Probiotics with sleep-aiding efficacy combined with american ginseng formula and preparation method

Abstract

The application provides a probiotic combined with American ginseng formula with sleep-aiding efficacy and a preparation method, relates to the technical field of biological medicine, and the probiotic combined with American ginseng formula provided by the embodiment of the application comprises lactobacillus rhamnosus AIML-553 with the preservation number of CGMCC No. 34977 and American ginseng ingredients. The application realizes in-vitro pre-transformation of probiotics through the probiotic combined with American ginseng formula, completes efficient metabolism of ginsenosides in advance, bypasses the restriction of differences of intestinal flora of human bodies, and enables different people to stably obtain high-activity rare ginsenosides, thereby significantly improving the consistency of product efficacy and the application universality.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a probiotic combined with American ginseng formula and preparation method that has sleep-aiding effects. Background Technology

[0002] American ginseng (Panax quinquefolius L.), a traditional and precious Chinese medicinal herb, possesses both medicinal and edible value and has received widespread attention in the fields of functional foods and nutritional interventions in recent years. It is rich in various active ingredients, including ginsenosides, polysaccharides, volatile oils, and amino acids, among which ginsenosides are its main pharmacologically active substances. Numerous studies have shown that American ginseng has multiple biological functions, including anti-fatigue, immune regulation, anti-inflammation, and neuroprotection, making it an important raw material source for the development of functional foods.

[0003] Ginsenosides belong to the triterpenoid class of saponins, and can be divided into two main categories based on their aglycone structure: protopanaxadiol (PPD) and protopanaxatriol (PPT). Pharmacokinetically, they can be further classified into progenitor saponins (such as Rb1, Re, Rg1, etc.) and secondary saponins (such as Compound K, Rh2, F2, etc.). However, American ginseng is predominantly composed of progenitor saponins. These molecules are characterized by their large molecular weight and high polarity, making them difficult to absorb directly through the small intestinal epithelium, which is one of the key factors limiting their bioavailability. The key to their efficacy lies in the metabolic transformation by intestinal microorganisms. Studies have shown that carbohydrate-metabolizing enzymes such as β-glucosidase and glycoside hydrolase synthesized by intestinal microorganisms can convert progenitor ginsenosides into secondary ginsenosides with smaller molecular weights and lower polarity. This process essentially involves metabolizing the predominantly hydrophilic ginsenosides into predominantly lipophilic metabolites, thereby significantly increasing their lipid solubility and membrane permeability, making them easier to absorb from the gastrointestinal tract and ultimately exert their biological activity. The gut microbiota-mediated mechanism of ginsenoside metabolism has been widely confirmed. Prototype ginsenosides are metabolized by gut microbiota into secondary ginsenosides, which are more bioactive and have higher bioavailability, exerting various pharmacological effects, including anti-tumor, anti-inflammatory, anti-diabetic, anti-allergic, and neuroprotective effects.

[0004] The metabolism and utilization of ginsenosides largely depend on the gut microbiota. However, the composition of the human gut microbiota varies considerably, and the gut strains of different individuals also have significantly different metabolic capabilities for ginsenosides. This limits the functional stability and application consistency of American ginseng in different populations. Summary of the Invention

[0005] This invention provides a probiotic combined with American ginseng formula and preparation method with sleep-aiding effects, in order to at least partially solve the problems existing in the prior art.

[0006] The first aspect of this invention provides a probiotic, wherein the probiotic is Lactobacillus rhamnosus AIML-553, and the preservation number of the probiotic is CGMCC No. 34977.

[0007] A second aspect of the present invention provides a probiotic combined with American ginseng formulation, the formulation comprising Lactobacillus rhamnosus AIML-553 and American ginseng as described in the first aspect.

[0008] Optionally, the total number of viable bacteria in the formula is 1×10⁻⁶. 4 CFU / mL ~ 1×10¹³ CFU / mL, or 1×10 4 CFU / g ~ 1×10¹³ CFU / g; the American ginseng components include any one or more of the following: whole American ginseng plant, American ginseng chunks / powder / slices, American ginseng extract, and American ginseng active extract.

[0009] Optionally, the dosage form of the formulation includes at least one of the following dosage forms: emulsion, suspension, oil drop, powder, wettable powder, granules, dispersant, tablet, capsule, suppository, lyophilized preparation, or liquid preparation.

[0010] Optionally, in the probiotic-American ginseng formula, after fermentation with Lactobacillus rhamnosus AIML-553, the contents of ginsenoside Rg2, (20R)-ginsenoside Rh2, ginsenoside Rh2, ginsenoside Rg3, ginsenoside Rg6, ginsenoside F1, pseudoginsenoside FII, and ginsenoside CK in the fermentation mixture are increased by ≥133%, ≥119%, ≥500%, ≥320%, ≥426%, ≥262%, ≥127%, and ≥154%, respectively, compared with unfermented American ginseng; the contents of 5-hydroxytryptophan, 5-hydroxytryptamine, and chlorogenic acid are increased by ≥6800%, ≥13800%, and ≥250%, respectively, compared with unfermented American ginseng.

[0011] The third aspect of this invention provides a method for preparing the probiotic combined with American ginseng formula described in the second aspect, the method comprising the following steps: The Lactobacillus rhamnosus AIML-553 described in the first aspect was activated to obtain a seed culture; The components of American ginseng were sterilized to obtain a fermentation substrate; The seed liquid was inoculated into the fermentation substrate for fermentation to obtain a probiotic combined with American ginseng formula.

[0012] Optionally, the seed liquid is inoculated into the fermentation substrate for fermentation, including: the inoculation amount is 0.5% to 5% of the fermentation substrate volume, the fermentation temperature is 25℃ to 40℃, and the fermentation time is 12h to 120h.

[0013] Optionally, the method further includes: The fermentation product obtained from fermentation is freeze-dried to obtain a probiotic combined with American ginseng formula. The freeze-drying protectant and dosage form excipients used in the freeze-drying process are selected from any one or at least two combinations of skim milk, gelatin, dextrin, resistant dextrin, gum arabic, sodium alginate, sucrose, lactose, trehalose, lacto-oligosaccharides, fructo-oligosaccharides, malto-oligosaccharides, sorbitol, xylitol, sodium carboxymethyl cellulose, microcrystalline cellulose, cysteine, carbomer, polyethylene glycol, and vitamin C.

[0014] The third aspect of this invention provides the application of the probiotic combined with American ginseng formula described in the second aspect, for the preparation of functional foods, special dietary foods, or health foods that improve sleep, regulate circadian rhythms, and relieve nervous anxiety.

[0015] Optionally, the dosage form of the functional food, special dietary food, or health food may be any one of oral liquid, solid beverage, compressed candy, soft capsule, hard capsule, or lyophilized powder.

[0016] Compared with the prior art, the technical solution provided by the embodiments of the present invention has the following outstanding advantages: The *Lactobacillus rhamnosus* AIML-553 strain, with accession number CGMCC No. 34977 provided by this invention, can maintain a high survival rate solely on American ginseng, enabling fermentation of the ginseng. This strain carries a complete and highly active glycosidase system, which can directionally convert the poorly absorbed proto-ginsenosides in American ginseng into rarer ginsenosides that are more lipid-soluble, have better membrane permeability, and higher bioactivity. Testing showed that the content of ginsenoside Rh2 increased by 500%, ginsenoside Rg6 by 426.4%, ginsenoside Rg3 by 320.1%, ginsenoside CK by 154.6%, and total saponins by 136.1%. This fundamentally solves the technical problem of the large molecular weight, high polarity, and difficulty in direct absorption of proto-ginsenosides in the small intestine, significantly improving the bioavailability of the active ingredients in American ginseng.

[0017] After fermentation with AIML-553, the content of active substances directly related to sleep regulation in the American ginseng system showed an explosive increase: 5-hydroxytryptophan (5-HTP) increased by 6867.3%, serotonin (5-HT) by 13854.3%, chlorogenic acid by 255%, and dihydrocaffeic acid by 174.6%, which can effectively inhibit anxiety, regulate circadian rhythms, shorten sleep onset time, and deepen sleep. Validated in a zebrafish insomnia model, the fermentation product of the probiotic combined with American ginseng formula provided in this invention significantly reduced the amount of wakefulness and total wakefulness time in insomniac zebrafish, demonstrating a definite sleep improvement effect comparable to the positive control drug estazolam, with a higher safety profile.

[0018] AIML-553 is isolated from the gut of healthy, long-lived humans. It boasts high safety and maintains a high survival rate even under ginseng-only nutritional conditions, failing to reach peak growth after 24 hours and retaining a high viable count after 48 hours. Compared to common probiotics such as LGG, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium bifidum, AIML-553 exhibits superior metabolic activity and a higher viable count in ginseng-containing culture media. It can achieve efficient fermentation without complex culture media, significantly reducing the fermentation process's dependence on the nutritional system.

[0019] The efficacy of American ginseng highly depends on the metabolic transformation of saponins by intestinal flora. However, the composition of human intestinal flora varies greatly, leading to inconsistent absorption and utilization of American ginseng among different individuals. This invention achieves in vitro probiotic pre-conversion through a probiotic-American ginseng formula, completing the efficient metabolism of ginsenosides in advance. This bypasses the limitations imposed by differences in human intestinal flora, enabling different populations to stably obtain highly active rare ginsenosides, significantly improving the consistency of product efficacy and its universality of application.

[0020] The preparation method of the probiotic combined with American ginseng formula of this invention adopts static fermentation at 25℃~40℃. The process conditions are mild, the operation is simple, and no special equipment is required. The product can be made into a variety of dosage forms such as oral liquid, powder, granules, tablets, capsules, and freeze-dried preparations. It is suitable for various product forms such as functional foods, dietary supplements, and health drinks. It has high industrialization feasibility and wide application scenarios.

[0021] The strains used in this invention are derived from the intestines of healthy humans, and the fermentation substrate is natural American ginseng. No toxic or harmful substances are generated during the fermentation process. According to the acute toxicity and tolerance tests of zebrafish, the formula of this invention is safe and non-toxic at effective concentrations, has good tolerability, can be taken daily for a long time, and is suitable for a wide range of people. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a colony morphology diagram of Lactobacillus rhamnosus AIML-553 provided in an embodiment of the present invention; Figure 2 The growth curve of Lactobacillus rhamnosus strain AIML-553 provided in the embodiments of the present invention; Figure 3 The viable cell count results of Lactobacillus rhamnosus AIML-553 in different culture media provided in the embodiments of the present invention; Figure 4 The viable count results of AIML-553 provided in this embodiment of the invention were compared with those of other lactobacilli on MRS total nutrient medium containing American ginseng. Figure 5 Provided for embodiments of the present invention Figure 6 This is a schematic diagram illustrating the experimental results of the effect of AIML553 American ginseng fermentation liquid on sleep-related behaviors provided in an embodiment of the present invention. Detailed Implementation

[0024] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents and other instruments whose manufacturers are not specified are all commercially available products.

[0026] Fermenting American ginseng with probiotics to pre-convert ginsenosides is an effective strategy to improve its bioavailability and functional stability.

[0027] Despite this, existing research and applications still have significant shortcomings: on the one hand, most studies focus only on single strains or single-step transformation reactions, lacking systematic screening of the complete β-glucosidase metabolic pathway; on the other hand, research on the "American ginseng-probiotic co-fermentation system" is mostly concentrated on formulation development, lacking research on screening functional strains at the gene level and combining metabolomics and in vivo models for systematic validation. Furthermore, the mechanisms by which specific lactic acid bacteria promote the production of rare ginsenosides (such as CK, Rh2, F2, etc.) during American ginseng fermentation and their functional evaluation remain insufficient.

[0028] This invention screened probiotic strains carrying the complete β-glucosidase metabolic pathway, verified their conversion ability of ginsenosides through in vitro American ginseng fermentation, and validated their pharmacological activity using a zebrafish insomnia model. This provides a new theoretical basis and technical approach for the development of functional foods based on American ginseng.

[0029] Specifically, this invention isolated a strain of Lactobacillus rhamnosus AIML-553 from the intestines of a healthy 93-year-old male centenarian. Lacticaseibacillus rhamnosus AIML-553 possesses a complete and highly active glycosidase system, enabling it to fully utilize American ginseng components under any nutritional conditions. It converts ginseng saponins into rarer saponins that are more easily absorbed and utilized by the human body. Specifically, the contents of ginsenoside RG2, (20R)-ginsenoside Rh2, ginsenoside Rh2, ginsenoside Rg3, ginsenoside Rg6, ginsenoside F1, pseudoginsenoside FII, and ginsenoside CK are increased by 133%, 119%, 500%, 320%, 426%, 262%, 127%, and 154%, respectively. Simultaneously, beneficial components that promote sleep and improve sleep quality are also significantly enhanced. The AIML-553 strain was deposited on June 24, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC 34977.

[0030] Specifically, the above-mentioned Lactobacillus rhamnosus AIML-553 was isolated using the following method in the embodiments of the present invention.

[0031] 1. Processing and preservation of fecal samples a. Use a sterile spoon to transfer the feces into a beaker and weigh it inside the biosafety cabinet.

[0032] b. Add the diluent (0.9% sodium chloride aqueous solution containing 0.05% L-cysteine ​​hydrochloride, the same below) at a ratio of 1:2 (w / v).

[0033] c. Use a magnetic stirrer to thoroughly stir the mixture to prepare a homogeneous fecal liquid, then filter it through sterile gauze. After filtration, add sterile glycerol at a ratio of 1:1 (v / v).

[0034] d. Mix using a vortex mixer, and aliquot 2-3 mL into 15 mL centrifuge tubes. The total number of aliquots should be at least 10.

[0035] e. Label the sample tubes with the fecal sample number, processing date, and the name of the experimenter, and seal them for storage at -80 ℃.

[0036] f. Fill out the fecal sample entry information form.

[0037] 2. Thawing and enrichment culture of fecal samples a. Take one sample of fecal matter.

[0038] b. Rapid thawing in a 37°C water bath.

[0039] c. Resuspend in 10 mL of MRS medium and dilute in 9 steps using MRS medium at a 10-fold ratio.

[0040] d. Transfer 10-100 μL of the bacterial solution of the required dilution gradient and streak or spread it onto 3-5 MRS medium plates.

[0041] The cells were isolated and cultured at 37℃, and their growth was observed.

[0042] f. After the culture is complete, seal the culture plate and store it at 4 ℃.

[0043] 3. Propagation, preservation and identification of strains a. Pick single colonies from each isolation culture medium and photograph and record them.

[0044] b. Inoculate a single colony into 5 mL of MRS medium, incubate at 37°C for proliferation, and observe the growth.

[0045] c. After the proliferation culture is completed, transfer 1 mL of bacterial culture to a 1.5 mL centrifuge tube and store at 4 °C for sequencing identification.

[0046] d. Add sterile glycerol to the remaining 4 mL of bacterial culture. The final concentration of glycerol should be 30-50%. Mix thoroughly and then seal and store temporarily at 4 °C.

[0047] e. After the strain identification is completed, all the bacterial culture of the target strain is aliquoted into 2 mL cryovials and stored at -80 ℃ or in liquid nitrogen.

[0048] 100 μL of AIML-553 bacterial suspension was streaked onto an MRS agar plate and incubated at 37°C for 24 hours to obtain the desired result. Figure 1 The diagram shows the colony morphology.

[0049] In this embodiment of the invention, the culture medium for the bacterial strain is specifically: a. The composition of MRS culture medium is: 52.24g MRS and 1000 mL purified water.

[0050] b. The composition of the MRS medium plate is: 52.24g MRS, 15g agar, and 1000mL purified water.

[0051] The method for preparing MRS culture medium is as follows: a. Accurately weigh / transfer the above reagents into a reagent bottle, add the corresponding volume of purified water, and stir until dissolved.

[0052] b. Autoclave at 118 ℃ for 15 min. Seal and store at 4 ℃. Use within 4 weeks.

[0053] The method for preparing MRS culture medium plates is as follows: a. Weigh 7.5 g of agar into a 1000 mL Erlenmeyer flask and add 484.8 mL of the solution from step “a” of the MRS culture medium preparation.

[0054] b. Autoclave at 118 ℃ for 15 min. After the culture medium has cooled to about 60 ℃, pour the culture medium into disposable petri dishes, 15-20 mL per dish. After the culture medium has solidified, invert the dishes and place them into resealable bags.

[0055] c. Store in a sealed container at 4 ℃ and use within 4 weeks.

[0056] In this embodiment of the invention, the preserved strain was sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for strain identification. The procedure was as follows: genomic DNA extraction, 16S amplification, PCR product detection and purification, sequencing, and result comparison. Gene amplification showed a single, clear target band. Sequencing results, compared with other strains via BLAST, showed a sequence similarity of: 1: Lacticaseibacillus rhamnosus(NR113332.1)100.000% 2: Lacticaseibacillus rhamnosus(NR043408.1)99.789% The 16S sequence of the strain is: >16S-L-0553.33672983 CTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAG GCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCC TACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCATTGTA GCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAG TCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCT CACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGAT CAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCC CCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTG AAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTAC CCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCA TTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAA GCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCA CCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTA CTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATC AGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCCAATGTGG CCGATCAACCTCTCAGTTCGGCTACGTATCATTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATACGCCGCGG GTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATTTATGCGGTATTAGCATC TGTTTCCAAATGTTATCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTCAAAATTA AATCAAGATGCAAGCACCTTTTCAATAATCAGAAC The final annotation result is Lacticaseibacillus rhamnosus (NR113332.1) 100.000%, which is Lactobacillus rhamnosus, internal number AIML-553.

[0057] In this embodiment of the invention, the growth curve of Lactobacillus rhamnosus strain AIML-553 was determined using the following experimental method: Specifically, one vial of frozen AIML-553 bacterial culture was thawed and incubated in 5 mL of MRS medium at 37°C for 16 hours. The culture was then inoculated into 20 mL of MRS medium at a 1% inoculation ratio. The OD600 absorbance, pH, and bacterial count were measured at 0, 6, 16, 20, and 24 hours of incubation. The bacterial cultures obtained at the above incubation time points were serially diluted 10-fold with 0.9% sodium chloride. 100 μL of the diluted culture was spread onto MRS agar plates and incubated at 37°C for 24 hours. The bacterial counts were then determined, and the growth data of *Lactobacillus rhamnosus* strain AIML-553 are shown in Table 1. Figure 2 The growth curve of Lactobacillus rhamnosus strain AIML-553 is shown.

[0058]

[0059] Table 1. Growth data of Lactobacillus rhamnosus strain AIML-553.

[0060] The embodiments of the present invention verify through experiments that the above-mentioned Lactobacillus rhamnosus AIML-553 can maintain a high survival rate under nutrient-poor culture conditions, relying solely on American ginseng.

[0061] The specific experimental method is as follows: 1. Thaw and reactivate one vial of AIML-351 frozen bacterial culture, and incubate at 37°C for 16 hours in 5 mL of MRS medium.

[0062] 2. Inoculate at a 1% inoculation ratio into 20 mL of MRS medium and incubate at 37°C for 16 hours.

[0063] 3. Weigh 52.24 g of MRS, add 1 L of purified water and mix well. Autoclave at 118 ℃ for 15 min to prepare MRS culture medium.

[0064] 4. Weigh 30 g of American ginseng powder, add 1 L of purified water and mix well. Sterilize at 118 ℃ for 15 min to prepare American ginseng culture medium.

[0065] 5. Weigh 54.24 g MRS and 30 g American ginseng powder, add 1 L of purified water and mix well. Sterilize at 118 ℃ for 15 min to prepare MRS+American ginseng culture medium.

[0066] 6. Inoculate 2% (v / v) into 20 mL MRS culture medium, 20 mL MRS + American ginseng culture medium, and 20 mL American ginseng culture medium respectively, and incubate at 37℃.

[0067] 7. The viable bacterial count was measured in the three culture media at 24 hours and 48 hours, respectively.

[0068] viable cell count results as follows Figure 3 As shown in the figure, the viable cell count results indicated that AIML-553 did not reach its peak growth after 24 hours of culture in a ginseng-only broth, and maintained a high viable cell count even after 48 hours of culture. This demonstrates that AIML-553 can survive under ginseng-only nutritional conditions, which provides a good foundation for the safety and process of fermentation.

[0069] The embodiments of this invention also experimentally verified that AIML-553 has better survival than other lactobacilli in MRS total nutrient medium containing American ginseng. The specific experimental methods are as follows: 1. Thaw and resuscitate one vial each of the following: Lacticaseibacillus rhamnosus AIML-553, Lacticaseibacillus rhamnosus GG (LGG), Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium animalis. Incubate each vial in 5 mL of MRS medium at 37°C for 16 hours. 2. Inoculate each of the five revived bacterial cultures at 1% (v / v) into 20 mL of MRS medium and incubate at 37°C for 16 hours. Set aside for later use.

[0070] 3. Weigh 52.24 g of MRS and 1 g of American ginseng powder, add 1 L of drinking water, and autoclave at 118 ℃ for 15 min.

[0071] 4. The above 5 bacterial cultures were inoculated at 2% (v / v) into MRS medium containing 1 g of American ginseng and cultured anaerobically at 37°C for 24 hours.

[0072] 5. Perform viable cell count; the count results are as follows: Figure 4 As shown, there are significant differences in the utilization of American ginseng by different probiotic strains. The activities of different strains or species in the total nutrient medium containing American ginseng also vary, and the differences in their ability to utilize American ginseng components directly reflect the differences in bacterial count and activity. AIML-553 exhibits better metabolic activity in the total nutrient medium containing American ginseng.

[0073] Therefore, in this embodiment of the invention, a probiotic combined with American ginseng formulation is further obtained based on the above-mentioned probiotics. This formulation contains the above-mentioned Lactobacillus rhamnosus AIML-553 and American ginseng components. Specifically, this formulation can be a mixture containing AIML-553 and / or American ginseng components, a supernatant, a precipitate, a fermentation mixture, a fermentation supernatant, a fermentation precipitate, inactivated substances and their fragments, metabolites, a mixture of bacterial cells and filtrate, a solid dry powder, etc.

[0074] Specifically, this formula can consist of two components: Lactobacillus rhamnosus AIML-553 and American ginseng. In practical applications, the formula can include Lactobacillus rhamnosus AIML-553 and American ginseng separately, and then mix them together before use. Alternatively, the formula can be a mixture of Lactobacillus rhamnosus AIML-553 and American ginseng, which is fermented before use; or it can be a fermented mixture of Lactobacillus rhamnosus AIML-553 and American ginseng, which can be used directly in practical applications.

[0075] Specifically, in the combined formulation, the total number of viable bacteria is not less than 1×10⁻⁶. 4 CFU / mL or 1×10 4 CFU / g, for example, could be: 1×10 4 CFU / mL (CFU / g), 5×10 4 CFU / mL (CFU / g), 1×10 5 CFU / mL (CFU / g), 5×10 5 CFU / mL (CFU / g), 1×10 6 CFU / mL (CFU / g), 5×10 6 CFU / mL (CFU / g), 1×10 7 CFU / mL (CFU / g), 5×10 7 CFU / mL (CFU / g), 1×10 8 CFU / mL (CFU / g), 5×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), 1×10 13 CFU / mL (CFU / g), etc.; other specific point values ​​within this range can be selected, which will not be elaborated here.

[0076] In this embodiment of the invention, the combined formula contains American ginseng components including, but not limited to, whole American ginseng plant, any part of American ginseng in any form such as block / powder / slice, American ginseng extract, and American ginseng active ingredients.

[0077] In this embodiment of the invention, the combined formulation may be provided in the form of solid, liquid, powder, or any combination thereof. For example, the dosage forms of the combined formulations disclosed herein include, but are not limited to, at least one of the following dosage forms: emulsion, suspension, oil drop, powder, wettable powder, granules, dispersant, tablet, capsule, suppository, lyophilized preparation, or liquid preparation.

[0078] In this embodiment of the invention, the preparation method of the above-mentioned combined formulation can be a conventional method in the industry, such as preparing the freeze-dried fermented preparation according to the process flow of strain activation—strain inoculation—American ginseng fermentation—pre-freezing—vacuum freeze-drying.

[0079] Specifically, the preparation method of the probiotic combined with American ginseng formula includes: 1. Preparation of a premixed formula combining probiotics and American ginseng 1.1. Preparation of probiotic components: AIML-553 is activated and cultured for fermentation. The resulting fermentation broth is centrifuged and resuspended in a solvent, or the resuspended bacterial solution is freeze-dried to obtain the bacterial agent.

[0080] 1.2. The freeze-drying protectant and formulation excipients used in the preparation of freeze-dried bacterial powder are selected from any one or a combination of at least two of the following: skim milk, gelatin, dextrin, resistant dextrin, gum arabic, sodium alginate, sucrose, lactose, trehalose, lacto-oligosaccharides, fructo-oligosaccharides, malto-oligosaccharides, sorbitol, xylitol, sodium carboxymethyl cellulose, microcrystalline cellulose, cysteine, carbomer, polyethylene glycol, and vitamin C.

[0081] 1.3. Mix the freeze-dried bacterial powder with solid American ginseng components in any proportion to obtain a probiotic and American ginseng premix product.

[0082] 2. Preparation of a probiotic combined with American ginseng fermentation formula 2.1. Preparation of probiotic components: AIML-553 bacterial solution was obtained by activating and culturing AIML-553.

[0083] 2.2. Mix American ginseng components and 1 g / L glucose in drinking water to obtain the pre-fermentation liquid of American ginseng. The concentration of American ginseng components in the culture medium is not limited; the amount of American ginseng components added per liter of culture medium can be freely set according to the requirements of conventional cell / strain / plant culture. The prepared liquid is then autoclaved.

[0084] 2.3. Inoculation of AIML-553. This invention does not impose a numerical limit on the inoculation amount of the fermentation strain; those skilled in the art can freely set it as needed.

[0085] 2.4. The fermentation time is flexibly determined according to the actual fermentation state and process requirements, and there is no fixed time limit.

[0086] The probiotics provided in this invention can survive and maintain high activity in a nutrient-poor environment containing only American ginseng, without the need for complex culture media. Compared with commonly used strains such as LGG and Bifidobacterium, they exhibit higher viable counts and stronger metabolic activity in the American ginseng system.

[0087] The preparation process of the probiotic combined with American ginseng formula provided in this invention is simple and requires no special equipment. The fermentation conditions are mild (25℃~40℃), and it can be flexibly prepared into various dosage forms such as oral liquid, powder, capsule, and granules.

[0088] In this embodiment of the invention, whole-genome analysis of AIML-553 revealed that AIML-553 possesses a complete enzyme system related to glycoside metabolism, which can fully utilize the original saponins in American ginseng to metabolize them into rare saponins with high activity and high bioavailability.

[0089] Specifically, the experimental method is as follows: 1. Activation of AIML-553 and preparation of working strain 1.1. Take one vial of AIML-553 frozen bacterial culture, thaw and resuscitate it, and incubate it in 5 mL of MRS medium at 37°C for 16 hours.

[0090] 1.2. Inoculate at a 1% (v / v) inoculation ratio into 20 mL of MRS medium and incubate at 37°C for 16 hours. Set aside for later use.

[0091] 2. Preparation of liquid before American ginseng fermentation 2.1. Weigh 0.5 g of American ginseng powder and 1 g of glucose, and add them to 1 L of drinking water.

[0092] 2.2.118 ℃ for 15 min, autoclave.

[0093] 2.3. Let stand until room temperature before use.

[0094] 3. Preparation of American ginseng fermentation broth 3.1. Inoculate the activated AIML-553 into the American ginseng pre-fermentation liquid at 2% (v / v) and mix well.

[0095] 3.2.37℃ static fermentation for 72 hours.

[0096] 3.3.121 ℃ for 15 min, autoclave to inactivate probiotics.

[0097] 3.4. Centrifuge at 6000 × g for 10 min and collect the supernatant for later use.

[0098] 4. Metabolomics analysis The pre-fermentation liquid and fermentation broth of American ginseng were sent to Sangon Biotech (Shanghai) Co., Ltd. for metabolomics analysis.

[0099] 5. Metabolomics Analysis Results The experimental results are shown in Tables 2 and 3:

[0100] Table 2. Twelve rare saponins whose content was significantly increased in American ginseng fermentation liquid.

[0101] Table 3. Five anti-aging components whose content was significantly increased in American ginseng fermentation liquid. The experimental results show that, compared with the liquid before American ginseng fermentation, the fermentation liquid of American ginseng has significantly increased the content of at least 12 rare saponins and 5 other substances beneficial to human health.

[0102] The AIML-553 provided in this invention carries a complete, highly active glycosidase system, which can efficiently convert the poorly absorbed proto-ginsenosides of American ginseng into small-molecule, highly lipophilic, and easily absorbed rare ginsenosides. The AIML-553 American ginseng fermentation broth shows a significant increase in key saponins: ginsenoside Rh2 increased by 500%, Rg6 by 426%, Rg3 by 320%, CK by 154%, and total saponins by 136.1%. Its bioactivity and absorption efficiency are far superior to those of ordinary American ginseng.

[0103] Experiments in this invention have shown that AIML-553-fermented American ginseng can significantly increase the content of beneficial sleep-aiding substances other than ginsenosides. Specifically, after fermentation with AIML-553, the beneficial components that promote sleep and improve sleep quality are significantly increased, except for rare saponins.

[0104] Specifically, the experimental method is as follows: 1. Activation of AIML-553 and preparation of working strain 1.1. Take one vial of AIML-553 frozen bacterial culture, thaw and resuscitate it, and incubate it in 5 mL of MRS medium at 37°C for 16 hours.

[0105] 1.2. Inoculate at a 1% (v / v) inoculation ratio into 20 mL of MRS medium and incubate at 37°C for 16 hours. Set aside for later use.

[0106] 2. Preparation of liquid before American ginseng fermentation 2.1. Weigh 15 g of American ginseng powder and 1 g of glucose, and add them to 1 L of drinking water.

[0107] 2.2.118 ℃ for 15 min, autoclave.

[0108] 2.3. Let stand until room temperature before use.

[0109] 3. Preparation of American ginseng fermentation broth 3.1. Inoculate the activated AIML-553 into the American ginseng pre-fermentation liquid at 2% (v / v) and mix well.

[0110] 3.2.37℃ static fermentation for 24 hours.

[0111] 3.3.121 ℃ for 15 min, autoclave to inactivate probiotics.

[0112] 3.4. Centrifuge at 6000 × g for 10 min and collect the supernatant for later use.

[0113] 4. Metabolomics analysis The pre-fermentation liquid and fermentation broth of American ginseng were sent to Sangon Biotech (Shanghai) Co., Ltd. for metabolomics analysis.

[0114] 5. Metabolomics Analysis Results The experimental results are shown in Table 4:

[0115] Table 4. Sleep-aiding components in American ginseng fermentation liquid Experimental results show that, compared with the liquid before fermentation of American ginseng, the content of components that can improve sleep in AIML0553 American ginseng fermentation liquid is significantly increased.

[0116] Furthermore, in this embodiment of the invention, the efficacy of the probiotic combined with American ginseng fermentation formula was verified based on the insomnia zebrafish model. The experimental method is as follows: This zebrafish efficacy verification experiment was commissioned to Hangzhou Huante Biotechnology Co., Ltd., a CRO company that has passed AAALAC international laboratory animal certification (certification number: 001458).

[0117] Zebrafish share a similar nervous system with mammals. Most of the molecules and cellular groups (monoaminers, cholinergics, hypothalamus-likes) that regulate sleep in mammals are conserved in zebrafish. The responses to various hypnotic drugs, such as H1 histamine antagonists, melatonin agonists, α-2 adrenergic agonists, and γ-aminobutyric acid (GABA) modulators, are also conserved in zebrafish. GABA is a naturally occurring non-protein amino acid and an important inhibitory neurotransmitter in the mammalian central nervous system. Pentyltetrazole (PTZ) is a GABA inhibitor; low doses of PTZ can induce bipolar disorder, characterized by both mania and depression, resulting in insomnia. Zebrafish suffering from insomnia exhibit abnormal behavior, including a significant increase in total movement distance, which can be observed using behavioral analyzers.

[0118] The experimental procedure is as follows: 1. Laboratory animals Zebrafish are all cultured in fish water at 28°C (water quality: 200 mg of instant sea salt is added to every 1 L of reverse osmosis water, with a conductivity of 450~550 uS / cm; pH of 6.5~8.5; hardness of 50~100 mg / L CaCO₃). The zebrafish are bred and provided by the Fish Culture Center of our company. The license number for the use of experimental animals is SYXK(Zhe) 2022-0004, the culture management meets the requirements of the international AAALAC certification (certification number: 001458), and the IACUC ethical review number is IACUC-2026-202601260009-01.

[0119] 2. Instruments, Consumables and Reagents Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, America); Behavior analyzer (Zebra Lab 3.22.3.31, Viewpoint, France); 6-well plates (Zhejiang Bellan Biotechnology Co., Ltd., China); 96-well plates (NestBiotech, China); Automatic rapid sample grinding instrument (JXFSTPRP-24L, Shanghai Jingxin Experimental Equipment Technology Department, China). Pentylenetetrazole (PTZ, batch number YH0171125, Shanghai Yihe Biotechnology Co., Ltd., China); Dimethyl sulfoxide (DMSO, batch number 20250624, Sinopharm Chemical Reagent Co., Ltd., China).

[0120] 3. Information of Test Samples Fermented American ginseng liquid; positive control: Estazolam tablets (batch number 221007, Shandong Xinyi Pharmaceutical).

[0121] 4. Preparation of Fermented American Ginseng Liquid Sample 4.1. Take 1 tube of AIML-553 frozen stock bacterial solution for thawing and resuscitation, and culture in 5 mL of MRS medium at 37°C for 16 hours.

[0122] 4.2. Inoculate into 20 mL of MRS medium at an inoculation ratio of 1%, and culture at 37°C for 16 hours, then set aside for use.

[0123] 4.3. Weigh 30 g of ground American ginseng powder and 1 g of glucose, add them to 1 L of drinking water.

[0124] 4.4. Autoclave at 118°C for 15 min. Let it stand until it cools to room temperature and set aside.

[0125] 4.5. Inoculate the activated AIML-553 into the pre-fermentation liquid of American ginseng at an inoculation amount of 2% (v / v), and mix evenly.

[0126] 4.6.37℃ static fermentation for 72 hours.

[0127] 4.7.121 ℃ for 15 min, autoclave to inactivate probiotics.

[0128] 4.8. Centrifuge at 6000 × g for 10 min and collect the supernatant for later use.

[0129] 5. MTC Measurement Wild-type AB strain zebrafish, 5 days post-fertilization (5 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble solution, and a normal control group was also included. Each well contained 3 mL of sample. After treatment at 28℃ for 1 day, except for the normal control group, all other experimental groups were treated with PTZ via water-soluble solution to establish a zebrafish insomnia model. The models were then treated at 28℃ for 1 hour to determine the mean chronotropic concentration (MTC) of the sample in the model zebrafish.

[0130] Under the conditions of this experiment, the maximum detectable concentration (MTC) of American ginseng fermentation broth for improving sleep was 62.5 μL / mL in zebrafish. It is safe, non-toxic, and well-tolerated at conventional dosage concentrations.

[0131] 6. Experimental Grouping Normal control group, model control group, estazolam 10 μg / mL group, and American ginseng fermentation broth 62.5 μg / mL group.

[0132] 7. Experimental and Detection Methods Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (concentrations shown in Table 5) were administered in water, with estazolam as a positive control at a concentration of 10.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. After treatment at 28°C for one day, 10 zebrafish from each experimental group were randomly transferred to 96-well plates. Except for the normal control group, all other experimental groups were given PTZ in water to establish a zebrafish insomnia model. The level of arousal and total wakefulness time of the zebrafish within 1 hour were measured using a behavioral analyzer. The sleep-improving efficacy of the samples was evaluated using statistical analysis of these indicators. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0133] 8. Experimental Test Results The experimental results are shown in Table 5. Figure 5 and Figure 6 As shown (compared with the model control group, **P<0.01, ***P<0.001)

[0134] Table 5. Experimental Results of the Effects of American Ginseng Fermentation Broth on Sleep-Related Behaviors As shown in Table 5, American ginseng fermentation liquid can significantly reduce the activity level and wakefulness time of the high arousal model, demonstrating a clear sedative and sleep-aiding effect. Moreover, the effect is better than that of the positive control drug estazolam, suggesting that it has the potential for development and application in improving sleep and relieving anxiety-induced arousal.

[0135] Figure 5 The diagram shows the movement trajectories of each group of zebrafish during the test, recorded using a behavior analyzer. Red lines represent fast movement, green lines represent medium-speed movement, and black lines represent slow movement. The total number of lines of each color indicates the zebrafish's state of insomnia and mania; fewer red lines or more black and green lines indicate a higher degree of relief from insomnia and mania. Figure 5 As shown, the fermented ginseng liquid fermented with AIML-553 can significantly improve insomnia and depression in zebrafish, and its effect is similar to that of estazolam.

[0136] Figure 6 The experimental results of the effects of AIML553 American ginseng fermentation liquid on sleep-related behaviors are shown. The left figure represents the activity levels of zebrafish in each group during insomnia / bipolar disorder. It can be seen that the activity levels in the model group significantly increased, indicating the successful establishment of the insomnia-bipolar disorder model. Estazolam significantly reduced activity levels, but the effect was limited. The American ginseng fermentation liquid group showed the largest decrease in activity levels, approaching the level of the normal control group, and its effect was far superior to the positive control drug estazolam. The right figure represents the total wake time of zebrafish in each group during insomnia / bipolar disorder. The wake time in the model group was significantly prolonged, verifying the effectiveness of the insomnia model. Estazolam can shorten wake time, but it remains at a high level. The wake time in the American ginseng fermentation liquid group decreased to an even lower level, very close to the normal control group, and its sleep-aiding effect was also significantly better than estazolam.

[0137] Experiments show that AIML553 American ginseng fermentation liquid can significantly reduce the activity level and wakefulness time of insomnia-induced bipolar zebrafish. Its sedative and sleep-aiding effects are not only clear but also far superior to estazolam under the same conditions, demonstrating a strong potential to improve sleep and relieve bipolar disorder.

[0138] The probiotics combined with American ginseng fermentation liquid provided in this invention have resulted in an explosive increase in key sleep-aiding substances. Specifically, 5-hydroxytryptophan (5-HTP) increased by 6867.3% (5-HT precursor, regulating mood rhythm), serotonin (5-HT) increased by 13854.3% (promoting sleep onset and deepening sleep), chlorogenic acid increased by 255%, and dihydrocaffeic acid increased by 174.6%, synergistically soothing nerves and shortening sleep onset time.

[0139] Furthermore, the zebrafish insomnia model showed that the fermentation broth could significantly reduce the amount of arousal activity and the total arousal time, with better effects than the positive control drug estazolam. It was also non-toxic, well-tolerated, and had stable and repeatable efficacy.

[0140] The probiotic combined with American ginseng formula provided in this invention uses Lactobacillus rhamnosus AIML-553 to ferment the American ginseng components, completing the pre-conversion of saponins by the intestinal flora in advance, solving the differences in human intestinal flora, and allowing different groups of people to stably absorb the active ingredients of American ginseng, greatly improving the consistency of efficacy.

[0141] In addition to the traditional effects of American ginseng in "anti-fatigue and immune regulation", the probiotic combined with American ginseng formula provided in this invention can achieve sleep-aiding effects. It can then be developed into a functional food, special dietary food or health food that improves sleep, regulates circadian rhythms and relieves nervous anxiety, with broad prospects for industrialization.

[0142] In the probiotic and American ginseng formula provided in this embodiment of the invention, the probiotics can be inactivated before use, fermented with natural substrates, and have been verified as safe and non-toxic using a zebrafish model, making them suitable for long-term daily consumption.

[0143] The above experimental results show that the probiotic and American ginseng co-fermentation formulation system provided by this invention uses American ginseng as the fermentation substrate and specific probiotics for synergistic fermentation. The probiotics can efficiently utilize the original saponins in American ginseng, achieving targeted biotransformation into rare saponins, significantly increasing the enrichment content of rare saponins in the fermentation system. Simultaneously, this co-fermentation system can further metabolize and transform the endogenous functional components of American ginseng, significantly upregulating the accumulation level of beneficial substances with sleep-aiding activity in the system. Pharmacodynamic verification using a zebrafish insomnia model demonstrates that the probiotic and American ginseng formulation of this invention has a clear and excellent sleep-improving effect. This invention optimizes the co-fermentation system of probiotics and American ginseng. AIML-553 has a complete glycoside metabolic enzyme system, enabling efficient fermentation of American ginseng without a complex nutritional environment and without complex processes. It simultaneously achieves efficient enrichment of rare saponins and increased generation of sleep-aiding active substances, resulting in products with clear activity and good safety. These products can be developed into oral liquids, functional beverages, and health foods for improving sleep, demonstrating significant application prospects and industrialization value.

[0144] Furthermore, this invention proposes the application of the above-mentioned probiotic combined with American ginseng formula for the preparation of functional foods, special dietary foods, or health foods that improve sleep, regulate circadian rhythms, and relieve nervous anxiety.

[0145] Furthermore, the dosage form of the functional food, special dietary food, or health food is any one of oral liquid, solid beverage, compressed candy, soft capsule, hard capsule, or lyophilized powder.

[0146] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0147] The above provides a detailed description of a probiotic combined with American ginseng formula and preparation method with sleep-aiding effects provided by the present invention. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A probiotic, characterized in that, The probiotic is Lactobacillus rhamnosus AIML-553, and the preservation number of the probiotic is CGMCC No. 34977.

2. A probiotic combined with American ginseng formula, characterized in that, The formula contains Lactobacillus rhamnosus AIML-553 as described in claim 1 and American ginseng.

3. The probiotic combined with American ginseng formula according to claim 2, characterized in that, The total number of viable bacteria in the formula is 1×10⁻⁶. 4 CFU / mL ~ 1×10¹³ CFU / mL, or 1×10 4 CFU / g ~ 1×10¹³ CFU / g; the American ginseng components include any one or more of the following: whole American ginseng plant, American ginseng chunks / powder / slices, American ginseng extract, and American ginseng active extract.

4. The probiotic combined with American ginseng formula according to claim 2, characterized in that, The dosage form of the formulation includes at least one of the following: emulsion, suspension, oil drop, powder, wettable powder, granules, dispersant, tablet, capsule, suppository, lyophilized preparation, or liquid preparation.

5. The probiotic combined with American ginseng formula according to any one of claims 2-4, characterized in that, In the probiotic-American ginseng formula, after fermentation with Lactobacillus rhamnosus AIML-553, the contents of ginsenoside Rg2, (20R)-ginsenoside Rh2, ginsenoside Rh2, ginsenoside Rg3, ginsenoside Rg6, ginsenoside F1, pseudoginsenoside FII, and ginsenoside CK in the fermentation mixture were increased by ≥133%, ≥119%, ≥500%, ≥320%, ≥426%, ≥262%, ≥127%, and ≥154%, respectively, compared with unfermented American ginseng; the contents of 5-hydroxytryptophan, 5-hydroxytryptamine, and chlorogenic acid were increased by ≥6800%, ≥13800%, and ≥250%, respectively, compared with unfermented American ginseng.

6. A method for preparing the probiotic combined with American ginseng formula according to any one of claims 2 to 5, characterized in that, The method includes the following steps: The Lactobacillus rhamnosus AIML-553 of claim 1 was activated to obtain a seed culture; The components of American ginseng were sterilized to obtain a fermentation substrate; The seed liquid was inoculated into the fermentation substrate for fermentation to obtain a probiotic combined with American ginseng formula.

7. The preparation method according to claim 6, characterized in that, The seed liquid is inoculated into the fermentation substrate for fermentation, including: the inoculation amount is 0.5% to 5% of the fermentation substrate volume, the fermentation temperature is 25℃ to 40℃, and the fermentation time is 12h to 120h.

8. The preparation method according to claim 6, characterized in that, The method further includes: The fermentation product obtained from fermentation is freeze-dried to obtain a probiotic combined with American ginseng formula. The freeze-drying protectant and dosage form excipients used in the freeze-drying process are selected from any one or at least two combinations of skim milk, gelatin, dextrin, resistant dextrin, gum arabic, sodium alginate, sucrose, lactose, trehalose, lacto-oligosaccharides, fructo-oligosaccharides, malto-oligosaccharides, sorbitol, xylitol, sodium carboxymethyl cellulose, microcrystalline cellulose, cysteine, carbomer, polyethylene glycol, and vitamin C.

9. The application of the probiotic combined with American ginseng formula according to any one of claims 2 to 5, characterized in that, It is used to prepare functional foods, special dietary foods, or health foods that improve sleep, regulate circadian rhythms, and relieve nervous anxiety.

10. The application according to claim 9, characterized in that, The dosage form of the functional food, special dietary food, or health food is any one of oral liquid, solid beverage, compressed candy, soft capsule, hard capsule, or lyophilized powder.

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