Xanthoceras sorbifolia bunge leaf fermented tea and preparation method thereof

Abstract

The application discloses a Xingguo fruit leaf fermented tea and a preparation method thereof, and the preparation method comprises the following steps: activating a strain, preparing a spore suspension, pretreating raw materials, inoculating, fermenting, and drying; the raw material ratio is Xingguo fruit leaf tea powder:new black aspergillus spore suspension=1g:0.5-1.5mL; the application directly uses Xingguo fruit leaves as a fermentation substrate, adopts a complete fungal microbial fermentation system, and does not rely on external auxiliary materials to improve the flavor, so that the product formula is more simple; the tea source new black aspergillus is used to adjust the component composition of the Xingguo fruit leaf tea through the fermentation effect; the new black aspergillus can produce various enzyme systems and metabolic effects in the growth process, and is more beneficial to the transformation of taste-related components; the application can improve the problems of obvious bitterness, insufficient coordination of taste, and poor palatability of the Xingguo fruit leaf tea, and obtain the Xingguo fruit fermented tea product which is more coordinated in flavor, more stable in quality, and suitable for tea drinking development.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a fermented tea made from the leaves of *Sapindus mukorossi* and its preparation method. Background Technology

[0002] *Xanthoceras sorbifolium* Bunge, a plant belonging to the genus *Xanthoceras* in the family Sapindaceae, is named for its fruit, which, before splitting open, resembles the hat of an ancient civil official. Its seeds are edible, with a flavor similar to chestnuts. The kernels are a promising source of oil from woody plants, with extremely high oil content (50%-72%) in both the seeds and kernels. The linoleic acid in the oil is a major component of the traditional Chinese medicine Yishou Ning, possessing excellent blood pressure-lowering effects. It can also be used to produce high-grade lubricants, high-grade paints, plasticizers, and cosmetics. Biodiesel prepared from *Xanthoceras sorbifolium* seed oil has a high content of hydrocarbon lipids and has significant industrial and nutritional value. Currently, the core development focus for *Xanthoceras sorbifolium* is primarily on its kernels.

[0003] The Chinese pistache (Xanthoceras sorbifolium) has a long history of medicinal use, but it was only a regional herb. Its leaves are rich in various bioactive components such as flavonoids, triterpenoid saponins, polyphenols, and coumarins. In traditional medicine, it has long been used to dispel wind and dampness, reduce swelling and relieve pain. Modern pharmacological studies have shown that it possesses various pharmacological activities, including antioxidant, anti-inflammatory, metabolic syndrome improvement, lipid regulation, and neuroprotective effects. Harvesting and drying the tender leaves, then steeping them in boiling water, is merely a herbal infusion and cannot be considered "tea." Its use in folk medicine was primarily for relieving discomfort, not as a daily beverage. Later, small-scale trial production began, mimicking traditional green tea processing methods, including harvesting tender leaves, fixing, rolling, and drying. Only then did the Chinese pistache leaf transition from a medicinal raw material to a food-grade tea product.

[0004] Unlike traditional tea leaves, *Xanthoceras sorbifolium* leaves do not contain caffeine or theophylline, making them suitable for nighttime consumption. However, due to their saponin and polyphenol content, they possess a pronounced bitterness and astringency, which some people may experience after drinking them, along with a sticky feeling in the mouth and a persistent astringent sensation on the tongue. High-temperature brewing drastically intensifies the bitterness and astringency, and the tea becomes significantly more pronounced after cooling, resulting in a poor cold-drinking experience. Conventional green tea processing methods, even with initial processing, often fail to completely eliminate the bitterness and astringency, and the tea has poor brewing endurance.

[0005] Subsequently, some attempts were made to alter the taste using the principles of black tea fermentation. However, due to the lack of mature and standardized processes, the palatability issue remained unresolved, resulting in low market recognition. Improper fermentation could also lead to the loss of some enzyme-active substances. Others combined *Xanthoceras sorbifolium* leaves and flowers with Fu brick tea to create fermented products similar to Fu brick tea. These products touted their health benefits and efficacy by adding only small amounts of *Xanthoceras sorbifolium* leaves and flowers, but their palatability was primarily determined by the Fu brick tea base (the main ingredient). Some products improved their flavor by adding fruit juice and pomace. However, fruit juice and pomace are prone to oxidation, leading to a darker color and a sour taste. The polyphenols in *Xanthoceras sorbifolium* leaves easily form flocculent precipitates with the proteins and pectin in the juice and pomace. The low pH of the fruit acids also accelerates the oxidation of polyphenols, resulting in poor tea color, cloudy tea soup, increased bitterness, and weakened aroma. The unique herbal aroma of *Xanthoceras sorbifolium* leaves clashes with the sweetness of the fruit juice, and the use of sugar to mask this creates a chaotic and unrefreshing taste. This method does not actually improve the taste of *Xanthoceras sorbifolium* leaf tea. Furthermore, the presence of bacteria in the juice and pomace, coupled with the high exogenous sugar content leading to microbial proliferation, not only hinders fermentation but also destroys natural nutrients and affects shelf life. Summary of the Invention

[0006] The purpose of this invention is to provide a fermented tea made from *Xanthoceras sorbifolium* leaves, and its preparation method, which optimizes and improves the taste and quality by regulating the flavor-related components contained in the tea.

[0007] This invention achieves its objective by employing the following technical solution: a method for preparing fermented tea from *Xanthoceras sorbifolium* leaves, comprising the following steps: (1) Activation of strain: Inoculate the new Aspergillus niger strain onto PDA medium plates and place them in an incubator with a certain temperature and humidity for cultivation. After activation, the strain is continuously passaged and cultured. The third generation strain with good growth and stable sporulation is selected as the subsequent fermentation strain for later use. (2) Preparation of spore suspension: Take the activated cultured fermentation strain, add sterile water or sterile physiological saline, gently scrape off the spores and filter to obtain spore suspension; (3) Raw material pretreatment: Select tender leaves of *Xanthoceras sorbifolium* that are free from mold, obvious insect infestation, and pesticide contamination as raw materials. After drying, crush them and pass them through a 60-mesh sieve to form uniform *Xanthoceras sorbifolium* leaf powder, so as to improve the uniformity of contact between the material and the spore suspension. Then, sterilize the tea powder. (4) Inoculation: Mix the sterilized Xanthoceras sorbifolium leaf powder from step (3) with the spore suspension prepared in step (2) and stir thoroughly to ensure that the spore suspension is evenly distributed in the tea powder to obtain a mixture. (5) Fermentation: Spread the mixture obtained in step (4) on a fermentation tray or aerated fermentation container, and place it under certain temperature and humidity conditions for solid-state fermentation. Keep the material aerated during the fermentation process, and stir it if necessary to ensure uniform fermentation. (6) Drying: The fermented material is freeze-dried to obtain fermented tea made from the leaves of the Chinese privet.

[0008] In step (1) of this invention, the incubation temperature in the incubator is 30℃~37℃, the relative humidity is 50%~70%, and the activation time is 3~5 days; in step (2), a hemocytometer is used to count spores under a microscope, and the concentration of the spore suspension is adjusted to 1×10⁻⁶. 7 CFU / mL, for later use; By directionally activating the strains to restore the physiological activity of the spores, and then quantitatively counting and calibrating the concentration of the spore suspension using a hemocytometer, the spores used for inoculation have stable and consistent physiological activity, while standardizing the number of inoculated spores. This eliminates experimental errors caused by differences in strain activity and inconsistent inoculation dosage, ensures the comparability and repeatability of subsequent experimental treatment groups, and guarantees accurate and reliable experimental results.

[0009] The sterilization process described in step (3) of this invention is high-pressure steam sterilization. The sterilization conditions are 0.11 Pa, 121 °C, and 25–40 min. After cooling to room temperature, it is ready for use. This process kills microorganisms mixed in the raw materials, avoids contamination by other microorganisms, ensures that the target strain becomes the dominant microbial group in the fermentation system, and guarantees the normal progress of fermentation.

[0010] In step (4) of this invention, the mixing ratio of tea powder to spore suspension is 1g: 0.5-1.5mL; Preferably, the mixing ratio of tea powder and spore suspension in step (4) is 1g:1mL; the inoculation amount is precisely controlled so that the target strain spores match the substrate nutrient level, so that the target strain forms a dominant microbial community, thereby achieving controllable fermentation process and ensuring fermentation effect and batch stability.

[0011] In step (5) of this invention, the spreading thickness is 1–8 cm, the fermentation temperature is 30–37°C, the relative humidity is 50%–70%, and the fermentation time is 10–15 days, which creates a suitable microenvironment for the microorganisms; the spreading thickness ensures aeration and heat and mass transfer of the material layer; the temperature and humidity maintain the enzyme activity and growth conditions of the microorganisms; the fermentation time ensures full conversion of the material; and the material fermentation is uniform, avoiding under-fermentation or over-fermentation, thus improving fermentation stability and batch consistency. In step (6), the drying method adopts freeze drying or low-temperature hot air drying to reduce the impact of high temperature on the aroma and heat-sensitive components of tea.

[0012] Preferably, the paving thickness described in step (5) of the present invention is 2 to 5 cm.

[0013] The fermented tea made from *Sapindus mukorossi* leaves obtained by this invention can be used directly as a tea beverage ingredient, or it can be further processed by crushing, sieving, packaging, or made into tea bags, solid tea beverages, or other product forms.

[0014] A fermented tea made from the leaves of the Chinese privet (Sapindus mukorossi), which is made from the following ingredients in the specified proportions: The ratio of *Xanthoceras sorbifolium* leaf tea powder to *Aspergillus niger* spore suspension is 1 g: 0.5–1.5 mL. The *Xanthoceras sorbifolium* leaf tea powder is obtained by drying and pulverizing young leaves of *Xanthoceras sorbifolium*, and the *Aspergillus niger* spore suspension has a concentration of 1 × 10⁻⁶. 7 CFU / mL.

[0015] Preferably, the mixing ratio of *Sapindus mukorossi* leaf tea powder and *Aspergillus niger* spore suspension in step (4) is 1 g: 1 mL.

[0016] Aspergillus neoniger belongs to the genus Aspergillus. Its colony morphology shows a brownish-red sporulation structure with radial wrinkles and a velvety texture. The conidiophores have smooth, transparent walls, and the apical vesicles are nearly spherical. The conidia are nearly spherical with a rough surface, approximately 3–4.5 μm in diameter. It is commonly cultured on Saccharomyces cerevisiae medium containing yeast extract, sucrose, sodium nitrate, etc., and pH and temperature must be strictly controlled to promote sporulation. Aspergillus neoniger, a tea-derived fungus isolated and sequenced from Pu-erh tea samples, can be used as a functional fermentation strain. Due to its powerful enzyme system and metabolic capacity, Aspergillus neoniger plays a role in multiple industrial fields and is an important industrial fermentation fungus, widely used in food, feed, and biofertilizer industries. This invention uses Aspergillus neoniger (catalog number: BCRC 32261) from the Taiwan Center for Biological Resources Conservation and Research.

[0017] PDA medium: Potato glucose agar medium, used for the culture and activation of new Aspergillus niger.

[0018] Spore suspension: A bacterial suspension formed by dispersing Aspergillus niger spores in sterile water or sterile saline, used for inoculating Xanthoceras sorbifolium leaf raw materials.

[0019] CFU / mL: Colony forming units per milliliter, used to express the number of viable bacteria in a spore suspension.

[0020] Solid-state fermentation: a microbial fermentation process carried out on solid Xanthoceras sorbifolium leaf material without significant free water.

[0021] By adopting the above technical solution, this invention achieves its objective well. It directly uses *Xanthoceras sorbifolium* leaves as the fermentation substrate and employs a complete fungal microbial fermentation system, without relying on exogenous auxiliary materials to improve flavor, resulting in a simpler product formula. Through the fermentation action of *Aspergillus niger*, the composition of *Xanthoceras sorbifolium* leaf tea itself is regulated. During its growth, *Aspergillus niger* can produce a variety of enzyme systems and metabolic activities, which are more conducive to the conversion of flavor-related components such as flavonoids, saponins, glycosides, and amino acids. This invention can improve the problems of obvious bitterness, insufficient taste coordination, and poor palatability of *Xanthoceras sorbifolium* leaf tea, resulting in a *Xanthoceras sorbifolium* fermented tea product with a more harmonious flavor, more stable quality, and suitable for tea beverage development. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the color change of tea soup before and after fermentation in an experimental example of the present invention; Figure 2 This is an electronic tongue taste change radar chart used in the experimental examples of this invention to describe the relative change trend of taste indicators before and after fermentation; Figure 2 In this context, AHS responds to sour substances, SCS responds to bitter substances, ANS responds to sweet substances, NMS responds to umami substances, CTS responds to salty substances, and CPS and PKS are complex cross-responses. These multiple responses are used for overall taste fingerprinting, sample differentiation, and auxiliary discrimination of differences. Figure 3 This is a PCA (principal component analysis) score diagram of metabolites before and after fermentation in the experimental examples of this invention; Figure 4 This is a schematic diagram of the chemical classification of 59 differential metabolites in the experimental examples of this invention; Figure 5 This is a comparison chart of the up-regulation / down-regulation of differential metabolites after fermentation by *Aspergillus cristatus* and *Aspergillus neonicotinus* in the experimental examples of this invention; Figure 6 This is a PCA score graph of the dynamic fermentation process at different time points in the experimental examples of this invention; Figure 7 The experiment in this invention uses the color change of tea soup fermented with *Eurotium cristatum* at different time points; Figure 8 The differential metabolites of *Aspergillus cristatus* at different time points in the experimental examples of this invention |log 10 (FC)|Distribution pattern diagram; Figure 9 The experiment in this invention uses Aspergillus niger to ferment tea at different time points to change the color of the tea soup. Figure 10 These are the differential metabolites of *Aspergillus niger* at different time points in the experimental examples of this invention |log 10 (FC)|Distribution pattern diagram; Figure 3 , Figure 6 In Chinese: QC group is a quality control sample group, which is made by mixing all samples in equal amounts and is used to test the system stability of the instrument during the sample testing process. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments. Example 1:

[0024] A method for preparing fermented tea from *Xanthoceras sorbifolium* leaves includes the following steps: (1) Activation of strain: Inoculate the new Aspergillus niger strain onto PDA medium plates and place them in an incubator with a certain temperature and humidity for cultivation. After activation, the strain is continuously passaged and cultured. The third generation strain with good growth and stable sporulation is selected as the subsequent fermentation strain for later use. (2) Preparation of spore suspension: Take the activated cultured fermentation strain, add sterile water or sterile physiological saline, gently scrape off the spores and filter to obtain spore suspension; (3) Raw material pretreatment: Select tender leaves of *Xanthoceras sorbifolium* that are free from mold, obvious insect infestation, and pesticide contamination as raw materials. After drying, crush them and pass them through a 60-mesh sieve to form uniform *Xanthoceras sorbifolium* leaf powder, so as to improve the uniformity of contact between the material and the spore suspension. Then, sterilize the tea powder. (4) Inoculation: Mix the sterilized Xanthoceras sorbifolium leaf powder from step (3) with the spore suspension prepared in step (2) and stir thoroughly to ensure that the spore suspension is evenly distributed in the tea powder to obtain a mixture. (5) Fermentation: Spread the mixture obtained in step (4) on a fermentation tray or aerated fermentation container (an aerated fermentation container in this embodiment), and place it under certain temperature and humidity conditions for solid-state fermentation. Keep the material aerated during the fermentation process, and stir it if necessary to ensure uniform fermentation. (6) Drying: The fermented material is freeze-dried to obtain fermented tea made from the leaves of the Chinese privet.

[0025] In step (1) of this invention, the incubation temperature in the incubator is 30℃~37℃ (30℃ in this embodiment), the relative humidity is 50%~70% (50% in this embodiment), and the activation time is 3~5 days (3 days in this embodiment); in step (2), a hemocytometer is used to count the spores under a microscope, and the concentration of the spore suspension is adjusted to 1×10⁻⁶. 7 CFU / mL, for later use; By directionally activating the strains to restore the physiological activity of the spores, and then quantitatively counting and calibrating the concentration of the spore suspension using a hemocytometer, the spores used for inoculation have stable and consistent physiological activity, while standardizing the number of inoculated spores. This eliminates experimental errors caused by differences in strain activity and inconsistent inoculation dosage, ensures the comparability and repeatability of subsequent experimental treatment groups, and guarantees accurate and reliable experimental results.

[0026] The sterilization process described in step (3) of this invention is high-pressure steam sterilization. The sterilization conditions are 0.11 Pa, 121 °C, and 25–40 min (25 min in this embodiment). After cooling to room temperature, it is ready for use. This process kills microorganisms mixed in the raw materials, avoids contamination by other microorganisms, ensures that the target strain becomes the dominant microbial group in the fermentation system, and guarantees the normal progress of fermentation.

[0027] In step (4) of this invention, the mixing ratio of tea powder to spore suspension is 1g:0.5-1.5 mL (1g:1mL in this embodiment); the inoculation amount is precisely controlled so that the target strain spores match the substrate nutrient level, so that the target strain forms a dominant microbial community, thereby achieving controllable fermentation process and ensuring fermentation effect and batch stability.

[0028] In step (5) of this invention, the spreading thickness is 1-8 cm (2 cm in this embodiment), the fermentation temperature is 30-37℃ (30℃ in this embodiment), the relative humidity is 50%-70% (55% in this embodiment), and the fermentation time is 10-15 days (10 days in this embodiment). This creates a suitable microenvironment for the microorganisms. The spreading thickness ensures aeration and heat and mass transfer of the material layer. The temperature and humidity maintain the enzyme activity and growth conditions of the microorganisms. The fermentation time ensures full material transformation. This achieves uniform fermentation of the material, avoids under-fermentation or over-fermentation, and improves fermentation stability and batch consistency. In step (6), the drying method adopts freeze drying or low-temperature hot air drying to reduce the impact of high temperature on the aroma and heat-sensitive components of tea.

[0029] The fermented tea made from *Sapindus mukorossi* leaves obtained by this invention can be used directly as a tea beverage ingredient, or it can be further processed by crushing, sieving, packaging, or made into tea bags, solid tea beverages, or other product forms.

[0030] A fermented tea made from the leaves of the Chinese privet (Sapindus mukorossi), which is made from the following ingredients in the specified proportions: The ratio of *Xanthoceras sorbifolium* leaf tea powder to *Aspergillus niger* spore suspension is 1g:0.5-1.5mL (1g:1mL in this example). The *Xanthoceras sorbifolium* leaf tea powder is prepared by drying and pulverizing young leaves of *Xanthoceras sorbifolium*, and the *Aspergillus niger* spore suspension has a concentration of 1×10⁻⁶. 7 CFU / mL. Example 2:

[0031] In this embodiment, the culture temperature in step (1) is 37°C, the relative humidity is 50%, and the activation time is 5 days.

[0032] The sterilization process described in step (3) of this embodiment is high-pressure steam sterilization, with sterilization conditions of 121°C for 40 minutes.

[0033] In step (4) of this embodiment, the mixing ratio of tea powder and spore suspension is 1g:1.5mL.

[0034] In step (5) of this embodiment, the mixture obtained in step (4) is spread in a fermentation pan with a spreading thickness of 1 cm, a fermentation temperature of 37°C, a relative humidity of 50%, and a fermentation time of 10 days.

[0035] A fermented tea made from the leaves of the Chinese privet (Sapindus mukorossi), which is made from the following ingredients in the specified proportions: Sapindus mukorossi leaf tea powder: Aspergillus niger spore suspension = 1g: 0.5-1.5 mL (in this example, it is 1g: 1.5mL).

[0036] Same as Example 1. Example 3:

[0037] In this embodiment, the culture temperature in step (1) is 35°C, the relative humidity is 60%, and the activation time is 3 days.

[0038] The sterilization process described in step (3) of this embodiment is high-pressure steam sterilization, with sterilization conditions of 121°C for 25 minutes.

[0039] In step (4) of this embodiment, the mixing ratio of tea powder and spore suspension is 1g:0.5mL.

[0040] In step (5) of this embodiment, the spreading thickness is 5cm, the fermentation temperature is 35℃, the relative humidity is 60%, and the fermentation time is 13 days.

[0041] A fermented tea made from the leaves of the Chinese privet (Sapindus mukorossi), which is made from the following ingredients in the specified proportions: Sapindus mukorossi leaf tea powder: Aspergillus niger spore suspension = 1g: 0.5-1.5 mL (in this example, it is 1g: 0.5mL).

[0042] Same as Example 1. Example 4:

[0043] In this embodiment, the culture temperature in step (1) is 30°C, the relative humidity is 70%, and the activation time is 4 days.

[0044] The sterilization process described in step (3) of this embodiment is high-pressure steam sterilization, with sterilization conditions of 121°C for 30 minutes.

[0045] In step (4) of this embodiment, the mixing ratio of tea powder to spore suspension is 1g:1mL.

[0046] In step (5) of this embodiment, the spreading thickness is 8cm, the fermentation temperature is 30℃, the relative humidity is 70%, and the fermentation time is 15 days.

[0047] A fermented tea made from the leaves of the Chinese privet (Sapindus mukorossi), which is made from the following ingredients in the specified proportions: Sapindus mukorossi leaf tea powder: Aspergillus niger spore suspension = 1g: 0.5-1.5 mL (1g: 1mL in this example).

[0048] Same as Example 1. Experimental example:

[0049] I. Experimental Grouping and Symbol Explanation According to the method in step (3) of the present invention, the leaves of *Xanthoceras sorbifolium* were prepared, and the samples were divided into groups. 2g of *Xanthoceras sorbifolium* leaves were weighed from each group as a single experimental sample for later use. Following the method and steps of Example 1 (take 2g of Xanthoceras sorbifolium leaf powder, autoclave, and then mix with a prepared concentration of 1×10⁻⁶), 7 After inoculating the CFU / mL spore suspension at a ratio of 1g:1ml, it was placed in a constant temperature and humidity chamber at 30℃ and 55% humidity for 10 days to ferment, which was used as the fermentation group and labeled as FAN10 group. The spore suspension was 1×10 7 The CFU / m *Aurotriarcha* strain was fermented for 10 days at 30℃ and 55% humidity. The samples were then frozen and stored as a fermentation control group, labeled as FAC10 group. Sterile water was used instead of spore suspension, and the samples were directly frozen and stored without culturing. This served as the unfermented control group and was labeled as the UF0 group. Using sterile water instead of spore suspension, the samples were fermented for 10 days at 30°C and 55% humidity. The samples were then frozen and stored as the unfermented control group, labeled as group UF10.

[0050] Each group underwent 6 parallel replicates (n=6), and the specific experimental groupings and treatments are shown in the table below: Table 1. Implementation of grouping symbols and processing conditions

[0051] Note: FAC0 / FAC5 / FAC10 / FAC15 and FAN0 / FAN5 / FAN10 / FAN15 represent the time points of 0d, 5d, 10d and 15d in the dynamic fermentation experiment, respectively (d is the number of days).

[0052] II. Experimental Results (a) Changes in the color of the tea soup For each group's single parallel repeated experiment, 0.6g of dried tea powder sample was taken, placed in an independent container, and 120mL of boiling water was added. The container was then placed in a 90℃ water bath for 20min.

[0053] like Figure 1 As shown, the tea liquor colors of the UF0 and UF10 groups in the unfermented group were generally similar, with no significant differences observed. Compared to the unfermented group, the tea liquor of the FAC10 group in the fermented group was redder and brighter; the tea liquor of the FAN10 group was further deepened, ranging from dark brown to almost black. The fermentation by *Aspergillus niger* caused a more significant change in tea liquor color, manifested as a marked deepening of color, indicating a stronger conversion effect of *Sapindus mukorossi* leaves on color-related components.

[0054] (ii) Changes in the pH value of tea infusion Table 2 pH values ​​of tea infusion before and after fermentation 1 5.94 5.80 6.46 5.88 2 5.80 5.92 6.50 5.86 3 5.72 5.77 6.52 5.78 4 5.92 5.80 6.56 5.83 5 5.80 5.85 6.54 5.84 6 5.80 5.77 6.49 5.78 average value 5.83 5.82 6.51 5.83 Note: The numbers represent the pH values ​​obtained from 6 parallel replicate experiments (corresponding to...). Figure 1 (6 bottles of samples from each group).

[0055] Table 2 shows that the pH of the tea infusion in each group was below 7, indicating an overall slightly acidic pH. The average pH values ​​of the UF0, UF10, and FAN10 groups were 5.83, 5.82, and 5.83, respectively, which were close to each other. The average pH value of the FAC10 group was 6.51, which was about 0.68 higher than that of the unfermented group. This indicates that the acidity of the tea infusion decreased after fermentation by *Aspergillus cristatus*.

[0056] (III) Changes in taste perception with electronic tongue like Figure 2 As shown in the electronic tongue radar chart, the two types of fungal fermentation have different effects on taste indicators. The FAC10 group showed a decrease in umami and saltiness, and an increase in bitterness and sweetness; the FAN10 group showed an increase in sourness, and a decrease in bitterness and saltiness. Unlike the increase in bitterness caused by *Aspergillus niger* fermentation, the saltiness and bitterness of *Aspergillus niger* decreased significantly after fermentation, while umami and sweetness remained unchanged compared to the original unfermented tea.

[0057] (iv) Metabolite profile PCA (principal component) analysis like Figure 3 As shown, there is a clear separation in the distribution areas of the unfermented group and the two fermented fungal groups in the PCA score map; at the same time, the FAC10 group and the FAN10 group are located in different PCA spatial regions. These results indicate that after 10 days of fermentation, the metabolite profiles of each group of samples underwent significant overall changes. The observed differences lie in the distribution of metabolite composition in two-dimensional space for each group of samples, and the different metabolite profile change patterns corresponding to the two fungal species.

[0058] (v) Chemical classification and trends of differential metabolites like Figure 4 As shown, a total of 59 differential metabolites were obtained. After classification by chemical category, flavonoids accounted for the highest proportion (33.90%), followed by triterpenoid saponins (13.56%) and other categories (11.86%). Other differential metabolites included amino acids, glycosides, alkaloids, phenolic acids, nucleosides and terpenes.

[0059] like Figure 5 As shown, the FAC10 group had 16 differentially regulated metabolites and 18 differentially regulated metabolites; the FAN10 group had 15 differentially regulated metabolites and 33 differentially regulated metabolites. In both fermentation treatments, the number of downregulated metabolites exceeded the number of upregulated metabolites, with the downregulation trend being more pronounced in the FAN10 group; the number of upregulated and downregulated metabolites in the FAC10 group was relatively close. The *Aspergillus niger* fermentation group showed a more prominent trend in metabolite changes, with most common bitter-tasting components (flavonoids and saponins) showing a decreasing trend, which is the reason for the decrease in bitterness after fermentation.

[0060] III. Dynamic Changes at Different Fermentation Time Points To observe the dynamic changes during fermentation, four time points were set at 0, 5, 10, and 15 days, and the results were combined with PCA, tea infusion color, and differential metabolites. 10 (FC)| distribution comparison.

[0061] (a) PCA changes during dynamic fermentation like Figure 6 As shown, the distribution areas of the samples from both bacterial species at day 0 were relatively close to those of the unfermented samples. With prolonged fermentation time, the two fermented groups gradually separated from the unfermented group. The PCA position of the *Aspergillus cristatus* group changed significantly between days 5 and 10, with the sample distributions at days 10 and 15 being relatively similar. The *Aspergillus niger* group had already clearly separated from both the day 0 and unfermented groups by day 5, and samples from days 5 to 15 were mainly distributed in similar PCA regions. These results suggest that there are differences in the dynamic changes of the metabolite profiles of the two bacterial species.

[0062] (II) Fermentation process of *Eurotium cristatum* like Figure 7 As shown, the tea soup color of the samples fermented by *Aspergillus cristatus* for 0 days and 5 days showed relatively small changes; however, the tea soup color darkened significantly and turned reddish-brown at 10 days and 15 days.

[0063] like Figure 8 As shown, the |log| values ​​for 5d, 10d, and 15d are... 10 The medians for (FC) are approximately 0.152, 0.655, and 0.634, respectively. Figure 8Statistical comparisons showed differences between 5d and 10d, and between 5d and 15d, while the difference between 10d and 15d was marked as "ns" (no statistical difference). Combined with the distribution pattern, it can be seen that the differential metabolite changes in *Aspergillus cristatus* fermentation tended to stabilize relatively after 10d.

[0064] (III) Fermentation process of Aspergillus niger like Figure 9 As shown, the tea soup color began to darken significantly after 5 days of fermentation with Aspergillus niger; by 10 and 15 days, the color had deepened further, turning dark brown to almost black.

[0065] like Figure 10 As shown, the |log| values ​​for 5d, 10d, and 15d are... 10 The median (FC) values ​​were approximately 1.282, 1.550, and 1.539, respectively, with statistical comparisons between the three time points labeled as "ns". These results indicate that the overall distribution of differential metabolites in the *Aspergillus niger* fermentation samples was quite similar after 5 days, with relatively limited variation up to 15 days. Compared to the *Aspergillus cristatus* group, the metabolite profile of the *Aspergillus niger* group showed a relatively stable distribution at earlier time points.

[0066] IV. Data Results Summary Based on the above experimental data, both fungal fermentations can cause changes in the color, taste indicators, and metabolite profile of *Xanthoceras sorbifolium* leaf tea, but the characteristics of these changes differ between the fungal species. After fermentation with *Aspergillus cristatus*, the tea soup color became reddish and the pH increased; the metabolite profile tended to stabilize after about 10 days. After fermentation with *Aspergillus niger*, the tea soup color darkened significantly, the pH was close to that of the unfermented group, and its metabolite profile showed a relatively stable distribution after 5 days.

[0067] V. Conclusion In summary, *Aspergillus niger* fermentation can significantly reshape the flavor characteristics of *Xanthoceras sorbifolium* leaf tea in a relatively short time, demonstrating high fermentation efficiency and strong flavor regulation capabilities. The bitterness and saltiness of the tea infusion are significantly reduced after *Aspergillus niger* fermentation, indicating that it can effectively weaken the relatively prominent bitter and salty characteristics in *Xanthoceras sorbifolium* leaf tea, thereby mitigating the potentially unpleasant taste of the raw material itself. Compared with *Aspergillus cristatus* fermentation, *Aspergillus niger* exhibits a faster metabolic transformation process and a stronger downregulation effect on unpleasant flavor metabolites, effectively regulating the flavor profile of the tea infusion within a shorter fermentation cycle. Overall, *Aspergillus niger* fermentation shows good application potential in reducing bitterness and saltiness, weakening unpleasant flavor characteristics of raw materials, and promoting the optimization of the flavor structure of the tea infusion. It can provide a new microbial fermentation pathway for improving the palatability of *Xanthoceras sorbifolium* leaf tea and developing fermented tea products with distinctive flavors.

Claims

1. A method for preparing fermented tea from *Sapindus mukorossi* leaves, characterized in that... Includes the following steps: (1) Activation of strain: Inoculate the new Aspergillus niger strain onto PDA medium plates and place them in an incubator with a certain temperature and humidity for culture. After activation, the strain is continuously passaged and cultured, and the third generation strain is selected as the fermentation strain for later use. (2) Preparation of spore suspension: Take the activated cultured fermentation strain, add sterile water or sterile physiological saline, gently scrape off the spores and filter to obtain spore suspension; (3) Raw material pretreatment: Select tender leaves of *Vernicia fordii* that are free from mold, insect infestation, and pesticide contamination as raw materials. After drying, crush them and pass them through a 60-mesh sieve to form uniform *Vernicia fordii* leaf powder. Then, sterilize the tea powder. (4) Inoculation: Mix the sterilized Xanthoceras sorbifolium leaf powder from step (3) with the spore suspension prepared in step (2) and stir thoroughly to obtain a mixture. (5) Fermentation: Spread the mixture obtained in step (4) on a fermentation tray or aerated fermentation container, and place it under certain temperature and humidity conditions for solid-state fermentation. Keep the material aerated during the fermentation process and stir it when necessary. (6) Drying: The fermented material is dried to obtain fermented tea made from the leaves of the Chinese privet.

2. The method for preparing fermented tea from *Xanthoceras sorbifolium* leaves according to claim 1, characterized in that... In step (1), the incubation temperature in the incubator is 30℃~37℃, the relative humidity is 50%~70%, and the activation time is 3~5 days; in step (2), the spore suspension concentration is adjusted to 1×10⁻⁶. 7 CFU / mL, for later use.

3. The method for preparing fermented tea from *Xanthoceras sorbifolium* leaves according to claim 1, characterized in that... The sterilization process described in step (3) is high-pressure steam sterilization, with sterilization conditions of 0.11 Pa, 121 °C, and 25–40 min. After cooling to room temperature, it is ready for use.

4. The method for preparing fermented tea from *Xanthoceras sorbifolium* leaves according to claim 2, characterized in that... The sterilization process described in step (3) is high-pressure steam sterilization, with sterilization conditions of 0.11 Pa, 121 °C, and 25–40 min. After cooling to room temperature, it is ready for use.

5. A method for preparing fermented tea from *Sapindus mukorossi* leaves according to claim 1, 2, 3, or 4, characterized in that... In step (4), the mixing ratio of tea powder to spore suspension is 1 g: 0.5-1.5 mL.

6. A method for preparing fermented tea from *Xanthoceras sorbifolium* leaves according to claim 1, 2, 3, or 4, characterized in that... The spreading thickness in step (5) is 1-8 cm, the fermentation temperature is 30-37℃, the relative humidity is 50%-70%, and the fermentation time is 10-15 days; the drying method in step (6) is freeze drying or low-temperature hot air drying.

7. The method for preparing fermented tea from *Xanthoceras sorbifolium* leaves according to claim 5, characterized in that... The spreading thickness in step (5) is 1-8 cm, the fermentation temperature is 30-37℃, the relative humidity is 50%-70%, and the fermentation time is 10-15 days; the drying method in step (6) is freeze drying or low-temperature hot air drying.

8. The method for preparing fermented tea from *Xanthoceras sorbifolium* leaves according to claim 6, characterized in that... The paving thickness mentioned in step (5) is 2 to 5 cm.

9. The method for preparing fermented tea from *Xanthoceras sorbifolium* leaves according to claim 7, characterized in that... The paving thickness mentioned in step (5) is 2 to 5 cm.

10. A fermented tea made from the leaves of *Sapindus mukorossi*, characterized in that... It is made from the following ingredients in the following proportions: The ratio of *Xanthoceras sorbifolium* leaf tea powder to *Aspergillus niger* spore suspension is 1 g: 0.5–1.5 mL. The *Xanthoceras sorbifolium* leaf tea powder is obtained by drying and pulverizing young leaves of *Xanthoceras sorbifolium*, and the *Aspergillus niger* spore suspension has a concentration of 1 × 10⁻⁶. 7 CFU / mL.

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