the extracted tea saponins were evaluated in terms of surface activity and
ep
489 emulsification. The extracted tea saponins were effective in reducing the interfacial tension at the
490 oil-water interface to 6.5 mN/m at its concentration of 0.05%. Compared to Tween 20 and Tween
Pr
491 80, the extracted tea saponins showed superior foamability and foam stability. In addition, the
23
492 extracted tea saponins were capable of forming nano-scale emulsions (d32 < 200 nm) with
ed
493 excellent stability at different pH values (3-11), temperatures (40-80 °C), and storage times (0-30
494 days), which are potentially appropriate for the application in various food and beverage products.
iew
495 Future research should focus on tea saponins' toxicological characteristics and biological activities
496 to ensure their safety and further explore the multifaceted applications of tea saponins.
497
v
498 CRediT authorship contribution statement
re
499 Xinjin Yu: Conceptualization, Methodology, Software, Investigation, Formal
500 analysis, Writing-original draft. Zhimei Zhao: Conceptualization, Methodology,

er
501 Investigation, Validation, Data curation. Xiaoli Yan: Investigation, Visualization,
pe
502 Supervision. Jianhua Xie: Supervision, Writing-review & editing. Qiang Yu:
503 Supervision, Writing-review & editing. Yi Chen: Conceptualization, Supervision,
504 Writing-review & editing, Project administration, Resources, Funding acquisition

ot
505 Declaration of Competing Interest

tn
506 The authors declare that they have no known competing financial interests or personal
507 relationships that could have appeared to influence the work reported in this paper.
508 Acknowledgments
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509 This work was supported by the Key projects of Natural Science Foundation of Jiangxi
510 Province (20212ACB205012), the Technology Innovation Leading Program of Jiangxi

ep
511 (20212BDH80001), and the Research Project of State Key Laboratory of Food Science and
512 Technology (SKLF-ZZB-202115) are gratefully acknowledged.

Pr
513
24
514 Reference
ed
515 Bai, L., & McClements, D. J. (2016). Formation and stabilization of nanoemulsions using
516 biosurfactants: Rhamnolipids. Journal of Colloid and Interface Science, 479, 71-79.
iew
517 https://doi.org/10.1016/j.jcis.2016.06.047.
518 Cai, C., Wang, Y., Yu, W., Wang, C., Li, F., & Tan, Z. (2020). Temperature-responsive deep eutectic
519 solvents as green and recyclable media for the efficient extraction of polysaccharides from
v
520 Ganoderma lucidum. Journal of Cleaner Production, 274, Article 123047.
re
521 https://doi.org/10.1016/j.jclepro.2020.123047.
522 Chen, Z., Zhang, J., Yang, F.,

er & Mostafa, K. (2022). Deep Eutectic Solvents
523 (DESs)-Ultrasonic-Assisted Extraction of Paeoniflorin and Paeonol from Moutan Cortex. Journal
pe
524 of Chemistry, 2022, 1-11. https://doi.org/10.1155/2022/5904038.
525 Cvjetko Bubalo, M., Curko, N., Tomasevic, M., Kovacevic Ganic, K., & Radojcic Redovnikovic, I.
526 (2016). Green extraction of grape skin phenolics by using deep eutectic solvents. Food Chemistry,
ot
527 200, 159-166. https://doi.org/10.1016/j.foodchem.2016.01.040.

tn
528 Di, T. M., Yang, S. L., Du, F. Y., Zhao, L., Xia, T., & Zhang, X. F. (2017). Cytotoxic and
529 Hypoglycemic Activity of Triterpenoid Saponins from Camellia oleifera Abel. Seed Pomace.
530 Molecules, 22(10), 1562. https://doi.org/10.3390/molecules22101562.

rin
531 Dong, J. N., Wu, G. D., Dong, Z. Q., Yang, D., Bo, Y. K., An, M., & Zhao, L. S. (2021). Natural deep
532 eutectic solvents as tailored and sustainable media for the extraction of five compounds from
ep
533 compound liquorice tablets and their comparison with conventional organic solvents. RSC
534 Advances, 11(59), 37649-37660. https://doi.org/10.1039/d1ra06338c.

Pr
535 Duan, L., Dou, L.-L., Guo, L., Li, P., & Liu, E. H. (2016). Comprehensive Evaluation of Deep Eutectic
25
536 Solvents in Extraction of Bioactive Natural Products. ACS Sustainable Chemistry & Engineering,
ed
537 4(4), 2405-2411. https://doi.org/10.1021/acssuschemeng.6b00091.
538 Gao, W., Jiang, Z., Du, X., Zhang, F., Liu, Y., Bai, X., & Sun, G. (2020). Impact of Surfactants on
iew
539 Nanoemulsions based on Fractionated Coconut Oil: Emulsification Stability and in vitro
540 Digestion. Journal of Oleo Science, 69(3), 227-239. https://doi.org/10.5650/jos.ess19264.
541 Guo, N., Tong, T., Ren, N., Tu, Y., & Li, B. (2018). Saponins from seeds of Genus Camellia:
v
542 Phytochemistry and bioactivity. Phytochemistry, 149, 42-55.
re
543 https://doi.org/10.1016/j.phytochem.2018.02.002.
544 He, J., Wu, Z.-y., Zhang, S., Zhou, Y., Zhao, F., Peng, Z.-q., & Hu, Z.-w. (2013). Optimization of
er
545 Microwave-Assisted Extraction of Tea Saponin and Its Application on Cleaning of Historic Silks.
pe
546 Journal of Surfactants and Detergents, 17(5), 919-928.
547 https://doi.org/10.1007/s11743-013-1523-8.
548 Jang, S., Lee, A. Y., Lee, A. R., Choi, G., & Kim, H. K. (2017). Optimization of ultrasound-assisted
ot
549 extraction of glycyrrhizic acid from licorice using response surface methodology. Integrative
tn
550 Medicine Research, 6(4), 388-394. https://doi.org/10.1016/j.imr.2017.08.003.
551 Jiang, Z.-M., Wang, L.-J., Gao, Z., Zhuang, B., Yin, Q., & Liu, E. H. (2019). Green and efficient
552 extraction of different types of bioactive alkaloids using deep eutectic solvents. Microchemical
rin
553 Journal, 145, 345-353. https://doi.org/10.1016/j.microc.2018.10.057.
554 Kumar, N., & Mandal, A. (2018). Thermodynamic and physicochemical properties evaluation for
ep
555 formation and characterization of oil-in-water nanoemulsion. Journal of Molecular Liquids, 266,
556 147-159. https://doi.org/10.1016/j.molliq.2018.06.069.

Pr
557 Lei, J., Wang, Y., Li, W., Fu, S., Zhou, J., Lu, D., . . . Wang, G. (2022). Natural green deep eutectic
26
558 solvents-based eco-friendly and efficient extraction of flavonoids from Selaginella moellendorffii:
ed
559 Process optimization, composition identification and biological activity. Separation and
560 Purification Technology, 283, Article 120203. https://doi.org/10.1016/j.seppur.2021.120203.
iew
561 Li, T., Zhang, H., & Wu, C.-e. (2014). Screening of antioxidant and antitumor activities of major
562 ingredients from defatted Camellia oleifera seeds. Food Science and Biotechnology, 23(3),
563 873-880. https://doi.org/10.1007/s10068-014-0117-1.
v
564 Lin, S., Meng, X., Tan, C., Tong, Y., Wan, M., Wang, M., . . . Ma, Y. (2022). Composition and
re
565 antioxidant activity of anthocyanins from Aronia melanocarpa extracted using an
566 ultrasonic-microwave-assisted natural deep eutectic solvent extraction method. Ultrason

er
567 Sonochem, 89, Article 106102. https://doi.org/10.1016/j.ultsonch.2022.106102.
pe
568 Liu, J.-Z., Lyu, H.-C., Fu, Y.-J., Jiang, J.-C., & Cui, Q. (2022a). Simultaneous extraction of natural
569 organic acid and flavonoid antioxidants from Hibiscus manihot L. flower by tailor-made deep
570 eutectic solvent. LWT - Food Science and Technology, 163, Article 113533.
ot
571 https://doi.org/10.1016/j.lwt.2022.113533.
tn
572 Liu, K., Tan, J.-N., Wei, Y., Li, C., Dou, Y., & Zhang, Z. (2022b). Application of choline
573 chloride-based deep eutectic solvents for the extraction of dopamine from purslane (Portulaca
574 oleracea L.). Results in Chemistry, 4. https://doi.org/10.1016/j.rechem.2022.100299.

rin
575 McClements, D. J., & Gumus, C. E. (2016). Natural emulsifiers - Biosurfactants, phospholipids,
576 biopolymers, and colloidal particles: Molecular and physicochemical basis of functional
ep
577 performance. Advances in Colloid and Interface Science, 234, 3-26.
578 https://doi.org/10.1016/j.cis.2016.03.002.
Pr
579 Shang, X., Tan, J. N., Du, Y., Liu, X., & Zhang, Z. (2018). Environmentally-Friendly Extraction of
27
580 Flavonoids from Cyclocarya paliurus (Batal.) Iljinskaja Leaves with Deep Eutectic Solvents and
ed
581 Evaluation of Their Antioxidant Activities. Molecules, 23(9), 2110.
582 https://doi.org/10.3390/molecules23092110.
iew
583 Shirsath, S. R., Sable, S. S., Gaikwad, S. G., Sonawane, S. H., Saini, D. R., & Gogate, P. R. (2017).
584 Intensification of extraction of curcumin from Curcuma amada using ultrasound assisted
585 approach: Effect of different operating parameters. Ultrason Sonochem, 38, 437-445.
v
586 https://doi.org/10.1016/j.ultsonch.2017.03.040.
re
587 Tang, Y., He, X., Sun, J., Liu, G., Li, C., Li, L., . . . Chen, X. (2021). Comprehensive evaluation on
588 tailor-made deep eutectic solvents (DESs) in extracting tea saponins from seed pomace of
er
589 Camellia oleifera Abel. Food Chemistry, 342, Article 128243.
pe
590 https://doi.org/10.1016/j.foodchem.2020.128243.
591 Wu, X., Jia, L., Wu, J., Liu, Y., Kang, H., Liu, X., . . . Li, B. (2019). Simultaneous Determination and
592 Quantification of Triterpene Saponins from Camellia sinensis Seeds Using

ot
593 UPLC-PDA-QTOF-MS/MS. Molecules, 24(20), 3794.

tn
594 https://doi.org/10.3390/molecules24203794.
595 Yang, W. S., Ko, J., Kim, E., Kim, J. H., Park, J. G., Sung, N. Y., . . . Cho, J. Y. (2014).
596 21-O-angeloyltheasapogenol E3, a novel triterpenoid saponin from the seeds of tea plants, inhibits
rin
597 macrophage-mediated inflammatory responses in a NF-kappaB-dependent manner. Mediators
598 Inflamm, 2014, Article 658351. https://doi.org/10.1155/2014/658351.

ep
599 Yang, Y., Leser, M. E., Sher, A. A., & McClements, D. J. (2013). Formation and stability of emulsions
600 using a natural small molecule surfactant: Quillaja saponin (Q-Naturale®). Food Hydrocolloids,
Pr
601 30(2), 589-596. https://doi.org/10.1016/j.foodhyd.2012.08.008.
28
602 Yang, Z., & Li, W. (2020). Extraction and surface activity of tea saponin from Pu’er tea seeds. IOP
ed
603 Conference Series: Earth and Environmental Science, 585(1), Article 012149.
604 https://doi.org/10.1088/1755-1315/585/1/012149.
iew
605 Yu, Z., Wu, X., & He, J. (2022). Study on the antifungal activity and mechanism of tea saponin from
606 Camellia oleifera cake. European Food Research and Technology, 248(3), 783-795.
607 https://doi.org/10.1007/s00217-021-03929-1.
v
608 Yuan, C., Li, Y., Li, Q., Jin, R., & Ren, L. (2018). Purification of Tea Saponins and Evaluation of its
re
609 Effect on Alcohol Dehydrogenase Activity. Open Life Sciences, 13, 56-63.
610 https://doi.org/10.1515/biol-2018-0008.
er
611 Yue, X., Cao, Y., Wang, Y., Bao, H., Xu, Y., & Peshev, D. (2022). Optimization of Deep Eutectic
pe
612 Solvents Extraction of Effective Components from Phellodendron chinense Schneid by Response
613 Surface Methodology. International Journal of Chemical Engineering, 2022, 1-12.
614 https://doi.org/10.1155/2022/3881551.
ot
615 Zhao, Q., Wu, C., Yu, C., Bi, A., Xu, X., & Du, M. (2021). High stability of bilayer nano-emulsions
tn
616 fabricated by Tween 20 and specific interfacial peptides. Food Chemistry, 340, Article 127877.
617 https://doi.org/10.1016/j.foodchem.2020.127877.
618 Zhao, W., Li, N., Zhang, X., Wang, W., Li, J., & Si, Y. (2015). Cancer chemopreventive theasaponin
rin
619 derivatives from the total tea seed saponin of Camellia sinensis. Journal of Functional Foods, 12,
620 192-198. https://doi.org/10.1016/j.jff.2014.11.017.

ep
621 Zhao, Y., Su, R., Zhang, W., Yao, G.-L., & Chen, J. (2020). Antibacterial activity of tea saponin from
622 Camellia oleifera shell by novel extraction method. Industrial Crops and Products, 153, Article
Pr
623 112604. https://doi.org/10.1016/j.indcrop.2020.112604.
29
624 Zhu, Q., Wang, C., Khalid, N., Qiu, S., & Yin, L. (2017). Effect of protein molecules and MgCl2 in the
ed
625 water phase on the dilational rheology of polyglycerol polyricinoleate molecules adsorbed at the
626 soy oil-water interface. Food Hydrocolloids, 73, 194-202.
iew
627 https://doi.org/10.1016/j.foodhyd.2017.06.030.
628 Zhu, Z., Wen, Y., Yi, J., Cao, Y., Liu, F., & McClements, D. J. (2019). Comparison of natural and
629 synthetic surfactants at forming and stabilizing nanoemulsions: Tea saponin, Quillaja saponin, and
v
630 Tween 80. Journal of Colloid and Interface Science, 536, 80-87.
re
631 https://doi.org/10.1016/j.jcis.2018.10.024.
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30
633 Table and Figure captions
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634 Table 1. Analysis of variance for regression model.
635 Table 2. Analysis of the composition of extracted tea saponins by UPLC-Q/TOF-MS.
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636 Fig. 1. Three-dimensional and two-dimensional response surface plots show the
637 interaction effect of experimental factors on the extraction yield of tea saponins.
638 Fig. 2. UV absorption (A), FT-IR spectra (B) and UPLC-Q/TOF-MS total ion
v
639 chromatogram (C) of tea saponins extracted from C. oleifera seed meal.
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640 Fig. 3. (A) Effect of the concentration of extracted tea saponins on the oil-water
641 interfacial tension. (B) Foamability and foam stability of extracted tea saponins at
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642 different concentrations. (C) Foamability and foam stability of different surfactants.
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643 Fig. 4. Particle diameter and stability analysis of emulsions. (A) Effect of surfactant
644 concentration on the particle diameter of emulsions. (B) Effect of PH on the particle
645 diameter of emulsions. (C) Effect of temperature on the particle diameter of

ot
646 emulsions. (D) Effect of concentration of extracted tea saponins on the storage
stability of emulsions.
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647
648 Table S1. Factors and levels for Box-Behnken Design.
649 Table S2. Box-Behnken experimental results.

rin
650 Fig. S1. Effects of extraction factors on the extraction yield of tea saponins. (A) DES
651 types, (B) Molar ratio, (C) Water content, (D) Liquid-material ratio, (E) Temperature,
ep
652 (F) Time. The different letters show significant differences between groups (p < 0.05).
653 Fig. S2. Mass fragments of peaks 1 (A) and 5 (B).

Pr
654 Fig. S3. Microstructure of emulsions stabilized by different surfactants.
31

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1 Extraction optimization of tea saponins from Camellia oleifera seed meal with

6 Nanchang 330047, People’s Republic of China

10 Professor Yi Chen, Ph.D.; E-mail address: chenyi-417@163.com

11 Tel.: 0791-88304449. Fax: 0791-88304449.

25 development of tea saponins.

27 UPLC-Q/TOF-MS, properties evaluation.

66 infrared (FT-IR) spectrum and ultra-performance liquid chromatography-quadrupole-time of

67 flight-mass spectrometry (UPLC-Q/TOF-MS). Furthermore, the surface activity and

72 2. Materials and methods

73 2.1. Chemicals and reagents

86 seed meal was stored in a closed desiccator after drying.

87 2.3. Extraction and isolation of tea saponins

88 2.3.1. Preparation and screening of DESs

94 to reduce the viscosity of the system after magnetic stirring at 80 °C for 1 h.

98 saponins extraction yield was selected for subsequent experiments.

110 2.3.4. Conventional extraction

114 2.3.5 Isolation and purification

122 2.4. Determination of total tea saponins content

130 2.5. Identification of the extracted tea saponins

131 2.5.1. UV analysis

133 (TU-1900, PGENERAL, Beijing, China) in the range of 190-600 nm.

134 2.5.2. FT-IR analysis

137 Scientific, USA) between 400 cm-1 and 4000 cm-1.

138 2.5.3. Identification of tea saponins by UPLC-Q/TOF-MS

152 2.6.1. Measurement of interfacial tension

158 calculated by fitting the Laplace-Young equation.

159 2.6.2. Determination of foamability and foam stability

166 2.7. Determination of emulsifying properties of the extracted tea saponins

167 2.7.1. Emulsion preparation

174 emulsions were homogenized with a high-pressure homogenizer (NCJJ0.007/200, Langfang

175 General Machinery Manufacturing Co., China).

176 2.7.2. Particle diameter analysis

178 (Zetasizer Nano ZS90, Malvern Instruments, UK).

179 2.7.3. Microstructure analysis

192 temperature and kept for 24 hours before being analyzed.

195 2.8. Statistical analysis

199 19.0 software.

200 3. Results and discussion

201 3.1. DESs Extraction

202 3.1.1. Preparation and screening of DESs

217 3.1.2. Analysis of single-factor experimental

226 performed with Chcl-Met at a molar ratio of 1:5.

242 liquid-material ratio for subsequent experiments was selected at 50 mL/g.

249 experiments were performed with an extraction temperature of 60 °C.

Y = 93.22 ― 2.57A + 7.27B ― 1.00C ― 6.43𝐴2 ― 9.08𝐵2 ― 5.75𝐶2 + 0.34𝐴𝐵 ― 1.32𝐴𝐶

280 3.1.4. Validation of optimal extraction conditions

289 3.1.5. Comparison of DES extraction and traditional solvent extraction

296 3.2. Identification of the extracted tea saponins

321 the mixtures of oleanane pentacyclic triterpene saponins.

327 formula and proposed compounds. A total of 20 saponins, including Teaseedsaponins,

328 Theasaponins, Oleiferasaponins, Assamsaponins, Floratheasaponins, and an unknown saponin

330 previous reports and saponin mass spectrometry database（http://47.92.73.208:8082）(Guo et al.,

331 2018; Wu et al., 2019).

337 theasaponin A1 (21-O-angeloyltheasapogenol A 3-O-β-D-galactopyranosyl

340 [M-H-arabinopyranosyl]-, [M-H-galactopyranosyl-arabinopyranosyl]-. Peak 5 showed molecular

343 with that of theasaponin A4 （ 21-O-angeloyltheasapogenol A

346 were [M-H-glucopyranosyl]-,

347 [M-H-glucopyranosyl-arabinopyranosyl]- and

372 ions at m/z 1037（-162-18 Da）、887（-162-132-18-18 Da）、761（-162-132-162 Da）. The only

375 to be a new saponin, and its oligosaccharide moiety was presumed to be

384 3.3. Surface activity of the extracted tea saponins

398 3.3.2. Foamability and foam stability