A momordica grosvenori fermentation liquor, a preparation method, composition and application thereof

Abstract

The application relates to a momordica grosvenori fermentation liquor and a preparation method, composition and application thereof, and belongs to the momordica grosvenori processing technical field. A preparation method of a momordica grosvenori fermentation liquor comprises the following steps: adding a composite enzyme into momordica grosvenori paste for ultrasonic enzymolysis to obtain momordica grosvenori enzymolysis liquor, inoculating the momordica grosvenori enzymolysis liquor with a composite bacteria for fermentation, and sterilizing, filtering and evaporating the fermented material; the composite enzyme comprises pectinase and cellulase, and the mass ratio of the pectinase to the cellulase is 1:1.2; the composite bacteria comprises aroma-producing yeast and saccharomyces cerevisiae, and the inoculation amount ratio of the aroma-producing yeast to the saccharomyces cerevisiae is 0.5:1.5:0.5-5. The momordica grosvenori fermentation liquor with a high momordica grosvenori saponin V content can be prepared, and the preparation method is simple and efficient.

Description

Technical Field

[0001] This application relates to the field of monk fruit processing technology, and particularly to a monk fruit fermentation broth, its preparation method, composition and application. Background Technology

[0002] Monk fruit is a precious plant used in both medicine and food, rich in medicinal and nutritional components, including abundant vitamin C, glycosides, fructose, glucose, protein, and lipids. Monk fruit saponins are the main triterpenoid compounds in monk fruit, unique chemical components found only in the fruit. Among them, mogroside V has the highest content, with a sweetness equivalent to about 300 times that of an equal amount of sucrose, making it an ideal natural sweetener. Current research on monk fruit mainly focuses on the purification process of mogroside V. Current extraction technology for mogroside V involves traditional processes such as pressing, plate and frame extraction, alcohol extraction, and steam concentration from fresh monk fruit. The yield and purity are generally low, and the extraction cost is high. The complex production process restricts the production of mogroside V. Although the above methods can still obtain mogroside V powder that meets market requirements, they increase production and operating costs, process risks, and environmental pollution. Monk fruit resources are not being fully utilized.

[0003] Monk fruit is primarily used in the food and health product sector, with less involvement in the cosmetics field. Research on monk fruit mainly focuses on fermentation broth obtained through physical or chemical methods. Currently, hot water extraction is the primary method for obtaining this fermentation broth, which suffers from low extraction rates of active ingredients and the incomplete utilization of monk fruit.

[0004] Chinese patent CN113272414B discloses "a fermented monk fruit flavored beverage and its preparation method," which involves adsorption chromatography and nanofiltration of monk fruit water extract to obtain a glycoside-free concentrate, followed by fermentation. This reduces the hydrolysis of glycosidic bonds and sugar chains in monk fruit glycosides, removing the main active ingredient, saponins, which would otherwise form bitter secondary glycosides or aglycones. However, the adsorption chromatography and nanofiltration processes are cumbersome and time-consuming. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present application aims to provide a monk fruit fermentation broth, its preparation method, composition, and applications. This method can prepare a monk fruit fermentation broth with a high content of monk fruit saponin V, and the preparation method is simple and efficient.

[0006] In a first aspect, embodiments of this application provide a method for preparing monk fruit fermentation broth. The preparation method includes: adding a compound enzyme to monk fruit pulp and performing ultrasonic enzymatic hydrolysis to obtain monk fruit enzymatic hydrolysate; then inoculating the monk fruit enzymatic hydrolysate with compound bacteria for fermentation; and sterilizing, filtering, and evaporating the fermented material obtained after fermentation. The compound enzyme includes pectinase and cellulase, with a mass ratio of pectinase to cellulase of 1:12. The compound bacteria include aroma-producing yeast and brewer's yeast, with an inoculation ratio of aroma-producing yeast to brewer's yeast of 0.5-1.5:0.5-5.

[0007] A combination of pectinase and cellulase in a specific ratio synergistically hydrolyzes monk fruit pulp, breaking down the cell walls and releasing mogroside V, thus increasing its content in the hydrolysate. Simultaneously, a combination of aroma-producing yeast and brewer's yeast in a specific ratio promotes the effective conversion of active substances in the hydrolysate into easily absorbed small molecules, enhancing product efficacy. Furthermore, the brewer's yeast effectively utilizes the nutrients in the hydrolysate and, in conjunction with the aroma-producing yeast, accumulates highly active substances such as organic acids, polyols, and small-molecule peptides. This process yields a monk fruit fermentation broth with a high mogroside V content and significant soothing, anti-inflammatory, antioxidant, and anti-aging effects.

[0008] In some embodiments of this application, the inoculation amount of the compound bacteria is 0.5%-5% of the mass of the monk fruit enzymatic hydrolysate.

[0009] In some embodiments of this application, the mass of the compound enzyme is 0.1% to 3% of the mass of the monk fruit pulp.

[0010] In some embodiments of this application, the fermentation conditions include fermentation at 2545°C for 1280 hours.

[0011] In some embodiments of this application, enzymatic hydrolysis includes ultrasonic enzymatic hydrolysis.

[0012] In some embodiments of this application, the conditions for ultrasonic enzymatic hydrolysis include: 40-70℃, pH 3.5-6.5, ultrasonic power 400-1000W, and ultrasonic enzymatic hydrolysis for 0.5-5 hours. Performing ultrasonication for an appropriate time within a suitable ultrasonic power range increases the interaction area between the compound enzyme and the cellulose and pectin in the monk fruit, accelerating the hydrolysis rate and promoting the release of mogroside V from the cell wall of the monk fruit; it also ensures the activity of the compound enzyme, guaranteeing the efficient execution of the enzymatic hydrolysis reaction.

[0013] In some embodiments of this application, the fermentation process also includes activating the complex microorganisms.

[0014] In some embodiments of this application, the process of filtering and sterilizing the monk fruit enzymatic hydrolysate before fermentation is further included.

[0015] Secondly, embodiments of this application provide a monk fruit fermentation broth prepared by the method provided in the first aspect. The monk fruit fermentation broth has a high content of monk fruit saponin V, exhibiting significant soothing, anti-inflammatory, antioxidant, and anti-aging effects.

[0016] Thirdly, embodiments of this application provide a composition of monk fruit fermentation broth, the composition comprising the following components by mass percentage: 0.1%-10% monk fruit fermentation broth provided in the second aspect, 0.5%-5% butylene glycol, 1%-3% hexanediol, 0.05%-0.1% ethylhexylglycerin, and the remainder being water.

[0017] The fermented broth of monk fruit has a high content of monk fruit saponin V. When combined with butylene glycol, hexanediol, and ethylhexylglycerin, it also has significant soothing, anti-inflammatory, antioxidant, and anti-aging effects.

[0018] Fourthly, embodiments of this application provide the application of the monk fruit ferment broth as provided in the second aspect or the composition provided in the third aspect in the preparation of skin care products.

[0019] When monk fruit fermentation broth or compositions with high levels of monk fruit saponin V are used in the preparation of skin care products, skin care products with significant soothing, anti-inflammatory, antioxidant, and anti-aging effects can be produced. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 A flowchart illustrating the preparation method of monk fruit fermentation broth provided in this application embodiment.

[0022] Figure 2 The image shows the intracellular ROS expression results provided in Experiment Example 5 of this application. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] This application provides a method for preparing monk fruit fermentation broth. Please refer to [link to relevant documentation]. Figure 1This includes the following steps:

[0025] (1) Wash fresh monk fruit and add 10 times the amount of sterile water to make monk fruit pulp.

[0026] (2) Add 0.1% to 3% of the mass of pectinase and cellulase compound enzyme to the monk fruit pulp, with a mass ratio of pectinase to cellulase of 1:12. Sonicate at 40-70℃, pH 3.5-6.5, and ultrasonic power of 400-1000W for 0.5-5 hours to obtain monk fruit enzymatic hydrolysate.

[0027] (3) After filtering the monk fruit enzymatic hydrolysate with gauze, sterilize it at 110℃ for 15 min; place it in a fermenter, inoculate it with 0.5%-5% of activated aroma-producing yeast and brewer's yeast compound of monk fruit enzymatic hydrolysate, with an inoculation ratio of aroma-producing yeast to brewer's yeast of 0.5-1.5:0.5-5, and ferment it in a sealed container at 25-45℃ for 12-80 h to obtain fermented material;

[0028] (4) Sterilize the fermented material obtained after fermentation at 110℃ for 15 minutes, then filter it using a hydraulic plate and frame filter press to separate the filter liquid. Evaporate the filter liquid using a rotary evaporator until the alcohol content of the liquid is measured to be zero, thus obtaining the Luo Han Guo fermented liquid.

[0029] A certain proportion of cellulase and pectinase can be combined to decompose plant cell walls and enzymatically hydrolyze monk fruit pulp. This process decomposes the monk fruit cell walls in the pulp, allowing mogroside V to be released from the cell walls. Simultaneously, the combined effect of ultrasonic enzymatic hydrolysis can more quickly and thoroughly decompose the monk fruit cell walls, further promoting the release of mogroside V from the cell walls and increasing the content of mogroside V in the monk fruit enzymatic hydrolysate. The synergistic effect of cellulase and pectinase can also reduce the pectin content in the monk fruit enzymatic hydrolysate, thereby reducing the amount of methanol produced by pectin during subsequent fermentation. Simultaneously, the synergistic fermentation of a certain proportion of aroma-producing yeast and brewer's yeast not only promotes the effective conversion of active substances in the monk fruit hydrolysate into easily absorbed small molecules, enhancing product efficacy, but also allows the brewer's yeast to effectively utilize the nutrients in the monk fruit hydrolysate and, together with the aroma-producing yeast, accumulate highly active substances such as organic acids, polyols, and small-molecule peptides. This results in a monk fruit fermentation liquid with a high content of monk fruit saponin V, exhibiting significant soothing, anti-inflammatory, antioxidant, and anti-aging effects. Furthermore, the aroma-producing yeast can ferment aromatic substances using ethanol and acetic acid as raw materials, thereby reducing the alcohol content of the monk fruit fermentation liquid, increasing its aroma, and reducing its sour and astringent taste after fermentation. This makes the monk fruit fermentation liquid suitable for use in the preparation of skincare products.

[0030] Optionally, a complex enzyme of pectinase and cellulase, comprising 1% to 3% of the mass of the monk fruit pulp, is added to the monk fruit pulp. As an example, the complex enzyme of pectinase and cellulase added to the monk fruit pulp may be, but is not limited to, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, or 3% of the mass of the monk fruit pulp.

[0031] As an example, the mass ratio of pectinase to cellulase may be, but is not limited to, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.

[0032] Optionally, the monk fruit enzymatic hydrolysate is inoculated with a complex of activated aroma-producing yeast and Saccharomyces cerevisiae, at 2%-5% of the mass of the monk fruit enzymatic hydrolysate. As an example, the inoculation of the monk fruit enzymatic hydrolysate with the complex of aroma-producing yeast and Saccharomyces cerevisiae can be, but is not limited to, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.5%, 4%, 4.5%, or 5% of the mass of the monk fruit enzymatic hydrolysate.

[0033] As an example, the inoculation ratio of aroma-producing yeast and brewer's yeast can be, but is not limited to, 0.5:0.5, 0.5:1, 0.5:1.5, 0.5:2, 0.5:2.5, 0.5:3, 0.5:3.5, 0.5:4, 0.5:4.5, 0.5:5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1.5:1.5, 1.5:2, 1.5:2.5, 1.5:3, 1.5:3.5, 1.5:4, 1.5:4.5, and 1.5:5.

[0034] This application also provides a monk fruit fermentation broth prepared by the above-described method. The monk fruit fermentation broth has a high content of monk fruit saponin V, exhibiting significant soothing, anti-inflammatory, antioxidant, and anti-aging effects.

[0035] This application also provides a composition of monk fruit fermentation liquid, comprising the following components by mass percentage: 0.1%-10% of the above-mentioned monk fruit fermentation liquid, 0.5%-5% of butylene glycol, 1%-3% of hexanediol, 0.05%-0.1% of ethylhexylglycerin, and the remainder being water. The monk fruit fermentation liquid has a high content of monk fruit saponin V, and when combined with butylene glycol, hexanediol, and ethylhexylglycerin, it also exhibits significant soothing, anti-inflammatory, antioxidant, and anti-aging effects.

[0036] As an example, the mass percentage of monk fruit fermentation broth in the composition may be, but is not limited to, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, and 10%.

[0037] This application also provides an application of the above-mentioned monk fruit fermentation liquid or composition in the preparation of skin care products. When monk fruit fermentation liquid or composition with a high content of monk fruit saponin V is used in the preparation of skin care products, skin care products with significant soothing, anti-inflammatory, antioxidant, and anti-aging effects can be obtained.

[0038] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0039] Example 1

[0040] This embodiment provides a method for preparing monk fruit fermentation broth, including the following steps:

[0041] (1) Wash fresh monk fruit and add 10 times the amount of sterile water to make monk fruit pulp.

[0042] (2) Add 1% of the mass of pectinase and cellulase compound enzyme to the monk fruit pulp, with a mass ratio of pectinase to cellulase of 1:1. Sonicate at 50℃, pH 4.0, and ultrasonic power of 800W for 1.5h to obtain monk fruit enzymatic hydrolysate.

[0043] (3) After filtering the Luo Han Guo enzymatic hydrolysate with gauze, sterilize it at 110℃ for 15 min; place it in a fermentation tank, inoculate it with 2% of the mass of Luo Han Guo enzymatic hydrolysate with a compound of activated aroma-producing yeast and brewer's yeast, with an inoculation ratio of 1:2, and ferment it in a sealed container at 35℃ for 48 h to obtain fermented material.

[0044] (4) Sterilize the fermented material obtained after fermentation at 110℃ for 15 minutes, then filter it using a hydraulic plate and frame filter press to separate the filter liquid. Evaporate the filter liquid using a rotary evaporator until the alcohol content of the liquid is measured to be zero, thus obtaining the Luo Han Guo fermented liquid.

[0045] The Luo Han Guo fermented broth prepared by the method provided in this embodiment is denoted as A.

[0046] Example 2

[0047] This embodiment provides a method for preparing monk fruit fermentation broth, including the following steps:

[0048] (1) Wash fresh monk fruit and add 10 times the amount of sterile water to make monk fruit pulp.

[0049] (2) Add 1% of the mass of pectinase and cellulase compound enzyme to the monk fruit pulp, with a mass ratio of pectinase to cellulase of 1:1. Sonicate at 50℃, pH 4.0, and ultrasonic power of 800W for 1.5h to obtain monk fruit enzymatic hydrolysate.

[0050] (3) After filtering the Luo Han Guo enzymatic hydrolysate with gauze, sterilize it at 110℃ for 15 min; place it in a fermentation tank, inoculate it with 0.5% of the mass of the Luo Han Guo enzymatic hydrolysate with a compound of activated aroma-producing yeast and brewer's yeast, with an inoculation ratio of 1:5, and ferment it in a sealed container at 25℃ for 80 h to obtain fermented material.

[0051] (4) Sterilize the fermented material obtained after fermentation at 110℃ for 15 minutes, then filter it using a hydraulic plate and frame filter press to separate the filter liquid. Evaporate the filter liquid using a rotary evaporator until the alcohol content of the liquid is measured to be zero, thus obtaining the Luo Han Guo fermented liquid.

[0052] The Luo Han Guo fermented broth prepared by the method provided in this embodiment is denoted as B.

[0053] Example 3

[0054] This embodiment provides a method for preparing monk fruit fermentation broth, including the following steps:

[0055] (1) Wash fresh monk fruit and add 10 times the amount of sterile water to make monk fruit pulp.

[0056] (2) Add 0.1% of the mass of pectinase and cellulase compound enzyme to the monk fruit pulp, with a mass ratio of pectinase to cellulase of 1:2. Sonicate at 40℃, pH 3.5, and ultrasonic power of 400W for 0.5h to obtain monk fruit enzymatic hydrolysate.

[0057] (3) After filtering the Luo Han Guo enzymatic hydrolysate with gauze, sterilize it at 110℃ for 15 min; place it in a fermentation tank, inoculate it with 2% of the mass of Luo Han Guo enzymatic hydrolysate with a compound of activated aroma-producing yeast and brewer's yeast, with an inoculation ratio of 1:2, and ferment it in a sealed container at 35℃ for 48 h to obtain fermented material.

[0058] (4) Sterilize the fermented material obtained after fermentation at 110℃ for 15 minutes, then filter it using a hydraulic plate and frame filter press to separate the filter liquid. Evaporate the filter liquid using a rotary evaporator until the alcohol content of the liquid is measured to be zero, thus obtaining the Luo Han Guo fermented liquid.

[0059] The Luo Han Guo fermentation broth prepared by the method provided in this embodiment is denoted as C.

[0060] Example 4

[0061] This embodiment provides a method for preparing monk fruit fermentation broth, including the following steps:

[0062] (1) Wash fresh monk fruit and add 10 times the amount of sterile water to make monk fruit pulp.

[0063] (2) Add 0.1% of the mass of pectinase and cellulase compound enzyme to the monk fruit pulp, with a mass ratio of pectinase to cellulase of 1:2. Sonicate at 40℃, pH 3.5, and ultrasonic power of 400W for 0.5h to obtain monk fruit enzymatic hydrolysate.

[0064] (3) After filtering the Luo Han Guo enzymatic hydrolysate with gauze, sterilize it at 110℃ for 15 min; place it in a fermentation tank, inoculate it with 0.5% of the mass of the Luo Han Guo enzymatic hydrolysate with a compound of activated aroma-producing yeast and brewer's yeast, with an inoculation ratio of 1:5, and ferment it in a sealed container at 25℃ for 80 h to obtain fermented material.

[0065] (4) Sterilize the fermented material obtained after fermentation at 110℃ for 15 minutes, then filter it using a hydraulic plate and frame filter press to separate the filter liquid. Evaporate the filter liquid using a rotary evaporator until the alcohol content of the liquid is measured to be zero, thus obtaining the Luo Han Guo fermented liquid.

[0066] The Luo Han Guo fermentation broth prepared by the method provided in this embodiment is denoted as D.

[0067] Example 5

[0068] This embodiment provides a method for preparing monk fruit fermentation broth, including the following steps:

[0069] (1) Wash fresh monk fruit and add 10 times the amount of sterile water to make monk fruit pulp.

[0070] (2) Add 3% of the mass of pectinase and cellulase compound enzyme to the monk fruit pulp, with a mass ratio of pectinase to cellulase of 1:2. Sonicate at 70℃, pH 6.5, and ultrasonic power of 1000W for 5.5h to obtain monk fruit enzymatic hydrolysate.

[0071] (3) After filtering the Luo Han Guo enzymatic hydrolysate with gauze, sterilize it at 110℃ for 15 min; place it in a fermentation tank, inoculate it with 5% of the mass of Luo Han Guo enzymatic hydrolysate with a compound of activated aroma-producing yeast and brewer's yeast, with an inoculation ratio of 3:10, and ferment it in a sealed container at 45℃ for 12 h to obtain fermented material.

[0072] (4) Sterilize the fermented material obtained after fermentation at 110℃ for 15 minutes, then filter it using a hydraulic plate and frame filter press to separate the filter liquid. Evaporate the filter liquid using a rotary evaporator until the alcohol content of the liquid is measured to be zero, thus obtaining the Luo Han Guo fermented liquid.

[0073] The Luo Han Guo fermentation broth prepared by the method provided in this embodiment is denoted as E.

[0074] Comparative Example 1

[0075] This comparative example provides a method for preparing monk fruit fermentation broth, including the following steps:

[0076] (1) Wash fresh monk fruit and add 10 times the amount of sterile water to make monk fruit pulp.

[0077] (2) After filtering the monk fruit pulp with gauze, sterilize it at 110℃ for 15 min; place it in a fermentation tank, inoculate it with 2% of the mass of monk fruit enzymatic hydrolysate of activated aroma-producing yeast and brewer's yeast compound, with an inoculation ratio of aroma-producing yeast and brewer's yeast of 1:2, and ferment it in a sealed container at 35℃ for 48 h to obtain fermented material.

[0078] (3) Sterilize the fermented material obtained after fermentation at 110℃ for 15 minutes, then filter it using a hydraulic plate and frame filter press to separate the filter liquid. Evaporate the filter liquid using a rotary evaporator until the alcohol content of the liquid is measured to be zero, thus obtaining the Luo Han Guo fermentation liquid.

[0079] The Luo Han Guo fermentation broth prepared by the method provided in this comparative example is denoted as M1.

[0080] Comparative Example 2

[0081] This comparative example provides a method for preparing monk fruit fermentation broth, which differs from Example 1 in that: the monk fruit enzymatic hydrolysate is filtered with gauze and sterilized at 110°C for 15 min; it is placed in a fermentation tank, inoculated with 2% of the mass of the monk fruit enzymatic hydrolysate of activated brewer's yeast, and fermented in a sealed container at 35°C for 48 h.

[0082] The Luo Han Guo fermentation broth prepared by the method provided in this comparative example is denoted as M2.

[0083] Comparative Example 3

[0084] This comparative example provides a method for preparing monk fruit fermentation broth, which differs from Example 1 in that: the monk fruit enzymatic hydrolysate is filtered with gauze and sterilized at 110°C for 15 min; it is placed in a fermentation tank, inoculated with 2% of the mass of the monk fruit enzymatic hydrolysate of activated aroma-producing yeast, and fermented in a sealed container at 35°C for 48 h.

[0085] The Luo Han Guo fermentation broth prepared by the method provided in this comparative example is denoted as M3.

[0086] Comparative Example 4

[0087] This comparative example provides a method for preparing monk fruit fermentation broth, which differs from Example 1 in that: 1% of the mass of monk fruit pulp containing a compound enzyme of pectinase and protease is added to the monk fruit pulp, with a mass ratio of pectinase to protease of 1:1, and ultrasonic enzymatic hydrolysis is performed.

[0088] The Luo Han Guo fermentation broth prepared by the method provided in this comparative example is denoted as M4.

[0089] Comparative Example 5

[0090] This comparative example provides a method for preparing monk fruit fermentation broth, which differs from Example 1 in that: the monk fruit enzymatic hydrolysate is filtered, sterilized, and then placed in a fermentation tank. 2% of the mass of the monk fruit enzymatic hydrolysate is inoculated with a compound of activated *Saccharomyces cerevisiae* and *Saccharomyces cerevisiae*, with an inoculation ratio of 1:2, and fermentation is carried out in a closed system.

[0091] The Luo Han Guo fermentation broth prepared by the method provided in this comparative example is denoted as M5.

[0092] Comparative Example 6

[0093] This comparative example provides a method for preparing monk fruit fermentation broth, which differs from Example 1 in that: the monk fruit enzymatic hydrolysate is filtered, sterilized, and then placed in a fermentation tank, and 2% of the mass of the monk fruit enzymatic hydrolysate is inoculated with a compound of activated aroma-producing yeast and Pichia pastoris, with an inoculation ratio of aroma-producing yeast to Pichia pastoris of 1:2, and fermentation is carried out in a closed system.

[0094] The Luo Han Guo fermentation broth prepared by the method provided in this comparative example is denoted as M6.

[0095] Comparative Example 7

[0096] This comparative example provides a method for preparing monk fruit fermentation broth, which differs from Example 1 in that the mass ratio of pectinase to cellulase is 2:1.

[0097] The Luo Han Guo fermentation broth prepared by the method provided in this comparative example is denoted as M7.

[0098] Comparative Example 8

[0099] This comparative example provides a method for preparing monk fruit fermentation broth, which differs from Example 1 in that the inoculation ratio of aroma-producing yeast and brewer's yeast is 5:1.

[0100] The Luo Han Guo fermentation broth prepared by the method provided in this comparative example is denoted as M8.

[0101] The preparation methods of the Luo Han Guo fermentation broth provided in Examples 1-5 and Comparative Examples 1-8 are shown in Table 1.

[0102] Table 1. Preparation method of Luo Han Guo fermentation broth

[0103]

[0104]

[0105]

[0106] Experimental Example 1

[0107] This experiment analyzed the components of the *Siraitia grosvenorii* fermentation broth AE prepared in Examples 1-5 and the *Siraitia grosvenorii* fermentation broths M1-M8 prepared in Comparative Examples 1-8. Specifically: the total saponin content in the *Siraitia grosvenorii* fermentation broth was determined using vanillin-glacial acetic acid method with *Siraitia grosvenorii* saponin V as a standard; the total phenolic content in the *Siraitia grosvenorii* fermentation broth was determined using Folin-Ciocalteu spectrophotometry with gallic acid as a standard; the flavonoid content in the *Siraitia grosvenorii* fermentation broth was determined using aluminum nitrate-sodium nitrite colorimetric method with rutin as a standard reference; and the total polysaccharide content in the *Siraitia grosvenorii* fermentation broth was determined using phenol-sulfuric acid method with glucose as a standard reference. The results are shown in Table 2.

[0108] Table 2. Composition of Luo Han Guo Fermentation Broth

[0109] A 402.3 0.525 0.566 0.438 B 384.7 0.505 0.548 0.402 C 341.6 0.489 0.531 0.390 D 323.1 0.451 0.511 0.383 E 307.8 0.412 0.509 0.367 M1 235.68 0.258 0.319 0.254 M2 262.15 0.265 0.326 0.265 M3 264.84 0.277 0.330 0.282 M4 274.40 0.284 0.337 0.297 M5 281.66 0.288 0.347 0.326 M6 288.55 0.295 0.351 0.332 M7 291.66 0.310 0.356 0.324 M8 291.84 0.331 0.382 0.354

[0110] As shown in Table 2, comparing the Luo Han Guo fermentation broth AE prepared in Examples 1-5 with the Luo Han Guo fermentation broths M1-M8 prepared in Comparative Examples 1-8, it can be seen that when Luo Han Guo is subjected to synergistic ultrasonic enzymatic hydrolysis with an appropriate ratio of pectinase and cellulase, and synergistic fermentation with an appropriate ratio of aroma-producing yeast and Saccharomyces cerevisiae, the total saponin content in the prepared Luo Han Guo fermentation broth increases several times, that is, the content of Luo Han Guo saponin V increases several times. This is because cellulase and pectinase can synergistically decompose the cell walls of Luo Han Guo in the Luo Han Guo pulp, and synergistic ultrasonic enzymatic hydrolysis can more quickly and thoroughly decompose the cell walls of Luo Han Guo, promote the release of Luo Han Guo saponin V from the cell walls, and increase the content of Luo Han Guo saponin V in the enzymatic hydrolysate. At the same time, the synergistic fermentation of aroma-producing yeast and Saccharomyces cerevisiae can promote the effective conversion of active substances in the Luo Han Guo enzymatic hydrolysate into small molecules that are easily absorbed by the human body, thus ensuring that the Luo Han Guo fermentation broth has a high content of Luo Han Guo saponin V.

[0111] Experimental Example 2

[0112] In this experiment, appropriate amounts of the *Siraitia grosvenorii* fermentation broth AE prepared in Examples 1-5 and the *Siraitia grosvenorii* fermentation broth M1-M8 prepared in Comparative Examples 1-8 were dissolved in distilled water to prepare a 100 μg / mL *Siraitia grosvenorii* fermentation broth test solution for antioxidant performance testing. The specific test method is as follows:

[0113] (1) Determination of hydroxyl radical scavenging rate: Add 1 mL of salicylic acid solution (9 mmol / L), 1 mL of Luo Han Guo fermentation broth test solution, 1 mL of ferrous sulfate solution (0.9 mmol / L), and 1 mL of hydrogen peroxide solution (8.8 mmol / L) to a stoppered test tube in sequence, mix well, incubate at 37℃ for 60 min, and measure the absorbance at 510 nm, which is recorded as A1. Use purified water as a control and perform the same operation, which is recorded as A2.

[0114] Hydroxyl radical scavenging rate (%) = (A2-A1) / A2×100%.

[0115] (2) Determination of superoxide anion free radical scavenging rate: Prepare TrisHCl EDTA buffer (pH 8.2), add 0.6 mL of Luo Han Guo fermentation broth test solution, 5.2 mL of buffer solution, and 0.2 mL of pyrogallol solution (0.045 mol / L) to a stoppered test tube, mix well, react for 5 min, and measure the absorbance value at 325 nm, recorded as A1. After reacting for 1 min, measure again, recorded as A2. Use purified water as a control, follow the same procedure, and record as W1 and W2.

[0116] Superoxide anion radical scavenging rate (%) = [(W2-W1)-(A2-A1)] / (W2-W1)×100%.

[0117] The test results are shown in Table 3.

[0118] Table 3 Antioxidant properties of Luo Han Guo fermentation broth

[0119] A 90.4 89.3 B 88.7 85.5 C 87.9 84.2 D 85.1 83.5 E 83.2 80.8 M1 51.5 50.0 M2 53.7 51.6 M3 55.2 52.1 M4 56.4 53.2 M5 58.4 55.4 M6 62.8 58.5 M7 66.3 65.4 M8 68.8 57.2

[0120] As shown in Table 3, comparing the Luo Han Guo fermentation broth AE prepared in Examples 1-5 with the Luo Han Guo fermentation broths M1-M8 prepared in Comparative Examples 1-8 reveals that when Luo Han Guo is subjected to synergistic ultrasonic enzymatic hydrolysis with an appropriate ratio of pectinase and cellulase, and synergistic fermentation with an appropriate ratio of aroma-producing yeast and Saccharomyces cerevisiae, the resulting Luo Han Guo fermentation broth exhibits significantly improved hydroxyl radical scavenging and superoxide anion radical scavenging rates, demonstrating better antioxidant capacity. This is because the synergistic ultrasonic enzymatic hydrolysis with an appropriate ratio of pectinase and cellulase, and the synergistic fermentation with an appropriate ratio of aroma-producing yeast and Saccharomyces cerevisiae, results in a higher content of mogroside V in the prepared Luo Han Guo fermentation broth, thus exhibiting better antioxidant capacity.

[0121] Experimental Example 3

[0122] This experiment used the MTT assay to investigate the effects of the *Siraitia grosvenorii* fermentation broth AE prepared in Examples 1-5 and the *Siraitia grosvenorii* fermentation broth M1-M8 prepared in Comparative Examples 1-8 on the viability of human skin fibroblasts (HSF) and human immortalized keratinocytes (HaCaT). Specific experimental methods: HSF and HaCaT at certain concentrations were seeded into 96-well plates and incubated for 24 h. After incubation, the solution in the wells was removed, and *Siraitia grosvenorii* fermentation broth was diluted to 2%, 5%, and 10% concentrations using DMEM and added to the 96-well plates, respectively. The plates were then incubated for 24 h, with a culture medium without *Siraitia grosvenorii* fermentation broth serving as a control. 20 μL of MTT solution was added to each well, and the plates were incubated for another 2 h. The culture medium was then removed, and 100 μL of isopropanol was added to each well. The plates were then shaken at 100 rpm for 30 min under light-shielded conditions at 25°C. Cell viability was detected by measuring the absorbance at 570 nm using a microplate reader. The MTT solution was prepared by dissolving 250 mg of thiazolyl blue (CAS: 298-93-1) in 50 ml of DPBS and then filtering it through a 0.22 μm filter for sterilization. The test results are shown in Table 4.

[0123] Table 4 Cell viability

[0124]

[0125]

[0126] As shown in Table 4, under the experimental concentration conditions, the Luo Han Guo fermentation broth resulted in a cell survival rate >100% after 24 hours of treatment with HSF and HaCaT cells, with no significant cytotoxicity. Furthermore, the Luo Han Guo fermentation broth AE prepared in Examples 1-5 exhibited significant cell proliferation-promoting activity; the proliferation-promoting ability increased with increasing concentration, but further increasing the concentration from 5% to 10% did not significantly enhance the proliferation effect.

[0127] Test Example 4

[0128] In this experiment, HSF at a certain concentration was seeded into 24-well plates and incubated for 24 hours. The solution in the wells was then removed, and TNF-α was added and cultured for another 24 hours. The *Siraitia grosvenorii* fermentation broth AE prepared in Examples 1-5 and the *Siraitia grosvenorii* fermentation broth M1-M8 prepared in Comparative Examples 1-8 were then diluted to 5% using DMEM and added to 24-well plates respectively. TNF-α was used as a model control, and culture medium without *Siraitia grosvenorii* fermentation broth served as a blank control. The plates were incubated for 24 hours. The levels of type I procollagen C-terminal peptide (PIP) and matrix metalloproteinase-1 (MMP-1) in the cell culture supernatant were detected using reagent kits. The MMP-1 kit was purchased from R&D Systems (product number DY901B-05), and the PIP kit was purchased from TARKA (product number TKR-MK101). The results are shown in Table 5.

[0129] Table 5. Soothing, anti-inflammatory, and anti-aging effects of monk fruit fermentation liquid.

[0130]

[0131]

[0132] As shown in Table 5, the inflammatory factor TNF-α causes abnormal collagen expression in skin cells, disrupting the redox system and affecting human skin, thus accelerating skin aging. Under TNF-α stimulation, the PIP secretion of cells in the model control group was reduced and the MMP-1 secretion was increased compared to the blank control, indicating the successful establishment of a chronic inflammatory aging model. Comparison of the Luo Han Guo fermentation broth AE prepared in Examples 1-5 with the model control and the Luo Han Guo fermentation broth M1-M8 prepared in Comparative Examples 1-8 shows that PIP secretion increased and MMP-1 secretion decreased. This is because the Luo Han Guo fermentation broth prepared by using an appropriate ratio of pectinase and cellulase for synergistic ultrasonic hydrolysis, and by using an appropriate ratio of aroma-producing yeast and Saccharomyces cerevisiae for synergistic fermentation, has a higher content of Luo Han Guo saponin V, which has the effect of inhibiting abnormal collagen expression caused by the inflammatory factor TNF-α, and has a soothing, anti-inflammatory, and anti-aging effect on the dermis.

[0133] Experimental Example 5

[0134] In this experiment, a certain concentration of HaCaT was inoculated into 24-well plates and incubated for 24 hours. The solution in the wells was then removed, and TNF-α was added and cultured for another 24 hours. Then, the *Siraitia grosvenorii* fermentation broth A provided in Example 1 was diluted to concentrations of 0.1%, 1%, 2%, 5%, and 10% using DMEM and added to the 24-well plates respectively. TNF-α was used as a model control, and culture medium without *Siraitia grosvenorii* fermentation broth was used as a blank control. The plates were incubated for 24 hours. Total reactive oxygen species (ROS) were detected using a kit purchased from Shanghai Yanyu Trading Co., Ltd., product code ml70634. The detection results are as follows: Figure 2 As shown.

[0135] Stimulation of keratinocytes (HaCaT) with the inflammatory factor TNF-α can induce oxidative stress leading to an increase in ROS, induce intracellular peroxidation and apoptosis, and accelerate the aging process. Under TNF-α stimulation, the expression level of ROS in HaCat cells stimulated by TNF-α increased, indicating that a chronic inflammatory aging model of HaCat cells was successfully established.

[0136] Depend on Figure 2The results showed that the intracellular fluorescence intensity in the model control group was significantly increased compared to the blank control group, indicating that TNF-α effectively induced an increase in intracellular reactive oxygen species (ROS) content in HaCat cells. A comparison of the Luo Han Guo fermentation broth A provided in Example 1 with the model control showed a decrease in intracellular fluorescence intensity, indicating that the Luo Han Guo fermentation broth inhibited ROS in HaCat cells induced by TNF-α, and this inhibition was concentration-dependent, demonstrating soothing, anti-inflammatory, and anti-aging effects on the epidermis.

[0137] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A method for preparing monk fruit fermentation broth, characterized in that, The preparation method includes: adding a compound enzyme to monk fruit pulp for enzymatic hydrolysis to obtain monk fruit enzymatic hydrolysate; inoculating the monk fruit enzymatic hydrolysate with compound bacteria for fermentation; sterilizing, filtering, and evaporating the fermented material obtained after fermentation until the alcohol content of the liquid is measured to be zero; the method also includes activating the compound bacteria before fermentation; the method also includes filtering and sterilizing the monk fruit enzymatic hydrolysate before fermentation; the compound enzyme is a combination of pectinase and cellulase, and the mass ratio of pectinase to cellulase is 1:1-2; the mass of the compound enzyme is 0.1%-3% of the mass of monk fruit pulp; the compound bacteria is a combination of aroma-producing yeast and brewer's yeast, and the inoculation ratio of aroma-producing yeast to brewer's yeast is 1:2-5; the inoculation amount of the compound bacteria is 0.5%-5% of the mass of monk fruit enzymatic hydrolysate; the monk fruit fermentation broth includes saponins, phenols, flavonoids, and polysaccharides.

2. The preparation method according to claim 1, characterized in that, The fermentation conditions include fermentation at 25-45℃ for 12-80 hours.

3. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis includes ultrasonic enzymatic hydrolysis.

4. The preparation method according to claim 3, characterized in that, The conditions for ultrasonic enzymatic hydrolysis include: 40-70℃, pH value 3.5-6.5, ultrasonic power 400-1000 W, and ultrasonic enzymatic hydrolysis for 0.5-5 h.

5. A Luo Han Guo fermentation broth prepared by the preparation method according to any one of claims 1-4.

6. A composition of monk fruit fermentation liquid, characterized in that, The composition comprises the following components in weight percentage: 0.1%-10% of the monk fruit fermentation broth according to claim 5, 0.5%-5% of butylene glycol, 1%-3% of hexanediol, 0.05%-0.1% of ethylhexylglycerin, and the remainder being water.

7. The use of the monk fruit ferment broth as described in claim 5 or the composition as described in claim 6 in the preparation of skin care products.

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