Fermented wild jujube leaf tea and preparation method thereof

Abstract

The invention belongs to the field of biological fermentation, and particularly relates to fermented wild jujube leaf tea and a preparation method thereof. The preparation method comprises the following steps: preparing wild jujube leaf raw tea from fresh wild jujube leaves; the wild jujube leaf raw tea is sterilized, the sterilized wild jujube leaf raw tea is inoculated with Eurotium cristatum for fermentation culture, a fermentation product is dried, the fermented wild jujube leaf tea is obtained, and the preservation number of the Eurotium cristatum is CICC 2099. According to the fermented wild jujube leaf tea prepared by the preparation method provided by the invention, the content of irritant, smelly and unpleasant volatile compounds is reduced, meanwhile, the content of volatile compounds with flower fragrance, costustoot, grass fragrance, fruit fragrance and the like is increased, the sensory quality of the fermented wild jujube leaf tea is improved, and the biological efficacy becomes good.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation, specifically relating to a fermented jujube leaf tea and its preparation method. Background Technology

[0002] Sour jujube, also known as thorn, wild jujube, and kudzu needle, is a plant belonging to the genus Ziziphus of the Rhamnaceae family. It is a pioneer tree species for windbreak and sand fixation, and soil and water conservation. Every part of the sour jujube is valuable. Its fruit is rich in nutrients and can be used to make beverages; the sour jujube kernel is a traditional Chinese medicine with effects such as nourishing the liver, calming the mind, soothing the nerves, and reducing sweating. The sour jujube leaves are rich in vitamins, triterpenoid acids, chlorogenic acid, and flavonoids, among other medicinal components. Extractable compounds such as "sour jujube leaf ketone" and rutin are effective treatments for coronary heart disease. Flavonoids possess significant antioxidant, anti-inflammatory, antibacterial, antiviral, and antitumor activities. Furthermore, sour jujube leaves have unique effects in calming the mind, soothing the nerves, and improving sleep quality, earning them the nickname "Oriental Sleeping Leaf." However, sour jujube leaves are often wasted due to a lack of effective utilization methods. Fermented tea is a tea-making process that involves the initial processing of tea buds and leaves, including withering, rolling, fermentation, and drying, to produce raw tea, which is then further refined into tea. The current production process for jujube leaf tea is generally as follows: timely leaf picking - appropriate fixation - proper rolling - timely roasting - packaging and storage. Although the traditional tea-making process is relatively simple, jujube leaves cannot fully realize their medicinal value, and their poor taste and flavor also make them uncompetitive in the market. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the defects of low dissolution of effective components and poor sensory quality of jujube leaf tea in the prior art, and to provide a fermented jujube leaf tea and its preparation method.

[0004] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0005] The first aspect of the present invention provides a method for preparing fermented jujube leaf tea, wherein fresh jujube leaves are processed into jujube leaf tea; after sterilizing the jujube leaf tea, Eurotium cristatum is inoculated into the sterilized jujube leaf tea for fermentation culture, and after drying, fermented jujube leaf tea is obtained.

[0006] In this invention, the strain number of *Aspergillus cristatus* is CICC 2099.

[0007] In some embodiments of the present invention, the *Aspergillus cristatus* is a suspension of *Aspergillus cristatus* spores, and the concentration of the *Aspergillus cristatus* spore suspension is 1-2 × 10⁻⁶. 7 CFU / mL.

[0008] In some embodiments of the present invention, the mass ratio of the *Aspergillus cristatus* spore suspension to jujube leaf tea is 4-10%.

[0009] In some embodiments of the present invention, the fermentation temperature is 28-32°C.

[0010] In some embodiments of the present invention, the fermentation time is 7-10 days.

[0011] In some embodiments of the present invention, the drying temperature is 50-65°C.

[0012] In some embodiments of the present invention, the drying time is 60-90 minutes.

[0013] In some embodiments of the present invention, the preparation method of the jujube leaf tea includes: withering, fixing, cooling, kneading, stir-frying, re-kneading, re-stir-frying and drying the fresh jujube leaves to obtain the jujube leaf tea.

[0014] A second aspect of the present invention provides fermented jujube leaf tea prepared by the aforementioned preparation method.

[0015] A third aspect of the invention provides the use of the fermented jujube leaf tea in the preparation of products for neuroprotection.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses *Aspergillus cristatus* and jujube leaves to co-ferment to prepare fermented jujube leaf tea, and explores the optimal fermentation process. It was found that the content of irritating, unpleasant, and volatile compounds in the fermented jujube leaf tea prepared by the preparation method provided in the present invention is reduced, while the content of volatile compounds with floral, woody, herbal, and fruity aromas is increased. Among them, the aroma components of alcohol compounds are the most significant, and its sensory quality is improved.

[0017] In vitro and in vivo antioxidant activity studies revealed that, compared with raw jujube leaf tea, fermented jujube leaf tea showed an enhanced trend in DPPH free radical scavenging rate and ferricyanide reduction capacity. In vivo antioxidant experiments showed that, compared with raw jujube leaf tea, fermented jujube leaf tea significantly improved the survival rate and duration of *C. elegans* under oxidative stress, significantly reduced ROS levels in *C. elegans*, increased the activity of the antioxidant enzyme SOD, and significantly reduced the content of the peroxidation product MDA in *C. elegans*. Furthermore, neuroprotective activity studies using *Aspergillus cristatus*-fermented jujube leaf tea showed that it significantly improved the behavioral abnormalities of the *C. elegans* HA759 disease model and provided good protection for ASH neurons.

[0018] The fermented jujube leaf tea and its preparation method provided by this invention open up new avenues for the comprehensive utilization of jujube leaf resources, provide theoretical guidance for the development of fermented health tea from medicinal plant leaf resources, promote the extension of the medicinal plant waste industry chain, and better serve the development of the Chinese medicinal materials industry. Attached Figure Description

[0019] Figure 1 This is a picture of fresh jujube leaves.

[0020] Figure 2 Image of the prepared jujube leaf tea.

[0021] Figure 3 Image of the prepared *Aspergillus cristatus* fermented jujube leaf tea.

[0022] Figure 4 Fingerprints of volatile substances in jujube leaf tea and jujube leaf tea fermented with *Aspergillus cristatus* are presented, where MT represents jujube leaf tea and IFT represents jujube leaf tea fermented with *Aspergillus cristatus*.

[0023] Figure 5 Bar chart showing the effect of jujube leaf tea fermentation on behavioral abnormalities in the HD model of *C. elegans*. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0025] Eurotium cristatum, strain number CICC 2099, was purchased from the China Industrial Microbial Culture Collection Center.

[0026] The existing processing method for jujube leaf tea is generally as follows: timely leaf picking - moderate fixation - proper rolling - timely roasting - packaging and storage. Problems: Traditional tea-making processes are simple, and jujube leaves cannot fully utilize their medicinal value, nor do they have a competitive market presence due to poor taste and flavor. This invention provides a method for preparing fermented jujube leaf tea, which involves processing fresh jujube leaves into raw jujube leaf tea; sterilizing the raw jujube leaf tea; inoculating the sterilized raw jujube leaf tea with *Eurotium cristatum* for fermentation; and drying to obtain fermented jujube leaf tea.

[0027] Preferably, the content of flavonoids, polyphenols, polysaccharides, amino acids, and spore count in the fermented tea is used as evaluation indicators for comprehensive assessment. The preparation conditions for fermented jujube leaf tea are determined as follows: *Aspergillus cristatus* is used as the spore suspension, and the concentration of the *Aspergillus cristatus* spore suspension is 1-2 × 10⁻⁶.7 The volume-to-mass ratio of *Aspergillus cristatus* spore suspension to jujube leaf tea was 4-10%; the fermentation temperature was 28-32℃, and the fermentation time was 7-10 days; the drying temperature was 50-65℃, and the drying time was 60-90 minutes. Under these conditions, the fermented jujube leaf tea had the highest overall score, and the *Aspergillus cristatus* fermented jujube leaf tea also had increased content of volatile compounds with floral, woody, herbaceous, and fruity aromas, such as perillyl alcohol, methyl salicylate, linalool, and linalool oxide. Among these, the aroma components of alcohol compounds were the most significant, resulting in a better taste and flavor of the fermented jujube leaf tea.

[0028] This invention also conducted in vitro and in vivo antioxidant studies on fermented jujube leaf tea. In vitro antioxidant experiments showed that, compared with raw jujube leaf tea, fermented jujube leaf tea increased the free radical scavenging rate by 5%. In vivo antioxidant experiments showed that *Aspergillus cristatus* fermented jujube leaf tea significantly improved the survival rate, maximum survival time, and median survival time of wild-type N2 nematodes under oxidative stress. *Aspergillus cristatus* fermented jujube leaf tea significantly reduced the ROS level in wild-type N2 nematodes by 56.9%, significantly increased the activity of the antioxidant enzyme SOD in wild-type N2 nematodes by 2.39 times, and significantly reduced the MDA content of peroxidation products in wild-type N2 nematodes by 69.8%. Furthermore, studies on the neuroprotective activity of *Aspergillus cristatus* fermented jujube leaf tea revealed that it significantly improved the behavioral abnormalities of the disease model *C. elegans* HA759 and provided good protection for ASH neurons.

[0029] Example 1: Preparation of Jujube Leaf Tea

[0030] The production process of jujube leaf tea:

[0031] Picking Figure 1 The process shown involves withering, fixing, cooling, kneading, stir-frying over low heat, kneading again, stir-frying again over high heat, and baking to produce raw jujube leaf tea.

[0032] Key points of the process:

[0033] (1) Harvest fresh jujube leaves from March to June.

[0034] (2) Withering: Place the selected jujube leaves in a cool and ventilated place and spread them out to wither for 1.5 hours.

[0035] (3) Blanching: Spread the withered jujube leaves evenly in an iron pot and blanch them with hot steam until the color of the fresh jujube leaves changes from light green to dark green, and remove as much moisture as possible from the leaves.

[0036] (4) Cooling: Let it cool to room temperature (15 min).

[0037] (5) Kneading: Knead the cooled jujube leaves by hand in a clockwise direction into curled strips.

[0038] (6) Stir-frying: After kneading, stir-fry the jujube leaves over low heat until the aroma is just beginning to emerge (5 minutes).

[0039] (7) Repeated kneading: Use your hands to repeatedly knead the jujube leaves in a clockwise direction into a curled strip shape, and make sure that they do not return to their original shape (10 minutes).

[0040] (8) Stir-fry again: Stir-fry the kneaded jujube leaves over high heat for 5 minutes until they are crisp and fragrant. Be careful not to burn them.

[0041] (9) Baking and Drying: Place the stir-fried jujube leaves in an oven and bake at 60℃ for 1 hour to enhance the aroma. Then, spread them out to cool to room temperature before packing and storing. Thus, as... Figure 2 As shown, the jujube leaf tea is now ready.

[0042] The contents of flavonoids, polyphenols, polysaccharides, and amino acids in jujube leaf tea were tested, and the contents of flavonoids, polyphenols, polysaccharides, and amino acids were 11.09%, 15.60%, 10.35%, and 15.99%, respectively.

[0043] Example 2: Preparation and Process Optimization of Ziziphus jujuba Leaf Fermentation by *Aspergillus cristatus*

[0044] 1. Preparation process of Ziziphus jujuba leaf tea fermented with Eurotium cristatum:

[0045] (1) Preparation method of spore suspension of *Eurotium cristatum*: scrape the golden yellow cleistothecia of a single colony from the culture medium with an inoculation loop, place them in sterile water (with glass beads), shake evenly, and adjust the concentration of the spore suspension with a hemocytometer.

[0046] (2) Preparation of Ziziphus jujuba leaf tea fermented with *Aspergillus cristatus*

[0047] An appropriate amount of the raw jujube leaf tea prepared in Example 1 was weighed and placed in a petri dish. After sterilization at 121°C for 20 minutes, a suspension of *Aspergillus cristatus* spores was inoculated under aseptic conditions, with the bacterial concentration controlled at 1×10⁻⁶. 7 The inoculated jujube leaf tea was cultured at 28℃ for one week to obtain *Aspergillus cristatus* fermented jujube leaf tea. The fermented tea was then dried at 60℃ for 75 minutes to obtain the desired product. Figure 3 The finished fermented tea shown.

[0048] 2. Optimization of the fermentation process for jujube leaf tea fermented with *Eurotium cristatum*

[0049] Single-factor and orthogonal experiments were conducted using four key factors: *Aspergillus cristatus* inoculum size, fermentation moisture content, fermentation temperature, and fermentation time. The content of flavonoids, polyphenols, polysaccharides, amino acids, and spore count in the fermented tea were used as evaluation indicators for comprehensive assessment. Based on the principles of the Analytic Hierarchy Process (AHP), using SPSSAU software, the comprehensive score was set as the decision objective, and the weight coefficients of flavonoids, polyphenols, polysaccharides, amino acids, and spore count were set as the scheme layer. A judgment matrix was established. Based on the judgment matrix consistency ratio (CR) < 0.1, the allocation coefficients of the above indicators were determined to be 17.45% for flavonoids, 23.37% for polyphenols, 5.39% for polysaccharides, 5.94% for amino acids, and 47.85% for spore count. The comprehensive score equation was determined as follows: Comprehensive score = [(Amino acid content / maximum amino acid content) × 5.94% + (flavonoid content / maximum flavonoid content) × 17.45% + (polyphenol content / maximum polyphenol content) × 23.37% + (polysaccharide content / maximum polysaccharide content) × 5.39% + (spore count / maximum spore count) × 47.85%] × 100.

[0050] The effects of different inoculum sizes on fermented jujube leaf tea were investigated, and the contents of flavonoids, polyphenols, polysaccharides, amino acids, and spore count in the fermented tea were measured. The results are shown in Table 1.

[0051] Table 1. Effect of inoculum size on fermented jujube leaf tea

[0052]

[0053] The effects of different moisture contents on fermented jujube leaf tea were investigated, and the contents of flavonoids, polyphenols, polysaccharides, amino acids, and spore count in the fermented tea were determined. The results are shown in Table 2.

[0054] Table 2. Effects of moisture content on fermented jujube leaf tea

[0055]

[0056]

[0057] The effects of different fermentation temperatures on fermented jujube leaf tea were investigated, and the contents of flavonoids, polyphenols, polysaccharides, amino acids, and spore count in the fermented tea were determined. The results are shown in Table 3.

[0058] Table 3. Effect of fermentation temperature on fermented jujube leaf tea

[0059]

[0060] The effects of different fermentation days on fermented jujube leaf tea were investigated, and the contents of flavonoids, polyphenols, polysaccharides, amino acids, and spores in the fermented tea were measured. The results are shown in Table 4.

[0061] Table 4. Effect of fermentation days on fermented jujube leaf tea

[0062]

[0063] Based on the results of the single-factor experiments above, a four-factor, three-level orthogonal experiment was conducted. The three levels for inoculum amount were 5%, 6%, and 7%; the three levels for moisture content of jujube leaf tea leaves were 25%, 30%, and 35%; the three levels for fermentation temperature were 28℃, 30℃, and 32℃; and the three levels for fermentation days were 6d, 7d, and 8d. The factor levels of the orthogonal experiment are shown in Table 5.

[0064] Table 5. Factor Level Table for Orthogonal Experiment

[0065] Horizontal factors A. Inoculation rate (%) B. Moisture content (%) C (temperature in °C) D days (d) 1 5 25 28 6 2 6 30 30 7 3 7 35 32 8

[0066] Table 6. Results of the Orthogonal Experiment

[0067]

[0068] Note: "-" indicates that the item is not available.

[0069] Based on the single-factor experiments, orthogonal experiments were conducted. The orthogonal experimental design is shown in Table 5, and the results are shown in Table 6. The optimal fermentation parameters were determined to be: inoculum size 7%, moisture content of the raw jujube leaf tea 30%, and fermentation at 32℃ for 7 days. Under these optimal fermentation conditions, the *Aspergillus cristatus* fermented jujube leaf tea contained 10.63% flavonoids, 14.72% polyphenols, 6.14% polysaccharides, 6.32% amino acids, and 7.6 × 10⁻⁶ spores, respectively. 8 CFU / g dry tea.

[0070] 3. Sensory flavor study of jujube leaf tea fermented with *Eurotium cristatum*

[0071] The volatile flavor compounds of raw jujube leaf tea and jujube leaf tea fermented with *Aspergillus cristatus* were detected using a GC-IMS flavor analyzer. Results... Figure 4 As shown.

[0072] Testing revealed a total of 65 volatile compounds in both raw jujube leaf tea and jujube leaf tea fermented with *Aspergillus cristatus*. These included 18 aldehydes (27.69%), 16 alcohols (24.62%), 12 ketones (18.46%), 10 esters (15.38%), 2 sulfur compounds and heterocyclic compounds (3.08%), 3 acids (4.62%), and 1 alkene and 1 ether (1.54%). The fermented tea showed a decrease in the content of irritating, unpleasant, and pungent volatile compounds such as 1-pentanol, butyraldehyde, and 1-penten-3-one. Meanwhile, the *Aspergillus cristatus*-fermented jujube leaf tea showed an increase in the content of volatile compounds with floral, woody, grassy, ​​and fruity aromas, such as perillol, methyl salicylate, linalool, and linalool oxide, with alcohols being the most prominent aroma component.

[0073] 4. Study on the antioxidant activity of *Aspergillus cristatus* fermented jujube leaf tea

[0074] (1) In vitro antioxidant activity study: The in vitro antioxidant activity was determined by measuring the DPPH free radical scavenging capacity and the ferricyanide reducing capacity. As shown in Table 7, in the DPPH free radical scavenging experiment, there was no significant difference in the free radical scavenging rate between the *Aspergillus cristatus* fermented jujube leaf tea and the raw jujube leaf tea. However, the free radical scavenging rate of the jujube leaf tea after *Aspergillus cristatus* fermentation increased by 5%, indicating that its DPPH free radical scavenging capacity was enhanced. In the ferricyanide reducing capacity experiment, the reducing capacity of the *Aspergillus cristatus* fermented jujube leaf tea and the raw jujube leaf tea was comparable.

[0075] Table 7. In vitro antioxidant activity experiment of *Aspergillus cristatus* fermented jujube leaf tea.

[0076] detection indicators Processing of jujube leaf tea Processing of Ziziphus jujuba leaves with Eurotium cristatum fermentation DPPH free radical scavenging capacity (%) 54.70 59.90 Ferricyanide reducing power (OD(700nm)) 0.374 0.393

[0077] (2) In vivo antioxidant activity experiment: Using the model organism N2 wild-type nematode, N2 wild-type nematodes were fed with 3 mg / mL of jujube leaf tea juice prepared in Example 1 and jujube leaf tea juice fermented with Eurotium cristatum prepared in Example 2. After treatment for 72 h, the survival rate of N2 wild-type nematodes under oxidative stress, as well as the ROS level, antioxidant enzyme SOD activity and peroxidation product MDA content in the nematodes were measured.

[0078] Table 8 shows that: a. Compared with raw jujube leaf tea, *Aspergillus cristatus* fermented jujube leaf tea significantly increased the survival rate, longest survival time, and median survival time of wild-type N2 nematodes under oxidative stress; b. Compared with raw jujube leaf tea, *Aspergillus cristatus* fermented jujube leaf tea significantly reduced the ROS level in wild-type N2 nematodes by 56.9%; c. Compared with raw jujube leaf tea, *Aspergillus cristatus* fermented jujube leaf tea significantly increased the activity of the antioxidant enzyme SOD in wild-type N2 nematodes by 2.39 times; d. Compared with raw jujube leaf tea, *Aspergillus cristatus* fermented jujube leaf tea significantly reduced the MDA content of peroxidation products in wild-type N2 nematodes by 69.8%.

[0079] Table 8. Experimental study on the in vivo antioxidant activity of *Aspergillus cristatus* fermented jujube leaf tea.

[0080]

[0081] 5. Study on the neuroprotective activity of *Aspergillus cristatus* fermented jujube leaf tea

[0082] ASH neurons in *C. elegans* control the worm's sensory responses to toxic chemical stimuli, one of which is avoidance of hypertonic solutions. When ASH neurons are normal, *C. elegans* will avoid hypertonic glycerol solutions. However, when ASH neurons are absent, the worm cannot avoid hypertonic glycerol solutions and will instead crawl across the glycerol solution towards the attractant. Therefore, the neuroprotective activity of drug treatment on *C. elegans* can be determined by detecting the worm's ability to avoid hypertonic glycerol solutions in the HA759 disease model worm.

[0083] Experimental results are as follows Figure 5 As shown in Table 9. Figure 5In the study, EGCG served as the positive control group. The Blank group consisted of N2 *C. elegans* with normal ASH neurons, exhibiting an avoidance rate of approximately 90% to hypertonic glycerol solution. The Control group consisted of HA759 *C. elegans* before drug administration, with an avoidance rate of approximately 47%, meaning that only about 47% of ASH neurons were functionally normal, resulting in poor avoidance of hypertonic glycerol solution. The figure shows that after feeding with raw jujube leaf tea (MT) and *Aspergillus cristatus* fermented jujube leaf tea (IFT), the avoidance rate of the disease model HA759 *C. elegans* to hypertonic glycerol solution improved. This was particularly evident after feeding with *Aspergillus cristatus* fermented jujube leaf tea (IFT), where the avoidance rate significantly increased by approximately 30% compared to the HA759 *C. elegans* before drug administration. Furthermore, the avoidance rate after feeding with *Aspergillus cristatus* fermented jujube leaf tea (IFT) significantly increased by nearly 20% compared to feeding with raw jujube leaf tea (MT). This demonstrates that *Aspergillus cristatus* fermented jujube leaf tea significantly improved the behavioral abnormalities of the disease model *C. elegans* HA759 and played a good protective role against ASH neurons.

[0084] Table 9. Results of the neuroprotective activity experiment of *Aspergillus cristatus* fermented jujube leaf tea.

[0085]

[0086] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0087] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing fermented jujube leaf tea, characterized in that, Fresh jujube leaves are processed into jujube leaf tea. After sterilization, Eurotium cristatum is inoculated into the sterilized jujube leaf tea for fermentation. The fermented product is dried to obtain the fermented jujube leaf tea. The preservation number of Eurotium cristatum is CICC2099.

2. The preparation method according to claim 1, characterized in that, The inoculation involves mixing a suspension of *Eurotium cristatum* spores with the raw jujube leaves, wherein the concentration of the spore suspension is 1-2 × 10⁻⁶. 7 CFU / mL.

3. The preparation method according to claim 2, characterized in that, The spore suspension contains 4-10% jujube leaf tea by mass.

4. The preparation method according to claim 1, characterized in that, The fermentation temperature is 28-32℃.

5. The preparation method according to claim 1, characterized in that, The fermentation time is 7-10 days.

6. The preparation method according to claim 1, characterized in that, The drying temperature is 50-65℃.

7. The preparation method according to claim 1, characterized in that, The drying time is 60-90 minutes.

8. The preparation method according to claim 1, characterized in that, The preparation of the sour jujube leaf tea includes: withering, fixing, cooling, kneading, stir-frying, re-kneading, re-stir-frying and drying the fresh sour jujube leaves to obtain the sour jujube leaf tea.

9. Fermented jujube leaf tea prepared by the preparation method according to any one of claims 1-8.

10. The use of the fermented jujube leaf tea according to claim 9 in the preparation of products for neuroprotection.

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