A medicinal and edible fermented product for regulating spleen and stomach and / or preventing precocious puberty in children and a preparation method thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a medicine-herbalism fermented product for regulating spleen and stomach and / or preventing precocious puberty of children and a preparation method thereof. The medicine-herbalism fermented product comprises 15-25 parts of cornus officinalis, 10-30 parts of mulberry, 10-20 parts of gardenia, 20-40 parts of poria cocos, 10-35 parts of radix asparagi, 5-12 parts of enzyme preparation and 0.06-0.15 parts of fermenting agent. The fermented product obtained after enzymolysis and fermentation of the above five medicine-herbalism traditional Chinese medicines by lactobacillus plantarum YSH08 and lactobacillus rhamnosus XS82 can reduce the contents of LH, FSH and E2 of precocious puberty rats, reduce the ovary and uterus coefficients, and achieve the effect of treating precocious puberty of children; on the other hand, the fermented product can treat spleen deficiency and phlegm-dampness.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine technology, specifically relating to a medicinal and edible fermented product for regulating the spleen and stomach and / or preventing precocious puberty in children, and its preparation method. Background Technology

[0002] Precocious puberty in children is a common developmental disorder of the endocrine system, characterized by the appearance of secondary sexual characteristics significantly earlier than in children of the same age. Clinically, it is divided into central precocious puberty, peripheral precocious puberty, and incomplete precocious puberty, among which central precocious puberty is the most common. Central precocious puberty (CPP) is caused by the premature activation of the hypothalamic-pituitary-gonadal axis, resulting in an earlier pulsatile peak in the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus. This stimulates the pituitary gland to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH further stimulate gonadal development and the secretion of sex hormones, leading to the development of internal and external genitalia and the appearance of secondary sexual characteristics, thus causing children to enter puberty prematurely.

[0003] Precocious puberty falls under the categories of "early onset of Tian Gui" and "breast lumps" in Traditional Chinese Medicine (TCM), which considers the disease location to be in the "kidney and liver," with kidney yin deficiency and excessive ministerial fire being the basic pathogenesis. It is classified into yin deficiency with excessive fire type, liver meridian stagnation with heat type, and phlegm-dampness stagnation type. For the yin deficiency with excessive fire type, Zhi Bai Di Huang Wan (Rehmannia and Phellodendron Formula) with modifications is commonly used; for the liver meridian stagnation with heat type, Dan Zhi Xiao Yao San (Gardenia and Gardenia Powder with modifications) is commonly used; and for the phlegm-dampness stagnation type, Er Chen Tang (Two-Ingredient Decoction with Modifications) is used. However, clinically, it often presents as a complex syndrome.

[0004] The core principle of the concept of food and medicine sharing the same origin lies in the fact that some raw materials possess both the safety of food and the conditioning effects of medicine. They can be used to regulate the body, prevent diseases, and assist in rehabilitation through daily dietary intake, without the need for deliberate medication, thus aligning with the essence of health preservation: "medicine in food." Modern bio-fermentation technology, through the fermentation and transformation of probiotics, can effectively solve the problems of insufficient dissolution and low absorption efficiency, enabling the in-depth development of raw materials that share the same origin as food and medicine. Summary of the Invention

[0005] To address the above problems, this invention provides a fermented product that is both food and medicine and its preparation method. This fermented product has the effects of regulating the spleen and stomach and preventing precocious puberty in children.

[0006] The technical content of this invention is as follows:

[0007] In one aspect, this invention provides a fermented food-medicine homology product for regulating the spleen and stomach and / or preventing precocious puberty in children. The food-medicine homology ingredients include Cornus officinalis, mulberry, gardenia, Poria cocos, and Asparagus cochinchinensis.

[0008] Preferably, the medicinal and edible fermented product, by weight, includes 15-25 parts of Cornus officinalis, 10-30 parts of mulberry, 10-20 parts of gardenia, 20-40 parts of Poria cocos, 10-35 parts of asparagus, 5-12 parts of enzyme preparation, and 0.06-0.15 parts of fermentation agent.

[0009] More preferably, the medicinal and edible fermented product, by weight, includes 20-25 parts of Cornus officinalis, 15-30 parts of mulberry, 15-20 parts of gardenia, 20-35 parts of Poria cocos, 10-25 parts of Asparagus cochinchinensis, 6-10 parts of enzyme preparation, and 0.08-0.13 parts of fermentation agent.

[0010] Preferably, the enzyme preparation includes cellulase, hemicellulase, pectinase and tanninase, and the weight ratio of cellulase, hemicellulase, pectinase and tanninase is (3-8):(2-5):(1-7):(0.5-3).

[0011] In some embodiments of the present invention, the weight ratio of cellulase, hemicellulase, pectinase and tanninase is 4:2:4:1 or 3:2:7:3 or 8:5:1:0.5 or 5:4:5:2.

[0012] More preferably, the cellulase has an enzyme activity of 10,000-100,000 U / g, the pectinase has an enzyme activity of 10,000-100,000 U / g, the hemicellulase has an enzyme activity of 100,000-500,000 U / g, and the tanninase has an enzyme activity of 30-300 U / g.

[0013] Preferably, the fermenting agent includes *Lactobacillus plantarum* and *Lactobacillus reuteri*.

[0014] More preferably, the *Lactobacillus plantarum* is *Lactobacillus plantarum* YSH08, with accession number CGMCC No. 36182; and the *Lactobacillus reuteri* is *Lactobacillus reuteri* XS82, with accession number CGMCC No. 36181.

[0015] Preferably, the weight ratio of Lactobacillus plantarum YSH08 and Lactobacillus reuteri XS82 in the fermentation agent is (2-4):(1-3).

[0016] In some embodiments of the present invention, the weight ratio of *Lactobacillus plantarum* YSH08 and *Lactobacillus reuteri* XS82 is 2:1, 2:3, 4:2, or 3:1.

[0017] More preferably, the viable count of the *Lactobacillus plantarum* YSH08 is 1.0 × 10⁻⁶. 11 CFU / g or higher; the viable count of the *Lactobacillus reuteri* XS82 is 1.0 × 10⁻⁶. 11 CFU / g or higher.

[0018] In another aspect, the present invention also provides a method for preparing the above-mentioned medicinal and edible fermented product, comprising the following steps: (1) mixing medicinal and edible raw materials asparagus, gardenia, poria cocos, cornus officinalis and mulberry with water to obtain a mixed liquid; (2) adding an enzyme preparation to the mixed liquid for ultrasonic enzymatic hydrolysis and sterilization to obtain a treated liquid; (3) inoculating the treated liquid with a fermenting agent for fermentation, separating the solid and liquid, and the supernatant is the fermented product.

[0019] Preferably, in step (1), the ratio of the total weight of the medicinal and edible raw materials to the water is 1:(15-30).

[0020] In some embodiments of the present invention, the ratio of the total weight of the medicinal and edible raw materials to water in step (1) is 1:25, 1:15, 1:20, or 1:30.

[0021] Preferably, in step (2), the ultrasonic enzymatic hydrolysis is performed under ultrasonic conditions. Specifically, the ultrasonic power is 180-220W, the ultrasonic frequency is 25-30kHz, the ultrasonic temperature is 40-50℃, and the ultrasonic time is 1-2h.

[0022] Preferably, the sterilization temperature in step (2) is 70-90℃ and the sterilization time is 20-40min.

[0023] Preferably, the fermentation temperature in step (3) is 30-42℃ and the fermentation time is 24-84h.

[0024] In some embodiments of the present invention, the fermentation temperature in step (3) is 37°C and the fermentation time is 72h; or the fermentation temperature is 30°C and the fermentation time is 84h; or the fermentation temperature is 42°C and the fermentation time is 24h; or the fermentation temperature is 35°C and the fermentation time is 48h.

[0025] In another aspect, the present invention provides the application of the above-mentioned medicinal and edible fermented products or the medicinal and edible fermented products obtained by the above-mentioned preparation methods in the preparation of products for regulating the spleen and stomach and / or preventing precocious puberty in children.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] 1. The medicinal and edible fermented product provided by this invention includes Cornus officinalis, mulberry, gardenia, Poria cocos, and Asparagus cochinchinensis. Among them, Cornus officinalis is sour and astringent, slightly warm, and enters the liver and kidney meridians, and has the effects of tonifying the liver and kidneys, and astringing and consolidating; Gardenia jasminoides is bitter and cold, and enters the heart, lung, and triple burner meridians, and has the effects of purging fire and relieving irritability, clearing heat and promoting diuresis, cooling blood and detoxifying, and protecting the liver and gallbladder; Poria cocos is sweet, bland, and neutral, and enters the heart, lung, spleen, and kidney meridians, and has the effects of promoting diuresis and removing dampness, and strengthening the spleen and calming the mind; Asparagus cochinchinensis is sweet, bitter, and cold, and enters the lung and kidney meridians, and has the effects of nourishing yin and moistening dryness, and clearing the lungs and generating fluids; Mulberry is sweet, sour, and cold, and enters the heart, liver, and kidney meridians. The five ingredients selected in this invention are suitable for children with complex precocious puberty caused by a combination of yin deficiency and excessive fire, liver stagnation, and phlegm-dampness stagnation. Among them, mulberry and cornus officinalis can nourish kidney water and clear ministerial fire (tonify kidney yin), solving the problem of kidney yin deficiency and excessive ministerial fire; poria cocos can strengthen spleen qi and resolve phlegm-dampness (tonify spleen yin), solving the source of phlegm-dampness and internal heat; gardenia clears liver heat and relieves liver stagnation (tonify liver yin); asparagus root moistens lung dryness and clears deficiency heat (tonify lung yin). Based on the traditional single-organ regulation of precocious puberty, this invention creatively adds asparagus root, an ingredient that tonifies lung yin. The addition of asparagus root in this formula is not simply a targeted treatment of the lungs, but rather it moistens lung dryness, clears internal heat, and smooths the body's qi flow; relying on the mutual generation of lung and kidney, it nourishes kidney yin while restraining excessive liver fire. This fermented product harmonizes multiple internal organs, restoring the balance of Yin and Yang within the body. It addresses the root cause of imbalances in children's constitution, such as Yin deficiency with excessive Yang and multiple pathogenic factors, thereby alleviating and improving precocious puberty in children, whether as a single symptom or a complex combination of Yin deficiency with excessive Yang, liver meridian stagnation, and phlegm-dampness stagnation. Furthermore, experimental verification has shown that the combination of these five ingredients comprehensively nourishes the Yin of the liver, spleen, lungs, and kidneys, slows the enlargement of the uterus and ovaries in precocious puberty rats, and reduces hormone levels such as LH, FSH, and E2, thus achieving the effect of improving precocious puberty in children.

[0028] 2. The food-medicine homologous fermentation product provided by this invention can improve the HDL-C level in the plasma of rats with spleen deficiency and phlegm-dampness syndrome, and reduce the levels of TG, TC and LDL-C, and enhance the expression of AQP3 in the colon of rats with spleen deficiency and phlegm-dampness syndrome, thereby achieving the treatment of spleen deficiency and phlegm-dampness syndrome.

[0029] 3. Traditional Chinese medicine decoction methods are complex and fail to concentrate the effective components. The fermentation preparation method provided by this invention uses a process of simultaneous ultrasonic enzymatic hydrolysis and fermentation. Four enzyme preparations, cellulase, pectinase, hemicellulase and tanninase, are selected for enzymatic hydrolysis to fully dissolve the effective components in the raw materials and improve efficacy. Furthermore, the applicant has found that the *Lactobacillus plantarum* YSH08 and *Lactobacillus reuteri* XS82 selected in this invention as fermentation agents can better improve the effects of fermentation on improving precocious puberty in children and regulating the spleen and stomach. Attached Figure Description

[0030] Figure 1The graph shows the plasma TC content of rats in the blank group, model group, Examples 1-4, and Comparative Examples 1-11. Compared with the blank group, +P<0.05, ++P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with Example 1, △P<0.05, △△P<0.01.

[0031] Figure 2 The graph shows the plasma TG content of rats in the blank group, model group, Examples 1-4, and Comparative Examples 1-11. Compared with the blank group, +P<0.05, ++P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with Example 1, △P<0.05, △△P<0.01.

[0032] Figure 3 The graph shows the plasma LDL-C content of rats in the blank group, model group, Examples 1-4, and Comparative Examples 1-11. Compared with the blank group, +P<0.05, ++P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with Example 1, △P<0.05, △△P<0.01.

[0033] Figure 4 The graph shows the HDL-C content in rat plasma in the blank group, model group, Examples 1-4, and Comparative Examples 1-11. Compared with the blank group, +P<0.05, ++P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with Example 1, △P<0.05, △△P<0.01.

[0034] Figure 5 Figure 1 shows the results of AQP3 expression in rat colon in the blank group, model group, Examples 1-4 and Comparative Examples 1-11. Compared with the blank group, +P<0.05, ++P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with Example 1, △P<0.05, △△P<0.01. Detailed Implementation

[0035] 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.

[0036] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels. Products from different manufacturers do not have a significant impact on the effectiveness.

[0038] Lactobacillus plantarum YSH08, Latin name Lactiplantibacillus plantarum The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 15, 2025, with accession number CGMCC No. 36182, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It has been published in Chinese Patent CN121736957A.

[0039] Lactobacillus reuteri XS82, Latin name Limosilactobacillus reuteri The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 15, 2025, with accession number CGMCC No. 36181, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It has been published in Chinese Patent CN121736956A.

[0040] Cornus officinalis, Gardenia jasminoides, Asparagus cochinchinensis, Poria cocos, Morus alba, Rehmannia glutinosa, Ophiopogon japonicus, Dioscorea opposita, and Cassia tora were purchased from Anhui Jiahe Traditional Chinese Medicine Technology Co., Ltd.; Cellulase and pectinase were purchased from Shandong Longket Enzyme Preparation Co., Ltd.; Tanninase was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.; Hemicellulase was purchased from Hubei Xinghengye Technology Co., Ltd.; Lactobacillus plantarum JYLP-326 was purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd.; and Lactobacillus rhamnosus NSL0401 was purchased from Qingdao Nuosen Biotechnology Co., Ltd.

[0041] Preparation Example 1

[0042] Lactobacillus plantarum YSH08 and Lactobacillus reuteri XS82 were inoculated into MRS medium and cultured at 35℃ for 24 h, followed by three consecutive subcultures. The bacterial culture was centrifuged (14000 r / min, 6 min) to collect the bacterial sludge. The sludge was mixed with a freeze-drying protectant at a ratio of 1:3 (dry weight of bacterial sludge to weight of protectant solution). The protectant formulation for Lactobacillus plantarum YSH08 consisted of 15% trehalose, 5% maltodextrin, and 1% glycerol; the freeze-drying protectant formulation for Lactobacillus reuteri XS82 consisted of 10% trehalose, 12% inositol, 10% skim milk, and 1.5% glutathione. The mixed mycelium sludge and protective agent solution were freeze-dried under a vacuum of 15 Pa. Pre-freezing: -40℃ for 1 hour; sublimation drying: -10℃ for 6 hours, 0℃ for 6 hours, 10℃ for 6 hours; desorption drying: 30℃ for 15 hours, ensuring a viable count of 1×10⁻⁶ cells in the mycelium powder. 11 CFU / g.

[0043] The MRS medium was prepared as follows: the medium formula consisted of 10.0 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2 g / L triammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween-80. 1 L of pure water was added to the above MRS medium, and the mixture was autoclaved at 121°C for 15 minutes.

[0044] Example 1

[0045] This embodiment provides a fermented product that is both food and medicine, comprising 20 parts of Cornus officinalis, 25 parts of mulberry, 15 parts of gardenia, 30 parts of Poria cocos, 25 parts of Asparagus cochinchinensis, 3.64 parts of cellulase, 1.82 parts of hemicellulase, 3.64 parts of pectinase, 0.91 parts of tanninase, 0.1 parts of Lactobacillus plantarum YSH08, 0.05 parts of Lactobacillus reuteri XS82, and 2875 parts of water; wherein the cellulase activity is 40,000 U / g, the hemicellulase activity is 300,000 U / g, the pectinase activity is 30,000 U / g, and the tanninase activity is 250 U / g.

[0046] Its preparation method is as follows:

[0047] (1) Grind the medicinal and edible raw materials asparagus, poria cocos, gardenia, cornus officinalis and mulberry into powder and pass them through a 100-mesh sieve; take 20 parts of cornus officinalis, 25 parts of mulberry, 15 parts of gardenia, 30 parts of poria cocos and 25 parts of asparagus, add 2875 parts of water to obtain a mixed liquid;

[0048] (2) Add 3.64 parts of cellulase, 1.82 parts of hemicellulase, 3.64 parts of pectinase and 0.91 parts of tanninase to the mixture. After ultrasonic enzymatic hydrolysis for 1 hour at 50℃, ultrasonic power of 200W and ultrasonic frequency of 28kHz, sterilize at 80℃ for 30 minutes to obtain the treatment solution.

[0049] (3) Inoculate the treatment liquid with 0.1 parts of Lactobacillus plantarum YSH08 (Preparation Example 1) and 0.05 parts of Lactobacillus reuteri XS82 (Preparation Example 1), ferment at 37°C for 72 h, separate the solid and liquid, and the supernatant is the fermentation product.

[0050] Example 2

[0051] This embodiment provides a fermented food-medicine homology product, comprising 15 parts of Cornus officinalis, 10 parts of mulberry, 10 parts of gardenia, 20 parts of Poria cocos, 30 parts of Asparagus cochinchinensis, 2.4 parts of cellulase, 1.6 parts of hemicellulase, 5.6 parts of pectinase, 2.4 parts of tanninase, 0.05 parts of Lactobacillus plantarum YSH08, 0.08 parts of Lactobacillus reuteri XS82, and 1275 parts of water; wherein the cellulase activity is 40,000 U / g, the hemicellulase activity is 300,000 U / g, the pectinase activity is 30,000 U / g, and the tanninase activity is 250 U / g.

[0052] Its preparation method is as follows:

[0053] (1) Grind the medicinal and edible raw materials asparagus, poria cocos, gardenia, cornus officinalis and mulberry into powder and pass them through a 100-mesh sieve; take 15 parts of cornus officinalis, 10 parts of mulberry, 10 parts of gardenia, 20 parts of poria cocos and 30 parts of asparagus, add 1275 parts of water to obtain a mixed liquid;

[0054] (2) Add 2.4 parts of cellulase, 1.6 parts of hemicellulase, 5.6 parts of pectinase and 2.4 parts of tanninase to the mixture. After ultrasonic enzymatic hydrolysis for 2 hours at 40℃, ultrasonic power of 180W and ultrasonic frequency of 25kHz, sterilize at 70℃ for 40 minutes to obtain the treatment solution.

[0055] (3) Inoculate the treatment liquid with 0.05 parts of Lactobacillus plantarum YSH08 (Preparation Example 1) and 0.08 parts of Lactobacillus reuteri XS82 (Preparation Example 1), ferment at 30°C for 84 h, separate the solid and liquid, and the supernatant is the fermentation product.

[0056] Example 3

[0057] This embodiment provides a fermented product that is both food and medicine, comprising 25 parts of Cornus officinalis, 30 parts of mulberry, 20 parts of gardenia, 35 parts of Poria cocos, 35 parts of Asparagus cochinchinensis, 2.76 parts of cellulase, 1.72 parts of hemicellulase, 0.34 parts of pectinase, 0.17 parts of tanninase, 0.04 parts of Lactobacillus plantarum YSH08, 0.02 parts of Lactobacillus reuteri XS82, and 4350 parts of water; wherein the cellulase activity is 40,000 U / g, the hemicellulase activity is 300,000 U / g, the pectinase activity is 30,000 U / g, and the tanninase activity is 250 U / g.

[0058] Its preparation method is as follows:

[0059] (1) Grind the medicinal and edible raw materials asparagus, poria cocos, gardenia, cornus officinalis and mulberry into powder and pass them through a 100-mesh sieve; take 25 parts of cornus officinalis, 30 parts of mulberry, 20 parts of gardenia, 35 parts of poria cocos and 35 parts of asparagus, add 4350 parts of water to obtain a mixed liquid;

[0060] (2) Add 2.76 parts of cellulase, 1.72 parts of hemicellulase, 0.34 parts of pectinase and 0.17 parts of tanninase to the mixture. After ultrasonic enzymatic hydrolysis for 1.5 h at 45 °C, ultrasonic power of 220 W and ultrasonic frequency of 30 kHz, sterilize at 90 °C for 20 min to obtain the treatment solution.

[0061] (3) Inoculate the treatment liquid with 0.04 parts of Lactobacillus plantarum YSH08 (Preparation Example 1) and 0.02 parts of Lactobacillus reuteri XS82 (Preparation Example 1), ferment at 42°C for 24 h, separate the solid and liquid, and the supernatant is the fermentation product.

[0062] Example 4

[0063] This embodiment provides a fermented food-medicine homology product, comprising 20 parts of Cornus officinalis, 15 parts of mulberry, 10 parts of gardenia, 40 parts of Poria cocos, 10 parts of Asparagus cochinchinensis, 1.88 parts of cellulase, 1.5 parts of hemicellulase, 1.88 parts of pectinase, 0.75 parts of tanninase, 0.06 parts of Lactobacillus plantarum YSH08, 0.02 parts of Lactobacillus reuteri XS82, and 1900 parts of water; wherein the cellulase activity is 40,000 U / g, the hemicellulase activity is 300,000 U / g, the pectinase activity is 30,000 U / g, and the tanninase activity is 250 U / g.

[0064] Its preparation method is as follows:

[0065] (1) Grind the medicinal and edible raw materials asparagus, poria cocos, gardenia, cornus officinalis and mulberry into powder and pass them through a 100-mesh sieve; take 20 parts of cornus officinalis, 15 parts of mulberry, 10 parts of gardenia, 40 parts of poria cocos and 10 parts of asparagus, add 1900 parts of water to obtain a mixed liquid;

[0066] (2) Add 1.88 parts of cellulase, 1.5 parts of hemicellulase, 1.88 parts of pectinase and 0.75 parts of tanninase to the mixture. After ultrasonic enzymatic hydrolysis for 2 hours at 40℃, ultrasonic power of 180W and ultrasonic frequency of 30kHz, sterilize at 80℃ for 40 minutes to obtain the treatment solution.

[0067] (3) Inoculate the treatment liquid with 0.06 parts of Lactobacillus plantarum YSH08 (Preparation Example 1) and 0.02 parts of Lactobacillus reuteri XS82 (Preparation Example 1), ferment at 35°C for 48 h, separate the solid and liquid, and the supernatant is the fermentation product.

[0068] Comparative Example 1

[0069] This comparative example provides a medicinal and edible fermented product, which differs from Example 1 only in that the fermentation raw materials are replaced with Cornus officinalis, specifically 45 parts Cornus officinalis, 15 parts Gardenia jasminoides, 30 parts Poria cocos, and 25 parts Asparagus cochinchinensis; the rest is the same as in Example 1.

[0070] Comparative Example 2

[0071] This comparative example provides a traditional Chinese medicine composition prepared using the method described in Example 1 of patent CN102114226B.

[0072] Specifically, the preparation of the traditional Chinese medicine composition is as follows: 100 parts by weight of Anemarrhena asphodeloides, 100 parts by weight of Phellodendron chinense, 100 parts by weight of Rehmannia glutinosa, 150 parts by weight of Cornus officinalis, 100 parts by weight of Paeonia lactiflora, 210 parts by weight of Alisma plantago-aquatica, 150 parts by weight of Poria cocos, 100 parts by weight of Rehmannia glutinosa (processed), 100 parts by weight of Scutellaria baicalensis, 100 parts by weight of Ligustrum lucidum, 210 parts by weight of Cyperus rotundus, 100 parts by weight of Gardenia jasminoides, 150 parts by weight of Curcuma longa, 100 parts by weight of Angelica sinensis, 3 parts by weight of sodium benzoate, and 0.5 parts by weight of total steviosides.

[0073] Soak the raw materials of the Chinese herbal medicine combination, Angelica sinensis, Curcuma longa, and Cyperus rotundus, in an appropriate amount of water for 10-30 minutes until fully moistened. Add water to cover the herbs, bring to a boil, and maintain a gentle simmer for about 15 minutes. Filter and store the filtrate separately. Soak the raw materials of Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Cornus officinalis, Paeonia lactiflora, Alisma plantago-aquatica, Poria cocos, Rehmannia glutinosa (processed), Scutellaria baicalensis, Ligustrum lucidum, and Gardenia jasminoides in water for 30 minutes until fully moistened. Combine these with the above-mentioned dregs, add water to cover the herbs by 2-4 cm, and decoct three times, maintaining a gentle simmer for 1.5 hours each time, stirring occasionally, until thoroughly cooked. Filter the decoction, combine the filtrates, and let stand for at least 12 hours. Concentrate the supernatant for about 4 hours to a suitable volume, add the previously prepared decoction, stir well, and then concentrate to an oral solution with a relative density of not less than 1.10. While still hot, add sodium benzoate and total steviosides to the above decoction, stir, and bring to a final volume.

[0074] Comparative Example 3

[0075] This comparative example provides a fermented product that is both food and medicine. The only difference from Example 1 is that the fermentation raw materials do not include Asparagus. Specifically, the raw materials are 20 parts of Cornus officinalis, 25 parts of Morus alba, 15 parts of Gardenia jasminoides, and 30 parts of Poria cocos. However, the total weight of Cornus officinalis, Morus alba, Gardenia jasminoides, and Poria cocos is the same as that of Cornus officinalis, Morus alba, Gardenia jasminoides, Poria cocos, and Asparagus officinalis in Example 1. The rest is the same as in Example 1.

[0076] Comparative Example 4

[0077] This comparative example provides a medicinal and edible fermented product, which differs from Example 1 only in that the fermentation raw material is replaced with Rehmannia glutinosa instead of mulberry. Specifically, it consists of 20 parts Cornus officinalis, 25 parts Rehmannia glutinosa, 15 parts Gardenia jasminoides, 30 parts Poria cocos, and 25 parts Asparagus cochinchinensis; the rest is the same as in Example 1.

[0078] Comparative Example 5

[0079] This comparative example provides a medicinal and edible fermented product, which differs from Example 1 only in that the fermentation raw materials are replaced with cassia seeds instead of gardenia. Specifically, the raw materials are 20 parts of cornus officinalis, 25 parts of mulberry, 15 parts of cassia seeds, 30 parts of poria cocos, and 25 parts of asparagus root; the rest is the same as in Example 1.

[0080] Comparative Example 6

[0081] This comparative example provides a fermented product that is both food and medicine. The only difference from Example 1 is that the fermentation raw materials are replaced with Ophiopogon japonicus instead of Asparagus cochinchinensis. Specifically, the raw materials are 20 parts of Cornus officinalis, 25 parts of Morus alba, 15 parts of Gardenia jasminoides, 30 parts of Poria cocos, and 25 parts of Ophiopogon japonicus. The rest is the same as in Example 1.

[0082] Comparative Example 7

[0083] This comparative example provides a fermented product that is both food and medicine. The only difference from Example 1 is that the fermentation raw materials are replaced with Poria cocos and Dioscorea opposita, specifically 20 parts of Cornus officinalis, 25 parts of Morus alba, 15 parts of Gardenia jasminoides, 15 parts of Poria cocos, 15 parts of Dioscorea opposita and 25 parts of Asparagus cochinchinensis; the rest is the same as in Example 1.

[0084] Comparative Example 8

[0085] This comparative example provides a medicinal and edible fermented product, which differs from Example 1 only in that the fermenting agent is replaced with Lactobacillus plantarum YSH08 and Lactobacillus plantarum JYLP-326; wherein the viable count of Lactobacillus plantarum JYLP-326 is 100 billion CFU / g; the rest is the same as Example 1.

[0086] Comparative Example 9

[0087] This comparative example provides a medicinal and edible extract, the raw materials of which include 20 parts of Cornus officinalis, 25 parts of mulberry, 15 parts of gardenia, 30 parts of Poria cocos, and 25 parts of Asparagus cochinchinensis.

[0088] Its preparation method is as follows:

[0089] (1) Grind the medicinal and edible raw materials asparagus, poria cocos, gardenia, cornus officinalis and mulberry into powder and pass them through a 100-mesh sieve; take 20 parts of cornus officinalis, 25 parts of mulberry, 15 parts of gardenia, 30 parts of poria cocos and 25 parts of asparagus, add 2875 parts of water to obtain a mixed liquid;

[0090] (2) The mixture was ultrasonicated at 50°C, 200W, and 28kHz for 1 hour, and then sterilized at 80°C for 30 minutes to obtain the treatment solution.

[0091] (3) The treatment solution was kept at 37°C for 72 hours to separate the solid and liquid. The supernatant was the extract.

[0092] Comparative Example 10

[0093] This comparative example provides a medicinal and edible fermented product. The difference from Example 1 is that the fermenting agent replaces Lactobacillus reuteri XS82 with Lactobacillus rhamnosus NSL0401; wherein the viable count of Lactobacillus rhamnosus NSL0401 is 100 billion CFU / g; the rest is the same as in Example 1.

[0094] Comparative Example 11

[0095] This comparative example provides a fermented product that is both food and medicine. The only difference from Example 1 is that the enzyme preparation only includes cellulase and pectinase, specifically 3.64 parts of cellulase and 3.64 parts of pectinase; however, the total weight of cellulase and pectinase is the same as the total weight of cellulase, hemicellulase, pectinase and tanninase in Example 1; the rest is the same as in Example 1.

[0096] Experimental Example 1: Prevention of Precocious Puberty in Children

[0097] 1.1 Experimental Samples

[0098] The supernatants obtained in Examples 1-4 and Comparative Examples 1-11.

[0099] 1.2 Laboratory Animals and Grouping

[0100] Experimental animals and grouping: Pups were obtained by mating clean-grade non-pregnant SD rats. Female pups born within 5 days were used. All pups were weaned at 22 days of age and acclimatized for 4 days. Then, 26-day-old female SD rats weighing (60±10.00) g were randomly divided into 17 groups: control group, precocious puberty model group, and experimental groups (Examples 1-4 and Comparative Examples 1-11), with 8 pups in each group. They were housed at room temperature of 22-26℃, with daily light hours from 06:00 to 18:00 and humidity controlled at 60%-70%. They were fed standard feed (purchased from Spiford (Beijing) Biotechnology Co., Ltd.), with free access to food and water, and their bedding was changed every two days.

[0101] 1.3 Modeling and Drug Administration

[0102] Model establishment: From 26 days of age until the day the vaginal opening opened, except for the control group, the remaining rat pups were subcutaneously injected with 40 mg / kg / rat of methyl-DL-aspartic acid (NMA) (dissolved in 0.2 mL of physiological saline) at 14:00 and 16:00 daily. The control group was subcutaneously injected with 0.2 mL of physiological saline twice daily at the same time. Using the model group as a control, the opening of the vaginal opening of the rats was observed at 8:00 AM every day. When the vaginal opening of the model group rats opened, the injection of NMA was stopped for that rat. The injection of NMA in the other groups was stopped at a 1:1 ratio. The injection of physiological saline in the control group was stopped. The age of the rats on the day the vagina opened was recorded.

[0103] Drug administration: At the same time as modeling, the blank group and the model group were given 1 mL of physiological saline at 9:00 am every day, and the experimental group was given the corresponding sample by gavage. The dosage was converted to 9 mL / kg according to the human and animal body surface area reduction method, once a day.

[0104] 1.4 Time of discontinuation of drug administration and time of sacrifice

[0105] For rats in the model group with open vaginal openings, vaginal cell smears should be taken daily to observe changes in their estrous cycle. When the vaginal cell smears are placed under a microscope and are in the interestrous phase, drug administration should be stopped. Drug administration should be stopped in a 1:1 ratio for the other groups. When the number of rats with open vaginal openings in the model group exceeds half of the total number of animals in the group, the experiment should be stopped and the rats should be sacrificed.

[0106] 1.5 Indicator Testing

[0107] 1.5.1 Organ Index

[0108] After anesthetizing the animals with 10% chloral hydrate, the uterus and ovaries of the rats were removed. The residual blood was washed away with physiological saline, the excess physiological saline was wiped dry, and the rats were weighed. The organ coefficient of each group of rats was calculated according to the formula: Organ coefficient = Wet weight of organ / Body weight of rat on the day of sampling.

[0109] 1.5.2 Detection of serum sex hormones and gonadotropins in rats

[0110] After collecting blood from the orbital cavity, the blood was allowed to stand for 1 hour, then centrifuged at 3000 rpm for 10 minutes to separate the serum, which was then stored at -20℃ for later testing. The levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol (E2) in rat serum were measured according to the kit instructions. The kits were purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., with catalog numbers ml002860, ml403115, and ml002869, respectively.

[0111] 1.6 Experimental Results

[0112] 1.6.1 Vaginal opening status in rats

[0113] The development and function of the normal reproductive system in female mice are regulated by the hypothalamic-pituitary-gonadal axis (HPGA axis). The hypothalamus secretes GnRH, which acts on the anterior pituitary gland to secrete LH and FSH, promoting ovarian development and secreting E2 and progesterone. When E2 rises to a certain level, sexual development begins, manifested by the development of external genitalia and the opening of the vulva.

[0114] At the end of the experiment, 5 rats in the model group showed vaginal opening. The age distribution of the rats at the time of the first vaginal opening was 1 at 29 days old, 1 at 30 days old, and 3 at 31 days old, with an average age of (30.4±0.89) days. In the control group, 1 rat in the 9 control groups showed vaginal opening at 31 days old; the remaining rats did not show vaginal opening. Compared with the control group, the model group showed earlier vaginal opening, indicating that the precocious puberty model was successfully established. Compared with the model group, the vaginal opening time of Examples 1-4 and Control Groups 1-8 and 10-11 was significantly delayed.

[0115] 1.6.2 Organ Index Results

[0116] Central precocious puberty is significantly positively correlated with the ovarian coefficient and uterine coefficient. The higher the value, the more active the gonadal development and the greater the possibility and risk of precocious puberty. The ovarian coefficient can determine whether the ovary has started development, and the uterine coefficient can determine the intensity and duration of hormone effects.

[0117] Table 1. Organ index results (n=8, mean ± variance)

[0118]

[0119] Note: Compared with the blank group, +P<0.05, ++P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with Example 1, △P<0.05, △△P<0.01.

[0120] The organ index results for each group are shown in Table 1. Compared with the blank group, the uterine coefficient and ovarian coefficient of the model group were significantly increased, indicating that the establishment of the precocious puberty model led to a significant increase in uterine volume and ovarian mass. Compared with the model group, the uterine coefficient and ovarian coefficient of Examples 1-4 were significantly lower than those of the model group, indicating that the medicinal and edible fermented product provided by the present invention can delay the enlargement of the uterus and ovary in central precocious puberty rats.

[0121] Compared with Example 1, Comparative Example 1 replaced mulberry, which has the effect of tonifying kidney yin, with cornus officinalis, which has the same effect, and increased the proportion of cornus officinalis; Comparative Example 4 replaced mulberry with rehmannia glutinosa (which nourishes yin and tonifies the kidney). As shown in Table 1, the ovarian coefficient and uterine coefficient of Comparative Example 1 and Comparative Example 4 are significantly higher than those of Example 1, indicating that the fermentation products obtained by Comparative Example 1 and Comparative Example 4 have a worse effect on delaying the enlargement of the uterus and ovaries in rats with precocious puberty. The mulberry and cornus officinalis selected in this invention, which tonify kidney yin, can achieve better results in delaying the enlargement of the uterine coefficient and ovarian coefficient in rats with precocious puberty.

[0122] Comparative Example 2 used the traditional Chinese medicine composition disclosed in patent CN102114226B. As shown in Table 1, its ovarian and uterine coefficients were significantly higher than those in Example 1, indicating that the fermented product prepared in this invention is more effective than the traditional Chinese medicine composition disclosed in patent CN102114226B in alleviating uterine and ovarian enlargement in rats with precocious puberty.

[0123] The difference between Comparative Example 5 and Example 1 is that Cassia Seed, which also has the effects of clearing liver heat and relieving liver stagnation (nourishing liver yin), was used instead of Gardenia. As shown in Table 1, the uterine coefficient and ovarian coefficient of Comparative Example 5 were significantly higher than those of Example 1, indicating that Gardenia, which nourishes liver yin, selected in this invention, together with other raw materials, can best delay the enlargement of the uterus and ovaries in precocious rats.

[0124] The difference between Comparative Example 3 and Example 1 is that it does not include Asparagus, a raw material for nourishing lung yin; the difference between Comparative Example 6 and Example 1 is that Ophiopogon japonicus, which has the same effects of moistening lung dryness, clearing deficiency heat, and nourishing lung yin, is used instead of Asparagus. As shown in Table 1, the ovarian and uterine coefficients of Comparative Example 6 and Comparative Example 3 are significantly higher than those of Example 1, indicating that the formula containing medicinal and edible ingredients lacks Chinese medicine with the effects of nourishing lung yin, moistening lung dryness, and clearing deficiency heat, thus reducing the formula's effect on alleviating ovarian and uterine enlargement. Adding raw materials for nourishing lung yin can significantly delay the increase in uterine and ovarian coefficients in precocious puberty rats. Asparagus selected in this invention, together with other medicinal and edible raw materials, can comprehensively nourish kidney yin, lung yin, and liver yin, and is the best at alleviating ovarian and uterine enlargement in precocious puberty rats.

[0125] The difference between Comparative Example 7 and Example 1 is that some of the Poria cocos was replaced with Dioscorea opposita, which has the same spleen-strengthening, phlegm-resolving, and spleen-yin-tonifying effects. As shown in Table 1, the effect of Comparative Example 7 was significantly lower than that of Example 1. This means that although the replaced Chinese medicine has the same efficacy, the effect on alleviating uterine and ovarian enlargement in rats with precocious puberty is completely different. This indicates that the Poria cocos and other food-medicine homologous raw materials selected in this invention have a synergistic effect. When Cornus officinalis, Morus alba, Gardenia jasminoides, Poria cocos, and Asparagus cochinchinensis are combined, they have a better effect on reducing the uterine and ovarian coefficients in rats with precocious puberty, thereby alleviating the symptoms of precocious puberty.

[0126] The difference between Comparative Example 8 and Example 1 is that *Lactobacillus plantarum* YSH08 was replaced with *Lactobacillus plantarum* JYLP-326. The difference between Comparative Example 10 and Example 1 is that *Lactobacillus reuteri* XS82 was replaced with *Lactobacillus rhamnosus* NSL0401. As shown in Table 1, the ovarian and / or uterine coefficients of Comparative Examples 8 and 10 were significantly higher than those of Example 1, indicating that the five medicinal and edible raw materials selected in this invention, after being fermented by mixed bacteria of *Lactobacillus plantarum* YSH08 and *Lactobacillus reuteri* XS82, have a better effect on alleviating ovarian and uterine enlargement in rats with precocious puberty.

[0127] The difference between Comparative Example 9 and Example 1 is that the five medicinal and edible raw materials were not enzymatically hydrolyzed or fermented. As shown in Table 1, the ovarian and uterine coefficients of Comparative Example 9 are significantly higher than those of Example 1, indicating that the enzymatic hydrolysis and fermentation technologies selected in this invention can enrich more of the effective components in the five medicinal and edible raw materials, thereby enhancing the efficacy and alleviating the symptoms of precocious puberty. The difference between Comparative Example 11 and Example 1 is that the enzyme preparation only contains cellulase and pectinase. As shown in Table 1, its uterine coefficient is significantly higher than that of Example 1, indicating that the mixed use of the four enzymes (cellulase, pectinase, hemicellulose, and tanninase) selected in this invention can fully dissolve the effective components in the medicinal and edible raw materials, achieving a better effect in alleviating precocious puberty.

[0128] 1.6.3 Hormone Levels

[0129] The anterior pituitary gland in the brain secretes luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which work together on the ovaries to promote the development and maturation of female follicles and trigger ovulation, playing a very important role in maintaining normal human reproductive function. Estradiol (E2) is the main component of estrogen, mainly secreted by ovarian follicle cells or increased exogenous uptake. It has the function of participating in the development and maturation of female reproductive organs and promoting the development of secondary sexual characteristics, and plays a key role in the regulation of the HPGA axis. High E2 levels are the main factors that induce precocious puberty and premature breast development in girls.

[0130] The specific sex hormone levels of each group are shown in Table 2. When the gonadal function of rats is activated, under the regulation of the anterior pituitary gland, the secretion of LH and FSH increases significantly, which acts on the gonadal tissue, increases the secretion of E2, and promotes the development of the gonads. Compared with the blank group, the LH, FSH, and E2 levels of the model group are significantly higher than those of the blank group, indicating that the precocious puberty model has been successfully constructed. Compared with the model group, the serum LH, FSH, and E2 levels of Examples 1-4 are significantly reduced, indicating that the medicinal and edible fermentation product provided by this invention can regulate the sex hormone levels of precocious puberty rats and alleviate the symptoms of precocious puberty.

[0131] Compared with Example 1, Comparative Example 1 replaced mulberry with Cornus officinalis, which also has the effect of tonifying kidney yin, and Comparative Example 4 replaced mulberry with Rehmannia glutinosa, which also has the effect of tonifying kidney yin. As shown in Table 2, the serum levels of LH, FSH and E2 in Comparative Example 1 and Comparative Example 4 were significantly higher than those in Example 1. This indicates that mulberry and Cornus officinalis, as kidney-tonifying drugs in the formula of food and medicine homology, can better reduce the levels of sex hormones LH, FSH and E2 in rats with precocious puberty by working together with other raw materials.

[0132] Comparative Example 2 used the traditional Chinese medicine composition disclosed in patent CN102114226B. As shown in Table 2, its serum LH, FSH, and E2 levels were significantly higher than those in Example 1, indicating that the fermentation product prepared in this invention is more effective than the traditional Chinese medicine composition disclosed in patent CN102114226B in alleviating serum LH, FSH, and E2 levels in rats with precocious puberty.

[0133] The difference between Comparative Example 5 and Example 1 is that Gardenia was replaced with Cassia Seed, which also has the effects of clearing liver heat and relieving liver stagnation (nourishing liver yin). As shown in Table 2, the serum LH, FSH and E2 levels of Comparative Example 5 were significantly higher than those of Example 1, indicating that the Gardenia selected in this invention, in combination with Cornus officinalis, Morus alba, Poria cocos and Asparagus cochinchinensis, has a better effect on reducing sex hormone levels.

[0134] The difference between Comparative Example 3 and Example 1 is that it does not include Asparagus. The difference between Comparative Example 6 and Example 1 is that Ophiopogon japonicus, which has the same effects of moistening lung dryness, clearing deficiency heat, and nourishing lung yin, is used instead of Asparagus. As shown in Table 2, the levels of sex hormones LH, FSH, and E2 in Comparative Example 3 and Comparative Example 6 are significantly higher than those in Example 1. This indicates that the formula containing both medicinal and edible herbs lacks Chinese medicine with the effects of nourishing lung yin, moistening lung dryness, and clearing deficiency heat, thus reducing the effect of the formula in alleviating the levels of sex hormones LH, FSH, and E2. In contrast, the Asparagus and other raw materials selected in this invention can work synergistically to reduce the levels of sex hormones LH, FSH, and E2 in rats with precocious puberty.

[0135] The difference between Comparative Example 7 and Example 1 is that Poria cocos was replaced with Poria cocos and Dioscorea opposita. As shown in Table 2, the levels of sex hormones LH, FSH, and E2 in Comparative Example 7 were significantly higher than those in Example 1. This indicates that even Chinese medicines with the same efficacy can have different effects when used in a formula. In contrast, the Poria cocos used in this invention, in combination with Cornus officinalis, Morus alba, Gardenia jasminoides, and Asparagus cochinchinensis, can better reduce the levels of sex hormones LH, FSH, and E2 in rats with precocious puberty and alleviate the symptoms of precocious puberty.

[0136] The difference between Comparative Example 8 and Example 1 is that *Lactobacillus plantarum* YSH08 was replaced with *Lactobacillus plantarum* JYLP-326. The difference between Comparative Example 10 and Example 1 is that *Lactobacillus reuteri* XS82 was replaced with *Lactobacillus rhamnosus* NSL0401. The difference between Comparative Example 9 and Example 1 is that the five medicinal and edible raw materials were not enzymatically hydrolyzed or fermented. The difference between Comparative Example 11 and Example 1 is that the enzyme preparation only includes cellulase and pectinase. As shown in Table 2, the levels of sex hormones LH, FSH, and E2 in Comparative Examples 8-11 were significantly higher than those in Example 1. This indicates that the fermentation technology, fermentation strains, enzymatic hydrolysis technology, and enzyme preparation selection all affect the regulatory effect of fermentation products on sex hormones LH, FSH, and E2 in precocious puberty rats. The four enzyme preparations (cellulase, hemicellulase, pectinase, and tanninase) and *Lactobacillus plantarum* YSH08 and *Lactobacillus reuteri* XS82 selected in this invention can maximize the release of active ingredients and achieve a better effect in regulating sex hormone levels.

[0137] In summary, the medicinal and edible raw materials selected in this invention, namely Cornus officinalis, mulberry, gardenia, Poria cocos, and Asparagus cochinchinensis, are synergistically combined and enzymatically hydrolyzed by a mixture of four enzymes: cellulase, hemicellulase, pectinase, and tanninase. The resulting fermentation product obtained by Lactobacillus plantarum YSH08 and Lactobacillus reuteri XS82 can delay the opening of the vaginal opening in NMA-induced CPP model rats and reduce the levels of sex hormones LH, FSH, and E2, thereby regulating central precocious puberty.

[0138] Table 2 Hormone level results for each group

[0139]

[0140] Note: Compared with the blank group, +P<0.05, ++P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with Example 1, △P<0.05, △△P<0.01.

[0141] Experimental Example 2: Spleen-strengthening effect

[0142] 2.1 Experimental Samples

[0143] The supernatants obtained in Examples 1-4 and Comparative Examples 1-11.

[0144] 2.2 Experimental animals, model construction, and grouping for drug administration

[0145] Animals: 5-week-old healthy male SD rats (SPF grade, body weight 130-150g) were used. The rats were kept at room temperature of 20-25℃ and humidity of (50±10)% for 12 / 12 hours of light / dark cycle, with free access to food and water. The experiment began after 7 days of acclimatization.

[0146] Model replication: Rats were randomly divided into a control group and a model group according to their body weight. The control group (n=8) was fed a normal diet daily, while the model group was fed a high-fat diet daily (diet formula: 69% normal diet, 15% lard, 15% whole milk powder, and 1% cholesterol) for four weeks. The control group's cages were filled with normal bedding, while the model group's cages were filled with normal bedding, then an appropriate amount of distilled water was added and stirred to create a moist bedding to simulate "living in a wetland". On odd-numbered days, each rat was given 2 mL of lard by gavage, and on even-numbered days, rats were given 20 mL / kg of 4°C cold water by gavage to simulate "irregular eating habits, excessive consumption of fatty, sweet, and cold foods", for 8 weeks (the high-fat diet was continued during this period). Throughout the experiment, the rats' food and water intake, physical and mental condition, fecal texture, fur color, and activity were observed daily. After 12 weeks of modeling, the model group rats exhibited obesity, greasy fur, lethargy, and slow movement. In addition, blood samples were collected from the orbital venous plexus of rats and tested. The results showed that the plasma levels of TC, TG, and LDL-C in the model rats increased, while the HDL-C level decreased, and there were significant differences compared with the blank group rats (P<0.05), indicating that the model was successfully established.

[0147] Grouping and administration: The successfully modeled rats were divided into the model group, Examples 1-4 and Comparative Examples 1-11, with 8 rats in each group. The rats were administered the drug for 4 consecutive weeks. The dosage was 9 mL / kg based on the body surface area. During the administration period, the rats had free access to food and water.

[0148] 2.3 Indicator Testing

[0149] 2.3.1 Blood Indicators

[0150] After four weeks of drug administration, rats were anesthetized by intraperitoneal injection of 20% urethane aqueous solution (0.3 mL / 100 g). Arterial blood was collected from the abdominal aorta and centrifuged at 3000 r / min for 10 min to detect the plasma TC, TG, LDL-C, and HDL-C levels (purchased from Nanjing Jiancheng Biotechnology Institute, catalog numbers A111-1-1, A110-1-1, A113-1-1, and A112-1-1).

[0151] 2.3.2 Immunohistochemical detection

[0152] The colon was dissected from rats, cleaned with physiological saline, dried with absorbent paper, and fixed with 4% paraformaldehyde solution. After fixation, cross-sectional sections of the colon were obtained, dehydrated with ethanol, cleared with xylene, and impregnated with paraffin. The paraffin-impregnated tissue was embedded in an embedding machine, then sectioned, and dewaxed in water. The sections were boiled in citrate buffer for 20 minutes, washed with water, treated with hydrogen peroxide for 10 minutes, rinsed with PBS buffer, treated with goat serum for 10 minutes, removed, incubated with diluted primary antibody for 2 hours, rinsed with PBS buffer, incubated with secondary antibody for 15 minutes, rinsed with PBS buffer, developed with DAB, washed with water, counterstained with hematoxylin, dehydrated, cleared, and mounted. The distribution of positive expression by immunohistochemical staining was observed under an optical microscope, and three fields of view were photographed under a 40x objective lens. The positive expression area (area) and absorbance (A) were measured using an Image Pro Plus 5.1 image analysis system. Positive signals were brown; the deeper the brown color, the more significant the aquaporin expression.

[0153] 2.4 Experimental Results

[0154] 2.4.1 Behavioral Indicators

[0155] During the experiment, no significant changes were observed in the coat color, urination and defecation, body shape, mental state, and activity of the control group rats, and their body weight steadily increased. However, compared with the control group, the model group rats had greasy and yellowish fur, loose and watery stools, were lethargic, preferred to curl up, arch their backs, and lie still. They were obese but had reduced food intake. This was due to spleen deficiency and impaired function of transporting and transforming fluids, leading to fluid stagnation in the body and the production of pathological products such as phlegm and fluid retention, resulting in diarrhea and other symptoms. However, after drug treatment, compared with the model group, the loose stools of the rats in Examples 1-4 improved, and they became more active, no longer curled up, and more restless; the rats in Comparative Examples 1-11 still showed loose stools.

[0156] 2.4.2 Blood Indicator Results

[0157] Traditional Chinese medicine (TCM) categorizes dyslipidemia under the "phlegm-dampness" syndrome, with its pathogenesis located in the blood vessels. Spleen deficiency weakens the body's ability to transform and transport fluids, leading to phlegm obstruction, which is the fundamental pathogenesis of dyslipidemia. Elevated levels of TC, TG, and LDL-C in plasma, and decreased levels of HDL-C, can serve as microscopic indicators for differentiating phlegm-dampness syndrome; blood lipid levels can also be used as microscopic indicators to assess the efficacy of medications for treating phlegm-dampness syndrome and to monitor disease progression. Therefore, this invention evaluates the efficacy of treatments for phlegm-dampness syndrome by observing plasma TC, TG, LDL-C, and HDL-C levels. The results of plasma TC, TG, LDL-C, and HDL-C levels for each group are shown below. Figure 1-4 ,Depend on Figure 1-4It can be seen that, compared with the blank group, the levels of TC, TG, and LDL-C in the model group were significantly increased, while the level of HDL-C was significantly decreased, indicating that the modeling was successful. Compared with the model group, the levels of TC, TG, and LDL-C in Examples 1-4 were significantly decreased, while the level of HDL-C was significantly increased, indicating that Examples 1-4 have a certain alleviating effect on spleen deficiency and phlegm-dampness syndrome.

[0158] Compared with Example 1, Comparative Example 3 does not include asparagus, and is composed of... Figure 1-4 It can be seen that the plasma TC, TG, and LDL-C levels in the three comparative groups were significantly higher than those in Example 1, while the HDL-C content was significantly lower than that in Example 1. This indicates that the five Chinese herbs in the herbal formula are indispensable. When the five medicinal and edible raw materials selected in this invention (Cornus officinalis, Morus alba, Gardenia jasminoides, Poria cocos, and Asparagus cochinchinensis) are combined together, the effect of relieving spleen deficiency and phlegm-dampness syndrome is the best.

[0159] Compared with Example 1, Comparative Example 1 replaced mulberry, which has the effect of tonifying kidney yin, with cornus officinalis, which has the same effect; Comparative Example 4 replaced mulberry with rehmannia glutinosa; Comparative Example 5 replaced gardenia with cassia seed, which has the effect of tonifying liver yin; Comparative Example 6 replaced asparagus root with ophiopogon japonicus, which has the effect of tonifying lung yin; and Comparative Example 7 replaced poria cocos, which has the same effect of invigorating spleen qi, resolving phlegm and dampness, and tonifying spleen yin, with poria cocos and yam. Figure 1-4 It can be seen that the plasma TC, TG, and LDL-C levels of Comparative Examples 1 and 4-7 were significantly higher than those of Example 1, while the HDL-C content was significantly lower than that of Example 1. This indicates that even if they have the same efficacy, the combined effect of these ingredients on relieving spleen deficiency and phlegm-dampness syndrome is completely different due to the synergistic and mutually exclusive phenomena among them. The five medicinal and edible ingredients selected in this invention—Cornus officinalis, Morus alba, Gardenia jasminoides, Poria cocos, and Asparagus cochinchinensis—have a synergistic effect. When combined, they have a better effect on reducing TC, TG, and LDL-C levels and increasing HDL-C levels, thereby relieving spleen deficiency and phlegm-dampness syndrome.

[0160] Comparative Example 2 uses the traditional Chinese medicine composition disclosed in patent CN102114226B, which is composed of... Figure 1-4 It can be seen that its TC, TG, and LDL-C levels are significantly higher than those of Example 1, while its HDL-C content is significantly lower than that of Example 1, indicating that the fermentation product prepared by the present invention has a greater advantage in relieving spleen deficiency and phlegm-dampness syndrome.

[0161] The difference between Comparative Example 8 and Example 1 is that *Lactobacillus plantarum* YSH08 was replaced with *Lactobacillus plantarum* JYLP-326, and in Comparative Example 10, *Lactobacillus reuteri* XS82 was replaced with *Lactobacillus rhamnosus* NSL0401. Figure 1-4The results showed that the levels of TC, TG, and LDL-C in Comparative Examples 8 and 10 were significantly higher than those in Example 1, while the HDL-C content was significantly lower than that in Example 1. This indicates that the five medicinal and edible raw materials selected in this invention, after being fermented by mixed bacteria of Lactobacillus plantarum YSH08 and Lactobacillus reuteri XS82, have a better effect in relieving spleen deficiency and phlegm-dampness syndrome.

[0162] The difference between Comparative Example 9 and Example 1 is that the five medicinal and edible raw materials were not enzymatically hydrolyzed or fermented; the difference between Comparative Example 11 and Example 1 is that the enzyme preparation only includes cellulase and pectinase, and is derived from... Figure 1-4 The results showed that the levels of TC, TG, and LDL-C in the plasma of Comparative Examples 9 and 11 were significantly higher than those in Example 1, while the HDL-C content was significantly lower than that in Example 1. This indicates that the fermentation technology, enzymatic hydrolysis technology, and enzyme preparations selected in this invention can enrich the effective components of the five traditional Chinese medicines, enhance their efficacy, and alleviate the symptoms of spleen deficiency and phlegm dampness.

[0163] 2.4.3 Immunohistochemical detection results

[0164] The spleen governs the transformation and transportation of fluids and the essence of food. If the spleen is weak and its function is impaired, the transformation and transportation of fluids and the essence of food will fail, leading to the inability of fluids to be properly distributed, resulting in symptoms such as dry mouth and thirst. This fluid retention and dampness can condense into phlegm, forming a spleen deficiency with phlegm-dampness syndrome. Aquaporins (AQPs), which regulate water transmembrane transport, are biological membrane channel proteins widely found in various human tissues and cells. Normal AQP expression may be the molecular biological basis for the spleen's function of transformation and transportation. Specifically, the expression level of AQP3 on the endothelial cells of the colonic basement membrane is related to intestinal water absorption. High AQP3 expression can increase water reabsorption in the colonic mucosa, leading to constipation, while low AQP3 expression can decrease water reabsorption in the colonic mucosa, leading to diarrhea or loose stools. Therefore, this invention uses AQP3 expression as an indicator of spleen function.

[0165] The expression of AQP3 in each group is shown in the figure. Figure 5 ,Depend on Figure 5 It can be seen that, compared with the blank group, the expression of AQP3 in the model group was significantly reduced. Low expression of AQP3 leads to loose stools, which is consistent with the results of behavioral indicators. Compared with the model group, the expression of AQP3 in the Example 1-4 groups was significantly increased, with no significant difference from the blank group. This indicates that the present invention selects medicinal and food homologous fermented products to increase the spleen's function of transportation and transformation by increasing the expression of AQP3, thereby achieving the effect of strengthening the spleen.

[0166] Compared to Example 1, Comparative Example 3 does not include asparagus, and is composed of... Figure 5The results showed that the AQP3 expression in the three comparative groups was significantly lower than that in Example 1, indicating that the five raw materials in the formula of food and medicine homology are indispensable. When the five food and medicine homology raw materials selected in this invention, namely Cornus officinalis, mulberry, gardenia, poria cocos and asparagus, are combined together, they can synergistically alleviate spleen deficiency and phlegm dampness syndrome to the greatest extent.

[0167] Comparative Example 2 uses the traditional Chinese medicine composition disclosed in patent CN102114226B, which is composed of... Figure 5 The results showed that its AQP3 expression was significantly lower than that of Example 1, indicating that the fermentation product prepared by the present invention has a better effect in relieving spleen deficiency and phlegm-dampness syndrome. The composition of Comparative Example 2 is mainly for yin deficiency and fire excess type of precocious puberty, while the fermentation product prepared by the present invention has more advantages in phlegm-dampness syndrome.

[0168] The difference between Comparative Example 1 and Example 1 is that mulberry is replaced with Cornus officinalis; the difference between Comparative Example 4 and Example 1 is that Rehmannia glutinosa is replaced with mulberry; the difference between Comparative Example 5 and Example 1 is that Cassia tora is replaced with Gardenia jasminoides; the difference between Comparative Example 6 and Example 1 is that Ophiopogon japonicus is replaced with Asparagus cochinchinensis; the difference between Comparative Example 7 and Example 1 is that Poria cocos is replaced with Poria cocos and Dioscorea opposita. Figure 5 It can be seen that the AQP3 of Comparative Examples 1 and 4-7 was significantly lower than that of Example 1, indicating that the fermented products obtained by Comparative Examples 1 and 4-7 had a worse effect on relieving spleen deficiency and phlegm-dampness syndrome than the fermented product obtained by Example 1. This shows that the five medicinal and edible raw materials selected in this invention have a synergistic effect. When Cornus officinalis, mulberry, gardenia, Poria cocos, and Asparagus cochinchinensis are combined, they have a better effect on relieving spleen deficiency and phlegm-dampness syndrome and achieve the effect of strengthening the spleen.

[0169] The difference between Comparative Example 8 and Example 1 is that *Lactobacillus plantarum* YSH08 was replaced with *Lactobacillus plantarum* JYLP-326, and the difference between Comparative Example 10 and Example 1 is that *Lactobacillus reuteri* XS82 was replaced with *Lactobacillus rhamnosus* NSL0401. Figure 5 It can be seen that the AQP3 expression in Comparative Examples 8 and 10 was significantly lower than that in Example 1, indicating that the five medicinal and edible herbs selected in this invention, after fermentation with Lactobacillus plantarum YSH08 and Lactobacillus reuteri XS82, have a better effect in relieving spleen deficiency and phlegm-dampness syndrome.

[0170] The difference between Comparative Example 9 and Example 1 is that the five medicinal and edible herbs were only extracted by ultrasound, without enzymatic hydrolysis or fermentation. Figure 5 The results showed that the AQP3 expression was significantly lower than that in Example 1, indicating that the enzymatic hydrolysis and fermentation technologies selected in this invention can enrich more of the effective components in the five kinds of medicinal and edible herbs, thereby improving the efficacy and relieving the symptoms of spleen deficiency and phlegm dampness.

[0171] The difference between Comparative Example 11 and Example 1 is that the enzyme preparation only includes cellulase and pectinase, which are derived from... Figure 5The results showed that the AQP3 expression was significantly lower than that in Example 1, indicating that the enzyme preparations selected in this invention are superior. The four enzyme preparations (cellulase, hemicellulase, pectinase, and tanninase) enabled more effective components of the food-medicine homologous raw materials to be dissolved, thereby relieving the symptoms of spleen deficiency and phlegm dampness.

[0172] In summary, the medicinal and edible fermented product provided by this invention treats central precocious puberty in children from four perspectives: nourishing lung yin, liver yin, kidney yin, and spleen yin. The selected ingredients—asparagus (for nourishing lung yin), gardenia (for nourishing liver yin), cornus officinalis and mulberry (for nourishing kidney yin), and poria cocos (for nourishing spleen yin)—have a synergistic effect, jointly achieving optimal reduction of sex hormone levels in rats with precocious puberty, thus achieving better therapeutic effects for central precocious puberty in children. Furthermore, the aforementioned medicinal and edible fermented product has been verified to also treat spleen deficiency and phlegm-dampness syndrome, and can regulate dyslipidemia and AQP3 expression caused by spleen deficiency and phlegm-dampness.

[0173] 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 fermented food-medicine homologous product for regulating the spleen and stomach and / or preventing precocious puberty in children, characterized in that, The raw materials, by weight, include 15-25 parts of Cornus officinalis, 10-30 parts of mulberry, 10-20 parts of gardenia, 20-40 parts of Poria cocos, 10-35 parts of Asparagus cochinchinensis, 5-12 parts of enzyme preparation, and 0.06-0.15 parts of fermentation agent.

2. The fermented product of medicinal and edible origin according to claim 1, characterized in that, The raw materials, by weight, include 20-25 parts of Cornus officinalis, 15-30 parts of mulberry, 15-20 parts of gardenia, 20-35 parts of Poria cocos, 10-25 parts of Asparagus cochinchinensis, 6-10 parts of enzyme preparation, and 0.08-0.13 parts of fermentation agent.

3. The fermented product of medicinal and edible origin according to claim 1, characterized in that, The enzyme preparation includes cellulase, hemicellulase, pectinase and tanninase; the weight ratio of cellulase, hemicellulase, pectinase and tanninase is (3-8):(2-5):(1-7):(0.5-3).

4. The fermented product of medicinal and edible origin according to claim 1, characterized in that, The fermentation agent includes *Lactobacillus plantarum* and *Lactobacillus reuteri*.

5. The fermented product of medicinal and edible origin according to claim 4, characterized in that, The *Lactobacillus plantarum* is *Lactobacillus plantarum* YSH08, with accession number CGMCC No. 36182; the *Lactobacillus reuteri* is *Lactobacillus reuteri* XS82, with accession number CGMCC No. 36181. The weight ratio of *Lactobacillus plantarum* YSH08 to *Lactobacillus reuteri* XS82 is (2-4):(1-3).

6. A method for preparing a medicinal and edible fermented product for regulating the spleen and stomach and / or preventing precocious puberty in children as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Mix the medicinal and edible raw materials asparagus, gardenia, poria, cornus officinalis and mulberry with water to obtain a mixture; (2) Add an enzyme preparation to the mixture and perform ultrasonic enzymatic hydrolysis and sterilization to obtain a treated solution; (3) Inoculate the treatment liquid with a fermentation agent for fermentation, separate the solid and liquid, and the supernatant is the fermentation product.

7. The preparation method according to claim 6, characterized in that, In step (1), the ratio of medicinal and edible raw materials to water is 1:(15-30).

8. The preparation method according to claim 6, characterized in that, In step (2), the ultrasonic enzymatic hydrolysis is performed under ultrasonic conditions, with an ultrasonic power of 180-220W and an ultrasonic frequency of 25-30kHz; the enzymatic hydrolysis temperature is 40-50℃ and the enzymatic hydrolysis time is 1-2h; the sterilization temperature is 70-90℃ and the sterilization time is 20-40min.

9. The preparation method according to claim 6, characterized in that, In step (3), the fermentation temperature is 30-42℃ and the fermentation time is 24-84h.

10. The application of the medicinal and edible fermented product according to any one of claims 1-5 or the medicinal and edible fermented product obtained by any one of claims 6-9 in the preparation of products for preventing precocious puberty in children and products for strengthening the spleen.

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