Colon-targeted slow-release probiotic amino acid composite microcapsules for improving sleep and a preparation method thereof

Abstract

The application relates to a colon-targeted slow-release probiotic amino acid composite microcapsule for improving sleep and a preparation method thereof. The composite microcapsule has a multilayer embedding structure and comprises, from inside to outside, an active substance core, a colon enzymatic slow-release layer and a gastric acid protection layer. The active substance core comprises probiotics and functional amino acids. The colon enzymatic slow-release layer comprises resistant starch material, and the resistant starch material comprises RS2 green banana powder and RS3 high-amylose corn retrograded starch. The gastric acid protection layer comprises polysaccharide gel material. The application is characterized in that the probiotic-amino acid symbiotic compound is matched with the three-layer slow-release structure as a whole, so that the in-vivo metabolism of exogenous amino acids and the rhythm of the continuous production of active metabolites by the probiotics after the colonization in the colon are connected in the time dimension, and the obtained preparation shows more persistent efficacy in improving sleep.

Description

Technical Field

[0001] This application relates to the field of functional food technology, and in particular to a colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep and its preparation method. Background Technology

[0002] Sleep disorders are increasingly impacting the quality of life for modern people. GABA, as the most important inhibitory neurotransmitter in the central nervous system, plays a crucial role in regulating the sleep-wake cycle; L-theanine has also been confirmed by numerous studies to have sedative and sleep-improving effects, and the combined effect of the two is superior to that of either ingredient alone. Therefore, amino acid-based sleep aid functional foods with GABA and L-theanine as their main active ingredients have become mainstream in the market.

[0003] Numerous sleep-aiding amino acid functional foods and compositions have been reported. For example, CN119817801A discloses a nutritional food containing GABA, theanine, jujube seed extract, and valerian extract, primarily targeting sleep and brain repair; CN121312830A presents a composition combining GABA with nerve-sedative components such as tea theanine. In recent years, some studies have also attempted to introduce probiotics into amino acid formulations, utilizing the fermentation of GABA-producing strains to help increase GABA supply levels.

[0004] However, each of the aforementioned amino acid-based sleep aid technologies has significant shortcomings. In ordinary powder and tablet products, GABA and theanine dissolve rapidly in the stomach, being destroyed by gastric acid or prematurely absorbed in the small intestine before reaching the colon. This results in a very low percentage of amino acids actually reaching their target sites, leading to insufficient bioavailability and difficulty in maintaining a long-lasting sleep-aiding effect. While some products use probiotics as an adjunct to GABA, their working rhythms are difficult to coordinate: exogenous GABA reaches peak blood concentration within 2 to 4 hours and then rapidly declines, while probiotic colonization and metabolite accumulation require a longer time. Therefore, the exogenous supplementation and endogenous synthesis of amino acids are not effectively coordinated in terms of time. The stability of amino acid preparations is also concerning. Free amino acids in the formula are prone to Maillard reactions with excipients during storage, and products containing probiotics experience a rapid decline in live bacteria count during room temperature storage, often failing to achieve the expected effects by the end of their shelf life.

[0005] In summary, the field of sleep-aid functional foods with amino acids as the core active ingredient still faces three mutually restrictive technical challenges: difficulty in controlling the release rate of active ingredients, mismatch between exogenous supplementation and endogenous synthesis time, and insufficient formulation stability. Therefore, there is an urgent need for a technical solution that can achieve a continuous and stable supply of amino acid active ingredients in the body, match the rhythm of exogenous amino acid synthesis with that of endogenous synthesis, and simultaneously ensure formulation stability, in order to overcome the application bottlenecks of amino acid-based sleep-aid functional foods. Summary of the Invention

[0006] In order to enhance the duration of action of active ingredients in vivo, achieve a time-match between exogenous supplementation and endogenous synthesis, and improve the shelf-life stability of amino acid preparations, this application provides a colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep and its preparation method.

[0007] In a first aspect, this application provides a colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep, employing the following technical solution: A colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep, wherein the complex microcapsule has a multi-layer encapsulation structure, comprising, from the inside out, an active substance core, a colonic enzymatic hydrolysis sustained-release layer, and a gastric acid protective layer; the active substance core comprises probiotics and functional amino acids; the colonic enzymatic hydrolysis sustained-release layer comprises resistant starch material, which includes RS2 green banana powder and RS3 high amylose corn retrograde starch; the gastric acid protective layer comprises polysaccharide gel material.

[0008] The inventors discovered that this application, through the overall coordination of the probiotic-amino acid symbiotic complex and the three-layer sustained-release structure, enables the in vivo metabolism of exogenous amino acids to be linked with the rhythm of the continuous production of active metabolites by probiotics after colonization in the colon in the time dimension.

[0009] Specifically, the polysaccharide gel material in the gastric acid protective layer maintains its structural integrity in the gastric juice environment, providing a gastric acid barrier for the inner active substances. When the composite microcapsules enter the small intestine, the polysaccharide gel material begins to dissolve, the outer structure gradually disintegrates, and the colonic enzymatic hydrolysis sustained-release layer is exposed to the small intestinal environment. In the colonic enzymatic hydrolysis sustained-release layer, RS2 green banana powder slowly swells in the proximal small intestine (pH 6.0-6.5), allowing some functional amino acids in the core of the active substances to be released in small amounts from the small intestine, achieving early onset of action. Meanwhile, RS3 high amylose corn retrograde starch resists amylase degradation in the small intestine, maintaining its structural integrity, thus protecting most of the active ingredients and allowing them to continue to be propelled with the chyme to the colon. This prevents the active ingredients from being prematurely metabolized and cleared in the small intestine, prolonging the duration of action of exogenous amino acids in the body.

[0010] Upon entering the colon, the amylase secreted by the intestinal flora specifically degrades RS3 high-amylose retrograde corn starch, causing the colonic enzymatic hydrolysis slow-release layer structure to disintegrate, exposing the active substance core and releasing functional amino acids and probiotics. The released probiotics colonize the colonic environment and continuously metabolize to produce endogenous active metabolites such as GABA. The release of GABA from the functional amino acids is rhythmically linked to the production of active metabolites after probiotic colonization. Exogenous supplementation takes effect in the early stages, while endogenous synthesis by probiotics continues in the later stages, resulting in a formulation that exhibits a more sustained effect in improving sleep.

[0011] In addition, the layered encapsulation structure of the composite microcapsules isolates the probiotics from the external environment, reducing the contact between oxygen and moisture and the bacteria, thus maintaining low metabolic activity of the probiotics during storage and slowing down the decline in the number of live bacteria.

[0012] In one specific implementation, the mass ratio of RS2 green banana powder to RS3 high amylose corn retrograde starch is 1:(1.5-3); more preferably, the mass ratio of RS2 green banana powder to RS3 high amylose corn retrograde starch is 1:2.

[0013] The inventors discovered that when the mass ratio of RS2 green banana powder to RS3 high amylose corn retrograde starch falls within the range of 1:(1.5-3), the two resistant starches in the colonic enzymatic hydrolysis sustained-release layer can complement each other in terms of release rhythm. If the proportion of RS2 green banana powder is too high, the active ingredient is released excessively in the pre-small intestine, and the amount remaining in the colon is insufficient for targeted delivery; if the proportion of RS3 high amylose corn retrograde starch is too high, the early release is too low, and exogenous amino acids are difficult to take effect quickly in the early stages of medication. Within the above ratio range, a balance can be formed between the early pre-release contributed by RS2 green banana powder and the colonic targeted release contributed by RS3 high amylose corn retrograde starch, so that the in vivo metabolism of exogenous amino acids and the rhythm of active metabolites produced by probiotics after colonization are linked in the time dimension. When the mass ratio is further optimized to 1:2, the swelling and permeation of RS2 green banana powder in the front of the small intestine is moderate, and the active ingredients delivered to the colon by RS3 high amylose corn retrograde starch are fully retained. The two are most closely linked, and the resulting formulation has a more lasting effect in improving sleep.

[0014] In one specific implementation, the probiotics are selected from one or more of *Lactobacillus plantarum* LP-28, *Bifidobacterium longum* BB-536, *Lactobacillus rhamnosus* LGG, and *Lactobacillus acidophilus* NCFM; preferably, the probiotics include *Lactobacillus plantarum* LP-28, *Bifidobacterium longum* BB-536, *Lactobacillus rhamnosus* LGG, and *Lactobacillus acidophilus* NCFM; more preferably, the amount of *Lactobacillus plantarum* LP-28 added per gram of compound microcapsule is 2 × 10⁻⁶. 9CFU; the amount of Bifidobacterium longum BB-536 added was 1.5 × 10⁻⁶. 9 CFU; the amount of *Lactobacillus rhamnosus* LGG added was 1×10⁻⁶. 9 CFU; the amount of Lactobacillus acidophilus NCFM added is 1×10 9 CFU.

[0015] By using probiotics formulated with the above four strains, they function in four dimensions: neurotransmitter synthesis, precursor supply, intestinal barrier maintenance, and mucosal repair. Lactobacillus plantarum LP-28 continuously metabolizes glutamate to produce GABA after colonization in the colon; Bifidobacterium longum BB-536 metabolizes tryptophan to produce indole derivatives, activating the 5-hydroxytryptamine (5-HT) precursor pathway; Lactobacillus rhamnosus LGG regulates intestinal barrier function and improves amino acid transport efficiency; and Lactobacillus acidophilus NCFM repairs the intestinal mucosa by producing short-chain fatty acids such as butyrate.

[0016] GABA, as the most important inhibitory neurotransmitter in the central nervous system, plays a core role in calming the mind and promoting sleep. *Lactobacillus plantarum* LP-28 is the direct synthesizer of GABA and is therefore given the highest dosage. *Bifidobacterium longum* BB-536 activates the 5-HT pathway indirectly, while *Lactobacillus rhamnosus* LGG and *Lactobacillus acidophilus* NCFM provide auxiliary support from the perspectives of intestinal barrier maintenance and mucosal repair, respectively. None of these three directly produce active neurotransmitters, and they can still exert their expected auxiliary functions even at slightly lower dosages. The four strains have complementary functions and clear division of labor, resulting in a more comprehensive effect in improving sleep.

[0017] In one specific embodiment, the functional amino acid is selected from one or more of L-theanine, GABA, L-tryptophan, glycine, and glutamine; preferably, the functional amino acid includes L-theanine, GABA, L-tryptophan, glycine, and glutamine; more preferably, per unit of composite microcapsule, the functional amino acid comprises: 80-120 mg L-theanine; 120-180 mg GABA; 60-100 mg L-tryptophan; 40-60 mg glycine; 150-250 mg glutamine; more preferably, per unit of composite microcapsule, the functional amino acid comprises: 100 mg L-theanine; 150 mg GABA; 80 mg L-tryptophan; 50 mg glycine; 200 mg glutamine.

[0018] By employing the aforementioned compound of functional amino acids, GABA directly exerts its inhibitory neurotransmission effect, L-theanine promotes the generation of alpha brain waves and synergizes with GABA-A receptors; together, they constitute the basis for rapid-acting exogenous sedation. L-tryptophan, as a 5-HT precursor, participates in circadian rhythm regulation, glycine helps lower core body temperature, and glutamine provides nitrogen support for probiotics while repairing the intestinal mucosa. These five amino acids work synergistically at five levels—neural inhibition, brain wave regulation, rhythm regulation, body temperature regulation, and intestinal support—achieving multi-dimensional coverage of sedation and sleep-aiding effects through an "exogenous supplementation" pathway.

[0019] Furthermore, compared to directly consuming single or simple combinations of amino acids, the functional amino acids in this application share the same release environment for the active substances as probiotics. When the colonic enzymatically hydrolyzed sustained-release layer disintegrates, the amino acids and probiotics are simultaneously exposed in the colon. The probiotics can then utilize substrates such as glutamine to continuously metabolize and produce active substances, ensuring that exogenous supplementation and endogenous synthesis are spatially aligned at the same release site. This further enhances the longevity of the formulation's efficacy in improving sleep.

[0020] In one specific implementation, the polysaccharide gel material is calcium alginate gel.

[0021] Calcium alginate gel is used as a gastric acid protective layer based on its pH-responsive structural stability in the gastric juice environment. Calcium alginate gel is formed by cross-linking sodium alginate with calcium ions, exhibiting a characteristic "eggbox" structure. In the gastric juice environment (pH 1.5-3.5), the carboxyl group in the calcium alginate gel has a low degree of protonation, and calcium ions maintain a stable chelate cross-linked state with the G units (guluronic acid) on the alginate molecular chain segments. The tightly cross-linked network structure effectively resists the penetration and erosion of gastric acid, providing a physical barrier for the inner active substances.

[0022] When the composite microcapsules enter the small intestine, the pH of the environment rises to 6.0-7.0. The carboxyl groups in the calcium alginate gel gradually undergo deprotonation, the electrostatic repulsion between molecular chains is enhanced, and the chelation and cross-linking of calcium ions and carboxyl groups is weakened. The gel structure begins to swell and gradually disintegrate, allowing the colonic enzyme hydrolysis sustained-release layer to be exposed to the small intestinal environment.

[0023] Secondly, this application provides a method for preparing colon-targeted sustained-release probiotic amino acid complex microcapsules for improving sleep, using the following technical solution: A method for preparing colon-targeted sustained-release probiotic amino acid complex microcapsules for improving sleep includes the following steps: S1. Preparation of active substance core: Probiotics are fermented and cultured, the bacterial cells are collected by centrifugation, washed and resuspended to obtain a mixed bacterial suspension; functional amino acids are dissolved in sterile water according to the ratio, mixed with the mixed bacterial suspension, freeze-dried and then pulverized and sieved to obtain active substance core powder; S2, Coating with Colon Enzyme Hydrolysis Sustained-Release Layer: The resistant starch material is prepared into a starch suspension, heated to gelatinize and then cooled; the active substance core powder obtained in step S1 is placed in a fluidized bed coating device, and the starch suspension is used as the coating liquid for spray coating. After drying, a colon enzyme hydrolysis sustained-release layer is formed, resulting in two-layer coated microspheres. S3. Gastric acid protective layer: The two-layer coated microspheres obtained in step S2 are dispersed in a polysaccharide gel material solution, cross-linked and cured to form a gastric acid protective layer. After washing and drying, the composite microcapsules are obtained.

[0024] The composite microcapsules prepared using the above steps exhibit clear interfaces and complete encapsulation between layers. The colonic enzymatic hydrolysis sustained-release layer uniformly coats the surface of the active substance core, while the gastric acid protective layer continuously and densely covers the outermost layer. The fluidized bed coating process enables the resistant starch material to form a uniformly thick coating layer on the surface of the active substance core, avoiding localized excessive thickness or exposure defects. This ensures the integrity of the colonic enzymatic hydrolysis sustained-release layer in resisting amylase degradation in the small intestine. The calcium alginate gel layer, through cross-linking and solidification, forms a pH-responsive three-dimensional network structure, maintaining structural stability in the gastric juice environment. Upon entering the small intestine, it gradually dissolves with increasing pH, allowing the inner structure to be exposed according to the expected time sequence. The resulting composite microcapsules possess reproducible interlayer binding forces and release behavior, providing a structural basis for the phased release of active ingredients in different segments of the digestive tract.

[0025] In summary, this application includes at least one of the following beneficial technical effects: 1. This application uses RS2 green banana powder and RS3 high amylose corn retrograde starch as a colonic enzymatic hydrolysis sustained-release layer. By utilizing the moderate swelling and penetration of RS2 in the pre-small intestine and the microbial-specific enzymatic hydrolysis of RS3 in the colon, a multi-stage release curve of "pre-release in the small intestine + targeted release in the colon" is formed. This allows the in vivo metabolism of exogenous amino acids and the rhythm of active metabolites produced by probiotics after colonization to be linked in the time dimension. The resulting formulation has a more lasting effect in improving sleep.

[0026] 2. This application uses a probiotic compound of four strains: Lactobacillus plantarum LP-28, Bifidobacterium longum BB-536, Lactobacillus rhamnosus LGG, and Lactobacillus acidophilus NCFM. These probiotics work in four dimensions: neurotransmitter synthesis, precursor supply, intestinal barrier maintenance, and mucosal repair. The four strains have complementary functions and clear division of labor, resulting in a more comprehensive effect in improving sleep.

[0027] 3. This application utilizes a combination of functional amino acids: L-theanine, GABA, L-tryptophan, glycine, and glutamine. GABA and L-theanine together form the basis for rapid-acting exogenous sedation, L-tryptophan participates in the regulation of circadian rhythms, glycine helps lower core body temperature, and glutamine provides nitrogen support for probiotics while repairing the intestinal mucosa. The five amino acids work synergistically to achieve multi-dimensional coverage of sedation and sleep-aiding effects. At the same time, the functional amino acids and probiotics share the same release site in the colon, allowing probiotics to continuously metabolize and produce active substances using substrates such as glutamine. Exogenous supplementation and endogenous synthesis work in a relay in time and space, further enhancing the longevity of the formulation's efficacy. Detailed Implementation

[0028] The present application will be further described in detail below with reference to embodiments and comparative examples: Some of the raw materials used in the examples and comparative examples: Lactobacillus plantarum LP-28 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 7.463, deposited on June 25, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Bifidobacterium longum BB-536 is deposited at the American Center for Type Culture Collection (ATCC) with accession number ATCC BAA-999. Lactobacillus rhamnosus LGG is deposited at the American Center for Type Culture Collection (ATCC) with accession number ATCC 53103. Lactobacillus acidophilus NCFM is deposited at the American Center for Type Culture Collection (ATCC) with accession number ATCC 700396. L-theanine, GABA (γ-aminobutyric acid), L-tryptophan, glycine, and glutamine all originated from Jiangsu Jinwei Amino Bioengineering Co., Ltd.

[0029] Unless otherwise specified, all raw materials used in the examples and comparative examples are commercially available products. Example Example 1

[0030] The preparation of composite microcapsules includes the following steps: S1. Preparation of the active substance core: *Lactobacillus plantarum* LP-28, *Bifidobacterium longum* BB-536, *Lactobacillus rhamnosus* LGG, and *Lactobacillus acidophilus* NCFM were inoculated into MRS liquid medium and activated by anaerobic culture at 37℃ for 24 h. The activated strains were then subjected to high-density fermentation at 37℃, with an initial pH of 6.2, and cultured anaerobically for 24 h. After fermentation, the bacterial cells of each strain were collected by centrifugation (4℃, 8000 rpm, 15 min), washed three times with sterile physiological saline (0.85% NaCl), and resuspended to obtain suspensions of each strain. The viable cell concentration of each strain suspension was determined by plate counting. Based on the pre-determined freeze-drying survival rate of each strain, the following feed amounts were used for mixing: The final product contained 2 × 10⁻⁶ viable *Lactobacillus plantarum* LP-28 per gram of composite microcapsule. 9 The viable count of CFU and Bifidobacterium longum BB-536 was 1.5 × 10⁻⁶. 9 The viable count of CFU and Lactobacillus rhamnosus LGG was 1×10⁻⁶. 9 The viable count of CFU and Lactobacillus acidophilus NCFM was 1×10⁻⁶. 9 CFU was used to obtain a mixed bacterial suspension.

[0031] For each batch of compound microcapsules, weigh out 100 mg of L-theanine, 150 mg of GABA, 80 mg of L-tryptophan, 50 mg of glycine, and 200 mg of glutamine, dissolve them in 100 mL of sterile water, and stir until completely dissolved to obtain an amino acid compound solution.

[0032] The amino acid compound solution and the mixed bacterial suspension were mixed at a volume ratio of 1:1. Trehalose with a final concentration of 10% (w / v) based on the total volume of the mixture was added as a freeze-drying protectant. The mixture was stirred evenly and then diluted to the preset volume with sterile deionized water. The mixture was placed in a freeze-drying tray and pre-frozen at -40℃ for 5 hours. Then, it was transferred to a vacuum freeze dryer and sublimation dried under a vacuum of 20 Pa. The shelf temperature was gradually increased from -40℃ to -10℃ and maintained for 16 hours. Subsequently, the temperature was increased to 28℃ for desorption drying for 8 hours until the moisture content of the material was less than 5%. The freeze-dried product was pulverized and passed through a 200-mesh sieve. The powder that passed through the 200-mesh sieve was collected to obtain the active substance core powder.

[0033] The particle size of the active ingredient core powder was determined to be 70 μm; the viable count of *Lactobacillus plantarum* LP-28 per gram of active ingredient core powder was 2 × 10⁻⁶. 9 The viable count of CFU and Bifidobacterium longum BB-536 was 1.5 × 10⁻⁶. 9 The viable count of CFU and Lactobacillus rhamnosus LGG was 1×10⁻⁶. 9 The viable count of CFU and Lactobacillus acidophilus NCFM was 1×10⁻⁶. 9CFU; each 0.5g of active ingredient core powder contains 100mg of L-theanine, 150mg of GABA, 80mg of L-tryptophan, 50mg of glycine, and 200mg of glutamine.

[0034] S2. Coating with a sustained-release colonic enzymatic layer: RS2 banana powder and RS3 high amylose corn retrograde starch were mixed at a mass ratio of 1:2 and dissolved in deionized water to prepare a 10% starch suspension. The suspension was heated to 80℃ and stirred for 30 minutes to fully gelatinize the starch. After cooling to room temperature, a starch coating solution was obtained. The active substance core powder obtained in step S1 was placed in a fluidized bed coating device, using a bottom-spray fluidized bed coating method. The inlet air temperature was set to 35℃, the material temperature to 40℃, the atomization pressure to 0.1MPa, and the inlet air flow rate to 10mL / min. The starch coating solution was used as the coating solution for spray coating at a spray flow rate of 2mL / min for 45 minutes. After coating, the microspheres were dried at 45℃ for 1.5 hours to obtain a two-layer coated microsphere.

[0035] The thickness of the colonic enzyme hydrolysis sustained-release layer was measured to be 150 μm.

[0036] S3. Coating with a gastric acid protective layer: Prepare a 2.0% sodium alginate aqueous solution. Take the bilayer coated microspheres obtained in step S2 at a solid-liquid ratio of 1:5 (w / v) and add them to the above sodium alginate aqueous solution (i.e., 100g of bilayer coated microspheres corresponds to 500mL of sodium alginate aqueous solution). Stir and disperse evenly to obtain a suspension. Drop the suspension into a 2.0% calcium chloride solution using a syringe, and cross-link and solidify for 20min to form a gastric acid protective layer. Filter and collect the solid microspheres, wash them three times with deionized water, and air-dry them at 30℃ for 3h to obtain composite microcapsules. The thickness of the gastric acid protective layer was measured to be 40μm. Example 2

[0037] The difference between Example 2 and Example 1 is that in step S2 of Example 2, instead of mixing RS2 green banana powder and RS3 high amylose corn retrograde starch at a mass ratio of 1:2, RS2 green banana powder and RS3 high amylose corn retrograde starch are mixed at a mass ratio of 1:1. Example 3

[0038] The difference between Example 3 and Example 1 is that in step S2 of Example 3, instead of mixing RS2 green banana powder and RS3 high amylose corn retrograde starch at a mass ratio of 1:2, RS2 green banana powder and RS3 high amylose corn retrograde starch are mixed at a mass ratio of 1:4.5. Example 4

[0039] The only difference between Example 4 and Example 1 is that in step S1 of Example 4, the probiotic component contains only *Lactobacillus plantarum* LP-28; the viable count of *Lactobacillus plantarum* LP-28 per gram of active substance core powder, as determined by plate count method, is 5.5 × 10⁻⁶. 9 CFU. Example 5

[0040] The only difference between Example 5 and Example 1 is that in step S1 of Example 5, the probiotic component contains only Bifidobacterium longum BB-536; the viable count of Bifidobacterium longum BB-53 in each gram of active substance core powder was determined by plate count method to be 5.5 × 10⁻⁶. 9 CFU. Example 6

[0041] The only difference between Example 6 and Example 1 is that in step S1 of Example 6, the probiotic component contains only *Lactobacillus rhamnosus* LGG; the viable count of *Lactobacillus rhamnosus* LGG per gram of active substance core powder, as determined by plate counting, is 5.5 × 10⁻⁶. 9 CFU. Example 7

[0042] The only difference between Example 7 and Example 1 is that in step S1 of Example 7, the probiotic component contains only Lactobacillus acidophilus NCFM; the viable count of Lactobacillus acidophilus NCFM per gram of active substance core powder, as determined by plate count method, is 5.5 × 10⁻⁶. 9 CFU. Example 8

[0043] The only difference between Example 8 and Example 1 is that in step S1 of Example 8, the functional amino acid component contains only L-theanine; each 0.5g of active substance core powder contains 580mg of L-theanine. Example 9

[0044] The only difference between Example 9 and Example 1 is that in step S1 of Example 9, the functional amino acid component contains only GABA; each 0.5g of active substance core powder contains 580mg of GABA. Example 10

[0045] The only difference between Example 10 and Example 1 is that in step S1 of Example 10, the functional amino acid component contains only L-tryptophan; each 0.5g of active substance core powder contains 580mg of L-tryptophan. Example 11

[0046] The only difference between Example 11 and Example 1 is that in step S1 of Example 11, the functional amino acid component contains only glycine; each 0.5g of active substance core powder contains 580mg of glycine. Example 12

[0047] The only difference between Example 12 and Example 1 is that in step S1 of Example 12, the functional amino acid component contains only glutamine; each 0.5g of active substance core powder contains 580mg of glutamine.

[0048] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that in step S2 of Comparative Example 1, RS2 green banana powder and RS3 high amylose corn retrograde starch are mixed at a mass ratio of 1:2 and replaced with an equal amount of RS2 green banana powder.

[0049] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that in step S2 of Comparative Example 2, RS2 green banana powder and RS3 high amylose corn retrograde starch are mixed at a mass ratio of 1:2 and replaced with an equal amount of RS3 high amylose corn retrograde starch.

[0050] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that no probiotic ingredients are added in step S1 of Comparative Example 3. The specific steps of step S1 are as follows: S1. Weigh out 100 mg of L-theanine, 150 mg of GABA, 80 mg of L-tryptophan, 50 mg of glycine, and 200 mg of glutamine per batch of composite microcapsules. Dissolve them in 100 mL of sterile water and stir until completely dissolved to obtain an amino acid compound solution. Add trehalose at a final concentration of 10% (w / v) based on the total volume of the mixture as a freeze-drying protectant, stir evenly, and bring the volume to the preset volume with sterile deionized water. Place the mixture in a freeze-drying pan and pre-freeze at -40°C for 5 hours. Then transfer it to a vacuum freeze dryer and perform sublimation drying under a vacuum of 20 Pa. Gradually increase the shelf temperature from -40°C to -10°C and maintain for 16 hours. Then raise the temperature to 28°C for desorption drying for 8 hours until the moisture content of the material is less than 5%. Crush the freeze-dried product and pass it through a 200-mesh sieve. Collect the powder that passes through the 200-mesh sieve to obtain the active substance core powder.

[0051] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is that in Comparative Example 4, step S1 does not involve the preparation of a symbiotic complex; instead, the probiotics and amino acids are freeze-dried separately and then physically mixed. The specific steps of step S1 are as follows: S1. Preparation of the active substance core: *Lactobacillus plantarum* LP-28, *Bifidobacterium longum* BB-536, *Lactobacillus rhamnosus* LGG, and *Lactobacillus acidophilus* NCFM were inoculated into MRS liquid medium and activated by anaerobic culture at 37℃ for 24 h. The activated strains were then subjected to high-density fermentation at 37℃, with an initial pH of 6.2, and cultured anaerobically for 24 h. After fermentation, the bacterial cells of each strain were collected by centrifugation (4℃, 8000 rpm, 15 min), washed three times with sterile physiological saline (0.85% NaCl), and resuspended to obtain suspensions of each strain. The viable cell concentration of each strain suspension was determined by plate counting. Based on the pre-determined freeze-drying survival rate of each strain, the following feed amounts were used for mixing: The final product contained 2 × 10⁻⁶ viable *Lactobacillus plantarum* LP-28 per gram of composite microcapsule. 9 The viable count of CFU and Bifidobacterium longum BB-536 was 1.5 × 10⁻⁶. 9 The viable count of CFU and Lactobacillus rhamnosus LGG was 1×10⁻⁶. 9 The viable count of CFU and Lactobacillus acidophilus NCFM was 1×10⁻⁶. 9 CFU was used to obtain a mixed bacterial suspension. Trehalose, with a final concentration of 10% (w / v) based on the total volume of the mixture, was added as a freeze-drying protectant. The mixture was stirred evenly and placed in a freeze-drying tray. It was pre-frozen at -40°C for 5 hours, and then transferred to a vacuum freeze dryer for sublimation drying under a vacuum of 20 Pa. The shelf temperature was gradually increased from -40°C to -10°C and maintained for 16 hours. Subsequently, the temperature was increased to 28°C for desorption drying for 8 hours until the moisture content of the material was less than 5%. The freeze-dried product was pulverized and passed through a 200-mesh sieve. The powder passing through the 200-mesh sieve was collected to obtain probiotic freeze-dried powder.

[0052] For each batch of composite microcapsules, weigh out 100 mg of L-theanine, 150 mg of GABA, 80 mg of L-tryptophan, 50 mg of glycine, and 200 mg of glutamine, dissolve them in 100 mL of sterile water, and stir until completely dissolved. Add trehalose at a final concentration of 10% (w / v) based on the total volume of the mixture as a freeze-drying protectant, stir evenly, place in a freeze-drying tray, and pre-freeze at -40°C for 5 hours. Then transfer to a vacuum freeze dryer and perform sublimation drying under a vacuum of 20 Pa. Gradually increase the shelf temperature from -40°C to -10°C and maintain for 16 hours, then raise the temperature to 28°C for desorption drying for 8 hours until the moisture content of the material is less than 5%. Crush the freeze-dried product, pass it through a 200-mesh sieve, and collect the powder passing through the 200-mesh sieve to obtain amino acid freeze-dried powder.

[0053] Mix the above-mentioned probiotic freeze-dried powder and amino acid freeze-dried powder evenly at a volume ratio of 1:1 to obtain the active substance core powder.

[0054] The composite microcapsules prepared in each embodiment and comparative example were subjected to performance tests according to the following standards: 1. In vitro simulated release test: Referring to the dissolution determination method in the Chinese Pharmacopoeia, the gastric juice, small intestinal juice and colonic juice environments were simulated respectively, and the cumulative release rate of GABA in each sample in different digestive stages was determined.

[0055] Simulated gastric juice: hydrochloric acid solution (containing pepsin) at pH 1.2, incubated at 37°C with shaking for 2 hours; simulated small intestinal juice: phosphate buffer (containing trypsin) at pH 6.8, incubated at 37°C with shaking for 4 hours; simulated colonic juice: phosphate buffer containing enzymatic hydrolysate of rat colonic flora, incubated anaerobically at 37°C for 6 hours.

[0056] The concentration of GABA in the release medium at each time point was determined by high performance liquid chromatography (HPLC), the cumulative release rate was calculated, and the data are recorded in Table 1 below: Table 1. Results of in vitro simulated release test

[0057] 2. Animal GABA Concentration-Time Curve Experiment: SPF-grade Kunming mice were randomly divided into groups of 10 each (half male and half female), weighing 18-22g. Each group was administered the composite microcapsules prepared in Example 1 and Comparative Examples 1-4 (dose of 150mg / kg GABA) by gavage, while the blank control group received an equal volume of physiological saline. Orbital blood was collected at 2h, 4h, 8h, and 12h after administration. Serum GABA concentration was detected by HPLC, and the data are recorded in Table 2 below: Table 2. Results of the GABA concentration-time curve experiment in animals

[0058] 3. Shelf-life stability test of viable bacteria: The composite microcapsules prepared in each example and comparative example were stored at room temperature (25±2℃, relative humidity 60±5%) for 180 days. Samples were taken on the day of preparation and at 180 days, and the total viable bacteria count was determined according to GB4789.35-2016. The data are recorded in Table 3 below: Table 3. Results of the shelf-life stability test of live bacteria

[0059] Based on Examples 1-12 and Tables 1-3, it can be seen that Example 1, using a RS2 to RS3 mass ratio of 1:2, resulted in only a trace release in the stomach, with the vast majority of the active ingredients successfully passing through the stomach and entering the intestines. Upon entering the small intestine, RS2 underwent moderate swelling, achieving a small pre-release. Upon reaching the colon, RS3 was specifically enzymatically hydrolyzed by the gut microbiota, achieving a large-scale targeted release. The three release phases were clearly distinguishable, presenting an ideal staged release curve. Regarding serum GABA concentration, Example 1 showed a rapid peak concentration in the early stages of administration, maintained a high level in the middle stages, and maintained an effective concentration in the later stages, significantly prolonging the effective duration of action. In contrast, Example 2 had a higher RS2 ratio, leading to an excessively high release ratio of the active ingredient in the small intestine, resulting in insufficient retention upon reaching the colon. The serum GABA concentration decreased significantly faster in the later stages compared to Example 1. Example 3 had a higher RS3 ratio, resulting in a low pre-release amount in the small intestine, and the serum GABA concentration was significantly lower than in Example 1 in the early stages, indicating a less rapid onset of action. The above results indicate that the ratio of RS2 to RS3 needs to be maintained within an appropriate range to achieve a balance between early pre-release and colon-targeted release, with 1:2 being the optimal ratio.

[0060] Examples 4-7 are preparations containing only a single probiotic strain. The serum GABA concentrations in each single-strain group were lower than those in Example 1, especially in the later stages of administration, where the serum concentration of Example 1 was significantly higher than that of any single-strain group. The *Lactobacillus plantarum* LP-28 single-strain group showed the best performance, which is consistent with the core function of LP-28 in directly synthesizing GABA. However, the serum concentrations of the four-strain combination in Example 1 were higher than any single-strain group in the later stages of administration, indicating that *Bifidobacterium longum* BB-536 supports gut-brain axis function from different dimensions by activating the 5-HT precursor pathway, *Lactobacillus rhamnosus* LGG by regulating intestinal barrier function, and *Lactobacillus acidophilus* NCFM by producing butyrate to repair the intestinal mucosa, forming an effective functional complement to the GABA synthesis of LP-28.

[0061] Example 9 (containing only GABA) showed a rapid peak plasma concentration in the early stages of administration, similar to Example 1, but the plasma concentration decreased rapidly in the middle and later stages of administration. Examples 8 (containing only L-theanine) and Examples 10-12 (containing only L-tryptophan, glycine, or glutamine) showed significantly lower plasma concentrations at all time points than Example 1. Example 1, with its five-amino acid complex, showed significantly higher plasma concentrations in the middle and later stages of administration than Example 9. This indicates that the synergistic effect of L-theanine on GABA-A receptors, the regulation of circadian rhythms by L-tryptophan, the auxiliary regulation of core body temperature by glycine, and the repair of intestinal mucosa by glutamine collectively support the sustained effect of GABA, maintaining a higher serum GABA concentration throughout the administration period.

[0062] Examples 1-12 all employed a three-layer encapsulation structure. After 180 days of storage at room temperature, the viable bacterial count in all examples remained well above the effective threshold, indicating that the three-layer encapsulation structure has a good protective effect on probiotics with different formulations, and the shelf-life stability does not change significantly due to adjustments in strain ratio or amino acid composition.

[0063] Combining Example 1 and Comparative Examples 1-4, and referring to Tables 1-3, it can be seen that in Comparative Example 1 (RS2 only), the active ingredient was released in large quantities in the small intestine, with a significantly insufficient release in the colon. Comparative Example 2 (RS3 only) showed a low release in the small intestine; although it delivered most of the active ingredient to the colon, it lacked the pre-release amount required for early onset of action. The serum GABA concentration in Comparative Example 1 decreased significantly and rapidly in the later stages of administration, while the blood drug concentration in Comparative Example 2 was significantly lower in the early stages of administration, indicating a slow onset of action. Example 1, through the combination of RS2 and RS3, maintained a high level of blood drug concentration in both the early and late stages of administration. These results indicate that neither RS2 nor RS3 alone can simultaneously achieve the dual goals of early onset of action and colon-targeted release; the combination of the two is key to achieving a multi-stage release curve.

[0064] Comparative Example 3 (containing only amino acids, without probiotics) showed similar blood drug concentrations to Example 1 in the early stages of administration, but its blood drug concentration rapidly decreased to near background levels in the middle and later stages of administration, resulting in a significantly shorter effective duration of action than Example 1. The only difference between the two was the presence or absence of probiotics, indicating that after exogenous amino acids are metabolically cleared, the endogenous GABA continuously synthesized by probiotics after colonization in the colon provides a crucial follow-up for serum GABA concentration, significantly prolonging the effective duration of action.

[0065] Comparative Example 4 (probiotics and amino acids were lyophilized separately and then physically mixed) showed similar blood drug concentrations to Example 1 in the early stages of drug administration, but significantly lower blood drug concentrations in the later stages of drug administration. The only difference between the two was the preparation method of the active substance core: in Example 1, probiotics and amino acids were co-lyophilized to form a symbiotic complex, while in Comparative Example 4, they were lyophilized separately and then physically mixed. Physical mixing resulted in the probiotics and amino acids being distributed as independent particles in the core. After colonic release, there was a microscopic separation between the two in space, which reduced the utilization efficiency of the amino acid substrate by the probiotics, leading to a less tight connection between exogenous supplementation and endogenous synthesis compared to Example 1.

[0066] In Example 1, the viable bacterial count remained well above the effective threshold after 180 days of storage at room temperature. Comparative Example 4, also employing a three-layer encapsulation structure, showed a viable bacterial count that, while remaining above the effective threshold, was significantly lower than that of Example 1, indicating that the symbiotic complex structure itself provides a certain protective and enhancing effect on probiotics. While the viable bacterial counts in Comparative Examples 1 and 2 were higher than in Comparative Example 4 after 180 days of storage at room temperature, they were significantly lower than those in Example 1, suggesting that the sustained-release layer formed by the combination of RS2 and RS3 provides better physical protection than a single resistant starch coating.

[0067] This application utilizes a compound of RS2 green banana powder and RS3 high amylose corn retrograde starch as a colonic enzymatic hydrolysis sustained-release layer. It leverages the moderate swelling and permeation of RS2 in the pre-small intestine and the microbial-specific enzymatic hydrolysis of RS3 in the colon to form a multi-stage release curve: low release in the stomach, pre-release in the small intestine, and targeted release in the colon. This ensures a temporal connection between the in vivo metabolism of exogenous amino acids and the rhythm of active metabolites produced by probiotics after colonization. Furthermore, the application uses a compound of four strains—Lactobacillus plantarum LP-28, Bifidobacterium longum BB-536, Lactobacillus rhamnosus LGG, and Lactobacillus acidophilus NCFM—to exert their effects in four dimensions: neurotransmitter synthesis, precursor supply, intestinal barrier maintenance, and mucosal repair, thereby enhancing the endogenous synthesis of probiotics. This invention effectively extends the duration of action by providing a continuous boost to exogenous supplementation. The combination of five amino acids—L-theanine, GABA, L-tryptophan, glycine, and glutamine—forms a rapidly acting exogenous sedative base. Simultaneously, the functional amino acids and probiotics form a symbiotic complex through co-freeze-drying, converging at the same release site in the colon. The probiotics can continuously metabolize and produce active substances using substrates such as glutamine. This creates a tight spatiotemporal connection between exogenous supplementation and endogenous synthesis. The three-layer concentric spherical encapsulation structure effectively isolates the probiotics from the external environment, reducing contact with oxygen and moisture, maintaining low metabolic activity during storage, and effectively improving the shelf-life stability of the formulation. In summary, this application effectively solves three mutually restrictive technical challenges: sustained release of active ingredients, matching the timing of exogenous supply and endogenous synthesis, and long-term survival of the strains. This results in a more lasting and comprehensive effect of the obtained composite microcapsules in improving sleep.

[0068] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep, characterized in that, The composite microcapsule has a multi-layer encapsulation structure, comprising, from the inside out, an active substance core, a colonic enzymatic hydrolysis sustained-release layer, and a gastric acid protective layer; the active substance core includes probiotics and functional amino acids; the colonic enzymatic hydrolysis sustained-release layer includes resistant starch material, which includes RS2 green banana powder and RS3 high amylose corn retrograde starch; the gastric acid protective layer includes polysaccharide gel material.

2. The colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep according to claim 1, characterized in that, The mass ratio of RS2 green banana powder to RS3 high amylose corn retrograde starch is 1:(1.5-3).

3. The colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep according to claim 1, characterized in that, The probiotics are selected from one or more of Lactobacillus plantarum LP-28, Bifidobacterium longum BB-536, Lactobacillus rhamnosus LGG, and Lactobacillus acidophilus NCFM.

4. The colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep according to claim 3, characterized in that, The probiotics include Lactobacillus plantarum LP-28, Bifidobacterium longum BB-536, Lactobacillus rhamnosus LGG, and Lactobacillus acidophilus NCFM.

5. The colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep according to claim 4, characterized in that, Based on per gram of composite microcapsule: the amount of *Lactobacillus plantarum* LP-28 added is 2 × 10⁻⁶. 9 CFU; the amount of Bifidobacterium longum BB-536 added was 1.5 × 10⁻⁶. 9 CFU; the amount of *Lactobacillus rhamnosus* LGG added was 1×10⁻⁶. 9 CFU; the amount of Lactobacillus acidophilus NCFM added is 1×10 9 CFU.

6. The colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep according to claim 1, characterized in that, The functional amino acid is selected from one or more of L-theanine, GABA, L-tryptophan, glycine, and glutamine.

7. The colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep according to claim 6, characterized in that, The functional amino acids include L-theanine, GABA, L-tryptophan, glycine, and glutamine.

8. The colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep according to claim 7, characterized in that, Based on each serving of composite microcapsule, the functional amino acids comprise: 80-120 mg L-theanine; 120-180 mg GABA; 60-100 mg L-tryptophan; 40-60 mg glycine; and 150-250 mg glutamine.

9. The colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep according to claim 1, characterized in that, The polysaccharide gel material is calcium alginate gel.

10. A method for preparing a colon-targeted sustained-release probiotic amino acid complex microcapsule for improving sleep according to any one of claims 1-9, characterized in that, Includes the following steps: S1. Preparation of active substance core: Probiotics are fermented and cultured, the bacterial cells are collected by centrifugation, washed and resuspended to obtain a mixed bacterial suspension; functional amino acids are dissolved in sterile water according to the ratio, mixed with the mixed bacterial suspension, freeze-dried and then pulverized and sieved to obtain active substance core powder; S2, Coating with Colon Enzyme Hydrolysis Sustained-Release Layer: The resistant starch material is prepared into a starch suspension, heated to gelatinize and then cooled; the active substance core powder obtained in step S1 is placed in a fluidized bed coating device, and the starch suspension is used as the coating liquid for spray coating. After drying, a colon enzyme hydrolysis sustained-release layer is formed, resulting in two-layer coated microspheres. S3. Gastric acid protective layer: The two-layer coated microspheres obtained in step S2 are dispersed in a polysaccharide gel material solution, cross-linked and cured to form a gastric acid protective layer. After washing and drying, the composite microcapsules are obtained.

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