Preparation method and application of probiotic preparation for targeting cognitive impairment caused by anti-navigation compound stress to microecological homeostasis

Abstract

This invention belongs to the field of biopharmaceutical technology, specifically relating to a method for preparing and applying a probiotic formulation targeting microecological homeostasis to combat cognitive impairment induced by combined maritime stress. This invention involves compounding Trichophyton spp., Butyric Acid Cocci, xylooligosaccharides, maltodextrin, sodium alginate, and low-methoxyl pectin in specific proportions. This significantly and precisely restores Trichophyton spp. homeostasis in the intestines of damaged animals, increasing in-situ butyrate production. These targeted metabolites cross the brain barrier, rapidly increasing the levels of BDNF (a key synaptic molecule) and the neurotransmitter 5-HT (a neurotransmitter) depleted by stress, forming a closed-loop brain-protective ecological mechanism. Furthermore, the probiotic compound formulation provided by this invention exhibits excellent stress resistance, maintaining extremely high activity after one month of intervention at 25°C and 85% RH high humidity.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a method for preparing and applying a probiotic preparation that targets microecological homeostasis to combat cognitive impairment caused by combined maritime stress. Background Technology

[0002] Crew members of large surface ships often face long-range deployments lasting several months. During this time, they are exposed to six-degree-of-freedom (6-DOF) rolling and pitching caused by severe sea conditions, continuous intense noise interference, and disruption of their circadian rhythms. This extreme combined stress environment at sea can lead to a decline in spatial cognitive function, impaired recognition and memory, and emotional anxiety among crew members.

[0003] Currently, drug interventions for motion sickness include anticholinergic drugs such as scopolamine. These drugs can alleviate some motion sickness symptoms, but may be accompanied by dry mouth, blurred vision, and certain degrees of central anticholinergic adverse reactions; in maritime operations requiring sustained alertness and concentration, long-term or repeated use of these drugs has certain limitations. With the introduction of the "microbe-gut-brain axis" concept, research has confirmed that the gut microbiota is one of the key factors regulating gut-axis function. The sympathetic and parasympathetic branches of the autonomic nervous system can affect the enteric nervous system circuits, leading to changes in intestinal motility, thereby affecting the entry of dietary fiber, prebiotics, or other key gut microbiota into the intestinal system and affecting the balance of gut microbiota. However, existing microbial intervention strategies are limited by the high salinity and humidity of the maritime environment, and stress can lead to delayed gastric emptying. Conventionally exposed or monolayer-embedded probiotics are easily deactivated by moisture absorption during storage, and cannot target and repair the loss of specific acid-producing anaerobic bacteria caused by strong neural stress. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying a probiotic preparation that targets microecological homeostasis to combat cognitive impairment caused by combined maritime stress. This probiotic preparation has high storage stability, can target and reshape specific gut microbiota, and achieves protection of brain cognitive function.

[0005] This invention provides a probiotic compound preparation comprising the following components by weight percentage: 20%~40% microbial composition, 30%~50% matrix and 25%~45% coating material; The microbial composition includes Trichophycetes (family Trichophyceae) Lachnospiraceae ) and butyric acid cocci ( Butyricicoccus sp.); the live bacteria ratio of the Trichophyceae family and Butyricococcus is (1~10):1, and not 1:1; The matrix comprises xylooligosaccharides and maltodextrin; The coating material includes sodium alginate and low-methoxyl pectin.

[0006] Preferably, the live bacteria ratio of *Trichophyton spp.* to *Butyricum coccidioides* is 5:1.

[0007] Preferably, the concentration of *Trichophyton spp.* in the microbial composition is 1 × 10⁻⁶. 9 ~5×10 10 CFU / g.

[0008] Preferably, the matrix comprises 100-300 g / L xylooligosaccharides, 100-300 g / L maltodextrin, and the balance being water.

[0009] Preferably, the coating material comprises 100-200 g / L sodium alginate, 100-200 g / L low-methoxyl pectin, and the balance being water.

[0010] Preferably, the probiotic compound preparation has the probiotic composition and matrix as the core and the coating material as the outer shell.

[0011] This invention provides a method for preparing the probiotic compound preparation described above, comprising the following steps: The microbial composition and the matrix are mixed and granulated to obtain a dry powder with a microbial core. The probiotic compound preparation is obtained by coating the bacterial core dry powder with a coating material.

[0012] Preferably, the granulation method includes freeze-drying and then pulverizing a mixture of microbial composition and matrix; the freeze-drying conditions include: freezing at -80°C for 2-4 hours followed by vacuum freeze-drying for 24-36 hours; The coating method includes: spraying the coating material onto the surface of the bacterial core dry powder and then drying it; the atomization pressure of the spraying is 0.15~0.2 MPa, and the air inlet temperature is 35℃~40℃; the drying temperature is 35℃~40℃.

[0013] This invention provides the application of the probiotic compound preparation described in the above technical solution or the probiotic compound preparation obtained by the preparation method described in the above technical solution in the preparation of drugs to improve cognitive impairment.

[0014] Preferably, the cognitive impairment is cognitive impairment caused by combined maritime stress; The aforementioned maritime composite stress includes complex motion stress, continuous noise stress, and photoperiod disorder stress; The parameters of the complex motion stress include pitch ±15°, sway ±25°, heave amplitude 1.5 m and frequency 0.15~0.3 Hz; The noise intensity of the continuous noise stress is 85 dB.

[0015] Beneficial effects: This invention provides a probiotic compound preparation comprising the following components by weight percentage: 20%–40% microbial composition, 30%–50% matrix, and 25%–45% coating material; the microbial composition includes *Trichophyton* and *Butylococcus*; the live bacteria ratio of *Trichophyton* and *Butylococcus* is (1–10):1, and not 1:1; the matrix includes xylooligosaccharides and maltodextrin; the coating material includes sodium alginate and low-methoxyl pectin. The inventors observed in a marine-induced stress animal model that prolonged exposure to repetitive multiaxial motion, noise, and diurnal rhythm disturbances was accompanied by decreased levels of *Trichophyton* and butyrate in the rat gut, along with decreased hippocampal 5-HT and BDNF levels and impaired learning and memory function. This invention combines *Trichophyton* bacteria and *Butylococcus* at a specific live bacteria ratio, and forms a bacterial-rich core with xylooligosaccharides and maltodextrin. A polymer coating layer containing sodium alginate and low-methoxyl pectin is further formed on the surface of this bacterial-rich core. Animal experiments showed that, compared with the combined maritime stress model group, the bacterial intervention group without a complete compound preparation, and the group with a non-preferred strain ratio preparation, the preparation of the present invention could more significantly increase the level of Trichophyton spp. and butyrate content in the rat gut, and improve the levels of 5-HT and BDNF in the hippocampus, as well as the Morris water maze and new object recognition test indicators, indicating that the probiotic compound preparation has the effect of improving cognitive function impairment caused by combined maritime stress. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 OD values ​​of reconstituted samples from each formulation before and after one month of high humidity treatment at 25°C and 85% RH were calculated. 600 value; Figure 2 The number of Trichophyceae in the feces of rats in each treatment group ( Lachnospiraceae Abundance; Figure 3 The butyric acid content in the intestinal contents of rats in each treatment group; Figure 4 The concentration of 5-HT in the hippocampus of rats in each treatment group; Figure 5 The concentration of BDNF in the hippocampus of rats in each treatment group; Figure 6 The results of the Morris water maze test for rats in each treatment group are shown below; where A is the rat trajectory map, Ctrl group is guided by the normal target, AO group is lost and wanders around at the edge, B is the total distance traveled, C is the escape latency, D is the number of times the platform is crossed, and E is the total distance traveled in the target quadrant. Figure 7The results of the novel object recognition ability test for rats in each treatment group are shown. Detailed Implementation

[0018] This invention provides a probiotic compound preparation comprising the following components by weight percentage: 20%~40% microbial composition, 30%~50% matrix, and 25%~45% coating material; the microbial composition comprises Trichophytonceae and Butyricococcus; the live bacteria ratio of Trichophytonceae and Butyricococcus is (1~10):1, and not 1:1; the matrix comprises xylooligosaccharides and maltodextrin; and the coating material comprises sodium alginate and low-methoxyl pectin.

[0019] In one embodiment, the probiotic compound preparation of the present invention comprises 30% a microbial composition, the mass of which is based on the dry weight of the microorganisms. In one embodiment, the live bacteria ratio of *Trichophyton spp.* and *Butyricum coccidioides* in the present invention is 5:1. In one embodiment, the concentration of *Trichophyton spp.* in the microbial composition of the present invention is 1 × 10⁻⁶. 9 ~5×10 10 CFU / g; As another embodiment, the concentration of *Hylocereus undatus* in the microbial composition of the present invention is 5 × 10⁻⁶. 9 ~1×10 10 CFU / g. This invention does not have strict requirements regarding the source of the Trichophyceae family; any Trichophyceae strain conventionally found in the art can be used, preferably commercially available or routinely obtained from a depository. Lachnospiraceae_NK4A136_group The safe strain used in this invention is the *Gnaphalaceae* strain with catalog number HZB640703 (DSM 24430). As one embodiment, the *Butyrica* strain used in this invention is the strain with German DSMZ accession number DSM 23266.

[0020] In one embodiment, the probiotic compound preparation of the present invention comprises 40% to 50% matrix. In one embodiment, the matrix of the present invention comprises 100 to 300 g / L xylooligosaccharides; in another embodiment, the matrix of the present invention comprises 200 g / L xylooligosaccharides. In one embodiment, the matrix of the present invention comprises 100 to 300 g / L maltodextrin; in another embodiment, the matrix of the present invention comprises 200 g / L maltodextrin. In one embodiment, the matrix of the present invention uses water as a solvent.

[0021] In one embodiment, the probiotic compound preparation of the present invention comprises 30% to 40% coating material; in another embodiment, the probiotic compound preparation of the present invention comprises 35% coating material. In one embodiment, the coating material of the present invention comprises 100 to 200 g / L sodium alginate; in another embodiment, the coating material of the present invention comprises 150 g / L sodium alginate. In one embodiment, the coating material of the present invention comprises 100 to 200 g / L low-methoxyl pectin; in another embodiment, the coating material of the present invention comprises 150 g / L low-methoxyl pectin. In one embodiment, the coating material of the present invention uses water as a solvent.

[0022] In one embodiment, the probiotic compound preparation of the present invention uses the probiotic composition and matrix as the core and the coating material as the outer shell. The coating material of the present invention can reduce the direct contact between the bacteria-rich core and the external high-humidity environment, thereby helping to maintain the stability of the preparation under high-humidity conditions.

[0023] The probiotic compound preparation obtained by this invention maintains extremely high activity even when stored in the high humidity environment of seafaring. Furthermore, this invention's research found that only when combined in specific proportions can the ability of affected rats to find targets in a water maze be dramatically restored, and the new object discrimination index show a highly significant rebound.

[0024] This invention provides a method for preparing the probiotic compound preparation described above, comprising the following steps: The microbial composition and the matrix are mixed and granulated to obtain a dry powder with a microbial core. The probiotic compound preparation is obtained by coating the bacterial core dry powder with a coating material.

[0025] In one embodiment, the present invention culturees Trichophytonceae and Butyrica spp. to the end-log phase or stationary phase, collects the bacterial solution, centrifuges it to obtain Trichophytonceae sludge and Butyrica spp., and then mixes them according to the live cell ratio of Trichophytonceae and Butyrica spp. of (1~10):1, and not 1:1, to obtain a microbial composition.

[0026] The present invention mixes the microbial composition with a matrix, granulates it, and obtains a dry powder with a rich microbial core.

[0027] In one embodiment, the granulation method of the present invention includes freeze-drying and then pulverizing a mixture of microbial composition and matrix; the freeze-drying conditions include: freezing at -80℃ for 2-4 hours followed by vacuum freeze-drying for 24-36 hours. Freezing at -80℃ for 2-4 hours followed by vacuum freeze-drying ensures complete solidification. In one embodiment, the cold trap temperature of the vacuum freeze-drying process is ≤-60℃, the vacuum degree is <10 Pa, and the partition heating program uses programmed temperature rise, slowly increasing from -40℃ to 25℃. In one embodiment, the moisture content of the microbial-enriched core powder of the present invention is ≤3%, and it can pass through a 60-80 mesh sieve.

[0028] After obtaining the bacterial-rich core dry powder, the present invention uses a coating material to coat the bacterial-rich core dry powder to obtain the probiotic compound preparation.

[0029] As one embodiment, the coating method of the present invention includes: spraying the coating material onto the surface of the bacterial core dry powder and then drying it; the atomization pressure of the spraying is 0.15~0.2 MPa, and the air inlet temperature is 35℃~40℃; the drying temperature is 35℃~40℃.

[0030] This invention provides the application of the probiotic compound preparation described in the above technical solution or the probiotic compound preparation obtained by the preparation method described in the above technical solution in the preparation of drugs to improve cognitive impairment.

[0031] In one embodiment, the cognitive impairment described in this invention is cognitive impairment caused by combined maritime stress. In another embodiment, the combined maritime stress described in this invention includes complex motion stress, persistent noise stress, and photoperiodic disturbance stress. In another embodiment, the parameters of the complex motion stress described in this invention include pitch ±15°, roll ±25°, heave amplitude 1.5 m, and frequency 0.15~0.3 Hz. In another embodiment, the noise intensity of the persistent noise stress described in this invention is 85 dB.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for preparing a probiotic preparation that targets microecological homeostasis and combats cognitive impairment caused by combined maritime stress, as well as its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Comparative Example 1 1. Inoculate *Trichophyton spp.* and *Butyricum coccidioides* separately into anaerobic liquid culture medium that has been deoxygenated (by purging with a 99.9% pure N2 / CO2 mixture to maintain strict anaerobic conditions). Ferment at a constant temperature of 37±0.5℃ and pH 6.5~6.8 for 18~24h until the bacterial community enters the late logarithmic or stationary phase. 2. Centrifuge at 6000-8000 rpm for 15 minutes using a tubular centrifuge at 4℃. Discard the supernatant, collect the bacterial sludge, and mix it with *Trichophyton spp.* and *Butyricum spp.* at a live bacteria ratio of 5:1 to obtain the core microecological composite bacterial sludge; wherein the live bacteria count of *Trichophyton spp.* is 5 × 10⁻⁶. 6 CFU / g, viable count of butyric acid cocci is 5×10⁻⁶. 6 CFU / g.

[0034] Example 1 1. Preparation of materials: 300g (dry weight) of core microecological composite bacterial mud obtained from Comparative Example 1, 200g xylooligosaccharide, 200g maltodextrin, 150g sodium alginate and 150g low methoxyl pectin.

[0035] 2. In sterile, deoxygenated purified water, add xylooligosaccharides and maltodextrin, stir to dissolve and form a matrix protection solution, and fully homogenize and suspend it with the core microecological composite bacterial mud to obtain a bacterial matrix suspension.

[0036] 3. Pre-freezing: Spread the above bacterial substrate suspension evenly in a freeze-drying tray and place it in an ultra-low temperature freezer at -80℃ or a pre-freezing machine for rapid freezing for 2~4 hours to ensure complete solidification.

[0037] 4. Freeze-drying: Transfer the pre-frozen material into a vacuum freeze dryer. Set the cold trap temperature to ≤-60℃ and the vacuum degree to <10 Pa. The partition heating program uses programmed temperature rise, slowly increasing from -40℃ to 25℃, with a drying time of 24~36 hours. Remove the material from the dryer when the moisture content drops to ≤3%. In a clean room with relative humidity (RH) <30%, pulverize the freeze-dried blocks and pass them through a 60~80 mesh sieve to obtain a dry powder rich in microbial cores.

[0038] 5. Dissolve sodium alginate and low-methoxyl pectin in purified water to prepare a coating solution.

[0039] 6. Add the above-mentioned enriched bacterial kernel dry powder to a bottom-spray fluidized bed (Wurster process), controlling the inlet air temperature at 35℃~40℃ and maintaining the material temperature at 30℃~32℃. Under an atomization pressure of 0.15~0.2 MPa, limit the spraying speed to 3~5 mL / min, spraying the above coating liquid onto the surface of the enriched bacterial kernel dry powder until the coating material completely covers the surface of the enriched bacterial kernel dry powder. After stopping the spraying, continue fluidized drying for 15~20 minutes while maintaining the same inlet air temperature to obtain the probiotic compound preparation. Pack it in aluminum foil bags filled with nitrogen or vacuum sealed for long-term maritime storage.

[0040] Comparative Example 2 1. Inoculate *Trichophyton spp.* and *Butyricum coccidioides* separately into anaerobic liquid culture medium that has been deoxygenated (by purging with a 99.9% pure N2 / CO2 mixture to maintain strict anaerobic conditions). Ferment at a constant temperature of 37±0.5℃ and pH 6.5~6.8 for 18~24h until the bacterial community enters the late logarithmic or stationary phase. 2. Centrifuge at 6000-8000 rpm for 15 minutes using a tubular centrifuge at 4℃. Discard the supernatant, collect the bacterial sludge, and mix it with *Trichophyton spp.* and *Butyricum spp.* at a 1:1 ratio to obtain the core microecological composite bacterial sludge; wherein the viable count of *Trichophyton spp.* is 5 × 10⁻⁶. 6 CFU / g, viable count of butyric acid cocci is 5×10⁻⁶. 6 CFU / g.

[0041] 3. Prepare probiotic compound preparations according to the method of Example 1, the only difference being the use of the core microecological compound bacterial mud obtained in step 1.

[0042] Test Example 1 Probiotic activity and stability testing The core microecological composite bacterial mud obtained in Comparative Example 1 (denoted as Form. 1), the probiotic compound preparation obtained in Comparative Example 2 (denoted as Form. 2), and the probiotic compound preparation obtained in Example 1 (denoted as Form. 3) were subjected to extreme high humidity environment (Env-Intv) intervention: they were placed in a constant temperature and humidity chamber (temperature 25℃, humidity 85% RH) to simulate bulk storage for 1 month. Samples before and after the intervention were reconstituted, and OD was measured. 600 value.

[0043] The results are as follows Figure 1 As shown, the core microecological composite bacterial mud obtained in Comparative Example 1 showed significant inactivation after one month of intervention in an extremely high humidity environment; the survival rate of the core microecological composite bacterial mud obtained in Comparative Example 2 also decreased significantly after one month of intervention in an extremely high humidity environment. The probiotic composite preparation obtained using the preparation method of Example 1 of this invention exhibits better stability under high humidity treatment.

[0044] Test Example 2 1. Experimental animals: 8-week-old SPF-grade male SD rats (n=6 per group).

[0045] 2. Experimental Methods (1) Setting up the modeling environment: The experimental animal cages were fixed on a six-degree-of-freedom (6-DOF) electric motion simulation platform. The measured spectrum parameters of sea state 6-8 were imported into the control system (pitch ±15°, sway ±25°, heave amplitude 1.5m, frequency 0.15-0.3Hz); at the same time, the cabin simulated the background noise of the engine room (85 dB); the ambient light was set to irregular variation (simulating 24-hour or three-shift work and rest disorder). Daily exposure duration: 2h / day, continuous modeling for 28 days (2h has been proven to be the best effective exposure time to induce chronic complex maritime motion central stress without causing non-specific death due to severe physical exhaustion).

[0046] (2) Group setup includes: randomly dividing the experimental animals into 5 treatment groups and performing the following treatments: Ctrl (normal control group): No stress treatment, daily gavage with normal saline.

[0047] AO (Combined Maritime Stress Model Group): The environment was modeled according to the above settings, and physiological saline was administered by gavage daily.

[0048] Example 1 Group (AO+Form.3): The probiotic compound preparation obtained in Example 1 was administered by gavage daily during the modeling process, with a gavage dose of 1×10⁻⁶. 9 CFU / each.

[0049] Example 2 (AO+Form.2): The probiotic compound preparation obtained in Comparative Example 2 was administered via gavage daily during the modeling process, with a gavage dose of 1×10⁻⁶. 9 CFU / each.

[0050] Example 3 (AO+Form.1): Following the environmental setup described above, the core microecological composite bacterial mud obtained in Comparative Example 1 was administered via gavage daily during the modeling process, with a gavage dose of 1×10⁻⁶. 9 CFU / each.

[0051] 3. Experimental Results (1) After the experiment, real-time quantitative PCR (qPCR) was used to quantitatively analyze the fecal microbiota of rats. The results are as follows: Figure 2 As shown, compared with the Ctrl group, the level of Trichophyceae in the feces of rats in the AO group was significantly lower, indicating that combined maritime stress can lead to a decrease in Trichophyceae levels. Compared with the AO group, the levels of Trichophyceae in all intervention groups were increased, with the most significant increase in the Example 1 group, which was significantly higher than that in the Example 2 and Example 3 groups. The results indicate that the probiotic compound preparation described in Example 1 of this invention can effectively improve the decrease in intestinal Trichophyceae levels caused by combined maritime stress, and its effect is superior to other control formulations.

[0052] (2) After the experiment, the butyric acid content in the rat intestinal contents was monitored. The detection results are as follows: Figure 3 As shown, the level of key short-chain fatty acids (butyric acid) in the intestines of the AO group was significantly decreased. After taking the probiotic compound preparation obtained in Example 1 of this invention, the butyric acid concentration in the intestines increased explosively, significantly higher than that of the AO group and other intervention groups. p <0.0001), which opens up a key metabolic node for gut-brain communication.

[0053] (3) After the experiment, the levels of 5-hydroxytryptamine (5-HT) and brain-derived neurotrophic factor (BDNF) in the hippocampus of rats in each treatment group were measured. The results are as follows: Figures 4-5 As shown, the expression levels of 5-HT and BDNF proteins in the hippocampus of the AO group experienced a precipitous decrease. The probiotic compound preparation obtained in Example 1, through a powerful ascending regulatory feedback loop along the gut-brain axis, resulted in a significant recovery in 5-HT expression and a highly significant restoration in BDNF expression. p The concentration of <0.0001 indicates that the effectiveness of the probiotic compound preparation of this invention in reversing central nervous system damage has been established at the molecular level. In contrast, the probiotic compound preparation obtained in Comparative Example 2, with its poor formulation, and the unprotected compound bacterial mud in Comparative Example 1, failed to achieve the same degree of repair.

[0054] (4) After the experiment, the Morris water maze experiment was conducted. The results are as follows: Figure 6 As shown, rats in the AO group exhibited severe spatial navigation system disorientation, displaying limb-oriented wandering; while rats in the AO+Form.3 group had a significantly shortened escape latency ( p <0.0001), the number of times the hidden platform was traversed and the distance spent in the target quadrant were significantly better than the AO group, and the platform-finding trajectory was restored to a short, target-oriented path.

[0055] (5) After the experiment, a new object recognition experiment was conducted. The results are as follows: Figure 7 As shown, the cognitive index (RI) of the Ctrl group was at a healthy high level of around 75; after combined stress, the exploratory motivation of the AO group rats significantly declined, with the RI value dropping to about 20%. The RI values ​​of the AO+Form.1 and AO+Form.2 groups showed no statistically significant difference (ns) compared to the AO group, indicating that a single naked bacterium or an inappropriate ratio could not salvage recognition memory. Treatment with the probiotic compound preparation obtained in Example 1 successfully activated the rats' motivation to explore new objects, and the RI value significantly recovered to the 50-60 range. p <0.0001).

[0056] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A probiotic compound preparation, characterized in that, It includes the following components by weight percentage: 20%–40% microbial composition, 30%–50% matrix, and 25%–45% coating material; The microbial composition includes Trichophycetes (family Trichophyceae) Lachnospiraceae ) and butyric acid cocci ( Butyricicoccus sp.); the live bacteria ratio of the Trichophyceae family and Butyricococcus is (1~10):1, and not 1:1; The matrix comprises xylooligosaccharides and maltodextrin; The coating material includes sodium alginate and low-methoxyl pectin.

2. The probiotic compound preparation according to claim 1, characterized in that, The live bacteria ratio of *Trichophyton spp.* to *Butyricum coccidioides* is 5:

1.

3. The probiotic compound preparation according to claim 1, characterized in that, The concentration of *Trichophyton spp.* in the microbial composition is 1 × 10⁻⁶. 9 ~5×10 10 CFU / g.

4. The probiotic compound preparation according to claim 1, characterized in that, The matrix comprises 100-300 g / L xylooligosaccharides, 100-300 g / L maltodextrin, and the balance being water.

5. The probiotic compound preparation according to claim 1, characterized in that, The coating material comprises 100-200 g / L sodium alginate, 100-200 g / L low-methoxyl pectin, and the balance being water.

6. The probiotic compound preparation according to any one of claims 1 to 5, characterized in that, The probiotic compound preparation uses the probiotic composition and matrix as the core and the coating material as the outer shell.

7. The method for preparing the probiotic compound preparation according to any one of claims 1 to 6, characterized in that, Includes the following steps: The microbial composition and matrix are mixed and granulated to obtain a dry powder with a microbial core. The probiotic compound preparation is obtained by coating the bacterial core dry powder with a coating material.

8. The preparation method according to claim 7, characterized in that, The granulation method includes freeze-drying and then pulverizing a mixture of microbial composition and matrix; the freeze-drying conditions include freezing at -80℃ for 2-4 hours followed by vacuum freeze-drying for 24-36 hours. The coating method includes: spraying the coating material onto the surface of the bacterial core dry powder and then drying it; the atomization pressure of the spraying is 0.15~0.2 MPa, and the air inlet temperature is 35℃~40℃; the drying temperature is 35℃~40℃.

9. The use of the probiotic compound preparation according to any one of claims 1 to 6 or the probiotic compound preparation obtained by the preparation method according to claim 7 or 8 in the preparation of a drug for improving cognitive impairment.

10. The application according to claim 9, characterized in that, The cognitive impairment mentioned is cognitive impairment caused by combined stress from seafaring; The maritime composite stress includes complex motion stress, continuous noise stress, and photoperiod disorder stress; The parameters of the complex motion stress include pitch ±15°, sway ±25°, heave amplitude 1.5 m and frequency 0.15~0.3Hz; The noise intensity of the continuous noise stress is 85 dB.

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