Probiotic medicinal and edible medicinal material composition as well as preparation method and application thereof

Abstract

The invention discloses a probiotic medicinal and edible medicinal material composition as well as a preparation method and application thereof. The composition comprises combined probiotics and medicinal and edible medicinal materials, the combined probiotics comprise Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002, and the medicinal and edible medicinal materials comprise cistanche deserticola, semen raphani, peach kernels, astragalus membranaceus, codonopsis pilosula and pericarpium citri reticulatae. The composition takes medicinal and edible medicinal materials as raw materials, has the effect of improving endocrine hormone disorder caused by chronic stress after being fermented by combined probiotics, and can be applied to preparation of products for improving endocrine hormone disorder caused by chronic stress; the composition is especially applied to preparation of products for reversing reduction of thyroid stimulating hormone release hormone, adrenocorticotropic hormone and / or testosterone secretion level caused by chronic stress, and meanwhile, the composition also meets the requirements of people for resisting fatigue and improving constipation due to intestinal dryness.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine fermentation technology, and more specifically, relates to a probiotic food-medicine composition, its preparation method and application. Background Technology

[0002] With the accelerating pace of modern life, the prevalence of sub-health has become a significant public health issue, among which kidney yang deficiency is a common symptom in Traditional Chinese Medicine (TCM). TCM believes that prolonged physiological or psychological stress (chronic stress) depletes the body's vital energy, especially "kidney essence" and "kidney yang," potentially leading to or exacerbating kidney yang deficiency. Clinical characteristics of kidney yang deficiency include aversion to cold, cold limbs, fatigue, decreased bodily functions, and functional gastrointestinal dysfunction, which are closely related to chronic fatigue, mild adrenal insufficiency, and reproductive endocrine disorders in modern medicine.

[0003] In modern medical research, dysfunction of the hypothalamus-pituitary-adrenal (HPA) axis is considered the core pathological basis of the TCM syndrome of "kidney yang deficiency." Chronic stress may affect the function of the HPA axis, leading to endocrine hormone secretion disorders. For example, under chronic stress, hypothalamic function is suppressed, resulting in a decrease in both the basal secretion and pulsatile secretion frequency of thyrotropin-releasing hormone (TRH). In the early stages of stress, adrenocorticotropic hormone (ACTH) rises sharply to cope with the stress. However, under long-term chronic stress, due to the negative feedback effect of glucocorticoids, the pituitary gland becomes less responsive to CRH, exhibiting a "desensitization" phenomenon. Chronic stress can also inhibit the release of gonadotropins (LH / FSH) through the HPA axis, leading to a decrease in sex hormone secretion. This is similar to the symptoms commonly seen in TCM "kidney yang deficiency," such as endocrine disorders, decreased immunity, and slowed energy metabolism.

[0004] Traditional Chinese medicine often achieves good results in improving kidney yang deficiency by "warming and tonifying kidney yang." For example, the core function of Jichuan Decoction is to warm the kidney and replenish essence, moisten the intestines and promote bowel movements, and it is mainly used to treat constipation caused by kidney deficiency and insufficient yang. The classic formula of Jichuan Decoction consists of Angelica sinensis, Achyranthes bidentata, Cistanche deserticola, Alisma plantago-aquatica, Cimicifuga foetida, and Citrus aurantium. However, Angelica sinensis, Achyranthes bidentata, Alisma plantago-aquatica, Cimicifuga foetida, and Citrus aurantium are common medicinal materials with relatively low safety. For example, long-term or excessive use of Alisma plantago-aquatica may damage liver and kidney function, and common side effects include gastrointestinal discomfort such as nausea, vomiting, abdominal pain, and diarrhea; Cimicifuga foetida has a certain degree of toxicity, and excessive use may lead to neurological symptoms such as dizziness, tremors, and limb contractures; Citrus aurantium has alkaloid toxicity, and large doses may cause gastrointestinal distension and excessive salivation. Therefore, the use of Jichuan Decoction must be under the guidance of a professional physician and modified according to the patient's specific condition. However, how to improve the safety of Jichuan Decoction while ensuring its efficacy is a difficult problem that needs to be solved in current research on traditional Chinese medicine. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a probiotic-based medicinal and edible herbal composition, its preparation method, and its application. The aim is to discover that replacing the traditional Chinese medicinal materials in the Jichuan Decoction formula with medicinal and edible herbs (radish seed, peach kernel, astragalus, codonopsis, and tangerine peel), and combining them with probiotics (preserved strains Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002) during fermentation and processing, can improve the safety of the traditional Jichuan Decoction while ensuring or enhancing its efficacy. This solves the technical problem that the safety of the traditional Jichuan Decoction still needs improvement.

[0006] To achieve the above objectives, according to a first aspect of the present invention, a probiotic-medicinal-food homology composition is provided, comprising a combination of probiotics and medicinal-food homology herbs; the combination of probiotics includes Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002, wherein the accession number of Lactobacillus plantarum JY001 is CCTCC NO:M 2025584, and the accession date is March 25, 2025; the accession number of Lactobacillus plantarum JY002 is CCTCC NO:M 2025585, and the accession date is March 25, 2025; the medicinal-food homology herbs include Cistanche deserticola, radish seed, peach kernel, Astragalus membranaceus, Codonopsis pilosula, and tangerine peel.

[0007] Preferably, the probiotic-medicinal-food homology composition includes 30 parts of Cistanche deserticola, 30 parts of radish seed, 15 parts of peach kernel, 20 parts of Astragalus membranaceus, 20 parts of Codonopsis pilosula, and 10 parts of dried tangerine peel.

[0008] Preferably, in the probiotic-medicinal-food homology composition, the viable count of the combined probiotics is 10. 8 CFU / ml or 10 8 CFU / g or higher.

[0009] According to a second aspect of the present invention, a combination probiotic is also provided, comprising Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002, wherein the accession number of Lactobacillus plantarum JY001 is CCTCC NO:M 2025584 and the accession date is March 25, 2025; and the accession number of Lactobacillus plantarum JY002 is CCTCC NO:M 2025585 and the accession date is March 25, 2025.

[0010] Preferably, the combined probiotics are Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002.

[0011] According to a third aspect of the present invention, a method for preparing a probiotic food-medicinal material composition as described in the present invention is also provided, comprising the following steps: The mixture was obtained by fermentation using medicinal and edible materials from Jichuan Decoction as the culture medium and combined probiotics Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002 as seed bacteria. The accession number of Lactobacillus plantarum JY001 is CCTCC NO:M 2025584; the accession number of Lactobacillus plantarum JY002 is CCTCC NO:M 2025585; the medicinal and edible materials of Jichuan Decoction include 30 parts of Cistanche deserticola, 30 parts of radish seed, 15 parts of peach kernel, 20 parts of Astragalus membranaceus, 20 parts of Codonopsis pilosula, and 10 parts of dried tangerine peel.

[0012] Preferably, the preparation method predicts the number of viable bacteria after fermentation of the combined probiotics according to the following formula: Y=-29.05+0.889X1+1.47X2+0.65X3-0.002X1X2-0.0068X1X3-0.0066X2X3-0.05X1 2 -0.021X2 2 -0.008X3 2 ; In the formula: Y is the number of viable bacteria, 10 9 CFU / g; X1 is the inoculum amount, %; X2 is the fermentation time, h; X3 is the fermentation temperature, ℃.

[0013] Preferably, in the preparation method, the fermentation is carried out at 25-35°C for 18-30 hours with an inoculation amount of 2%-8% of the combined probiotics.

[0014] According to a fourth aspect of the present invention, an application is also provided of a probiotic food-medicinal material composition prepared according to the method described in the present invention, wherein the probiotic food-medicinal material composition is used in the preparation of a product for improving endocrine hormone imbalance caused by chronic stress; wherein the endocrine hormones are endocrine hormones regulated by the hypothalamic-pituitary-target gland axis, including thyrotropin-releasing hormone, adrenocorticotropic hormone and testosterone.

[0015] Preferably, the application refers to the use of the probiotic food-grade medicinal material composition in the preparation of a product for reversing the decrease in secretion levels of thyrotropin-releasing hormone, adrenocorticotropic hormone, and / or testosterone caused by chronic stress.

[0016] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: The probiotic-medicinal herb composition provided by this invention comprises a combination of probiotics and medicinal herbs of the same origin. The combined probiotics include *Lactobacillus plantarum* JY001 and *Lactobacillus plantarum* JY002. The medicinal herbs of the same origin retain the principal herb *Cistanche deserticola* from the traditional Jichuan Decoction formula, and transform the original "kidney-warming" effect into a "qi-tonifying" effect as the main or auxiliary principle. This improves the safety of the composition while simultaneously enhancing the efficacy of the probiotics. Chronic stress model experiments have confirmed that it not only reverses the decrease in thyrotropin-releasing hormone and testosterone secretion levels caused by chronic stress, but also improves the behavioral behavior of model mice to a level comparable to that of the traditional Jichuan Decoction. By combining medicinal herbs of the same origin with probiotics, the safety of the traditional Jichuan Decoction is improved while maintaining its efficacy, achieving food-grade safety. This approach balances the efficacy and safety of the traditional Jichuan Decoction, avoiding the reduction in efficacy that occurs when replacing traditional Jichuan Decoction herbs with medicinal herbs of the same origin.

[0017] Using this combination of probiotics as the fermentation strain, the Jichuan Decoction, a combination of medicinal and edible herbs, after fermentation, has the effect of reversing the decrease in secretion levels of thyrotropin-releasing hormone, adrenocorticotropic hormone, and / or testosterone caused by chronic stress. It can be applied to the preparation of products for improving endocrine hormone imbalance caused by chronic stress.

[0018] In addition, this probiotic food and medicine combination uses Cistanche deserticola, Astragalus membranaceus, Codonopsis pilosula and other Chinese medicinal and food homologous herbs as the main raw materials. It can be produced as a functional fermented beverage by artificial inoculation and pure culture, and the combination of probiotics separated and screened by itself. While fully maintaining the nutritional quality of the raw materials, it is rich in probiotics and also meets people's needs for fighting fatigue and improving dry intestines and constipation. Attached Figure Description

[0019] Figure 1 This is a pectinase activity assay.

[0020] Figure 2 It is a cellulase activity assay.

[0021] Figure 3 This study determined the activity of single and combined bacteria in the culture medium of the Jichuan Decoction, a traditional Chinese medicine and food formula.

[0022] Figure 4 The effects of single and combined bacteria on the dissolution of active ingredients in the Jichuan Decoction, a formula containing both medicinal and edible ingredients.

[0023] Figure 5 This relates to the effect of fermentation time on the activity of the combined bacteria.

[0024] Figure 6 This study investigated the effect of the ratio of medicinal and edible herbs to water in Jichuan Decoction on the activity of the combined bacteria.

[0025] Figure 7 This relates to the effect of fermentation temperature on the activity of the combined bacteria.

[0026] Figure 8 This relates to the effect of inoculum size on the activity of the combined bacteria.

[0027] Figure 9 It involves response surface optimization and contour plotting.

[0028] Figure 10 The effect of different formulations of Jichuan Decoction administered by gavage on the body weight of mice.

[0029] Figure 11 The effect of different formulations of Jichuan Decoction administered by gavage on the amount of food consumed in mice.

[0030] Figure 12 The effects of different formulations of Jichuan Decoction on fecal water content, time to first defecation, number of fecal pellets at 6 hours, and small intestinal transit rate in mice were investigated.

[0031] Figure 13 The effects of different formulations of Jichuan Decoction on the activity level of mice.

[0032] Figure 14 The activity status of mice with different formulations of Jichuan Decoction.

[0033] Figure 15The effect of different formulations of Jichuan Decoction on the swimming time of mice.

[0034] Figure 16 The effects of different formulations of Jichuan Decoction on the thymus index, spleen index, kidney index, seminal vesicle index, and testis index in mice were investigated.

[0035] Figure 17 This study compares the morphological characteristics of testicular tissue in mice administered Jichuan Decoction via gavage with different formulations.

[0036] Figure 18 The effect of different formulations of Jichuan Decoction administered by gavage on serum indicators in mice. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0038] The essence of the traditional Jichuan Decoction formula lies in its approach: addressing the root cause by warming the kidneys, replenishing essence, and nourishing blood; and addressing the symptoms by moistening the intestines, reducing turbidity, and regulating qi. In the traditional Jichuan Decoction formula, Cistanche deserticola is the principal herb, warming and tonifying kidney yang, replenishing essence and nourishing blood, and moistening dryness and lubricating the intestines; Angelica sinensis is the assistant herb, nourishing blood and moistening dryness, and assisting the principal herb in relieving constipation; Achyranthes bidentata and Alisma plantago-aquatica are the adjuvant herbs, with Achyranthes bidentata tonifying the liver and kidneys and guiding the qi downward, and Alisma plantago-aquatica draining turbidity and eliminating water retention; Cimicifuga foetida and Citrus aurantium are the guiding herbs, with Cimicifuga foetida promoting the upward movement of clear yang and Citrus aurantium widening the intestines and lowering qi. However, the traditional Jichuan Decoction has relatively low safety, and large-scale use can lead to certain side effects.

[0039] To improve the safety and reduce the side effects of traditional Jichuan Decoction, this invention uses medicinal and edible herbs with similar medicinal properties to replace the assistant, adjuvant, and guiding herbs in the original Jichuan Decoction formula. Although the principal herb, Cistanche deserticola, is retained, the formulation environment of the medicinal and edible herbs has changed, and its "warming the kidney" effect is now dominated or assisted by the "tonifying qi" principle of the new formula. Astragalus membranaceus and Codonopsis pilosula are used as assistant herbs to greatly tonify the qi of the spleen and lungs, which is a fundamental change; Prunus persica and Citrus reticulata are used as adjuvant herbs (Prunus persica replaces Achyranthes bidentata, and Citrus reticulata replaces Alisma plantago-aquatica); and Raphanus sativus is used as the guiding herb to lower qi, resolve phlegm, promote digestion, and relieve constipation. The "ascending qi" combination has been completely abandoned (Cimicifuga foetida is removed), and the "lowering qi and clearing the bowels" effect has been strengthened (by using a large amount of Raphanus sativus), which is also a fundamental change.

[0040] The specific formula of Jichuan Decoction, obtained according to the above-mentioned compatibility principles, consists of 30 parts of Cistanche deserticola, 30 parts of Raphanus sativus seed, 15 parts of Prunus persica kernel, 20 parts of Astragalus membranaceus, 20 parts of Codonopsis pilosula, and 10 parts of Citrus reticulata peel. According to the animal model verification results in Example 5, although there was no statistically significant difference in biological indicators (such as adrenocorticotropic hormone and thyrotropin-releasing hormone) between mice given this Jichuan Decoction formula and mice in the traditional Jichuan Decoction group, the total distance traveled and swimming time of mice given this Jichuan Decoction formula were significantly lower than those of mice in the traditional Jichuan Decoction group (see Appendix). Figures 13 to 15 This indicates that the Jichuan Decoction, a formula derived from both food and medicine, is not as effective as the traditional Jichuan Decoction in improving the behavior of model mice. Chronic stress-induced endocrine hormone imbalance is a complex process that affects the secretion levels of multiple endocrine hormones. It is speculated that the limited number of endocrine hormones tested in this experiment may have prevented the biochemical indicators from clearly showing a difference between the Jichuan Decoction and the traditional Jichuan Decoction, although significant behavioral differences were observed.

[0041] However, the inventors discovered that the probiotic-based medicinal and edible herbal composition obtained by fermenting the above-mentioned Jichuan Decoction formula with combined probiotics (preserved strains Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002) not only maintains a comparable level of adrenocorticotropic hormone (ACTH) secretion compared to the traditional Jichuan Decoction, but also significantly increases the secretion levels of thyrotropin-releasing hormone (TRH) and testosterone. This has the effect of improving endocrine hormone imbalance caused by chronic stress, especially reversing the decrease in TRH, ACTH, and / or testosterone secretion levels caused by chronic stress. The regulatory effect on TRH and testosterone secretion levels is significantly better than that of the traditional Jichuan Decoction (see appendix). Figure 18 It can be used to prepare products that reverse the decrease in the secretion levels of thyrotropin-releasing hormone, adrenocorticotropic hormone and / or testosterone caused by chronic stress.

[0042] More importantly, behavioral experimental data in animal models showed that the probiotic-based medicinal herb composition had a similar effect on improving the behavior of model mice as the traditional Jichuan Decoction. Specifically, data from the total distance experiment showed that mice given the probiotic-based medicinal herb composition had a total distance comparable to or even slightly better than those given the traditional Jichuan Decoction (see...). Figure 13 Swimming time data from mice showed that the swimming time of mice given this probiotic-based food-grade herbal combination was comparable to that of mice given the traditional Jichuan Decoction (see...). Figure 15 ), active state of motion (see Figure 14Furthermore, since the medicinal and edible materials and probiotics in this composition have a safety level as high as food grade, this probiotic medicinal and edible material composition can achieve the safety level of food grade and can be used for a long time or in large quantities.

[0043] In addition, fermentation of the Jichuan Decoction's medicinal and edible components using a combination of probiotics (preserved strains Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002) can promote the dissolution and release of the effective active ingredients (flavonoids) in the formula (see...). Figure 4 Numerous studies have shown that many plant-derived flavonoids and polysaccharides can regulate and improve endocrine hormone disorders or decreases caused by chronic stress through multiple pathways. It is speculated that this probiotic-food homology composition may also work in conjunction with other factors to synergistically improve endocrine hormone disorders caused by chronic stress, thereby promoting the recovery of endocrine hormone secretion levels.

[0044] Based on this, the present invention provides a combination probiotic, comprising Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002, wherein the accession number of Lactobacillus plantarum JY001 is CCTCC NO:M 2025584 and the accession date is March 25, 2025; and the accession number of Lactobacillus plantarum JY002 is CCTCC NO:M 2025585 and the accession date is March 25, 2025.

[0045] A probiotic-medicinal-food homology composition is also provided, comprising the combined probiotics and medicinal-food homology materials as described in this invention; the medicinal-food homology materials include Cistanche deserticola, radish seed, peach kernel, Astragalus membranaceus, Codonopsis pilosula, and tangerine peel. Cistanche deserticola is the principal ingredient, Astragalus membranaceus and Codonopsis pilosula are the assistant ingredients, peach kernel and tangerine peel are the adjuvant ingredients, and radish seed is the guiding ingredient.

[0046] In some embodiments, the medicinal and edible herbs specifically include: 30 parts of Cistanche deserticola, 30 parts of Raphanus sativus seeds, 15 parts of Prunus persica kernels, 20 parts of Astragalus membranaceus, 20 parts of Codonopsis pilosula, and 10 parts of Citrus reticulata peel. The combined probiotics are Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002, with a live bacteria count of 10-1. 8 CFU / ml or 10 8 CFU / g or higher.

[0047] In addition, the present invention also provides a method for preparing a probiotic food-medicinal material composition as described in the present invention, which includes the following steps: The mixture was obtained by fermentation using medicinal and edible materials from Jichuan Decoction as the culture medium and combined probiotics Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002 as seed bacteria. The medicinal and edible herbs used in Jichuan Decoction include Cistanche deserticola, radish seed, peach kernel, astragalus, codonopsis, and tangerine peel. Among them, Cistanche deserticola is the principal herb, astragalus and codonopsis are the assistant herbs, peach kernel and tangerine peel are the adjuvant herbs, and radish seed is the guiding herb.

[0048] The preparation method is as follows: Preparation of the medicinal and edible herbal culture medium for Jichuan Decoction: 30 parts of Cistanche deserticola, 30 parts of radish seed, 15 parts of peach kernel, 20 parts of Astragalus membranaceus, 20 parts of Codonopsis pilosula, and 10 parts of dried tangerine peel were mixed, pulverized into powder, and sterilized at 121℃ for 20 min according to the ratio of powder mass to water volume of the formula.

[0049] Probiotic fermentation: Probiotic seed liquid is inoculated into the culture medium of medicinal and edible materials at an inoculation rate of 2%~8%, and fermented at 25~35℃ for 18~30h to obtain a probiotic-medicinal and edible material composition. The probiotic seed liquid is the seed liquid of Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002.

[0050] In this invention, the biomass of probiotics can be predicted using the following formula: Y = -29.05 + 0.889X1 + 1.47X2 + 0.65X3 - 0.002X1X2 - 0.0068X1X3 - 0.0066X2X3 - 0.05X1 2 -0.021X2 2 -0.008X3 2 ; In the formula: Y is the predicted response value, i.e., the number of viable bacteria, (10 9 (CFU / g); X1 is the inoculum amount, %; X2 is the fermentation time, h; X3 is the fermentation temperature, ℃.

[0051] In some embodiments, the viable count of the seed culture of the probiotics Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002 is preferably 10. 9 The inoculum concentration was 6%~7% (CFU / ml), the fermentation temperature was 30~31℃, and the fermentation time was 25~26h.

[0052] In addition, the present invention also provides the application of a probiotic food-medicine composition prepared according to the method described in the present invention in the preparation of a product for improving endocrine hormone imbalance caused by chronic stress, wherein the endocrine hormones are endocrine hormones regulated by the hypothalamic-pituitary-target gland axis, including thyrotropin-releasing hormone (TRH), adrenocorticotropic hormone (ACTH) and testosterone (T).

[0053] In some embodiments, the probiotic food-grade medicinal material composition is used in the preparation of a product for reversing the decrease in serum testosterone levels caused by chronic stress.

[0054] In some embodiments, the probiotic food-grade medicinal material composition is used in the preparation of a product for reversing the decrease in serum thyrotropin-releasing hormone levels caused by chronic stress.

[0055] In some embodiments, the probiotic food-grade medicinal material composition is used in the preparation of a product for reversing the decrease in serum adrenocorticotropic hormone levels caused by chronic stress.

[0056] The following are examples. The strains involved in this invention are derived from healthy human subjects and obtained through routine isolation and screening. The isolated strains are deposited with the China Center for Type Culture Collection (CCTCC). In use, all microorganisms undergo three consecutive cultures, followed by incubation in sterile MRS broth at an inoculum size of 5%. Fresh fermentation broth is obtained after 24 hours of incubation at 37°C for testing.

[0057] Example 1: Isolation, purification, and identification of strains (1) Isolation of strains Fecal samples were prepared by mixing fecal matter with physiological saline. The fecal concentration in the fecal sample solution was 5 g / ml. The fecal sample solution was then serially diluted with physiological saline to a concentration of 10 g / ml. -1 10 -2 10 -3 10 -4 10 -5 Diluted samples were spread onto MRS basal broth medium and placed directly in an aerobic environment (ordinary incubator) at 37°C for 3-5 days.

[0058] Single colonies were selected based on their morphology, color, and size and streaked on three zones of MRS basal broth agar plates. Single colonies were repeatedly selected for purification until pure strains were isolated and numbered.

[0059] (2) Molecular biological identification The obtained single colonies were scraped and subjected to 16s identification for preservation. The morphological characteristics and molecular biological identification of the strains were combined as follows: Genomic DNA of the isolated bacterial strain was extracted strictly according to the steps of the bacterial DNA extraction kit, and polymerase chain reaction (PCR) amplification was performed using it as a template. The forward primer for the amplification reaction was 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), and the reverse primer was 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0060] PCR reaction system: 25 μL of 2×EcoTaqPCRSuper-Mix, 1 μL each of forward and reverse primers, 1 μL of genomic DNA, and ddH2O to make up to 50 μL. PCR reaction program: 95℃ for 10 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, for a total of 30 cycles; final extension at 72℃ for 10 min.

[0061] The PCR products were directly sequenced by Qingke Biotechnology Co., Ltd., and the gene sequences were compared using BLAST in NCBI. The gene sequences of the strains with the highest homology were then identified in GenBank for further identification and confirmation of the isolated strains. The 16S rDNA sequence of strain number 177 is shown in SEQ ID NO:1, and the 16S rDNA sequence of strain number 191 is shown in SEQ ID NO:2.

[0062] The 16S rDNA sequence of strain 177 was compared with that of Lactobacillus plantarum (KP976095.1) using BLAST on NCBI. The results showed that the 16S rDNA sequence of strain 177 was 99.79% similar to that of Lactobacillus plantarum (KP976095.1), thus identifying strain 177 as a new strain of Lactobacillus plantarum.

[0063] The 16S rDNA sequence of strain 191 was compared with that of Lactobacillus plantarum (OR502092.1) using BLAST on NCBI. The results showed that the 16S rDNA sequence of strain 191 was 99.89% similar to that of Lactobacillus plantarum (OR502092.1), thus identifying strain 191 as another new strain of Lactobacillus plantarum.

[0064] Performance of the strain in Example 2 This embodiment uses two new Lactobacillus plantarum strains, numbered 177 and 191, isolated and purified in Example 1 as experimental subjects to study the performance of these two new strains, as detailed below: (1) Pectinase activity A screening medium with pectin as the sole carbon source was prepared (K₂HPO₄: 1g, MgSO₄: 0.5g, NaNO₃: 3g, FeSO₄·7H₂O: 0.01g, pectin 2g, agar 15g, distilled water 1000mL, pH 5.5). Using the Congo red staining method, the purified strain was inoculated into pectin agar plates and incubated at 37℃ for 3 days. The diameter of each colony was measured. Then, 1mg / mL Congo red staining solution was added to the plates, and staining was performed for approximately 40 minutes. The Congo red staining solution was discarded, and a thin layer of surface Congo red was rinsed off with a small amount of distilled water. Distilled water was added again for destaining for 10 minutes, and the plates were observed under light to check for the presence of a distinct hydrolysis zone. The diameter of the hydrolysis zone was also measured; a larger diameter indicates higher pectinase activity. The results of the pectinase activity assay are shown below. Figure 1 .

[0065] Depend on Figure 1 The results showed that both new strains of *Lactobacillus plantarum*, numbered 177 and 191, exhibited obvious hydrolysis zones. The diameter of the hydrolysis zone of strain 177 was larger than that of strain 191, indicating that both strains possessed pectinase activity, with strain 177 exhibiting higher pectinase activity.

[0066] (2) Cellulase activity A screening medium with cellulose as the sole carbon source was prepared (5g yeast extract, 5g carboxymethyl cellulose, 15g agar powder, 1000mL distilled water). Using the Congo red staining method, the purified strain was inoculated into pectin agar plates and incubated at 37℃ for 3 days. The diameter of each colony was measured. Then, 1mg / mL Congo red staining solution was added to the plates, and staining was performed for approximately 40 minutes. The Congo red staining solution was discarded, and a thin layer of Congo red was rinsed off with a small amount of distilled water. Distilled water was then added again for destaining for 10 minutes. The presence of a distinct hydrolysis zone was observed under light, and its diameter was measured. A larger hydrolysis zone diameter indicates higher cellulase activity. The cellulase activity measurement results are shown below. Figure 2 .

[0067] Depend on Figure 2 The results showed that only the Lactobacillus plantarum strain numbered 177 and 191 exhibited a clear hydrolysis zone, indicating that the Lactobacillus plantarum strain numbered 177 also possessed cellulase activity.

[0068] (3) Ligninase activity Prepare a lignin screening medium with lignin as the sole carbon source: 4g lignin, 0.266g ammonium nitrate, 0.1g magnesium sulfate, 2g potassium dihydrogen phosphate, 0.04g disodium hydrogen phosphate, 4g agar, 200mL distilled water, pH 7.5-8.0.

[0069] Strain activation: The strains to be screened were removed from the -80°C freezer. Before use, all microorganisms were activated by three consecutive cultures, and then cultured in sterile MRS broth at an inoculum of 5% for 24 hours at 37°C.

[0070] ① Initial screening of ligninase: Use a disposable inoculation loop to dip the activated bacterial solution of the strain, streak it on the lignin screening medium plate, place it in a constant temperature incubator, set the temperature to 37℃ and incubate for 1-7 days. Observe whether colonies grow on each plate every 24 hours. The results are shown in Table 1.

[0071] Table 1. Results of initial screening for ligninase

[0072] ② Ligninase rescreening: Prepare aniline blue agar medium (aniline blue 0.2g, potassium dihydrogen phosphate 6g, magnesium sulfate 3g, agar 40g, distilled water 2000mL, pH 7.5-8.0), and use the aniline blue plate decolorization test.

[0073] The screened and purified strains 177 and 191 were inoculated into aniline blue agar plates and cultured in a constant temperature incubator at 30°C for 3 days. The diameter of the decolorization zone was measured, and the results are shown in Table 2.

[0074] Manganese chloride agar medium (MnCl2·4H2O 20.3g, potassium dihydrogen phosphate 6g, magnesium sulfate 3g, agar 40g, distilled water 2000mL, pH 7.5-8.0) was prepared, and the colorimetric assay was performed using manganese chloride plates. The screened and purified strain 191 was spotted into manganese chloride agar plates and incubated at 30℃ for 3 days. The diameter of the colorimetric zone was then measured, and the results are shown in Table 2.

[0075] Table 2 Ligninase Activity

[0076] The experimental results above show that the newly selected Lactobacillus plantarum strain numbered 177 has both pectinase and cellulase activities; while the newly selected Lactobacillus plantarum strain numbered 191 has both ligninase and pectinase activities.

[0077] The new strain, numbered 177, obtained through isolation and purification, was deposited at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on March 25, 2025. Its accession number is CCTCC NO:M 2025584, the deposit date is March 25, 2025, and its classification name is Lactobacillus plantarum JY001.

[0078] The newly isolated strain of Lactobacillus plantarum, numbered 191, was deposited at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on March 25, 2025. Its accession number is CCTCC NO:M 2025585, the deposit date is March 25, 2025, and it is classified and named Lactobacillus plantarum JY002.

[0079] Example 3: Combined probiotic fermentation to enhance the medicinal and edible ingredients of Jichuan Decoction In this embodiment, Jichuan Decoction is an improved formulation of medicinal and edible herbs. The specific formulation of Jichuan Decoction consists of 30 parts of Cistanche deserticola, 30 parts of radish seed, 15 parts of peach kernel, 20 parts of Astragalus membranaceus, 20 parts of Codonopsis pilosula, and 10 parts of dried tangerine peel. The study investigated the effects of fermentation with different probiotics on the active ingredients of Jichuan Decoction. The probiotics were Lactobacillus plantarum JY001 (abbreviated as 177), Lactobacillus plantarum JY002 (abbreviated as 191), and a combination of Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002 (abbreviated as 177+191).

[0080] The culture medium for the Jichuan Decoction, a formula containing both medicinal and edible ingredients, consists of: 30g of Cistanche deserticola, 30g of radish seed, 15g of peach kernel, 20g of Astragalus membranaceus, 20g of Codonopsis pilosula, and 10g of dried tangerine peel. Mix and grind the ingredients into powder, weigh out 5g, add 20ml of pure water, sterilize at 121℃ for 20 minutes, and then cool for later use.

[0081] The Jichuan Decoction, a formula combining medicinal and edible ingredients, was used as a control. All probiotics underwent three consecutive cultures, followed by incubation in sterile MRS broth at an inoculum of 5% for 24 hours at 37°C. The concentrations of 177, 191, and 177+191 were adjusted to 10... 9 CFU / ml was inoculated into the Jichuan Decoction (a traditional Chinese medicine and food homology formula) culture medium at a 6% inoculation rate. After static incubation at 37℃ for 24 hours, 1 ml of fermentation product was taken, serially diluted, and plate counted. The results are as follows. Figure 3 As shown. The remaining fermentation product was centrifuged at 5000 rpm for 6 min, the supernatant was collected, and the content of active ingredients in the supernatant was detected. The results are shown below. Figure 4 As shown.

[0082] Depend on Figure 4 The results showed that, compared with the control group, the dissolution of active ingredients flavonoids and polysaccharides in the traditional Chinese medicine composition decreased after fermentation with a single strain of *Lactobacillus plantarum* JY001, and the dissolution of active ingredient polysaccharides decreased after fermentation with a single strain of *Lactobacillus plantarum* JY002. However, after fermentation with a combination of *Lactobacillus plantarum* JY001 and *Lactobacillus plantarum* JY002, the content of active ingredient polysaccharides in the traditional Chinese medicine composition showed no significant difference compared with the control group, while the flavonoid content increased significantly. Therefore, fermentation of Jichuan Decoction with a combination of *Lactobacillus plantarum* JY001 and *Lactobacillus plantarum* JY002 can significantly increase the dissolution of flavonoids without reducing polysaccharides.

[0083] Example 4: Optimization of the combined probiotic fermentation process (1) Effect of fermentation time on strain activity The powder of the Jichuan Decoction (a traditional Chinese medicine formula) was added to a 250mL Erlenmeyer flask at a powder-to-water ratio of 1:4. The mixture was then pressure-cooked at 121℃ and 0.1MPa for 20 minutes. A combination of *Lactobacillus plantarum* JY001 and JY002 was used as the fermentation seed culture. The combined probiotic seed culture was inoculated at a 6% inoculum and fermented at 37℃ for 12h, 18h, 24h, 30h, and 36h, respectively. The effect of fermentation time on the activity of the strains was studied, and the results are as follows: Figure 5 As shown.

[0084] (2) Effect of moisture content on bacterial activity The powder of the Jichuan Decoction (a traditional Chinese medicine formula) was added to 250 mL Erlenmeyer flasks, and prepared according to the following powder-to-water ratios: 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, and 1:12. The mixtures were then pressure-cooked at 121℃ and 0.1 MPa for 20 min. A 6% inoculum of the combined probiotic seed culture was then inoculated, and the mixtures were allowed to ferment at 37℃ for 24 h. The effect of fermentation moisture content on bacterial activity was investigated, and the results are shown below. Figure 6 As shown.

[0085] (3) Effect of fermentation temperature on strain activity The powder of the Jichuan Decoction (a traditional Chinese medicine formula) was added to 250mL Erlenmeyer flasks, and the powder was mixed with water at a ratio of 1:4. The mixture was then pressure-cooked at 121℃ and 0.1MPa for 20 minutes. A 6% inoculum of the combined probiotic seed culture was then inoculated, and fermented at 25℃, 30℃, 35℃, and 40℃ for 24 hours each. The effect of fermentation temperature on the activity of the strain was studied, and the results are as follows: Figure 7 As shown.

[0086] (4) Effect of inoculum size on bacterial viability The powder of the Jichuan Decoction (a traditional Chinese medicine formula) was added to 250 mL Erlenmeyer flasks, and the powder was mixed with water at a ratio of 1:4. The mixture was then pressure-cooked at 121℃ and 0.1 MPa for 20 min. Inoculation with probiotic seed culture at inoculum rates of 2%, 4%, 6%, 8%, and 10% was carried out, and fermentation was allowed to proceed at 37℃ for 24 h. The effect of inoculum rate on bacterial activity was studied, and the results are as follows: Figure 8 As shown.

[0087] (5) Response surface optimization Based on the single-factor experiments, response surface optimization experiments were conducted. Three factors with significant influence were selected: inoculum size (factor X1), fermentation temperature (factor X2), and fermentation time (factor X3). Using strain activity as an indicator, a Box-Behnken response surface optimization experimental design with 3 factors and 3 levels was carried out using DesignExpert 8.0.6 software, as shown in Table 3 below.

[0088] Table 3. Factor Levels in the Box-Behnken Experiment

[0089] Based on the optimization results of the single-factor experiment, a three-factor, three-level Box-Behnken response surface experiment was conducted according to the Design-ExpertVersion 8.0.6 experimental design. The results are shown in Table 4 below.

[0090] Table 4 Box-Behnken test protocol and results

[0091] The table above contains 17 test points. To detect experimental error, the center point test was repeated 5 times. Then, the standard polynomial regression method was used for analysis and fitting. Response surface design analysis was performed on the response values ​​obtained from the above 17 test points to obtain the Box-Behnken test variance table and the quadratic polynomial of the relationship between the response value and the independent variable, as shown in Table 5.

[0092] Table 5. Analysis of Variance in the Box-Behnken Trial

[0093] Note: The difference was extremely significant (P < 0.01); the difference was significant (P < 0.05).

[0094] Obtain the quadratic polynomial relating the response value to the independent variable: Y=-29.05+0.889X1+1.47X2+0.65X3-0.002X1X2-0.0068X1X3-0.0066X2X3-0.05X1 2 -0.021X2 2 -0.008X3 2 ; In the formula: Y is the predicted response value, i.e., the number of viable bacteria, (10 9 (CFU / g); X1 is the inoculum amount, %; X2 is the fermentation time, h; X3 is the fermentation temperature, ℃.

[0095] As shown in the table above, the model R 2 =0.9253 > 0.9, indicating that the model fits well and the linear relationship between the independent variable and the response value reaches a significant level. The optimal fermentation component of this probiotic can be determined using this regression equation. Based on the response surface and contour lines... Figure 9 It can be seen that when the temperature remains constant, the number of viable bacteria first increases and then decreases with increasing inoculum size and time; when the time remains constant, the number of viable bacteria increases and then decreases with increasing temperature and inoculum size; when the inoculum size remains constant, the number of viable bacteria first increases and then decreases with increasing temperature and time. Setting the first-order partial derivatives of each variable (X1, X2, X3) to 0, and differentiating the fitted system of three linear equations, we obtain the model's predicted optimal point as: inoculum size 6.6%, time 25.2 h, and temperature 30.78℃. Substituting these values ​​into the regression equation, we obtain the theoretical biomass Y = 4.7 × 10⁻⁶. 9 CFU / g.

[0096] Model validation results: Fermentation was carried out using the optimal fermentation conditions obtained above, and viable cell counts were performed to verify the reliability of the model predictions. The results are shown in Table 6 below: Table 6 Validation of the prediction model

[0097] The optimized enzyme activity showed a 96.4% agreement with the model prediction, indicating that the optimization results using this model are accurate and reliable.

[0098] Example 5: Chronic Stress Animal Model Experiment Continuous administration of exogenous glucocorticoids (hydrocortisone) simulated a state of chronic stress. It inhibited the function of the hypothalamus-pituitary-adrenal (HPA) axis through negative feedback, leading to endogenous hormone secretion disorders, and subsequently triggering a series of syndromes including systemic hypoenergy metabolism, thermoregulatory imbalance, immunosuppression, and digestive dysfunction. This example investigates the effects of a probiotic-based food-grade herbal combination on endocrine hormone imbalances induced by chronic stress, as detailed below: 1. Materials and Methods 1.1 Reagents and Methods Hydrocortisone solution: Dissolve 0.05g / vial of hydrocortisone sodium succinate (Tianjin Lisheng Pharmaceutical Co., Ltd.) in 10ml of physiological saline to achieve a final concentration of 50mg / 10ml.

[0099] Jichuan Decoction Granules: Jiangsu Kangyuan Pharmaceutical Co., Ltd., based on the conversion between animal weight and human dosage (referencing Professor Xu Shuyun's "Pharmacological Experimental Methodology"), is designated as a positive control drug and abbreviated as Jichuan Decoction Group.

[0100] Jichuan Decoction, a formula combining medicinal and edible ingredients: 30g of Cistanche deserticola, 30g of Raphanus sativus seeds, 15g of Prunus persica kernels, 20g of Astragalus membranaceus, 20g of Codonopsis pilosula, and 10g of Citrus reticulata peel. Mix and grind into powder, then sterilize at 121℃ for 20 minutes. Weigh 125g of the Jichuan Decoction formula and place it in a ceramic pot. Add 1000ml of water. After boiling, reduce to a simmer and cook for 45 minutes, concentrating to 200ml. The final concentration is 125g / 200mL. Store at -20℃ for subsequent feeding to mice (after equilibration to room temperature after removal from the refrigerator). This formula is abbreviated as the formula group.

[0101] Jichuan Decoction, a fermented formula of medicinal and edible herbs, formula 1: 30g of Cistanche deserticola, 30g of radish seed, 15g of peach kernel, 20g of Astragalus membranaceus, 20g of Codonopsis pilosula, and 10g of dried tangerine peel. Mix and grind into powder. Add 20ml of pure water per 5g of powder and sterilize at 121℃ for 20 minutes. Inoculate with a 6% inoculum of probiotic seed liquid. Ferment at 37℃ for 24 hours to obtain a probiotic medicinal and edible herbal composition. Weigh 125g of the probiotic medicinal and edible herbal composition and place it in a ceramic pot. Add 1000ml of water. After boiling, simmer over low heat for 45 minutes, concentrating to 200ml. The final concentration is 125g / 200mL. Store at -20℃ for subsequent mouse feeding (after equilibration to room temperature after removal from the refrigerator). This formula is abbreviated as Fermentation Formula 1.

[0102] Jichuan Decoction Fermentation Formula 2: Dilute the above Jichuan Decoction Fermentation Formula 1 by 1 time, and abbreviate it as Fermentation Formula 2.

[0103] 1.2 Animals and Experimental Design ① Experimental animals: Kunming mice (18±2)g, 10 males per group. The mice were kept at a constant temperature of 25±2℃ and a relative humidity of 50±5%, with a day / night cycle of 12 / 12h.

[0104] ② Model establishment and group design: After the experimental mice had adapted to the environment for one week, they were housed in separate cages, weighed, and randomly divided into 6 groups: Healthy control group: fed with normal feed for 0-3 weeks, and administered PBS by gavage for 2-3 weeks; Kidney Yang Deficiency Syndrome Model Group: Feeded with normal feed for 0-3 weeks, and administered hydrocortisone by gavage at a dose of 25mg / kg for 2-3 weeks.

[0105] Positive drug group: For the first 0-3 weeks, the mice were fed normal diet plus Jichuan Decoction Granules (1.82g / kg). For the second and third weeks, they were given hydrocortisone at a dose of 25mg / kg by gavage. The positive drug was commercially available. The dosage of Jichuan Decoction Granules was calculated based on the weight of the finished product. The dosage of Jichuan Decoction Granules was 1.82g / kg based on the weight of the mice (kg).

[0106] Food and medicine homology formula group: Feeding with normal feed + Jichuan decoction food and medicine homology formula (1.6g / ml / kg) for 0-3 weeks, and administering hydrocortisone at a dose of 25mg / kg by gavage in 2-3 weeks; the dosage of Jichuan decoction food and medicine homology formula is converted according to the weight of medicinal materials.

[0107] Food and medicine homology fermented formula group 1: Feeding with normal feed + Jichuan decoction food and medicine homology fermented formula 1 (1.6g / ml / kg) for 0-3 weeks, and administering hydrocortisone at a dose of 25mg / kg by gavage in the 2nd-3rd week; Food and medicine homology fermentation formula group 2: Feeding with normal feed + Jichuan decoction food and medicine homology fermentation formula 2 (0.8g / ml / kg) for 0-3 weeks, and administer hydrocortisone at a dose of 25mg / kg by gavage in the 2nd-3rd week.

[0108] 1.3 Sample Collection and Index Detection (1) Measurement of body weight From the start of the intervention, mouse body weight was measured at fixed times each week, and changes in body weight were recorded. The percentage change in body weight for each group of mice was calculated as (current body weight × 100) / body weight before modeling. The results are as follows: Figure 10 As shown.

[0109] (2) Food intake measurement From the start of the intervention, the food intake of the mice was measured at fixed times each week, and the average food intake of each mouse was recorded weekly. The results are as follows: Figure 11 As shown. The calculation formula is as follows: Average food intake (water intake) / (g / d) = (AB) / (C × D); In the formula: A is the total feed or water mass within one week / g; B is the remaining feed or water mass on the day of recording / g; C is the time / d; D is the number of mice per cage / mice.

[0110] After software analysis of the differences, the results showed that there was no significant difference in food intake between the model group and each treatment group.

[0111] (3) Detection of fecal water content, duration of first black stool, and small intestinal transit rate Fecal moisture content detection: Empty sterile EP tubes were weighed first. Feces were collected and weighed at the same time period to obtain wet weight. The samples were then dried in an incubator and weighed again to obtain dry weight. The calculation method was: Fecal relative moisture content (%) = ((wet weight − dry weight) / wet weight) × 100%. Duration and number of fecal pellets of the first black stool test: Mice were administered 250 µL of activated charcoal solution by gavage. The time of the first black stool and the number of fecal pellets after 6 hours were recorded. Small intestinal transit rate test: On the last day of the experiment, mice were orally administered 250 µL of 10% activated charcoal suspension by gavage. Mice were sacrificed 30 minutes later, and the small intestine was harvested. The total length of the intestine and the distance the activated charcoal traveled in the intestine were measured. The intestinal transit rate was calculated as: (distance traveled by activated charcoal in the intestine / length of the small intestine) × 100%. Results are as follows: Figure 12 As shown.

[0112] (4) General behavioral observation During the experiment, the general condition of the mice in each group was observed. Animals treated with the modeling method showed sluggishness, arched backs, curled up, huddled together, and were sensitive to cold and preferred slow movements. Mouse activity was assessed using OFT as follows: Mice were gently placed in an open area, in a white plastic box (46×46×40cm), occupying 3 / 5 of the length and width. Another area was defined as the perimeter along the walls. All mice were acclimatized to the test room for at least 2 hours before the formal behavioral test. Mouse movements were digitally recorded and analyzed using SMART 3.0 video tracking software. The results are as follows: Figure 13 and Figure 14 As shown.

[0113] (5) Anti-fatigue experiment in mice The following day, a mouse fatigue resistance experiment was conducted. Water was added to a 50cm×30cm×25cm glass tank, with a depth of 20cm and a water temperature maintained at (20±0.5)℃. Mice were placed in the tank with a 5.5g weight on their tails and swam. Timing began immediately; mice whose heads were submerged for 10 seconds and could not resurface were considered exhausted, and the swimming time was recorded as that. The results were as follows: Figure 15 As shown.

[0114] (6) Mice were fasted for 12 hours after the last administration of the drug, but allowed free access to water, and were then weighed. After sacrifice, the thymus, spleen, kidneys, seminal vesicles, and testes were harvested, weighed, and the organ index was calculated. The organ index = organ weight (mg) / mouse body weight (g). The results are as follows: Figure 16 As shown.

[0115] (7) HE staining to observe testicular tissue morphology Mouse testicular tissue fixed in 10% formaldehyde was dehydrated, embedded, and sectioned to obtain 4μm paraffin sections. These sections underwent routine HE staining, were mounted with neutral resin, dried, and then the morphological changes of the testicular tissue were observed under a light microscope. The results are as follows: Figure 17 As shown.

[0116] Depend on Figures 11 to 17 The results showed that, compared with the healthy control group, the model group mice had a decrease in body weight; a significantly reduced fecal water content; a significantly increased time to first defecation; a significantly reduced number of fecal pellets in 6 hours; a significantly slowed small intestinal transit rate; a significantly reduced total distance of movement; a significantly reduced swimming time and easy fatigue; and significantly reduced testicular and kidney indices, resulting in weight loss.

[0117] After treatment with this model, mice exhibited weight loss, reduced activity, sluggishness, arched back and curled-up posture, huddling together, aversion to cold and a preference for slow movement, sparse and dull fur with piloerection, scrotal shrinkage, testicular retraction, perianal discoloration, reduced food intake, and emaciation. The weight of the testes, prepuce glands, and adrenal glands was reduced. This indicates the successful establishment of a chronic stress model, and that long-term chronic stress leads to dysfunction and depletion of the hypothalamic-pituitary-adrenal (HPA) axis.

[0118] Depend on Figure 12 The results showed that, compared with the model group, the traditional Jichuan Decoction group had significantly increased fecal water content, a significantly reduced first defecation time, a significantly increased number of stool particles at 6 hours, and a significantly accelerated small intestinal transit rate, demonstrating a significant laxative effect. Compared with the existing traditional Jichuan Decoction group, this food-medicine homologous fermented formula showed comparable efficacy in terms of fecal water content and small intestinal transit rate, while the first defecation time was significantly lower (P < 0.05), and the number of stool particles at 6 hours was also significantly increased (P < 0.05). Overall, this food-medicine homologous fermented formula has a better laxative effect than the traditional Jichuan Decoction and can be used to prepare products that improve functional gastrointestinal dysfunction caused by chronic stress.

[0119] Depend on Figure 13 The results showed that, compared with the model group, the total distance of movement of mice in the traditional Jichuan Decoction group was significantly increased. There was no statistical difference between the traditional Jichuan Decoction group and the fermented group 2. However, the total distance of movement of mice in the fermented group 1 was longer and the amount of movement was greater than that in the traditional Jichuan Decoction group.

[0120] Depend on Figure 14 and Figure 15 The results showed that, compared with the model group, the swimming time of mice in both the formula group and the fermented formula group was significantly increased, indicating significant anti-fatigue effects. Furthermore, compared with the traditional Jichuan Decoction, the anti-fatigue effect of this medicinal and edible fermented formula, under certain low-dose conditions, was comparable to that of the existing traditional Jichuan Decoction.

[0121] Figure 17 The results showed that the normal control group had intact testicular tissue structure, well-developed and tightly arranged seminiferous tubules, abundant interstitium, and normal morphology and quantity of spermatogenic cells at all levels, with regular arrangement and high cell maturity. Compared with the normal control group, the model control group showed testicular tubule wall atrophy, reduced number of cell layers in the seminiferous tubules, loose arrangement, significantly reduced number of interstitial cells, disordered arrangement of spermatogenic cells at all levels within the tubules, reduced sperm count in the lumen, and decreased cell maturity. In contrast, the symptoms of kidney yang deficiency and the degree of testicular pathological damage in mice treated with this formula and the high- and low-dose fermented formula were all alleviated compared with the model group, manifested as thickening of the seminiferous tubule wall, increased number of cell layers, increased spermatogenesis and sperm production, and recovery of interstitial cell count; the fermented formula 2 showed better effects than the traditional Jichuan Decoction group.

[0122] (8) Serum markers After a 24-hour fast following the last dose, blood was collected from the eyeballs on day 21 of the experiment. The serum was separated by centrifugation at 3500 rpm for 15 minutes, and thyrotropin-releasing hormone (TRH), adrenocorticotropic hormone (ACTH), and testosterone (T) were measured. Results are as follows: Figure 18 As shown.

[0123] Hydrocortisone is an exogenous glucocorticoid. Long-term, high-dose use can inhibit the function of the hypothalamus-pituitary-adrenal (HPA) axis through a negative feedback mechanism, leading to endogenous hormone secretion disorders. For example, under chronic stress, hypothalamic function is suppressed, resulting in a decrease in both the basal secretion and pulsatile secretion frequency of thyrotropin-releasing hormone (TRH). In the early stages of stress, adrenocorticotropic hormone (ACTH) rises sharply to cope with stress, but under long-term chronic stress, due to the negative feedback effect of glucocorticoids, the pituitary gland becomes less responsive to CRH, exhibiting a "desensitization" phenomenon. Chronic stress also inhibits the release of gonadotropins (LH / FSH) through the HPA axis, leading to a decrease in sex hormone secretion.

[0124] Depend on Figure 18 The results showed that, compared with the control group, the model group had significantly lower levels of thyrotropin-releasing hormone (TRH), adrenocorticotropic hormone (ACTH), and testosterone (T), indicating that chronic stress led to endocrine hormone imbalance and reduction.

[0125] Compared to the model group, the TRH levels in both fermented formulation group 1 and fermented formulation group 2 were significantly increased after treatment with traditional Chinese medicine. This indicates that the combination of this probiotic and food-derived medicinal materials alleviated the inhibition of the hypothalamus and activated the highest command center. The combination of this food-derived and fermented formulation can take effect from the highest center (hypothalamus) and reverse the decrease in the secretion level of thyrotropin-releasing hormone caused by chronic stress.

[0126] Compared to the model group, ACTH levels in both fermentation groups 1 and 2 were significantly increased, while ACTH secretion after administration of traditional Jichuan Decoction showed no statistically significant difference compared to the model group. This indicates that, under TRH stimulation, the pituitary gland's ACTH secretion function is enhanced after administration of this fermented formula. Therefore, this fermented formula can restore pituitary function and reverse the decrease in adrenocorticotropic hormone (ACTH) secretion caused by chronic stress. Furthermore, increased ACTH levels stimulate the recovery of adrenal cortex function; although corticosterone was not detected in this example, this is an essential pathway.

[0127] Compared with the model group, after treatment with traditional Chinese medicine, the serum testosterone (T) levels of the Jichuan Decoction (a formula based on the same source of medicinal and edible ingredients), fermented formula group 1, and fermented formula group 2 were significantly increased. The increase in serum testosterone (T) indicates that the formula and the fermented formula reversed the decrease in testosterone secretion caused by chronic stress and improved gonadal (testicular) function.

[0128] This fermentation formula not only restores pituitary function but also improves the function of downstream target glands (adrenal glands and gonads), thus restoring the function of the hypothalamus-pituitary-target gland axis. It can be used to regulate endocrine hormone disorders caused by chronic stress, especially to reverse the decrease in endocrine hormones.

[0129] In modern medical research, dysfunction of the hypothalamus-pituitary-adrenal (HPA) axis is considered the core pathological basis of the "kidney yang deficiency" syndrome in Traditional Chinese Medicine (TCM). That is, the essence of kidney yang deficiency is the inhibition or imbalance of the hypothalamus-pituitary-adrenal (HPA) axis. In TCM theory, kidney yang deficiency manifests as aversion to cold, fatigue, decreased sexual function, and delayed growth and development. Warming and tonifying kidney yang herbs (such as Yougui Wan and Jinkui Shenqi Wan) are the treatment methods. After administering the fermented formula of Benjichuan Decoction (a blend of medicinal and edible herbs), ACTH and TRH levels increased, indirectly restoring adrenal function. Adrenal cortex hormones correspond to the body's "firepower" and stress response; the restoration of adrenal function corresponds to the improvement of "yang deficiency leading to cold" (such as improvement in symptoms like aversion to cold, cold limbs, lethargy, and decreased immunity). The increase in testosterone (T) directly corresponds to the restoration of the kidney's function of "governing reproduction," improving reproductive system symptoms such as impotence, seminal emission, and infertility.

[0130] This Jichuan Decoction, a fermented formula combining medicinal and edible ingredients, not only improves endocrine hormone imbalances caused by chronic stress and enhances the overall symptoms of kidney yang deficiency, but its safety has also been upgraded to food-grade standards. Furthermore, this probiotic-based medicinal and edible herbal combination can be fermented into a functional fermented beverage using self-isolated probiotics. While fully preserving the nutritional quality of the raw materials, it is rich in probiotics, meeting people's needs for combating fatigue and improving constipation.

[0131] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A probiotic-based food-grade medicinal material composition, characterized in that, The combination probiotics comprise Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002, wherein the Lactobacillus plantarum The preservation number of JY001 is CCTCC NO: M 2025584, and the preservation date is March 25, 2025; the Lactobacillus plantarum The preservation number of JY002 is CCTCC NO: M 2025585, and the preservation date is March 25, 2025. The medicinal and edible medicinal materials include Cistanche, Raphanus sativus, peach kernel, Astragalus, Codonopsis, dried tangerine or orange peel.

2. The probiotic-medicinal-food homology composition as described in claim 1, characterized in that, The medicinal and edible medicinal materials include Cistanche 30 parts, Raphanus sativus 30 parts, peach kernel 15 parts, Astragalus 20 parts, Codonopsis 20 parts and dried tangerine or orange peel 10 parts.

3. The probiotic-medicinal-food homology composition as described in claim 2, characterized in that, The viable cell number of the combination probiotic is 10 8 CFU / ml or 10 8 CFU / g or more.

4. A combination probiotic, characterized in that, include Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002, the aforementioned Lactobacillus plantarum JY001 has the accession number CCTCC NO: M 2025584 and the accession date is March 25, 2025; the aforementioned Lactobacillus plantarum JY002 has the accession number CCTCC NO: M 2025585 and the accession date is March 25, 2025.

5. The combination probiotic of claim 4, wherein the combination probiotic is a combination of Bifidobacterium longum AHNA 323 and Bifidobacterium longum AHNA 324. For Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002.

6. A method of preparing the probiotic food-grade material composition according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: Jichuan decocting medicinal food material as culture medium, with combined probiotics Lactobacillus plantarum JY001 and Lactobacillus plantarum JY002 are seed bacteria, obtained by fermentation; The Lactobacillus plantarum JY001 has a preservation number of CCTCC NO: M 2025584; the Lactobacillus plantarum JY002 has a preservation number of CCTCC NO: M 2025585; the Jichuan decoction homologous medicinal materials include Cistanche 30 parts, Raphanus sativus 30 parts, Semen Persicae 15 parts, Astragalus 20 parts, Codonopsis 20 parts and Pericarpium Citri Reticulatae 10 parts.

7. The production method according to claim 6, wherein The number of live bacteria after fermentation of the combined probiotics is predicted according to the following formula: Y = -29.05 + 0.889X1+ 1.47X2+ 0.65X3- 0.002X1X2- 0.0068X1X3- 0.0066X2X3- 0.05X1 2 - 0.021X2 2 - 0.008X3 2 ; wherein: Y is the number of viable bacteria, 10 9 CFU / g; X1 is the inoculum, %; X2 is the fermentation time, h; X3 is the fermentation temperature, °C.

8. The production method according to claim 7, wherein The fermentation is carried out at 25-35 DEG C for 18-30h according to the inoculation amount of the combined probiotics being 2%-8%.

9. Use of a probiotic functional food composition prepared according to the method of any one of claims 6 to 8, wherein, The application of the probiotic medicinal and edible medicinal material composition in preparing products for improving endocrine hormone disorder caused by chronic stress; the endocrine hormone is an endocrine hormone regulated by hypothalamic-pituitary-targeted gland axis function, including thyrotropin releasing hormone, adrenocorticotropic hormone and testosterone.

10. Use according to claim 9, wherein The application of the probiotic medicinal and edible medicinal material composition in preparing products for reversing the decrease of secretion level of thyrotropin releasing hormone, adrenocorticotropic hormone and / or testosterone caused by chronic stress.

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