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2024 Volume 4
Article Contents
Abstract
Introduction
Materials and methods
Materials
Preparation of mixed surimi samples
Rheological characteristics of mixed surimi samples
Apparent viscosity
Dynamic rheological behavior
Strength of mixed surimi gels
Textural properties of mixed surimi gels
Cooking loss of mixed surimi gels
Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels
Color difference of mixed surimi gels
Scanning electron microscopy (SEM) of mixed surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Statistical analysis
Results and discussion
Rheological properties of surimi gels
Steady-state shear properties
Dynamic elastic properties during gelation
Strength of surimi gels
Textural properties of surimi gels
Cooking loss of mixed surimi gels
Color of surimi gels
Changes in moisture distribution of surimi gels
Scanning electron microscopy of surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
Rights and permissions
References
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ARTICLE   Open Access    

Metabolome profiling unveil the composition differences of quality of different tea cultivars

  • 1.

    Key Laboratory of Biology, Genetics and Breeding of Special Economic Animals and Plants, Ministry of Agriculture and Rural Affairs; Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China

  • 2.

    Tea Research Institute, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China

More Information
  • The tea plant is abundant in bioactive compounds, including flavonoids, amino acids, alkaloids, terpenoids, and lipids, which greatly affect tea quality and flavors. Despite there are many studies of metabolites about different tea cultivars, the composition differences of their biosynthesis and regulation still largely unknown. In this study, 505 metabolites were detected from the apical bud and 1st leaf of 'Shuchazao' (SCZ), 'Huangkui' (HK) and 'Zijuan' (ZJ) using widely-targeted metabolomics, including 192 flavonoids, 45 lipids, 59 amino acids and derivatives, and 28 phenolamides. Metabolite analysis showed that flavonols and anthocyanins are mainly distributed in the form of glycosides in three cultivars. Notably, anthocyanins and their glycosides are mainly accumulated in 'ZJ', indicating a correlation with the color attributes. EGCG emerged as the most abundant flavan-3-ols compound among the three cultivars. Simultaneously, L-theanine represented the predominant free amino acid, mainly concentrated in the apical bud compared to the 1st leaf. Similarly, lipids, akin to free amino acids, predominantly accumulated in the apical buds of all three cultivars. These findings offer valuable insights into the genetic and metabolite diversity, augmenting our understanding of the biosynthesis of specialized metabolites in tea plants.

  • Introduction

    Surimi gel, known as 'concentrated myofibrillar protein'[1], is a kind of gel prepared by processing fish tissue according to fixed steps, such as rinsing, dehydration, and chopping, then adding a certain number of auxiliary materials for crushing, molding, heating and cooling. Salt-soluble myofibrillar protein (mainly myosin) of surimi denatures and unfolds after heating, and then re-crosslinks and polymerizes to form large protein aggregates[2], which is the internal mechanism of forming the gel structure. To enhance the texture and taste of surimi gel products, 2%−3% salt is added to promote the formation of the protein gel network structure and enhance the solubility and functional properties of the products[3,4]. Nonetheless, numerous studies have confirmed that excessive salt intake may result in risks of disease to human health, such as hypertension, and coronary heart disease[5]. Therefore, the development of low-salt surimi products will be widely focused on in future research.

    Recently, two strategies have been innovatively proposed to improve or maintain the gel properties of low-salt surimi products: one is to use exogenous additives or sodium salt substitutes[6−8], and the other is to exploit new processing technologies[9−11]. Glutamine transaminase (TGase) is the most effective surimi quality improver to enhance the functional properties of protein, which could catalyze acyl donors in proteins (γ-Hydroxylamine group) and acyl receptors (lysine residue, primary amine compound, etc.) undergo acyl transfer reaction to form cross-linked structures[12−14]. Previous studies by Jiang et al.[15] revealed that the cross-linking effect of TGase catalysis depends on the content and spatial distribution of available substrates. In addition, lysine, as an ideal exogenous additive, has been widely used in different meat protein gel systems, which can effectively improve the gel properties of low-salt surimi gel[16] and emulsified chicken sausage[17]. Many researchers have spent a lot of time to obtain higher-quality surimi gel products through the combination of two or more exogenous additives. Many scientists devoted themselves to obtaining higher quality surimi gel products through the combination of two or more exogenous additives. For example, Cao et al.[18] demonstrated that lysine (Lys) could bring about the dissociation of actin under the condition of the presence of TGase, promoting the cooking yield and gel properties of oxidative damaged MP. Similar findings reported by Cando et al.[19] indicated that Lys could induce the changes in protein structure, which were favorable terms to heighten the cross-linking effect of TG and improve the strength of surimi gel.

    Recently, the term ε-Poly-lysine (ε-PL) was followed with interest since it's a natural amino acid polymer produced by microbial fermentation, and was speculated that it has a similar molecular structure and potential effect to Lys[20]. ε-PL, which is generally composed of 25−30 lysine residues connected by amide bonds (by ε-Amino and α-Carboxyl group), was often used as an antibacterial agent for the preservation of meat products and aquatic products[21]. Li et al.[21] explored the effect of preservative coating with ε-PL on the quality of sea bass fillets during storage. It turned out that, ε-PL treatment evidently reduced the thiobarbituric acid and volatile base nitrogen values of bass slices during storage, inhibited the growth of microorganisms, and improved the water retention, texture, and flavor characteristics of the fish. A previous study by Cai et al.[20] proved the bacteriostatic and fresh-keeping effect of the composite coating of protein and sodium alginate on Japanese sea bass, and found that the composite coating had an effect on inhibiting the proliferation of sea bass microorganisms (Escherichia coli, lactic acid bacteria, yeast, etc.), fat oxidation, protein degradation, and nucleotide decomposition. As a natural cationic polypeptide, ε-PL has the advantages of no biological toxicity and low viscosity of aqueous solution. It is noteworthy that it can produce strong electrostatic adsorption with negatively charged amino acids[22]. Its effective modification of protein helps it fully interact with gel component molecules and groups[23], which may play a role in enhancing the texture and water retention of protein gel.

    Nevertheless, few reports are based on the addition of ε-PL to explore the influence mechanism on TGase-catalyzed surimi gel properties. The current study sought to shed light on the effects of different additions of ε-PL on TGase-induced cross-linking effect and the performance of composite surimi gel, to provide a theoretical basis and reference for further research and development of 'low salt', efficient and healthy surimi products.

    Materials and methods

    Materials

    Testing materials, including marine surimi, transglutaminase (TG, enzyme activity 100 IU/g), and ε-Polylysine (ε-PL, ≥ 99% purity) were supplied by Jiangsu Yiming Biological Technology Co., Ltd (Jiangsu, China). Egg white protein was purchased from Henan Wanbang Chemical Technology Co., Ltd (Henan, China). All other chemicals were from Shanghai Yuanye Biochemical Co., Ltd (Shanghai, China) and were at least analytical grade. The ingredients of the surimi gel are shown in Table 1.

    Table 1.  Surimi samples with different treatments.
    GroupsSurimi
    (g)    		TGase
    (w/w, %)    		Egg white
    Protein
    (w/w, %)    		ε-PL
    (w/w, %)    		NaCl
    (w/w, %)
    CK300−−−0.5
    TE3000.47.0−0.5
    TE + P13000.47.00.0050.5
    TE + P23000.47.00.010.5
    TE + P33000.47.00.020.5
    TE + P43000.47.00.040.5
    TE + P53000.47.00.060.5
     | Show Table
    DownLoad: CSV

    Preparation of mixed surimi samples

    The prepared block surimi was firstly put into the chopping machine (or tissue masher) and chopped for 2 min, then different mass fractions of ingredients were added, while the chopping time was extended to 5 min (the temperature should be controlled below 10 °C during this process). The exhausted surimi paste was poured into the special mold (50 mm × 20 mm), which was heated in two stages to form the heat-induced surimi gel (40 °C water bath for 40 min; 90 °C water bath for 20 min). Above prepared surimi gel underwent an ice water bath for 30 min and finally stored overnight at 4 °C for further use.

    Rheological characteristics of mixed surimi samples

    Mixed surimi samples with different treatments were determined under a Haake Mars 60 Rheometer (Thermo Fisher Scientific, Germany) with 35 mm stainless steel parallel plates referring to the method described by Cao et al.[18], and each determination was repeated three times. After centrifugation (1,000× g, 3 min) and 4 °C, the degassed surimi sol (~2 g) was equilibrated at 4 °C for 3 min before measurement.

    Apparent viscosity

    The shear stress of the mixed surimi sol was measured at shear rates between 0−100 s−1.

    Dynamic rheological behavior

    The rheological properties of the mixed surimi sols were measured in an oscillatory mode of CD-Auto Strain at 0.02% and 0.1 Hz frequency, respectively. The heating temperature range is set to 20~90 °C while the heating rate is 1 °C·min−1.

    Strength of mixed surimi gels

    The TA-XT Plus physical property analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) was used to analyze the mixed surimi gel strength of each group of samples (n ≥ 3) at room temperature, and the cube-shaped surimi gel samples are measured through the P/0.5 probe. Referring to the method previously described by Fang et al.[24], the test parameters are as follows, pre-test and test rate (1 mm·s−1); rate after measurement (5 mm·s−1); depressing degree (30%); trigger force (5 g); data acquisition rate (400 p/s).

    Textural properties of mixed surimi gels

    Concerning the method of Jirawat et al.[25], texture profile analysis (TPA) was employed to record the hardness, elasticity, cohesion, chewiness, and resilience of mixed surimi gels. It is well known that TPA can explore the textural properties of food through the texture analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) equipped with a P/75 probe to simulate human oral chewing action and obtain the texture characteristic values related to human sensory evaluation. The physical property parameter settings were as follows: downforce (5 g), compression degree (50%), pre-test speed, test speed, and post-test speed (1.0 mm·s−1).

    Cooking loss of mixed surimi gels

    The cooking loss of surimi gel under different treatments was determined referring to the protocol of Dong et al.[26]. The cooked surimi gel sample was instantly absorbed dry and weighed (W5). The cooking loss (CL) is calculated as follows:

    CL(g/100g)=(M1−M2)/M1×100 (1)

    Where M1, weight of the sample before cooking; M2, weight of the sample after cooking.

    Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels

    The LF-NMR of the mixed surimi gel was detected by a PQ001-20-025V NMR analyzer (Niumag Analytical Instruments Co., Ltd, Suzhou, China) referring to the previous method conducted by Li et al.[27]. Relevant characteristic parameters were set as follows: sampling frequency (200 KHZ), echo time (0.3 ms), and cumulative number (8). Keeping the surimi gel sample (approximately 2 g) at room temperature for 30 min, they were carefully put into a cylindrical nuclear magnetic tube (15 mm in diameter), and finally, the relaxation time (T2) was recorded using the Carr Purcell Meiboom Gill (CPMG) pulse sequence.

    Color difference of mixed surimi gels

    CM-5 colorimeter (Konica Minolta Sensing, Inc., Tokyo, Japan) was used for surimi gel with different treatments following to the procedure of Wang et al.[28]. Several 1 cm thick slices were cut from surimi samples (n = 3) selected from different treatment groups, and the gel whiteness was calculated according to the following formula:

    Whiteness=100−√(100−L∗)2+a∗2+b∗2 (2)

    Scanning electron microscopy (SEM) of mixed surimi gels

    According to the previously described procedure by Gao et al.[29], the square-shaped surimi gels (4 mm × 4 mm × 4 mm) were immersed in 0.1 mol·L−1 phosphate buffer (pH 7.2) containing 2.5% (v/v) glutaraldehyde for 24 h. The above samples were washed using phosphate buffer (0.1 mol·L−1, pH 7.2) three times and then subsequently dehydrated in a series of alcohol solutions. The microstructures of the mixed surimi gels were imaged using an FEI Verios 460 SEM (FEI Inc., Hillsboro, OR, USA).

    Thiobarbituric acid-reactive substances (TBARS) determination

    The TBARS value was analyzed by the method described by Hu et al.[30] with slight modification. Thiobarbituric acid solution (1.5 mL) and 8.5 mL of trichloroacetic acid solution were added to the sample in turn, the mixture was bathed in water at 100 °C for 30 min. The supernatant was extracted and centrifuged twice (3,000× g, 5 min): (1) The original supernatant (5 mL) was taken and the same amount of chloroform for mixed centrifugation; (2) The second supernatant (3 mL) and petroleum ether (1.5 mL) were taken for mixed centrifugation. Finally, a small amount of lower liquid was taken to determine the absorbance (A532 nm). The final result was expressed in malondialdehyde equivalent (mg·kg−1).

    Statistical analysis

    All statistical analyses of data were investigated by statistical product and service solutions IBM SPSS Statistics version 23.0 (IBM SPSS Inc., Chicago, USA). The LSD all-pairwise multiple comparison method was used for the least significance analysis, and p < 0.05 was considered to indicate significance. The experimental data are expressed as mean ± standard deviation (SD) and plotted using Origin 2019 (Origin Lab, Northampton, MA, USA) software.

    Results and discussion

    Rheological properties of surimi gels

    Steady-state shear properties

    The steady-state shear flow curve can be used to characterize the interaction between proteins. The steady shear flow changes of apparent viscosity (Pa·s) of composite surimi with shear rate (s−1) under different treatments are shown in Fig. 1a. The apparent viscosity of all samples decreased significantly (p < 0.05) with increasing shear rate, exhibiting shear-thinning behavior[31]. In the range of shear rates from 0.1 to 100 s−1, the samples with TGase addition were always higher than the control, indicating that the induction of TGase enhanced the cross-linking of surimi proteins and formed a more stable structure. The surimi samples added with a lower proportion of ε-PL (0.005% and 0.01%) were regarded as lower viscosity fluid (Fig. 1a). The possible reason was that the low concentration of ε-PL provided a weak effect on the pH value of the surimi system. As a consequence, the content of the net charge provided was relatively small and the electrostatic interaction between the surimi proteins was weakened[20].

    Figure 1.  Effect of different treatments on rheological properties of surimi.
    DownLoad: Full-Size Img PowerPoint
    Dynamic elastic properties during gelation

    The elastic modulus (G′) mainly depends on the interaction between protein molecules, which can reflect the change of elasticity in the heat-induced surimi gel. As shown in Fig. 1b, the gel process of surimi in the control group was a thermodynamic process consisting of two typical stages. In the first stage, the value of G' showed a gradual downward trend in the range of 20~50 °C. This is mainly because, (1) the activity of endogenous protease in surimi is increased; (2) the myosin light chain subunits of surimi are dissociated under the action of protease, forming myosin and actin[24]; (3) heat-induced hydrogen bonds break between protein molecules, thus enhancing the fluidity of the gel system[31]. The second stage: From 50 to 73 °C, the G' value increases rapidly. As the temperature continues to rise to 90 °C, the G 'value keeps rising steadily. At this time, the gel network structure gradually became stable and irreversible.

    Compared with several curves in Fig. 1b, TGase treatment could significantly increase the G' value. The change trend of G' value of the samples added with TGase and ε-PL were similar to that of the control group, but the former increased faster and the maximum value of G' was also significantly higher. The temperature corresponding to the first peak of G' gradually decreased (PL1≈PL2 < PL4≈TE < PL3≈PL5 < CK), indicating that the process of protein denaturation and aggregation was advanced. This result showed that the proper amount of ε-PL (0.04%) in combination with TGase can reduce the thermal denaturation temperature of protein-forming gel and improve the forming ability of composite gel, which was unanimous with the changes in texture and properties of surimi gel (Table 2). Furthermore, the addition of a small amount (0.005%) or an excessive amount (0.06%) of ε-PL made the mixed surimi protein system more unstable, affecting the ability of TG-induced gel formation as the last resort[32].

    Table 2.  Effect of different treatments on the textural properties of surimi gels.
    GroupsHardness (g)SpringinessCohesivenessChewiness (g)Resilience
    CK811.40 ± 45.28de0.81 ± 0.01a0.53 ± 0.02b498.36 ± 21.58c0.21 ± 0.01e
    TE1105.80 ± 61.83c0.83 ± 0.01a0.56 ± 0.01b510.40 ± 26.04c0.24 ± 0.00bcd
    TE + P1736.01 ± 18.49e0.80 ± 0.03a0.61 ± 0.00a348.78 ± 18.36d0.23 ± 0.01cde
    TE + P2777.45 ± 39.61de0.80 ± 0.01a0.60 ± 0.02a307.03 ± 12.97e0.22 ± 0.00de
    TE + P3869.66 ± 45.82d0.83 ± 0.02a0.63 ± 0.01a485.06 ± 22.73c0.28 ± 0.00a
    TE + P41492.80 ± 77.13a0.83 ± 0.01a0.60 ± 0.01a721.74 ± 31.55a0.26 ± 0.01ab
    TE + P51314.60 ± 84.10b0.81 ± 0.00a0.60 ± 0.02a652.36 ± 30.24b0.25 ± 0.01bc
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Strength of surimi gels

    Gel strength is one of the vital indicators to test the quality of surimi products, which directly affects the texture characteristics and sensory acceptance of the products. The strength of mixed surimi gel with TGase was significantly enhanced by 23.97% (p < 0.05) compared with the control group after heat-inducing. This enhancement is probably caused by the crosslinking promotion of TGase (Fig. 2a). A deeper explanation is that under the catalysis of TGase, the ε-amino group on lysine and the γ-amide group on glutamic acid residues undergo acylation reaction inside or between proteins, forming ε-(γ-Glutamyl)-lysine covalent cross-linking bond which promotes the production of the protein gel network[26].

    Figure 2.  (a) Catalytic reaction of glutamine transaminase and (b) effect of different treatments on gel strength and cooking loss of surimi gel. a−d/A−D: values with different lowercase letters indicate significant difference (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    On the basis of adding TGase, the strength of the mixed gel with ε-PL concentration ranging from 0.005% to 0.03% was lower than that of TE (Fig. 2b). It was speculated that the surimi sample with low concentration ε-PL was a fluid with lower viscosity, and the interaction between protein and water is strengthened, while the electrostatic interaction between proteins is further weakened[33]. This was also consistent with the change in rheological properties in Fig. 1a. Nevertheless, with the increase of ε-PL content to 0.04%, the gel strength of surimi gel significantly increased and reached the highest value (781.63 g·cm), which was about 21.03% higher than that of the TE group (p < 0.05) (Fig. 2b). The increase of gel strength in this process was obtained due to the following three possible reasons, (1) ε-PL is a cationic amino acid with positive charge, which can improve the pH value of protein or meat product system; (2) The interaction between the ε-amino group of ε-PL and the aromatic residue of protein, namely the cation-π interaction, can change the structure of meat protein[34]; (3) Protein (mixed surimi system) with high ε-amino group content, TGase has strong gel improving ability[35].

    Such synergy (ε-PL = 0.04%), as a comprehensive result of different impactors, is that after the surimi was mixed under the optimal ratio conditions, the synthesis of TGase enzyme catalysis, pH value shift, changes in protein and amino acid composition, etc., increased the strength of various forces, thus forming a denser stereoscopic network structure. Note that when the content of ε-PL reached 0.06%, the gel strength of the corresponding surimi gel decreased by 10% compared with 0.04%. Similarly, others reported the result that alkaline amino acids led to the reduction of the strength of the myosin gel of bighead carp[36].

    Textural properties of surimi gels

    TPA, also known as whole texture, is a comprehensive parameter that determines the sensory quality of surimi gel, including hardness, elasticity, cohesion, chewiness, and resilience[37]. As shown in Table 2, the addition of TGase significantly improved the texture properties of surimi gel (p < 0.05), which is related to TGase's ability to induce surimi protein to form more ε-(γ-Glu)-Lys covalent bond during heating[38] (Fig. 2a). Compared with the cross-linking induced by TGase alone, the gel hardness of the mixed surimi gel treated with ε-PL showed a similar change to the gel strength (Fig. 2b). As the concentration of ε-PL increased from 0.005% to 0.04%, the hardness of surimi gel gradually increased until it reached the maximum (1,492.80 g). At the same time, we also observed that the elasticity, cohesion, and chewiness of the composite gel reached the highest values with the 0.04% ε-PL, and these were higher than those of the TGase-only group. Similar to the findings of Ali et al.[39], the author found that the combination of ε-PL and beetroot extract can effectively replace nitrite, which produced a marked effect in maintaining the color of Frankfurt sausage and improving the texture and performance. However, the data we collated above (Table 2) also showed a clear phenomenon, that is, the addition of ε-PL with lower concentration (0.005%−0.02%) or highest concentration (0.06%) was not conducive to the combination with TGase to improve the texture characteristics of mixed surimi gel.

    Cooking loss of mixed surimi gels

    Cooking loss indicates the water holding capacity of heated surimi, which usually represents the stability of the three-dimensional network structure of surimi gel[40]. The 0.4% TGase significantly reduced the cooking loss of surimi samples (p < 0.05), which was 4.91% lower than that of the control group (Fig. 2b). When the addition of PL (combined with TGase) gradually increased from 0.005% to 0.04%, the cooking loss of surimi showed a decreasing trend and reached the minimum at 0.04%. The ε-PL-added surimi gel samples had higher cooking loss compared with that of only with TGase. Most notably, this may be due to ε-PL is able to further exert the cross-linking effect of TGase. A similar finding was reported in the study by Ma et al.[22], adding ε-PL is able to improve the solubility of myofibrillar proteins. Proteins with high solubility are more easily induced by TGase during heating, which aggravates the cross-linking between ε-PL-protein or protein-protein and forms a disordered gel network structure with relatively weaker water-holding capacity.

    Color of surimi gels

    As shown in Table 3, the high lightness (L*), low yellowness (b*), and high whiteness of surimi in the control group were relatively low, with values of 72.49, 10.42, and 70.56 respectively. After TGase was added to induce cross-linking, these values increased significantly (p < 0.05), which may be related to the photochromic effect of water molecules released from gel matrix under TGase-induced surimi protein cross-linking reported in the previous study[41]. Furthermore, on the basis of adding TGase, it was interesting to see that the L* and whiteness values of surimi gel first increased and then gradually decreased with the increase in ε-PL concentration. The influence of ε-PL combined with TGase on the whiteness of gel can be attributed to three reasons: (1) The addition of ε-PL can effectively increase the substrate content of TGase, and promote the cross-linking between proteins, so as to obtain a much more compact surimi gel structure, which affects the refractive index of light; (2) The water retention performance of the high surimi gel was significantly improved with the addition of ε-PL[42] (Fig. 2b), at this time, the L* value (positively related to the whiteness) value showed a decreasing trend with the decrease of the surface free water content of the gel sample; (3) With the increase of ε-PL concentration, some colored substances, formed due to the accelerated Maillard reaction rate during the preparation of gel, may have an adverse effect on the improvement of gel whiteness[43]. After adding TGase to induce cross-linking, although the whiteness value of composite surimi was significantly enhanced, ε-PL was not enough to further improve the whiteness value of final mixed surimi gel, and high concentration so far as to have a negative impact on the whiteness value.

    Table 3.  Effect of different treatments on the color of surimi gels.
    GroupsL*a*b*Whiteness
    CK72.49 ± 0.82e−0.92 ± 0.01e10.42 ± 0.33d70.56 ± 0.65d
    TE77.82 ± 0.34ab−0.23 ± 0.02c12.29 ± 0.29bc74.64 ± 0.26ab
    TE + P178.39 ± 0.40a−0.14 ± 0.01a12.46 ± 0.12ab75.05 ± 0.41a
    TE + P278.42 ± 0.43a−0.17 ± 0.0b12.46 ± 0.15ab75.07 ± 0.32a
    TE + P377.48 ± 0.16b−0.18 ± 0.02b12.37 ± 0.09abc74.31 ± 0.11b
    TE + P476.34 ± 0.05c−0.22 ± 0.01c12.73 ± 0.02a73.13 ± 0.04c
    TE + P575.48 ± 0.18d−0.26 ± 0.01d12.01 ± 0.14c72.70 ± 0.19c
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Changes in moisture distribution of surimi gels

    The water distribution in surimi gel products was measured by LF-NMR[44], and the results were inverted to obtain the transverse relaxation time (T2), which reflects the strength of water fluidity[45]. There are four fitted wave peaks which are assigned to four different water distribution states and wave peak ranges (Fig. 3, Table 4): strong bound water T21 (0.1−1 ms), weak bound water T22 (1−10 ms), non-flowing water T23 (10−100 ms) and free water T24 (100−1,000 ms)[46]. At the same time, similar characteristics (the main peak is centered on T23) are possessed by the water distribution of all surimi gel samples. The addition of TGase increased the proportion of non-flowing water, accompanying the significant decrease in the content of free water (p < 0.05), compared with the control group. It proved that the addition of TGase brought this phenomenon, namely the bound water in surimi gel was partially transferred to the non-flowing, which also indicated the reduction of cooking loss of surimi samples induced by TGase (Fig. 2). On the basis of adding TGase, the low level of ε-PL (0.005%, 0.01% and 0.02%) inhibited the transition of free water in surimi gel to non-flowing water, in which the proportion of T23 decreased by 2.05%, 2.74%, and 1.42% respectively compared with the surimi gel only with TGase, while T24 increased by 15.76%, 14.17%, and 4.43% respectively. The proportion of P23 in the surimi gel sample (ε-PL = 0.04%) was equivalent to that of the TE group, implying that the addition of appropriate ε-PL was not directly connected with the changes in water distribution in surimi after TGase-induced cross-linking, and slight addition would have a negative impact, which was unanimous with the above research results of cooking loss (Fig. 2a).

    Figure 3.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    DownLoad: Full-Size Img PowerPoint
    Table 4.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    GroupsP21 (%)P22 (%)P23 (%)P24 (%)
    CK2.175 ± 0.186c1.198 ± 0.012e82.863 ± 0.691bc13.764 ± 0.091a
    TE1.938 ± 0.095d2.166 ± 0.043a85.144 ± 1.336a10.752 ± 0.033e
    TE + P12.705 ± 0.067b1.414 ± 0.009d83.435 ± 1.616b12.446 ± 0.105c
    TE + P23.005 ± 0.003a1.845 ± 0.056b82.874 ± 1.739bc12.276 ± 0.108c
    TE + P33.107 ± 0.063a1.715 ± 0.033c83.950 ± 1.587ab11.228 ± 0.086d
    TE + P41.770 ± 0.018d2.099 ± 0.059a85.021 ± 1.756a11.110 ± 0.144d
    TE + P53.121 ± 0.007a1.758 ± 0.107bc82.136 ± 1.091c12.985 ± 0.018b
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Scanning electron microscopy of surimi gels

    The effect of different treatment methods on the microstructure of surimi gel was observed by SEM. As shown in Fig. 4, the microstructure of the control surimi gel (Fig. 4a) was observed to be enormously loose, uneven, and irregular, with large pores, which explained the reason why the control surimi gel had low strength and large cooking loss (Fig. 2b). In addition, the morphological properties of another group of surimi gel (TGase-induced) are appreciably different from the scanning electron micrograph mentioned above (Fig. 4b). Numerous compact and evenly distributed small pores were observed, instead of the loose structure in the control sample, which accounted for the fact that TGase could interact with proteins to form a more uniform and orderly three-dimensional network structure in the process of gel formation[47]. Moreover, similar findings were also reported in the previous studies of Dong et al.[26]. When treated with TGase and ε-PL (from 0.05% to 0.04%), the surface of the surimi gel sample gradually became flat through observation (Fig. 4f). We found that some smaller pores were formed and some larger pores were slightly supplemented visually. Remarkably, when the addition of ε-PL increased to 0.06% (Fig. 4g), the structure of surimi gel was not as uniform as before, and some large and irregular holes appeared obviously. This phenomenon might be related to moderate ε-PL, especially 0.02%, which can promote the cross-linking of TGase with protein and the aggregation/denaturation of different components, which agreed with the change of coagulation strength of surimi samples.

    Figure 4.  Effect of different treatments on the microstructure of surimi.
    DownLoad: Full-Size Img PowerPoint

    Thiobarbituric acid-reactive substances (TBARS) determination

    TBARS value, which can reflect the degree of lipid oxidation and rancidity of surimi, is an important indicator for the occurrence of oxidation[48]. Some substances in surimi, such as unsaturated fatty acids, will inevitably undergo oxidative decomposition to produce a large amount of malondialdehyde (MDA), the latter reacts easily with thiobarbituric acid (TBA) reagent to produce red compounds with maximum absorbance at 532 nm. As illustrated in Fig. 5, when TGase and a certain amount of PL (from 0.005% to 0.06%) were added, the TBARs value of surimi gels decreased to 2.03 and 1.67 mg/kg respectively, compared with the control group (2.26 mg/kg). This shows that the addition of both can significantly reduce the TBARS value of surimi gel (p < 0.05), considered to be a cue for the fat oxidation of surimi being inhibited. In addition, we also found that the antioxidant effect of PL was dose-dependent. The ε-PL can be chelated by ferrous ions along with the ability to scavenge free radicals like hydroxyl radicals, ensuring its antioxidant properties[33]. The ε-PL was prospectively considered as an antioxidant additive to prevent surimi protein from deteriorating induced by oxidation[49]. Similarly, making use of lysine or ε-PL as antioxidants in plant protein and meat protein has been widely reported[33, 50]. Fan et al.[42] confirmed that ε-PL addition has a significant effect on maintaining a high total phenolic content and vitamin C levels in fresh lettuce, which also shows that it has certain antioxidant activity.

    Figure 5.  Effect of different treatments on TBARS values of surimi gel. a−f: values with different lowercase letters indicate significant difference (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    Conclusions

    The addition of ε-PL at different concentrations had an apparent impact on the characteristics of TGase-induced mixed surimi gel, accompanied by the following situations: The 0.04% ε-PL provided a synergistic effect to promote the aggregation and crosslinking of surimi proteins induced by TGase; The rheology, LF-NMR and SEM results showed that the appropriate concentration (0.04%) of ε-PL apparently enhanced the initial apparent viscosity and elasticity of surimi samples, which was conducive to the formation of a more dense and uniform three-dimensional network structure, further limiting the flow of water in surimi and the exudation of hydrophilic substances; Simultaneously, the network strength was strengthened along with the texture properties of the mixed surimi gel. Furthermore, ε-PL had a strong ability to inhibit lipid oxidation in mixed surimi gel, showing a concentration dependence. When the content of ε-PL ran to lowest (0.005%−0.01%) or highest (0.6%), the improvement of the quality of surimi mixed gel was opposite to the above. The results show that ε-PL can be perceived as a prospective polyfunctional food additive to improve the texture properties, nutritional advantages, and product stability of surimi products.

  • The authors confirm contribution to the paper as follows: study conception and design: Cao Y, Xiong YL, Yuan F, Liang G; data collection: Liang G, Cao Y, Li Z, Liu M; analysis and interpretation of results: Li Z, Cao Y, Liang G, Liu Z, Liu M; draft manuscript preparation: Li Z, Cao Y, Liu ZL. All authors reviewed the results and approved the final version of the manuscript.

  • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    • Acknowledgments

    This work was financially supported by the Natural Science Basic Research Program of Shaanxi (No. 2023-JC-YB-146), the fund of Cultivation Project of Double First-Class Disciplines of Food Science and Engineering, Beijing Technology & Business University (No. BTBUKF202215), the Innovation Capability Support Plan of Shaanxi (No. 2023WGZJ-YB-27), the Agricultural Technology Research and Development Project of Xi'an Science and Technology Bureau (No. 22NYYF057), and Jiangsu Yiming Biological Technology Co., Ltd. in China.

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1 Metabolite classification.
    Supplemental Table S2 Related parameters of Volcano plots.
    Supplemental Table S3 Differential metabolite analysis of Volcano plots.
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    Zhao X, Chi N, Xu X, Lai J, Chen J, et al. 2024. Metabolome profiling unveil the composition differences of quality of different tea cultivars. Beverage Plant Research 4: e023 doi: 10.48130/bpr-0024-0012
    Zhao X, Chi N, Xu X, Lai J, Chen J, et al. 2024. Metabolome profiling unveil the composition differences of quality of different tea cultivars. Beverage Plant Research 4: e023 doi: 10.48130/bpr-0024-0012
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Metabolome profiling unveil the composition differences of quality of different tea cultivars

  • 1.

    Key Laboratory of Biology, Genetics and Breeding of Special Economic Animals and Plants, Ministry of Agriculture and Rural Affairs; Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China

  • 2.

    Tea Research Institute, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China

  • Received: 05 January 2024
  • Revised: 01 February 2024
  • Accepted: 22 February 2024
  • Published online: 04 July 2024
Beverage Plant Research  4,  Article number: e023  (2024)  |  Cite this article

Abstract: The tea plant is abundant in bioactive compounds, including flavonoids, amino acids, alkaloids, terpenoids, and lipids, which greatly affect tea quality and flavors. Despite there are many studies of metabolites about different tea cultivars, the composition differences of their biosynthesis and regulation still largely unknown. In this study, 505 metabolites were detected from the apical bud and 1st leaf of 'Shuchazao' (SCZ), 'Huangkui' (HK) and 'Zijuan' (ZJ) using widely-targeted metabolomics, including 192 flavonoids, 45 lipids, 59 amino acids and derivatives, and 28 phenolamides. Metabolite analysis showed that flavonols and anthocyanins are mainly distributed in the form of glycosides in three cultivars. Notably, anthocyanins and their glycosides are mainly accumulated in 'ZJ', indicating a correlation with the color attributes. EGCG emerged as the most abundant flavan-3-ols compound among the three cultivars. Simultaneously, L-theanine represented the predominant free amino acid, mainly concentrated in the apical bud compared to the 1st leaf. Similarly, lipids, akin to free amino acids, predominantly accumulated in the apical buds of all three cultivars. These findings offer valuable insights into the genetic and metabolite diversity, augmenting our understanding of the biosynthesis of specialized metabolites in tea plants.

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    Introduction

    • The tea plant (Camellia sinensis) is an important global commercial crop and tea is primarily consumed as a non-alcoholic beverage made from processed leaves[1,2]. The tea plant is abundant in secondary metabolites, including, flavonoids, alkaloids, amino acids, and terpenoids[3−5], and various health-promoting functions of teas are largely attributable to these bioactive natural products. Flavonoids are one of the main secondary metabolites in tea, including chalcones, flavones, flavonols, flavanols, anthocyanins, condensed tannins or proanthocyanidins (PAs), and other specialized forms of flavonoids[6]. Of these, catechins (flavan-3-ols) are best characterized molecularly and biochemically[7−9]. The six main types of flavan-3-ols include epicatechin (EC), catechin (C), epigallocatechin (EGC), gallocatechin (GC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG), with EGCG being the representative catechin with the highest content in tea plants, playing essential roles in imparting bitterness and astringency[10−14]. Alongside flavan-3-ols, flavones, flavonols, and flavonol glycosides are also important secondary metabolites that affect the bitterness and astringency of tea soup[15−17]. Several typical flavones and flavonols in tea soup are apigenin, luteolin, kaempferol, quercetin, and myricetin[10]. Anthocyanins are also a branch of flavonoids, which mainly have protective effects such as antioxidant, anti-inflammatory, and anti-cancer properties, and they are mainly present in the form of glycosylation or acylation, and are generally abundant in purple plants[18].

      Free amino acids constitute the principal chemical components in tea, playing a pivotal role in enhancing its pleasant flavor. Among these amino acids, L-Theanine (γ-glutamyl-L-ethylamide) a non-protein amino acid, is particularly important in affecting tea flavor[19,20]. Concomitantly, L-theanine was verified to promote the umami taste of tea infusions by neutralizing catechins, flavonol glucosides, and caffeine[21]. To date, about 26 free amino acids have been identified in tea plants[22−26], wherein L-theanine is highly accumulated in tea plants[27−28]. L-theanine can be produced by β- Oxoglutamate synthesized through GOGAT, which catalyzes the β-Oxoglutamate production of L-Glutamate, followed by L-Glutamate being catalyzed by TS to produce L-theanine[5].

      The aroma of tea is also one of the important factors affecting its quality, among which lipid substances play an important role in the volatile[29]. Several important volatiles, jasmine, cis-jasmone, methyl jasmonate, hexanal, pentanal, nonanal, heptanal, (E)-2-hexenal, (E, E)-2,4-hexadienal, 1-penten-3-ol, (Z)-3-hexen-1-ol, (E)-2-Hexen-1-ol, and nerolidol, are lipid-derivative aroma compounds[30]. Furthermore, during the processing of oolong tea, extracellular lipids can also be transformed into aromatic substances and released[29]. Lipids mainly exist in the forms of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides in plants[31]. To date, about 170 lipids and their derivatives have been identified in tea plants[32].

      China possesses abundant germplasm resources, and the genetic and metabolite diversity within its tea population significantly mirrors the global tea diversity[33]. Nevertheless, different types of secondary metabolites vary greatly in different tea cultivars[10]. Notably, 'ZJ' and 'P113' are abundant in anthocyanins[34,35], and the theanine concentration in the yellow cultivar was significantly higher than that in the green cultivar[36−38]. To better regulate and improve tea quality, it is very important to study the composition differences of different tea cultivars. Here, we integrate metabolomic analyses to study the apical bud and the 1st leaf of 'SCZ', 'HK', and 'ZJ', and the results provide metabolic markers for tea breeding, and advance our understanding of the biosynthesis of specific metabolites in tea plants.

    Materials and methods

      Plant materials

    • Tea cultivars 'Shuchazao' (SCZ), 'Huangkui' (HK) and 'Zijuan' (ZJ) were grown in the experimental tea garden of Anhui Agricultural University (31°55' North, 117°12′ East; Hefei City, Anhui Province, China). The apical bud and the 1st leaf of five-year-old tea plants were sampled in April.

      Sample preparation and extraction

    • Fresh tea samples were collected, ground into powder in liquid nitrogen, extracted with a solution of methanol, acetonitrile, and water (2:2:1), and vortexed and mixed for 30 s. Steel balls were added to the mixture, and then treated with a 45-Hz grinder for 10 min, sonicated for 10 min (in an ice water bath), and left for 1 h at −20 °C. After centrifugation at 12,000 rpm for 15 min, 500 μL of the supernatant was transferred to a new centrifuge tube. The extraction solution was dried in a vacuum concentrator, and 160 μL of solution (acetonitrile : water =1:1) was added to the metabolite extraction solution after drying and then redissolved in a whirlpool bath for 30 s and an ice water bath for 10 min. After centrifugation at 12,000 rpm for 15 min, 120 μL of supernatant was placed in an injection bottle[39]. Additionally, lidocaine was used as the internal standard.

      Instrument platform and chemicals

    • Metabolites detection by Waters UPLC Acquity I-Class PLUS and Waters UPLC Xevo G2-XS QTof. Acquity UPLC HSS T3 1.8 um 2.1 * 100 mm, Waters; Methanol, acetonitrile, and acetic acid were purchased from Shanghai Guo Mei Pharmaceutical Co. UPLC-grade water was prepared from distilled water using a Milli-Q system (Millipore Laboratory, Bedford, MA, USA).

      Metabolite detection parameters

    • The liquid chromatography-mass spectrometry system used for metabolomics analysis was composed of a Worth Equity I-Class PLUS ultra-high performance liquid chromatograph in series with a Worth Xevo G2-XS QTof high-resolution mass spectrometer. The chromatographic column used was the purchased Waters Equity UPLC HSS T3 column (1.8 μm 2.1 mm × 100 mm). Positive ion mode: mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile. Negative ion mode: mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile. The samples (2 μL injection volume) were loaded on a UPLC HSS T3 column and eluted at a flow rate of 400 μL/min. The elution program was as follows: starting with 95% and 5%, a linear gradient from 0−5.5 min, from 5% to 50%, B for 5.5−9.0 min, from 50% to 95%, B for 9.0−12 min, and B from 95% to 5% were performed, followed by washing and equilibration of the column.

      Metabolome data analysis

    • Simca-P software (version 13.0) was used for unsupervised principal component analysis (PCA)[40], and orthogonal projections to latent structure-discriminant analysis (OPLS-DA) as previously described[41]. The heatmaps for metabolites relative content were conducted using the 'heatmap' package implemented in R.

    Results

      Construction of a metabolomic database in tea plants

    • To assess variations in metabolites among three tea cultivars, we collected the apical bud and 1st leaf from three tea cultivars, including the green cultivar 'SCZ', the yellow cultivar 'HK', and the purple cultivar 'ZJ', as shown in Fig. 1a. Clustering analysis of all metabolites showed significant differences in the chromatograms of different tissues and cultivars (Fig. 1b). A total of 505 metabolites were identified in tea plants (Supplemental Table S1). The identified metabolites can be divided into ten main categories, including flavonoids (38.03%), terpenoids (1.39%), alkaloids (2.57%), phenolamides (5.54%), vitamins (2.57%), amino acids and derivatives (11.68%), nucleotides and derivatives (9.51%), Organic acid (3.56%), lipids (8.91%), and others (16.24%) (Fig. 1c). In the PCA score plot, the cumulative contribution rate of two principal components (t1 59.5% and t 20.2%) reached 79.7% (Fig. 1d). PCA analysis results showed that variance and distinguished samples according to the different apical bud and 1st leaf of three tea cultivars, and were appropriate for further difference assessments. The current results further revealed that the tea plants are rich in secondary metabolites, and the compositions differences between different cultivars and tissues.

      Figure 1. 

      Detection and identification of specialized metabolites. (a) The phenotypes of two tissues of the three cultivars. 'Shuchazao' (SCZ), 'Huangkui' (HK) and 'Zijuan' (ZJ). (b) Clustering heatmap tree of total metabolites of two tissues of the three cultivars. Z-scores normalize the value. Red indicates a high abundance, and blue indicates a low relative abundance of metabolites. (c) Composition and proportion of different metabolites in different tea cultivars. (d) PCA of the metabolites in different tissues of tea plants. SB: SCZ-bud; ZB: ZJ-bud; HB: HK-bud; SL: SCZ-leaf; ZL: ZJ-leaf; HL: HL-leaf.

      Multivariate analysis of differential metabolites

    • To further elucidate the variances in metabolites among different cultivars, multivariate statistical methods were adopted to analyze metabolic differences. Pairwise comparisons were performed among SB vs HB, ZB vs SB, ZB vs HB, SL vs HL, ZL vs SL, and ZL vs HL of the three tea cultivars by the OPLS-DA model to identify the metabolites responsible for the observed differences (Fig. 2a, b). Consequently, high predictability (Q2Y) and strong goodness of fit (R2X, R2Y) of the OPLS-DA models were observed in the comparison between SB vs HB (R2X = 0.956, R2Y = 0.999, Q2Y = 0.995), as well as between ZB vs SB (R2X = 0.954, R2Y = 1, Q2Y = 0.999), ZB vs HB (R2X = 0.811, R2Y = 1, Q2Y = 0.997), and SL vs HL (R2X = 0.955, R2Y = 1, Q2Y = 0.998), ZL vs SL (R2X = 0.987, R2Y = 1, Q2Y = 1 ), and ZL vs HL (R2X = 0.973, R2Y = 1, Q2Y = 0.999). These parameters underscore the stability and appropriateness of the models, signifying distinct metabolic profiles among the three tea cultivars. To gain more insight into the metabolic differences between SB vs HB, ZB vs SB, ZB vs HB, SL vs HL, ZL vs SL, and ZL vs HL of the three tea cultivars, respectively, differential metabolites screening was performed by Volcano plots, and the screening results are shown in Fig. 3 and Supplemental Table S2. Approximately 505 differential metabolites were analyzed through Volcano plots, revealing 91 up-regulated and 42 down-regulated metabolites between SB and HB (Fig. 3a, Supplemental Table S3), 68 up-regulated and 52 down-regulated metabolites between SB and ZB (Fig. 3b, Supplemental Table S3), and 82 up-regulated and 61 down-regulated metabolites between ZB and HB (Fig. 3c, Supplemental Table S3). Similar results were also observed in the comparison of three different leaf groups (Fig. 3d−f, Supplemental Table S3). Attention-worthy, most differential metabolites were flavonoids (Supplemental Table S3), thus, in the following analysis process, we focus on analyzing the differences of flavonoids in three tea cultivars.

      Figure 2. 

      Differential metabolite analysis by OPLS-DA.

      Figure 3. 

      Volcano plots showing the differential metabolite expression levels.

      Specific analysis of differential metabolites

    • Figure 3 showed that the main differential metabolites in the six tissues of the three cultivars are flavonoids. Flavonoids are derived from the phenylpropanoids pathway, which is catalyzed by a series of enzymes to produce flavonols, anthocyanins, and flavan-3-ol (Fig. 4a). To identify specific differences in flavonoid metabolites, we conducted a more detailed analysis using cluster analysis (Fig. 4b,c). Flavan-3-ols are the main flavonoids, and their content directly affects the quality of the tea. The EGCG has the highest content of six types of catechins, and compared to 'SCZ' and 'HK', the relative content of EGCG in 'ZJ' is slightly lower in both the apical bud and the 1st leaf, as shown in part P1 of Fig. 4b. The accumulation of two other typical catechins, EC and C, in 'HK' is inferior to that in 'ZJ' and 'SCZ', as shown in part P2 of Fig. 4b. In contrast to flavan-3-ols, anthocyanins mainly accumulate in the form of glycosides in 'ZJ', as shown in part P3 of Fig. 4b, except for cyanidin 3,5-O-digglucoside, indicating a correlation with the color attributes. Additionally, beyond catechins and anthocyanins, flavonols and glycosides are also important factors affecting tea quality. Among the six tissues of three cultivars, flavonoids mainly exist in the form of glycosides, mainly flavonol glycosides (Fig. 4c). Furthermore, flavonol glycosides, similar to anthocyanin glycosides, accumulate in 'ZJ', as shown in part P4 of Fig. 4c. In addition to the above characteristics, the accumulation of flavonols and flavonol glycosides in the apical buds of the three varieties is slightly lower than that in the leaves (Fig. 4c). These findings indicate that 'SCZ' mainly accumulates a large number of flavan-3-ols, such as EGCG, EC, and C, while anthocyanins, anthocyanin glycosides, and flavonol glycosides are mainly accumulated in 'ZJ'. In comparison to the other two cultivars, the synthesis, and accumulation of flavonoid metabolites in 'HK' are comparatively less, suggesting a potential correlation with genetic factors.

      Figure 4. 

      Phenylpropanoid pathways toward the biosynthesis of flavonoids in tea plants. (a) Flavonoid biosynthesis pathways in tea plant. (b), (c) Catechins, flavonoids and anthocyanin relative content in different tissues of SCZ, HK and ZJ plants. CHS: Chalcone synthase; CHI: Chalcone isomerase; C4H: Cinnamate 4-hydroxylase; DFR: Dihydroflavonol reductase; EGC: Epigallocatechin; ECG: Epicatechin-3-gallate; F3H: flavonoid 3-hydroxylase; F3'H: Flavonoid 3'-hydroxylase; F3'5'H: Flavonoid 3'5'-hydroxylase; FNS: flavone synthase; FLS: Flavonol synthase; LAR: Leuacoanthocyanidin reductase; PAL: Phenylalanine ammonialyase; 4CL: 4-Coumarate:CoA ligase; HCT: Hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase1; SCPL: Serine carboxypeptidase-like Clade 1A; UGT: UDP-Glucose flavonoid 3-O-glucosyl transferase; C: Catechin; EC: Epicatechin; EGCG: Epigallocatechin-3-gallate.

      Metabolic analysis of free amino acids

    • Free amino acids are also the main compounds that affect the quality of tea, especially L-theanine. Amino acids are synthesized through branched pathways in tea plants (Fig. 5a). Glucose serves as the foundational element for all amino acid metabolism and is synthesized diversely to generate essential and non-essential amino acids in plants. Glycolysis produces 3-phosphoglycerate, which is the substrate of serin, glycine, and cysteine synthesis. Furthermore, a further reaction of glycerate 3-phosphate resulted in the generation of phosphoenolpyruvate, which is the initiator of shikimate. Pyruvate is the starting point for the synthesis of L-theanine and other free amino acids. Alanine, ethylamine, and L-glutamate are several important prerequisite amino acids for the synthesis of L-theanine, and those undergo different catalytic enzyme reactions to produce L-theanine, such as TS, and GGT (Fig. 5a). A cluster analysis of amino acids and derivatives reveals that L-theanine exhibits the highest content of free amino acids, as shown in the P5 section of Fig. 5b. It is worth noting that the content of most free amino acids in the apical buds of the three cultivars is higher than in the leaves, which is contrary to the synthesis pattern of flavonoids (Fig. 4c, 5b). Clustering analysis results indicate a marginally higher accumulation of several amino acids and derivatives in 'HK' compared to the other two cultivars, including L-glutamine, L-theanine, 2-aminoisobutyric acid, and L-glutamic acid (Fig. 5b). Consequently, based on the above results, it can be seen that 'HK' did not accumulate too many flavonoids but instead accumulated a large number of free amino acids, mainly in the apical buds.

      Figure 5. 

      L-theanine and other free amino acid metabolism pathways in tea plants. (a) L-theanine biosynthesis pathways in tea plant. (b) Other free amino acid and L-theanine relative content in different tissues of SCZ, HK and ZJ plants. OGAT: Glutamate synthase; ALDA: Alanine decarboxylase; GGT: γ-glutamyltranspeptidase; TS: Theanine synthetase.

      Accumulation patterns of lipids in different tea cultivars

    • It was necessary to analyze lipids to compare the differences in metabolites of different tea qualities, as lipids are precursors of volatile aroma compounds. Targeted lipid metabolite detection was conducated on six samples from three different cultivars. Cluster analysis revealed that Octadeca-11E,13E,15Z-trienoic acid, LysoPC 18:3 (2n isomer), LysoPC 18:2 (2n isomer), MAG (18:3) isomer3, and α-linolenic acid show the highest content of all the detected lipids and derivatives in three tea cultivars (Fig. 6). Additionally, Lysopc, lysope, and MAG have a large number of isomers present (Fig. 6). Comparison between apical buds and leaves of the same cultivar unveiled that the majority of lipid metabolites accumulated in the apical buds, with similar patterns observed in all three cultivars (Fig. 6). It is worth noting that the accumulation pattern of lipids is similar to the accumulation pattern of free amino acids (Fig. 5b). Further examination of lipid accumulation patterns across three tea cultivars indicated that the 'ZJ' variety's apical buds accumulated relatively higher lipid content than the other two cultivars, as shown in Part P6 in Fig. 6. Conversely, there is not much difference in the accumulation amount in the leaves of Fig. 6. The observed accumulation patterns suggest distinctions in lipid accumulation among various tea cultivars, with a notable tendency for higher lipid accumulation in apical buds compared to leaves.

      Figure 6. 

      Differential analysis of lipid metabolites. (MGDG - monogalactosyldiacylglycerol, GDG - digalactosyldiacylglycerol, C12:0 - lauric acid, C14:0 - myristic acid, C16:0 - palmitic acid, C16:1 - palmitoleic acid, C18:0 - stearic acid, C18:1 - oleic acid, C18:2 - linoleic acid, C18:3 - linolenic acid, C20:0 - arachidic acid, C20:1 - eicosenoic acid, C20:2 - eicosadienoic acid, C20:3 - eicosatrienoic acid, C20:4 - eicosatetraenoic acid, C22:0 - docosanoic acid, C24:0 - tetracosanoic acid, C24:1 - tetracosenoic acid, C26:0 - hexacosanoic acid, C28:0 - octacosanoic acid; DG - diacylglycerol, TG - triacylglycerol; PC - phosphatidylcholine; LPC - lysophosphatidylcholine, PE - phosphatidylethanolamine, PA - phosphatidic acid).

    Discussion

    • Tea is favored by consumers due to its abundance of various bioactive substances. Presently, there are about 3000 tea accessions in China[33]. Confronted with such a vast variety of tea cultivars, analyzing the quality metabolites of each tea cultivar remains a formidable challenge. Previous studies showed that flavonoids, amino acids, and lipids affect the quality of tea[1,12,28]. In 2020, Yu et al.[10] analyzed differential metabolites in 136 cultivars, the results showed that CSA tea accessions feature a high accumulation of diverse classes of flavonoid compounds, such as flavanols, flavonol mono-/di-glycosides, proanthocyanidin dimers, and phenolic acid.

      In this study, We selected three tea cultivars with different color phenotypes (Fig. 1a), and analyzed the different metabolites through widely targeted metabolomics. Notably, flavonoids exert a significant influence on the bitterness and astringency of tea, and we can derive from analyzing the data that flavan-3-ols, flavones, flavonols, and glycosides mainly accumulate in green ('SCZ') and purple ('ZJ') cultivars Fig. 2. This result to some extent indicates that under the same processing technology, the bitterness and astringency of 'SCZ' and 'ZJ' cultivars exhibit a slightly greater intensity than that of 'HK'. In addition, 'ZJ' has accumulated a large number of anthocyanins, which is also the reason for the high bitterness and astringency of 'ZJ' (Fig. 4b). Free amino acids mainly provide the pleasant flavor of tea soup, especially L-theanine. By comparative analysis, the free amino acid content in the apical buds and 1st leaf of 'HK' is marginally higher than that of the two cultivars (Fig. 5), and this result indicates that 'HK' may have a more pleasant flavor, particularly in the apical buds. Lipids and their derivatives provide the aroma of tea, which directly affects the quality of tea. Fig. 6 showed that the accumulated lipids in 'ZJ' are slightly higher than those in 'SCZ' and 'HK', especially the apical buds. Drawing on the accumulation pattern of flavonoids and lipids, we infer that 'ZJ' not only exhibits pronounced bitterness and astringency but also boasts a heightened aromatic profile.

      By comparative analysis of various metabolites of the three cultivars, we infer that there are significant differences in the accumulation of metabolites of different tea cultivars, and the main reason should be genetic differences[1,10]. Furthermore, under the same genetic background, there are also differences in metabolite accumulation in different tissues, leading to this result mainly due to differences in the transcription level of key genes involved in metabolite synthesis[42]. The above results provide insights into metabolite diversity and are useful for accelerated tea plant breeding.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and design: Chen L, Zhao X; analysis and interpretation of results: Zhao X, Chi N, Xu X, Lai J, Chen J; draft manuscript preparation and revision: Chen L, Zhao X. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All experimental data were from at least three independent experiments. The majority of data that support the findings of this study are available in the supplementary material of this article, others will be available upon request from the corresponding author, who will also be responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors.

      Acknowledgments

      • This work was supported by the Hainan Province postdoctoral surface funding project (RZ2300001314), the National Natural Science Foundation of China (U19A2030, 32072631), the China Agricultural Research System of MOF and MARA (CARS-019), and the Chinese Academy of Agricultural Sciences through the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2021-TRICAAS) to LC.

      Conflict of interest

      • The authors declare that they have no conflict of interest. Liang Chen is the Editorial Board member of Beverage Plant Research who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

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      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  References (42)
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    Zhao X, Chi N, Xu X, Lai J, Chen J, et al. 2024. Metabolome profiling unveil the composition differences of quality of different tea cultivars. Beverage Plant Research 4: e023 doi: 10.48130/bpr-0024-0012
    Zhao X, Chi N, Xu X, Lai J, Chen J, et al. 2024. Metabolome profiling unveil the composition differences of quality of different tea cultivars. Beverage Plant Research 4: e023 doi: 10.48130/bpr-0024-0012
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  • Introduction
  • Materials and methods

  • Plant materials

  • Sample preparation and extraction

  • Instrument platform and chemicals

  • Metabolite detection parameters

  • Metabolome data analysis

  • Results

  • Construction of a metabolomic database in tea plants

  • Multivariate analysis of differential metabolites

  • Specific analysis of differential metabolites

  • Metabolic analysis of free amino acids

  • Accumulation patterns of lipids in different tea cultivars

  • Discussion
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
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