Flos lonicerae japonicae black tea and preparation method thereof

Abstract

This invention provides a Sichuan honeysuckle black tea and its preparation method, belonging to the field of black tea preparation technology. The preparation method, targeting the tissue structure and component characteristics of Sichuan honeysuckle, effectively transforms chlorogenic acid and polyphenols that cause bitterness in Sichuan honeysuckle through withering, rolling, fermentation, and drying processes, reducing the raw, grassy taste and astringency. Simultaneously, it stimulates the generation of floral, fruity, and sweet aromas, resulting in a Sichuan honeysuckle black tea with superior characteristics such as tightly rolled leaves, bright red liquor, harmonious aroma, and mellow taste, overcoming the shortcomings of directly brewing Sichuan honeysuckle, which results in a bitter taste and bland aroma. Through coordinated control of each step, the preparation method ensures flavor transformation while enhancing and preserving bioactive components such as quinic acid, amino acids, total flavonoids, and saponins in Sichuan honeysuckle, giving the Sichuan honeysuckle black tea both the palatability of black tea and the nutritional and health benefits of Sichuan honeysuckle.

Description

Technical Field

[0001] This invention belongs to the field of black tea preparation technology, specifically relating to a Sichuan honeysuckle black tea and its preparation method. Background Technology

[0002] Sichuan honeysuckle is the fine felt-haired honeysuckle ( Lonicera similis Honeysuckle (Hemsl.) is a traditional Chinese medicinal herb that is also used in food. It is rich in chlorogenic acid, flavonoids, saponins, and amino acids, among other bioactive components, and possesses properties such as clearing heat and detoxifying, and antibacterial and anti-inflammatory effects. With the upgrading of health-conscious consumption demands, the development of medicinal and edible ingredients into tea drinks has become a research hotspot, and Sichuan honeysuckle stands out due to its potential in both nutrition and flavor. However, when honeysuckle is brewed directly, it suffers from a bitter taste, a bland aroma, and a low extraction rate of active ingredients, limiting its widespread application in the tea beverage industry. Currently, existing technologies for developing honeysuckle tea beverages primarily focus on simple processing methods such as flower tea and herbal tea; no mature reports have been found regarding specialized preparation processes for honeysuckle black tea. Traditional black tea processing techniques are mainly designed for fresh tea leaves, but the tissue structure and active ingredient composition of honeysuckle differ significantly from those of fresh tea leaves. Directly applying traditional black tea processing methods can easily lead to uneven fermentation, mixed aromas, and lingering bitterness in the product. For example, improper control of temperature, humidity, and time during the withering process can cause honeysuckle to lose water too quickly or too slowly, affecting the cell damage rate and fermentation efficiency during subsequent rolling. If the rolling speed, pressure, and time parameters are not adapted to the material characteristics of honeysuckle, it can result in loose tea leaves, insufficient release of effective components, or excessive breakage and loss of flavor substances. An unreasonable combination of temperature, humidity, and time during the fermentation stage can lead to insufficient or excessive oxidation of polyphenols, thereby affecting the color, aroma, and flavor harmony of the tea soup. In addition, the flowering rate of honeysuckle, the pretreatment method, and the parameter settings of the drying process all directly affect the final product's appearance, soup color, aroma, flavor, and retention rate of active ingredients. Therefore, developing a method for preparing Sichuan honeysuckle black tea that can balance flavor quality and nutritional benefits, based on the raw material characteristics of Sichuan honeysuckle, to realize the high-value utilization of Sichuan honeysuckle resources and meet consumers' demand for healthy tea drinks, has important practical significance and market value. Summary of the Invention

[0003] In view of the deficiencies in the prior art, the purpose of this invention is to provide a method for preparing Sichuan honeysuckle black tea, which can improve the quality of Sichuan honeysuckle black tea.

[0004] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing Sichuan honeysuckle black tea, comprising the following steps: After the honeysuckle flowers are withered, they are kneaded to obtain kneaded honeysuckle flowers. The kneaded honeysuckle is fermented to obtain fermented honeysuckle; Fermented honeysuckle flowers are dried to obtain honeysuckle black tea.

[0005] Preferably, the fermentation temperature is 25~32℃; the fermentation time is 1.5~4.5h; and the fermentation humidity is 55%~65%.

[0006] Preferably, the honeysuckle includes honeysuckle with a flowering rate of 40% to 60%; the withering includes air withering and natural withering; the temperature of air withering is 15 to 20°C; the time of air withering is 1 to 3 hours; the temperature of natural withering is 20 to 25°C; the time of natural withering is 3 to 5 hours; and the kneading time is 15 to 20 minutes.

[0007] Preferably, the drying method includes high-temperature drying and low-temperature drying; the high-temperature drying temperature is 160~200℃ and the time is 30~50min; the low-temperature drying temperature is 80~90℃ and the time is 2~3h.

[0008] This invention provides a honeysuckle black tea prepared by the preparation method described in the above technical solution.

[0009] This invention provides the application of the preparation method described in the above technical solution in improving the quality of Sichuan honeysuckle black tea.

[0010] Preferably, improving the quality of Sichuan honeysuckle black tea includes improving the sensory quality and / or the content of active ingredients in Sichuan honeysuckle black tea; the improvement of the sensory quality of Sichuan honeysuckle black tea includes improving any one or more of the following: appearance, liquor color, aroma, taste and infused leaf. Improving the content of active ingredients in Sichuan honeysuckle black tea includes improving the content of any one or more of the following: chlorogenic acid, quinic acid, amino acids, polyphenols, soluble sugars, soluble proteins, total flavonoids, saponins, and volatile components.

[0011] This invention provides a method for increasing the content of saponins and / or quinic acid in honeysuckle black tea, comprising the following steps: After the honeysuckle flowers are withered, they are kneaded to obtain kneaded honeysuckle flowers. The kneaded honeysuckle flowers are fermented to obtain fermented honeysuckle flowers; the fermentation temperature is 25~32℃; the fermentation time is 2.5~3.5h; and the fermentation humidity is 55%~65%. Fermented honeysuckle flowers are dried to obtain honeysuckle black tea.

[0012] Preferably, the withering includes air withering and natural withering; the temperature of air withering is 15~20℃; the time of air withering is 1~3h; the temperature of natural withering is 20~25℃; and the time of natural withering is 3~5h.

[0013] Preferably, the drying method includes high-temperature drying and low-temperature drying; the high-temperature drying temperature is 160~200℃ and the time is 30~50 min; the low-temperature drying temperature is 80~90℃ and the time is 2~3 h.

[0014] This invention provides a method for preparing Sichuan honeysuckle black tea, comprising the following steps: withering Sichuan honeysuckle and then kneading it to obtain kneaded Sichuan honeysuckle; fermenting the kneaded Sichuan honeysuckle to obtain fermented Sichuan honeysuckle; and drying the fermented Sichuan honeysuckle to obtain Sichuan honeysuckle black tea. The preparation method provided by this invention, targeting the tissue structure and component characteristics of Sichuan honeysuckle, effectively transforms chlorogenic acid and polyphenols that cause bitterness in Sichuan honeysuckle through the withering, kneading, fermentation, and drying processes, reducing the raw, grassy taste and astringency, while simultaneously stimulating the generation of floral, fruity, and sweet aromas. This results in a Sichuan honeysuckle black tea with the characteristics of a high-quality black tea: tightly rolled appearance, bright red liquor, harmonious aroma, and mellow taste, overcoming the shortcomings of directly brewing Sichuan honeysuckle, which results in a bitter taste and bland aroma. The preparation method described herein, through coordinated control of each step, ensures flavor transformation while reducing the content of substances that easily lead to bitterness, such as chlorogenic acid, flavonoids, and polyphenols. It also enhances or retains bioactive components in honeysuckle, such as quinic acid, amino acids, soluble sugars, soluble proteins, and saponins. Furthermore, it improves the volatile components in black tea, resulting in a complex flavor profile of floral, fruity, nutty, and sweet notes. Simultaneously, it reduces irritating off-flavor components such as diallyl trisulfide, solving the problem of traditional honeysuckle tea having a monotonous flavor and a pronounced spiciness. In summary, the preparation method provided by this invention enables honeysuckle black tea to possess both the palatability of black tea and the nutritional and health benefits of honeysuckle. This invention transforms honeysuckle, a food and medicine source, into a flavorful and palatable black tea product, breaking through the limitations of honeysuckle's application as only a traditional Chinese medicine or simple flower tea. It expands its application scenarios in the tea beverage field, increases the added value of honeysuckle resources, and meets consumers' diversified needs for healthy tea drinks, possessing significant economic value and market potential. Attached Figure Description

[0015] Figure 1 A diagram illustrating the tea sample evaluation process; Figure 2 Two images showing the tea sample evaluation process; Figure 3 The images show the external appearance of tea samples from each treatment group. From left to right, the images represent ck, 1.5h fermentation, 2.5h fermentation, 3.5h fermentation, and 4.5h fermentation; the same applies below. Figure 4 Images of tea infusions from each treatment group; Figure 5 Background images of tea leaves from each treatment group; Figure 6 Principal component analysis (PCA) score plots of total volatile components in tea samples from each treatment group; Figure 7 Cluster heatmaps of tea samples from each treatment group based on GC-MS volatile component data; Figure 8 Venn diagram for differential volatile components in multiple comparisons; Figure 9 Volcano diagrams showing differential volatile metabolites; A: 1.5h fermentation time vs. control group; B: 2.5h fermentation time vs. 1.5h fermentation time; C: 3.5h fermentation time vs. 2.5h fermentation time; D: 4.5h fermentation time vs. 3.5h fermentation time. Figure 10 Bubble diagram for KEGG enrichment analysis of differentially volatile metabolites; Top left: fermentation time 1.5 h vs. control group; Bottom left: fermentation time 2.5 h vs. 1.5 h; Top right: fermentation time 3.5 h vs. 2.5 h; Bottom right: fermentation time 4.5 h vs. 3.5 h. Figure 11 Radar charts showing the sensory flavor characteristics of each treatment group; A represents fermentation at 1.5 h and the control group; B represents fermentation at 2.5 h and 1.5 h; C represents fermentation at 3.5 h and 2.5 h; D represents fermentation at 4.5 h and 3.5 h. Figure 12 Principal component analysis (PCA) score plots of microbial communities for tea samples with different treatments; Figure 13 A heatmap of species abundance of microbial composition at the species level; Figure 14 LEfSe clade diagram of differentially expressed microbial species in different treatment groups; Figure 15 Venn diagram for screening key microbial species; Figure 16 Bar chart classifying the secondary functional pathways of KEGG genes in microbial communities; Figure 17 The graphs show the differential enrichment analysis of KEGG pathway function in microbial genes at different fermentation times compared to the control group. The top left graph shows the differential enrichment analysis of KEGG pathway function in microbial genes at 1.5 h of fermentation compared to the control group; the top right graph shows the differential enrichment analysis of KEGG pathway function in microbial genes at 2.5 h of fermentation compared to the control group; the bottom left graph shows the differential enrichment analysis of KEGG pathway function in microbial genes at 3.5 h of fermentation compared to the control group; and the bottom right graph shows the differential enrichment analysis of KEGG pathway function in microbial genes at 4.5 h of fermentation compared to the control group. Figure 18Stacked bar chart of eggNOG functional classification composition of microbial communities; Figure 19 Spearman correlation heatmap of differential metabolites and differential microorganisms at 3.5 h fermentation time and CK. Detailed Implementation

[0016] This invention provides a method for preparing Sichuan honeysuckle black tea, comprising the following steps: After the honeysuckle flowers are withered, they are kneaded to obtain kneaded honeysuckle flowers. The kneaded honeysuckle is fermented to obtain fermented honeysuckle; Fermented honeysuckle flowers are dried to obtain honeysuckle black tea.

[0017] This invention involves withering and then kneading *Lonicera japonica* to obtain kneaded *Lonicera japonica*. As an optional embodiment of this invention, the *Lonicera japonica* can be *Lonicera japonica* with a flowering rate of 40%~60%, or it can be *Lonicera japonica* with a flowering rate of 50%. After obtaining the *Lonicera japonica*, this invention preferably involves withering. The spreading thickness during withering can be 3 cm. In this invention, withering includes air-blowing withering and natural withering; the air-blowing withering method can be cold air blowing; the temperature of the cold air blowing can be 15~20℃, or it can be 15, 16, 17, 18, 19, or 20℃; the duration of the cold air blowing can be 1~3 hours, or it can be 1 hour. This invention primarily uses cold air to regulate the moisture content of tender leaves, control fermentation, and enhance tea aroma and quality. Specifically, cold air achieves a balanced moisture level and even water removal, effectively allowing residual moisture inside the leaves to slowly and evenly diffuse to the surface and then evaporate, promoting uniform water removal. Cold air also prevents stuffiness, fixing quality and preventing the "dry outside, wet inside" phenomenon, which can lead to a "musty" taste and affect aroma, thus avoiding quality loss due to "heat and stuffiness." During the withering process, if the quantity of fresh honeysuckle flowers is large and the spread thickness is high, it is preferable to turn them over every hour.

[0018] After the air-drying process, the resulting honeysuckle flowers are naturally withered. As an optional embodiment of this invention, the natural withering temperature can be 20-25℃, or 20, 21, 22, 23, 24, or 25℃; the natural withering time can be 3-5 hours, or 3, 4, or 5 hours. This invention reduces the moisture content of the flowers through natural withering, making them softer and more resilient. This makes them less prone to breakage during subsequent rolling processes and easier to curl into strips. This invention activates enzymatic reactions during withering, breaking down proteins into amino acids. Amino acids are not only the main source of the fresh and crisp flavor but also precursors for subsequent aroma formation; starch breaks down into soluble sugars, increasing the sweet and mellow taste; and some low-boiling-point aromatic substances with grassy notes (such as quinol) evaporate and dissipate. Simultaneously, other substances with floral and fruity aromas begin to form or accumulate.

[0019] After withering, the honeysuckle is kneaded. In an optional embodiment, the kneading time can be 15-20 minutes, or 15, 16, 17, 18, 19, or 20 minutes. In another optional embodiment, the kneading includes three processes: a first light kneading, a heavy kneading, and a second light kneading. The first light kneading time can be 3-5 minutes, or 4 minutes; no pressure is applied during this process. The heavy kneading time can be 6-10 minutes, or 7, 8, or 9 minutes; specifically, the heavy kneading process involves pressing the honeysuckle to 2 / 3 of its original volume. The second light kneading time can be 3-5 minutes, or 4 minutes. The first light kneading initially forms the honeysuckle into strips and causes the flowers to curl slightly without breaking them. The heavy kneading breaks down the cells, extracting tea juice and providing a sufficient material basis for fermentation. The second light kneading tightens and shapes the strips, allowing the kneaded flowers to naturally retract, resulting in a more compact and aesthetically pleasing shape.

[0020] In this invention, the kneading is preferably performed until just as the juice is released. The kneading time specified in this invention is the optimal kneading time for Sichuan honeysuckle. Compared with the corresponding time in this invention, if the kneading time is too short, the damage to the tea leaves will be incomplete, affecting the release of flavor substances from the Sichuan honeysuckle; if the kneading time is too long, it will lead to excessive loss of effective substances. In this invention, the kneading is performed in a tea kneading machine. The kneading process of this invention first breaks down the tea leaves, causing the tea juice and internal substances to be released; secondly, it tightly rolls the tea leaves to shape a regular shape for black tea; at the same time, it allows polyphenols and enzymes to fully contact, creating conditions for fermentation and oxidation; and it also enhances the concentration and aroma of the subsequent tea soup. After the kneading is completed, this invention yields kneaded Sichuan honeysuckle.

[0021] After obtaining the kneaded honeysuckle, this invention ferments it to obtain fermented honeysuckle. The thickness of the spread honeysuckle during fermentation can be 8-10 cm, or 9 cm. As an optional embodiment of this invention, the fermentation temperature can be 25-32℃, or 25, 26, 27, 28, 29, 30, 31, or 32℃; the fermentation time can be 1.5-4.5 hours, or 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 hours; the fermentation humidity can be 55%-65%, or 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65%. After fermentation, fermented honeysuckle is obtained. This invention changes the color of honeysuckle from yellowish-white to red or reddish-brown through fermentation. The oxidative environment during fermentation degrades and transforms anthocyanins, contributing to a more stable and pleasing soup color. The tea liquor changes from yellowish-green to orange-red, the grassy aroma dissipates, floral and fruity aromas begin to form, bitterness decreases, and the mellow, fresh, and sweet flavor increases. Furthermore, this invention, through research, discovered that fermenting Sichuan honeysuckle black tea for 3.5 hours yields a sensory weighted score of 90.82 points. The tea leaves are curled and bright reddish-brown, the tea liquor is bright orange-yellow, the aroma combines sweetness and ripe fruit notes, the taste is mellow and harmonious, and the infused leaves are soft, bright, and uniform, achieving an optimal balance across all quality indicators. The Sichuan honeysuckle black tea prepared under these conditions achieves a quality upgrade through controlled fermentation, avoiding the astringent taste and monotonous aroma of unfermented Sichuan honeysuckle, while also mitigating the defects of over-fermentation (4.5 hours) such as aroma loss and insufficient mellowness.

[0022] After obtaining fermented honeysuckle, this invention dries the fermented honeysuckle to obtain honeysuckle black tea. As an optional embodiment of this invention, the drying method includes high-temperature drying and low-temperature drying; the high-temperature drying temperature is 160-200℃, or 160, 170, 180, 190, or 200℃; the high-temperature drying time is 30-50 minutes, or 30, 40, or 50 minutes; the low-temperature drying temperature is 80-90℃, or 85℃; the low-temperature drying time is 2-3 hours, or 2.5 hours. This invention, through the drying process, obtains honeysuckle black tea with a moisture content ≤10%. In the drying process of this invention, high-temperature drying completely terminates fermentation, preventing over-fermentation that leads to sourness and a darkened tea liquor, and rapidly evaporating moisture to quickly reduce the moisture content below a safe level, which is beneficial for long-term storage. High temperature promotes the Maillard reaction and caramelization reaction of amino acids and sugars in the flowers, producing a strong aroma of roasted fruit, sweetness, and chestnut. High temperatures allow the rolled tea leaves to quickly solidify and become less prone to crumbling. In the drying process of this invention, low-temperature drying achieves the advantages of preserving aroma, color, and activity. Low-temperature drying helps retain the delicate floral fragrance of honeysuckle itself, preventing the aroma from being masked or destroyed by high temperatures. Initial high-temperature drying rapidly deactivates enzymes and fixes quality. Low-temperature slow drying promotes a deep fusion of floral and tea aromas while avoiding excessive aroma loss, ultimately resulting in a unique quality characterized by high aroma, mellow taste, and bright liquor.

[0023] The preparation method of this invention targets the tissue structure and component characteristics of honeysuckle. Through withering, rolling, fermentation, and drying, it effectively transforms chlorogenic acid and polyphenols in honeysuckle that cause bitterness, reduces the raw, grassy taste and astringency, and simultaneously stimulates the generation of floral, fruity, and sweet aromas. This results in a high-quality honeysuckle black tea with a tightly rolled appearance, bright red liquor, harmonious aroma, and mellow taste, completely overcoming the shortcomings of directly brewing honeysuckle, which results in a bitter taste and bland aroma. Through coordinated control of each step, the preparation method ensures flavor transformation while preserving bioactive components such as quinic acid, amino acids, total flavonoids, and saponins in honeysuckle. This allows the honeysuckle black tea to possess both the palatability of black tea and the nutritional and health benefits of honeysuckle. This invention transforms honeysuckle, a plant with both medicinal and edible properties, into a flavorful and palatable black tea product. It breaks through the limitations of honeysuckle's application as a traditional Chinese medicine or simple flower tea, expands its application scenarios in the tea beverage field, enhances the added value of honeysuckle resources, and meets consumers' diversified needs for healthy tea beverages, thus possessing significant economic value and market potential.

[0024] This invention provides a Sichuan honeysuckle black tea prepared by the method described in the above-mentioned technical solution. In this invention, the flavor and quality of the Sichuan honeysuckle black tea are significantly improved. The Sichuan honeysuckle black tea has a curled, reddish-brown, and uniform appearance; a bright orange-yellow liquor; a pure, long-lasting sweet aroma; a mellow, sweet taste with good harmony; and bright, uniform tea leaves. The content of quinic acid, saponins, amino acids, polyphenols, soluble sugars, soluble proteins, and total flavonoids in the Sichuan honeysuckle black tea is efficiently retained, and even increased; the volatile components are improved, resulting in a significant improvement in quality.

[0025] This invention provides the application of the preparation method described in the above technical solution in improving the quality of Sichuan honeysuckle black tea. As an optional embodiment of this invention, improving the quality of Sichuan honeysuckle black tea includes improving its sensory quality and / or the content of active ingredients; the improvement of the sensory quality includes improving any one or more of the following: appearance, liquor color, aroma, taste, and infused leaf content; the improvement of the active ingredient content includes improving any one or more of the following: chlorogenic acid, quinic acid, amino acids, polyphenols, soluble sugars, soluble proteins, total flavonoids, saponins, and volatile components.

[0026] Regarding the improvement of volatile content, the results of the embodiments of this invention show that the preparation method, through fermentation treatment, identified a total of 1849 volatile substances, covering esters, terpenes, heterocyclic compounds, etc., and added a large number of flavor precursor substances compared with the unfermented group (CK), providing material guarantee for the formation of complex flavors. The preparation method can effectively weaken off-flavor components: during the fermentation process, the content of irritating sulfur-containing compounds such as diallyl trisulfide and 2-methyl-1-propanethiol decreases, and the concentration of undesirable flavor substances such as 2-ethylhexanoic acid and acetic anhydride decreases, solving the problem of the traditional Sichuan honeysuckle's spiciness and strong off-flavor. The preparation method can enrich characteristic aroma substances, specifically, the content of pleasant flavor components such as phenylethanol, (E)-4-nonenal, and 2,3,5-trimethylpyrazine is greatly increased, among which the content of phenylethanol increases more than 10 times after fermentation, becoming the core substance that dominates the sweet and fruity aroma.

[0027] This invention targets and screens differential metabolites to identify key aroma-contributing factors. Through precise identification of highly active flavor compounds, 904 differential metabolites were screened using a p-value < 0.05 and a VIP > 1. Among them, ultra-highly active components with an OAV > 10000, such as 2-furfuryl mercaptan (ROAV = 100), 1-octen-3-one (fungal aroma), and benzyl mercaptan (meat aroma), although present in low concentrations, have extremely low aroma thresholds and become the core targets for shaping the complex flavors of Sichuan honeysuckle black tea, including roasted, floral, fruity, and nutty aromas. The biosynthesis of secondary metabolites, monoterpene biosynthesis, and phenylalanine metabolism pathways are significantly enriched, driving the biotransformation of flavor compounds such as terpenes, aldehydes, and heterocyclic compounds. For example, monoterpenes (geraniol, menthol) construct a fresh herbal base, while sesquiterpenes (β-caryophyllene) add spicy-woody layers, achieving a scientific enhancement of flavor complexity.

[0028] Furthermore, metagenomic analysis revealed that the preparation method provided by this invention can significantly reshape the microbial community structure of Sichuan honeysuckle black tea, directionally inhibit native bacteria and harmful microorganisms in the raw materials, and substantially reduce undesirable flavor substrates. Gradual regulation of fermentation time modulates microbial diversity; the 3.5-hour fermentation group exhibits the highest microbial diversity and fully activated microbial metabolic functions, with significant enrichment of key pathways such as sugar metabolism, amino acid metabolism, and secondary metabolic synthesis. At 4.5 hours, the microbial community becomes functionally specialized, facilitating the formation of a caramel aroma. Different fermentation stages enrich specific differential microbial communities and core metabolic pathways. Core functional bacteria, through efficient material transformation and aroma precursor synthesis, synergistically drive the accumulation of excellent flavor substances such as floral, fruity, nutty, and roasted aromas. Simultaneously, the stable microbial community and well-developed functional metabolic system ensure a stable and controllable fermentation process, improving the inherent bitterness and grassy flavor of the raw materials. This confirms 3.5 hours as the optimal fermentation process, providing microbiological theoretical support for the standardized fermentation processing and flavor quality improvement of Sichuan honeysuckle black tea.

[0029] This invention provides a method for increasing the content of saponins and / or quinic acid in honeysuckle black tea. The steps are the same as the preparation method of honeysuckle black tea, and the specific method is the same as above, so it will not be repeated here.

[0030] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] The normalized peak data described in the following scheme were obtained by qualitative and quantitative analysis using high-resolution liquid chromatography-mass spectrometry (LC-MS) metabolomics.

[0032] Example 11: Research on the Processing Technology of Sichuan Honeysuckle 1.1 Processing techniques of Sichuan honeysuckle black tea with different fermentation times Take 30 catties of each of four groups of honeysuckle with a flowering rate of 40%~50%, and blow them with cold air for 1 hour at a temperature of 15~20℃. After blowing with cold air, turn off the fan and let them wither naturally at 20~25℃ for 3 hours. The thickness of the spread during air withering and natural withering is 3 cm. After natural withering, put the withered honeysuckle into a rolling machine and roll it for 15~20 minutes. Ferment it for 1.5 hours, 2.5 hours, 3.5 hours and 4.5 hours respectively at 25~32℃ and 55%~65% humidity. The thickness of the spread during fermentation is 8~10 cm. Treat it at 180℃ for 30 minutes for initial drying, and then dry it at 85℃ for 2.5 hours to obtain honeysuckle black tea samples.

[0033] 1.2 Preparation of dried honeysuckle samples: The honeysuckle samples were prepared by microwave fixation at 300KW for 3-5 minutes and then dried at 85℃ to the same degree of dryness as the honeysuckle black tea mentioned above. These samples were used as a control and were designated as CK.

[0034] 2. Sensory Evaluation 2.1 Effects of Different Fermentation Times on the Quality of Sichuan Honeysuckle Black Tea 2.1.1 Effects of different fermentation times on the sensory quality of Sichuan honeysuckle black tea Sensory quality assessment was conducted in accordance with the quality evaluation criteria for Gongfu black tea. The terminology used in sensory evaluation followed GB / T 14487 "Tea Sensory Evaluation Terminology," and the evaluation procedure followed GB / T 23776-2018 "Tea Sensory Evaluation Method." This method comprehensively evaluates the tea sample from five aspects: appearance, aroma, taste, liquor color, and infused leaf color. The Gongfu black tea standard: Product requirements (including sensory quality) are based on GB / T 13738.2 "Black Tea Part 2: Gongfu Black Tea." Honeysuckle from Sichuan, treated with different fermentation times, showed differences in appearance, liquor color, aroma, taste, and infused leaf color. The sensory evaluation results for each quality factor are shown in Table 1. The sensory quality evaluation process is as follows: Figures 1-5 As shown.

[0035] Table 1. Sensory Quality Evaluation Results of Black Tea

[0036] From Table 1 and Figures 1-5It can be seen that the appearance of Sichuan honeysuckle black tea with different fermentation times is not significantly different, with scores ranging from 86.8 to 88.8. The scores are relatively high and close, indicating that the appearance score is not significantly affected by fermentation time. All teas are characterized by "curled, reddish-brown and bright," with the main difference being in uniformity. The 1.5h and 3.5h groups received the highest scores, followed by the 2.5h and 4.5h groups. The appearance before and after fermentation is significantly different, with the color changing from green and yellow to reddish-brown due to fermentation. Regarding the quality of the tea liquor, it initially increases and then decreases with fermentation time. The "bright orange-yellow" liquor of 1.5h and 2.5h is less bright than the "transparent orange-yellow" liquor of 3.5h and 4.5h. This indicates that the ratio of theaflavins and thearubigins is more balanced at the 3.5h fermentation level, resulting in a bright and lustrous liquor, which is the key to forming the best liquor color. The CK group is a light green and bright, indicating the formation of polyphenol oxidation products in the tea liquor before and after fermentation. Fermentation greatly enriches and enhances aroma, with different fermentation times having the greatest impact, ranging from 81.8 to 93.2 in scores. At 1.5 hours of fermentation, aroma characteristics are not fully formed, resulting in poor persistence. Fermentation deepens at 2.5 hours, the sweet aroma becomes purer and more persistent, reaching its highest score at 3.5 hours, where a pleasant ripe fruit aroma develops on top of the pure sweet aroma, with excellent persistence. At 4.5 hours of fermentation, while retaining the pure, sweet, and ripe fruit aroma, persistence decreases, and aroma compounds may begin to dissipate or transform due to over-fermentation. The aroma types change significantly before and after fermentation. Taste, as a key indicator, has the highest scoring coefficient. Among the fermentation groups, quality first increases and then decreases with fermentation time, reaching its highest score at 3.5 hours, characterized by "mellow and sweet, with good harmony." Various taste compounds reach optimal balance; beyond 3.5 hours, over-fermentation is indicated, and the mellowness decreases. Taste changes significantly before and after fermentation; the control group (CK) is mellow but astringent, indicating that fermentation significantly enhances the taste of the honeysuckle, resulting in good palatability. The leaf base of Sichuan honeysuckle black tea was minimally affected by fermentation time, with no significant difference in scores. However, the leaf base changed significantly before and after fermentation. Using a weighted scoring method (appearance 25%, liquor color 10%, aroma 25%, taste 30%, leaf base 10%), the Sichuan honeysuckle black tea fermented for 3.5 hours received the highest score and had the best sensory quality.

[0037] The above results, obtained through professional sensory evaluation, revealed that fermentation significantly altered the original flavor of honeysuckle. A preliminary assessment identified 3.5 hours of fermentation time as the optimal window for sensory quality. Moving beyond traditional sensory and simple component determination, modern analytical techniques were employed to construct a four-dimensional data network encompassing "components-metabolism-microorganisms-flavor," providing scientific evidence for the optimal processing point.

[0038] 3. Composition determination: Chlorogenic acid, polyphenols, amino acids, and other active components were determined in the control group (CK) before fermentation and in groups fermented for 1.5h, 2.5h, 3.5h, and 4.5h. This revealed the biochemical impact of the black tea fermentation process on the composition of honeysuckle.

[0039] 3.1 Chlorogenic acid (1) Determination method Referring to the method under the Honeysuckle section of Part I of the Chinese Pharmacopoeia (2025 edition), ultraviolet spectrophotometry was used with 50% methanol as the extraction solvent (solid-to-liquid ratio 1:20). Ultrasonic extraction was performed for 30 min (300W power), and the absorbance was measured at 327 nm. The results were obtained using a chlorogenic acid standard curve (Y=0.0461X+0.0089, relationship coefficient R0). 2 The content is calculated using the formula (=0.9870).

[0040] (2) The results of chlorogenic acid detection in each treatment group are detailed in Table 4.

[0041] Chlorogenic acid is the core active ingredient, possessing antiviral, antibacterial, anti-inflammatory, and antioxidant activities. It is a hallmark component of honeysuckle, but also a source of bitterness. Table 4 shows that the content decreases after fermentation, stabilizing around 3.5 hours. Qualitative and quantitative analysis using a high-resolution UPLC-MS / MS platform yielded the normalized peak areas of chlorogenic acid and quinic acid in the tea samples of the CK treatment group and the 3.5-hour fermentation group, as shown in Table 2.

[0042] Table 2. Results of chlorogenic acid and quinic acid detection in tea samples from the CK treatment group and the 3.5h fermentation treatment group.

[0043] Raw honeysuckle (unfermented honeysuckle) has a high chlorogenic acid content, but chlorogenic acid is quite cold in nature and has a greater side effect on people with weak spleen and stomach. The content decreases after fermentation, indicating that chlorogenic acid undergoes oxidative decomposition and is transformed into quinic acid, which is more beneficial to the body. (The normalized peak areas of CK and 3.5h fermentation treatment are 53300729.43 and 429137892.3, respectively. The content of chlorogenic acid in 3.5h fermentation is 8 times that of CK. The lower chlorogenic acid content indicates more complete oxidative decomposition.)

[0044] 3.2 Amino Acids (1) Determination Method: Refer to GB / T 8314-2002 "Determination of Total Free Amino Acids in Tea": Weigh 0.2 g of sample, add 30 mL of boiling water, boil in a water bath for 30 min, cool and filter, and then make up to 50 mL. Take the test solution, add 0.5 mL of pH 8.0 phosphate buffer and 0.5 mL of 2% ninhydrin solution, boil in a water bath for 15 min, cool and then make up to 50 mL with distilled water, measure the absorbance at 570 nm, and calculate the total amino acid content.

[0045] (2) The results of amino acid content detection in each treatment group are detailed in Table 3.

[0046] Amino acids determine the freshness and sweetness of tea soup, which is crucial to its flavor quality. After fermentation, the amino acid content increased from 9.14% to 10.55-10.69% (Table 3), indicating an improvement in flavor, consistent with the evaluation results. However, excessive fermentation exceeding 3.5 hours leads to a decrease in amino acid content. Amino acid content remained high at 2.5 and 3.5 hours, highlighting the freshness. A comparison of the relevant amino acid content of Sichuan honeysuckle black tea fermented for 3.5 hours with that of the control group (CK) revealed the formation of 15 new substances during fermentation, while 7 substances were undetectable after fermentation. This indicates significant changes in the composition of amino acids and their derivatives after fermentation, which is the fundamental reason for the substantial changes in flavor.

[0047] Table 3. Amino acid detection results of tea samples from the CK treatment group and the 3.5h fermentation treatment group.

[0048] 3.3 Polyphenols (1) Determination method: The polyphenol content in the sample was determined by the Folin-Ciocalteu method. Accurately weigh 0.2 g of sample and place it in a test tube. Add 2.4 mL of 50% ethanol at a material-to-liquid ratio of 1:12. Extract by sonication for 20 minutes. Centrifuge at 3000 rpm for 10 minutes. Take 0.3 mL of supernatant into a 10 mL volumetric flask, add 1 mL of Folin-Ciocalteu reagent, shake well, add 2 mL of 15% sodium carbonate solution, shake well, let stand for 2 minutes, dilute with water to the mark, heat in a 50℃ water bath for 30 minutes, and cool to room temperature. Measure the absorbance at 763 nm. Plot a standard curve (y = 11.874X + 0.1404, R) using gallic acid standard. 2 =0.9946), calculate the polyphenol content. The experiment was performed in triplicate, and the average value was taken. A blank experiment was also performed simultaneously.

[0049] (2) The polyphenol detection results for each treatment group are detailed in Table 4. Polyphenols are key flavor and health components, determining the astringency, aftertaste, and antioxidant capacity of tea. As shown in Table 4, the content decreased after fermentation, reaching its lowest point at 3.5 h, and then tended to stabilize. Fermentation may have promoted the conversion of polyphenols. After 3.5 hours of fermentation, chlorogenic acid (3-O-caffeoylquinic acid), 4-p-coumarylquinic acid, diethyl 2-hydroxyphenylphosphonic acid, [4-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxoalkyl-2-yl]oxoalkyl-2-yl]oxo-2-hydroxy-6-methoxyphenyl]benzophenone, and (1R)-1alpha,3alpha-Dihydroxy-4alpha,5beta-bis(3,4-dihydroxycinnamoyloxy)cyclohexanecarboxylic acid methyl Eight types of substances, including ester, (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2S,3S)-3-hydroxy-4-phenylbut-2-yl]oxacyclohexane-3,4,5-triol, 1,3,4-tri-O-caffeoylquinic acid, and 2-O-galloyl-6-O-caffeoyl glucoside, disappeared, while nine new types of substances were generated, including trans-5-O-caffeoyl-D-quinic acid salt, phenoxyacetic acid ester, terephthalate ester, [6]-paragingerone methyl, tetrahydrocurcumin, demethoxycurcumin, methyl 3,4,5-trimethoxybenzoate, 1-O-caffeoyl monoglyceride, and [8]-paragingerone.

[0050] 3.4 Soluble Sugar (1) Determination Method The total soluble sugar content in the sample was determined by the anthrone colorimetric method. Accurately weigh 0.1 g of sample, add 5 mL of 80% ethanol, and extract in an 80℃ water bath for 30 minutes. Rinse the glass rod with a small amount of 80% ethanol, cool the solution to room temperature, centrifuge at 3500 rpm for 10 minutes, and transfer the supernatant to a 25 mL volumetric flask; repeat the above method twice, combine the supernatants in a 25 mL volumetric flask, and dilute to the mark. Take 2.0 mL of extract, evaporate to dryness in a 100℃ water bath, add 10.0 mL of distilled water, stir thoroughly to dissolve the sugar, and centrifuge. Take 1.0 mL of the test solution, 1.0 mL of distilled water and 5.0 mL of anthrone reagent, develop color in a boiling water bath for 10 min, cool in an ice bath, and measure the absorbance at 620 nm. Plot a standard curve (Y=0.006X-0.0067, R) using glucose standard. 2 =0.9942), calculate the soluble sugar content. The experiment was performed in triplicate, and the average value was taken.

[0051] (2) The results of the soluble sugar content test of tea samples in each treatment group are detailed in Table 4. Soluble sugar affects sweetness and mellowness. The determination of soluble sugar content is an important indicator for evaluating the degree of tea fermentation, sweetness and mellowness, and rationality of the process. As shown in Table 4, the soluble sugar content generally showed a downward trend, but slightly rebounded after 4.5 h. It decreased significantly after fermentation, and the sugar may have been utilized by microorganisms. After 3.5 h of fermentation, new 4-decanoyl sucrose was formed, while D-glycerol-L-galactose-octofructose, methyl acetate (3,4,5,6-tetrahydroxyoxane-2-yl)acetate, and 3'-galactose-galactose disappeared. Gluconic acid increased from 64100085.28 before fermentation to 313610009.4, and glucose increased from 56553393.22 before fermentation to 518936176, which is also the reason for the significant increase in sweetness.

[0052] 3.5 Soluble Protein (1) Determination Method: The content was determined by staining with Coomassie Brilliant Blue G-250. Accurately weigh 0.5g of sample and dissolve it in distilled water. Transfer the solution to a 10mL volumetric flask and bring the volume up to the 10mL mark. Pour the homogenate into a 10mL centrifuge tube and centrifuge at 4000rpm for 10min. Collect the supernatant. Take 0.1mL of the supernatant, add 0.9mL of distilled water and 5mL of Coomassie Brilliant Blue G-250 staining solution, mix, and let stand at room temperature for 2min. Measure the absorbance at 595nm. Plot a standard curve (Y=0.0088X+0.0293, R) using bovine serum albumin (BSA) as the standard. 2 =0.9949), calculate the soluble protein content. The experiment was repeated 3 times, and the average value was taken. A blank experiment was also performed.

[0053] (2) The results of the determination of soluble protein content in tea samples of each treatment group are detailed in Table 4. Soluble protein content is related to the mellowness and smoothness of tea soup. As shown in Table 4, the content increases significantly after fermentation and then tends to stabilize. The 3.5h group reaches the peak and then decreases slightly. This confirms that the 3.5h fermentation time results in the best mellowness and flavor fullness of the tea soup.

[0054] 3.6 Total Flavonoids (1) Determination Method The total flavonoid content in the sample was determined by spectrophotometry. Accurately weigh 0.4 g of sample powder, add 60% ethanol to make up to 10 ml, transfer to a 10 mL volumetric flask, and extract by sonication for 30 minutes. Take 1.0 mL of supernatant, add 1 mL of 60% ethanol and 0.5 mL of 5% sodium nitrite and shake well (let stand for 6 minutes), add 0.5 mL of 10% aluminum nitrate and shake well (let stand for 6 minutes), add 4 mL of 4% sodium hydroxide solution and shake well, then add 60% ethanol to make up to 10 mL, let stand for 15 min, and measure the absorbance at 510 nm. Plot a standard curve using rutin as a standard (Y = 0.005X + 0.0147, R 2=0.999), calculate the total flavonoid content. The experiment was performed in triplicate, and the average value was taken. A blank experiment was also performed simultaneously.

[0055] (2) The results of total flavonoid detection in tea samples from each treatment group are detailed in Table 4. Flavonoids are important antioxidants and anti-inflammatory components, affecting the color of the tea soup and certain flavors, but they are also components that bring bitterness and irritation. As shown in Table 4, the content decreased after fermentation, and slightly rebounded at some time points. The fermentation process effectively improved the bitter taste of honeysuckle. The reduction in total flavonoid content can reduce bitterness, enhance freshness, and create a unique flavor. It balances "health" and "taste".

[0056] 3.7 Determination of saponins (1) The vanillin-sulfuric acid method was adopted, with 80% ethanol as the extraction solvent (solid-liquid ratio 1:30), ultrasonic extraction for 40 min (power 300W), 5% vanillin-glacial acetic acid solution and concentrated sulfuric acid were added for color development, and the absorbance was measured at a wavelength of 544 nm. The standard curve of ginsenoside Rg1 (Y=5.8573X-0.0053, correlation coefficient R) was used. 2 Calculate the saponin content using (=0.9999).

[0057] (2) The results of saponin detection in tea samples from each treatment group are detailed in Tables 4 and 5. Total saponins mainly contribute to bitterness and are one of the important sources of the complexity of tea-like flavor. A suitable ratio can increase the layering and structure of the tea soup, making the tea soup fuller and more robust. During high-temperature roasting or fermentation, saponins may undergo transformation or combine with other substances, thus making their strong bitterness milder and more mellow. The results in Table 4 show that the saponin content reaches its highest level at 2.5h and 3.5h through fermentation.

[0058] Table 4. Content of active ingredients in different fermentation treatments determined by ultraviolet spectrophotometry.

[0059] Table 5. Material transformation of saponins before and after fermentation

[0060] 4. Key Aroma Components Analysis of Sichuan Honeysuckle Black Tea Based on GC-MS 4.1 Total Volatile Components Analysis of Sichuan Honeysuckle Black Tea The total volatile components of Sichuan honeysuckle black tea from each treatment group were analyzed, and the results are as follows: Figures 6-8 As shown in Table 6. In the figure, Treat-1h, Treat-2h, Treat-3h, and Treat-4h represent the treatment groups with fermentation treatments of 1.5, 2.5, 3.5, and 4.5h, respectively; in the table, H-1, H-2, H-3, and H-4 represent the treatment groups with fermentation treatments of 1.5, 2.5, 3.5, and 4.5h, respectively.

[0061] Table 6 Key aroma active components of Sichuan honeysuckle black tea in each treatment group

[0062] To investigate the dynamic changes of volatile components during the fermentation of Sichuan honeysuckle black tea, targeted metabolomics analysis was performed using gas chromatography-mass spectrometry (GC-MS). A total of 1849 volatile substances were identified in all samples, mainly including esters (18.13%), terpenoids (17.26%), ketones (11.26%), alcohols (9.09%), and heterocyclic compounds (10.34%). To assess the impact of processing on the flavor of Sichuan honeysuckle black tea, PCA and HCA were used for analysis. PCA effectively distinguished between samples before and after processing. Figure 6 This confirms the transformative effect of processing on the sample composition. For example... Figure 7 The clustering heatmap shown further verifies this distinction. The content of volatile components in Sichuan honeysuckle black tea was screened based on a volatile component content greater than 0.2%, resulting in 56 volatile substances. After removing substances without aroma descriptions, 23 aroma substances remained, as detailed in Table 6.

[0063] After fermentation, diallyl trisulfide, which has a pungent, garlic-like flavor, decreased significantly. 2-ethylhexanoic acid (paint, varnish) and acetic anhydride (sharp, vinegar) also decreased to some extent. 2-methyl-1,3-dithiopentane, which has a sulfurous, smoky aroma, increased significantly after 2.5 hours of fermentation, with little change after other treatments. (E,E)-2,4-heptadialdehyde, which has a roasted, fatty aroma, and 2,3,5-trimethylpyrazine, which has a roasted, nutty aroma, both increased significantly after fermentation. Phenylene alcohol increased markedly after fermentation, with its content increasing more than tenfold. Different fermentation times have a significant impact on the main volatile substances, including phenylethanol, (E)-4-nonenal, (E,E)-2,4-heptanedial, and 2,3,5-trimethylpyrazine. These substances are characterized by floral and fruity aromas, roasted meat aromas, and nutty aromas. These substances are the material basis for the floral, fruity, and sweet aromas of fermented honeysuckle black tea, which is consistent with the results of aroma sensory evaluation.

[0064] 4.2 Identification and analysis of differential metabolites in Sichuan honeysuckle black tea are shown in Tables 7 and 8. In Tables 7 and 8, the same number corresponds to the same substance.

[0065] Table 7 Differential metabolites of honeysuckle black tea

[0066] Table 8. Differential metabolites of honeysuckle black tea in each treatment group

[0067] To further screen characteristic aroma compounds in Sichuan honeysuckle black tea fermented at different times, 904 differential metabolites in 15 categories were identified based on the criteria of P < 0.05 and VIP > 1, accounting for 48.89% of the total volatile metabolites. Esters (164, 18.1%) were the most abundant, followed by terpenes (159, 17.5%), heterocyclic compounds (93, 10.2%), ketones (90, 9.9%), alcohols (90, 9.9%), and aldehydes (77, 8.5%). The sum of these 15 categories accounted for 48.89% of the total metabolites. The aroma of Sichuan honeysuckle black tea is determined by both the flavor threshold and concentration of volatile components. OAV (Organic Aspect Ratio) is commonly used to evaluate the contribution of individual aroma components to the overall aroma of the tea. Among them, 1-octen-3-one (fungal aroma) and benzyl mercaptan (meaty, garlicky, caramel, and sulfurous aroma) both had an OAV > 10,000 in the control (CK) and various fermentation treatments, forming the basis of complex food flavors. After fermentation, some of the (E,E)-3,5-octen-2-one, ethyl 3-cyclohexenecarboxylate, 2,4-nonadienal, 2-furfuryl mercaptan, 5-methyl-2-furanthiol, (E)-4-nonenal, and tert-butyl butyrate had OAVs > 10,000 under different fermentation times, but all showed significant increases compared to the control (CK). These components showed outstanding relative content and aroma intensity under different fermentation treatments, mostly exhibiting fatty, meaty, and floral / fruity aromas, contributing significantly to the aroma characteristics of fermented honeysuckle black tea. Further calculations of ROAV were used to quantify the contribution of each volatile component to the overall aroma of Sichuan honeysuckle black tea. The top 40 components were selected for comparative analysis (Tables 6-7). In Sichuan honeysuckle black teas with different treatments, 1-octen-3-one, benzyl mercaptan, (E,E)-3,5-octadien-2-one, 2,4-nonadienal, 2-furfuryl mercaptan, 3-octen-2-one, 5-methyl-2-furanthiol, and (E)-4-nonenal were likely common key aroma components in Sichuan honeysuckle black teas with different fermentation times. These components made significant contributions to the overall aroma characteristics and mostly exhibited characteristics of light, sweet, fruity, and milky aromas. Therefore, the aromas of the five varieties of black tea were mainly characterized by fungal, fatty, fruity, and sulfurous aromas, which is consistent with the sensory evaluation results. 2-Furfuryl mercaptan (OAV ≥ 45494.73) had the highest OAV, reaching 100 in Sichuan honeysuckle black tea. Its content in the four black tea varieties ranged from 0.204 to 0.362 μg / g. Although the OAV of 2-furfuryl mercaptan is relatively low compared to other volatile compounds, its low aroma threshold due to sulfur compounds results in a small amount but a strong aroma, contributing a minty fragrance to the overall aroma. This substance plays a crucial role in food flavor mics, and its characteristic roasted, coffee, and meaty aromas significantly influence the sensory quality of the final product.2-Furfuryl mercaptan is a sulfur-containing furan compound with strong olfactory activity. It produces unique roasted and coffee-like aromas in a variety of heat-processed and fermented foods, making it an important target for flavor mics research.

[0068] Differential analysis of volatile metabolites, such as Figures 9-10 As shown, the complex dynamic changes of volatile components during fermentation are influenced by multiple factors, including physical evaporation, chemical transformation, and degradation of volatile compounds. For example... Figure 9 As shown in the figure, the OPLS-DA score plot clearly illustrates the differences between the groups, indicating that the metabolites of honeysuckle are significantly different before and after processing. Figure 10The study showcases the top 20 pathways identified by KEGG enrichment analysis. Compared to the control (CK) at 1.5 h of fermentation, the biosynthesis of secondary metabolites, monoterpenoid biosynthesis, tropane, piperidine, and pyridinealkaloid biosynthesis, phenylalanine metabolism, phenylpropanoid biosynthesis, D-Amino acid metabolism, and butanoate metabolism were the most significantly enriched pathways based on P-value and the number of enriched metabolites. This indicates that the secondary metabolite biosynthesis pathway is enriched with a large number of differential metabolites, possibly due to the complex reactions of microorganisms to metabolite catabolism during fermentation. The monoterpene biosynthesis pathway shows a large amount of monoterpenes produced, indicating that fermentation promotes the conversion of multiple substances. Comparative analysis between 2.5 h and 1.5 h of fermentation shows that the secondary metabolite biosynthesis pathway has the highest enrichment level, with 8 metabolites; followed by the biosynthesis of phenylalanine metabolites and secondary metabolites, both with 3 metabolites; the biosynthesis of various alkaloids has 2; and other biosynthetic pathways have 1. The biosynthesis pathways of secondary metabolites are enriched with a large number of differential metabolites, possibly due to the complex reactions of microorganisms to the catabolism of metabolites during fermentation. Compared with 2.5 hours of fermentation, the metabolic pathways, biosynthesis of secondary metabolites, sesquiterpenoid and triterpenoid biosynthesis, phenylpropanoid biosynthesis, and phenylalanine metabolism were the most significant enrichment pathways based on P-value and the number of enriched metabolites. Phenylacetic acid in the phenylalanine metabolic pathway has a strong and unique honey aroma, and after dilution, it also has a rose, civet, and sweet chocolate and strawberry-like sweetness. Its concentration increases with the extension of fermentation time; while the concentration of trans-cinnamic acid decreases with the extension of fermentation time. Four differential substances in the biosynthesis of sesquiterpenes and triterpenes are synthesized through the mevalonate pathway, etc., mainly contributing "woody", "pine", and "fruity" aromas. The concentration of these flavors decreases with the extension of fermentation time.Compared to fermentation time of 3.5 h, fermentation time of 4.5 h resulted in the synthesis of six metabolites in monoterpenoids: geraniol, neomenthol, menthone, (1S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene, L-menthol, and [1S-(1α,2α,5β)]-5-methyl-2-(1-methylethyl)cyclohexanol. These are the core substances that construct fresh herbal and floral flavor profiles. With prolonged fermentation time, the contents of L-menthone, L-menthol, and neomenthol all decreased to varying degrees. The sensory flavor characteristics associated with the differential metabolites in each treatment group are shown in the radar chart below. Figure 11 As shown in the figure. The results indicate that, compared with the unfermented (CK) sample, the top 10 sensory flavors with the highest number of annotations—sweet, fruity, minty, green, fatty, floral, waxy, herbal, fresh, and woody—all showed a significant increase after 1.5 hours of fermentation. Compared with the 1.5-hour fermentation sample, the sweet, fruity, and berry flavors remained balanced after 2.5 hours of fermentation, while the grassy flavor decreased significantly. The floral, woody, violet, and fresh flavors increased to some extent, and the spicy flavor increased significantly, with a new vinegar aroma added. After 3.5 hours of fermentation, the sweetness of the sample decreased slightly, and the woody, grassy, ​​and fleshy flavors decreased significantly, but the fruity and floral flavors increased. The balsamic, mild, and nutty flavors became more prominent. The herbal aroma remained relatively balanced. Compared to fermentation time of 3.5 hours, the main flavor impact of 4.5 hours of fermentation is in sweetness; the longer fermentation time reduces the sweet aroma. Secondly, fruity aromas also decrease with longer fermentation time. Next, floral and grassy aromas also slightly decrease with longer fermentation time. However, woody and fleshy aromas are enhanced. Herbal aromas remain largely unchanged. Balsamic vinegar aroma is significantly reduced, spicy aroma remains balanced, and waxy aroma is slightly reduced.

[0069] 5. Metagenomic analysis Principal component analysis (PCA) score plots of microbial communities for tea samples with different treatments are shown below. Figure 12 As shown in the figure. Heatmaps of microbial species abundance at the phylum, genus, and species levels are shown in the figure. Figure 13 As shown in the figure. The LEfSe evolutionary clade diagram of the differentially expressed microbial species in different treatment groups is as follows. Figure 14 As shown in the Venn diagram for screening key microbial species. Figure 15 A bar chart showing the classification of KEGG secondary functional pathways in microbial communities is shown below. Figure 16 As shown in the figure. The differential enrichment analysis of KEGG pathway microbial gene function in tea samples from each treatment group is illustrated in the figure below. Figure 17 As shown.

[0070] The five groups of samples showed significant differences in microbial community composition. The fermentation time of 4.5 hours resulted in the lowest microbial diversity (lowest Shannon index), which was conducive to the formation of caramel aroma. The control group (CK) showed the highest microbial diversity and more diverse flavors. Among the four fermentation treatments, the group fermented for 3.5 hours exhibited the highest microbial diversity, indicating a richer flavor profile.

[0071] Principal coordinate analysis (PCoA) showed that the five community structures were clearly separated. Linear discriminant analysis (LEfSe) identified 11 differential biomarkers. Each aroma group has its own characteristic species. Core functional bacteria (such as Bacillus daquensis) drive aroma formation through enzyme production and synthesis of volatile compounds.

[0072] The microbial community distribution at the cultivar level in Sichuan honeysuckle black tea under different fermentation time conditions is shown. A total of 3792 species were annotated. At 1.5 h of fermentation, Sphingomonas bacteria were the dominant fermenting microorganisms. After 2.5 h, Rhodobulobacterium CPCC 204708 and Sphingomonas bacteria were the dominant fermenting microorganisms. After 3.5 h, the abundance of these two microorganisms decreased slightly, but they remained dominant fermenting microorganisms. Meanwhile, *Tatumorum* OPLPL6 and *Roseola epidermidis* showed significant increases. After 4.5 h of fermentation, the abundance of *Tatumorum* OPLPL6, *Roseola epidermidis*, *Rhodobulobacterium* CPCC 204708, and Sphingomonas bacteria all decreased significantly. The abundance of these 10 microorganisms was lower in the control (CK) treatment. The results indicate that fermentation time has a significant regulatory effect on the microbial community structure. Functional annotation of microbial genes in honeysuckle black tea fermented at different times was performed using the KEGG database. Among the top 25 graded metabolic pathways, the abundance of microbial metabolism in diverse environments, glycolate and dicarboxylate metabolism, sulfur metabolism, beta-lactam resistance, and peptidoglycan biosynthesis was high at 1.5 h of fermentation. At 2.5 h, no significant enrichment was observed, and all pathways showed varying degrees of decrease compared to the control (CK). At 3.5 h of fermentation, the abundance of microbial metabolism under different environments, binary component systems, decomposition of valine, leucine, and isoleucine, oxalic acid and dicarboxylic acid metabolism, pyruvate metabolism, tryptophan metabolism, carbon metabolism, nicotinic acid and nicotinamide metabolism, sulfur metabolism, β-lactam resistance, and peptidoglycan biosynthesis was high. At 4.5 h of fermentation, only furfural degradation was significantly abundant among the 27 metabolic pathways. The eggNOG functional classification stacked bar chart of the microbial community is shown below. Figure 18As shown. Through comparison with the eggNOG database, a total of 298,005 genes were annotated into 24 COG functional categories. Among them, the 'Metabolism' category had the highest proportion (45.53%), with the most abundant genes related to amino acid transport and metabolism (E), carbohydrate transport and metabolism (G), and inorganic ion transport and metabolism (P), indicating that the samples have active basal metabolic activity; followed by 'Cellular Processes and Signal Transduction' (24.73%) and 'Information Storage and Processing' (9.70%), while the category with poor features accounted for only 9.21%. The functional abundance (default top 10) of the eggNOG database's Functional Category is integrated and displayed. Unfermented honeysuckle showed higher levels of all functional categories compared to fermented honeysuckle. However, gene abundance varied significantly with different fermentation times, generally exhibiting a trend of decrease-increase-sharp decrease with prolonged fermentation time. The 3.5-hour fermentation treatment showed the highest gene abundance across all categories, indicating that 3.5 hours of fermentation had the greatest impact on the microbial community. In the 3.5-hour fermentation group, all 10 COG categories showed consistent upregulation. This comprehensive enhancement of the functional spectrum suggests that the microbial community (or strain) has entered a highly active physiological state.

[0073] 6. A heatmap showing the correlation between the top 20 differentially metabolites and microorganisms, obtained through combined GC-MS and metagenomic analysis, is shown below. Figure 19shown. Marortus_luteolus, Plectosphaerella_plurivora, Rhodococcus_tukisamuensis, Tatumella_sp_JGM94, Botryotinia_calthae, Malassezia_restri cta, Nodosilinea_nodulosa, Rubripirellula_obstinata, Candidatus_Brocadia_pituitae, Rheinheimera_baltica, Amanita_inopinata and 2,3-dimethylpyridine respectively Pyridine, ethylhexanol, d-menthol, dl-menthol, (1α,2β,5β)-5-methyl-2-(1-methylethyl)cyclohexanol, (1α,2α,5α)-5-methyl-2-(1-methylethyl)cyclohexanol, L-menthol, [1S-(1α,2α,5β)]-5-methyl-2-(1-methylethyl)cyclohexanol, (1α,2α,5β)-5-methyl-2-(1-methylethyl)cyclohexanol, ethyl benzoate, ethyl 2-hydroxybenzoate, cis-5-methyl-2-(1-methylethyl)cyclohexanone, 2-methylpropyl butenoate, caryophyllein, N-cyclohexylformamide, 2-nonenal, E-2-nonenal, 1-methylethyl phenylacetate, triethylene glycol, and 3-methylhexanol were significantly positively correlated (P < 0.05). 0.05); Kosakonia_quasisacchari, Butyrivibrio_fibrisolvens, Roseimaritima_ulvae, Rouxiella_silvae, Limisphaera_ngatamarikiensis, except for KMW0643 caryophyllin and WAMW2089 N-cyclohexylformamide, all showed significant positive correlations with the other 18 substances.Acinetobacter_portensis, Xanthomonas_sp_MWU16_30325, Paenibacillus_sp_VKM_B_2647, Brevundimonas_mediterranea, Acinetobacter_sp_SM1B, Paenibacillus_physcomitrellae, Sphaerimonospora_thailandensis, Geodermatophilus_sp_TF02_6, Methylobacterium_gregans, and Xenorhabdus_innexi showed a significant negative correlation with the above substances (P < 0.05), indicating that these 10 microorganisms have a significant negative impact on the above flavor substances; they may have an important influence on the flavor substances of Sichuan honeysuckle black tea, thus affecting the quality of the product.

[0074] Comparative Example 1: A method for preparing Sichuan honeysuckle black tea, comprising the following steps: Take 30 jin (15 catties) of Sichuan honeysuckle with a flowering rate of 40%~50%, and blow it with cold air for 4 hours at a temperature of 15~20℃. After the cold air blowing is completed, the spreading thickness for cold air withering is the same as in Example 1. Turn off the fan and place it in a rolling machine for 15~20 minutes. Ferment for 2.5 hours and 3.5 hours respectively under conditions of 25~32℃ and 55%~65% humidity, with the spreading thickness for fermentation being the same as in Example 1. Treat it at 180℃ for 30 minutes for initial firing, and then dry it at 85℃ for 2.5 hours to obtain Sichuan honeysuckle black tea samples.

[0075] Comparative Example 2: A method for preparing Sichuan honeysuckle black tea, the steps of which are as follows: Take 30 jin of Sichuan honeysuckle with a flowering rate of 40%~50%, wither under natural conditions for 5 hours, with the withering thickness being the same as in Example 1, knead in a kneading machine for 15~20 minutes, ferment for 2.5 hours and 3.5 hours respectively under conditions of 25~32℃ and 55%~65% humidity, with the fermentation spreading thickness being the same as in Example 1, treat at 180℃ for 30 minutes for roughing treatment, and then dry at 85℃ for 2.5 hours to obtain Sichuan honeysuckle black tea samples.

[0076] Comparative Example 3: A method for preparing Sichuan honeysuckle black tea, the steps of which are as follows: Take 30 catties each of Sichuan honeysuckle with a flowering rate of 40%~50%, and blow them with cold air for 1 hour at a temperature of 15~20℃. After blowing with cold air, turn off the fan and let them wither naturally at 20~25℃ for 3 hours, with the same spreading thickness as in Example 1. After natural withering, place the withered Sichuan honeysuckle in a rolling machine and roll it for 15~20 minutes. Ferment for 2.5 hours and 3.5 hours respectively at 25~32℃ and 85%~95% humidity, with the same spreading thickness as in Example 1. Treat them at 180℃ for 30 minutes for initial drying, and then dry them at 85℃ for 2.5 hours to obtain Sichuan honeysuckle black tea samples.

[0077] The sensory quality of the honeysuckle black tea prepared by comparative examples 1-3 was evaluated and the saponins were detected. The results are shown in Tables 9-10.

[0078] Table 9 Sensory qualities of Sichuan honeysuckle black tea

[0079] Table 10. Saponin content of Sichuan honeysuckle black tea

[0080] During the withering process of Sichuan honeysuckle black tea, cold air is blown to dissipate surface moisture and prevent the hot, humid conditions from damaging the fresh flowers. If the airflow is too strong, uneven withering can occur, leading to a reddish discoloration. Without natural withering, relying solely on cold air can result in a dry surface and uneven internal moisture, making subsequent rolling difficult and causing tea juice to leak out and hindering shaping. However, reducing pressure and controlling cell damage can impede subsequent fermentation. During fermentation after rolling, the high moisture content restricts airflow, hindering normal oxidation and resulting in poor internal quality, a strong grassy aroma, and a bitter taste. Withering, on the surface, involves water loss, flower shrinkage, softening of the flower, and darkening of the leaves. As internal moisture is lost, the internal decomposition process intensifies, altering the internal components. Therefore, insufficient surface water loss can also lead to inadequate internal cell water loss, affecting cell sap concentration and enzyme activity.

[0081] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing Sichuan honeysuckle black tea, characterized in that, Includes the following steps: After the honeysuckle flowers are withered, they are kneaded to obtain kneaded honeysuckle flowers. The kneaded honeysuckle is fermented to obtain fermented honeysuckle; Fermented honeysuckle flowers are dried to obtain honeysuckle black tea.

2. The preparation method according to claim 1, characterized in that, The fermentation temperature is 25~32℃; the fermentation time is 1.5~4.5h; and the fermentation humidity is 55%~65%.

3. The preparation method according to claim 1, characterized in that, The honeysuckle mentioned above includes honeysuckle with a flowering rate of 40% to 60%; the withering includes air withering and natural withering; the temperature of air withering is 15 to 20°C; the time of air withering is 1 to 3 hours; the temperature of natural withering is 20 to 25°C; the time of natural withering is 3 to 5 hours; and the kneading time is 15 to 20 minutes.

4. The preparation method according to claim 1, characterized in that, The drying method includes high-temperature drying and low-temperature drying; the high-temperature drying temperature is 160~200℃ and the time is 30~50 min; the low-temperature drying temperature is 80~90℃ and the time is 2~3 h.

5. A Sichuan honeysuckle black tea prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the preparation method according to any one of claims 1 to 4 in improving the quality of Sichuan honeysuckle black tea.

7. The application according to claim 6, characterized in that, Improving the quality of Sichuan honeysuckle black tea includes improving the sensory quality and / or the content of active ingredients in Sichuan honeysuckle black tea; the improvement of the sensory quality of Sichuan honeysuckle black tea includes improving any one or more of the following: appearance, liquor color, aroma, taste and infused leaf. Improving the content of active ingredients in Sichuan honeysuckle black tea includes improving the content of any one or more of the following: chlorogenic acid, quinic acid, amino acids, polyphenols, soluble sugars, soluble proteins, total flavonoids, saponins, and volatile components.

8. A method for increasing the content of saponins and / or quinic acid in honeysuckle black tea, characterized in that, Includes the following steps: After the honeysuckle flowers are withered, they are kneaded to obtain kneaded honeysuckle flowers. The kneaded honeysuckle flowers are fermented to obtain fermented honeysuckle flowers; the fermentation temperature is 25~32℃; the fermentation time is 2.5~3.5h; and the fermentation humidity is 55%~65%. Fermented honeysuckle flowers are dried to obtain honeysuckle black tea.

9. The method according to claim 8, characterized in that, The withering includes air withering and natural withering; the temperature of air withering is 15~20℃; the time of air withering is 1~3h; the temperature of natural withering is 20~25℃; the time of natural withering is 3~5h.

10. The method according to claim 8, characterized in that, The drying method includes high-temperature drying and low-temperature drying; the high-temperature drying temperature is 160~200℃ and the time is 30~50 min; the low-temperature drying temperature is 80~90℃ and the time is 2~3 h.

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