Pu'er tea containing pandan leaves as well as preparation method and application of Pu'er tea

Abstract

The invention discloses pandan leaf kangpu tea as well as a preparation method and application thereof, and belongs to the technical field of food processing. In order to solve the technical problems that an existing kangpu tea preparation technology is single in raw material, sour and astringent in taste, bad in smell, limited in flavor innovation and the like, the preparation method of the pandan leaf kangpu tea is characterized by comprising the following steps: (1) mixing pandan leaves and water to obtain a mixed solution, then adding white granulated sugar, stirring and sterilizing to obtain a pandan leaf culture medium; and (2) inoculating kombucha pellicle and strain liquid into the pandan leaf culture medium, activating, fermenting, adding ethanol during fermentation to obtain fermentation liquor, and finally centrifuging and collecting supernate to obtain the kombucha leaf culture medium.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food processing, and particularly relates to a spearmint leaf kombucha tea and a preparation method and application thereof. BACKGROUND

[0002] For understanding the technical content of the application: Kombucha tea is a kind of fermented tea beverage naturally fermented by the symbiotic culture of bacteria and yeast and the fermentation substrate of sugar tea water, also known as "black tea fungus" or "sea treasure". Kombucha tea is rich in various probiotics, vitamins, amino acids, organic acids and antioxidant substances, etc. These components make kombucha tea have various effects beneficial to human health such as promoting intestinal health, enhancing immunity, helping digestion, regulating metabolism, etc. Due to its unique flavor, warm taste, benefits to the stomach and natural health properties, kombucha tea is favored by many consumers and becomes one of the most representative sub-categories in fermented beverages.

[0003] At present, in the traditional preparation method of kombucha tea, black tea is used as the main raw material, and fermentation is carried out after filtering the tea water. The product prepared by this method has problems such as sour and astringent, irritating odor and other undesirable flavors, resulting in uneven quality and poor consumer acceptance. Therefore, developing new kombucha tea flavors is an urgent problem to be solved.

[0004] Retrieved relevant patent documents: The disclosure country is China, the disclosure number is CN114376040A, and the disclosure date is April 22, 2022. The document discloses: a Xinhui citrus kombucha tea, and a preparation method thereof, which comprises the following steps: S1, preparation of a symbiotic bacteria culture medium: A1. Preparation of tea liquid; A2. Preparation of Xinhui citrus juice; A3. Compounding; A4. Sterilization; S2, inoculation: B1. First-stage seed liquid culture; B2. Second-stage seed liquid culture; B3. Bacterial strain compounding; B4. Inoculation and fermentation; S3, pasteurization. The Xinhui citrus kombucha tea prepared by the invention can fully improve the utilization rate of Xinhui citrus resources, combine green tea, probiotics and the unique nutritional value of Xinhui citrus, improve the original bad taste of Xinhui citrus pulp, and meet the health needs of modern people for nutritious and healthy beverages.

[0005] Retrieved relevant non-patent documents: The journal titled *Food Industry Technology*, and the article titled "Sensory-Based Construction of Synthetic Microbial Community and Optimization of Fermentation Process for Kombucha Fermentation," Volume 1-18, published on May 22, 2025, discloses the screening and identification of eight yeast strains and four acetic acid bacteria from kombucha strains of different origins. Two strains of *Zygosaccharomyces bisporus* (Y1-1 and Y3-2) and one strain of *Gluconobacter oxydans* (A3) were selected to reconstruct a synthetic microbial community (SMC) for kombucha fermentation on a raw Pu-erh tea substrate. The optimal fermentation conditions for kombucha, based on sensory evaluation, were: inoculum size 1%, sucrose concentration 8%, tea leaf concentration 0.8%, and fermentation time 5 days. Under these conditions, the kombucha achieved a sensory score of 8.38, with acetic acid and ethanol contents of 65.55±3.53 μg / L and 1.71±0.22 μg / L, respectively. The total phenols and total flavonoids reached 1074.86 mg GAE / L and 1076.31 mg RE / L, respectively, and showed significant in vitro antioxidant activity.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: There is still room for improvement in the diversity and uniqueness of kombucha's flavor and functional components. Evidence suggests that the content of total acids, total flavonoids, and total phenols is not high, and the content of antioxidant active ingredients and sensory scores are also low.

[0007] Pandanus amaryllifolius Roxb., commonly known as saffron leaf, is a tropical plant widely distributed in Southeast Asia. Its leaves possess a unique natural aroma and are rich in nutrients and functional substances. Studies have shown that the volatile aroma compound 2-acetyl-1-pyrrolline, abundant in pandanus leaves, gives it its distinctive aroma reminiscent of bamboo leaves. It also contains vitamins, minerals, dietary fiber, polysaccharides, and antioxidants. In traditional cuisine, pandanus leaves are often used as a natural spice and ingredient in pastries, desserts, and beverages, adding unique flavors and offering certain health benefits, such as clearing heat and detoxifying, and promoting metabolism. Therefore, it has high potential for development and utilization.

[0008] Current kombucha preparation techniques suffer from problems such as limited raw materials, a sour and astringent taste, unpleasant aroma, and limited flavor innovation, resulting in poor consumer acceptance. Therefore, developing new flavors of kombucha is an urgent problem to be solved. Summary of the Invention

[0009] The purpose of this invention is to provide: A pandan leaf kombucha, its preparation method and application, and related technologies, to solve the technical problems of existing kombucha preparation technologies, such as single raw materials, sour and astringent taste, unpleasant aroma and limited flavor innovation, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this invention have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and disclosures referenced throughout this invention are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this invention, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definition of the standard chemical term can be found in the reference "Functional Foods: China Agricultural University Press, January 2024, 3rd Edition".

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as static fermentation, sterilization, and microbial activation, shall be used.

[0014] Unless specifically defined, the use of various commercially available products in this invention employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the reagent kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0016] The term "sterilization" as used in this invention refers to the elimination of all microorganisms, including pathogenic and non-pathogenic microorganisms and spores, using physical or chemical methods to achieve a level of sterility. Items that have not been contaminated after sterilization are called sterile items; areas that have not been contaminated are called sterile areas.

[0017] The term "activation" as used in this invention refers to restoring the physiological activity of a dormant bacterial strain through a series of operations. After the strain is revived, its physiological activity may not be fully restored due to the effects of prolonged dormancy and storage conditions, requiring further activation operations.

[0018] The term "fermentation" as used in this invention refers to the process of preparing microbial cells themselves, or their direct or secondary metabolites, by means of the life activities of microorganisms under aerobic or anaerobic conditions.

[0019] In a first aspect, the present invention provides: A method for preparing pandan leaf kombucha includes the following steps: (1) Mix the pandan leaves with water to obtain a mixture, then add white sugar, stir, sterilize, and obtain pandan leaf culture medium; (2) Inoculate kombucha film and inoculum into pandan leaf culture medium, activate and ferment, add ethanol during fermentation to obtain fermentation broth, centrifuge and collect supernatant to obtain the final product.

[0020] The technical features include: the need to crush pandan leaves, the mass-to-volume ratio of pandan leaves to water, the final mass concentration of white sugar in the mixture, the stirring speed and time, the sterilization temperature and time, the inoculation amount of kombucha film and inoculum, and the activation process.

[0021] Among them, the technical feature is that "the pandan leaves mentioned in step (1) need to be crushed to 200-600 mesh".

[0022] The preferred technical feature “the pandan leaves in step (1) need to be pulverized to 200-600 mesh” is 200 mesh, 300 mesh, 400 mesh, 500 mesh, or 600 mesh.

[0023] The technical feature “the pandan leaves in step (1) need to be crushed to 200-600 mesh” is further preferably 200-500 mesh.

[0024] The technical feature “the pandan leaves in step (1) need to be crushed to 200-600 mesh” is further preferably 200 mesh, 400 mesh, or 500 mesh.

[0025] The technical feature “the mass-volume ratio of pandan leaves to water in step (1)” is selected from: 1 g: 20-50 mL.

[0026] The preferred technical feature “the mass-to-volume ratio of Pandanus leaves to water in step (1)” is 1 g: 20 mL, 1 g: 25 mL, 1 g: 30 mL, 1 g: 35 mL, 1 g: 40 mL, 1 g: 45 mL, or 1 g: 50 mL.

[0027] The preferred technical feature is the mass-to-volume ratio of Pandanus leaves to water in step (1): 1 g: 25-35 mL.

[0028] The preferred technical feature “the mass-to-volume ratio of Pandanus leaf to water in step (1)” is 1 g: 25 mL, 1 g: 30 mL, or 1 g: 35 mL.

[0029] The technical feature “final mass concentration of white sugar in the mixture in step (1)” is selected from: 8%-10%.

[0030] The preferred values ​​for the technical feature “final mass concentration of white sugar in the mixture in step (1)” are: 8%, 8.5%, 9%, 9.5%, and 10%.

[0031] The technical feature “final mass concentration of white sugar in the mixture in step (1)” is further preferably 9%-10%.

[0032] The technical feature “final mass concentration of white sugar in the mixture in step (1)” is further preferably 9%, 9.5%, or 10%.

[0033] In this context, the technical feature "white sugar" can be replaced with "glucose".

[0034] The technical feature “stirring speed in step (1)” is selected from 150-300 rpm.

[0035] The preferred speed of stirring in step (1) is 150 rpm, 200 rpm, 250 rpm, or 300 rpm.

[0036] The technical feature “stirring speed in step (1)” is further preferably 200-300 rpm.

[0037] The technical feature “stirring speed in step (1)” is further preferably 200 rpm, 250 rpm, or 300 rpm.

[0038] The technical feature “stirring time in step (1)” is selected from 5-12 min.

[0039] The preferred stirring time in step (1) is 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, or 12 min.

[0040] The technical feature “stirring time in step (1)” is further preferably 6-10 min.

[0041] The technical feature “stirring time in step (1)” is further preferably 6 min, 7 min, 8 min, 9 min, or 10 min.

[0042] The technical feature “the sterilization temperature described in step (1)” is selected from: 120-130℃.

[0043] The preferred temperature for sterilization in step (1) is 120℃, 121℃, 122℃, 123℃, 124℃, 125℃, 126℃, 127℃, 128℃, 129℃, or 130℃.

[0044] The sterilization temperature described in step (1) is further preferably 120-125℃.

[0045] The technical feature “sterilization temperature in step (1)” is further preferably 120℃, 121℃, 122℃, 123℃, 124℃, or 125℃.

[0046] The technical feature “sterilization time in step (1)” is selected from 15-20 min.

[0047] The preferred sterilization time in step (1) is 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min.

[0048] The sterilization time described in step (1) is further preferably 15-18 min.

[0049] The sterilization time described in step (1) is further preferably 15 min, 16 min, 17 min, or 18 min.

[0050] The technical feature “the mass-to-volume ratio of kombucha film to pandan leaf culture medium in step (2)” is selected from: 10-20 g: 1 L.

[0051] The preferred mass-to-volume ratio of kombucha film to pandan leaf culture medium in step (2) is 10 g:1 L, 11 g:1 L, 12 g:1 L, 13 g:1 L, 14 g:1 L, 15 g:1 L, 16 g:1 L, 17 g:1 L, 18 g:1 L, 19 g:1 L, or 20 g:1 L.

[0052] The preferred technical feature is the mass-to-volume ratio of kombucha film to pandan leaf culture medium in step (2): 10 g-15 g: 1 L.

[0053] The preferred technical feature “the mass-to-volume ratio of kombucha film to pandan leaf culture medium in step (2)” is 10 g: 1 L, 11 g: 1 L, 12 g: 1 L, 13 g: 1 L, 14 g: 1 L, or 15 g: 1 L.

[0054] The technical feature “the volume ratio of the kombucha culture liquid to the pandan leaf culture medium in step (2)” is selected from 5%-10%.

[0055] The preferred ratio of the volume of kombucha culture medium to pandan leaf culture medium in step (2) is 5%, 6%, 7%, 8%, 9%, 10%.

[0056] The preferred technical feature is the volume ratio of kombucha culture medium to pandan leaf culture medium in step (2): 5%-8%.

[0057] The preferred ratio of the volume of kombucha culture medium to pandan leaf culture medium in step (2) is 5%, 6%, 7%, or 8%.

[0058] The technical feature “the activation process described in step (2)” includes: static activation culture at 26-30℃ for 13-15 days, and repeated activation 3-4 times.

[0059] The preferred technical feature “the activation process described in step (2)” is: static activation culture at 28-30℃ for 14-15 days, and repeated activation 3-4 times.

[0060] The technical feature “the activation process described in step (2)” is further preferably: static activation culture at 28°C for 14 days, and repeated activation 3 times.

[0061] The technical feature “the fermentation temperature in step (2)” is selected from: 26-30℃.

[0062] The preferred temperature for fermentation in step (2) is 26°C, 27°C, 28°C, 29°C, or 30°C.

[0063] The technical feature “the fermentation temperature in step (2)” is further preferably 28-30℃.

[0064] The technical feature “the fermentation temperature in step (2)” is further preferably 28°C, 29°C, or 30°C.

[0065] The technical feature “fermentation time in step (2)” is selected from: 4-8 days.

[0066] The preferred fermentation time in step (2) is 4 days, 5 days, 6 days, 7 days, or 8 days.

[0067] The fermentation time described in step (2) is further preferably 4-6 days.

[0068] The fermentation time described in step (2) is further preferably 4 days, 5 days, or 6 days.

[0069] Among them, the technical feature “in step (2), ethanol is added on the third day of fermentation, and the volume ratio of ethanol to pandan leaf culture medium” is selected from: 0.05%-0.1%.

[0070] Among them, the technical feature “in step (2), ethanol is added on the third day of fermentation, and the volume ratio of ethanol to pandan leaf culture medium” is preferably 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%.

[0071] Among them, the technical feature “in step (2), ethanol is added on the third day of fermentation, and the volume ratio of ethanol to pandan leaf culture medium” is further preferably 0.05%-0.08%.

[0072] Among them, the technical feature “in step (2), ethanol is added on the third day of fermentation, and the volume ratio of ethanol to pandan leaf culture medium” is further preferred to be: 0.05%, 0.06%, 0.07%, 0.08%.

[0073] The technical feature “centrifugation speed in step (2)” is selected from 8000-12000 rpm.

[0074] The preferred speed of centrifugation in step (2) is 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm, or 12000 rpm.

[0075] Among them, the technical feature “centrifugation speed in step (2)” is further preferably 9000-11000 rpm.

[0076] The preferred speed of centrifugation in step (2) is 9000 rpm, 10000 rpm, or 11000 rpm.

[0077] The technical feature “centrifugation time in step (2)” is selected from 8-12 min.

[0078] The preferred time for centrifugation in step (2) is 8 min, 9 min, 10 min, 11 min, or 12 min.

[0079] The preferred option for the technical feature “centrifugation time in step (2)” is 9-11 min.

[0080] The preferred option for the technical feature “centrifugation time in step (2)” is 9 min, 10 min, or 11 min.

[0081] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred solution is as follows: In step (1), the pandan leaves need to be pulverized to 200-600 mesh; the mass-to-volume ratio of the pandan leaves to water is 1 g: 20-50 mL. This technical solution not only solves the technical problem of "providing a method for preparing pandan leaf kombucha", but also further solves the technical problem of "existing kombucha preparation technology has the problems of single raw materials, sour and astringent taste, unpleasant smell and limited flavor innovation".

[0082] The second preferred option: In step (1), the final mass concentration of the white sugar in the mixture is 8%-10%; the white sugar can be replaced with glucose. This technical solution, based on solving the technical problem of "providing a method for preparing pandan leaf kombucha", further solves the technical problem of "existing kombucha preparation technology has the problems of single raw materials, sour and astringent taste, poor aroma and limited flavor innovation".

[0083] The third preferred option: In step (1), the stirring speed is 150-300 rpm and the stirring time is 5-12 min; the sterilization temperature is 120-130℃ and the sterilization time is 15-20 min. This technical solution, based on solving the technical problem of "providing a method for preparing pandan leaf kombucha", further solves the technical problem of "existing kombucha preparation technology has the problems of single raw materials, sour and astringent taste, unpleasant smell and limited flavor innovation".

[0084] The fourth preferred option: In step (2), the mass-to-volume ratio of the kombucha spawn to the pandan leaf culture medium is 10-20 g: 1 L; the volume ratio of the kombucha inoculum to the pandan leaf culture medium is 5%-10%. This technical solution, based on solving the technical problem of "providing a method for preparing pandan leaf kombucha", further solves the technical problem of "existing kombucha preparation technology has the problems of single raw materials, sour and astringent taste, unpleasant aroma and limited flavor innovation".

[0085] The fifth preferred option: In step (2), the activation process includes: static activation culture at 26-30℃ for 13-15 days, and repeated activation 3-4 times; the fermentation temperature is 26-30℃, and the fermentation time is 4-8 days. This technical solution, based on solving the technical problem of "providing a method for preparing pandan leaf kombucha", further solves the technical problem of "existing kombucha preparation technology has the problems of single raw materials, sour and astringent taste, poor aroma and limited flavor innovation".

[0086] The sixth preferred option: In step (2), ethanol is added on the third day of fermentation, and the volume ratio of ethanol to pandan leaf culture medium is 0.05%-0.1%. This technical solution solves the technical problem of "providing a method for preparing pandan leaf kombucha" and further solves the technical problem of "existing kombucha preparation technology has the problems of single raw materials, sour taste, bad smell and limited flavor innovation".

[0087] The seventh preferred option: The centrifugation speed in step (2) is 8000-12000 rpm and the centrifugation time is 8-12 min. This technical solution solves the technical problem of "providing a method for preparing pandan leaf kombucha" and further solves the technical problem of "existing kombucha preparation technology has the problems of single raw materials, sour taste, bad smell and limited flavor innovation".

[0088] Secondly, the present invention provides: The pandan leaf kombu tea prepared by the above method.

[0089] Thirdly, the present invention provides: Application of pandan leaf kombucha prepared by the above method in the preparation of functional beverages.

[0090] Embodiments 1-3 of this invention at least support the protection scope of claim 1.

[0091] Regarding the claim 1: The technical feature “The preparation method of pandan leaf kombucha includes: (1) mixing pandan leaves with water to obtain a mixture, then adding white sugar, stirring, sterilizing, and obtaining pandan leaf culture medium; (2) inoculating kombucha film and inoculum into the pandan leaf culture medium, activating, fermenting, adding ethanol during fermentation to obtain fermentation liquid, and finally centrifuging and collecting the supernatant” is summarized from the foregoing explanation and / or Examples 1-3 of this invention through common features. Therefore, by reasonable presumption, those skilled in the art can recognize the subordinate concept of the technical feature “the preparation method of pandan leaf kombucha includes: (1) mixing pandan leaves with water to obtain a mixture, then adding white sugar, stirring, sterilizing, and obtaining pandan leaf culture medium; (2) inoculating kombucha film and inoculum into the pandan leaf culture medium, activating, fermenting, adding ethanol during fermentation, obtaining fermentation liquid, and finally centrifuging and collecting the supernatant, thus obtaining”, and the sub-concept of ... The following technical means are basically equivalent to the following: (1) mixing pandan leaves with water to obtain a mixture, then adding white sugar, stirring, sterilizing, and obtaining pandan leaf culture medium; (2) inoculating kombucha film and inoculum into the pandan leaf culture medium, activating, fermenting, adding ethanol during fermentation, obtaining fermentation broth, and finally centrifuging and collecting the supernatant to obtain the pandan leaf culture medium. The following technical means can replace the following within the scope of conventional technical means and common knowledge based on the existing technical level: (1) mixing pandan leaves with water to obtain a mixture, then adding white sugar, stirring, sterilizing, and obtaining pandan leaf culture medium; (2) inoculating kombucha film and inoculum into the pandan leaf culture medium, activating, fermenting, adding ethanol during fermentation, obtaining fermentation broth, and finally centrifuging and collecting the supernatant to obtain the pandan leaf culture medium. All of these technical means should fall within the protection scope of claim 1.

[0092] Embodiments 1-3 of this invention at least support the protection scope of claim 2.

[0093] Regarding claim 2: The technical feature "the pandan leaves need to be pulverized to 200-600 mesh" is summarized from the foregoing explanation and / or the corresponding technical features in Examples 1-3 of this invention, such as mixing 400-mesh pandan leaf powder with purified water at a material-to-liquid ratio of 1:30 (g / mL); mixing 200-mesh pandan leaf powder with purified water at a material-to-liquid ratio of 1:50 (g / mL); and mixing 600-mesh pandan leaf powder with purified water at a material-to-liquid ratio of 1:20 (g / mL), etc., through the common feature "the pandan leaves need to be pulverized". Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "the pandan leaves need to be crushed to 200-600 mesh", the technical means that are basically equivalent to "the pandan leaves need to be crushed to 200-600 mesh", and the technical means that can replace "the pandan leaves need to be crushed to 200-600 mesh" based on the existing technical level and common knowledge should all fall within the protection scope of claim 2. For example, if other technical features remain unchanged, replacing "the pandan leaves need to be crushed to 200-600 mesh" with "the pandan leaves need to be crushed to 300 mesh" or "the pandan leaves need to be crushed to 500 mesh" still falls within the protection scope of claim 2 of this invention.

[0094] The technical feature “the mass-to-volume ratio of pandan leaves to water is 1 g: 20-50 mL” is derived from the aforementioned explanation and / or the corresponding technical features in Examples 1-3 of this invention, such as mixing 400-mesh pandan leaf powder with purified water at a material-to-liquid ratio of 1:30 (g / mL); mixing 200-mesh pandan leaf powder with purified water at a material-to-liquid ratio of 1:50 (g / mL); and mixing 600-mesh pandan leaf powder with purified water at a material-to-liquid ratio of 1:20 (g / mL), etc., through the common feature “mass-to-volume ratio of pandan leaves to water”. Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "the mass-to-volume ratio of the pandan leaves to water is 1 g: 20-50 mL", the essentially equivalent technical means of "the mass-to-volume ratio of the pandan leaves to water is 1 g: 20-50 mL", and the technical means that can replace "the mass-to-volume ratio of the pandan leaves to water is 1 g: 20-50 mL" based on the existing technical level and common knowledge should all fall within the protection scope of claim 2. For example, if other technical features remain unchanged, replacing "the mass-to-volume ratio of the pandan leaves to water is 1 g: 20-50 mL" with a mass-to-volume ratio of pandan leaves to water of 1 g: 40 mL or 1 g: 25 mL, still falls within the protection scope of claim 2 of this invention.

[0095] Embodiments 1-3 of this invention at least support the protection scope of claim 3.

[0096] Regarding claim 3: The technical feature “the final mass concentration of the white sugar in the mixture is 8%-10%” is summarized from the common feature “the final mass concentration of the white sugar in the mixture” by adding white sugar with a final mass concentration of 10%, 9%, or 8% as described in the foregoing explanation and / or the corresponding technical features in Examples 1-3 of this invention. Therefore, those skilled in the art can reasonably presume that the subordinate concepts of the technical feature "the final mass concentration of the white sugar in the mixture is 8%-10%", the essentially equivalent technical means of "the final mass concentration of the white sugar in the mixture is 8%-10%", and the technical means that can replace "the final mass concentration of the white sugar in the mixture is 8%-10%" based on existing technology and common knowledge should all fall within the protection scope of claim 3. For example, if other technical features remain unchanged, replacing "the final mass concentration of the white sugar in the mixture is 8%-10%" with a final mass concentration of 8.5% or 9.5% still falls within the protection scope of claim 3 of this invention.

[0097] The technical feature "the white sugar can be replaced with glucose" is summarized from the foregoing explanation and / or the corresponding technical feature in Embodiment 3 of this invention. Therefore, those skilled in the art can reasonably infer that the technical feature "the white sugar can be replaced with glucose", the subordinate concept of "the white sugar can be replaced with glucose", the essentially equivalent technical means of "the white sugar can be replaced with glucose", and the technical means that can replace "the white sugar can be replaced with glucose" based on the existing technical level and within the scope of conventional technical means and common knowledge should all fall within the protection scope of claim 3.

[0098] Embodiments 1-3 of this invention at least support the protection scope of claim 4.

[0099] Regarding claim 4: The technical feature “the stirring speed is 150-300 rpm and the stirring time is 5-12 min” is summarized from the common feature “stirring speed and stirring time” in the foregoing explanation and / or the corresponding technical features in Examples 1-3 of this invention, such as stirring at 200 rpm for 8 min until completely dissolved; stirring at 300 rpm for 5 min until completely dissolved; and stirring at 150 rpm for 12 min until completely dissolved. Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "the stirring speed is 150-300 rpm and the stirring time is 5-12 min", "the stirring speed is 150-300 rpm and the stirring time is 5-12 min", the essentially equivalent technical means of "the stirring speed is 150-300 rpm and the stirring time is 5-12 min", and the technical means that can replace "the stirring speed is 150-300 rpm and the stirring time is 5-12 min" based on the existing technical level and conventional technical means and common knowledge should all fall within the protection scope of claim 4. For example, if other technical features remain unchanged, replacing "the stirring speed is 150-300 rpm and the stirring time is 5-12 min" with the stirring speed of 250 rpm and the stirring time of 6 min, or the stirring speed of 180 rpm and the stirring time of 10 min, still falls within the protection scope of claim 4 of this invention.

[0100] The technical feature “the sterilization temperature is 120-130℃ and the sterilization time is 15-20 min” is summarized by further high-temperature sterilization at 121℃ for 15 min based on the corresponding technical features in the foregoing explanation and / or Examples 1-3 of this invention. Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "the sterilization temperature is 120-130℃ and the sterilization time is 15-20 min", "the sterilization temperature is 120-130℃ and the sterilization time is 15-20 min", the essentially equivalent technical means of "the sterilization temperature is 120-130℃ and the sterilization time is 15-20 min", and the technical means that can replace "the sterilization temperature is 120-130℃ and the sterilization time is 15-20 min" based on the existing technical level and conventional technical means and common knowledge should all fall within the protection scope of claim 4. For example, if other technical features remain unchanged, replacing "the sterilization temperature is 120-130℃ and the sterilization time is 15-20 min" with the sterilization temperature of 125℃ and the sterilization time of 18 min, or the sterilization temperature of 128℃ and the sterilization time of 16 min, still falls within the protection scope of claim 4 of this invention.

[0101] Embodiments 1-3 of this invention at least support the protection scope of claim 5.

[0102] Regarding claim 5: The technical feature “the mass-to-volume ratio of kombucha film to pandan leaf culture medium in step (2) is 10-20 g:1L” is derived from the aforementioned explanation and / or the corresponding technical features in Examples 1-3 of this invention, such as inoculating 20 g / L kombucha film and 10% (v / v) kombucha inoculum into pandan leaf culture medium; inoculating 15 g / L kombucha film and 7% (v / v) kombucha inoculum into pandan leaf culture medium; and inoculating 10 g / L kombucha film and 5% (v / v) kombucha inoculum into pandan leaf culture medium, etc., through the common feature “mass-to-volume ratio of kombucha film to pandan leaf culture medium”. Therefore, based on reasonable presumption, those skilled in the art can determine that the subordinate concepts of the technical feature "the mass-to-volume ratio of the kombucha film to the pandan leaf culture medium in step (2) is 10-20 g:1 L", the technical means that are basically equivalent to "the mass-to-volume ratio of the kombucha film to the pandan leaf culture medium in step (2) is 10-20 g:1 L", and the technical means that can replace "the mass-to-volume ratio of the kombucha film to the pandan leaf culture medium in step (2) is 10-20 g:1 L" within the scope of conventional technical means and common knowledge based on the existing technical level, should all fall within the protection scope of claim 5. For example, if other technical features remain unchanged, replacing "the mass-to-volume ratio of the kombucha film to the pandan leaf culture medium in step (2) is 10-20 g:1 L" with a mass-to-volume ratio of 18 g:1 L of the kombucha film to the pandan leaf culture medium should all fall within the protection scope of claim 5. L. The mass-to-volume ratio of the kombucha film to the pandan leaf culture medium is 12 g:1 L, etc., which are still within the protection scope of claim 5 of this invention.

[0103] The technical feature “the volume ratio of kombucha culture medium to pandan leaf culture medium in step (2) is 5%-10%” is derived from the aforementioned explanation and / or the corresponding technical features in Examples 1-3 of this invention, such as inoculating 20 g / L kombucha film and 10% (v / v) kombucha culture medium; inoculating 15 g / L kombucha film and 7% (v / v) kombucha culture medium; and inoculating 10 g / L kombucha film and 5% (v / v) kombucha culture medium, etc., through the common feature “volume ratio of kombucha culture medium to pandan leaf culture medium”. Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature “the volume ratio of the kombucha inoculum to the pandan leaf culture medium in step (2) is 5%-10%”, the technical means that are basically equivalent to “the volume ratio of the kombucha inoculum to the pandan leaf culture medium in step (2) is 5%-10%”, and the technical means that can replace “the volume ratio of the kombucha inoculum to the pandan leaf culture medium in step (2) is 5%-10%” based on the existing technical level and conventional technical means and common knowledge, should all fall within the protection scope of claim 5. For example, if “the volume ratio of the kombucha inoculum to the pandan leaf culture medium in step (2) is 5%-10%” is replaced with “the volume ratio of the kombucha inoculum to the pandan leaf culture medium is 8%” or “the volume ratio of the kombucha inoculum to the pandan leaf culture medium is 9%”, while other technical features remain unchanged, it still falls within the protection scope of claim 5 of this invention.

[0104] Embodiments 1-3 of this invention at least support the protection scope of claim 6.

[0105] Regarding claim 6: The technical feature “the activation process includes: static activation culture at 26-30℃ for 13-15 days, repeated activation 3-4 times” is summarized from the common feature “activation process” in the foregoing explanation and / or the corresponding technical features in Examples 1-3 of this invention, such as static fermentation in an incubator at 28℃ for 14 days, repeated activation 3 times; static fermentation in an incubator at 30℃ for 14 days, repeated activation 3 times; static fermentation in an incubator at 26℃ for 15 days, repeated activation 4 times. Therefore, those skilled in the art, through reasonable presumption, can recognize the subordinate concepts of the technical feature "the activation process includes: static activation culture at 26-30℃ for 13-15 days, repeated activation 3-4 times" and "the activation process includes: static activation culture at 26-30℃ for 13-15 days, repeated activation 3-4 times," as well as the essentially equivalent technical means of "the activation process includes: static activation culture at 26-30℃ for 13-15 days, repeated activation 3-4 times," and the alternatives to "the activation process" based on existing technical levels and conventional technical means and common knowledge. The technical means, including "static activation culture at 26-30℃ for 13-15 days, repeated activation 3-4 times", should all fall within the protection scope of claim 6. For example, if other technical features remain unchanged, replacing "the activation process includes: static activation culture at 26-30℃ for 13-15 days, repeated activation 3-4 times" with "the activation process includes: static activation culture at 29℃ for 13 days, repeated activation 4 times" or "the activation process includes: static activation culture at 26℃ for 15 days, repeated activation 3 times" still falls within the protection scope of claim 5 of this invention.

[0106] The technical feature “the fermentation temperature is 26-30℃ and the fermentation time is 4-8 days” is summarized from the corresponding technical features in the foregoing explanation and / or Examples 1-3 of this invention, such as static fermentation in a 26℃ incubator for 4 days; static fermentation in a 28℃ incubator for 6 days; static fermentation in a 30℃ incubator for 8 days, etc., through the common feature “fermentation temperature and fermentation time”. Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "the fermentation temperature is 26-30℃ and the fermentation time is 4-8 days", the essentially equivalent technical means of "the fermentation temperature is 26-30℃ and the fermentation time is 4-8 days", and the technical means that can replace "the fermentation temperature is 26-30℃ and the fermentation time is 4-8 days" based on the existing technical level and conventional technical means and common knowledge should all fall within the protection scope of claim 6. For example, if other technical features remain unchanged, replacing "the fermentation temperature is 26-30℃ and the fermentation time is 4-8 days" with "the fermentation temperature is 26℃ and the fermentation time is 4 days" or "the fermentation temperature is 29℃ and the fermentation time is 8 days" still falls within the protection scope of claim 6 of this invention.

[0107] Embodiments 1-3 of this invention at least support the protection scope of claim 7.

[0108] Regarding claim 7: The technical feature "adding ethanol on the third day of fermentation, wherein the volume ratio of ethanol to pandan leaf culture medium is 0.05%-0.1%" is summarized from the corresponding technical features in the foregoing explanation and / or Examples 1-3 of this invention, such as adding edible ethanol with a final volume concentration of 0.08%, 0.05%, or 0.1% on the third day of fermentation, through the common feature "volume ratio of ethanol to pandan leaf culture medium". Therefore, those skilled in the art can reasonably presume that the technical feature "adding ethanol on the third day of fermentation, wherein the volume ratio of ethanol to pandan leaf culture medium is 0.05%-0.1%" is a subordinate concept, and that "adding ethanol on the third day of fermentation, wherein the volume ratio of ethanol to pandan leaf culture medium is 0.05%-0.1%" is a fundamentally equivalent technical means. Furthermore, based on the existing level of technology and within the bounds of conventional technical means and common knowledge, "adding ethanol on the third day of fermentation" can be replaced by other technical means. The technical means of adding ethanol, wherein the volume ratio of ethanol to pandan leaf culture medium is 0.05%-0.1%, should all fall within the protection scope of claim 7. For example, if other technical features remain unchanged, replacing "adding ethanol on the third day of fermentation, wherein the volume ratio of ethanol to pandan leaf culture medium is 0.05%-0.1%" with adding ethanol on the third day of fermentation, wherein the volume ratio of ethanol to pandan leaf culture medium is 0.06%, or adding ethanol on the third day of fermentation, wherein the volume ratio of ethanol to pandan leaf culture medium is 0.07%, etc., still falls within the protection scope of claim 7 of this invention.

[0109] Embodiments 1-3 of this invention at least support the protection scope of claim 8.

[0110] Regarding claim 8: The technical feature "the centrifugation speed is 8000-12000 rpm and the centrifugation time is 8-12 min" is summarized from the common feature "centrifugation speed and centrifugation time" in the foregoing explanation and / or the corresponding technical features in Examples 1-3 of this invention, such as centrifuging the fermentation broth at 10000 rpm for 10 min; centrifuging the fermentation broth at 8000 rpm for 12 min; and centrifuging the fermentation broth at 12000 rpm for 8 min. Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "the centrifugation speed is 8000-12000 rpm and the centrifugation time is 8-12 min" and "the centrifugation speed is 8000-12000 rpm and the centrifugation time is 8-12 min", the essentially equivalent technical means of "the centrifugation speed is 8000-12000 rpm and the centrifugation time is 8-12 min", and the technical means that can replace "the centrifugation speed is 8000-12000 rpm and the centrifugation time is 8-12 min" based on the existing technical level and conventional technical means and common knowledge should all fall within the protection scope of claim 8. For example, if other technical features remain unchanged, replacing "the centrifugation speed is 8000-12000 rpm and the centrifugation time is 8-12 min" with "the centrifugation speed is 8000 rpm and the centrifugation time is 12 min" or "the centrifugation speed is 12000 rpm and the centrifugation time is 8 min" still falls within the protection scope of claim 8 of this invention.

[0111] Embodiments 1-3 of this invention at least support the protection scope of claim 9.

[0112] Embodiments 1-3 of this invention at least support the protection scope of claim 10.

[0113] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: Compared with existing technologies, the present invention has better technical effects in terms of volatile flavor substances, total sugar content, total acid content, and sensory evaluation.

[0114] According to experimental tests, this invention increases the number of volatile flavor compounds from 25 in the prior art to 37; reduces the total sugar content from 145.77 mg / mL to below 120.97 mg / mL; increases the total acid content from 5.9 mg / mL to above 16.04 mg / mL; and improves the overall sensory evaluation acceptance from 7 points in the prior art to 7.7 points.

[0115] Furthermore, based on the present invention: Based on the comparison of Examples 1-3 and Comparative Examples 1-6, this invention employs a combination of techniques, including "first mixing pandan leaves with water to obtain a mixture, then adding white sugar, stirring, sterilizing, and obtaining a pandan leaf culture medium; then inoculating the pandan leaf culture medium with kombucha film and inoculum, activating, fermenting, adding ethanol during fermentation to obtain a fermentation broth, and finally centrifuging and collecting the supernatant," achieving new technical effects: pandan leaf kombucha has more types of volatile flavor compounds, lower total sugar content, and higher total acid content and sensory score. The combined technical effect is superior to the sum of the effects of each individual technique. Attached Figure Description

[0116] Figure 1 This is a flowchart illustrating the process for preparing pandan leaf kombucha according to the present invention.

[0117] Figure 2 The graph shows the changes in total sugar content of pandan leaf kombucha.

[0118] Figure 3 The graph shows the change in total acid content of pandan leaf kombucha.

[0119] Figure 4 Sensory rating radar chart for pandan leaf kombucha.

[0120] Note: Figures 2-3 In the table, different letters indicate significant differences between groups (p < 0.05), and the same letter indicates no significant difference (p > 0.05). Detailed Implementation

[0121] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0122] The present invention will be further described below with reference to specific embodiments. All instruments, devices, equipment, reagents, and products used in the embodiments of the present invention, unless otherwise specified, were obtained through conventional commercial channels. Specifically, the kombucha mulch and inoculum were purchased from Liaocheng Zhishi Weibao Biotechnology Co., Ltd.

[0123] Example 1 A method for preparing pandan leaf kombucha, the flowchart of which is as follows: Figure 1 As shown, the specific steps are as follows: (1) Mix 400 mesh pandan leaf powder with purified water at a ratio of 1:30 (g / mL), add 10% white sugar, stir at 200 rpm for 8 min until completely dissolved, obtain a mixture, sterilize at 121℃ for 15 min, cool, and obtain pandan leaf culture medium.

[0124] (2) Inoculate 10 g / L kombucha film and 5% (v / v) kombucha inoculum into the pandan leaf culture medium, and activate it by static incubation at 28℃ for 13 days. Repeat the activation 3 times, and then put it into a 26℃ incubator for static fermentation for 4 days. On the 3rd day of fermentation, add 0.05% edible ethanol to obtain fermentation broth. Finally, centrifuge the fermentation broth at 8000 rpm for 12 min and take the supernatant to obtain pandan leaf kombucha.

[0125] Example 2 A method for preparing pandan leaf kombucha, comprising the following steps: (1) Mix 200 mesh pandan leaf powder with purified water at a ratio of 1:50 (g / mL), add 9% white sugar, stir at 300 rpm for 5 min until completely dissolved, obtain a mixture, sterilize at 121℃ for 15 min, cool, and obtain pandan leaf culture medium.

[0126] (2) Inoculate 15 g / L kombucha film and 7% (v / v) kombucha inoculum into pandan leaf culture medium, and activate it by static incubation at 30℃ for 14 days. Repeat the activation 3 times, and then put it into a 28℃ incubator for static fermentation for 6 days. On the 3rd day of fermentation, add edible ethanol with a final volume concentration of 0.08% to obtain fermentation broth. Finally, centrifuge the fermentation broth at 10000 rpm for 10 min and take the supernatant to obtain pandan leaf kombucha.

[0127] Example 3 A method for preparing pandan leaf kombucha, comprising the following steps: (1) Mix 600 mesh pandan leaf powder with purified water at a ratio of 1:20 (g / mL), add glucose with a final mass concentration of 8%, stir at 150 rpm for 12 min until completely dissolved, obtain a mixture, sterilize at 121℃ for 15 min, cool, and obtain pandan leaf culture medium.

[0128] (2) Inoculate 20 g / L kombucha film and 10% (v / v) kombucha inoculum into pandan leaf culture medium, and activate it by static incubation at 26℃ for 15 days. Repeat the activation 4 times, and then put it into a 30℃ incubator for static fermentation for 8 days. On the 3rd day of fermentation, add 0.1% edible ethanol to obtain fermentation broth. Finally, centrifuge the fermentation broth at 12000 rpm for 8 min and take the supernatant to obtain pandan leaf kombucha.

[0129] Comparative Example 1 A method for preparing a pandanus leaf culture medium, comprising the following steps: Pandanus leaf powder of 400 mesh was mixed with purified water at a material-to-liquid ratio of 1:30 (g / mL), and then white sugar with a final mass concentration of 10% was added. The mixture was stirred at 200 rpm for 8 min until completely dissolved to obtain a mixture. The mixture was then sterilized at 121℃ for 15 min and cooled to obtain pandanus leaf culture medium.

[0130] Comparative Example 2 Compared with Example 1, the only difference is the amount of edible ethanol added in step (3), specifically: edible ethanol with a final volume concentration of 0.01% is added on the 3rd day of fermentation.

[0131] Comparative Example 3 Compared with Example 1, the only difference is the amount of white sugar added in step (1), specifically: white sugar with a final mass concentration of 5% is added.

[0132] Comparative Example 4 Compared with Example 1, the only difference is the raw materials used, specifically: pandan leaves are replaced with Keemun black tea.

[0133] Comparative Example 5 Compared with Example 1, the only difference is the amount of edible ethanol added in step (3), specifically: edible ethanol with a final volume concentration of 0.3% is added on the 3rd day of fermentation.

[0134] Comparative Example 6 Compared with Example 1, the only difference is the amount of white sugar added in step (1), specifically: white sugar with a final mass concentration of 15% is added.

[0135] Example 1: Determination of Volatile Flavor Compounds First, headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used to analyze the flavor changes of the products prepared in Examples 1-3 and Comparative Examples 1-6; then, the Nist 20 database was used for substance matching, and the content of volatile flavor compounds was calculated using the internal standard method, with the following formula:

[0136] In the formula: Wi The mass concentration of the volatile component to be tested, in μg / g; Cs The concentration of the internal standard, in μg / mL; Vs The volume of the internal standard added, in mL; m The weight of the product taken, in grams; APi Peak area of ​​volatile components; APs Peak area of ​​internal standard.

[0137] Verification of technical effectiveness and / or analysis of solutions to technical problems: The test results are shown in Table 1. Comparative Example 1 detected 24 volatile aroma compounds, with ketones and alkenes accounting for the largest proportion, exhibiting characteristic aromas of freshness, woodiness, and floral and fruitiness. Comparative Example 2 detected 22 volatile aroma compounds, with ketones accounting for the largest proportion, exhibiting characteristic aromas of slight coolness, light citrus, and a small amount of woodiness. Due to the low addition of ethanol, the flavor is mainly cool, lacking acidity and overall blandness, and lacking the fermentation characteristics of pandan leaf kombucha. Comparative Example 3 detected 22 volatile aroma compounds, mainly ketones, with a slight enhancement of faint rose and woody aromas, but lacking a rich floral and fruity aroma, resulting in a relatively simple flavor profile. Comparative Example 4 detected 25 volatile aroma compounds, mainly alcohols and ketones. Phenylethanol is the main aroma contributor, bringing a faint floral scent; the content of acidic substances is low, with no obvious sour aroma; the overall flavor is mainly a light tea aroma and a faint floral scent, lacking the unique herbal aroma of pandan leaves and the complex flavor layers formed by kombucha fermentation; Comparative Example 5 detected 20 volatile aroma compounds, lacking characteristic aroma substances, with an overall light woody scent and a slight cooling sensation, and a bland and simple flavor; Comparative Example 6 detected 23 volatile aroma compounds, with an overall high sweetness and rich floral and fruity aroma, although the flavor layers are better than most comparative examples, but it lacks characteristic flavor substances.

[0138] A total of 37 volatile aroma compounds were detected in Examples 1-3. Compared with Comparative Examples 1-6, the content of acidic substances in Examples 1-3 was significantly increased, indicating that by optimizing the raw materials, the amount of white sugar added, and the amount of ethanol added, flavor substances continuously accumulated during the fermentation process, bringing a unique complex flavor to the pandan leaf kombucha. In Examples 1-3, with the increase of fermentation time, temperature, inoculum amount, and amount of ethanol added, the content of damastones continuously increased, bringing a unique floral and fruity sweet aroma to the pandan leaf kombucha. In Example 3, 2-acetylpyrrole was detected, which brought a rice aroma of bamboo leaves to the pandan leaf kombucha.

[0139] Table 1. Volatile compound content (μg / g)

[0140] Note: "-" indicates a content of 0.

[0141] Example 2: Determination of total sugar content Take 1 mL of the products prepared in Examples 1-3 and Comparative Examples 1-6 respectively, dilute them 1000 times with deionized water, and then add 1 mL of the diluted product, 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid (98% concentration) to the stoppered test tubes respectively. Shake well and incubate in a water bath at 40°C for 10 min, then in a cold water bath for 5 min. Finally, measure the absorbance at 490 nm using an ELISA reader. Perform the measurements in triplicate and establish a standard curve using 0.1 mg / mL glucose solution.

[0142] Verification of technical effectiveness and / or analysis of solutions to technical problems: Test results as follows Figure 2 As shown, Comparative Example 1 had the highest total sugar content at 157.14 mg / mL, Example 1 had a total sugar content of approximately 120.97 mg / mL, and Examples 2 and 3 had similar total sugar contents.

[0143] This is because during fermentation, yeast and bacteria convert sugars into organic acids, carbon dioxide, and other metabolic products, leading to a decrease in total sugar content. Increased fermentation time, temperature, inoculum size, and ethanol addition result in more significant sugar consumption and a lower total sugar content. In the early stages of fermentation, sugar content is abundant, and microbial metabolism is active, leading to rapid sugar consumption in pandan leaf kombucha. However, by day 6, most of the sugar may have been consumed, leaving limited sugar, and the microbial metabolic rate decreases significantly, resulting in smaller subsequent changes in sugar content. In the later stages of fermentation, the dominant microbial community may shift from yeast to acetic acid bacteria, leading to a decrease in the sugar metabolism rate.

[0144] Furthermore, the total sugar content of Comparative Examples 2, 3, 5, and 6 was significantly higher than that of Examples 1-3 (p<0.05), indicating that only specific amounts of ethanol or white sugar added can reduce the total sugar content.

[0145] Example 3: Determination of total acid content Take 1 mL of the products prepared in Examples 1-3 and Comparative Examples 1-6, respectively, and dilute them 100 times with deionized water. Using the acid-base titration method, take 25 mL of the diluted product and place it in an Erlenmeyer flask. Add 3 drops of phenolphthalein indicator solution and titrate with 0.1 mol / L NaOH standard titration solution until a faint pink color persists for 30 seconds. Record the volume of NaOH standard titration solution consumed. Perform three parallel determinations. Use carbon dioxide-free water as a blank control. The formula for calculating the total acid content is shown below:

[0146] In the formula: X Total acid content, g / L; c The concentration of the NaOH standard titration solution, in mol / L; V 1. The volume of NaOH standard titration solution consumed during titration, in mL; V 2. The volume of NaOH standard titration solution consumed in the blank test, in mL; k The conversion factor for acids, in acetic acid form, is 0.060. F Product dilution factor; m , the volume of the product drawn, mL; 1000, conversion factor.

[0147] Verification of technical effectiveness and / or analysis of solutions to technical problems: Test results as follows Figure 3 As shown, the total acid content of Comparative Examples 1-6 was lower than that of Examples 1-3, with Comparative Example 1 having the lowest total acid content, almost close to 0; and Example 3 having the highest total acid content, reaching approximately 16.04 mg / mL. Examples 1-3 demonstrate that the total acid content increased significantly with increasing fermentation time, temperature, inoculum size, and ethanol addition. This is because during the fermentation of pandan leaf kombucha, yeast consumes sugars to produce ethanol, which is then further oxidized by acetic acid bacteria to produce acidic substances such as acetic acid. The continuous production of acidic metabolites by microorganisms in a more suitable environment leads to the continuous accumulation and significant increase in total acid content, a key step in the formation of kombucha flavor.

[0148] Example 4: Sensory evaluation determination The products prepared in Examples 1-3 and Comparative Examples 1-6 were evaluated using a nine-point scale. The sensory evaluation was conducted by a panel of 10 untrained members (5 males and 5 females, aged 20-25 years) who primarily assessed the color, aroma, taste, and overall acceptability of the kombucha. The rating scale ranged from 1 to 9: (1) extremely dislike; (2) very dislike; (3) somewhat dislike; (4) slightly dislike; (5) neither like nor dislike; (6) slightly like; (7) moderately like; (8) very like; (9) extremely like.

[0149] Verification of technical effectiveness and / or analysis of solutions to technical problems: Sensory evaluation results such as Figure 4 As shown, Examples 1-3 scored similarly and relatively high, ranging from 7.5 to 7.7, while Comparative Example 1 scored the lowest. This indicates that the kombucha from Examples 1-3 performed better than all the comparative examples in terms of color, which may be due to the optimization of conditions such as fermentation time, temperature, inoculum amount, and ethanol addition, making the color more in line with consumer preferences.

[0150] In terms of aroma, Comparative Example 5 scored the lowest; Comparative Example 1 scored slightly lower, relying solely on the volatile substances of the pandan leaves themselves, resulting in a simple aroma; Comparative Examples 2-4 and 6 showed moderate aroma performance; Examples 1-3 scored higher in aroma, indicating that more complex volatile substances were produced during the fermentation process of Examples 1-3, resulting in a richer aroma profile (such as floral and fruity aromas and layered acidic aromas), which significantly enhanced the aroma fullness of the pandan leaf kombucha.

[0151] In terms of taste, Example 2 scored the highest, demonstrating the best effect in optimizing the taste. The acidity and residual sweetness after fermentation were balanced, resulting in a rich flavor. Example 1 was second best, with some sugar being consumed during fermentation, leading to initial acidity and a moderate taste. Example 3 scored slightly lower than Examples 1 and 2, but still higher than all comparative examples. Comparative example 1 scored the lowest because no fermentation products were produced, retaining only the original bitter taste of pandan leaves. Comparative example 2 scored the lowest in aroma, with slow sugar consumption during fermentation and an inconspicuous acidity. Although the taste scores of Comparative examples 3-6 were better than those of Comparative examples 1 and 2, they were still inferior to the Example 1 group. Furthermore, the aroma scores of Examples 1-2 were higher than those of Comparative example 4, with a richer flavor profile. In addition to the floral and fruity aromas and acidity brought by fermentation, they also retained the unique herbal aroma of pandan leaves, effectively avoiding the problem of "single flavor and lack of memorable notes" in kombu tea made from Qimen black tea, and better meeting consumers' expectations for the flavor of natural tea drinks.

[0152] In terms of overall acceptability, Example 2 scored the highest, with a balanced sweet and sour taste that retained the original aroma of pandan leaves while also incorporating the floral, fruity, and sour notes of fermentation, resulting in the highest acceptability. Example 3 scored lower because prolonged fermentation led to excessive total acidity, resulting in a sour and astringent taste that masked other flavors, leading to lower palatability. Examples 1-3 all scored higher than Comparative Example 1, and Examples 1-3 also scored higher than Comparative Examples 2-6, indicating that kombucha fermentation made the original pandan leaf flavor more diverse and the aroma richer, significantly improving the overall palatability of the tea. Examples 1-3 all scored higher than Comparative Example 4, indicating that kombucha made with pandan leaves performed better than kombucha made with Qimen black tea in terms of aroma intensity and taste harmony, better meeting consumers' expectations for kombucha flavor.

[0153] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing pandan leaf kombucha, characterized in that, Includes the following steps: (1) Mix the pandan leaves with water to obtain a mixture, then add white sugar, stir, sterilize, and obtain pandan leaf culture medium; (2) Inoculate kombucha film and inoculum into pandan leaf culture medium, activate and ferment, add ethanol during fermentation to obtain fermentation broth, centrifuge and collect supernatant to obtain the final product.

2. The preparation method according to claim 1, characterized in that, In step (1), the pandan leaves need to be pulverized to 200-600 mesh; the mass-to-volume ratio of the pandan leaves to water is 1 g: 20-50 mL.

3. The preparation method according to claim 1, characterized in that, In step (1), the final mass concentration of the white sugar in the mixture is 8%-10%; the white sugar can be replaced with glucose.

4. The preparation method according to claim 1, characterized in that, In step (1), the stirring speed is 150-300 rpm and the stirring time is 5-12 min; the sterilization temperature is 120-130℃ and the sterilization time is 15-20 min.

5. The preparation method according to claim 1, characterized in that, In step (2), the mass-to-volume ratio of the kombucha spawn to the pandan leaf culture medium is 10-20 g: 1 L; the volume ratio of the kombucha inoculum to the pandan leaf culture medium is 5%-10%.

6. The preparation method according to claim 1, characterized in that, In step (2), the activation process includes: static activation culture at 26-30℃ for 13-15 days, and repeated activation 3-4 times; the fermentation temperature is 26-30℃ and the fermentation time is 4-8 days.

7. The preparation method according to claim 6, characterized in that, In step (2), ethanol is added on the third day of fermentation, and the volume ratio of ethanol to pandan leaf culture medium is 0.05%-0.1%.

8. The preparation method according to claim 1, characterized in that, The centrifugation speed in step (2) is 8000-12000 rpm, and the centrifugation time is 8-12 min.

9. A pandan leaf kombucha prepared by the preparation method according to any one of claims 1-8.

10. The application of pandan leaf kombucha prepared by the preparation method according to any one of claims 1-8 in the preparation of functional beverages.

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