A product combination for improving sleep and its preparation method

Abstract

This application relates to the fields of microbial fermentation and traditional Chinese medicine processing technology, and discloses a product combination and preparation method for improving sleep, including Gastrodia elata powder, Ziziphus jujuba seed powder, Rosa rugosa juice, honey, and a compound microbial agent. The compound microbial agent contains *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Pichia pastoris*, and *Saccharomyces cerevisiae*. The preparation method includes steps such as fermentation culture medium preparation, autoclaving, inoculation with the compound microbial agent for constant-temperature fermentation, separation and filtration, and autoclaving. This invention utilizes the synergistic metabolic action of multiple microbial groups to disrupt plant cell walls, converting macromolecular polar glycosides into free aglycones and γ-aminobutyric acid (GABA) that easily penetrate the blood-brain barrier, thereby enhancing the efficacy of central sedation and sleep improvement. In the fermentation system, the metabolites of lactic acid bacteria and yeast undergo esterification to synthesize volatile flavor esters, masking the inherent earthy and bitter tastes of the raw materials, thus improving the sensory quality and drinking compliance of the product.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation and traditional Chinese medicine processing technology, specifically to a product combination and preparation method for improving sleep. Background Technology

[0002] In recent years, with the accelerated pace of life, sleep disorders have become increasingly common. Gastrodia elata and Ziziphus jujuba var. spinosa, as traditional medicinal and edible substances, possess clear sedative and tranquilizing pharmacological activities and are frequently used in the development of health products to improve sleep. However, existing product development and processing typically employ traditional water decoction extraction or simple physical mixing processes. Because plant raw materials such as Gastrodia elata and Ziziphus jujuba var. spinosa have tough cell wall structures, conventional physical and chemical methods are insufficient to completely destroy them, resulting in the inability to fully release the bound active substances encapsulated within the cells into the solvent, thus limiting the extraction rate.

[0003] Based on this, the main sedative and tranquilizing active ingredients in Gastrodia elata and Ziziphus jujuba var. spinosa, such as gastrodin and jujube seed saponins, mostly exist in the form of large-molecule polar glycosides. These large-molecule polar substances have poor lipid solubility, making it difficult for them to directly penetrate the blood-brain barrier and enter the central nervous system to exert their effects. They usually require a long metabolic degradation process by the human gut microbiota to be converted into small-molecule aglycones to take effect. This results in low bioavailability and slow onset of action in existing products, failing to meet the need for rapid intervention in sleep disorders.

[0004] Furthermore, the raw materials of Gastrodia elata and Ziziphus jujuba var. spinosa contain secondary metabolites such as alkaloids and tannins, which give the extracts or mixtures processed conventionally a strong earthy and bitter taste. Existing flavoring methods are mostly limited to simply adding sweeteners such as sucrose or artificial sweeteners to physically mask these off-flavors, failing to degrade or eliminate these odorous substances from a chemical perspective. This results in poor sensory quality and an abrupt taste in the final product, severely reducing consumer compliance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a product portfolio and preparation method for improving sleep, solving the problems of discontinuous control trajectory, static and unadjustable emotion mapping, lack of user feedback loop, weak adaptive ability, and insufficient fusion of multi-dimensional control information in existing technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a product combination for improving sleep, which adopts the following technical solution: a product combination for improving sleep, wherein the product combination is fermented from raw materials comprising the following amounts: 1200-2000g of Gastrodia elata powder; 300-500g of Ziziphus jujuba seed powder; 15-25L of prickly pear juice; 400-600g of honey; and a compound microbial agent with an inoculum amount of 0.5%-1.5% of the total weight of the fermentation substrate.

[0008] By adopting the above technical solution, using prickly pear juice and honey as the fermentation base, combined with Gastrodia elata powder and Ziziphus jujuba seed powder, and inoculated with a compound microbial agent for fermentation, the effects of breaking through plant cell wall limitations, achieving targeted conversion of macromolecular prodrugs, and reshaping product flavor are achieved. The specific reaction process and innovative mechanism are as follows: Step 1, cell wall degradation and substance release. The compound microbial community grows and multiplies in a multi-substrate system, secreting a complex hydrolytic enzyme system including cellulase, hemicellulase, and pectinase. This enzyme system specifically cleaves the polysaccharide cross-linked backbone in the cell walls of Gastrodia elata and Ziziphus jujuba seed powder, promoting the swelling and disintegration of plant cell walls. During this process, the bound active ingredients and lipid-soluble components encapsulated inside the protoplast are fully released into the fermentation broth, increasing the dissolution rate of total solids and active ingredients. Step 2, targeted enzymatic hydrolysis and targeted conversion of macromolecules. The active ingredients in Gastrodia elata and Ziziphus jujuba seed are mostly macromolecular polar glycosides, which are difficult to penetrate the blood-brain barrier. In the fermentation system, glycoside hydrolases mediate a targeted deglycosylation reaction, cleaving the structure of gastrodin, a large molecular weight glycoside, and converting it into the highly lipid-soluble, low molecular weight free gastrodinogenin p-hydroxybenzyl alcohol. The reaction formula is as follows:

[0009] ;

[0010] Step 3: Central nervous system inhibition of neurotransmitter synthesis. Amino acid decarboxylases secreted by the complex microbial flora act on the protein substrates and precursors such as free glutamate produced by the degradation of jujube seeds, undergoing a decarboxylation reaction to generate γ-aminobutyric acid (GABA), which has a targeted sedative effect on the central nervous system. The reaction formula is as follows:

[0011] ;

[0012] Step four: Secondary metabolism and flavor reshaping. The fermentation metabolic network produces a large number of secondary products. Lactic acid bacteria undergo glycolysis to generate free short-chain fatty acids, including lactic acid and acetic acid. These short-chain fatty acids enter the human body with the product, lowering the local pH of the intestine to inhibit harmful bacteria and transmitting regulatory signals to the central nervous system through the enteroendocrine system. Simultaneously, organic acids in the system spontaneously undergo esterification with trace amounts of ethanol produced by yeast in an acidic environment, synthesizing volatile flavor molecules such as ethyl lactate and ethyl acetate. Taking the synthesis of ethyl lactate as an example, the reaction formula is as follows:

[0013] ;

[0014] The generated ester molecules have floral and fruity aromas, which chemically mask the earthy and bitter tastes of the raw materials, such as Gastrodia elata and Ziziphus jujuba, thus improving drinking compliance.

[0015] Preferably, the mass ratio of Gastrodia elata powder to Ziziphus jujuba seed powder is fixed at 4:1, and both Gastrodia elata powder and Ziziphus jujuba seed powder are 80-100 mesh powders formed through ultrafine grinding. By adopting the above technical solution, the 80-100 mesh ultrafine powder provides a huge solid-liquid contact surface area, which is conducive to the efficient binding of the complex hydrolytic enzyme system with the cell wall, while avoiding the sudden increase in system viscosity and agglomeration caused by excessively fine powder. The fixed mass ratio of 4:1 conforms to the formulation logic of calming the mind and stabilizing the spirit, ensuring that the ratio of p-hydroxybenzyl alcohol and γ-aminobutyric acid generated by conversion is in the optimal synergistic range.

[0016] Preferably, the composite microbial agent is a composition comprising *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Pichia pastoris*, and *Saccharomyces cerevisiae*. By adopting the above technical solution, the yeast community and lactic acid bacteria community form a mutually beneficial symbiotic system. In the early stage of fermentation, *Saccharomyces cerevisiae* and *Pichia pastoris* utilize the monosaccharides in honey for aerobic respiration, consuming free oxygen and releasing carbon dioxide, thus constructing an anaerobic habitat suitable for lactic acid bacteria colonization and acid production. The lactic acid bacteria community continuously produces acid using polysaccharide degradation products, while the yeast community provides the higher alcohols and trace amounts of ethanol necessary for esterification reactions, achieving seamless linkage of different substrate metabolic pathways.

[0017] Preferably, in the compound microbial agent, the ratio of viable bacteria *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Pichia pastoris*, and *Saccharomyces cerevisiae* is 1:1:1:(0.5-1):(0.5-1). By adopting the above technical solution, the initial abundance matrix of acid-producing and alcohol-producing bacteria is controlled, preventing excessive yeast proliferation in the early stage of fermentation, which could lead to excessive ethanol concentration or excessive carbon source consumption. This viable bacteria ratio ensures that the lactic acid bacteria community maintains a dominant microecological position in the middle and late stages of fermentation, maintaining a dynamic balance between the amount of organic acid produced and the amount of substrate consumed in the esterification reaction.

[0018] Preferably, the physicochemical properties of the fermentation endpoint of the product combination are: pH value controlled between 3.3 and 3.8, and refractive index controlled between 2.5 and 3.5. By adopting the above technical solution, the endpoint of biotransformation is accurately anchored using both pH value and refractive index. This range indicates that the carbon source in the system has completed appropriate metabolism, ensuring sufficient accumulation of short-chain fatty acids while avoiding over-fermentation that could lead to system rancidity or secondary degradation of the active ingredients.

[0019] Secondly, the present invention provides a method for preparing a product combination for improving sleep, which adopts the following technical solution: A method for preparing a product combination for improving sleep includes the following steps: adding weighed Gastrodia elata powder and Ziziphus jujuba seed powder to prickly pear juice, and adding honey as an auxiliary material, stirring and mixing thoroughly to prepare a fermentation culture medium; subjecting the fermentation culture medium to high-pressure sterilization, and then cooling; under aseptic conditions, inoculating the cooled fermentation culture medium with a compound microbial agent, stirring evenly, and then placing it in a fermentation tank for constant-temperature fermentation; after fermentation, terminating the fermentation state, and sequentially performing coarse filtration, centrifugation separation to collect the supernatant, and finally high-pressure sterilization to obtain the prickly pear, Gastrodia elata, and Ziziphus jujuba seed combination fermentation stock solution.

[0020] By adopting the above technical solution, a complete closed-loop multi-strain synergistic fermentation process was established. The sterilization process eliminated the interference of endogenous bacteria in the raw materials on the symbiotic fermentation network; the isothermal fermentation process provided the core habitat for targeted biotransformation and secondary metabolite synthesis; the subsequent coarse filtration, centrifugation and sterilization operations effectively removed fermentation waste and bacterial residues, so that free small molecule active ingredients and ester flavor substances were stably retained in the supernatant.

[0021] Preferably, the autoclaving temperature is 115-125℃, and the time is 10-20 minutes; after sterilization, it is rapidly cooled to 33-37℃. By adopting the above technical solution, the need to completely eliminate resistant spores and protect the substrate nutrients is met. Rapid cooling to 33-37℃ directly matches the suitable inoculation temperature for subsequent compound microbial agents, shortening the lag period of the bacterial community.

[0022] Preferably, the isothermal fermentation temperature is 33-37℃, the fermentation time is 144-192 hours, and gentle stirring is performed every 24 hours during the fermentation process. By adopting the above technical solution, the fermentation system is maintained within the optimal temperature range for the proliferation of various microbial communities and enzyme production. The deep fermentation cycle of 144-192 hours ensures the complete cleavage of macromolecular glycoside structures and the sufficient accumulation of secondary metabolic flavor molecules. Intermittent gentle stirring promotes uniform mass transfer between the substrate, oxygen, and microbial communities, increasing the probability of contact between extracellular enzyme systems and plant cell walls.

[0023] Preferably, after the pH and refractive index of the fermentation broth reach the target range, the fermentation broth is rapidly cooled to 0-10℃ to terminate fermentation; the coarse filtration is performed using an 80-120 mesh sieve, and the centrifugation speed is 5000-8000 rpm for 10-15 minutes. By adopting the above technical solution, the rapid cooling operation instantly inactivates the metabolic enzyme activity of the microbial system, locking in the final component profiles of the active pharmaceutical ingredients and flavor molecules. The combination of physical sieving and medium-to-high-speed centrifugation removes suspended large particles of plant fiber and dead bacterial aggregates, improving the clarity and physical stability of the fermentation broth.

[0024] Preferably, the final high-pressure sterilization process parameters for the separated supernatant fermentation broth are: sterilization temperature 110-120℃, sterilization time 20-40 minutes. By adopting the above technical solution, residual microorganisms and heat-resistant degrading enzymes are completely inactivated. The combination of preset temperature and time can achieve a commercially sterile state for the product without damaging the small molecule active structures such as gastrodin and γ-aminobutyric acid.

[0025] This invention provides a product combination and preparation method for improving sleep. It has the following beneficial effects:

[0026] 1. This invention utilizes a multi-substrate fermentation process using a complex microbial agent composed of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Pichia pastoris*, and *Saccharomyces cerevisiae*. The complex hydrolytic enzyme system secreted by the complex microbial community can disrupt the cell wall structure of *Gastrodia elata* powder and *Ziziphus jujuba* seed powder, promoting the release of bound components into the fermentation broth. Glycoside hydrolysates and amino acid decarboxylases within the fermentation system trigger targeted enzymatic hydrolysis reactions, converting large-molecule polar glycosides that are difficult to directly penetrate the blood-brain barrier into highly lipid-soluble, low-molecular-weight free gastrodin, p-hydroxybenzyl alcohol, and γ-aminobutyric acid. This alters the physical state of the active ingredients, which are difficult to absorb in traditional water extraction processes, and improves the efficiency of the active substances entering the brain and exerting a central sedative effect.

[0027] 2. This invention utilizes the symbiotic metabolic network of yeast and lactic acid bacteria in prickly pear juice and honey base to synthesize a large amount of free short-chain fatty acids and trace amounts of ethanol. Then, in the fermentation environment, esterification reaction generates volatile components mainly composed of ethyl lactate and ethyl acetate. These ester molecules with fruit wine aromas, combined with the partial degradation of tannins by microorganisms, chemically mask the earthy and bitter taste of the raw materials of Gastrodia elata and Ziziphus jujuba. This is different from the traditional Chinese medicine oral liquid that simply relies on adding sweeteners to mask off-flavors, and fundamentally improves the sensory quality and drinking compliance of the product.

[0028] 3. The fermentation product of this invention accumulates high concentrations of short-chain free organic acids such as lactic acid and acetic acid. After entering the body with the product, it can reduce the local pH value of the intestine, inhibit the proliferation of harmful bacteria in the intestine to maintain the microecological environment. The generated short-chain fatty acids also act on intestinal endocrine cells, transmit physiological regulatory signals through the enterobrain axis to the central nervous system, and form a multi-target synergistic effect with free small molecule pharmacological components, showing a definite synergistic effect in improving sleep rhythm and suppressing abnormal central nervous system excitation. Attached Figure Description

[0029] Figure 1 This is a graph showing the comprehensive test results of various test indicators for embodiments and comparative examples of the present invention. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Preparation Examples 1-3:

[0032] Preparation Example 1: This preparation example provides a method for preparing a compound microbial agent, including the following steps:

[0033] Under aseptic conditions, weigh 10 grams of *Lactobacillus plantarum* freeze-dried powder, 10 grams of *Lactobacillus rhamnosus* freeze-dried powder, 10 grams of *Lactobacillus casei* freeze-dried powder, 5 grams of *Pichia pastoris* freeze-dried powder, and 5 grams of *Saccharomyces cerevisiae* freeze-dried powder. Place the above five powders in an aseptic mixing device and continuously mix and stir for 20 minutes in an aseptic environment at 25°C to ensure that the powders of each strain are fully and evenly mixed. Collect the mixed powder to obtain the composite microbial agent. The ratio of viable cells of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Pichia pastoris*, and *Saccharomyces cerevisiae* in the composite microbial agent is 1:1:1:0.5:0.5.

[0034] Preparation Example 2: This preparation example provides a method for preparing a compound microbial agent, including the following steps:

[0035] Under aseptic conditions, 10 grams of *Lactobacillus plantarum* freeze-dried powder, 10 grams of *Lactobacillus rhamnosus* freeze-dried powder, 10 grams of *Lactobacillus casei* freeze-dried powder, 8 grams of *Pichia pastoris* freeze-dried powder, and 8 grams of *Saccharomyces cerevisiae* freeze-dried powder were weighed and placed in an aseptic mixing device. The mixture was stirred continuously for 20 minutes at a temperature of 25°C to ensure that the powders were thoroughly and evenly mixed. The mixed powder was collected to obtain the composite microbial agent. The ratio of viable cells of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Pichia pastoris*, and *Saccharomyces cerevisiae* in the composite microbial agent was 1:1:1:0.8:0.8.

[0036] Preparation Example 3: This preparation example provides a method for preparing a compound microbial agent, including the following steps:

[0037] Under aseptic conditions, weigh 10 grams of *Lactobacillus plantarum* freeze-dried powder, 10 grams of *Lactobacillus rhamnosus* freeze-dried powder, 10 grams of *Lactobacillus casei* freeze-dried powder, 10 grams of *Pichia pastoris* freeze-dried powder, and 10 grams of *Saccharomyces cerevisiae* freeze-dried powder. Place the above five powders in an aseptic mixing device and continuously mix and stir for 20 minutes in an aseptic environment at 25°C to ensure that the powders of each strain are fully and evenly mixed. Collect the mixed powder to obtain the composite microbial agent. The ratio of viable cells of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Pichia pastoris*, and *Saccharomyces cerevisiae* in the composite microbial agent is 1:1:1:1:1.

[0038] Examples 1-3:

[0039] Example 1: This example provides a method for preparing a sleep-improving stock solution of a prickly pear, gastrodia elata, and jujube seed composition based on compound microbial fermentation, including the following steps:

[0040] Gastrodia elata and Ziziphus jujuba seeds from Dafang, Bijie, were washed, dried, and then pulverized to 100 mesh using an ultrafine pulverizer. 1600g of Gastrodia elata powder and 400g of Ziziphus jujuba seed powder were accurately weighed and set aside. The powders were added to 20 liters of prickly pear juice, along with 500g of honey as an auxiliary ingredient, and thoroughly mixed to prepare a fermentation medium. This fermentation medium was placed in a sterilizer and autoclaved at 121℃ for 15 minutes. After sterilization, it was rapidly cooled to 35℃. Under aseptic conditions, the compound microbial agent obtained in Preparation Example 1 was inoculated into the cooled medium at an inoculation rate of 1% of the total weight of the fermentation substrate and stirred thoroughly. The inoculated medium was placed in a fermenter and fermented at a constant temperature of 35℃ for 168 hours, with gentle stirring every 24 hours during fermentation. After fermentation, the pH of the fermentation broth was measured to be 3.5, the refractive index to be 3, and the taste to be slightly bitter. The fermentation was then rapidly cooled to below 10℃ to terminate the fermentation. The fermentation broth was first coarsely filtered through a 100-mesh sieve. The filtrate was then centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. Finally, the separated fermentation broth was autoclaved at 115℃ for 30 minutes to obtain the fermentation stock broth of the prickly pear, gastrodia elata, and jujube seed combination.

[0041] Example 2: This example provides a method for preparing a sleep-improving stock solution of a prickly pear, gastrodia elata, and jujube seed composition based on compound microbial fermentation, including the following steps:

[0042] Gastrodia elata and Ziziphus jujuba seeds from Dafang, Bijie, were washed, dried, and then pulverized to 80 mesh using an ultrafine pulverizer. 1200g of Gastrodia elata powder and 300g of Ziziphus jujuba seed powder were accurately weighed and set aside. The powders were added to 15 liters of prickly pear juice, along with 400g of honey as an auxiliary ingredient, and thoroughly mixed to prepare a fermentation medium. This fermentation medium was placed in a sterilizer and autoclaved at 115℃ for 20 minutes. After sterilization, it was rapidly cooled to 33℃. Under aseptic conditions, the compound microbial agent obtained in Preparation Example 2 was inoculated into the cooled medium at an inoculation rate of 0.5% of the total weight of the fermentation substrate, and stirred thoroughly. The inoculated medium was placed in a fermenter and fermented at a constant temperature of 33℃ for 144 hours, with gentle stirring every 24 hours during fermentation. After fermentation, the pH of the fermentation broth was measured to be 3.8, and the refractive index was 3.5. The fermentation was then rapidly cooled to below 10℃ to terminate the fermentation. The fermentation broth was first coarsely filtered through a 100-mesh sieve. The filtrate was then centrifuged at 5000 rpm for 15 minutes, and the supernatant was collected. Finally, the separated fermentation broth was autoclaved at 110℃ for 40 minutes to obtain the fermentation stock broth of the prickly pear, gastrodia elata, and jujube seed combination.

[0043] Example 3: This example provides a method for preparing a sleep-improving stock solution of a prickly pear, gastrodia elata, and jujube seed composition based on compound microbial fermentation, including the following steps:

[0044] Gastrodia elata and Ziziphus jujuba seeds from Dafang, Bijie, were washed, dried, and then pulverized to 100 mesh using an ultrafine pulverizer. 2000g of Gastrodia elata powder and 500g of Ziziphus jujuba seed powder were accurately weighed and set aside. The powders were added to 25 liters of prickly pear juice, along with 600g of honey as an auxiliary ingredient, and thoroughly mixed to prepare a fermentation medium. This fermentation medium was placed in a sterilizer and autoclaved at 125℃ for 10 minutes. After sterilization, it was rapidly cooled to 37℃. Under aseptic conditions, the compound microbial agent obtained in Preparation Example 3 was inoculated into the cooled medium at an inoculation rate of 1.5% of the total weight of the fermentation substrate, and stirred thoroughly. The inoculated medium was placed in a fermenter and fermented at a constant temperature of 37℃ for 192 hours, with gentle stirring every 24 hours during fermentation. After fermentation, the pH of the fermentation broth was measured to be 3.3, and the refractive index was 2.5. Fermentation was then rapidly terminated by cooling the broth to below 10℃. The fermentation broth was first coarsely filtered through a 100-mesh sieve. The filtrate was then centrifuged at 6500 rpm for 12 minutes, and the supernatant was collected. Finally, the separated fermentation broth was autoclaved at 120℃ for 20 minutes to obtain the fermentation stock broth of the prickly pear, gastrodia elata, and jujube seed combination.

[0045] Comparative Examples 1-5:

[0046] Comparative Example 1: Compared with Example 1, the difference is that the inoculation and compound microbial fermentation steps are not performed. Instead, the prepared fermentation culture medium is directly boiled in water at 100°C for 2 hours, cooled, coarsely filtered, centrifuged and sterilized to obtain the stock solution. All other steps are the same.

[0047] Comparative Example 2: Compared with Example 1, the difference is that the inoculated microbial agent is only a mixture of equal amounts of lactic acid bacteria, including Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei, without the addition of Pichia pastoris and Saccharomyces cerevisiae, while all other aspects are the same.

[0048] Comparative Example 3: Compared with Example 1, the difference is that the inoculated microbial agent is only a yeast group of equal amounts of Pichia pastoris and Saccharomyces cerevisiae, without the addition of Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus casei, and the rest are the same.

[0049] Comparative Example 4: Compared with Example 1, the difference is that no prickly pear juice was added to the fermentation medium, which was replaced with an equal volume of sterile purified water, and an equal amount of glucose was added to maintain the basic carbon source. All other aspects were the same.

[0050] Comparative Example 5: Compared with Example 1, the difference is that no heating sterilization, inoculation with compound microbial agents and constant temperature fermentation steps were performed. Gastrodia elata powder, jujube seed powder, prickly pear juice and honey were simply physically stirred and mixed evenly at room temperature and used directly as the control sample. All other steps were the same.

[0051] Test Examples 1-4:

[0052] Test Example 1: Validation of Targeted Biotransformation and High Dissolution Rate of Active Ingredients

[0053] This test case is used to verify the conversion of macromolecular active ingredients into free small molecule active substances in each group of samples and the dissolution efficiency of total solids.

[0054] Take 50 mL of each of the stock solutions prepared in Examples 1 to 3 and Comparative Examples 1 to 5, centrifuge at 10,000 rpm for 15 minutes at 4°C, and filter the supernatant through a 0.22 μm microporous membrane as the sample solution to be tested. The sample of Comparative Example 5 needs to be coarsely filtered through defatted cotton before repeating the above centrifugation operation.

[0055] Accurately weigh the standards of gastrodin, p-hydroxybenzyl alcohol, jujube seed saponin A, and γ-aminobutyric acid, dissolve them in chromatographic grade methanol and dilute to volume to prepare mixed standard solutions with different concentration gradients for plotting standard curves.

[0056] High-performance liquid chromatography (HPLC) was used for detection. A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used. For the detection of gastrodin and p-hydroxybenzyl alcohol, the mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with isocratic elution, and the detection wavelength was set to 220 nm. For the detection of jujuboside A, an evaporative light scattering detector was used, with an acetonitrile-water gradient elution mobile phase. For the detection of γ-aminobutyric acid (GABA), the sample was pre-derivatized with phenyl isothiocyanate, and the detection wavelength was set to 254 nm. The column temperature was maintained at 30 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

[0057] Each sample solution was injected and analyzed separately, and the peak area was recorded. The mass concentrations of gastrodin, p-hydroxybenzyl alcohol, jujube seed saponin A, and γ-aminobutyric acid in each sample were calculated by substituting them into the standard curve using the external standard method.

[0058] Accurately pipette 20 ml of the supernatant from each sample into a pre-weighed evaporating dish, evaporate to dryness in a water bath, and then transfer to a 105°C drying oven to dry to constant weight. Weigh and calculate the total solids dissolution rate.

[0059] Test results:

[0060] Table 1. Results of determination of effective ingredient concentration and solid dissolution rate in each example and comparative sample

[0061] Group Gastrodin (mg / L) p-Hydroxybenzyl alcohol (mg / L) Jujube seed saponin A (mg / L) γ-Aminobutyric acid (mg / L) Total solids dissolution rate (%) Example 1 124.36 487.62 89.21 214.53 42.64 Example 2 142.18 451.27 98.45 198.71 40.28 Example 3 115.82 512.44 76.53 231.96 44.15 Comparative Example 1 412.55 18.41 345.62 12.35 26.83 Comparative Example 2 287.64 204.38 210.47 145.22 33.51 Comparative Example 3 376.21 56.73 298.14 45.86 29.47 Comparative Example 4 168.45 395.21 124.76 156.34 38.72 Comparative Example 5 85.42 Not detected 62.18 4.21 12.86

[0062] Results analysis:

[0063] Note: Not detected means below the instrument's detection limit.

[0064] According to Table 1 and Appendix Figure 1 The data from Examples 1 to 3 show that the total solids dissolution rate reached over 40%, which is an improvement compared to the water decoction process in Comparative Example 1 and the physical mixing process in Comparative Example 5. Physical pulverization and conventional water extraction cannot effectively destroy the cell wall structure of Gastrodia elata and Ziziphus jujuba var. spinosa, resulting in limited release of intracellular substances. In the examples, the complex microbial community secreted hydrolytic enzymes such as cellulase and pectinase during fermentation, which cleaved the polysaccharide skeleton of the plant cell wall, allowing the bound active ingredients and lipid-soluble components to be fully released into the liquid phase system, thus verifying the cell wall degradation and deep release mechanism of substances.

[0065] Meanwhile, in the examples, the concentrations of gastrodin and jujube seed saponin A, representing macromolecular polar glycosides, were at low levels, while the concentrations of gastrodin aglycone p-hydroxybenzyl alcohol and the central nervous system depressant neurotransmitter γ-aminobutyric acid (GABA) increased significantly. In Comparative Examples 1 and 5, the concentration of gastrodin was high, and almost no p-hydroxybenzyl alcohol was generated. This fundamental change in the composition of substances confirms that the composite fermentation system can mediate targeted enzymatic hydrolysis. Glycoside hydrolases secreted by microorganisms cleave the glycosidic bonds of gastrodin, converting it into p-hydroxybenzyl alcohol, which is more lipid-soluble and has a smaller molecular weight; amino acid decarboxylases convert free amino acids in the fermentation substrate into GABA. The macromolecular precursor is converted into a free, active molecule that easily crosses the blood-brain barrier, verifying the technical principle of targeted biotransformation.

[0066] The concentrations and dissolution rates of the transformation products in Comparative Example 2 (yeast deficiency) and Comparative Example 3 (lactic acid bacteria deficiency) were significantly lower than those in Examples 1 to 3. Single-type bacterial communities have limited metabolic pathways and cannot provide sufficient extracellular enzyme systems. Pichia pastoris and Saccharomyces cerevisiae consume oxygen in the early stages of fermentation to create a microaerobic environment, promoting the proliferation and enzyme production efficiency of the lactic acid bacteria community. Different strains form a cascade effect in substrate utilization and enzymatic reactions. The conversion rate decreased after omitting prickly pear juice in Comparative Example 4, indicating that prickly pear juice not only serves as a carbon source, but its internal active substances also participate in the coenzyme composition or redox potential regulation of the fermentation system, playing a supporting role in the biochemical transformation process. The above data comparisons comprehensively demonstrate the necessity of yeast-lactic acid bacteria symbiotic systems and multi-substrate formulations in achieving efficient biotransformation.

[0067] Test Example 2: Determination of Secondary Metabolites and Flavor Remodeling Material Basis

[0068] This test case is used to verify the accumulation of short-chain fatty acids and the generation level of volatile flavor esters in the fermentation systems of each group of samples.

[0069] Accurately transfer 10 mL of each of the original sample solutions from Examples 1 to 3 and Comparative Examples 1 to 5 into a 20 mL headspace vial, add 3 g of sodium chloride solid and an appropriate amount of 2-octanol internal standard solution of known concentration, and perform headspace extraction for 40 minutes in a constant temperature water bath at 50 °C using a polydimethylsiloxane solid phase microextraction head.

[0070] The extraction head was removed and inserted into the injection port of the gas chromatograph-mass spectrometer (GC-MS), and desorption was performed at 250°C for 5 minutes. Chromatographic separation was performed using a DB-WAX polar capillary column. The temperature program was set as follows: initial temperature 40°C, held for 3 minutes, increased to 150°C at a rate of 5°C / min, then increased to 230°C at a rate of 10°C / min and held for 5 minutes. The mass spectrometry system used an electron impact ion source, and the mass scan range was set to a mass-to-charge ratio of 35 to 350. Ethyl acetate and ethyl lactate compounds were identified by searching and comparing mass spectrometry libraries, and their mass concentrations were calculated using the internal standard method.

[0071] Five mL of each sample stock solution was taken, diluted 10-fold with ultrapure water, and filtered through a 0.45 μm aqueous microporous membrane. Short-chain fatty acids were quantitatively determined using high-performance liquid chromatography (HPLC). An organic acid analytical column was selected, with a mobile phase of 0.01 mol / L potassium dihydrogen phosphate aqueous solution, a flow rate controlled at 0.8 mL / min, and a column temperature maintained at 35 °C. The concentrations of lactic acid and acetic acid were measured at 210 nm using a UV detector.

[0072] Record and process gas chromatography and liquid chromatography data, substitute the obtained peak areas into the standard curve equations of each target analyte, and calculate the absolute concentrations of lactic acid, acetic acid, ethyl acetate, and ethyl lactate in the original solution.

[0073] Test results:

[0074] Table 2. Results of determination of secondary metabolites and characteristic flavor compounds in samples from each example and comparative example.

[0075] Group Lactic acid (g / L) Acetic acid (g / L) Ethyl acetate (mg / L) Ethyl lactate (mg / L) Example 1 14.62 4.81 48.74 36.52 Example 2 12.38 3.94 41.29 31.86 Example 3 15.11 5.23 52.41 39.04 Comparative Example 1 0.84 0.12 Not detected 0.45 Comparative Example 2 13.56 4.15 2.18 3.67 Comparative Example 3 2.47 1.05 11.43 8.26 Comparative Example 4 8.53 2.61 19.82 15.34 Comparative Example 5 0.41 Not detected Not detected Not detected

[0076] Results analysis:

[0077] Note: Not detected means below the instrument's detection limit.

[0078] According to Table 2 and Appendix Figure 1 Data from Examples 1 to 3 showed high concentrations of lactic acid, acetic acid, and volatile esters such as ethyl acetate and ethyl lactate in the original sample solutions. Based on the technical mechanism of this invention, *Lactobacillus plantarum* and *Lactobacillus casei* in the complex microbial community utilize carbohydrate carbon sources for glycolysis metabolism within the fermentation system, synthesizing a large amount of short-chain fatty acids, primarily lactic acid and acetic acid. The increased concentration of short-chain fatty acids constitutes the material basis for altering the intestinal microecological environment. After entering the body, high concentrations of organic acids inhibit the proliferation of harmful bacteria by lowering the local pH of the intestine, and simultaneously act on intestinal endocrine cells to trigger the vagus nerve signaling pathway, indirectly transmitting regulatory signals to the central nervous system, thus verifying the pharmacological pathway of the product's targeted regulation of sleep rhythms through the gut-brain axis.

[0079] Comparative Examples 1 and 5, lacking microbial communities, showed only trace amounts of endogenous organic acids from the raw materials, with no detectable characteristic esters that masked flavors. Comparative Example 2, lacking both Pichia pastoris and Saccharomyces cerevisiae in its formulation, maintained high levels of lactic acid and acetic acid, but extremely low concentrations of ethyl acetate and ethyl lactate. Trace amounts of ethanol and other higher alcohols produced by yeast in the fermentation symbiosis system are precursors for esterification reactions. The absence of yeast metabolites prevented the synthesis of floral and fruity flavor molecules through alcohol-esterification reactions of free organic acids, resulting in the product retaining earthy and bitter tastes reminiscent of Gastrodia elata and Ziziphus jujuba seeds. Comparative Example 3, lacking lactic acid bacteria, hindered organic acid production, and the resulting decrease in substrate concentration also limited ester synthesis. Comparative Example 4, without the addition of prickly pear juice, reduced initial carbon sources and nutrients, leading to a general decrease in the concentrations of various secondary metabolites due to substrate scarcity. Measurement data confirms that the multi-strain joint metabolic network ensures the accumulation of free short-chain fatty acids and generates a variety of volatile esters through the enzymatic collaboration of acid-producing and alcohol-producing bacteria, thereby changing the sensory properties of raw materials and reshaping the flavor characteristics of the product from a chemical perspective.

[0080] Test Example 3: Comparative Verification of Core Sleep-Promoting Efficacy in Animals

[0081] This test case utilizes a zebrafish insomnia model induced by pentylenetetrazol to evaluate the differences in the sedative effects of the original solutions of each group on the central nervous system and their efficacy in improving sleep.

[0082] Healthy, wild-type AB strain adult zebrafish were selected and randomly divided into a normal control group, a model control group, drug treatment groups of Examples 1 to 3, and drug treatment groups of Comparative Examples 1 to 5, with 12 zebrafish in each group. Before the experiment, the zebrafish were placed in standard culture water for 7 days for acclimatization, maintaining a water temperature of 28±1℃ and a light-dark cycle of 14 hours of light and 10 hours of darkness.

[0083] Configure the modeling and drug administration water bodies. Pentylenetetrazole solution was added to the culture water of the model control group and each drug administration group to achieve a final concentration of 5 mmol / L, inducing central nervous system excitation and insomnia phenotypes in zebrafish. Simultaneously, the stock solutions of samples from Examples 1 to 3 and Comparative Examples 1 to 5 were added to the water of each drug administration group to ensure a uniform volume concentration of 1% for the test samples. The normal control group received only an equal volume of standard culture water. An equal volume of blank solvent was added to both the normal control group and the model control group.

[0084] Zebrafish were continuously exposed to the aforementioned modeling and drug administration water for 24 hours. After exposure, each group of zebrafish was transferred individually to a 24-well plate, and 2 ml of the corresponding group's test water was added to each well.

[0085] The 24-well plate was placed in the isolated dark chamber of the DanioVision zebrafish behavioral trajectory tracking and analysis system for a 10-minute acclimatization period. The system then automatically recorded and analyzed the movement trajectories of the zebrafish within 10 minutes after entering the dark phase.

[0086] Export behavioral tracking data and statistically analyze the total distance moved by each group of zebrafish during the 10-minute observation period, i.e., the amount of arousal activity, and the total time spent in an active state, i.e., the arousal time.

[0087] Test results:

[0088] Table 3. Effects of each example and comparative example on behavioral indicators of the pentylenetetrazol-induced zebrafish insomnia model.

[0089] Group Arousal activity level (mm) Awakening time (s) normal control group 1482.35 118.62 Model control group 4315.78 462.31 Example 1 1856.42 145.28 Example 2 2013.67 172.54 Example 3 1795.81 139.15 Comparative Example 1 3214.56 351.47 Comparative Example 2 2587.12 268.93 Comparative Example 3 2764.89 291.55 Comparative Example 4 2305.44 242.18 Comparative Example 5 3981.25 425.66

[0090] Results analysis:

[0091] According to Table 3 and the appendix Figure 1 The data showed that the arousal activity and wakefulness time of the model control group were significantly higher than those of the normal control group, indicating that pentylenetetrazol successfully induced central nervous system excitation and insomnia in zebrafish. The arousal activity and wakefulness time of groups 1 to 3 in Examples 1 to 3 decreased compared to the model control group, with the data returning to levels close to those of the normal control group, confirming that the composition of this invention has a clear and potent central sedative and sleep-improving effect.

[0092] Based on the technical mechanism of this invention, the behavioral indicators of Comparative Examples 1 and 5 showed only slight improvement, with persistently high arousal levels. Traditional water extraction and simple physical mixing methods cannot break down the tough cell wall layer of plants, resulting in insufficient release of large molecular bound components such as gastrodin and jujube seed saponins. Furthermore, the polar macromolecules have difficulty penetrating the blood-brain barrier, leading to low efficacy in the brain. This embodiment utilizes a complex microbial community that secretes a complex hydrolytic enzyme system during growth, triggering cell wall disintegration and releasing active ingredients from deep protoplasts. Subsequently, through glycoside hydrolase-mediated targeted enzymatic hydrolysis, the slow-acting macromolecular prodrugs are converted into highly lipid-soluble, low-molecular-weight free aglycones. The resulting secondary metabolites, such as γ-aminobutyric acid, act directly on central nervous system receptors at multiple targets, fundamentally solving the problem of low bioavailability of traditional Chinese medicine extracts.

[0093] The improvement in indicators in Comparative Example 2 (yeast deficiency) and Comparative Example 3 (lactic acid bacteria deficiency) was significantly less than that in the Example. This indicates that symbiotic interactions among the microbial communities are indispensable. Saccharomyces cerevisiae and Pichia pastoris consume free oxygen and release carbon dioxide through aerobic respiration, creating a suitable microaerobic environment for the lactic acid bacteria. The lack of microbial community structure leads to insufficient secretion of extracellular enzyme systems, which not only reduces the efficiency of macromolecular targeted conversion into free aglycones but also weakens the accumulation of short-chain fatty acids and secondary small molecule peptides. The sedative effect was also reduced in Comparative Example 4 after the absence of prickly pear juice, confirming that prickly pear juice not only provides a basic carbon source in the system, but its polyphenols and antioxidants also participate in synergistic conversion within a multi-substrate network. The multi-substrate and multi-strain synergistic fermentation system of the Example realizes the cascading and integration of multiple pharmacodynamic pathways, producing a synergistic effect in promoting sleep.

[0094] Test Example 4: Flavor and Compliance Comparison Test Based on Sensory Evaluation Matrix

[0095] This test case is used to verify the differences in multidimensional sensory indicators of the fermentation broth of each group of samples, and to evaluate the masking effect of compound microbial fermentation on the characteristic unpleasant odors of Chinese medicinal materials and its sensory quality reshaping effect.

[0096] A sensory evaluation team of 15 professionals with experience in sensory evaluation of food or traditional Chinese medicine was selected. The team was balanced in gender and avoided contact with strong irritating foods or odors for 24 hours before the evaluation.

[0097] The original solutions of the samples from Examples 1 to 3 and Comparative Examples 1 to 5 were placed in a refrigerated environment at 4°C for 24 hours to equilibrate. Before testing, they were removed and warmed to 20°C at room temperature. Each group of samples was poured into a colorless and transparent tasting cup with a uniform capacity of 20 ml. The tasting cups were blind-sampled using three random numbers.

[0098] A sensory evaluation scale was established, including dimensions for earthy aroma intensity, bitterness intensity, fruit wine aroma richness, and overall acceptability. Earthy and bitterness intensity were scored on a scale of 0 to 10, with higher scores indicating stronger unpleasant odors or tastes; fruit wine aroma richness was scored on a scale of 0 to 10, with higher scores indicating richer and more harmonious aromas; and overall acceptability was scored on a scale of 0 to 100 to comprehensively assess the drinking compliance of the samples.

[0099] The evaluators took turns smelling and tasting the blind samples. After each tasting, the evaluators were required to spit out the sample, rinse their mouths thoroughly with purified water, and rest for 3 minutes before evaluating the next sample to avoid sensory fatigue and cross-contamination of tastes.

[0100] Collect all the score sheets from the evaluators, remove the highest and lowest scores for each dimension, and calculate the arithmetic mean of each indicator as the final sensory score.

[0101] Test results:

[0102] Table 4. Sensory evaluation score matrix of fermentation broth for each embodiment and comparative example.

[0103] Group earthy smell intensity Intensity of bitterness Fruit wine aroma richness Overall acceptance Example 1 1.3 2.4 8.7 91.5 Example 2 1.8 2.1 8.2 88.4 Example 3 1.1 2.8 9.1 93.2 Comparative Example 1 8.5 7.9 0.4 34.6 Comparative Example 2 5.6 6.8 2.3 52.8 Comparative Example 3 6.2 5.4 3.5 48.1 Comparative Example 4 4.9 5.2 4.6 63.7 Comparative Example 5 9.6 8.5 0.1 21.8

[0104] Results analysis:

[0105] According to Table 4 and Appendix Figure 1 The data showed that Comparative Examples 1 and 5 scored highly in terms of earthy and bitterness intensity, but had the lowest overall acceptability. Gastrodia elata and Ziziphus jujuba var. spinosa, as traditional Chinese medicinal herbs, contain a large number of secondary metabolites (such as some alkaloids and tannins) with strong earthy and bitter tastes. These components cannot be removed by unconverted water extraction or physical mixing processes, resulting in a lack of consumer compliance. Examples 1 to 3 reduced the earthy and bitterness intensity to low levels, significantly improving the aroma richness and overall acceptability of the fruit wine, confirming the definite effect of this invention in flavor reshaping.

[0106] Based on the innovative mechanism of this invention, the flavor improvement is not simply achieved by adding sweeteners to mask the taste, but rather is built upon the deep metabolism and material transformation of the microbial community. In the fermentation system, *Saccharomyces cerevisiae* and *Pichia pastoris* produce trace amounts of ethanol and higher alcohols through sugar metabolism, while *Lactobacillus plantarum* and *Lactobacillus casei* synthesize large quantities of short-chain free organic acids such as lactic acid and acetic acid. In the slightly acidic, microaerobic fermentation environment, organic acids and alcohols undergo esterification reactions, spontaneously synthesizing volatile ester molecules such as ethyl lactate and ethyl acetate, which possess distinct floral and fruity aromas. These ester molecules have high volatility and low odor thresholds, creating a strong masking effect on the inherent earthy taste of traditional Chinese medicine at both spatial and sensory levels. Simultaneously, the enzyme system secreted during the multi-microbial metabolism partially degrades the tannin precursors that cause astringency, reducing the bitterness.

[0107] In Comparative Example 2, the yeast-deficient group lacked precursors such as ethanol and higher alcohols, preventing the accumulated organic acids in the system from undergoing esterification. This not only failed to generate the masking fruit wine aroma but also resulted in a jarring sour and astringent taste, failing to mask the earthy, herbal flavor. Comparative Example 3, lacking lactic acid bacteria, also lacked sufficient free organic acid substrates, similarly blocking the material basis for esterification and resulting in a thin aroma. In Comparative Example 4, the absence of prickly pear juice led to the loss of natural fruit aroma precursors and polyphenols in the fermentation substrate, resulting in a decrease in ester aroma abundance and impaired overall flavor harmony. These differences in test data confirm that the secondary metabolic network generated during fermentation in the yeast-lactic acid bacteria symbiotic system can fundamentally alter the sensory properties of the product at a chemical level, achieving a complete reshaping of the flavor of traditional Chinese medicine preparations.

Claims

1. A product portfolio for improving sleep, characterized in that, The product mix is ​​made by fermentation of raw materials containing the following amounts: 1200-2000g of Gastrodia elata powder; 300-500g of jujube seed powder; 15-25L of prickly pear juice; 400-600g of honey; The inoculum amount is 0.5%-1.5% of the total weight of the fermentation substrate, using a compound microbial agent.

2. The product combination for improving sleep according to claim 1, characterized in that, The mass ratio of Gastrodia elata powder to Ziziphus jujuba seed powder is fixed at 4:1, and both Gastrodia elata powder and Ziziphus jujuba seed powder are 80-100 mesh powders formed by ultra-fine grinding.

3. The product combination for improving sleep according to claim 1, characterized in that, The compound microbial agent is a composition comprising Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Pichia pastoris, and Saccharomyces cerevisiae.

4. The product combination for improving sleep according to claim 3, characterized in that, In the compound microbial agent, the ratio of viable bacteria of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Pichia pastoris* and *Saccharomyces cerevisiae* is 1:1:1:(0.5-1):(0.5-1).

5. The product combination for improving sleep according to claim 1, characterized in that, The fermentation endpoint physicochemical properties of the product combination are as follows: The pH value should be controlled between 3.3 and 3.8, and the refractive index should be controlled between 2.5 and 3.

5.

6. A method for preparing a product combination for improving sleep according to any one of claims 1-5, characterized in that, Includes the following steps: Add the weighed Gastrodia elata powder and Ziziphus jujuba seed powder to the prickly pear juice, and add honey as an auxiliary ingredient. Stir and mix thoroughly to prepare a fermentation culture medium. The fermentation medium was autoclaved and then cooled. Under aseptic conditions, a compound microbial agent was added to the cooled fermentation medium, stirred evenly, and then placed in a fermenter for constant temperature fermentation. After fermentation is completed, the fermentation process is terminated, and the fermentation liquid is subjected to coarse filtration, centrifugation to collect the supernatant, and finally sterilized under high pressure to obtain the fermentation stock solution of prickly pear, gastrodia elata and jujube seed combination.

7. The method for preparing a product combination for improving sleep according to claim 6, characterized in that, The autoclaving temperature is 115-125℃ and the time is 10-20 minutes; After sterilization, cool rapidly to 33-37℃.

8. The method for preparing a product combination for improving sleep according to claim 6, characterized in that, The constant temperature fermentation is 33-37℃, the fermentation time is 144-192 hours, and a slight stirring is performed every 24 hours during the fermentation process.

9. The method for preparing a product combination for improving sleep according to claim 6, characterized in that, Once the pH and refractive index of the fermentation broth reach the target range, the fermentation broth should be rapidly cooled to 0-10℃ to terminate the fermentation. The coarse filtration is performed using an 80-120 mesh sieve, and the centrifugation speed is 5000-8000 rpm, with a centrifugation time of 10-15 minutes.

10. The method for preparing a product combination for improving sleep according to claim 6, characterized in that, The final process parameters for high-pressure sterilization of the separated supernatant fermentation broth are as follows: Sterilization temperature 110-120℃, sterilization time 20-40 minutes.

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