AU2020101673A4 - Method for identifying "almond aroma" black tea resource at seedling stage - Google Patents

Abstract

The present invention relates to a method for identifying "almond aroma" black tea resources at seedling stage. The method of the present invention includes the following steps: (1) extracting aromatic components from fresh tea leaves by headspace solid-phase microextraction (HS SPME): freeze-drying and grinding a fresh leaf sample, adding 90-100°C water, soaking at 40 70°C, and inserting an extraction fiber into the space above the liquid level for aroma adsorption; (2) after adsorption, inserting the extraction fiber into an injection port of a gas chromatograph (GC) to desorb and separate, and detecting by mass spectrometry (MS), and analyzing data; and (3) determining relative percentage content of benzaldehyde in aromatic components. The method can detect fresh leaves of any tea cultivar and determine whether black tea products thereof feature "almond aroma" without tea preproduction, providing a rapid and accurate method for identifying "almond aroma" black tea resources at seedling stage. Fresh tea leaves Extraction of aromatic components from tea leaves by headspace solid-phase microextraction (HS-SPME) Separation by gas chromatography (GC) and detection by mass spectrometry (MS) Match retrieval and data analysis by MS Identification of an aroma indicator for almond aroma black tea, "benzaldehyde", providing a method for early identification of almond aroma black tea resources FIG. 1 RT: 0.00 - 99.53 100- 47.23 NL: 8.00E7 TIC MS 90- JF180967 2 80 70 60 50 40 66.20 30 4994 72.43 94.32 20 10 43.37 0- ~40.6 60.74____ 89.03_________ 3.00 3.64 17.87 24.81 33.64 406 5291 6074 77.84 0 10 20 30 40 50 60 70 80 90 Time (min) FIG. 2 1 / 1

Description

Fresh tea leaves

Extraction of aromatic components from tea leaves by headspace solid-phase microextraction (HS-SPME)

Separation by gas chromatography (GC) and detection by mass spectrometry (MS)

Match retrieval and data analysis by MS

Identification of an aroma indicator for almond aroma black tea, "benzaldehyde", providing a method for early identification of almond aroma black tea resources

FIG. 1

RT: 0.00 - 99.53 100- 47.23 NL: 8.00E7 TIC MS 90- JF180967 2 80

70

60

50

40 66.20 30 4994 72.43 94.32 20

10 43.37 0- ~40.6 60.74____ 89.03_________ 3.00 3.64 17.87 24.81 33.64 406 5291 6074 77.84 0 10 20 30 40 50 60 70 80 90 Time (min)

FIG. 2

1/ 1

METHOD FOR IDENTIFYING "ALMOND AROMA" BLACK TEA RESOURCE AT SEEDLING STAGE TECHNICAL FIELD The present invention relates to the technical field of breeding of special tea resources, and in particular to a method for identifying "almond aroma" black tea resources at seedling stage. BACKGROUND Tea germplasm resources are identified by means of morphology, cytology, isozyme, chemistry, molecular biology, etc. Herein, chemical identifications determine the content of related compounds in a tea cultivar by scientific instrumentation; the advantage thereof is that special compounds can be determined at seedling stage; thus, researchers can accurately determine whether this cultivar satisfies the breeding objectives in regard to quality, yield, or resistance at early stage of breeding, substantially shortening breeding cycle and improving breeding efficiency. For example, a ratio of tea polyphenols to amino acids is measured to determine whether the tea cultivar is suitable to make green or black tea; and the content of a specific catechin is assayed to determine the adaptability of the tea cultivar. In previous research, inventors found that black tea made from a local wild tea resource in Luokeng, Guangdong featured strong "almond aroma" (JIANG XH, WU HL, CHEN D, et al. Sensory quality evaluation and biochemical composition of Luokeng black tea [J]. Chin Agric Sci Bull, 2014, 30(31):126-131). The "almond aroma" is a very rare and unique type of aroma in black teas, which has not been found in other black teas made from other tea cultivars, with a good prospect for development. However, in Luokeng tea groups, individual plants able to make "almond aroma" black tea are extremely scarce in quantity, and the amount of tea buds harvestable from a single plant is quite small as well, leading to difficulty in processing. Therefore, it is necessary to breed an excellent clonal black tea cultivar with "almond aroma" from the wild tea groups as soon as possible, and to enable vigorous propagation, popularization and application thereof. However, conventional systematic breeding of tea plants includes a plurality of steps, such as scion plucking from individual plant, cutting propagation, transplantation, planting into a tea garden, and adaptability of processing. The whole breeding process needs a long period of 8-10 years, which is time and energy consuming. Undoubtedly, this is a huge challenge for the breeding and industrialization of "almond aroma" black tea cultivars. Therefore, developing a method for identifying "almond aroma" black tea cultivars rapidly and accurately at seedling stage is of important and practical significance for accelerating the breeding process of new cultivars. SUMMARY In view of this, the present invention provides a method for identifying "almond aroma" black tea resources at seedling stage. The method can determine any fresh tea leaves and judge whether black tea products feature "almond aroma" without tea preproduction. A specific technical solution is provided as follows: A method for identifying "almond aroma" black tea resources at seedling stage is provided, including the following steps: (1) extracting aromatic components from fresh tea leaves by headspace solid-phase microextraction (HS-SPME): freeze-drying and grinding a fresh leaf sample, adding 90-100°C water, and soaking at 40-70°C, and inserting an extraction fiber into the space above the liquid level for aroma adsorption; (2) after adsorption, inserting the extraction fiber into an injection port of a gas chromatograph (GC) to desorb and separate; and detecting by mass spectrometry (MS), and analyzing data; and (3) determining relative percentage content of benzaldehyde in aromatic components. Compared with the prior art, the present invention has the following beneficial effects: Through a large number of experimental studies, the inventors of the present invention creatively use benzaldehyde as an indicator for early identification of "almond aroma" black tea resources; aromatic components are extracted simply and rapidly from fresh tea leaves by HS SPME, and content of benzaldehyde in aromatic components of fresh leaves is rapidly determined by gas chromatography-mass spectrometry (GC-MS); when the benzaldehyde content is >38%, black tea made from the fresh leaves is predicted to feature "almond aroma", and therefore this tea tree is further determined as an "almond aroma" black tea resource. The identification method provided by the present invention can be applied to detect fresh leaves of any tea cultivar and determine whether the black tea products thereof feature "almond aroma" without tea preproduction. This method reduces the long duration and tracking process for breeding and identification of tea resources, and provides a rapid and accurate method for identifying "almond aroma" black tea resources at seedling stage. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flowchart of the technology of Example 1; FIG. 2 is a total ion chromatography of the volatile components in fresh leaves of XR-1 by GC-MS. DETAILED DESCRIPTION To facilitate the understanding of the present invention, the invention will be more fully described below with reference to the examples, and preferred examples of the invention are given as follows. This invention may, however, be embodied in many different forms and should not be construed as limited to the examples set forth herein. These examples are provided so that this disclosure will be thorough and complete. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular examples only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more related items listed. In some examples, a method for identifying "almond aroma" black tea resources at seedling stage is provided, including the following steps: (1) extracting aromatic components from fresh tea leaves by headspace solid-phase microextraction (HS-SPME): freeze-drying and grinding a fresh leaf sample, adding 90-100°C water, and soaking at 40-70°C, and inserting an extraction fiber into the space above the liquid level for aroma adsorption; (2) after adsorption, inserting the extraction fiber into an injection port of a gas chromatograph (GC) to desorb and separate, and detecting by mass spectrometry (MS), and analyzing data; and (3) determining relative percentage content of benzaldehyde in aromatic components. In some examples, in step (1), a ratio of the fresh leaf sample to water is 1 g: (8-12) ml. In some examples, in step (1), the soaking temperature is 40-70 °C, preferably for 50-60°C. In some examples, in step (1), the adsorption lasts for 50-80 min, preferably for 60-70 min. In some examples, in step (1), the extraction fiber is 50/30pm DVB/Car/PDMS SPME fiber. In some examples, in step (2), the apparatus used in separating by GC and detecting by MS is TRACE DSQ GC-MS; a GC column is HP-5MS elastic quartz capillary column; preferably, the HP-5MS elastic quartz capillary column has a specification of 30 m x 0.25 mm ID x 0.25 tm film thickness. In some examples, in step (2), the injection port has a temperature of 220-240°C; the desorption is conducted at 220-240°C, and lasts for 4-6 min; preferably, the temperature of both the injection port and the desorption is 230°C, and the desorption lasts for 5 min. In some examples, in step (2), MS conditions include: ionization mode, electron ionization (EI); ion source temperature, 220-240°C; electron energy, (70±2) eV; scanning range, 50-650 amu; carrier gas, high purity helium (He) with a percentage purity of>99.999%; flow rate, (1±0.2) mL/min, splitless; electron multiplier voltage, (1,800±200) V; and intensity of total ion current: (100±20) mA. Specifically, the MS conditions include: ionization mode, El; ion source temperature, 230°C; electron energy, 70 eV; scanning range, 50-650 amu; carrier gas, high purity He with a percentage purity of >99.999%; flow rate, 1 mL/min, splitless; electron multiplier voltage, 1,800 V; and intensity of total ion current, 100 mA.

In some examples, in step (2), a method for analyzing the data includes: using a GC data processing system to scan all peaks in a total ion chromatogram (TIC) by MS, to obtain a mass spectrum; retrieving via random Xcalibur software NIST11MS database, and identifying chemical components of all the chromatographic peaks with reference to retention time, matching degree of material, and literature reports; and determining relative percentage content of each aromatic component by the peak area normalization method. In some examples, in step (3), when relative content of benzaldehyde in aromatic components is >38%, a tea tree is identified as an "almond aroma" black tea resource accordingly. The present invention will be described in detail below with reference to the examples. Example 1 1 Materials and methods 1.1 Experimental materials Selection of fresh tea leaves: Three kinds of fresh tea leaves were selected, two were "almond aroma" tea resources, XR-1 and XR-3 (experimental groups), and one was not an "almond aroma" tea resource, BM (control group). Preparation of black tea products: After harvesting, fresh leaves were spread and withered for 2 h; after withering, the leaves were rolled for 45 min, fermented for 5-6 h, and dried for 1 h to obtain black tea products. An important point was that fresh tea leaves and final black tea products were used as analytical materials; samples were freeze-dried, ground, and stored in a refrigerator at 4°C for further use. 1.2 Experimental method 1.2.1 Apparatus TRACE DSQ GC-MS 1.2.2 Extraction and identification of aromatic components Extraction of aromatic components: HS-SPME. 10.0 g of tea sample was placed in a 500 mL extraction flask and soaked in 100 mL of boiling water; the extraction flask was heated in a water bath at 50°C; after aromatic components in the extraction flask achieved an equilibrium, an extraction fiber (DVB/CAR/PDMS-50/30 tm) was inserted into the space above the liquid level, and the extraction fiber was pushed out for adsorption; after adsorption for 60 min, SPME needle enriching volatile components of tea was rapidly into an injection port of GC for desorption. GC conditions: HP-5MS elastic quartz capillary columns (30 m x 0.25 mm ID x 0.25 tm film thickness) were used; injector temperature was 230°C. After injection, desorption was conducted for 5 min at 230°C. MS conditions were shown as follows: ionization mode was El, ion source temperature was 230°C, electron energy was 70 eV, scanning range was 50-650 amu, carrier gas was high purity He (with a purity of >99.999%); flow rate was 1 mL/min, splitless; electron multiplier voltage was 1,800 V; and intensity of total ion current was 100 mA. The flowchart of the experimental method is shown in FIG. 1. 2 Results and analysis The total ion chromatogram of volatile components in tea samples was obtained by GC-MS; after retrieving via random Xcalibur software NIST 1MS database, chemical components attributable to all chromatographic peaks were identified with reference to retention time, matching degree of material, and literature reports; relative content of each aromatic component was determined by the peak area normalization method. Results are shown in Table 1. Table 1 Main aromatic components in fresh leaves of different tea germplasm resources and black tea products (in %) Serial Nameof Fresh Black tea Fresh Black tea Fresh Black tea number aromatic leaves of product of leaves of product of leaves of product of component XR-1 XR-1 XR-3 XR-3 BM BM 1 Benzaldehyde 77.30 68.08 54.07 53.51 6.25 4.96 2 Geraniol 4.75 8.67 9.16 12.07 6.96 14.46 3 Methyl salicylate 4.64 6.05 12.31 8.94 34.93 33.76 4 §-Linalool 2.53 2.01 7.81 2.81 10.73 7.78 Trans-3,7 5 dimethyl-2,6 butadienoic 2.50 4.81 3.70 7.93 1.01 1.53 acid 6 Linalool oxide II (furanoid) 0.88 0.97 1.56 0.65 2.17 2.02 7 Olivetol 0.87 0.22 0.26 0.59 0.45 0.36 8 Phenethyl alcohol 0.57 0.81 0.66 1.37 0.62 1.78 9 Benzyl alcohol 0.56 0.67 0.40 0.58 0.42 0.33 10 p-Ionone 0.52 0.98 0.75 1.72 1.08 1.51 11 Nerolidol 0.48 0.40 0.65 0.85 1.54 1.41 12 Linalool oxide I (furanoid) 0.41 0.52 0.77 0.36 1.64 1.87

13 3,7-Dimethyl 2,6-octadienal 0.38 0.57 0.33 0.60 0.83 1.25

14 Nerol 0.22 0.53 0.58 0.78 0.86 1.75 15 Methyl geranate 0.18 0.47 0.47 0.39 0.53 0.78 16 Terpinen-4-ol 0.16 0.26 0.63 0.45 3.94 6.81

Sixteen main aromatic components are listed in Table 1, including aldehydes, esters, alcohols, and ketones. The table does not list aromatic components that contribute little to the overall aroma characteristics of tea. Specifically, FIG. 2 is a total ion chromatogram of volatile components in fresh leaves of the tea resource XR-1 by GC-MS; the component peaked at 18.19 min is benzaldehyde, with a peak area of 173,829,395 and a relative content of 77.30%, which has the highest content in all aromatic components of fresh leaves. In fresh leaves of the tea resource XR 3, benzaldehyde accounted for 54.07% of the total aromas, which has the highest content in all aromatic components of fresh leaves. In fresh leaves of the tea resource BM, the benzaldehyde content is merely 6.25%, far lower than methyl salicylate and p-linalool. Further, the present invention determined the content of benzaldehyde in the black tea processed from fresh leaves of XR-1, XR-3, and control sample BM (Table 1). It was found that the benzaldehyde content was high in black tea products of XR-1 and XR-3, the benzaldehyde content was 68.08% in the black tea product of XR-1, and the retention of benzaldehyde was more than 88% of that in fresh leaves. The benzaldehyde content was 53.51% in the black tea product of XR-3, and the retention was more than 98% of that in fresh leaves. The benzaldehyde content was very low (only 4.96%) in the black tea product of the control sample BM, and the retention thereof (79.4%) was lower than the first two samples. Thus, it is further indicated that the aromatic component of the black tea, benzaldehyde, will be retained substantially during processing and will not disappear due to black tea processing. Therefore, benzaldehyde can serve as an indicator for early identification of "almond aroma" black tea resources. Benzaldehyde is the simplest aromatic aldehyde, commonly known as bitter almond oil, also known as phenylaldehyde. The molecular formula thereof is C7H60. Benzaldehyde is a colorless to pale yellow volatile liquid, with a special almond odor; there is an aroma when burned. Benzaldehyde has a very low odor threshold (0.085); when benzaldehyde dominates total aromas of the tea, feature of almond aroma exhibited by benzaldehyde represents the aroma characteristics of the whole tea. Sniffing with GC-MS has found that benzaldehyde really has a bitter almond aroma. The accuracy of benzaldehyde as a characteristic indicator for "almond aroma" is further verified. 3. Sensory quality evaluation and verification of aromas of black tea samples Sensory quality evaluation was conducted on the final black tea samples of the above three tea resources. Results are shown in Table 2. The sensory quality evaluation found that XR-1 and XR-3 featured obvious "almond aroma"; due to the high benzaldehyde content (low threshold), the almond aroma exhibited by benzaldehyde becomes a dominant aroma characteristic of the black tea. However, BM is evaluated as floral and fruity aromas due to low benzaldehyde content and the almond aroma exhibited by benzaldehyde does not serve as an aroma characteristic of this black tea. The evaluation result in Table 2 is consistent with the result of GC-MS, further verifying the accuracy of the method in the present invention from a sensory point of view. Table 2 The sensory quality evaluation result of aromas of the black tea XR-1 XR-3 BM Aroma characteristic Strong almond aroma Obvious almond Floral and fruity aroma aromas Example 2 Method validation Fresh leaves were harvested from 19 tea cultivars, and the contents of benzaldehyde in fresh leaves were identified according to the method provided by the present invention, and sensory quality evaluation of aromas was conducted to the black teas. Results are shown in Table 3. Table 3 Content of benzaldehyde in fresh leaves of 19 tea cultivars and sensory quality evaluation of aromas of the black teas Content of Serial Name of the benzaldehyde in Sensory quality evaluation of aromas number tea tree fresh tea leaves

Fresh and long-lasting sweet cane aroma with 1 HH 1.22 floral aroma

2 YD-2 0.83 Fresh and long-lasting sweet aroma with faint floral aroma 3 YD-3 1.22 Fresh and long fruity (sugar) and sweet aromas 4 ASM 1.05 Sweet and delicate aromas, with minty aroma 5 YN 1.37 High and long sweet aroma with faint floral aroma 6 WW 0.03 Strong, fine, sharp, and long-lasting minty aroma 7 JQMM 1.48 High and strong aroma 8 SH 1.34 Sweet and sugar aromas 9 QH 3.03 Fruity and sweet aromas 10 DH 1.62 Mellow and long-lasting aroma 11 GH 0.9/ Rose and floral aromas 12 HNH 1.6/ - -High and long-lasting aroma 13 SK 2.42 Red mango aroma and rich aromas 14 YZ 2./9 Long-lasting sweet floral aroma 15 BSY 3. /U Strong and long-lasting sweet aroma 16 YJ U. /1 Strong and long-lasting Iloral aroma 17 SX 1.6 Strong and long-lasting sweet aroma 18 LX 38./6 Long-lasting almond aroma 19 HJY 46.59 Strong and long-lasting almond aroma Results showed that the benzaldehyde content was low in 17 fresh leaves of 19 identified tea cultivars, ranging between 0.03% and 3.70%, and it was deduced therefrom that these black tea samples did not feature "almond aroma". After processing, sensory evaluation found that these 17 black tea samples featured floral, sweet, fruity, and rose aromas, but not "almond aroma". The benzaldehyde content ranged between 38.76% and 46.59% in fresh leaves of the other two tea resources, "LX" and "HJY", and the cultivars were identified as "almond aroma" black tea resources accordingly. After making fresh leaves of "LX" and "HJY" into the black teas, both black tea samples featured "long-lasting and strong almond aroma", respectively, further verifying the accuracy of the method. After assay and verification of aromatic components in fresh leaves of a plurality of tea cultivars, when benzaldehyde accounts for 38% of total aromas in fresh leaves, the made black tea features "almond aroma", and the tea resource can therefore be identified as an "almond aroma" black tea resource. When the benzaldehyde content is below 38% in fresh leaves, the black tea does not feature "almond aroma", and the tea resource is not an "almond aroma" black tea resource. Therefore, XR-1 and XR-3 in Example 1 and "LX" and "HJY"in Example 2 are "almond aroma" black tea resources. The above experimental results further verify the practicability and operability of the present invention. That is, identification of the benzaldehyde content in aromatic components of fresh tea leaves at seedling stage can determine whether the made black tea features "almond aroma". The present invention provides a reliable method and means for early identification of "almond aroma" black tea resources. The technical characteristics of the above examples can be employed in arbitrary combinations. In an effort to provide a concise description of these examples, all possible combinations of all technical characteristics of the examples may not be described; however, these combinations of technical characteristics should be construed as disclosed in the description as long as no contradiction occurs. Several examples of the present invention are merely described more in detail above, but they should not therefore be construed as limiting the scope of the invention. It should be noted that several variations and improvements can be made by one of ordinary skill in the art without departing from the conception of the present invention and are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims. In the claims which follow, and in the preceding description, except where the context requires otherwise due to express language or necessary implication, the word "comprise" and variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the apparatus and method as disclosed herein.

Claims (5)

1. What is claimed is: 1. A method for identifying "almond aroma" black tea resources at seedling stage, comprising the following steps: (1) extracting aromatic components from fresh tea leaves by headspace solid-phase microextraction (HS-SPME): freeze-drying and grinding a fresh leaf sample, adding water at 90 100°C, soaking at 40-70°C, and inserting an extraction fiber into the space above the liquid level for aroma adsorption; (2) after adsorption, inserting the extraction fiber into an injection port of a gas chromatograph (GC) to desorb and separate, and detecting by mass spectrometry (MS), and analyzing data; and (3) determining relative percentage content of benzaldehyde in aromatic components.
2. 2. The method according to claim 1, wherein, in step (1), a ratio of the fresh leaf sample to water is 1 g: (8-12) ml.
3. 3. The method according to claim 1, wherein, in step (1), the soaking is conducted at 50 60°C; wherein, in step (1), the adsorption lasts for 50-80 min; wherein, in step (1), the extraction fiber is 50/30 pm DVB/Car/PDMS SPME fiber.
4. 4. The method according to claim 1, wherein, in step (2), the gas chromatograph-mass spectrometer (GC-MS) used is TRACE DSQ GC-MS; a GC column is HP-5MS elastic quartz capillary column; wherein, in step (2), the injection port of the GC has a temperature of 220-240°C; the desorption is conducted at 220-240 0C, and lasts for 4-6 min; wherein, in step (2), MS conditions comprise: ionization mode, electron ionization (EI); ion source temperature, 220-240°C; electron energy, (70±2) eV; scanning range, 50-650 amu; carrier gas, high purity helium (He) with a purity of >99.999%; flow rate, (1±0.2) mL/min, splitless; electron multiplier voltage, (1,800±200) V; and intensity of total ion current, (100±20) mA.
5. 5. The method according to any one of claims 1 to 4, wherein, in step (2), a method for analyzing the data comprises: using a GC data processing system to scan all peaks in a total ion chromatogram (TIC) by MS, to obtain a mass spectrum; retrieving via random Xcalibur software NIST11 MS database, and identifying chemical components attributable to all chromatographic peaks with reference to retention time, matching degree of material, and literature reports; and determining percentage content of each aromatic component by the peak area normalization method.

   Fresh tea leaves

   Extraction of aromatic components from tea leaves by headspace solid-phase microextraction (HS-SPME)

   Separation by gas chromatography (GC) and detection by mass spectrometry (MS) 2020101673

   Match retrieval and data analysis by MS

   Identification of an aroma indicator for almond aroma black tea, "benzaldehyde", providing a method for early identification of almond aroma black tea resources

   FIG. 1

   RT: 0.00 - 99.53 47.23 NL: NL: 100 8.00E7 8.00E7 TIC MS TIC MS 90 JF 180967- JF180967- 2 2

   80

   70

   60

   50

   40 66.20 30 72.43 94.32 49.94 20

   10 43.37 40.96 60.74 77.84 89.03 3.00 17.87 24.81 33.64 40.63 52.91 3.64 0 0 10 20 30 40 50 60 70 80 90 Time (min)

   FIG. 2

   1/1