A drink for improving sleep and a method of preparation

Abstract

The application discloses a sleep-improving beverage and a preparation method thereof, and belongs to the technical field of fermentation engineering and health food. The application is characterized in that natural raw material extract, sodium glutamate and hop extract are co-fermented by a specific probiotic fermentation system, endogenous GABA is efficiently converted and formed into a multi-form composite system together with functional components such as pachyman, lily polysaccharide and jujube seed saponin. The prepared beverage has high bioavailability and good stability of GABA, and various components are synergistically combined through multiple pathways such as the gut-brain axis regulation and neurotransmitter regulation, so that the sleep is effectively improved, and the beverage has good flavor and storage stability.

Description

Technical Field

[0001] This invention belongs to the fields of fermentation engineering technology and health food technology, specifically relating to a beverage for improving sleep and its preparation method. Background Technology

[0002] With the fast pace of modern life and increasing work pressure, sleep disorders have become a common health problem, affecting approximately 30% of the global population. Chronic sleep deprivation leads to emotional imbalance, impacting not only quality of life but also potentially triggering a series of diseases such as endocrine disorders and weakened immunity. Therefore, developing safe and effective sleep-aiding health foods has become a hot research topic in the food industry.

[0003] In recent years, with in-depth research into traditional herbal medicines and health foods, many natural ingredients have been found to improve sleep. For example, gamma-aminobutyric acid (GABA) is a naturally occurring non-protein amino acid and an important inhibitory neurotransmitter in the mammalian central nervous system; approximately 50% of central nervous system synapses use GABA as a neurotransmitter. It plays a crucial role in the human cerebral cortex, hippocampus, thalamus, basal ganglia, and cerebellum, and regulates various bodily functions. Sufficient GABA in the brain can make people feel relaxed and happy, while low levels may lead to anxiety, tension, depression, and insomnia.

[0004] In existing technologies, γ-aminobutyric acid (GABA) is mostly added directly to product formulations, making it an exogenous form. Exogenous GABA is mostly a free macromolecule, which is easily competitively inhibited by intestinal epithelial cell GABA transporters (GAT1 / GAT3) during intestinal absorption. Furthermore, it has difficulty penetrating the blood-brain barrier (BBB)—the capillary walls of the BBB are tightly connected, allowing only small molecules, lipid-soluble substances, or substances mediated by specific carriers to pass through. The penetration rate of exogenous free GABA is usually less than 10%, resulting in limited central nervous system regulatory effects. Moreover, directly added exogenous GABA (especially chemically synthesized products) is prone to oxidative degradation in beverage systems (affected by pH, temperature, and oxygen), and may interact with sweeteners, stabilizers, and other components. This can lead to a degradation rate of 15-25% in solution within 6 months, resulting in a significant decrease in the product's efficacy over its shelf life. Summary of the Invention

[0005] To address the aforementioned technical problems in the prior art, the present invention aims to provide a method for preparing a beverage that improves sleep, characterized in that the preparation method includes: (1) After crushing jujube seed, lily bulb, poria cocos and jujube, a natural raw material extract is prepared, wherein the jujube seed saponin content is ≥1.2mg / mL, the poria cocos polysaccharide content is ≥0.8mg / mL, and the lily bulb polysaccharide content is ≥0.9mg / mL; (2) Preparation of fermentation substrate First, mix oat flour and maltodextrin in a mass ratio of 1-3:1, then add purified water in a volume of 8-12 times the total mass of oat flour and maltodextrin, heat to 80-90℃ to dissolve for 30-60 minutes, then sterilize at 121℃ for 15-20 minutes, and then cool to 37℃. Then, the natural raw material extract, monosodium glutamate and hop extract are added and mixed evenly to obtain the fermentation substrate; (3) Probiotic fermentation Lactobacillus delbrueckii subsp. bulgaricus CGMCC 1.1480, Lactobacillus acidophilus CGMCC 1.1854, and Bifidobacterium animalis subsp. animalis CGMCC 1.1852 were inoculated into a fermentation substrate for fermentation to obtain a fermentation broth. (4) Mixing and shaping Add one or more of the following to the fermentation broth: walnut peptide, theanine, sweetener, or stabilizer. Stir and dissolve, then homogenize and sterilize in sequence to finally produce a drink that improves sleep.

[0006] Preferably, in the fermentation substrate, the natural raw material extract accounts for 10-20% of the total volume of the fermentation substrate, the monosodium glutamate accounts for 0.8-1.2% of the dry weight of the fermentation substrate, and the hop extract accounts for 0.06-0.12% of the dry weight of the fermentation substrate. The hop extract is prepared from dried hops (Humulus lupulus L.) by water extraction or alcohol extraction. On a dry weight basis, the hop extract contains not less than 2.0% flavohumol and not less than 1.5% humulone.

[0007] Preferably, the mass ratio of monosodium glutamate to hop extract is 10-18:1.

[0008] Preferably, the probiotic fermentation employs a staged sequential fermentation process, including: First stage fermentation: Lactobacillus delbrueckii subsp. bulgaricus CGMCC 1.1480 was separately inoculated into the fermentation substrate at an inoculum volume of 2-3% of the fermentation substrate volume. Anaerobic fermentation was carried out at 37±0.5℃ and pH 5.5-6.0 for 10-14 hours to obtain the first stage fermentation broth. Second stage fermentation: Simultaneously inoculate Lactobacillus acidophilus CGMCC 1.1854 and Bifidobacterium animalis subsp. animalis CGMCC 1.1852 into the first stage fermentation broth, with the inoculation amount of Lactobacillus acidophilus being 1.5-2.5% of the first stage fermentation broth volume and the inoculation amount of Bifidobacterium being 2.5-3.5% of the first stage fermentation broth volume; adjust the fermentation temperature to 38±0.5℃, and dynamically maintain the pH at 4.2-4.5 by using a small amount of calcium carbonate powder, and continue anaerobic fermentation for 20-28 hours.

[0009] Preferably, the endpoint of the first stage of fermentation is when the pH value drops to 5.6 ± 0.1.

[0010] Preferably, during the second stage of fermentation, the pH is monitored every 4 hours, and when the pH is < 4.3, 0.1-0.3% (w / v) calcium carbonate buffer is added to maintain the pH at 4.2-4.5.

[0011] Preferably, after the probiotic fermentation in step (3) is completed and before the preparation and molding in step (4) begins, an enzymatic hydrolysis step is also included: adding a complex protease to the fermentation broth, wherein the amount of the complex protease added is 0.1-0.5% of the mass of the fermentation broth, and hydrolyzing for 30-60 minutes at pH 6.5-7.0 and 45°C, and then inactivating the enzyme at 85°C for 5-10 minutes.

[0012] Preferably, the complex protease is a flavor protease and trypsin in a mass ratio of 1-2:1.

[0013] Preferably, the homogenization pressure in step (4) is 25-30 MPa, and the number of homogenization cycles is 1-3.

[0014] A second objective of this invention is to provide a beverage for improving sleep, which is obtained by any of the preparation methods described above.

[0015] The beneficial effects of this invention include: (1) Endogenous GABA with high bioavailability and good stability was successfully prepared by probiotic fermentation.

[0016] By utilizing the glutamate decarboxylase (GAD) of a specific bacterial strain, monosodium glutamate (MSG) in the fermentation substrate is efficiently converted into gamma-aminobutyric acid (GABA). The GABA generated in this process is endogenous and coexists in the fermentation broth in three forms: free, complex, and intracellular bound, forming a diverse "GABA library." This presence of endogenous and complex forms effectively avoids the problems of exogenous free GABA being susceptible to competitive inhibition by intestinal transporters and having low blood-brain barrier penetration, potentially improving absorption efficiency. Furthermore, GABA is protected in the complex fermentation microenvironment, and its chemical stability is far higher than that of directly added exogenous GABA, significantly reducing the degradation rate of the product during its shelf life.

[0017] (2) The efficacy was significantly enhanced through the multi-functional synergistic effect of the fermentation system.

[0018] First, during fermentation, probiotics not only produce GABA, but also generate a variety of beneficial metabolites such as short-chain fatty acids (SCFAs), bioactive peptides, and B vitamins. SCFAs, as key signaling molecules of the gut-brain axis, work synergistically with GABA to regulate nervous system function and exert a calming effect.

[0019] Secondly, the fermentation process itself biotransforms the components in the natural raw material extract, such as converting some macromolecular polysaccharides into low molecular weight polysaccharides or oligosaccharides, and converting saponins into saponins with higher bioavailability, thereby further optimizing the activity and absorption of these components.

[0020] Finally, the hop extract in the fermentation substrate works synergistically with the entire fermentation system. Not only do its own active ingredients (xanthumol, humulone, etc.) enhance the effect of GABA at its target site (GABA_A receptor), but it also promotes the efficient synthesis and accumulation of GABA through multiple mechanisms, such as regulating GAD enzyme activity and inhibiting GABA degradation. This "compound" system, composed of endogenous GABA, probiotic metabolites, transformed plant components, and hop extract, achieves a synergistic effect in improving sleep through the combined action of multiple targets and pathways (neurotransmitter regulation, gut-brain axis regulation, etc.).

[0021] (3) Through comprehensive process optimization, the flavor and physical stability of the product have been effectively improved.

[0022] The probiotic fermentation process transforms unpleasant flavor precursors in hop extract and plant materials, generating lactic acid, esters, and other substances with pleasant flavors, effectively masking the inherent off-flavors of functional ingredients. Subsequent blending (addition of sweeteners, theanine, walnut peptides, etc.) further balances the sweet-sour ratio, enhancing umami and palatability. Furthermore, homogenization and the addition of stabilizers work together to ensure that the bacteria, plant particles, and other solids in the fermentation broth remain uniformly and stably suspended, preventing sedimentation and stratification during storage, resulting in a final product with a uniform texture, smooth taste, and high stability. Detailed Implementation

[0023] The following description includes certain specific details to provide a comprehensive understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments can be implemented without employing one or more of these specific details, but using other methods, components, materials, etc.

[0024] Unless otherwise required by the present invention, throughout the specification and the following claims, the words “comprising” and “including” shall be interpreted in an open-ended, inclusive sense, meaning “including but not limited to”.

[0025] Throughout this specification, the terms "an embodiment," "an embodiment," "a preferred embodiment," or "some embodiments" refer to including, in at least one embodiment, a specific reference element, structure, or feature associated with that embodiment. Therefore, the phrases "in an embodiment," "in a preferred embodiment," or "in some embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features may be combined in one or more embodiments in any suitable manner.

[0026] According to a first aspect of the present invention, a method for preparing a beverage for improving sleep is provided, the method comprising: (1) After crushing jujube seed, lily bulb, poria cocos and jujube, a natural raw material extract is prepared. The natural raw material extract contains jujube seed saponin content ≥1.2mg / mL, poria cocos polysaccharide content ≥0.8mg / mL and lily bulb polysaccharide content ≥0.9mg / mL.

[0027] In this invention, the preparation of the natural raw material extract in step (1) uses an enzymatic-ultrasonic synergistic extraction process, including: Raw material pretreatment: Sour jujube kernels are ground to 60-80 mesh, Poria cocos and lily bulbs are ground to 80-100 mesh respectively, and jujubes are ground to 100-120 mesh.

[0028] Enzymatic hydrolysis: Ziziphus jujuba seeds were hydrolyzed in water using cellulase and hemicellulase at a mass ratio of 2:1 to obtain ziziphus jujuba seed hydrolysate; Poria cocos and lily bulbs were mixed and hydrolyzed in water using pectinase and β-glucanase at a mass ratio of 3:2 to obtain Poria cocos and lily bulb hydrolysate; jujubes were extracted in water at 70-75℃ and filtered to obtain jujube extract.

[0029] Specifically, the enzymatic hydrolysis of jujube kernels includes: adding purified water at 8-10 times the mass of jujube kernels, adjusting the temperature to 50-55℃ and pH to 5.5-6.0, adding a first compound enzyme preparation, wherein the mass ratio of cellulase to hemicellulase in the first compound enzyme preparation is 2:1, and the amount of the first compound enzyme preparation added is 0.3-0.5% of the mass of jujube kernels, stirring at 120-150 r / min for 1.5-2 h, to obtain the enzymatic hydrolysate of jujube kernels.

[0030] Preferably, the enzyme activity of the first compound enzyme preparation meets the following requirements: cellulase ≥ 5000 U / g, hemicellulase ≥ 3000 U / g; during the enzymatic hydrolysis process, the reducing sugar content and the viscosity of the hydrolysate are monitored online to control the end point of the enzymatic hydrolysis and ensure that the cell wall destruction rate of the raw material is ≥ 90%.

[0031] Enzymatic hydrolysis of Poria cocos and lily: Mix Poria cocos and lily at a mass ratio of 1-2:1, add purified water at 10-12 times the total mass of Poria cocos and lily, adjust the temperature to 45-50℃ and pH to 4.8-5.2, add the second compound enzyme preparation, in which the mass ratio of pectinase to β-glucanase is 3:2, and the amount of the second compound enzyme preparation added is 0.25-0.4% of the total mass of Poria cocos and lily, stir at 120-150 r / min for 1-1.5 h to obtain the enzymatic hydrolysate of Poria cocos and lily.

[0032] Preferably, the enzyme activity of the second compound enzyme preparation meets the following requirements: pectinase ≥ 4000 U / g, β-glucanase ≥ 2000 U / g; during the enzymatic hydrolysis process, the reducing sugar content and the viscosity of the enzymatic hydrolysate are monitored online to control the end point of enzymatic hydrolysis and ensure that the cell wall destruction rate of the raw material is ≥ 90%.

[0033] Jujube extraction: Add purified water at 8-10 times the weight of jujubes, extract at 70-75℃ for 1-1.5 hours, and filter to obtain jujube extract.

[0034] Ultrasonic extraction: Mix the enzymatic hydrolysate of jujube seed, enzymatic hydrolysate of poria cocos and lily bulb, and jujube extract. Add purified water to the extraction system until the total water volume is 12-15 times the total mass of the dried jujube seed, poria cocos, lily bulb, and jujube. Extract at 65-70℃ with stirring, using continuous ultrasound at a frequency of 25-30kHz and an ultrasound power of 350-400W for 1-1.5 hours. Concentration: After filtration, the filtrate is concentrated at 60-70℃ and 0.06-0.08MPa until the solid content is 15-20%, thus obtaining a natural raw material extract with jujube seed saponin content ≥1.2mg / mL, poria cocos polysaccharide content ≥0.8mg / mL, and lily polysaccharide content ≥0.9mg / mL.

[0035] (2) Preparation of fermentation substrate First, mix oat flour and maltodextrin in a mass ratio of 1-3:1, then add purified water in a volume of 8-12 times the total mass of oat flour and maltodextrin, heat to 80-90℃ to dissolve for 30-60 minutes, then sterilize at 121℃ for 15-20 minutes, and cool to 37℃. Then, the natural raw material extract, monosodium glutamate and hop extract are added and mixed evenly to obtain the fermentation substrate.

[0036] In this invention, oat flour serves as a complex carbon source in the fermentation substrate, providing not only starch but also protein, fat, and valuable β-glucan. β-glucan, as a prebiotic, optimizes the microenvironment of the fermentation system, enhances the metabolic efficiency of probiotics, and helps regulate the balance of intestinal flora, indirectly influencing the gut-brain axis and aiding in sleep improvement. Maltodextrin is a simple carbon source easily metabolized and utilized by probiotics, rapidly providing energy for their initial growth and metabolism. The mass ratio of oat flour to maltodextrin is 1-3:1, balancing rapid energy supply with sustained nutrition and prebiotic function (oat flour). Too much oat flour may lead to excessive viscosity, affecting fermentation and mass transfer; too much maltodextrin may result in overly rapid fermentation, excessive acid production, and a lack of prebiotic benefits. At 80-90℃, oat starch is fully gelatinized, and the gelatinized starch molecular chains unfold, making them more readily utilized by enzymes produced by probiotics, thus improving the fermentability of the substrate. Simultaneously, the heating process also allows for the preliminary extraction of some water-soluble nutrients from the oats. Sterilize at 121℃ for 15-20 minutes to kill bacteria and spores in raw materials and water.

[0037] In this invention, the natural raw material extract, on the one hand, infuses the fermentation matrix with pre-extracted sedative and tranquilizing active ingredients (jujube seed saponins, lily polysaccharides, and poria polysaccharides). These ingredients, together with GABA produced during subsequent fermentation, exert a multi-target, synergistic effect. On the other hand, Monosodium glutamate (MSG) is a precursor to GABA. Probiotics such as *Lactobacillus delbrueckii* and *Lactobacillus acidophilus* contain abundant glutamate decarboxylase, which converts MSG into GABA through microbial fermentation. The resulting GABA is endogenous and bound to the bacterial cells and their metabolites. This form is more stable and can be facilitated by microbial carriers or form more easily absorbed small peptide-GABA complexes.

[0038] Hops extract mainly contains active ingredients such as xanthohumol, humulone, and 8-isopentenylnaringenin. On the one hand, it can form a synergistic effect with endogenous GABA and other natural extracts in the product to enhance the overall sleep-aiding effect through multiple mechanisms of action; on the other hand, it can regulate the synthesis, degradation, and metabolic microenvironment of GABA through multiple targets, thereby promoting GABA synthesis, inhibiting degradation, and optimizing the efficiency of GABA production by probiotics.

[0039] The core rate-limiting step in GABA synthesis is the decarboxylation of monosodium glutamate (MSG) to GABA under the catalysis of glutamate decarboxylase (GAD). GAD activity depends on pyridoxal phosphate (PLP, vitamin B6) as a coenzyme and is regulated by enzyme conformation and gene expression. 8-Isoprenylnaringenin can allosterically activate GAD: it binds to the regulatory subunit of GAD, inducing a conformational shift from an inactive to an active state, thus increasing the affinity of GAD for its substrate, MSG. Simultaneously, 8-isoprenylnaringenin enhances the binding stability of GAD to the coenzyme PLP, reducing PLP shedding and thus maintaining the catalytic activity of GAD. Xanthohumol from hop extract can penetrate the cell membrane of probiotics and bind to the promoter region of the gadB gene (the core gene encoding GAD), activating the binding efficiency of RNA polymerase, upregulating the transcriptional level of the gadB gene, directly increasing the amount of GAD protein synthesized, and enhancing the GABA synthesis rate.

[0040] GABA degradation primarily relies on GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Hops extract can block GABA decomposition by targeting and inhibiting the activity of these degrading enzymes. Xanthohumol, with a molecular structure similar to GABA (both containing amino and carboxyl groups), can competitively bind to the active site of GABA-T, thus reducing the catalytic efficiency of GABA-T for GABA. Humus can bind to the coenzyme NAD of SSADH. + The binding domain alters the enzyme's spatial conformation, preventing SSADH from effectively catalyzing the oxidation of succinate semialdehyde and indirectly blocking downstream pathways of GABA degradation.

[0041] Hop extract can also indirectly improve GABA conversion efficiency by regulating the growth status of probiotics and the fermentation system environment. The polysaccharides in hop extract can act as specific prebiotics for *Bifidobacterium animalis* subspecies CGMCC 1.1852 and *Lactobacillus acidophilus* CGMCC 1.1854. These polysaccharides cannot be utilized by harmful bacteria (such as *E. coli* and *Salmonella*), but can be hydrolyzed into monosaccharides by glycosidases secreted by GABA-producing probiotics, thus increasing the viable count of probiotics. The phenolic components in hop extract (such as xanthohumol) have antioxidant properties, which can scavenge reactive oxygen species in the fermentation system and maintain the redox potential within a suitable range. Simultaneously, the organic acids in hop extract (such as hop acid) can assist calcium carbonate in dynamically maintaining the pH value of the fermentation system, further adapting to GABA activity.

[0042] (3) Probiotic fermentation Lactobacillus delbrueckii subsp. bulgaricus CGMCC 1.1480, Lactobacillus acidophilus CGMCC 1.1854, and Bifidobacterium animalis subsp. animalis CGMCC 1.1852 were inoculated into the fermentation substrate for fermentation to obtain the fermentation broth.

[0043] In this invention, during fermentation, oat flour and maltodextrin in the fermentation substrate serve as the core carbon source. Under the action of *Lactobacillus delbrueckii* subsp. bulgaricus CGMCC 1.1480, complex carbohydrates are hydrolyzed into smaller sugar molecules such as glucose and maltose through the secreted amylase and β-glucanase. *Lactobacillus acidophilus* CGMCC 1.1854 and *Bifidobacterium animalis* subsp. animal CGMCC 1.1852 further utilize these smaller sugar molecules for glycolysis (EMP pathway) or pentose phosphate pathway, producing lactic acid, acetic acid, short-chain fatty acids (SCFAs, such as propionic acid and butyric acid), and ATP. Lactic acid can regulate the pH of the fermentation system to the suitable range for probiotics (4.2-6.0), maintaining the metabolic activity of the bacterial community. Short-chain fatty acids not only provide energy for the proliferation of probiotics themselves but also serve as energy substrates for intestinal cells, improving intestinal barrier function.

[0044] During fermentation, all three probiotic strains expressed functional glutamate decarboxylase (GAD), catalyzing the decarboxylation of monosodium glutamate to generate endogenous γ-aminobutyric acid (GABA). Among them, *Lactobacillus acidophilus* CGMCC 1.1854 exhibited the highest GAD activity, *Bifidobacterium animalis* subspecies *CGMCC 1.1852* helped enhance the conversion efficiency, and *Lactobacillus delbrueckii* subspecies *Bulgaria* provided coenzymes (such as vitamin B6) for GAD through metabolic regulation, ensuring the continuous conversion process.

[0045] Through probiotic fermentation, monosodium glutamate (MSG) is efficiently converted into GABA. GABA exists in the fermentation broth primarily in three forms: free, complex, and intracellular bound. The free form exists as free ions in the fermentation broth; it has a small molecular weight, carries an amphoteric charge, and is easily recognized by GABA transporters (GAT1 / GAT3) in the intestine, but is also easily inhibited by competition from other amino acids. The complex form includes complexes formed with polysaccharides from Poria cocos and lily bulbs via hydrogen bonds and van der Waals forces, as well as complexes formed with small peptides produced by probiotic metabolism; it is highly stable and not easily degraded. During fermentation, intracellular bound GABA is formed by probiotics actively taking up extracellularly synthesized GABA into the cell via GABA permease. This form remains stable after sterilization and can form sustained-release GABA, prolonging its duration of action.

[0046] Furthermore, hop extract can promote the transport of GABA into cells, while xanthohumol can upregulate the expression of GABA permease on the cell membrane of probiotics and activate proton-driven transmembrane transport channels, accelerating the transport of extracellular GABA into cells, forming a cycle of "synthesis-accumulation-re-release", and ultimately increasing the total GABA yield in the fermentation broth.

[0047] Secondly, fermentation completes the biotransformation and reshaping of flavor compounds. The enzyme system of probiotics can break down precursors of unpleasant flavor compounds in hops and plant-based ingredients, consuming them as carbon or nitrogen sources and reducing off-flavors at their source. The small-molecule off-flavor compounds released during fermentation (such as isovaleric acid) can be used by microorganisms as substrates to resynthesize pleasant flavor compounds such as fruit flavor esters (e.g., ethyl acetate, ethyl hexanoate). Fermentation itself produces a wealth of flavor compounds, such as: lactic acid and acetic acid, which bring a fresh, mellow sourness and mask bitterness; acetaldehyde and dimethyl ethyl ketone, which impart typical yogurt and cream aromas; and amino acids and small peptides, which bring a rich, full-bodied taste and sweetness, improving the mouthfeel. These rich fermentation aromas collectively mask any remaining unpleasant odors.

[0048] Thirdly, fermentation produces a variety of beneficial metabolites, which can achieve multifunctional synergy.

[0049] Short-chain fatty acids (SCFAs): Primarily produced by Bifidobacteria via the bifidogenic pathway, such as acetic acid, propionic acid, and butyric acid. They are key signaling molecules in the gut-brain axis. They can regulate intestinal barrier function, inhibit harmful bacteria, and transmit signals directly or indirectly (via the vagus nerve) to the brain, regulating the nervous system and exerting a powerful synergistic sedative effect with GABA.

[0050] Bioactive peptides: These are produced by the hydrolysis of oat and microbial proteins by microbial proteases. These small peptides may possess opioid-like or sedative activities and can act directly on the nervous system. More importantly, they serve as a more readily absorbed nitrogen source, providing nutrition to the body and gut microbiota.

[0051] Vitamins: Lactic acid bacteria and bifidobacteria can synthesize B vitamins (such as B12 and folic acid) during fermentation, further supplementing the nutrients needed for nerve regulation.

[0052] Fourthly, through fermentation, the polysaccharides of Poria cocos and lily bulb in the natural raw material extract undergo enzymatic modification under the action of Bifidobacteria, improving their water solubility and bioavailability, thereby enhancing their immunomodulatory and antioxidant functions. Saponins, under the action of lactic acid bacteria, cleave the glycosyl groups on the saponin molecules to produce secondary saponins or aglycones, which have smaller molecular weights, higher lipid solubility, and higher bioavailability, making them easier to absorb by the intestines and cross the blood-brain barrier, thus enhancing their sedative and hypnotic effects. Flavonoids also undergo minor structural modifications, such as demethylation and glycosylation, thereby enhancing their activity and absorption. Amino acids and organic acids are directly integrated into the nutrient pool of the fermentation substrate. Amino acids can be used as a nitrogen source by the bacteria, and some (such as tryptophan) are precursors to the neurotransmitter serotonin, which is beneficial for sleep. Organic acids (such as malic acid and citric acid) contribute to the acidity of the system and can also serve as a carbon source.

[0053] (4) Mixing and shaping Add one or more of the following to the fermentation broth: walnut peptide, theanine, sweetener, or stabilizer. Stir and dissolve, then homogenize and sterilize in sequence to finally produce a drink that improves sleep.

[0054] In this invention, walnut peptides, being small molecules, can penetrate the intestinal barrier and synergistically act on the nervous system with GABA and theanine to help improve sleep. Simultaneously, the amino acids they contain (such as tryptophan and serotonin precursors) can supplement the nutrients needed for nerve regulation. Furthermore, walnut peptides themselves are odorless and have a slightly sweet taste, which can synergistically work with theanine and sucralose to further balance the acidity of the fermentation liquid and enhance the richness of the beverage. Moreover, walnut peptides are water-soluble small molecules and do not antagonize the pectin and gelatin in the original system; instead, they can bind to polysaccharides through hydrogen bonds, enhancing system stability and reducing the risk of precipitation during storage. The final concentration of the walnut peptides is 0.2-0.5%.

[0055] The theanine concentration is 0.08-0.12%. Theanine can penetrate the blood-brain barrier and act directly on the brain. GABA can form a complex with theanine through intermolecular interactions, which is beneficial for GABA absorption. Theanine itself has a savory sweet taste and can also improve the overall flavor profile.

[0056] The fermentation broth contains a large amount of lactic acid and acetic acid, resulting in a sharp sour taste. Sweeteners provide sweetness, creating a pleasant "sweet-sour ratio" that greatly improves palatability. The sweetness also effectively masks any slight bitterness, astringency, or medicinal taste that may result from fermentation and the plant-based ingredients. For example, sucralose is used as a sweetener at a final concentration of 0.01-0.03%.

[0057] The stabilizer forms a stable three-dimensional network structure through thickening, emulsification, and suspension, uniformly suspending various solids in the liquid and ensuring product homogeneity. The stabilizer includes one or more of pectin, gelatin, and xanthan gum, for example, pectin at a final concentration of 0.1-0.3% and gelatin at 0.1-0.2%.

[0058] In a preferred embodiment of the present invention, the natural raw material extract constitutes 10-20% of the total volume of the fermentation substrate, the monosodium glutamate constitutes 0.8-1.2% of the dry weight of the fermentation substrate, and the hop extract constitutes 0.06-0.12% of the dry weight of the fermentation substrate. The hop extract is prepared from dried hops (Humulus lupulus L.) by water extraction or alcohol extraction. On a dry weight basis, the hop extract contains not less than 2.0% xanthohumol and not less than 1.5% humulone.

[0059] The dry matter mass of the fermentation substrate refers to the total mass of all solid components remaining after water removal. For example, if the fermentation substrate consists of oat flour, maltodextrin, natural ingredient extract, monosodium glutamate (MSG), hop extract, and purified water, the dry matter mass of the fermentation substrate is the sum of the dry weights of oat flour, maltodextrin, natural ingredient extract, MSG, and hop extract. The dry weight of the natural ingredient extract should be calculated based on its solids content: Dry weight = Extract volume × Density × Solids content, where the liquid density is approximately 1 kg / L. Oat flour, maltodextrin, MSG, and hop extract are themselves dry powders and are directly included in the dry matter mass based on their added amounts in the formula.

[0060] In this invention, the proportion of natural raw material extract is 10-20%. This ensures that the final concentration of active ingredients such as jujube seed saponins, poria cocos polysaccharides, and lily polysaccharides in the fermentation substrate reaches the effective threshold, and also facilitates multi-target synergistic effects with GABA produced during subsequent fermentation. At the same time, it avoids the increase in viscosity of the fermentation substrate due to excessive addition, which would affect the mass transfer efficiency and metabolic activity of probiotics, or the antagonistic effect caused by excessive active ingredients, such as high concentrations of polysaccharides potentially encapsulating probiotic cells and hindering nutrient absorption. Furthermore, it helps the prebiotic components in the extract (such as poria cocos polysaccharides and lily polysaccharides) to optimize the microenvironment of the fermentation system, improve the metabolic conversion efficiency of probiotics, and ensure the fermentation effect.

[0061] The optimal addition of monosodium glutamate (MSG) is 0.8-1.2%, which helps ensure the rate and yield of GABA synthesis. If the ratio is <0.8%, insufficient substrate leads to low GABA production. If the ratio is >1.2%, the osmotic pressure of the fermentation substrate increases, inhibiting probiotic growth, and excessive glutamate can cause a "MSG taste" in the fermentation broth, affecting product palatability. At this ratio, the decomposition rate of MSG matches the growth and metabolic rate of probiotics, and the ammonia produced neutralizes some of the fermentation acid, helping to maintain a stable pH level and preventing GAD enzyme inactivation due to excessively low pH, thus ensuring the continuity of GABA synthesis.

[0062] A hop extract concentration of 0.06-0.12% is beneficial for achieving effective concentrations of xanthohumulin and humulone in the hop extract to regulate GABA metabolism: xanthohumulin activates GAD enzyme activity, and 8-isopentenylnaringenin upregulates GAD B gene expression. If the hop extract concentration is <0.06%, the regulatory effect is weak; if the hop extract concentration is >0.12%, the distinctive aroma of the hop extract will mask the natural flavor of the fermented product.

[0063] In a preferred embodiment of the present invention, the mass ratio of monosodium glutamate to hop extract is 10-18:1.

[0064] In this invention, monosodium glutamate (MSG) is the substrate for GABA synthesis, and hop extract is the activator and environment maker for GAD, the key enzyme in GABA synthesis. If the proportion is too low, and the hop extract is relatively excessive, certain antibacterial components (such as humulone) may slightly inhibit the growth of probiotics at high concentrations, thus slowing down fermentation and affecting the final GABA yield. If the proportion is too high, and the hop extract is relatively insufficient, it means that the amount of hop extract used is insufficient, and the initial acidity and stress signals it provides are not strong enough, easily leading to slow GABA synthesis initiation and low efficiency, resulting in excess and waste of MSG.

[0065] When the mass ratio of monosodium glutamate (MSG) to hop extract is 10-18:1, the amount of hop extract is sufficient to fully activate the fermentation system without inhibiting the activity of the microorganisms. This provides a favorable catalytic environment for the conversion of MSG, ensuring that the substrate is efficiently and completely converted into GABA. Furthermore, within the 10-18:1 range, the final product also benefits from the synergistic effect of both components on the nervous system, producing a good sedative and sleep-inducing effect.

[0066] Because the three strains described in this invention have different growth characteristics, *Lactobacillus delbrueckii* grows extremely rapidly and has a strong acid-producing capacity. After simultaneous inoculation, it multiplies rapidly and uncontrollably, producing large amounts of lactic acid, causing a sharp and uncontrollable drop in the pH of the fermentation broth. For *Lactobacillus acidophilus*, during the rapid pH drop, its growth is inhibited by the premature arrival of a strongly acidic environment before it has had sufficient time to fully proliferate and initiate GABA synthesis. Although its GABA synthase (GAD) requires acid activation, the bacteria themselves need time to grow; a rapid pH drop can easily disrupt both of these rhythms. *Bifidobacterium* is a strict anaerobe, and its tolerance to acid is relatively the worst. The rapid acid production of *Lactobacillus delbrueckii* easily leads to the death of a large number of *Bifidobacterium*. Due to the complex interactions and competition between the strains, problems such as large fluctuations in GABA content, imbalances in the proportion of viable cells, and abnormal flavor can easily occur.

[0067] In a preferred embodiment of the present invention, the probiotic fermentation employs a staged sequential fermentation process, including: First stage fermentation: Lactobacillus delbrueckii subsp. bulgaricus CGMCC 1.1480 was separately inoculated into the fermentation substrate at an inoculum volume of 2-3% of the fermentation substrate volume. Anaerobic fermentation was carried out at 37±0.5℃ and pH 5.5-6.0 for 10-14 hours to obtain the first stage fermentation broth.

[0068] In this invention, *Lactobacillus delbrueckii* subsp. bulgaricus CGMCC 1.1480 is introduced alone for the first stage of fermentation. During fermentation, the fermentation broth rapidly acidifies, and this slightly acidic environment effectively inhibits most other bacteria, creating a pure and safe fermentation environment for the subsequent two probiotic strains. Simultaneously, *Lactobacillus delbrueckii* exhibits strong protease activity, hydrolyzing large protein molecules in raw materials such as oats into polypeptides and free amino acids. This provides easily absorbed nutrients for the subsequent bacterial flora, especially *Lactobacillus acidophilus* and *Bifidobacterium*, which require amino acids and peptides as nitrogen sources. *Lactobacillus delbrueckii* is a facultative anaerobic bacterium; its metabolic activities consume all the residual oxygen in the fermenter, and it also creates a favorable anaerobic environment for the strictly anaerobic *Bifidobacterium*.

[0069] Second stage fermentation: Simultaneously inoculate Lactobacillus acidophilus CGMCC 1.1854 and Bifidobacterium animalis subsp. animalis CGMCC 1.1852 into the first stage fermentation broth, with the inoculation amount of Lactobacillus acidophilus being 1.5-2.5% of the first stage fermentation broth volume and the inoculation amount of Bifidobacterium being 2.5-3.5% of the first stage fermentation broth volume; adjust the fermentation temperature to 38±0.5℃, dynamically maintain the pH at 4.2-4.5, and continue anaerobic fermentation for 20-28 hours.

[0070] In this invention, *Lactobacillus acidophilus* is the main force in GABA synthesis, and its cells are rich in glutamate decarboxylase (GAD), which is the core engine for converting monosodium glutamate (MSG) into GABA. *Bifidobacterium* mainly plays a multifunctional auxiliary role, utilizing metabolites such as short-chain fatty acids (SCFAs) produced during prebiotic growth, which can participate in gut-brain axis regulation and enrich product functions. The inoculum size of *Bifidobacterium* (2.5-3.5%) is slightly higher than that of *Lactobacillus acidophilus* (1.5-2.5%) because *Bifidobacterium* grows relatively slowly and is more sensitive to the environment. Slightly raising the temperature to 38°C during this stage is the optimal or near-optimal growth temperature for both *Lactobacillus acidophilus* and *Bifidobacterium*, significantly improving their metabolic activity and GABA yield. Dynamically controlling the pH to 4.2-4.5 is a high-activity zone for GAD enzymes, ensuring rapid GABA synthesis and a tolerable and healthy growth range for the cells, which helps avoid premature termination of fermentation due to uncontrolled pH drops and achieves a stable output of high GABA yield.

[0071] In summary, this invention, through a phased sequential fermentation process, addresses several key advantages. First, it effectively resolves the competition and inhibition issues between bacterial strains. By physically separating the fermentation stages, it avoids the inhibition of early growth of *Lactobacillus acidophilus* and *Bifidobacterium* by the rapid acid production of *Lactobacillus delbrueckii*. Second, it improves the conversion rate and stability of GABA. This is achieved by creating an optimal biochemical environment for GABA synthesis (sufficient amino acid precursors, optimal acidic pH, and optimal temperature), and by ensuring that the produced GABA is endogenous and coexists with high-density bacterial cells, exhibiting significantly higher stability than exogenously added GABA. Third, it facilitates synergistic effects and gut-brain axis regulation. It yields not only high concentrations of GABA but also highly active *Lactobacillus acidophilus* and *Bifidobacterium*, as well as short-chain fatty acids produced by *Bifidobacterium*. These components collectively constitute a system that improves sleep through a dual pathway: neurotransmitter (GABA) + gut-brain axis (probiotic metabolites + SCFAs). This cleverly circumvents the challenge of low blood-brain barrier penetration of exogenous GABA, as gut-brain axis signals do not require direct GABA entry into the brain.

[0072] In a preferred embodiment of the present invention, the endpoint of the first stage of fermentation is when the pH value drops to 5.6 ± 0.1.

[0073] In this invention, when the pH drops to 5.6, *Lactobacillus delbrueckii* has already undergone rapid proliferation, reached a high cell density, and produced a sufficient amount of lactic acid. If fermentation continues, the pH will further decrease, causing it to enter a decline or stationary phase, and potentially producing excessive acidic substances or undesirable metabolites. For *Lactobacillus acidophilus* and *Bifidobacterium*, this creates a near-perfect inoculation environment. A pH of 5.6 is the ideal starting point for *Lactobacillus acidophilus* to begin proliferation and also the critical point for *Bifidobacterium* to successfully initiate growth without severe inhibition. During the process of reaching pH 5.6, *Lactobacillus delbrueckii* has already undergone preliminary hydrolysis of macromolecules such as proteins, preparing an easily usable nitrogen source for subsequent bacterial strains. GABA synthase GAD requires low pH for activation; its optimal activity range is pH 4.0-5.0. The pH of 5.6 in the first stage does not directly activate GAD, but it creates the basic conditions for the rapid acid reduction in the second stage and entry into the highly efficient activity range of GAD.

[0074] In a preferred embodiment of the present invention, during the second stage of fermentation, the pH is monitored every 4 hours. When the pH is < 4.3, 0.1-0.3% (w / v) of calcium carbonate buffer (i.e., 0.1-0.3g of calcium carbonate per 100mL of buffer) is added to maintain the pH at 4.2-4.5.

[0075] In this invention, intervention is initiated when the pH is <4.3 to prevent the pH from falling further into the range that strongly inhibits bacterial cell growth (e.g., <4.0). Monitoring every 4 hours helps avoid excessive pH fluctuations. By preventing excessive acidification, Lactobacillus acidophilus and Bifidobacterium can continue to proliferate and maintain a high survival rate.

[0076] These probiotics are not only biotransformation tools for GABA, but their fermentation produces short-chain fatty acids, bioactive peptides, and other metabolites that can regulate the nervous system through the gut-brain axis and work synergistically with GABA to improve sleep.

[0077] In a preferred embodiment of the present invention, after the probiotic fermentation in step (3) is completed and before the preparation and molding in step (4) begins, an enzymatic hydrolysis step is also included: adding a complex protease to the fermentation broth, wherein the amount of the complex protease added is 0.1-0.5% of the mass of the fermentation broth, and hydrolyzing for 30-60 minutes at pH 6.5-7.0 and 45°C, and then inactivating the enzyme at 85°C for 5-10 minutes.

[0078] In this invention, the fermentation broth contains a large amount of microbial protein (from probiotic cells) and incompletely utilized plant-derived proteins (from oats, poria cocos, etc.). The complex protease can cleave these large protein molecules into small peptides and free amino acids that can be directly absorbed by the intestines. Some of these bioactive small peptides can synergistically suppress GABA, further enhancing the product's core efficacy. The complex protease can also further hydrolyze some bitter peptides, eliminating or reducing the unpleasant bitterness caused by proteins. The free amino acids (such as glutamic acid and aspartic acid) and small peptides produced by enzymatic hydrolysis are natural flavoring substances that impart a rich umami and full-bodied taste to the product, making the overall flavor more saturated and harmonious, and reducing reliance on sweeteners. Enzymatic hydrolysis breaks down large protein molecules and microbial fragments into soluble small molecules, significantly reducing the system's instability, maintaining a homogeneous and clear beverage, significantly improving its appearance, and synergistically enhancing the physical stability of the beverage with subsequent stabilizers and homogenization processes. Proteases can break down the cell walls of probiotics that have become fragile due to fermentation, promoting the release of cell contents.

[0079] In a preferred embodiment of the present invention, the complex protease is a flavor protease and a trypsin in a mass ratio of 1-2:1.

[0080] In this invention, trypsin is an endopeptidase that specifically targets peptide bonds formed by the carboxyl groups of lysine or arginine within protein molecules. It rapidly cleaves large protein molecules into numerous polypeptide fragments of varying lengths, exhibiting high hydrolysis efficiency and ensuring deep hydrolysis. The flavor protease is a mixture of telopeptidases that further hydrolyzes the polypeptides produced by trypsin into smaller peptides and free amino acids. It specifically removes the terminal hydrophobic amino acids (sources of bitterness), thereby effectively degrading bitter peptides and significantly improving the product flavor.

[0081] A 1-2:1 mass ratio of flavor protease to trypsin ensures complementary benefits and maximizes the effectiveness of both enzymes. Setting the proportion of flavor protease slightly higher than trypsin ensures sufficient exonuclease activity to promptly remove bitter peptides generated by trypsin hydrolysis. Excessive trypsin leads to the rapid and excessive formation of bitter peptides, exceeding the processing capacity of the flavor protease and resulting in flavor degradation. Conversely, excessive flavor protease reduces hydrolysis efficiency, increases costs, and may produce excessive free amino acids, which is not always beneficial for flavor.

[0082] In a preferred embodiment of the present invention, the homogenization pressure in step (4) is 25-30 MPa, and the number of homogenization cycles is 1-3.

[0083] According to a second aspect of the present invention, a beverage for improving sleep is provided, characterized in that the beverage is obtained by the preparation method described above.

[0084] In this invention, due to the use of probiotic fermentation technology, the GABA in the beverage is endogenous and exists in multiple forms, including free, intracellular bound, and complex forms. This helps to solve the problems of exogenous GABA absorption being competitively inhibited by intestinal transporters and low blood-brain barrier penetration.

[0085] The GABA produced during fermentation is protected within the complex fermentation substrate. Subsequent enzymatic hydrolysis, homogenization, and sterilization processes collectively construct a system with highly stable physical and chemical properties. This ensures that functional components such as GABA and theanine exhibit extremely low degradation rates during their shelf life, completely resolving the pain point of easily oxidized degradation of exogenous GABA.

[0086] The beverage is not a simple mixture, but a synergistic functional body formed through biotransformation. It contains: (1) endogenous GABA, which acts directly on the nervous system; (2) probiotic metabolites, such as short-chain fatty acids, which act as signaling molecules of the gut-brain axis; (3) fermented and modified plant active ingredients, such as deglycosylated jujube seed saponins (enhancing absorption) and active peptides produced by enzymatic hydrolysis; (4) added synergistic ingredients, such as theanine and walnut peptides. The various components work together to form a multi-functional mechanism of neurotransmitter regulation + gut-brain axis regulation + multi-target plant ingredients.

[0087] By transforming unpleasant flavors through fermentation, eliminating bitterness through enzymatic hydrolysis, optimizing the mouthfeel through homogenization, and flavoring with sweeteners, this beverage successfully minimizes the off-flavors of various ingredients, providing a smooth, harmonious, and palatable drink.

[0088] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0089] In the following embodiments, unless otherwise specified, all raw material components are commercially available products.

[0090] Example 1 I. Raw material formula (based on 100L of beverage) Raw materials for preparing natural raw material extract: 5 kg of jujube seed, 3 kg of lily bulb, 4 kg of poria cocos, and 6 kg of jujube; Compound enzyme preparations: cellulase (≥5000U / g), hemicellulase (≥3000U / g), pectinase (≥4000U / g), β-glucanase (≥2000U / g); Fermentation substrate ingredients: 8 kg oat flour, 4 kg maltodextrin (mass ratio 2:1), 0.12 kg monosodium glutamate, 0.009 kg hop extract (mass ratio of monosodium glutamate to hop extract 13.3:1), and appropriate amount of purified water; Hops extract powder: prepared by water extraction from dried hops (Humulus lupulus L.), with a dry weight content of 2.4% xanthohumol and 1.6% humulone; Probiotic strains: Lactobacillus delbrueckii subsp. bulgaricus CGMCC 1.1480, Lactobacillus acidophilus CGMCC 1.1854, Bifidobacterium animalis subsp. animalis CGMCC 1.1852; Ingredients for preparation: 0.1 kg theanine, 0.02 kg sucralose, 0.2 kg pectin, 0.15 kg gelatin.

[0091] II. Preparation Steps (1) Preparation of natural raw material extract Raw material pretreatment: Sour jujube seeds are ground to 70 mesh, Poria cocos and lily bulbs are ground to 90 mesh respectively, and jujubes are ground to 110 mesh; Enzymatic hydrolysis: Enzymatic hydrolysis of jujube kernels: Take 5 kg of crushed jujube kernels, add 45 kg of purified water (8.5 times the amount), adjust the temperature to 52℃ and pH to 5.8, add the first compound enzyme preparation (the mass ratio of cellulase and hemicellulase is 2:1, and the amount added is 0.4% of the mass of jujube kernels), and stir at 135 r / min for 1.8 h for enzymatic hydrolysis; Enzymatic hydrolysis of Poria cocos and lily: Mix 4 kg of Poria cocos and 3 kg of lily, add 77 kg of purified water (11 times the volume), adjust the temperature to 48℃ and pH to 5.0, add the second compound enzyme (the mass ratio of pectinase and β-glucanase is 3:2, and the amount added is 0.3% of the total mass of Poria cocos and lily), and stir at 140 r / min for 1.2 h for enzymatic hydrolysis; Jujube extraction: Take 6 kg of crushed jujubes, add 54 kg of purified water (9 times the amount), extract at 72℃ for 1.2 h, and filter to obtain jujube extract; Ultrasonic extraction: The enzymatic hydrolysate of jujube seed, the enzymatic hydrolysate of poria cocos and lily bulb, and the jujube extract were mixed, and 40 kg of purified water was added. At this time, the total water volume of the extraction system was 216 kg (12 times the total mass of the dried jujube seed, poria cocos, lily bulb, and jujube). Under the conditions of 68℃ and stirring, continuous ultrasound with a frequency of 28 kHz and an ultrasonic power of 375 W was used for 1.3 h. Concentration: The filtrate after filtration was concentrated at 65℃ and 0.07MPa to a solid content of 18%, yielding approximately 15L of natural raw material extract. The extract contained 1.4mg / mL jujube seed saponins, 1.0mg / mL Poria cocos polysaccharides, and 1.1mg / mL lily polysaccharides.

[0092] (2) Preparation of fermentation substrate Carbon source dissolution: Mix 8 kg of oat flour with 4 kg of maltodextrin (total mass 12 kg), add 132 kg of purified water (11 times the total mass), heat to 85°C to dissolve for 45 min, sterilize at 121°C for 18 min, and cool to 37°C; Ingredient mixing: Add 15L of natural raw material extract (accounting for 15% of the total volume of fermentation substrate), 0.12kg of monosodium glutamate, and 0.009kg of hop extract, stir well, and obtain approximately 150L of fermentation substrate.

[0093] (3) Staged sequential probiotic fermentation First stage fermentation: Lactobacillus delbrueckii subsp. bulgaricus CGMCC 1.1480 bacterial culture was inoculated into the fermentation substrate at an inoculation amount of 2.5% of the fermentation substrate volume. Anaerobic fermentation was carried out at 37℃ and pH 5.8 for 12 hours until the pH dropped to 5.6 to obtain the first stage fermentation broth. Second stage fermentation: Simultaneously inoculate Lactobacillus acidophilus CGMCC 1.1854 (inoculation amount of 2.0% of the first stage fermentation broth volume) and Bifidobacterium animalis subsp. animalis CGMCC 1.1852 (inoculation amount of 3.0% of the first stage fermentation broth volume) into the first stage fermentation broth. Adjust the temperature to 38℃, and check the pH every 4 hours. When the pH < 4.3, add 0.2% (w / v) calcium carbonate buffer to maintain the pH at 4.2-4.5. Continue anaerobic fermentation for 24 hours to obtain the fermentation broth.

[0094] (4) Mixing and shaping Preparation: Add 0.1 kg theanine, 0.02 kg sucralose, 0.2 kg pectin, and 0.15 kg gelatin to the fermentation broth, and stir until completely dissolved; Homogenization: Homogenize twice under a pressure of 28 MPa to ensure the system is homogeneous and stable; Sterilization: Ultra-high temperature sterilization (UHT) at 135℃ for 5 seconds; Filling: Filling under aseptic conditions yields a beverage that improves sleep.

[0095] Example 2 The difference between Example 2 and Example 1 is that the probiotic fermentation uses a simultaneous fermentation process: Inoculation preparation: Mix the bacterial cultures of *Lactobacillus delbrueckii* subsp. bulgaricus CGMCC 1.1480, *Lactobacillus acidophilus* CGMCC 1.1854, and *Bifidobacterium animalis* subsp. animalis CGMCC 1.1852 in the following proportions: *Lactobacillus delbrueckii* subsp. bulgaricus inoculation amount is 2.5% of the fermentation substrate volume; *Lactobacillus acidophilus* inoculation amount is 2.0% of the fermentation substrate volume; *Bifidobacterium animalis* inoculation amount is 3.0% of the fermentation substrate volume; total inoculation amount is 7.5% of the fermentation substrate volume.

[0096] Simultaneous fermentation: The mixed bacterial culture was inoculated into the fermentation substrate at once and anaerobic fermentation was carried out at 37.5℃. The pH was measured every 4 hours. When the pH was <4.3, 0.2% calcium carbonate buffer was added to maintain the pH at 4.2-4.5. Fermentation was continued for 36 hours to obtain the fermentation broth.

[0097] Everything else is the same as in Example 1.

[0098] Example 3 The difference between Example 3 and Example 1 is that, after the probiotic fermentation in step (3) is completed, an enzymatic hydrolysis step is added before the preparation and molding in step (4). The enzymatic hydrolysis compound protease: flavor protease and trypsin are added in a mass ratio of 1.5:1, and the total amount added is 0.3% of the fermentation liquid mass. Add a complex protease to the fermentation broth, adjust the pH to 6.8, and enzymatically hydrolyze for 45 minutes at 45℃, then inactivate the enzyme at 85℃ for 8 minutes.

[0099] Everything else is the same as in Example 1.

[0100] Example 4 The difference between Example 4 and Example 1 is that the amount of monosodium glutamate used is 0.12 kg, the amount of hop extract used is 0.012 kg, and the mass ratio of monosodium glutamate to hop extract is 10:1.

[0101] Everything else is the same as in Example 1.

[0102] Example 5 The difference between Example 5 and Example 1 is that the amount of monosodium glutamate used is 0.12 kg, the amount of hop extract used is 0.01 kg, and the mass ratio of monosodium glutamate to hop extract is 12:1.

[0103] Everything else is the same as in Example 1.

[0104] Example 6 The difference between Example 6 and Example 1 is that the amount of monosodium glutamate used is 0.12 kg, the amount of hop extract used is 0.005 kg, and the mass ratio of monosodium glutamate to hop extract is 24:1.

[0105] Everything else is the same as in Example 1.

[0106] Example 7 The difference between Example 7 and Example 1 is that the amount of monosodium glutamate used is 0.12 kg, the amount of hop extract used is 0.006 kg, and the mass ratio of monosodium glutamate to hop extract is 20:1.

[0107] Everything else is the same as in Example 1.

[0108] Example 8 The difference between Example 8 and Example 1 is that the natural raw material extract is concentrated to a solid content of 16%, and the amount added is 12% of the total volume of the fermentation substrate.

[0109] Everything else is the same as in Example 1.

[0110] Example 9 The difference between Example 9 and Example 1 is that the anaerobic fermentation time in the second stage of fermentation is 19 hours.

[0111] Everything else is the same as in Example 1.

[0112] Example 10 The difference between Example 10 and Example 1 is that 0.35 kg of walnut peptide was added to the raw materials used in the preparation; In the preparation and molding process, the walnut peptide mother liquor is prepared first: take 0.35 kg of walnut peptide powder, add 3.5 kg of purified water, stir at 180 r / min for 8 min at room temperature, and dissolve into a uniform mother liquor without lumps. Then, add walnut peptide mother liquor, 0.1 kg theanine, 0.02 kg sucralose, 0.2 kg pectin, and 0.15 kg gelatin to the fermentation broth in sequence, and stir until completely dissolved; The homogenization, sterilization, and filling processes are the same as in Example 1; Everything else is the same as in Example 1.

[0113] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that exogenous GABA (purity ≥99%) was added directly, without the addition of monosodium glutamate. In the formulation and molding steps, GABA was added directly, with a final concentration of 23.5 mg / 100 mL.

[0114] Everything else is the same as in Example 1.

[0115] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that hop extract was not used; otherwise, they are the same as in Example 1.

[0116] Performance testing I. Physicochemical Indicators The total solids content and pH value were tested according to GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food" (reduced pressure drying method). Determination of GABA, theanine, and jujube seed saponin content in the final product: (1) The GABA content was determined by high performance liquid chromatography; (2) The content of Poria cocos polysaccharide and lily polysaccharide was determined by gel filtration chromatography and phenol-sulfuric acid colorimetric method: the extract was separated by Sephadex G-100 gel column, the corresponding elution peaks were collected and concentrated, and then determined according to GB / T 15672-2009 standard. The degradation rate of GABA after 6 months of storage. Storage conditions: room temperature (below 25℃), relative humidity (RH) 60%±5%, opaque packaging, completely sealed.

[0117] The test results are shown in Table 1.

[0118] Table 1 shows the physicochemical test results of Examples 1-10 and Comparative Examples 1-2.

[0119] As shown in Table 1, the initial GABA content in the beverages prepared in Examples 1-10 ranged from 16.5 to 23.8 mg / 100 mL. Among them, Example 3 had the best performance with 23.8 mg / 100 mL and Example 10 had the best performance with 23.3 mg / 100 mL, which was much higher than the 18.6 mg / 100 mL in Comparative Example 2. This proves that the technical solution described in this invention can efficiently activate GABA synthesis.

[0120] In Examples 1-10, the GABA degradation rate was only 5.2-7.6%, with Example 10 showing the lowest degradation rate at 5.2%. In contrast, Comparative Example 1 showed a GABA degradation rate of 20%, demonstrating that the technical solution described in this invention has advantages in system stability.

[0121] II. Microbiological Indicators The testing of coliform bacteria, molds, and yeasts was conducted in accordance with GB 4789.35-2016, "National Food Safety Standard: Microbiological Examination of Food - Examination of Lactic Acid Bacteria". The testing of pathogenic bacteria (Salmonella, Staphylococcus aureus, etc.) is based on the GB 4789 series standards; The test results are shown in Table 2.

[0122] Table 2 shows the test results of microbial indicators for Examples 1-10 and Comparative Examples 1-2.

[0123] As shown in Table 2, in all examples, the coliform count was <10 CFU / mL, the mold and yeast count was <50 CFU / mL, and no pathogenic bacteria were detected, proving that the process was hygienic and safe.

[0124] III. Flavor and Sensory Experience The flavor and sensory rating scale (total score 100 points) is shown in Table 3.

[0125] Table 3 Flavor and Sensory Rating Table

[0126] The blind test method was used, with 50 participants aged 20-50 years, and a male-to-female ratio of 1:1. Sensory evaluation room: temperature 23±2℃, humidity 50±5%, no odor, no direct sunlight, and good ventilation; Evaluation equipment: White ceramic cups and disposable straws are used uniformly, and sample codes are randomized (three-digit blind evaluation); All test samples were placed in a constant temperature environment of 25℃ for 30 minutes to equilibrate. 30 mL of each sample was then placed into a coded ceramic cup. After scoring according to the scoring sheet, excluding outliers (deviations from the mean ± 2 standard deviations), calculate the average score for each sample (retaining one decimal place).

[0127] The scoring results are shown in Table 4.

[0128] Table 4. Flavor and sensory score results of Examples 1-10 and Comparative Examples 1-2

[0129] As shown in Table 4, the total score ranking is: Example 3 (91.6 points) > Example 10 (89.9 points) > Example 1 (87.9 points), all of which are higher than the comparative examples (81.9-84.8 points).

[0130] Example 10 scored 34.8 points in the "taste" dimension and 27.2 points in the "aroma" dimension, proving that the sweet taste of walnut peptides and the herbal aroma of hops work synergistically to improve the richness of the beverage without any unpleasant off-flavors.

[0131] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, nor does it mean that the present invention must rely on the above process steps for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a beverage to improve sleep, characterized in that, The preparation method includes: (1) After crushing jujube seed, lily bulb, poria cocos and jujube, a natural raw material extract is prepared, wherein the jujube seed saponin content is ≥1.2mg / mL, the poria cocos polysaccharide content is ≥0.8mg / mL, and the lily bulb polysaccharide content is ≥0.9mg / mL; (2) Preparation of fermentation substrate First, mix oat flour and maltodextrin in a mass ratio of 1-3:1, then add purified water in a volume of 8-12 times the total mass of oat flour and maltodextrin, heat to 80-90℃ to dissolve for 30-60 minutes, then sterilize at 121℃ for 15-20 minutes, and then cool to 37℃. Then, the natural raw material extract, monosodium glutamate and hop extract are added and mixed evenly to obtain the fermentation substrate; The mass ratio of monosodium glutamate to hop extract is 10-18:1; based on dry weight, the hop extract contains not less than 2.0% xanthohumol and not less than 1.5% humulone. (3) Probiotic fermentation Lactobacillus delbrueckii subsp. bulgaricus CGMCC 1.1480, Lactobacillus acidophilus CGMCC 1.1854, and Bifidobacterium animalis subsp. animalis CGMCC 1.1852 were inoculated into a fermentation substrate for fermentation to obtain a fermentation broth. The probiotic fermentation employs a staged sequential fermentation process, including: First stage fermentation: Lactobacillus delbrueckii subsp. bulgaricus CGMCC 1.1480 was separately inoculated into the fermentation substrate at an inoculation amount of 2-3% of the fermentation substrate volume. Anaerobic fermentation was carried out at 37±0.5℃ and pH 5.5-6.0 for 10-14 hours to obtain the first stage fermentation broth. Second stage fermentation: Simultaneously inoculate Lactobacillus acidophilus CGMCC 1.1854 and Bifidobacterium animalis subsp. animalis CGMCC 1.1852 into the first stage fermentation broth, with the inoculation amount of Lactobacillus acidophilus being 1.5-2.5% of the first stage fermentation broth volume and the inoculation amount of Bifidobacterium animalis being 2.5-3.5% of the first stage fermentation broth volume; adjust the fermentation temperature to 38±0.5℃, and dynamically maintain the pH at 4.2-4.5 by using a small amount of calcium carbonate powder, and continue anaerobic fermentation for 20-28 hours; (4) Mixing and shaping Add one or more of the following to the fermentation broth: walnut peptide, theanine, sweetener, or stabilizer. Stir and dissolve, then perform homogenization and sterilization sequentially to produce a drink that improves sleep. The homogenization pressure is 25-30 MPa, and the homogenization is performed 1-3 times. Among them, after the probiotic fermentation in step (3) is completed and before the preparation and molding in step (4) begins, the enzymatic hydrolysis step is also included: adding a complex protease to the fermentation broth, wherein the amount of the complex protease added is 0.1-0.5% of the mass of the fermentation broth, and hydrolyzing for 30-60 minutes at pH 6.5-7.0 and 45℃, and then inactivating the enzyme at 85℃ for 5-10 minutes; The complex protease is a flavor protease and a trypsin in a mass ratio of 1-2:

1.

2. The preparation method according to claim 1, characterized in that, In the fermentation matrix, the natural raw material extract accounts for 10-20% of the total volume of the fermentation matrix, the monosodium glutamate accounts for 0.8-1.2% of the dry matter of the fermentation matrix, and the hop extract accounts for 0.06-0.12% of the dry matter of the fermentation matrix. The hop extract is prepared from dried hops (Humulus lupulus L.) through water extraction or alcohol extraction.

3. The preparation method according to claim 1, characterized in that, The endpoint of the first stage of fermentation is when the pH value drops to 5.6 ± 0.

1.

4. The preparation method according to any one of claims 1-3, characterized in that, During the second stage of fermentation, the pH was monitored every 4 hours. When the pH was less than 4.3, 0.1-0.3% (w / v) of calcium carbonate buffer was added to maintain the pH at 4.2-4.

5.

5. A beverage for improving sleep, characterized in that, The beverage is obtained by the preparation method described in any one of claims 1-4.

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