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2023 Volume 3
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    

Analysis of fragrance compounds in flowers of Chrysanthemum genus

  • 1.

    Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture, China Agricultural University, Beijing 100193, China

  • 2.

    Institute of Agri-food Processing and Nutrition (IAPN), Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China

  • # These authors contributed equally: Zhiling Wang, Xin Zhao

More Information
  • Chrysanthemum is one of the four major cut flowers in the world, with high ornamental and economic value. Fragrance is an important ornamental character of chrysanthemum flowers, especially those consumed as tea and other foods, and the flower fragrance is the major determinant of the commercial value of chrysanthemum cultivars. Currently, however, the research on chrysanthemum flower fragrance is mainly focused on the composition and content of fragrant compounds, and a clear classification of fragrance types is lacking. Here, we divided chrysanthemum fragrance into six categories based on sensory evaluation and determined the identity and content of fragrant compounds of chrysanthemum accessions representative of each fragrance type by GC-MS. In addition, we analyzed the conserved aromatic substances responsible for the fruity fragrance type chrysanthemum with multi-functional development potential, providing a theoretical basis for creating new chrysanthemum germplasm with specific fragrance types. The results of this study can accelerate the breeding process of chrysanthemum accessions with new fragrance types.

  • 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 Statistics of various volatile substances in Chrysanthemum with different aroma types.
    Supplemental Table S2 Statistical table of sensory evaluation of chrysanthemum flavor.
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    Wang Z, Zhao X, Tang X, Yuan Y, Xiang M, et al. 2023. Analysis of fragrance compounds in flowers of Chrysanthemum genus. Ornamental Plant Research 3:12 doi: 10.48130/OPR-2023-0012
    Wang Z, Zhao X, Tang X, Yuan Y, Xiang M, et al. 2023. Analysis of fragrance compounds in flowers of Chrysanthemum genus. Ornamental Plant Research 3:12 doi: 10.48130/OPR-2023-0012
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Analysis of fragrance compounds in flowers of Chrysanthemum genus

  • 1.

    Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture, China Agricultural University, Beijing 100193, China

  • 2.

    Institute of Agri-food Processing and Nutrition (IAPN), Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China

  • Received: 14 February 2023
  • Accepted: 19 April 2023
  • Published online: 27 June 2023
Ornamental Plant Research  3,  Article number: 12  (2023)  |  Cite this article

Abstract: Chrysanthemum is one of the four major cut flowers in the world, with high ornamental and economic value. Fragrance is an important ornamental character of chrysanthemum flowers, especially those consumed as tea and other foods, and the flower fragrance is the major determinant of the commercial value of chrysanthemum cultivars. Currently, however, the research on chrysanthemum flower fragrance is mainly focused on the composition and content of fragrant compounds, and a clear classification of fragrance types is lacking. Here, we divided chrysanthemum fragrance into six categories based on sensory evaluation and determined the identity and content of fragrant compounds of chrysanthemum accessions representative of each fragrance type by GC-MS. In addition, we analyzed the conserved aromatic substances responsible for the fruity fragrance type chrysanthemum with multi-functional development potential, providing a theoretical basis for creating new chrysanthemum germplasm with specific fragrance types. The results of this study can accelerate the breeding process of chrysanthemum accessions with new fragrance types.

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    Introduction

    • Chrysanthemum (Chrysanthemum × morifolium [Ramat.] Kitamura) is native to China and is cultivated for sale as fresh cut flowers, planting in ornamental gardens, landscaping, and medicinal use. Floral fragrance is an important trait which mediates the intraspecific and interspecific interactions of plants[1]. Floral volatiles can attract pollinators, which promotes sexual reproduction, as well as natural enemies of phytophagous insects, which prevents attack by insect pests[2]. In addition, volatile compounds protect plants from abiotic stresses, such as strong light, high temperature, and oxidative stress[3,4]. The importance of floral fragrance is receiving increased research attention.

      Floral fragrance is determined by the type and content of volatile organic compounds (VOCs)[5]. A variety of VOCs are synthesized in plants. Depending on their source, VOCs are classified into three categories: terpenoids, phenylpropanoids/benzenoids, and fatty acid derivatives[1].

      Terpenoids, which form the largest class of VOCs are composed of several isoprene (C5) structural units. Depending on the number of structural units, terpenoids are classified as monoterpene (C10), sesquiterpene (C15), and diterpene (C20) compounds[6]. For example, monoterpenoids are the main aromatic substances in rose (Rosa × hybrida) flowers[7] , while linalool and ionone are the main compounds in Osmanthus fragrans flowers[8].

      Phenylpropanoids/benzenoids for the second largest class of plant VOCs[9], however, the complete biosynthetic pathway of phenylpropanoid compounds remains unclear. According to current knowledge, the direct precursor of phenylpropanoid/benzenoid compounds is phenylalanine, which is synthesized mainly through the shikimate pathway[10]. The flower fragrance of Petunia (Petunia hybrida) is mainly attributable to phenylpropanoid/benzenoid compounds, among which benzaldehyde, phenylacetaldehyde and methyl benzoate are the most abundant[11].

      Fatty acid derivatives are the third group of plant volatile substances. Acetyl coenzyme A (acetyl CoA) is the precursor of fatty acid derivatives. Acetyl CoA enters the lipoxygenase (LOX) pathway, and produces volatile substances through a series of reactions. According to a recent study, (E) -2-hexenal is one of the main compounds responsible for the floral fragrance of carnation[12].

      Previous research on the floral fragrance of chrysanthemum has mainly focused on the identification of aromatic compounds. In chrysanthemum and its wild relatives, monoterpenoids and oxygenated monoterpenoids, including camphor, α-pinene, laurene, and eucalyptus alcohol, are the predominant volatile components[13]. Monoterpenoids and sesquiterpenoids, including hydrocarbons, esters, aldehydes, ketones, phenols, and organic acids, are the predominant compounds of chrysanthemum volatile oil[14]. Investigation of the relationship between the accumulation and release of terpenoids in 44 related species and cultivars of chrysanthemum revealed that the release of terpenoids is strongly correlated with their internal concentration, whereas the concentration of terpenoids is associated with the release of the compound and the size of the capitulum. Tubular florets have a greater impact on the release of volatile substances than ligulate florets. In addition, the involucre and receptacle serve as the main sites for the accumulation of terpenoids[15]. The volatiles of chrysanthemum cultivar 'Boju' are mainly eucalyptus alcohol, filifon, pyrethrone, and trans- and cis-pyrethroid acetates[16]. An aromatic wild species, Dendranthema indicum (Chrysanthemum indicum var. aromaticum) was introduced to breed aphid-resistant offspring through hybridization with Chrysanthemum nankingense. Nineteen compounds of aphid resistant lines were selected and cis-4-thujanol was confirmed to be an effective aphid repellent[17]. Thus, the composition and content of volatiles differ substantially among chrysanthemum species. Although the volatile substances of chrysanthemum have been researched, the classification of chrysanthemum fragrance has not yet been reported.

      As stated above, the aroma of flowers determines the commercial value of chrysanthemum cultivars, especially those used for tea and edible purposes. Because of long-term natural selection and evolution, the fragrance type of chrysanthemum is highly diverse. Nevertheless, previous research on chrysanthemum floral fragrance mainly focused on the determination of the volatile compounds and their contents, and research on the classification of chrysanthemum fragrance types is lacking. In this study, the aroma type of among a large sample of chrysanthemums was investigated using a sensory evaluation method, and volatile substances of representative chrysanthemums of each aroma type were analyzed by gas chromatography–mass spectrometry (GC–MS). Based on the aroma type, chrysanthemum accessions were classified into six categories, providing a theoretical basis for the accelerated breeding of new chrysanthemum germplasm with specific aroma types.

    Materials and methods

      Plant materials

    • Chrysanthemum materials used for fragrance classification were collected from major parks in Beijing (China) and the chrysanthemum resource garden at the Shangzhuang Experimental Station of the China Agricultural University, Beijing, China.

      Chrysanthemums used for GC-MS determination were Chrysanthemum × morifolium 'Qihuang', C. indicum L., C. × morifolium 'Bairuixiang', C. × morifolium 'Quehuan', C. × morifolium 'Xiaokuixiang', and C. × morifolium 'Sigong'.

      Artificial pollination

    • Rooted cuttings of 'Xiaokuixiang' were planted at the Shangzhuang Experimental Station (Beijing, China), and reproductive isolation was carried out. Artificial self-pollination was conducted at the onset of flowering. The seeds were collected when mature.

      Open hybridization with 'Xiaokuixiang' as the female parent

    • A chrysanthemum cultivar with the same flowering period as 'Xiaokuixiang' was planted on either side of the female parent ('Xiaokuixiang'). Sterilized tweezers were used to remove the stamens at the onset of flowering of 'Xiaokuixiang', and the upper portion of the corolla of the outer florets in the capitulum was removed to expose the pistils. The seeds were collected when mature.

      Sensory evaluation

    • The aroma type of chrysanthemum was determined by means of a questionnaire. The members of the research group randomly distributed questionnaires to recipients. The aroma types were determined after statistical analysis. On the basis of the questionnaire, 24 students and teachers who were familiar with chrysanthemums and had the ability to distinguish aroma types were invited as sensory evaluators to screen representative cultivars of each fragrance type.

      Solid phase microextraction

    • Flowering stems of chrysanthemum were cut with secateurs, immediately placed in a bucket containing clean water, and transported to the laboratory for sampling. The capitulum (0.2–1.0 g) of each chrysanthemum was placed in a sampling bottle, with three replicates per cultivar, and then 15 μL of the internal standard (43.25 ng/g ethyl decanoate) was added to each bottle.

      Solid phase extraction head comprised 50/30 μm divinylbenzene/carbon/polydimethyl siloxane. The sample was placed in a 15 ml glass bottle in a 45 °C water bath, the extraction head was inserted, and the headspace was extracted for 30 min. The extraction head was analyzed in the 250 °C injection port for 3 min.

      Gas chromatography–mass spectrometry

    • The GC-MS analysis was conducted using a GCMS-QP2010 mass spectrometer (Shimadzu, Kyoto, Japan). The chromatographic conditions were as follows: injection port temperature, 250 °C; injection mode, split flow; total flow rate, 27.4 mL/min; split ratio, 20; ion source temperature, 200 °C; and interface temperature, 250 °C.

      The total analysis time was 30 min. The initial temperature was 40 °C, held for 1 min, then increased to 280 °C at 10 °C/min, held for 5 min, and the solvent delay time was 2.5 min. Mass spectrum conditions were: detector, 1 kV; mass scanning range 30–500 m/z; and full scanning mode.

      Statistical analysis

    • All determinations were performed with three biological replicates. Microsoft Excel and Graphpad Prism 8 were used to process and analyze the data. The results are expressed as the mean ± standard deviation (SD). Statistical significance was assessed using one-way analysis of variance (p < 0.05).

    Results

      Sensory evaluation and analysis of chrysanthemum flavor aroma

    • To classify the fragrance types of chrysanthemums, we performed sensory evaluation of the aroma characteristics of 520 chrysanthemum accessions. The fragrance of chrysanthemums could be grouped into six types: chrysanthemum fragrance, artemisia, medicinal, sweet, perfume fragrance, and fruity (Fig. 1). Among these types, chrysanthemum fragrance accounted for 30% of the cultivars, artemisia for 27.5%, medicinal for 20.0%, sweet for 6.0%, perfume fragrance for 6.5%, fruity for 3.5%, and others for 6.5%.

      Figure 1. 

      Statistical distribution of fragrance types in chrysanthemum accessions.

      According to the sensory evaluation results, the chrysanthemum accessions with the highest score in each fragrance type was selected as the representative of that category. The results showed that 91.67% of the evaluators considered that 'Sigong' was the most typical chrysanthemum cultivar with chrysanthemum fragrance, 91.67% considered that wild chrysanthemum (Chrysanthemum indicum L.) was the most typical chrysanthemum with artemisia fragrance, 87.50% considered that 'Qihuang' was the most typical cultivar chrysanthemum with medicinal fragrance, 75.00% considered that 'Quehuan' was the most typical chrysanthemum cultivar with sweet fragrance, 79.17% considered that 'Bairuixiang' was the most typical chrysanthemum cultivar with perfume fragrance, and 83.33% considered 'Xiaokuixiang' was the most typical chrysanthemum cultivar with fruity fragrance (Table 1). Therefore, to further analyze the aroma components of chrysanthemums of different fragrance types, the identity components and contents of the volatile substances were determined by selecting 'Sigong', C. indicum, 'Qihuang', 'Quehuan', 'Bairuixiang', and 'Xiaokuixiang' as the representative accessions of the chrysanthemum fragrance, artemisia, medicinal, sweet, perfume fragrance, and fruity fragrance, respectively (Fig. 2).

      Table 1.  Statistics foruation of chrysanthemum fragrance.

      Chrysanthemum cultivarsChrysanthemum
      fragrance (%)      		Artemisa
      fragrance (%)      		Medicinal
      fragrance (%)      		Sweet
      fragrance (%)      		Perfume
      fragrance (%)      		Fruity
      fragrance (%)
      Sigong91.678.330000
      Chrysanthemum indicum8.3391.670000
      Qihuang012.5087.50000
      Quehuan16.670075.0008.33
      Bairuixiang00012.5079.178.33
      Xiaokuixiang00012.504.1783.33

      Figure 2. 

      Chrysanthemum materials used in the experiment. (a) Chrysanthemum indicum L.; (b) C. × morifolium 'Xiaokuixiang'; (c) C. × morifolium 'Quehuan'; (d) C. × morifolium 'Bairuixiang'; (e) C. × morifolium 'Qihuang'; (f) C. × morifolium 'Sigong'.

      Identity and content of VOCs in chrysanthemum accessions of different aroma types

    • To explore the biochemical basis of different chrysanthemum fragrance types, the identity and content of volatile substances of representative chrysanthemum accessions were determined by GC–MS. Terpenoids were predominant in C. indicum (artemisia fragrance) and fatty acid derivatives were the most in 'Xiaokuixiang' (fruity fragrance), and the number of phenylpropanoid/benzenoid compounds was low in accessions of all fragrance types (Supplemental Table S1). As shown in Fig. 3, accessions with chrysanthemum fragrance, artemisia, medicinal, sweet, and perfume fragrance were dominated by terpenoids, accounting for more than 50% of all VOCs, followed by fatty acid derivatives, however, no significant difference was observed in the types and proportions of terpenoids and fatty acid derivatives in the of 'Xiaokuixiang' (fruity fragrance).

      Figure 3. 

      Proportions of volatile organic compounds in chrysanthemum with different fragrance types.

      To compare the differences among chrysanthemum accessions of different aroma types, the VOCs of different fragrance types were analyzed quantitatively. As shown in Fig. 3, except for 'Xiaokuixiang', other representative chrysanthemum accessions showed the highest content of terpenoids. Fatty acid derivatives were the most volatile substances.

      In 'Sigong', terpenoids were the most abundant, followed by fatty acid derivatives, and lastly phenylpropanoids/benzenoids. Eucalyptol was the main terpenoid, (E)-2-hexenal was the main fatty acid derivative, and o-cymene was the main phenylpropanoid/benzenoid compound (Fig. 4a). The compounds with the highest contents in 'Sigong' were eucalyptol, 2-pinene-6-one, and α-pinene, with the contents of 2,158.89, 849.00 , and 743.28 ng/µL/g, respectively (Fig. 4a).

      Figure 4. 

      Analysis of main volatile substances in Chrysanthemum with different fragrance types. (a) Analysis of main volatile substances in 'Sigong' with chrysanthemum fragrance; (b) Analysis of main volatile substances in Chrysanthemum indicum with artemisia fragrance; (c) Analysis of main volatile substances in 'Qihuang' with medicinal fragrance; (d) Analysis of main volatile substances in 'Bairuixiang' with perfume fragrance; (e) Analysis of main volatile substances in 'Quehuan' with sweet fragrance; (f) Analysis of main volatile substances in 'Xiaokuixiang' with fruity fragrance. FW: fresh weight.

      In C. indicum (artemisia fragrance), terpenoids were the most abundant, followed by fatty acid derivatives and phenylpropanoid/benzenoid compounds (Fig. 4b). D-camphor, β-myrcene, and eucalyptol were the main terpenoids (595.18, 214.92, and 143.53 ng/µL/g, respectively), and o-cymene was the main phenylpropanoid/benzenoid (Fig. 4b).

      In 'Qihuang' (medicinal fragrance), the content of terpenoids was the highest, followed by fatty acid derivatives and phenylpropanoid/benzenoid compounds (Fig. 4c). Eucalyptol was the main terpenoid compound, and (E)-2-hexenal and o-cymene were the main fatty acid derivatives and phenylpropanoid/benzenoid compounds, respectively (Fig. 4c). Eucalyptol, β-phellandrene, and (1S)-(−)-β-pinene showed the highest concentrations in 'Qihuang' (430.24, 109.42, and 105.67 ng/µL/g, respectively) (Fig. 4c).

      In 'Bairuixiang' (perfume fragrance), the content of fatty acid derivatives was the highest, followed by terpenoids, and that of phenylpropanoid/benzenoid compounds was lowest (Fig. 4d). (E)-2-hexenal was the main component of fatty acid derivative, whereas ocimene, β-myrcene, and linalool were the main components among terpenoids (Fig. 4d). (E)-2-hexenal, ocimene and β-myrcene showed the high concentrations in 'Bairuixiang' (504.72, 136.79, and 133.58 ng/µL/g, respectively) (Fig. 4d).

      In 'Quehuan' (sweet fragrance), the content of terpenoids were the most abundant, followed by fatty acid derivatives and phenylpropanoid/benzenoid compounds (Fig. 4e). β-myrcene, α-thujene and umbellulonl were the main components among terpenoids, (E)-2-hexenal were the main fatty acid derivatives, and o-cymene was the main phenylpropanoid/benzenoid compounds (Fig. 4e). The compounds with the high contents were β-myrcene, o-cymene, and α-thujene (1,236.08，604.88 and 537.86 ng/µL/g, respectively) (Fig. 4e).

      In 'Xiaokuixiang' (fruity fragrance) fatty acid derivatives were the most abundant, followed by terpenoids (Fig. 4f). (E)-2-hexenal and methyl salicylate were the main fatty acid derivatives, and (E)-β-farnesene and (E)-β-ocimene were the main terpenoids (Fig. 4f). The compounds with the high contents in 'Xiaokuixiang' were (E)-2-hexenal, (E)-β-farnesene, and methyl salicylate (78.94, 49.94, and 26.88 ng/µL/g, respectively) (Fig. 4f).

      Volatile organic compounds specific to accessions of different fragrance types

    • The preceding analysis showed that (E)-2-hexenal was the main volatile substance associated with the chrysanthemum, medicinal, perfume, and fruity fragrance types, and that eucalyptol was the main volatile substance associated with chrysanthemum, artemisia, and medicinal fragrance types. 2-Pinene-6-one, α-pinene and sabenene were the main volatile substances peculiar to the fragrance of 'Sigong', bornyl acetate was the main volatile substance peculiar to the artemisia fragrance of C. indicum. (1S)-(−)-β-pinene and β-phellandrene were the main volatile substances peculiar to the medicinal fragrance of 'Qihuang', ocimene and linalool were the main volatile substances peculiar to the perfume fragrance of 'Bairuixiang', 3-thujene and umbellulon were the main volatile compounds unique to the sweet fragrance of 'Quehuan', and methyl salicylate, (E)-β-ocimene, and 1-octene were the main volatile compounds unique to the fruity fragrance of 'Xiaokuixiang' (Fig. 5).

      Figure 5. 

      Bubble chart of volatile organic compounds specific in different fragrance types of chrysanthemum accessions.

      Analysis of aroma compounds of 'Xiaokuixiang' at different development stages

    • As shown in Fig. 6a, 'Xiaokuixiang' flowers were collected at three stages: bud stage, early flower stage and full flower stage. The aroma substances released by flowers at these three stages were divided into four categories: terpenoids (47%), fatty acid derivatives (32%), phenylpropanoid/benzenoid compounds (5%) and others (16%) (Fig. 6b). Quantitative analysis of different volatile substances showed that the content compounds were (E)-β-farnesene, (E)-2-hexenal, methyl salicylate and hexanal were high (Fig. 6c).

      Figure 6. 

      Analysis of volatile substances in different stage of flower in 'Xiaokuixiang' . (a) Different stage of flower in 'Xiaokuixiang'; (b) Proportions of volatile organic compounds in three stages; (c) Analysis of main volatile substances in different stage of flower in 'Xiaokuixiang'.

      Analysis of fragrance types in 'Xiaokuixiang' offspring

    • To analyze the genetic heritability of fruity fragrance type, we obtained 248 self-pollinated progenies and 383 hybrid progenies from 'Xiaokuixiang' as the female parent. Then, we determined the volatile substances of eight fruit-scented progenies by GC-MS (Supplemental Table S2), and compared the results with the volatile substances identified in 'Xiaokuxiang' . Ten volatile compounds were identified in nine fruit-scented chrysanthemums (Fig. 7), namely (E)-β-farnesene, 1-octene, caryophyllene, α-bergamotene, 1-hexanol, butanoic acid 2-methyl ethyl ester, butanoic acid 2-methyl propyl ester, butanoic acid 3-methyl hexyl ester, hexanoic acid ethyl ester and hexanal (Fig. 8).

      Figure 7. 

      Analysis of conserved volatile compounds in 'Xiaokuixiang' and its offsprings. Different colors represent different accessions.

      Figure 8. 

      A model for the fragrance types of chrysanthemum and main volatile substances of fruity fragrance. There is the classification and representative chrysanthemum of every fragrance type on the left, main volatile substances of fruity fragrance are on the right.

    Discussion

    • Flower fragrance is an important trait of flowering plants. It not only attracts pollinators for sexual reproduction but also promotes the interaction between plants and the environment, thus protecting plants from attack by pathogens, parasites, and herbivores[18,19]. Chrysanthemum is an important commercial floriculture crop. After long-term interspecific hybridization and artificial selection, a variety of chrysanthemum types have been developed, which are enriched in secondary metabolites that affect the floral fragrance of chrysanthemum.

      Aroma, as a trait perceptible by humans is particularly suitable for determining the fragrance type of chrysanthemum accessions through sensory evaluation. At present, the sensory evaluation method is used more systematically for the perception of food flavors. Although the sensory evaluation procedure for ornamental plants is not perfect, we used a relatively simple and convenient sensory evaluation method, employing a questionnaire survey to directly evaluate the perception of chrysanthemum fragrance. Through sensory evaluation of the fragrance of a large collection of accessions, we classified the accessions into six fragrance types: chrysanthemum fragrance represented by 'Sigong', artemisia fragrance represented by wild chrysanthemum, medicinal fragrance represented by 'Qihuang', sweet fragrance represented by 'Quehuan', perfume fragrance represented by 'Bairuixiang', and fruity fragrance represented by 'Xiaokuixiang'. Ongoing research will help improve the sensory evaluation of the aroma of ornamental plants.

      Aroma is dependent on volatile substances perceived by olfactory organs[20]. Therefore, the fragrance of plants is determined by the type and content of volatile substances. Detection of volatile substances responsible for the floral fragrance of chrysanthemum by GC-MS indicated that the main volatile substances in most chrysanthemums accessions were terpenoids (Fig. 3). The finding that terpenoids are important components of floral fragrance in chrysanthemum is consistent with previous studies[15].

      Further analysis showed that eucalyptol, 2-pinene-6-one, and α-pinene were the main volatile substances responsible for the fragrance of 'Sigong' (Fig. 4a). D-camphor, myrcene, and eucalyptol for artemisia fragrance (Fig. 4b), eucalyptol, β-phellandrene and (1S)-(-)-β-pinene for the medicinal fragrance of 'Qihuang' (Fig. 4c) and β-myrcene, o-cymene, and α-thujene for the sweet fragrance of 'Quehuan' (Fig. 4e). Previous studies have reported that the main volatile compounds of German chamomile (Matricaria recutita) are sesquiterpenes and monoterpenes, including (−)-γ-elemene, β-elemene, piperone, o-cymene, 3-perylene, and γ-terpene. The main volatile compounds reported for Roman chamomile (Chamaemelum nobile) are esters, including 3-methyl-2-butenoic acid, 3-methyl-2-alkenyl ester, 3-methyl-2-enoic acid, 2-methyl butyl ester, and 3-methyl-2-butenoic acid allyl ester[21]. These results indicate that the differences in volatile substances also exist among the different species of chamomile. 'Xiaokuixiang' is a novel chrysanthemum cultivar with a unique fragrance (fruity type) developed in the laboratory, whereas 'Bairuixiang' with perfume fragrance is a hybrid offspring of 'Xiaokuixiang'. The main volatile substances of 'Bairuixiang' are D-camphor, ocimene, and β-myrcene (Fig. 4d), and those of 'Xiaokuixiang' are (E)-2-hexenal, (E)-β-farnesene, and methyl salicylate (Fig. 4f). Analysis of the components of the various fragrance types by GC-MS revealed that the identity and content of the main volatile substances differed considerably among chrysanthemum cultivars.

      However, the content of volatile substances alone cannot confirm the characteristic volatiles of each chrysanthemum accession. Given the diversity and complex composition of volatile substances, their absolute content is not the only standard to measure their contribution to fragrance, an additional important factor is the aroma threshold of volatile substances[22]. Aroma threshold is a quantitative expression of aroma[23]. At a certain concentration, the lower the aroma threshold, the stronger the aroma of the substance, and vice versa. Furthermore, the aroma threshold of a of volatile substance changes under different conditions and in different solvents Therefore, based on the quantitative data obtained for the volatile substances in the present study, we could only determine the chrysanthemum fragrance type through sensory evaluation, and the main volatile substances that contribute to each fragrance type. Determination of the characteristic aromatic substances responsible for each fragrance type requires further investigation. Overall, we divide chrysanthemum accessions into six categories based on the sensory evaluation of floral fragrance, and found a novel fragrance type (fruity) chrysanthemum. Furthermore, this work provides a theoretical basis for the accelerated breeding of new chrysanthemum germplasm with specific aroma types.

    Conclusions

    • Chrysanthemum is an important ornamental and horticultural crop. However, there is no clear classification of its fragrance types. The results of this study divided chrysanthemum fragrance into six categories by sensory evaluation. We determined the types and content of VOCs in each chrysanthemum accession representative of different fragrance types by GC-MS. Furthermore, through genetic analysis, we determined the heritable aromatic substances of fruity fragrance chrysanthemum. Our present findings systematically classified the fragrance types of chrysanthemums and improved classification of chrysanthemum aroma types, providing a theoretical basis for the accelerated breeding of new chrysanthemum germplasm with specific aroma types.

      Acknowledgments

      • This work was supported by National Natural Science Foundation (Grant no. 32002072).

      Conflict of interest

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

      • # These authors contributed equally: Zhiling Wang, Xin Zhao

      Supplementary information
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      • Copyright: © 2023 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 (8)  Table (1) References (23)
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    Cite this article
    Wang Z, Zhao X, Tang X, Yuan Y, Xiang M, et al. 2023. Analysis of fragrance compounds in flowers of Chrysanthemum genus. Ornamental Plant Research 3:12 doi: 10.48130/OPR-2023-0012
    Wang Z, Zhao X, Tang X, Yuan Y, Xiang M, et al. 2023. Analysis of fragrance compounds in flowers of Chrysanthemum genus. Ornamental Plant Research 3:12 doi: 10.48130/OPR-2023-0012
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  • Introduction
  • Materials and methods

  • Plant materials

  • Artificial pollination

  • Open hybridization with 'Xiaokuixiang' as the female parent

  • Sensory evaluation

  • Solid phase microextraction

  • Gas chromatography–mass spectrometry

  • Statistical analysis

  • Results

  • Sensory evaluation and analysis of chrysanthemum flavor aroma

  • Identity and content of VOCs in chrysanthemum accessions of different aroma types

  • Volatile organic compounds specific to accessions of different fragrance types

  • Analysis of aroma compounds of 'Xiaokuixiang' at different development stages

  • Analysis of fragrance types in 'Xiaokuixiang' offspring

  • Discussion
  • Conclusions
  • Acknowledgments
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
  • About this article
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