Influencing Factors on the Physicochemical Characteristics of Tea Polysaccharides

Tea polysaccharides (TPSs) are one of the main bioactive constituents of tea with various biological activities such as hypoglycemic effect, antioxidant, antitumor, and immunomodulatory. The bioactivities of TPSs are directly associated with their structures such as chemical composition, molecular weight, glycosidic linkages, and conformation among others. To study the relationship between the structures of TPSs and their bioactivities, it is essential to elucidate the structure of TPSs, particularly the fine structures. Due to the vast variation nature of monosaccharide units and their connections, the structure of TPSs is extremely complex, which is also affected by several major factors including tea species, processing technologies of tea and isolation methods of TPSs. As a result of the complexity, there are few studies on their fine structures and chain conformation. In the present review, we aim to provide a detailed summary of the multiple factors influencing the characteristics of TPS chemical structures such as variations of tea species, degree of fermentation, and preparation methods among others as well as their applications. The main aspects of understanding the structural difference of TPSs and influencing factors are to assist the study of the structure and bioactivity relationship and ultimately, to control the production of the targeted TPSs with the most desired biological activity.


Introduction
Tea, an important agricultural product made from the fresh leaves and buds of plant Camellia sinensis, is the most consumed functional beverage in the world [1]. It has a long history of dietary and medicinal application, especially in Asian countries-such as China, Japan, India, and Thailand-for more than five thousand years [2]. According to the manufacturing process, tea can be categorized as unfermented green, white, and yellow teas, partly-fermented oolong and raw pu-erh teas, fully fermented black, pu-erh and dark teas, and post-fermented dark tea [3]. The processing methods including withering, rolling, fermentation, post-fermentation, and roasting of tea, and other factors such as cultivars, degree of ripeness, geographical location and agricultural practices will affect the content and structure of active compounds, resulting in the changes of biological activities. Tea possesses multiple biological functions, including antioxidant, hypoglycemic effect, anti-microbial, lowering blood lipids, and anticancer [1,4,5]. These biological activities have been attributed to the variety of chemical ingredients of tea, mostly to tea polyphenols such as catechins, theaflavins, thearubigins, theasinensins and other flavonoids, but also polysaccharides, alkaloids (caffeine, theobromine and theophylline), proteins, lipids, and inorganic elements (selenium, iron, manganese, etc.) among others [1,[4][5][6].
nearly 13% [7]. Figure 1 illustrates a general structure of TPSs, but is noninclusive due to the complexity of TPS structures. Recently, TPSs have attracted increasing levels of attention due to their various biological activities, including antioxidant [3,[8][9][10][11][12], antitumor [13,14], anti-diabetes [12,[15][16][17], antifatigue [18], anticoagulant [19], anti-obesity [20], hypoglycemic [16,21,22], and immunomodulatory activities [23,24]. According to the reports, TPSs contained 2-10 monosaccharides such as glucose (Glc), rhamnose (Rha), arabinose (Ara), mannose (Man), ribose (Rib), xylose (Xyl), galactose (Gal), fucose (Fuc), galacturonic acid (GalA), and glucuronic acid (GluA), and the monosaccharides linked by multiple glycosidic linkages such as 1→2, 1→3, 1→4, 1→6, leading to a wide range of molecular weight (Mw) distribution (MWD) [4,7]. The molecular structure of a TPS is composed of multiple monosaccharide units. Similar to amino acids in proteins, the composition and linkage of individual monosaccharides have many different ways, resulting in variety of TPSs. Due to the nature of poly-monosaccharide unit composition, multiple sites of connection and chain conformation, the structures of TPSs vary dramatically in molecular weight, chain length and connection type, configuration and others. Hence the composition and connections of monosaccharide units are the defining parameters of TPSs. The molecular weight (Mw) of a TPS is determined by the number of single monosaccharide units and the conformation of a TPS is directly associated with the types of monosaccharides and their linking position between two adjacent monosaccharides. Therefore, the components of monosaccharides and their connecting style are the basic foundations of TPSs. Factors affecting the composition and connection of monosaccharides are thereby influencing the structures of TPSs, which in turn influence the biological activities.
Therefore, to further study the biological property of TPSs and to effectively correlate the bioactivity and the structure of TPSs, it is also essential to understand and elucidate the fine structure of TPSs and the factors contributing to their variety, particularly the monosaccharide units and their connection. There are multiple factors that can cause the differentiation of monosaccharide composition and linkage, but the main ones are tea species, tea process, and isolation method of TPSs. The aim of this review is to provide a comprehensive summary of structural variety of TPSs and factors affecting the polymorphism of TPS structures, to associate the influential factors, TPS structures and bioactivity, and to enhance the characteristic understanding of TPS structure and to enhance the application of TPSs in the fields of bioactive polysaccharides and functional foods.  Therefore, to further study the biological property of TPSs and to effectively correlate the bioactivity and the structure of TPSs, it is also essential to understand and elucidate the fine structure of TPSs and the factors contributing to their variety, particularly the monosaccharide units and their connection. There are multiple factors that can cause the differentiation of monosaccharide composition and linkage, but the main ones are tea species, tea process, and isolation method of TPSs. The aim of this review is to provide a comprehensive summary of structural variety of TPSs and factors affecting the polymorphism of TPS structures, to associate the influential factors, TPS structures and bioactivity, and to enhance the characteristic understanding of TPS structure and to enhance the application of TPSs in the fields of bioactive polysaccharides and functional foods.
A schematic diagram of extraction and purification of TPSs is shown in Figure 2. Different types of teas-including green, white, yellow, oolong, black, and dark tea-are obtained by different manufacture processes. Different extraction methods-such as hot water extraction (HWE), boiling water extraction (BWE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), enzyme extraction (EE), and supercritical liquid extraction (SLE)-are used to extract TPSs from tea leaves, seeds, and flowers [9,16,25,26]. After decolorization, deproteinization and dialysis of the extracted solution, alcohol precipitation is used to obtain the crude TPSs. The crude TPSs are further purified by ultrafiltration or column chromatography filled with separating materials including DEAE-cellulose, Anion exchange resin D315, DEAE Sepharose, Sephadex G-100 gel, Superdex-200, Sephacryl S-300, and others [9,17,23,[27][28][29]. The purified TPSs are usually lyophilized for readiness in characterization and biological activities study.
The traditional water extraction (WE) method is widely used because of its simple operation and low cost. However, due to its long extraction time and low yield, various assisted extraction methods such as UAE, MAE, and EE are developed to improve the yield of TPS. UAE and MAE have the advantages of simple, rapid, energy-saving and high efficiency, but they will have a certain impact on the bioactivity of TPS. The reaction conditions of EE are mild, which will not affect the bioactivity of TPS, but the cost is relatively high. In addition, a novel extraction method, SLE has the advantages of low energy consumption, high efficiency, mild, and environmental-friendly, but the equipment is expensive and the extraction time is long.  The traditional water extraction (WE) method is widely used because of its simple operation and low cost. However, due to its long extraction time and low yield, various assisted extraction methods such as UAE, MAE, and EE are developed to improve the yield of TPS. UAE and MAE have the advantages of simple, rapid, energy-saving and high efficiency, but they will have a certain impact on the bioactivity of TPS. The reaction conditions of EE are mild, which will not affect the bioactivity of TPS, but the cost is relatively high. In addition, a novel extraction method, SLE has the advantages of low energy consumption, high efficiency, mild, and environmental-friendly, but the equipment is expensive and the extraction time is long.

Physicochemical Characterization of TPSs
The physicochemical characterization of TPSs includes monosaccharide composition, molecular weight (M w ), sequence of monosaccharides, location of glycosidic linkages, degree of branches, configuration, and conformation of the entire molecule. The monosaccharide composition of TPSs is usually analyzed using gas chromatography (GC) and GCmass spectroscopy (GC-MS) after the hydrolysis of glycosidic linkages by trifluoroacetic acid (TFA) and derivatization with acetic anhydride. Gel permeation chromatography (GPC), gel-filtration chromatography (GFC), and/or multiangle laser light-scattering instrument (MLLS) are used to determine the M w of TPSs. The chemical structures of TPSs are complex and determined by UV-vis, Fourier transform infrared spectroscopy (FT-IR), GC, GC-MS, 1D and 2D nuclear magnetic resonance spectroscopy (NMR), transmission electron microscopy (TEM), atomic force microscopy (AFM) combined with monosaccharide composition analysis, periodate oxidation, Smith degradation, partial acid hydrolysis, and methylation analysis.
To date, more than 120 TPSs have been extracted and isolated from various types of tea. The main chemical characteristics of TPSs, such as the composition of monosaccharides, average M w and chemical structure, are summarized in Tables 1 and 2. As illustrated in Table 1, TPSs are heteropolysaccharides, consisting of 2-10 monosaccharides which contain Glc, Rha, Ara, Man, Rib, Xyl, Gal, Fuc, GalA, and GluA, with the average M w ranged from 1.02 to 4940 kDa. There are only several studies about the chemical structure and chain conformation of TPSs, the main monosaccharides are Glc, Gal, Rha, Xyl, Ara, and Fuc and the linkages of main chain are 1→2, 1→3, 1→4, and 1→6 as shown in Figure 1. The conformation of TPSs in solution is characterized as sphere-like, random coil, and/or ordered helix-coil shapes [30,31]. The differences in monosaccharide composition, average M w and chemical structure are closely related with the material, manufacture processes, extraction, and isolation methods of TPSs.           The structures of TPSs vary due to different tea materials, harvest years, processing methods as well as extraction and purification methods among others. Tables 1 and 2 summarized the factors including tea raw materials, processing technologies and isolation methods that affect the main chemical characteristics of TPSs, such as monosaccharide composition, M w , and chemical structure.

Tea Material
The monosaccharide composition and M w of TPSs differ from different parts of tea (i.e., leaves, flowers, seeds) and different species even in the same category of tea [10,11,40]. For instance, the M w and monosaccharides of green tea TPSs were found different among species of Xihu Longjing, Huizhoulvcha, Chawentianxia, and others [11].
Tissues of tea-The leaves, flowers and seeds of tea had different profiles of monosaccharide composition and molar ratio of TPSs. Wang [10]. It was found that the in TPS from leaves had the most types of monosaccharides (nine) than that of TFPS (eight) and TSPS (seven). The molecular weight distribution (MWD) of TLPS, TFPS, and TSPS were ranged from 3.67 to 758 kDa, 2.56 to 1460 kDa, and 3.66 to 961 kDa, respectively, indicating that the M w of polysaccharide from tea flowers was the highest [10]. In the same study, they also found that TFPS had higher M w than TLPS [45].
Species of tea-TPSs extracted from different species of the same category of tea also had different monosaccharide composition and M w . For example, the monosaccharide composition of three species of green tea polysaccharides extracted from Xihu Longjing (XTPS), Chawentianxia (CTPS), and Huizhoulvcha (HTPS) was different. XTPS and CTPS were mainly consisted of Rha, Ara, Gal, Glc, Xyl, Man, and GalA with mole ratios of 9. 50 [40]. There was only difference in the mole percentages for three TPSs from green tea and two TPSs from Oolong tea.
The M w of TPSs is also different among tea species. It was found that the M w of TPSs from three green tea (Shufeng, Longjin, and Jialaoshan) and two oolong tea species (Fenghuangdanzong and Tieguanyin) were 127, 106, 121, 107, and 95, respectively. The M w of polysaccharides from Tieguanyin oolong tea was the lowest [40]. Asides from the composition, the M w of three green tea polysaccharides extracted from above mentioned XTPS, CTPS, and HTPS was also different as described in the following. XTPS was mainly consisted of three kinds of polysaccharides with the M w of 810, 54.5, and 1.26 kDa, respectively; CTPS was mainly consisted of four kinds of polysaccharides with the M w of 805, 138, 19, and 12 kDa, respectively; and HTPS was composed of four kinds of polysaccharides with the M w of 771, 137, 11, and 1.2 kDa, respectively [11]. It was observed that the M w of CTPS was generally higher than that of XTPS and HTPS in corresponding TPS range.
There is very limited research on the chemical structures of TPSs related to tea materials. In Scoparo's study, the chemical structures of two kinds of polysaccharides from green (GSP) and black (BSP) teas were characterized and found that they both consisted of rhamnogalacturonan as the backbone containing a long sequence of →4)-6-O-Me-α-GalpA-(1→ and interrupted by α-L-Rhap residues. The difference was that GSP contained 65% GalA residues in comparison to only one third of GalA from BSP [35]. It is likely resulted from the oxidation during the processing of black tea, leading to the degradation of uronic acid.

Processing Technologies
Technology of tea process is another influencing factor on physicochemical characterization of TPSs. The monosaccharide composition and M w can vary among TPSs extracting from teas with different processing procedures such as fermentation, aging, extrusion processing and selenium-rich technologies [8,12,28,43,50].  [8]. It was found that OTPS and BTPS contained no D-Xyl and D-Man, suggesting fermentation had a significant impact on these two monosaccharides. The MWD of GTPS, OTPS, and BTPS was ranged from 9.2 to 251.5 kDa, from 5.3 to 100.9 kDa and from 3.8 to 32.7 kDa, respectively [8]. MWD was decreased with the increase of the fermentation degree, indicating that the glycosidic bonds in the backbone were cleaved during the fermentation process, and the larger degree of fermentation, the more cleavage. Moreover, the different degree of fermentation in the same category of oolong tea also resulted in discrepancies among corresponding TPSs. For instance, the polysaccharides from three oolong teas, namely, Tieguanyin ( [50]. Therefore, the fermentation degree of tea effectively affected the molar ratio of monosaccharides of TPS, and the content of D-Xyl and D-Man decreased with the increasing of fermentation degree. Aside from that, the M w of TTPS, FTPS, and DTPS was different: TTPS contained one major peak of 92.9% with M w of 8.17 × 10 5 Da and two minor peaks with M w of 0.25 × 10 5 (4.5%) and 0.07 × 10 5 Da (2.7%); FTPS had two peaks with M w of 9.30 × 10 5 (34.2%) and 0.14 × 10 5 (65.8%) Da; and DTPS consisted of one major peak 94.4% with M w of 26.4 × 10 5 Da and two minor peaks with M w of 1.10 × 10 5 (4.3%) and 0.42 × 10 5 Da (1.4%), respectively. The largest M w of oolong tea polysaccharides came from Dahongpao with the highest degree of fermentation [50]. The increased degree of fermentation decreased the M w (or MWD), an opposite pattern from above mentioned TPSs from green, oolong, and black tea. Within the category of oolong tea, the reported data from the measurement of three oolong tea TPSs revealed that the increased M w with the increased degree of fermentation, may result from three different species of tea prior to fermentation process, because the three brands of oolong tea in this example came from different regions of China. Species of tea plays more important roles in the composition and molecular weight than process in general.
Aging time-One of the characteristics of pu-erh tea is aging.  [12]. The monosaccharide composi-tion of PTPS-1, PTPS-3, and PTPS-5 was the same, while the content of each monosaccharide was different. PTPS-1, PTPS-3, and PTPS-5 had different Mw. PTPS-1 had one major fraction of 92% with the M w of 2.7 × 10 6 Da; PTPS-3 had two major fractions with M w of 6.31 × 10 5 Da (52%) and 1.93 × 10 6 Da (47%), respectively; PTPS-5 also had two major fractions with M w of 1.16 × 10 6 (60%) and 3.9 × 10 6 Da (33%) Da, respectively [12]. Time of aging changed TPSs in different directions in terms of molecular weight, but more study was required to measure the molecular weight and amount of TPSs in the same time and then to correlate the MWD and the aging time, which could yield a conclusive pattern and reveal the relationship between aging or degree of fermentation with M w in the comparison of same species.
Selenium enrichment-Selenium also has impact on the composition and structure of TPS. The monosaccharide composition of two Se-enriched tea polysaccharides (Se-TPS) extracted from artificial (ASe-TPS2) and natural Se-enriched teas (NSe-TPS2) were analyzed and found that ASe-TPS2 was composed of Rha, Ara, Glc, Xyl, and GalA in the molar ratio of 1.93:7.05:1.00:1.05:26.12, whereas NSe-TPS2 was consisted of Fuc, Rha, Ara, Gal, Glc, GlcA, and GalA in the molar ratio of 0.07:0.28:0.59:1.00:0.10:0.49:1.24 [28]. The main monosaccharide compositions of ASe-TPS2 were Ara and GalA, and the galacturonic acid was the highest among all five groups of monosacchrides. The main monosaccharides of NSe-TPS2 were Ara, Gal, GlcA, and GalA. The M w of ASe-TPS2 and NSe-TPS2 was 6.73 × 10 3 Da and 2.44 × 10 5 Da, respectively, indicating that the M w of Se-TPS from artificially Se-enriched green tea was smaller than that of Se-TPS from naturally Se-enriched green tea [28].

Isolation Methods
The monosaccharide composition, molar contents, M w and chemical structures of TPSs are different under different isolation methods, which are listed in Tables 1 and 2. Extraction methods-Tea polysaccharide conjugates can be extracted with water or alkali solution, containing neutral sugars, uronic acid, and protein. Water-soluble TPS conjugates (TPC-W) and alkali-soluble TPS conjugates (TPC-A) were extracted from green tea by hot water and alkali solution respectively [39]. The TPC-W and TPC-A were both composed of seven monosaccharides, namely Rha, Fuc, Ara, Xyl, Man, Glc, and Gal with different molar ratios of 8  [39]. The difference in monosaccharide composition between them was not much, but there was a significant difference in molecular weight. TPC-W had three homogeneous components with the M w of 6.62 × 10 3 (56.07%), 4.85 × 10 4 (6.54%), and 4.55 × 10 6 (37.38%) Da, respectively; TPC-A was consisted of four homogeneous components with the M w of 4.13 × 10 3 (17.14%), 1.12 × 10 4 (11.43%), 6.77 × 10 4 (2.86%), and 4.94 × 10 6 (68.57%) Da, respectively. Hence, the higher M w of TPC was more efficiently extracted by alkali solution than water [39]. Wang, Yang, and Wei studied the monosaccharide composition of polysaccharides from leaves and flowers of green tea obtained by different extraction methods [45].  [45]. It can be seen that the M w of polysaccharides using enzyme extraction were decreased, indicating that enzyme catalyzed cleavage of TPS bonds occurred in the extraction process. Three tea flower polysaccharides (TFPS) were prepared by traditional water extraction (TWE-TFPS), microwave assisted extraction (MAE-TFPS), and ultrasound assisted extraction (UAE-TFPS), respectively. The peak M w from TWE-TFPS was 4.4 and 31 kDa. Comparing with TWE-TFPS, the peak M w from UAE-TFPS decreased with the increasing ultrasonic power, whereas the peak M w from MAE-TFPS increased with the augment of microwave power, demonstrating that radio waves have significant effects on the M w of TPS [56].
Drying methods-Four polysaccharides were isolated from tea leaves using freezedrying (TPS-F), vacuum-drying (TPS-V), spray-drying (TPS-S), and microwave-vacuum drying (TPS-M), respectively. These four crude tea polysaccharides were all composed of Rha, Rib, Ara, Glc, Xyl, Gal, Man, GalA, and GluA with different molecular ratios of 1.0:0. 58 [44]. The content of GalA and GluA in TPS-S and TPS-M were lower than TPS-F and TPS-V. TPS-F, TPS-V, TPS-S, and TPS-M showed similar molecular weight distribution and mainly contained four distinct peaks with groups of molecular weights around 9.2 × 10 5 , 2.2 × 10 5 , 3.0 × 10 4 , and 0.34 × 10 4 Da, respectively [44]. The difference of monosaccharide ratio and MWD can be resulted from the different drying methods of TPSs.

Applications of TPSs
TPSs have a variety of biological activities, such as antioxidant [3], hypoglycemia [15], anti-fatigue [18], anti-obesity [20], prebiotics [57], and immunomodulatory effect [24], it can be added to food as a functional ingredient to prepare health products. At the same time, TPS is a good emulsifier, which can be used in food, cosmetic and pharmaceutical industry. In Chen's study, an alkali-extracted tea polysaccharide conjugates (TPC-A) was used to stabilize oil-in-water emulsions, and found TPC-A had a favorable protective effect on catechins and can be used as a natural emulsifier [58]. Li et al. (2021) also obtained a natural antioxidant emulsifier from Chin brick tea, tea polysaccharide conjugate (TPC) possessed a good emulsifying properties with excellent antioxidant activity, which can be used as dual-purpose antioxidant emulsifiers [59]. In addition, TPSs can be used as feed additives in the poultry and feed industry to enhance animal immunity and improve meat quality. For example, a green tea polysaccharide conjugates (GTPC) was extracted from Yingshan Yunwu tea could improve immune status, intestinal microflora and meat quality in chickens [60]. Moreover, in the biomedical industry, TPSs can be used as a drug delivery agent. In Li's work, a biodegradable, non-toxic and environmental-friendly PTX loaded nanoparticle was prepared using TPS as the shell and zein as the core, it was found that TPS would be a promising agent in the drug delivery system [61]. Wu et al. (2018) also synthesized a cationic branched tea polysaccharide derivative (CTPSA), which exhibited lower cytotoxicity and can be used as a nonviral vector for the delivery of siRNA to the liver [62]. However, the application of TPSs are few or even a blank in other fields such as wound treatment, antiviral preparations, fertilizers, etc. which may be related with the complexity of TPSs structure and unclear structure-activity relationship of TPSs. Therefore, a lot of work needs to be done to develop the potential application of TPSs in the future.

Conclusions
The different structures of TPSs obtained in various reports are due to different tea raw materials, processing technologies, and isolation methods. The structural features of TPSs differ from different teas, even different parts and categories of the same tea. The structures of TPSs obtained with different degree of fermentation and extraction methods could have vast differences. Therefore, it is critical to clarify how the fermentation process affects the structure of TPSs and find an optimal method to obtain targeted TPSs with higher bioactivities. Due to the multiple influencing factors discussed in this review and the complex structures of TPSs, it is currently impossible to predict and also quite difficult to determine the structures of TPSs, especially high-level structures. Moreover, it is unrealistic to speculate the efficacy of TPSs without the characterization and actual biological testing of isolated TPSs. Thus, a large number of experiments are required to identify the complete structures of TPSs and to further evaluate the bioactivity of characterized TPSs. Furthermore, most of the researches focused on the polysaccharides from green tea, whereas the studies about polysaccharides from other teas are scarce. Therefore, more studies should be also focused on the characterization, identification, and comparison of the structures of TPSs from different teas with different processing technologies and isolation methods to have a comprehensive evaluation and understanding of the factors influencing the structures and biological properties of TPSs and broaden their applications in various fields.

Conflicts of Interest:
The authors declare no conflict of interest.