Bifidobacterium longum subsp. longum, double-layer microspheres of the same and application thereof in improving sleep

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Bifidobacterium longum* subsp. *longum*, its bilayer microsphere formulation, and its application in improving sleep. The *Bifidobacterium longum* subsp. *longum* of this invention, named *Bifidobacterium longum* Slp99, is deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 27, 2026, with accession number GDMCC No: 68726. The Slp99 strain obtained by this invention can effectively shorten the sleep latency, prolong sleep duration, alleviate anxiety behaviors associated with insomnia, and possesses good activity in improving sleep quality. The bilayer microsphere formulation further developed by this invention can effectively improve the encapsulation rate and gastrointestinal fluid tolerance of the strain, while significantly enhancing the sleep improvement effect, providing a technical basis for the development of safe and reliable sleep aid products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bifidobacterium longum subsp. longum, its bilayer microsphere formulation, and its application in improving sleep. Background Technology

[0002] Poor sleep quality is a prevalent health problem in modern populations, typically manifested as prolonged sleep latency, increased nighttime awakenings, shortened sleep duration, decreased deep sleep percentage, and morning fatigue. Chronic sleep deprivation can disrupt neuroendocrine homeostasis, suppress immune function, impair cognitive abilities, and induce mood disorders. Current research confirms a close correlation between sleep disorders and gut microbiota dysbiosis, chronic low-grade inflammation, and excessive activation of the hypothalamic-pituitary-adrenal axis.

[0003] Currently, products used to improve sleep mainly include melatonin, sedatives, and plant extracts. However, chemical sedatives are prone to dependence and can induce daytime sleepiness; melatonin exhibits individual variability in response, and its long-term safety is controversial; plant extracts have a slow onset of action, and the effective dosage is difficult to control. Therefore, developing mild, safe, and long-term suitable probiotic sleep-regulating products is of significant value.

[0004] The gut microbiota can regulate neurotransmitter synthesis, short-chain fatty acid production, tryptophan metabolism, bile acid metabolism, immune factor release, and vagal nerve signal transduction through the gut-brain axis. Among these, *Bifidobacterium longum* is an endogenous symbiotic probiotic in the human gut and is highly safe for consumption. However, probiotic functions are strictly strain-specific, with significant functional differences between different strains. Furthermore, *Bifidobacterium longum* is sensitive to oxygen, gastric acid, bile salts, processing shear stress, and moisture. During freeze-drying, storage, and gastrointestinal transport, the viable bacteria count of ordinary probiotic powders decreases significantly, resulting in insufficient effective viable bacteria reaching the intestines and limiting their efficacy in vivo. In addition, current probiotic encapsulation systems often use wall materials such as calcium alginate and protein membranes, which have limited oxygen and moisture protection capabilities during storage, making industrial-scale process control difficult.

[0005] Therefore, it is urgent to screen a Bifidobacterium longum strain with a clear function of improving sleep and to develop a novel encapsulation delivery system to improve the strain's processing stability and gastrointestinal tolerance, while effectively improving its sleep-aiding effect. Summary of the Invention

[0006] To overcome the problems of unclear probiotic strains for improving sleep, poor stability of common live bacteria, low gastrointestinal survival rate, repeated use of conventional embedding materials, difficulty in industrialization control, and unsatisfactory sleep-aiding effects in existing technologies, the purpose of this invention is to provide a strain of *Bifidobacterium longum* subsp. *longum*, its bilayer microsphere formulation, and its application in improving sleep. The *Bifidobacterium longum* subsp. *longum* Slp99 provided by this invention can effectively improve sleep quality and related anxiety-like behaviors, making it suitable for development and application in sleep aid products. Furthermore, this invention utilizes a bilayer microsphere formulation constructed from this *Bifidobacterium longum* subsp. *longum*. Through the synergistic effect of the inner bacterial cellulose-fructan layer and the outer polyphenol-calcium coordination protective layer, the stability of Slp99 during freeze-drying, storage, and gastrointestinal digestion can be improved, while significantly enhancing its effectiveness in improving sleep.

[0007] To achieve the above objectives, the first aspect of the present invention adopts the following technical solution:

[0008] A strain of Bifidobacterium longum subsp. longum Slp99 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 27, 2026, with accession number GDMCC No: 68726.

[0009] The second aspect of this invention is the following technical solution:

[0010] A bilayer microsphere formulation encapsulating Bifidobacterium longum subsp. longum as described above, the bilayer microsphere formulation comprising an inner microsphere core encapsulating Bifidobacterium longum subsp. longum and a polyphenol-calcium coordination protective layer coating the surface of the inner microsphere core.

[0011] The inner microsphere core is mainly formed by encapsulating Bifidobacterium longum subsp. longum with bacterial cellulose and fructan; the polyphenol-calcium coordination protective layer is mainly formed by the coordination of epigallocatechin gallate and calcium salt.

[0012] More preferably, the calcium salt is calcium lactate. The use of the above-mentioned food-grade calcium salt in this invention helps ensure the safety of the bilayer microsphere formulation.

[0013] The third aspect of this invention is the following technical solution:

[0014] A method for preparing the above-mentioned bilayer microsphere formulation encapsulating Bifidobacterium longum subsp. longum includes the following steps:

[0015] Prepare a bacterial suspension of the aforementioned *Bifidobacterium longum* subsp. *longum*; separately, uniformly disperse bacterial cellulose and fructan in water to obtain a bacterial cellulose-fructan dispersion; dissolve epigallocatechin gallate in water to obtain a coating solution; dissolve calcium salt in water to obtain a calcium salt complex solution;

[0016] The bacterial suspension was mixed with a bacterial cellulose-fructose dispersion to obtain a bacterial dispersion; the bacterial dispersion was prepared into microdroplets, which were then frozen and freeze-dried to obtain an inner microsphere core.

[0017] The coating solution and the calcium salt coordination solution are sequentially sprayed onto the surface of the inner microsphere core to form a polyphenol-calcium coordination protective layer. Finally, the mixture is freeze-dried to obtain the bilayer microsphere formulation containing Bifidobacterium longum subsp. longum.

[0018] The inner microsphere core of this invention uses bacterial cellulose and fructan to immobilize the bacterial cells, buffer against freeze-drying dehydration damage, and provide a basis for rehydration and release. The outer layer uses epigallocatechin gallate (EGCG) and calcium salt, which can coordinate to form a polyphenol-calcium protective layer, improving oxygen barrier, moisture protection, and gastrointestinal fluid tolerance. The synergistic use of the inner and outer layers can improve the stability of Slp99 during freeze-drying, storage, and gastrointestinal digestion, and enhance its effectiveness in exerting a sleep-regulating effect in the intestine.

[0019] To further enhance the synergistic effect between the inner and outer layers, preferably, the bacterial cellulose-fructose dispersion has a mass concentration of 1.0% and a mass concentration of 0.8%; the coating solution has a mass concentration of 0.5%; and the calcium salt coordination solution has a mass concentration of 0.3%.

[0020] More preferably, the volume ratio of the bacterial suspension to the bacterial cellulose-fructose dispersion is 1:9.

[0021] More preferably, the microdroplets are prepared using a micro-injection pump; the micro-injection pump has a propulsion speed of 0.6 mL / min and an inner diameter of 0.3 mm for the outlet channel.

[0022] More preferably, the freezing and shaping is performed by instantaneous freezing with liquid nitrogen; the single addition volume of the coating liquid and the calcium salt coordination liquid is 5% of the mass of the microspheres; and the spraying is performed twice.

[0023] The fourth aspect of this invention is the following technical solution:

[0024] The application of the above-mentioned bilayer microsphere formulation or the bilayer microsphere formulation prepared by the above-mentioned preparation method in the preparation of products that improve sleep, wherein the improvement of sleep includes at least one of the following: prolonging sleep time, shortening sleep latency, alleviating anxiety-like behavior caused by insomnia, and repairing hypothalamic neuronal damage caused by insomnia.

[0025] More preferably, the product is a health supplement or a medicine.

[0026] The technical solution of the present invention has the following beneficial effects:

[0027] The Bifidobacterium longum subspecies Slp99 provided by this invention can significantly shorten the sleep latency and prolong the sleep time in insomnia model mice, while improving anxiety behaviors associated with insomnia. Its effect is superior to other Bifidobacterium longum subspecies strains, demonstrating excellent sleep regulation potential.

[0028] The bilayer microsphere formulation of Bifidobacterium longum subsp. longum provided by the present invention uses dietary bacterial cellulose and microbial fructan as the inner wall material, and uses epigallocatechin gallate (EGCG) and calcium salt to construct the outer polyphenol-calcium coordination protective layer.

[0029] On the one hand, bacterial cellulose nanofibers possess a high aspect ratio, a three-dimensional entangled network, and water-holding capacity; fructan has excellent film-forming, adhesive, and moisture-regulating properties, capable of filling the fiber gaps. Together, they form an inner microsphere core with functions of fixation, water retention, and rehydration release, thereby reducing bacterial cell damage during freeze-drying, improving encapsulation efficiency, and providing a matrix for bacterial rehydration release. On the other hand, unlike traditional calcium alginate gel crosslinking systems, this invention uses ortho-phenolic hydroxyl groups of EGCG to form a reversible coordination network with calcium ions. This dense network adheres to the surface of the inner microsphere core through hydrogen bonding and hydrophobic interactions, forming an outer protective structure different from conventional protein membranes or alginate gels. The resulting coordination structure reduces the rate at which oxygen and water enter the microspheres, while remaining stable in acidic gastric juice and gradually loosening in the gastrointestinal environment, thus balancing storage stability and intestinal release performance.

[0030] This invention combines the functional bacterial strain Slp99 with a double-layer encapsulation system. Slp99 serves as the core active strain for improving sleep quality, while the double-layer microspheres act as a delivery method to increase the effective bacterial count reaching the intestines. Experiments show that the encapsulation rate of the double-layer microsphere formulation of this invention can reach over 95%, and the number of viable bacteria after simulated gastrointestinal digestion is significantly higher than that of unencapsulated microspheres and control microsphere formulations, effectively protecting Slp99's ability to pass smoothly through the gastrointestinal environment. In particular, animal experiments show that the double-layer microsphere formulation of this invention can significantly prolong sleep duration and significantly shorten sleep latency in sleep model mice, and its effect in alleviating anxiety-like behaviors caused by insomnia is close to that of the normal control group. Simultaneously, it effectively reduces hypothalamic neuronal damage, and its overall effect is superior to unencapsulated bacterial suspensions and other single- or double-layer encapsulated microsphere formulations.

[0031] Furthermore, the preparation method of the bilayer microsphere formulation of the present invention is process-controllable and suitable for industrialization. The use of droplet preparation combined with liquid nitrogen freezing and vacuum freeze-drying technology enables the homogeneous preparation of microspheres; the outer coating employs coordination deposition, which is simple to operate under mild conditions and avoids damage to bacterial activity caused by organic solvents and high-temperature treatment.

[0032] Therefore, the Bifidobacterium longum subsp. Slp99 and its bilayer microsphere formulation provided by this invention can provide new strain evidence for the application of Bifidobacterium longum subsp. Slp99 in sleep improvement products, and can also provide a technical basis for the development of safe and reliable sleep aid products. Attached Figure Description

[0033] Figure 1 These are the test results of sleep latency and sleep duration in mice of different Bifidobacterium longum subspecies intervention groups in this invention; where A is sleep duration and B is sleep latency.

[0034] Figure 2 The figures represent the total movement distance and central zone dwell time of mice in different Bifidobacterium longum subspecies intervention groups in the open field experiment and the number of times they entered the open arm in the elevated cross maze test; where A is the total movement distance, B is the central zone dwell time, and C is the number of times they entered.

[0035] Figure 3 The results show the test results of sleep latency and sleep duration of mice in the bacterial suspension, Example 3, and Comparative Examples 1-4 of this invention; where A is sleep duration and B is sleep latency.

[0036] Figure 4 The values ​​represent the total movement distance and central zone dwell time of the mice in the intervention groups of the bacterial suspension, Example 3, and Comparative Examples 1-4 in this invention, as well as the number of times they entered the open arm in the elevated cross maze test; where A is the total movement distance, B is the central zone dwell time, and C is the number of times they entered.

[0037] Figure 5 The images show the morphological sections of the bacterial suspension, Example 3, and the intervention group mouse brain tissues of Comparative Examples 1-4 in this invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] The biological preservation information of this invention is as follows:

[0040] Bifidobacterium longum subsp. longum, Latin taxonomic name, is deposited at Guangdong Provincial Center for Microbial Culture Collection on July 27, 2026, with accession number GDMCC No: 68726, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0041] Unless otherwise specified, all raw materials and equipment used in this invention are commercially available products in the art. Bacterial cellulose was purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd. Fructose (levan) was purchased from Sigma-Aldrich, purity ≥98%, catalog number L8647. Epigallocatechin gallate (EGCG) was purchased from Dalian Meilun Biotechnology Co., Ltd. Calcium lactate was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Artificial saliva, artificial gastric juice, and artificial intestinal juice were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The microinjection pump, model LSP01-1A, was from Baoding Lange Constant Flow Pump Co., Ltd.

[0042] In this invention, the MRS liquid culture medium is a common culture medium in the art and can be prepared by referring to the following basic formula: Take 12g peptone, 6g yeast extract, 6g beef extract, 18g glucose, 5g sodium acetate, 2.15g ammonium citrate, 1g Tween 80, 0.58g magnesium sulfate, 0.05g manganese sulfate, 2g dipotassium hydrogen phosphate, and 0.5g L-cysteine ​​hydrochloride, and make up to 1L with pure water. Adjust the pH to 6.5, and sterilize in an autoclave at 121℃ for 15min to obtain the MRS liquid culture medium. When preparing the MRS solid culture medium, add 15g agar to the above liquid culture medium raw materials, mix well, and keep other steps unchanged. After solidification, the MRS solid culture medium is obtained.

[0043] In this invention, the experimental data were tested using one-way ANOVA to examine differences among three or more groups. A p-value < 0.05 was used to determine whether a difference was statistically significant, and different lowercase letters were used to represent significant differences between different groups.

[0044] Example 1: Screening and identification of Bifidobacterium longum subsp. Slp99

[0045] This embodiment provides a strain of Bifidobacterium longum subsp. longum, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 27, 2026, with accession number GDMCC No: 68726.

[0046] The above-mentioned strain was isolated from a fecal sample of a healthy child in Harbin, Heilongjiang Province. The fecal sample was serially diluted and spread onto MRS solid medium, and cultured anaerobically at 37°C for 48 hours. After culture, typical colonies that were milky white, round, with neat edges and moist surfaces were selected for repeated streak purification. The purified strain was Gram-positive and non-spore-forming upon Gram staining. Finally, 16S rRNA gene sequencing confirmed that the strain belonged to *Bifidobacterium longum* subsp. *Longum*, and was named *Bifidobacterium longum* subsp. *Longum* Slp99.

[0047] The nucleotide sequence of the identified *Bifidobacterium longum* subspecies Slp99 is as shown in SEQ ID No. 1:

[0048]

[0049] Example 2: Functional verification of Bifidobacterium longum subsp. Slp99

[0050] In this embodiment, Bifidobacterium longum subsp. Slp99 and nine other strains of Bifidobacterium longum (EASY12, EASY17, EASY19, EASY21, EASY27, EASY38, EASY42, EASY46, and EASY49) isolated from other samples were used as candidate strains to investigate the application potential of different strains in improving sleep in mice.

[0051] (1) Candidate strains and establishment of insomnia model

[0052] The activation culture method for *Bifidobacterium longum* subsp. *slp99* was as follows: *Bifidobacterium longum* subsp. *slp99*, stored in a -80℃ glycerol storage tube, was rapidly thawed and inoculated into MRS liquid medium at a 2% v / v inoculum. The medium was then anaerobically cultured at 37℃ for 18 h. After two consecutive subcultures, the logarithmic growth phase bacterial culture was centrifuged at 4000×g and 4℃ for 10 min. The supernatant was discarded, and the culture was washed twice with sterile phosphate buffer to obtain Slp99 bacterial sludge. The Slp99 bacterial sludge was resuspended in sterile deoxygenated water, and the viable cell concentration was adjusted to 1.0×10⁻⁶. 8 CFU / mL was used to obtain a bacterial suspension of Bifidobacterium longum subsp. Slp99 for later use.

[0053] The other nine strains of *Bifidobacterium longum* subsp. *longum* (EASY12, EASY17, EASY19, EASY21, EASY27, EASY38, EASY42, EASY46, and EASY49) were isolated from fecal samples of different healthy children in Harbin, Heilongjiang Province. The isolation and identification processes were basically the same as for *Bifidobacterium longum* subsp. *longum* Slp99, and will not be described in detail here. Furthermore, the activation culture methods and bacterial suspension concentrations of the nine *Bifidobacterium longum* subsp. *longum* strains were the same as those for *Bifidobacterium longum* subsp. *longum* Slp99.

[0054] The insomnia model was established as follows: SPF-grade C57 male mice aged 6-8 weeks and weighing 18-22g were selected and kept in an environment with a temperature of 20-23℃, humidity of 54-56%, and a 12h / 12h light / dark cycle, with free access to food and water. After 7 days of acclimatization, they were randomly divided into four groups: control group (NC), model group (MC), *Bifidobacterium longum* subsp. *longum* Slp99 group, *Bifidobacterium longum* subsp. *longum* EASY12 group, *Bifidobacterium longum* subsp. *longum* EASY17 group, *Bifidobacterium longum* subsp. *longum* EASY19 group, *Bifidobacterium longum* subsp. *longum* EASY21 group, *Bifidobacterium longum* subsp. *longum* EASY27 group, *Bifidobacterium longum* subsp. *longum* EASY38 group, *Bifidobacterium longum* subsp. *longum* EASY42 group, *Bifidobacterium longum* subsp. *longum* EASY46 group, and *Bifidobacterium longum* subsp. *longum* EASY49 group, with 9 mice in each group. Except for the NC group, all other groups were injected intraperitoneally with p-chlorophenylalanine (PCPA) at a dose of 300 mg / kg for 3 consecutive days to establish an insomnia mouse model. Starting from day 4, gavage intervention was initiated, with mice in each strain intervention group receiving 1×10 mg / kg of PCPA daily via gavage. 8 CFU-corresponding Bifidobacterium longum subsp. longum bacterial suspension was administered to the NC and MC groups via gavage with an equal volume of sterile saline for 21 days.

[0055] (2) Sodium pentobarbital-induced sleep experiment

[0056] Thirty minutes after the last intervention, mice were injected intraperitoneally with sodium pentobarbital (45 mg / kg) to induce sleep. Sleep was defined as ending when the righting reflex returned within 1 minute. The time of loss and recovery of the righting reflex was recorded for each mouse, i.e., sleep latency and sleep duration. Sleep latency is the time from the injection of sodium pentobarbital to the loss of the righting reflex; sleep duration is the time from the loss of the righting reflex to its recovery. Figure 1 The results show the sleep latency and sleep duration of mice in different intervention groups of Bifidobacterium longum subsp. longum; where A is the sleep duration and B is the sleep latency.

[0057] like Figure 1 As shown in Figures A and B, the sleep duration of mice in the MC group was significantly shorter than that in the NC group, while the sleep latency was significantly longer (P < 0.05), indicating that sodium pentobarbital induced the typical characteristics of the insomnia model, and the insomnia model was successfully established. Compared with the MC group, the sleep duration of each intervention group of *Bifidobacterium longum* subsp. *longum* was significantly prolonged, and the sleep latency was significantly shortened (P < 0.05). Among them, *Bifidobacterium longum* subsp. *longum* Slp99 showed the most significant effect, with a significantly longer sleep duration and a significantly shorter sleep latency compared to the other nine control strains of *Bifidobacterium longum* subsp. *longum* (P < 0.05).

[0058] (3) Open field experiment and elevated cross maze test

[0059] Open field test: The open field test was conducted the day after the sleep latency test. The test used a cubic test chamber with a base area of ​​50cm × 50cm and a wall height of 40cm, with the floor divided into a central area and a peripheral area. Mice were placed alone in the center of the chamber, and their spontaneous activities over 5 minutes were recorded, including total movement distance and time spent in the central area, to reflect motor ability and anxiety-like behavior.

[0060] Elevated Cross Maze Test: In the elevated cross maze test, the frequency and duration of mice entering the open arms reflect their anxiety levels. Two hours after completing the open field experiment, the apparatus was thoroughly cleaned with 75% ethanol to remove odor interference. The elevated cross maze test was then conducted. The elevated cross maze consisted of two open arms (30cm × 5cm) and two closed arms (30cm × 5cm), with the entire apparatus 50cm above the ground. During the experiment, mice were placed on the central platform with their heads facing the open arms and allowed to explore freely for 5 minutes. The number of times a mouse entered an open arm was recorded using an infrared video tracking system.

[0061] Figure 2 The total movement distance and central zone dwell time of mice in different intervention groups of Bifidobacterium longum subsp. longum in the open field experiment, as well as the number of times they entered the open arm in the elevated cross maze test; where A is the total movement distance, B is the central zone dwell time, and C is the number of times they entered.

[0062] like Figure 2 As shown in Figures A and B, in the open field experiment, the activity trajectories of the NC group mice were widely distributed in the open field, with relatively dense movement paths in the central area, demonstrating normal levels of spontaneous activity and exploratory motivation. In contrast, the MC group mice's trajectories clearly tended to move in the peripheral areas of the open field, with significantly sparse path distribution in the central area, and the time spent in the central area was significantly reduced compared to the NC group (P < 0.05), consistent with anxiety-like behaviors commonly seen in insomnia models, indicating that their spontaneous activity and spatial exploration range were significantly limited. Furthermore, the activity trajectory characteristics of the *Bifidobacterium longum* subsp. *slp99* group contrasted with other intervention groups, showing increased trajectory density and coverage throughout the open field. The total movement distance was significantly increased compared to other *Bifidobacterium longum* subsp. *slp99* intervention groups, and the time spent in the central area was also significantly increased (P < 0.05), similar to the distribution pattern of the NC group.

[0063] like Figure 2As shown in Figure C, in the elevated cross maze test, the NC group mice exhibited clear exploration trajectories in both the open and closed arms, reflecting their normal exploratory activity and low anxiety levels. Compared with the NC group, the MC group mice entered the open arms significantly less frequently (P < 0.05), and their movement trajectories were significantly concentrated in the closed arms, with very few movement paths in the open arms, indicating a significantly enhanced avoidance behavior towards novel open environments and high anxiety-like behavioral characteristics. Compared with the MC group, the number of times the mice in the Bifidobacterium longum subsp. longum intervention group entered the open arms significantly increased (P < 0.05), with Bifidobacterium longum subsp. longum Slp99 showing the best effect.

[0064] The above results indicate that, compared to other control strains of *Bifidobacterium longum* subsp. *longum*, *Bifidobacterium longum* Slp99 effectively improves sleep quality and anxiety-like behavior in insomnia model mice, demonstrating the best application potential. Therefore, the following section uses *Bifidobacterium longum* subsp. *longum* Slp99 to construct an encapsulation system.

[0065] Example 3: Preparation of a bilayer microsphere formulation encapsulating Bifidobacterium longum subsp. Slp99

[0066] This embodiment provides a bilayer microsphere formulation encapsulating Bifidobacterium longum subsp. Slp99, which consists of a bacterial cellulose-fructan composite core + EGCG-Ca 2+ Bilayer freeze-dried microspheres with a coordinating outer layer. The specific preparation method includes the following steps:

[0067] (1) Material preparation

[0068] Weigh 1.00 g of bacterial cellulose and 0.80 g of fructan (levan), add sterile deoxygenated water to a final volume of 100 mL, and homogenize at room temperature (25±5℃) and a rotation speed of 10000 r / min for 8 min to ensure uniform dispersion of bacterial cellulose and fructan, thus obtaining a bacterial cellulose-fructan dispersion. The mass concentration (w / v) of cellulose was 1%, and the mass concentration of fructan was 0.8%.

[0069] Alternatively, take 0.50 g of epigallocatechin gallate (EGCG), add sterile deoxygenated water to a final volume of 100 mL, and stir at 4°C in the dark until completely dissolved to obtain an EGCG coating solution with a mass concentration of 0.50%.

[0070] Take 0.30g of calcium lactate, add sterile deoxygenated water to a final volume of 100mL, and stir at room temperature until completely dissolved to obtain a calcium lactate complex solution with a mass concentration of 0.30%.

[0071] (2) Preparation of freeze-dried microspheres

[0072] The bacterial suspension of *Bifidobacterium longum* subsp. Slp99 prepared in Example 2 (1×10⁻⁶) was used. 8 The bacterial cellulose-fructose dispersion (CFU / mL) was mixed with the bacterial cellulose-fructose dispersion at a volume ratio of 1:9 and gently stirred for 10 min at 4℃ and 100 r / min to uniformly disperse the Slp99 cells in the bacterial cellulose-fructose dispersion, thus obtaining a bacterial dispersion.

[0073] The prepared bacterial dispersion was drawn into a sterile syringe, which was then attached to a microinjection pump. The injection rate was set to 0.6 mL / min, and the dispersion was extruded through an outlet channel with an inner diameter of 0.3 mm, forming uniform microdroplets. Maintaining a vertical distance of 8 cm between the end of the outlet channel and the liquid nitrogen surface below, the extruded microdroplets were allowed to fall directly into the liquid nitrogen under gravity for instantaneous freezing and shaping, resulting in molded microspheres encapsulating Slp99.

[0074] Finally, the shaped microspheres were collected and spread in a freeze-drying tray with a thickness of 6±2 mm. They were pre-frozen at -40℃ for 2 hours. Then, the pre-frozen shaped microspheres were placed in a vacuum freeze dryer for vacuum freeze-drying. The cold trap temperature was set at -55℃, the vacuum degree was 20 Pa, and the freeze-drying time was 24 hours to obtain freeze-dried microsphere cores embedded with Slp99 (i.e., bacterial cellulose-fructan composite cores).

[0075] (3) Preparation of bilayer microsphere formulation

[0076] The lyophilized microspheres prepared in step (2) were placed in a sterile coating device. At 4°C, based on 100g of lyophilized microspheres, 5.00g of the EGCG coating solution prepared in step (1) was sprayed evenly to fully wet and adsorb the EGCG onto the surface of the microspheres. After stirring at a low speed of 20r / min for 5min, 5.00g of the calcium lactate coordination solution prepared in step (1) was sprayed evenly, and the mixture was stirred at the same speed for 10min to allow the polyphenolic hydroxyl groups in the EGCG molecules to react with the Ca released from the calcium lactate. 2+ A coordination reaction occurs, forming EGCG-Ca on the surface of the microsphere core. 2+ Coordination outer layer. The single addition volume of EGCG coating solution and calcium lactate coordination solution was the same, both being 5.00% of the mass of the lyophilized microsphere core.

[0077] After completing the above operations, repeat the operation once in the complete sequence of "spraying in EGCG coating solution - stirring for 5 minutes - spraying in calcium lactate complex solution - stirring for 10 minutes", that is, perform a total of 2 complete coating cycles, thereby obtaining double-layer wet microspheres.

[0078] The obtained bilayer wet microspheres were spread in a freeze-drying tray to a thickness of 6±2 mm and pre-frozen at -40℃ for 2 hours. Subsequently, they were placed in a vacuum freeze dryer for vacuum freeze-drying. The vacuum freeze-drying process conditions were as follows: primary drying temperature -35℃, vacuum degree 20 Pa, drying time 12 hours; secondary drying temperature 20℃, vacuum degree 10 Pa, drying time 12 hours, for a total time of 24 hours. After freeze-drying, the microspheres were collected under aseptic drying conditions and sealed for storage, thus obtaining the bilayer microsphere formulation of *Bifidobacterium longum* subsp. *slp99* encapsulated in this embodiment.

[0079] Comparative Example 1

[0080] This comparative example provides a monolayer microsphere formulation encapsulating Bifidobacterium longum subsp. Slp99, which is a monolayer EGCG-Ca 2+ Coordinated freeze-dried microspheres, without bacterial cellulose-fructan composite core.

[0081] The preparation method of the comparative monolayer microsphere formulation includes the following steps:

[0082] EGCG coating solution and calcium lactate complex solution were prepared according to step (1) of Example 3. Then, the bacterial suspension of *Bifidobacterium longum* subsp. *Slp99* prepared in Example 2 (1×10⁻⁶) was added. 8 The bacterial EGCG mixture was mixed with the EGCG coating solution at a volume ratio of 1:9 and stirred at 4℃ and 100r / min for 10min to obtain the bacterial EGCG mixture.

[0083] The prepared bacterial EGCG mixture was drawn into a sterile syringe, which was then attached to a microinjection pump. The injection speed was set to 0.6 mL / min, and the bacterial EGCG mixture was extruded through an outlet channel with an inner diameter of 0.3 mm to form uniform microdroplets.

[0084] Maintain a vertical distance of 8 cm between the end of the outlet channel and the surface of the calcium lactate complex solution below, allowing the extruded microdroplets to drip directly into the calcium lactate complex solution; the volume ratio of the bacterial EGCG mixture to the calcium lactate complex solution is 1:10. After the microdroplets enter the calcium lactate complex solution, the polyphenolic hydroxyl groups in the EGCG molecules react with Ca... 2+ Coordination occurs, causing EGCG-Ca to form on the droplet surface. 2+ Coordination protection layer.

[0085] After the above steps are completed, the formed microspheres are further immersed in calcium lactate coordination solution and cured at 4℃ and 100 r / min for 20 min. After curing, the microspheres are collected using a sterile filter and washed three times with sterile physiological saline to remove uncoordinated EGCG and calcium lactate from the surface of the microspheres, obtaining a monolayer of EGCG-Ca 2+ Wet microspheres.

[0086] The obtained monolayer EGCG-Ca 2+ The wet microspheres were evenly spread in a freeze-drying tray and pre-frozen and vacuum freeze-dried according to step (3) of Example 1. After freeze-drying, the microspheres were collected under aseptic drying conditions and sealed for storage to obtain the monolayer microsphere formulation of Bifidobacterium longum subsp. longum Slp99 embedded in the comparative example. The other unspecified operating conditions were the same as in Example 1.

[0087] Comparative Example 2

[0088] This comparative example provides a monolayer microsphere formulation encapsulating *Bifidobacterium longum* subsp. *slp99*, which is a lyophilized microsphere with a monolayer bacterial cellulose-fructan composite core, and does not contain EGCG-Ca. 2+ Coordination outer layer.

[0089] The preparation method of the comparative monolayer microsphere formulation is similar to that of Example 3. The specific steps are as follows: prepare bacterial cellulose-fructose dispersion according to step (1) of Example 3; prepare lyophilized microsphere cores encapsulated with Slp99 according to step (2) of Example 3. Step (3) is adjusted to: directly spread the lyophilized microsphere cores encapsulated with Slp99 in a lyophilization tray, and directly perform pre-freezing and vacuum freeze-drying according to step (3) of Example 3. After freeze-drying, collect the microspheres under aseptic drying conditions and seal them for storage to obtain the comparative monolayer microsphere formulation encapsulated with Bifidobacterium longum subsp. longum Slp99.

[0090] Comparative Example 3

[0091] This comparative example provides a bilayer microsphere formulation encapsulating Bifidobacterium longum subsp. Slp99, which consists of a single fructan core and EGCG-Ca. 2+ Double-layer freeze-dried microspheres with a coordinated outer layer.

[0092] The preparation method of the comparative bilayer microsphere formulation is similar to that of Example 3, except that bacterial cellulose is omitted, and fructan dispersion is used instead of bacterial cellulose-fructan dispersion for the preparation of the bilayer microsphere formulation. The preparation method of fructan dispersion is as follows: 1.80 g of fructan (levan) is weighed and added to sterile deoxygenated water to a final volume of 100 mL. The mixture is then homogenized at high speed for 8 min at room temperature (25±5℃) to ensure uniform dispersion of the fructan, thus obtaining the fructan dispersion. All other raw material compositions and process parameters are the same as in Example 3.

[0093] Comparative Example 4

[0094] This comparative example provides a bilayer microsphere formulation encapsulating Bifidobacterium longum subsp. Slp99, which consists of a bacterial cellulose-fructan composite core + sodium alginate-Ca 2+ Double-layer freeze-dried microspheres with a coordinated outer layer.

[0095] The preparation method of the comparative bilayer microsphere formulation is similar to that of Example 3. The specific steps are as follows: freeze-dried microsphere cores encapsulated with Slp99 are prepared according to steps (1) and (2) of Example 3. Step (3) is adjusted as follows: the freeze-dried microsphere cores encapsulated with Slp99 are added to a sodium alginate solution with a mass concentration of 1.5% and stirred at 100 r / min for 10 min at room temperature to allow sodium alginate to be uniformly adsorbed on the surface of the microspheres; then the microspheres adsorbed with sodium alginate are slowly added to a calcium chloride solution with a mass concentration of 2.0% and crosslinked at 100 r / min for 20 min at room temperature to allow the carboxyl groups in sodium alginate to react with the calcium chloride. 2+ Ionic crosslinking occurs, forming an outer layer of calcium alginate gel. After crosslinking, the microspheres are washed three times with sterile saline to remove uncrosslinked sodium alginate and residual calcium chloride, resulting in bilayer wet microspheres. The obtained bilayer wet microspheres are spread in a freeze-drying pan and pre-frozen and then freeze-dried under vacuum according to step (3) of Example 1. After freeze-drying, the microspheres are collected under sterile drying conditions and sealed for storage, thus obtaining the bilayer freeze-dried microsphere formulation of this comparative example.

[0096] Example 4: Encapsulation rate determination

[0097] 1.00 g of each of the microsphere formulations prepared in Example 3 and Comparative Examples 1-4 were added to sterile phosphate buffer, and subjected to thorough rehydration and mechanical dispersion to fully release the bacteria. After serial dilution, the microspheres were spread onto MRS solid medium and incubated at 37°C under anaerobic conditions for 48 h. The viable cell count was then determined. The encapsulation efficiency was calculated as the percentage of actual viable cells within the microspheres relative to the total number of viable cells added. The encapsulation efficiency results for different formulations are shown in Table 1. Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0098] Table 1. Encapsulation efficiency test results for different formulations

[0099] As shown in Table 1, the encapsulation rate of the double-layer freeze-dried microspheres in Example 3 of this invention was 95.58 ± 0.55%, significantly higher than that of the comparative examples (P < 0.05). This indicates that the bacterial cellulose-fructan composite core constructed in this invention is compatible with EGCG-Ca 2+ The outer coordination layer effectively enhances the encapsulation effect of Bifidobacterium longum subsp. Slp99. This indicates that the inner bacterial fiber-fructan network facilitates the fixation of bacteria within the microspheres during the instantaneous shaping of droplets; simultaneously, the outer EGCG-calcium coordination layer forms a continuous protective structure on the surface of the inner microsphere core, reducing bacterial escape during transfer, freeze-drying, and rehydration.

[0100] Example 5: Simulated gastrointestinal digestion survival rate determination

[0101] This example examines the in vitro simulated gastrointestinal survival rate of the bilayer microsphere formulation of Example 3 and the microsphere formulations of Comparative Examples 1-4. A non-encapsulated bacterial suspension control group (Slp99 bacterial suspension, 1×10⁻⁶) was also included. 8 CFU / mL). The experimental procedure was as follows: 2g of the microsphere formulations from Example 3 and Comparative Examples 1-4 and 2mL of Slp99 bacterial suspension (1×10⁻⁶ CFU / mL) were respectively added. 8 Microspheres (CFU / mL) were inoculated into a shake flask containing 20 mL of artificial saliva and incubated at 37°C for 2 min, followed by centrifugation at 1000 rpm for 2 min to obtain saliva-digested microspheres and bacterial suspensions. The saliva-digested microspheres or bacterial suspensions were then added to a shake flask containing 20 mL of artificial gastric fluid and treated on a shaker (37°C, 3 rpm) for 2 h, followed by centrifugation at 1000 rpm for 2 min to obtain gastric fluid-digested microspheres and bacterial suspensions. These gastric fluid-digested microspheres and bacterial suspensions were then added to a shake flask containing 20 mL of artificial intestinal fluid and treated on a shaker (37°C, 2 rpm) for 3 h, followed by centrifugation at 1000 rpm for 2 min to harvest the microspheres and bacterial suspensions. At the end of each digestion step, the viability of the strains was determined by viable cell counting, and the results are shown in Table 2. Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0102] Table 2. Survival results of live bacteria after simulated gastrointestinal digestion of different formulations (Log CFU / mL)

[0103] As shown in Table 2, the viable bacterial count of the unencapsulated Slp99 bacterial suspension decreased sharply after simulated gastrointestinal digestion, with only 2.48 Log CFU / mL in the intestinal fluid. Meanwhile, all encapsulation formulations protected the bacteria from passing through the gastrointestinal environment to varying degrees. Among them, the bilayer microsphere formulation of Example 3 of this invention showed significantly higher viable bacterial counts in saliva, gastric juice, and intestinal juice than Comparative Examples 1-4 (P < 0.05), indicating that the synergistic effect of the bacterial cellulose-fructan inner layer and the EGCG-calcium coordination protective outer layer effectively improves the survival rate of Slp99 during gastrointestinal digestion.

[0104] Example 6: Animal experiments on improving sleep quality

[0105] (1) Animal grouping and establishment of insomnia model

[0106] SPF-grade male C57 mice, aged 6-8 weeks and weighing 18-22g, were selected and kept in an environment with a temperature of 20-23℃, humidity of 54-56%, and a 12h / 12h light-dark cycle, with free access to food and water. After 7 days of acclimatization, they were randomly divided into four groups: control group (NC), model group (MC), Slp99 bacterial suspension group, comparative example 1, comparative example 2, comparative example 3, comparative example 4, and example 3 group, with 9 mice in each group. Except for the NC group, all mice were intraperitoneally injected with p-chlorophenylalanine (PCPA) at a dose of 300mg / kg for 3 consecutive days to establish an insomnia mouse model. Starting from day 4, gavage intervention was initiated, with mice in each bacterial strain intervention group being administered either the bacterial suspension or the microsphere formulation of example 3 and comparative examples 1-4 daily. In the Slp99 bacterial suspension group, the daily gavage dose per mouse was 1×10⁻⁶. 8 CFU. In Comparative Examples 1-4 and Example 3, the viable count was 1×10⁻⁶. 8 The CFU dosage was converted to the corresponding microsphere formulation and dissolved in an equal volume of physiological saline for gavage. The NC group and MC group were administered an equal volume of physiological saline by gavage for 21 days.

[0107] (2) Sodium pentobarbital-induced sleep experiment

[0108] After the last intervention, the pentobarbital sodium-induced sleep experiment was performed using the same method as in Example 2, and the sleep latency and sleep duration of mice in each group were recorded. Figure 3 The results show the sleep latency and sleep duration of mice in the bacterial suspension, Example 3, and Comparative Examples 1-4; where A represents sleep duration and B represents sleep latency.

[0109] Depend on Figure 3 As shown in Figures A and B, the sleep duration of mice in the MC group was significantly shorter than that in the NC group, while the sleep latency was significantly longer (P < 0.05), indicating that the insomnia model was successfully established. Compared with the MC group, the sleep duration of all intervention groups was significantly prolonged, and the sleep latency was significantly shortened (P < 0.05). Among them, compared with the microsphere formulation groups of Comparative Examples 1-4, the effect of Group 3 of this invention was the best, close to the level of the NC group.

[0110] (3) Open field experiment and elevated cross maze test

[0111] The open field experiment was conducted using the same method as in Example 2, and the spontaneous activities of the mice over 5 minutes were recorded, including the total distance traveled and the time spent in the central area. Additionally, the elevated cruciate maze test was conducted using the same method as in Example 2, and the number of times the mice entered the open arm was recorded using an infrared video tracking system. Figure 4The total distance traveled and the time spent in the central area of ​​the mice in the intervention groups of bacterial suspension, Example 3, and Comparative Examples 1-4, as well as the number of times they entered the open arm in the elevated cross maze test; where A is the total distance traveled, B is the time spent in the central area, and C is the number of times they entered.

[0112] like Figure 4 As shown in Figures A and B, in the open field experiment, the trajectories of mice in the MC group clearly tended towards the periphery of the open field, with significantly sparser paths in the central area. The total movement distance and time spent in the central area were significantly reduced compared to the MC group (P < 0.05). The activity trajectory density of each intervention group increased and spread throughout the open field, similar to the distribution pattern of the NC group, with the best results observed in Group 3 of Example 1.

[0113] like Figure 4 As shown in Figure C, in the elevated cross maze test, the activity trajectory of mice in the MC group was significantly concentrated in the closed arms, and the number of times they entered the open arms was significantly reduced compared to the NC group (P < 0.05). The number of times mice in each intervention group entered the open arms was significantly increased compared to the MC group (P < 0.05). Among them, the number of times mice in Example 3 entered the open arms was significantly increased compared to the comparative examples 1-4, showing the most outstanding performance.

[0114] (4) Pathological tissue section analysis of mouse brain tissue

[0115] Whole brain tissue from each group of mice was collected and placed in EP tubes, and fixed with sufficient 4% paraformaldehyde. Then, the tissue was dehydrated, trimmed, embedded, sectioned, stained with hematoxylin and eosin (HE), and mounted. Finally, the sections of the mouse brain tissue were examined under a microscope. Figure 5 Morphological images of pathological tissue sections of bacterial suspension, brain tissue of mice in the intervention groups of Example 3 and Comparative Examples 1-4.

[0116] Depend on Figure 5 It was found that the hypothalamus structure in the NC group was intact, with no obvious pathological changes. Neuronal morphology within the visual field was normal, without degeneration, edema, or inflammatory cell infiltration. The hypothalamus structure in the MC group showed moderate damage, with numerous neuronal degenerations visible in the visual field, characterized by deeply stained cell bodies, blurred structures, and a certain degree of structural disorder. In the intervention groups, the hypothalamus structure in Example 3 group remained largely intact, with only a few neuronal degenerations observed, and no edema or inflammatory cell infiltration. The degree of pathological changes in the hypothalamus in the other comparative intervention groups (Examples 1-4) varied; some groups showed more neuronal degeneration, while others showed only a few degenerated neurons, but none showed significant edema or inflammatory cell infiltration. Overall, the improvement effect of Example 3 group was superior to the other comparative intervention groups.

[0117] The above results collectively demonstrate that the bilayer microsphere formulation of the present invention can effectively protect Slp99 from passing through the gastrointestinal environment and exert its effects in vivo in improving sleep quality, reducing anxiety-like behaviors, and protecting against hypothalamic tissue damage. The effects are significantly better than those of unencapsulated bacterial suspensions and other single-layer or bilayer microsphere formulations.

[0118] In summary, the Slp99 strain screened in this invention can effectively shorten the sleep latency, prolong sleep duration, alleviate anxiety-related behaviors associated with insomnia, and possess activity to improve sleep quality. The bilayer microsphere formulation further developed in this invention can significantly improve the strain encapsulation rate and gastrointestinal fluid tolerance, while significantly enhancing the sleep-improving effect, providing a technical foundation for the development of safe and reliable sleep aid products.

[0119] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of *Bifidobacterium longum* subsp., characterized in that, The specimen, named *Bifidobacterium longum subsp. longum* Slp99, is deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 27, 2026, with accession number GDMCC No: 68726.

2. A bilayer microsphere formulation encapsulating *Bifidobacterium longum* subsp. as described in claim 1, characterized in that, The bilayer microsphere formulation includes an inner microsphere core encapsulating Bifidobacterium longum subsp. longum and a polyphenol-calcium coordination protective layer coating the surface of the inner microsphere core. The inner microsphere core is mainly formed by encapsulating Bifidobacterium longum subsp. longum with bacterial cellulose and fructan; the polyphenol-calcium coordination protective layer is mainly formed by the coordination of epigallocatechin gallate and calcium salt.

3. The bilayer microsphere formulation encapsulating *Bifidobacterium longum* subsp. *longum* according to claim 2, characterized in that, The calcium salt is calcium lactate.

4. A method for preparing a bilayer microsphere formulation encapsulating *Bifidobacterium longum* subsp. as described in claim 2, characterized in that, Includes the following steps: Prepare a bacterial suspension of the aforementioned *Bifidobacterium longum* subsp. *longum*; separately, uniformly disperse bacterial cellulose and fructan in water to obtain a bacterial cellulose-fructan dispersion; dissolve epigallocatechin gallate in water to obtain a coating solution; dissolve calcium salt in water to obtain a calcium salt complex solution; The bacterial suspension was mixed with a bacterial cellulose-fructose dispersion to obtain a bacterial dispersion; the bacterial dispersion was prepared into microdroplets, which were then frozen and freeze-dried to obtain an inner microsphere core. The coating solution and the calcium salt coordination solution are sequentially sprayed onto the surface of the inner microsphere core to form a polyphenol-calcium coordination protective layer. Finally, the mixture is freeze-dried to obtain the bilayer microsphere formulation containing Bifidobacterium longum subsp. longum.

5. The method for preparing the bilayer microsphere formulation encapsulating *Bifidobacterium longum* subsp. *longum* according to claim 4, characterized in that, In the bacterial cellulose-fructose dispersion, the mass concentration of bacterial cellulose is 1.0%, the mass concentration of fructose is 0.8%, the mass concentration of the coating solution is 0.5%, and the mass concentration of the calcium salt complex solution is 0.3%.

6. The method for preparing the bilayer microsphere formulation encapsulating *Bifidobacterium longum* subsp. *longum* according to claim 4, characterized in that, The volume ratio of the bacterial suspension to the bacterial cellulose-fructose dispersion is 1:

9.

7. The method for preparing the bilayer microsphere formulation encapsulating *Bifidobacterium longum* subsp. *longum* according to claim 4, characterized in that, The microdroplets were prepared using a micro-injection pump; the micro-injection pump had a propulsion speed of 0.6 mL / min and an inner diameter of 0.3 mm for the outlet channel.

8. The method for preparing the bilayer microsphere formulation encapsulating *Bifidobacterium longum* subsp. *longum* according to claim 4, characterized in that, The freezing and shaping process uses liquid nitrogen for instantaneous freezing and shaping; the single addition volume of the coating solution and the calcium salt coordination solution is 5% of the mass of the microspheres; the spraying is performed twice.

9. The use of a bilayer microsphere formulation as described in claim 2, or a bilayer microsphere formulation prepared by any one of the preparation methods described in claims 4 to 8, in the preparation of a product for improving sleep, characterized in that, The improvement of sleep includes at least one of the following: prolonging sleep time, shortening sleep latency, alleviating anxiety-like behaviors caused by insomnia, and repairing hypothalamic neuronal damage caused by insomnia.

10. The application of the bilayer microsphere formulation according to claim 9 in the preparation of products for improving sleep, characterized in that, The product in question is a health supplement or a medicine.

Images