Probiotic micro-pellets for improving emotional disorders caused by shipboard complex stress, and preparation method and application thereof

Abstract

The application provides a probiotic pellet for improving emotional disorders caused by shipborne complex stress and a preparation method and application thereof, and belongs to the technical field of microbial preparations. The probiotic composition for resisting shipborne complex stress provided by the application is composed of lactobacillus rhamnosus, yellow rumen coccus and veillonella dispar with a viable count ratio of (3-5):(1-2):1. The probiotic composition provided by the application precisely intervenes in the intestinal metabolism damaged by shipborne complex stress through the scientifically screened specific triple probiotic group, prevents the pathological deviation of endogenous tryptophan to kynurenine (neurotoxin), restores the hypothalamic tryptophan content from the root, and realizes the cross-organ double effects of resisting constipation and resisting depression.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, specifically relating to a probiotic microsphere for improving mood disorders caused by shipboard compound stress, its preparation method and application. Background Technology

[0002] Modern naval warfare and aircraft carrier operations pose extreme physiological and psychological challenges to specialized professions such as pilots. These individuals often need to endure complex "sea-air composite environments" for missions lasting several months at sea: they must withstand high +Gz (positive acceleration) overloads during takeoff and landing, as well as long-term exposure to six-degree-of-freedom (6-DOF) spatial turbulence, high-decibel engine room noise, and disrupted day-night cycles on the aircraft carrier platform in sea states 6-8.

[0003] Existing epidemiological and maritime medical surveys indicate that the long-term cumulative effects of the aforementioned combined stressors can easily induce severe physical and psychological damage among officers and soldiers. On the one hand, this manifests as severe psychological disorders (such as generalized anxiety, cognitive decline, slowed reaction time, and sleep disturbances); on the other hand, over 60% of personnel on long-distance voyages suffer from gastrointestinal motility disorders (such as intractable constipation). Traditional physical protection (such as anti-G suits and shock-absorbing seats) and routine psychological counseling cannot fundamentally resolve this systemic illness caused by endocrine and neurometabolic reprogramming.

[0004] In a previous animal model simulating the complex environment of an aircraft carrier (+Gz exposure plus a 6-DOF rocking platform and noise), the applicant's research team discovered that complex stress significantly alters the gut microbiota structure. This dysbiosis triggers a lethal cascade reaction in the "gut-brain axis (MGB Axis): stress-induced hypercortisol activates indoleamine 2,3-dioxygenase (IDO), leading to a pathological shift in tryptophan metabolism in the diet—a large amount of tryptophan is metabolized into neurotoxic kynurenine, which crosses the blood-brain barrier and induces neuroinflammation and anxiety in the brain; at the same time, it leads to a sharp depletion of the synthesis of 5-hydroxytryptamine (5-HT) and indole-3-acetic acid (IAA) in the gut and central nervous system, directly causing intestinal motility stagnation (constipation) and decreased BDNF expression in the hippocampus (cognitive and emotional disorders).

[0005] Most probiotic products on the market are designed for simple bowel movements or gut health, lacking precise formulations targeting "tryptophan metabolic diversion." Furthermore, conventional probiotic powders are easily deactivated in the high-temperature, high-humidity environment of ships. Simultaneously, the extremely high levels of abnormal gastric acid secretion associated with stress cause commercially available single probiotics to die rapidly after entering the body, failing to survive, colonize, and exert their medicinal effects. They also struggle to resist the abnormal gastric acid secretion caused by combined stress in the human body. Therefore, there is an urgent need to develop a special neuropsychiatric probiotic formulation that can precisely block the conversion of tryptophan to kynurenine, promote the synthesis of 5-HT and IAA, and possess extremely high environmental stability. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a probiotic composition that resists shipboard complex stress, which has a protective effect against stress in complex and harsh environments such as ships, can specifically reverse the tryptophan metabolic pathway and has extremely high environmental and digestive tract resistance.

[0007] The second objective of this invention is to provide a probiotic freeze-dried powder that can resist gastric acid attack and is resistant to shipboard compound stress. During the freeze-drying process, the probiotics are not inactivated and have extremely high environmental and digestive tract resistance.

[0008] The third objective of this invention is to provide a probiotic enteric-coated microcapsule with targeted sustained-release function to combat shipboard combined stress.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a probiotic composition for resisting shipboard compound stress, which is composed of Lactobacillus rhamnosus, Ruminococcus flavomarginata and Brontë brevis, wherein the live bacteria ratio of Lactobacillus rhamnosus, Ruminococcus flavomarginata and Brontë brevis is (3~5):(1~2):1.

[0010] The present invention also provides a probiotic freeze-dried powder for resisting shipboard combined stress, which is composed of the above-mentioned probiotic composition and an anti-stress matrix carrier; the anti-stress matrix carrier is composed of skim milk powder, trehalose and sodium ascorbate.

[0011] Preferably, the mass ratio of the skim milk powder, trehalose, and sodium ascorbate is (9~11):(4~6):(0.8~1.2).

[0012] Preferably, the mass ratio of the probiotic composition to the stress-resistant matrix carrier is 20~35:65~80.

[0013] The present invention also provides a probiotic pill for resisting shipboard combined stress, which is made from the above-mentioned probiotic freeze-dried powder, tryptophan metabolite substrate, neurotransmitter synthesis cofactor and prebiotic carrier; the tryptophan metabolite substrate is composed of L-tryptophan and α-lactalbumin; the neurotransmitter synthesis cofactor is composed of vitamin B6 and glycine magnesium; the prebiotic carrier is composed of water-soluble dietary fiber and microcrystalline cellulose.

[0014] Preferably, the weight proportions of each raw material are: 15-35 parts of probiotic freeze-dried powder, 10-25 parts of tryptophan metabolism substrate, 2-8 parts of neurotransmitter synthesis cofactor, and 30-60 parts of prebiotic carrier.

[0015] Preferably, the mass ratio of L-tryptophan to α-lactalbumin is 1:3; the mass ratio of vitamin B6 to magnesium glycine is 1:10; and the mass ratio of water-soluble dietary fiber to microcrystalline cellulose is (3~4):1.

[0016] The present invention also provides a method for preparing the above-mentioned probiotic pellets, comprising the following steps: culturing *Lactobacillus rhamnosus*, *Ruminococcus xanthophyll*, and *Brutella westerlensis* in a culture medium containing 0.5%–1.5% L-tryptophan, collecting the bacterial sludge of the three bacteria and mixing it, then mixing it with an aqueous solution of an anti-stress matrix carrier, and freeze-drying to obtain probiotic lyophilized powder; mixing tryptophan metabolic substrate, neurotransmitter synthesis cofactor, and prebiotic carrier, and adding an aqueous binder solution to form spherical pellet cores; mixing the probiotic lyophilized powder with anhydrous ethanol binder solution, and spraying it onto the surface of the spherical pellet cores to obtain microspheres with a probiotic active layer; mixing acrylic resin, triethyl citrate, and talc to prepare an enteric coating solution; spray coating the microspheres with the probiotic active layer, and drying to obtain probiotic pellets.

[0017] Preferably, the culture medium for culturing Lactobacillus rhamnosus is MRS liquid medium, and the culture medium for culturing Ruminococcus luteus and Broutbacter vegetans is anaerobic BHI medium; the adhesive includes hydroxypropyl methylcellulose, and the mass-volume concentration of the adhesive in the aqueous solution of the adhesive is 3% to 5%; the acrylic resin includes Eudragit L30D-55, and the coating weight gain is 8% to 15%.

[0018] The present invention also provides the application of the above-mentioned probiotic composition, the above-mentioned probiotic freeze-dried powder, or the above-mentioned probiotic pills in the preparation of anti-shipborne combined stress products, wherein the shipborne combined stress includes psychological anxiety, cognitive decline, and / or intestinal constipation.

[0019] The beneficial effects of this invention are: Current probiotics are mostly limited to regulating the local intestinal environment. The probiotic composition provided by this invention, through a scientifically selected specific triple probiotic group, precisely intervenes in intestinal metabolism damaged by shipborne compound stress, preventing the pathological shift of endogenous tryptophan to kynurenine (a neurotoxin), and fundamentally restoring the hypothalamic tryptophan content, achieving a dual-organ effect of anti-constipation and anti-depression. Experiments have confirmed that Lactobacillus rhamnosus, Ruminococcus xanthorum, and Broutbacillus brevis, at the live bacteria ratio specified in this invention, can produce a very strong metabolic synergistic effect, rapidly reshaping the intestinal micro-inflammatory microenvironment under stress.

[0020] The probiotic freeze-dried powder provided by this invention has excellent stress resistance and colonization stability. It can form a dense protective layer after freeze-drying using only a specific composite protective framework (stress-resistant matrix carrier), so that the bacterial community can still survive and colonize even when subjected to extreme gastric acid abnormalities caused by stress. The preparation process is simple and the product yield is high.

[0021] The probiotic pills provided by this invention disintegrate in intestinal fluid, releasing substrates (L-tryptophan), coenzymes (VB6, magnesium), and a specially induced probiotic flora. *Brutella westermani* and *Ruminococcus xanthophyll* rapidly colonize and improve intestinal micro-inflammation, while *Lactobacillus rhamnosus* utilizes coenzymes to efficiently catalyze the production of indoleacetic acid (IAA) and 5-hydroxytryptamine precursors from tryptophan. This mechanism effectively steals tryptophan substrates, reversing the excessive production of kynurenine (a neurotoxin) under stress, fundamentally achieving the dual effects of anti-anxiety and promoting intestinal motility (anti-constipation). The high salt spray and high humidity / heat environment of aircraft carriers / long-haul voyages makes traditional powders highly susceptible to moisture absorption and inactivation. The probiotic pills provided by this invention have a dense structure, providing strong physical barrier against humidity and heat, extending the shelf life at room temperature by more than two times compared to ordinary powders. Moreover, the probiotic pills provided by this invention have a precise gastric avoidance-intestinal slow release targeted colonization effect. Complex physical stress often leads to a surge in gastric acid secretion in animals or humans (stress-induced ulcer tendency). The probiotic pills provided by this invention do not dissolve in gastric juice with pH < 4.5 for 2 hours (zero loss of live bacteria), while they can rapidly dissolve and disintegrate in intestinal juice with pH 6.8 within 45 minutes, achieving efficient and targeted release of active ingredients in the small intestine and colon. Attached Figure Description

[0022] Figure 1 The graph shows the ELISA results of gastrointestinal function and mucosal protection indicators in rats of each group. A represents the PG I / PG II ratio, B represents the motilin MTL, C represents the gastrin GAS, and D represents the prostaglandin E2 (PGE2) result. ns indicates p > 0.05. This indicates that p < 0.05. This indicates that p < 0.01. This means p < 0.0001.

[0023] Figure 2 The image shows the LC-MS precise quantitative detection results of L-tryptophan content in the hypothalamus tissue of rats in each group. ns indicates p > 0.05. This indicates that p < 0.05. This means p < 0.0001.

[0024] Figure 3 The graphs show the results of emotional and behavioral tests on rats in each group. A represents the open field trajectory, B represents the total distance traveled in the open field, C represents the total distance traveled in the four corner regions of the open field, D represents the time spent stationary in the open field, E represents the time spent immobile during the tail suspension test, and F represents the sugar water preference rate. This indicates that p < 0.05. This indicates that p < 0.01. ns indicates p < 0.0001, and ns indicates p > 0.05. Detailed Implementation

[0025] This invention provides a probiotic composition for resisting shipboard combined stress, comprising Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), xanthophyllococcus ( Ruminococcus flavefaciens ) and Brontë ( Blautia wexlerae The composition is as follows: the ratio of viable bacteria of Lactobacillus rhamnosus, Luteinized Ruminococcus and Broutbacter vegetans is (3~5):(1~2):1.

[0026] This invention does not specifically limit the source of the aforementioned bacteria; commercially available products commonly used in the field are acceptable. In this invention, the preferred live bacteria ratio of *Lactobacillus rhamnosus*, *Ruminococcus flavinatus*, and *Brutella westerlens* is 3:2:1.

[0027] The present invention also provides a probiotic freeze-dried powder for resisting shipboard combined stress, which is composed of the above-mentioned probiotic composition and an anti-stress matrix carrier; the anti-stress matrix carrier is composed of skim milk powder, trehalose and sodium ascorbate.

[0028] In this invention, the preferred mass ratio of skim milk powder, trehalose, and sodium ascorbate (sodium vitamin C) is (9-11):(4-6):(0.8-1.2), more preferably 10:5:1. The stress-resistant matrix carrier provided by this invention can provide a glassy skeletal support for three environmentally sensitive strains during freeze-drying, resisting gastric acid attack. In this invention, the preferred mass ratio of the probiotic composition to the stress-resistant matrix carrier is 20-35:65-80, more preferably 25-30:70-75.

[0029] The preferred method for preparing the probiotic freeze-dried powder of the present invention includes the following steps: *Lactobacillus rhamnosus*, *Ruminococcus xanthophyll*, and *Brutella westermani* are cultured separately in a medium containing 0.5%–1.5% L-tryptophan; the bacterial sludge from the three strains is collected and mixed; then mixed with an aqueous solution of an anti-stress matrix carrier; and freeze-dried to obtain the probiotic freeze-dried powder. In this invention, the culture medium for culturing *Lactobacillus rhamnosus* is preferably MRS liquid medium, and the culture medium for culturing *Ruminococcus xanthophyll* and *Brutella westermani* is preferably anaerobic BHI medium. The present invention does not have specific limitations on the specific composition of the MRS liquid medium and the anaerobic BHI medium; conventional commercially available products in the art can be used. The anaerobic BHI medium is preferably an anaerobic BHI medium that has undergone deoxygenation treatment. In this invention, the culture temperature is preferably 37°C, and the culture time is preferably 18–24 hours, until the bacterial community enters the early stage of the stationary phase. This invention induces the culture of *Lactobacillus rhamnosus*, *Ruminococcus xanthophyll*, and *Brutella westermani* in a medium containing L-tryptophan, thereby enhancing the enzyme activity of tryptophan-to-indole derivatives (IAA) produced by these strains. Pre-application of L-tryptophan significantly induces the overexpression of tryptophan decarboxylase and amino acid transferase within the strains. After culture, the bacterial sludge is preferably collected by centrifugation at 5500-6500 rpm for 10-15 minutes. After collection, the sludge is preferably washed once with sterile PBS buffer before the three types of sludge are mixed. In this invention, the freeze-drying method is preferably as follows: pre-freezing and solidification, sublimation drying, and finally desorption drying. The pre-freezing and solidification preferably involves spreading the suspension evenly in a freeze-drying tray with a thickness of 1.0~1.5 cm, and then placing it in an ultra-low temperature freezer at -75℃~-80℃ for deep freeze-drying for 4 hours. The sublimation drying preferably uses a vacuum freeze dryer with a cold trap temperature ≤-65℃, a vacuum degree <10Pa, and a partition temperature maintained at -22℃~-20℃ for 12 hours. The desorption drying preferably involves heating to 25℃ at a rate of 2℃ / h and maintaining this temperature for 10 hours to remove bound water. After the freeze-drying steps are completed, the product is pulverized through a 60-mesh sieve under a relative humidity of <30% to obtain a freeze-dried probiotic powder with a moisture content of <4.0% to combat complex stress.

[0030] The present invention also provides a probiotic pill for resisting shipboard combined stress, which is made from the above-mentioned probiotic freeze-dried powder, tryptophan metabolite substrate, neurotransmitter synthesis cofactor and prebiotic carrier; the tryptophan metabolite substrate is composed of L-tryptophan and α-lactalbumin; the neurotransmitter synthesis cofactor is composed of vitamin B6 and glycine magnesium; the prebiotic carrier is composed of water-soluble dietary fiber and microcrystalline cellulose.

[0031] The existing maritime and special aviation medical protection systems lack specific biological interventions for central nervous system damage and intestinal motility disorders caused by "shipborne compound stress." Furthermore, conventional commercially available probiotic preparations are easily inactivated in the high-temperature and high-humidity environment of long-distance voyages, are unable to resist gastric acid invasion under stress, and single strains cannot reverse host neurometabolic disorders. This invention provides an anti-shipborne compound stress probiotic pill targeting the tryptophan metabolic pathway. This overcomes the limitations of traditional single-strain live bacteria supplementation by proposing a synergistic formula involving specific fermentation-retentive bacteria (Lactobacillus rhamnosus, Ruminococcus xanthipes, and Broutbacillus vesiculosus), tryptophan metabolic substrates, and neurotransmitter synthesis cofactors. This formula precisely intervenes in the "gut-brain axis," forcibly blocking the pathological shift of tryptophan to the neurotoxic kynurenine pathway induced by shipboard compound stress (high +Gz superimposed 6-DOF swing), prompting it to be targeted and converted into beneficial 5-hydroxytryptamine (5-HT) and indole-3-acetic acid (IAA). This achieves the dual purpose of alleviating severe psychological anxiety, cognitive decline, and intractable intestinal constipation in special operations personnel, providing a novel internal environment metabolic regulation product for medical support.

[0032] In the probiotic pellets of this invention, the total live bacteria count of the finished micro-pellets is preferably ≥2×10⁻⁶. 10 CFU / g; the preferred weight proportions of each raw material are: 15-35 parts probiotic freeze-dried powder, 10-25 parts tryptophan metabolic substrate, 2-8 parts neurotransmitter synthesis cofactor, and 30-60 parts prebiotic carrier; more preferably, 20-30 parts probiotic freeze-dried powder, 12-20 parts tryptophan metabolic substrate, 3-7 parts neurotransmitter synthesis cofactor, and 35-55 parts prebiotic carrier. In this invention, the mass ratio of L-tryptophan to α-lactalbumin is preferably 1:3. In the neurotransmitter synthesis cofactor of this invention, vitamin B6 is an essential coenzyme for tryptophan decarboxylase, and magnesium ions in glycine magnesium help antagonize the neurotoxicity of kynurenine; the preferred mass ratio of the two is 1:10. In this invention, the prebiotic carrier is preferably a mixture of water-soluble dietary fiber (polydextrose) and microcrystalline cellulose (MCC). The prebiotic carrier serves as both a core material for pellets and can also relieve physical constipation by expanding in the intestines. The preferred mass ratio of water-soluble dietary fiber to microcrystalline cellulose is (3~4):1, and more preferably 4:1.

[0033] This invention also provides a method for preparing the above-mentioned probiotic pellets, comprising the following steps: mixing tryptophan metabolic substrate, neurotransmitter synthesis cofactor and prebiotic carrier, and adding an aqueous binder solution to form a spherical pellet core; mixing the probiotic freeze-dried powder obtained above with anhydrous ethanol binder solution, and spraying it onto the surface of the spherical pellet core to obtain microparticles with a probiotic active layer; mixing acrylic resin, triethyl citrate and talc to prepare an enteric coating solution; spray coating the microparticles with the probiotic active layer, and drying to obtain probiotic pellets.

[0034] The method for preparing the above-mentioned probiotic compound microspheres provided by this invention aims to fundamentally solve the problem of storage stability of live bacteria preparations in the harsh environment of high salt spray and high humidity and heat on ships by introducing the "extrusion-spheronization granulation" process combined with the "fluidized bed enteric polymer coating" process in the field of modern pharmaceutical formulation. At the same time, by utilizing the unique pH-dependent solubility characteristics of the enteric coating, it perfectly avoids the invasion of abnormal gastric acid induced by compound stress, ensuring the efficient, targeted and synergistic release of neurotrophic probiotics and coenzyme substrates in specific parts of the intestine, and maximizing their anti-stress efficacy and bioavailability.

[0035] This invention prepares spherical pellet cores based on an extrusion-spheronization process, preferably including the following steps: Premix: Add tryptophan metabolism substrate, neurotransmitter synthesis cofactor and prebiotic carrier into a high-shear mixing granulator and dry mix for 5-10 minutes until uniformly mixed.

[0036] To prepare the soft material: While stirring, spray in an aqueous solution of hydroxypropyl methylcellulose (HPMC, viscosity specification E5) at a concentration of 2%~5% (3% is optimal) as a binder, and stir for 3~5 minutes to prepare a suitable soft material that can be formed into a ball by hand and crumbles when lightly pressed.

[0037] Extrusion: Add the soft material to the oscillating extruder, select an extrusion screen with an aperture of 0.8~1.2mm (1.0mm is optimal), and set the extrusion speed to 30~50rpm to obtain a smooth cylindrical strip.

[0038] Rounding and Drying: Quickly transfer the strip-shaped material to a centrifugal granulator and rounding machine. Set the toothed disc speed to 600~1000 rpm (optimal 800 rpm) and the rolling time to 3~8 minutes to break the strip-shaped material and round it into spherical cores with uniform surface height. Then transfer the cores to a bottom-spray fluidized bed, set the inlet air temperature to 40℃~45℃, and fluidize and dry for 30~45 minutes until the moisture content of the cores is <3%. Collect and set aside for later use.

[0039] This invention employs a bottom-spray fluidized bed process to load a probiotic active layer, preferably including the following steps: Preparation of coating solution: The probiotic freeze-dried powder obtained above is uniformly suspended in an anhydrous ethanol / purified water mixed solvent containing 2% HPMC (as a film-forming binder) (the volume ratio of anhydrous ethanol to purified water is preferably 7:3), and continuously stirred at low speed with magnetic force to obtain the drug-containing coating solution.

[0040] • Active Layer Loading: The spherical pellets prepared above are placed in a Wurster bottom-spray fluidized bed coating machine. The core process parameters are controlled as follows: the inlet air temperature is strictly controlled at 30℃~35℃ (optimal is 32℃, this low-temperature environment ensures that the fragile live bacteria are not thermally inactivated), the material temperature is maintained at 28℃~30℃, the atomizing air pressure is set to 0.15~0.25MPa (optimal is 0.18MPa), and the spraying rate is 2~5g / min. The atomized live bacteria coating liquid uniformly adheres to the outer surface of the spherical pellets, and the moisture evaporates rapidly, forming dense, dry microspheres with a beneficial bacteria active layer.

[0041] The present invention preferably employs the following coating steps (forming process): Preparation of enteric coating solution: Select Eudragit-type acrylic resin aqueous dispersion (model Eudragit L30D-55, which dissolves precisely in the intestinal environment with pH>5.5), add 10% by weight of the dry polymer (referring to the solid component of the acrylic resin in Eudragit L30D-55 after removing 70% of the water) of triethyl citrate (as a plasticizer) and 5% of talc (to prevent adhesion), dilute with water to a solid content of about 15%, and stir evenly.

[0042] Enteric coating: Continue spray coating of the microspheres with the probiotic active layer in the fluidized bed bottom spray coating machine. Preferred process parameters are: inlet air temperature slightly increased to 38℃~42℃ (optimal 40℃), atomization pressure set to 0.20~0.30MPa (optimal 0.25MPa). Continue spray coating until the physical coating weight gain of the microspheres reaches 8%~15% (optimal setting is 12%; too low a weight gain will result in intolerance to extreme gastric acid stress, while too high a weight gain will delay intestinal disintegration and release).

[0043] Curing and Sieving: After coating, continue fluidization (curing) at the original inlet air temperature for 10-15 minutes to ensure complete fusion and densification of the polymer film. Cool and discharge the material, then sieve it through a 14-20 mesh (approximately 0.85-1.4 mm) standard sieve to obtain probiotic pellets with uniform particle size, smooth and dense surface, and targeted sustained-release function.

[0044] This invention also provides the application of the above-mentioned probiotic composition, the above-mentioned probiotic freeze-dried powder, or the above-mentioned probiotic pills in the preparation of products for combating shipboard combined stress, wherein the shipboard combined stress includes psychological anxiety, cognitive decline, and / or intestinal constipation. In this invention, the products preferably include pharmaceuticals and / or food products.

[0045] The probiotic composition provided by this invention is a probiotic composition that targets and regulates tryptophan metabolism in the gut-brain axis. The probiotic composition provided by this invention has the effect of preventing and improving gastrointestinal motility disorders and severe mood disorders caused by shipboard compound stress (high + Gz superimposed spatial rocking).

[0046] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Unless otherwise specified, the following embodiments are all conventional methods.

[0048] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0049] Lactobacillus rhamnosus in the following examples ( Lactobacillus rhamnosus Purchased from the German Microbial Culture Collection (DSMZ), accession number DSM 20079, *Ruminococcus luteus* (…). Ruminococcus flavefaciens Purchased from the German Microbial Culture Collection (DSMZ), accession number DSM 25089, *Brutella wechslerae* (…). Blautia wexlerae Purchased from the German Microbial Culture Collection (DSMZ), accession number DSM 19850.

[0050] Example 1 A probiotic composition for resisting shipboard combined stress, comprising Lactobacillus rhamnosus, Ruminococcus flavomarginata, and Broutella westeriensis, wherein the live count ratio of Lactobacillus rhamnosus, Ruminococcus flavomarginata, and Broutella westeriensis is 3:2:1.

[0051] Example 2 A probiotic composition for resisting shipboard combined stress, comprising Lactobacillus rhamnosus, Ruminococcus flavomarginata, and Broutella westeriensis, wherein the live count ratio of Lactobacillus rhamnosus, Ruminococcus flavomarginata, and Broutella westeriensis is 3:1:1.

[0052] Example 3 A probiotic composition for resisting shipboard combined stress, comprising Lactobacillus rhamnosus, Ruminococcus flavomarginata, and Broutella westeriensis, wherein the live count ratio of Lactobacillus rhamnosus, Ruminococcus flavomarginata, and Broutella westeriensis is 5:2:1.

[0053] Example 4 A lyophilized probiotic powder for resisting shipboard combined stress is composed of the probiotic composition described in Example 1 and an anti-stress matrix carrier; the mass ratio of the probiotic composition to the anti-stress matrix carrier is 30:70; the anti-stress matrix carrier is composed of skim milk powder, trehalose and sodium ascorbate, and the mass ratio of the skim milk powder, trehalose and sodium ascorbate is 10:5:1.

[0054] The preparation method of the probiotic freeze-dried powder is as follows: Lactobacillus rhamnosus was inoculated into MRS liquid medium containing 1.0% L-tryptophan and cultured. Ruminococcus luteus and Brontë vegetans were inoculated separately into deoxygenated anaerobic BHI medium containing 1.0% L-tryptophan and cultured under strictly anaerobic conditions by introducing a mixed gas (80% N2, 10% CO2, 10% H2). The culture conditions were: 36°C for 20 h, followed by cooling to 4°C, centrifugation at 6000 rpm for 13 minutes, discarding the supernatant, washing once with sterile PBS buffer, collecting the viable bacterial sludge separately, and mixing to obtain the probiotic composition bacterial sludge for later use.

[0055] Skim milk powder, trehalose, and sodium ascorbate were added to purified water. The mixture was stirred in a 41°C water bath for 20 minutes until completely dissolved. Subsequently, it was autoclaved at 121°C for 15 minutes and rapidly cooled to 5°C to obtain an aqueous solution of the stress-resistant matrix carrier, which was then ready for use.

[0056] Under aseptic conditions, the probiotic composition sludge was added to an aqueous solution of an anti-stress matrix carrier. The ratio of the total live bacteria count (CFU) of the three sludge species—Lactobacillus rhamnosus, Ruminococcus luteus, and Broutella westeriensis—was 3:2:1. The mixture was magnetically stirred at 100 rpm for 45 minutes at 4°C to form a highly homogeneous suspension.

[0057] The suspension was spread evenly on a freeze-drying tray (1.0 cm thick) and placed in an ultra-low temperature freezer at -75℃ to -80℃ for deep freezing and solidification for 4 hours. Then, it was transferred to a vacuum freeze dryer with a cold trap temperature ≤ -65℃, a vacuum degree <10Pa, and a partition temperature maintained at -22℃ to -20℃ for 12 hours. The temperature was then increased to 25℃ at a rate of 2℃ / h and maintained for 10 hours to remove bound water. After removal from the freeze dryer, the product was pulverized through a 60-mesh sieve under conditions of relative humidity <30% to obtain a probiotic freeze-dried powder product with a moisture content <4.0%.

[0058] Example 5 A lyophilized probiotic powder for resisting shipboard combined stress is composed of the probiotic composition described in Example 2 and an anti-stress matrix carrier; the mass ratio of the probiotic composition to the anti-stress matrix carrier is 35:65; the anti-stress matrix carrier is composed of skim milk powder, trehalose and sodium ascorbate, and the mass ratio of the skim milk powder, trehalose and sodium ascorbate is 11:4:1.2.

[0059] The preparation method of the probiotic freeze-dried powder is as follows: Lactobacillus rhamnosus was inoculated into MRS liquid medium containing 1.5% L-tryptophan and cultured. Ruminococcus luteus and Broutbacterium velutipes were inoculated separately into deoxygenated anaerobic BHI medium containing 1.5% L-tryptophan and cultured under strictly anaerobic conditions by introducing a mixed gas (80% N2, 10% CO2, 10% H2). The culture conditions were: 37°C for 24 h, followed by cooling to 4°C, centrifugation at 6500 rpm for 10 minutes, discarding the supernatant, washing once with sterile PBS buffer, collecting the viable bacterial sludge separately, and mixing to obtain the probiotic composition bacterial sludge for later use.

[0060] Skim milk powder, trehalose, and sodium ascorbate were added to purified water. The mixture was stirred in a 40°C water bath for 20 minutes until completely dissolved. Subsequently, it was autoclaved at 121°C for 15 minutes and rapidly cooled to 6°C to obtain an aqueous solution of the stress-resistant matrix carrier, which was then ready for use.

[0061] Under aseptic conditions, the probiotic composition sludge was added to an aqueous solution of an anti-stress matrix carrier. The ratio of the total live bacteria count (CFU) of the three sludge species—Lactobacillus rhamnosus, Ruminococcus xanthophyll, and Broutella westeriensis—was 3:1:1. The mixture was magnetically stirred at 120 rpm for 45 minutes at 4°C to form a highly homogeneous suspension.

[0062] The suspension was freeze-dried under vacuum (cold trap -60℃, vacuum degree <15Pa, 24h), and then pulverized through an 80-mesh sieve to obtain the finished probiotic freeze-dried powder.

[0063] Comparative Example 1 The difference from Example 4 is that the ratio of total live bacteria (CFU) in the probiotic composition is 1:1:1, while the rest are the same as in Example 4.

[0064] Comparative Example 2 The difference from Example 4 is that no stress-resistant matrix carrier was added, and the mixture was directly mixed with water and then freeze-dried. All other aspects are the same as in Example 4.

[0065] Experimental Example 1 To verify the stress protection effect of the present invention on ships and other complex and harsh environments, the following in vivo animal verification experiments were completed using a special simulation platform.

[0066] 1. Experimental Grouping and Model Construction SPF-grade male SD rats were used. Parameters for shipboard combined stress modeling: +10 Gz exposure was applied using an animal centrifuge, while the rats were placed on a six-DOF simulation platform to simulate high sea state rolling and background noise for 28 consecutive days.

[0067] The experiment was set up with 5 groups: Ctrl group (healthy control group): No stress modeling, daily routine feeding and saline gavage.

[0068] AO group (complex stress model group): received shipboard complex stress modeling and daily saline gavage.

[0069] AO+Form.3 group: stress modeling + daily gavage administration of the probiotic freeze-dried powder obtained in Example 4 of this invention.

[0070] AO+Form.2 group: probiotic freeze-dried powder obtained from comparison group 1, which was created by stress modeling and daily gavage.

[0071] AO+Form.1 group: The probiotic freeze-dried powder obtained by the comparison example 2 (naked bacterial raw powder without any added stress-resistant matrix carrier) was created by stress modeling and daily gavage.

[0072] 2. Evaluation of core indicators and experimental results (1) Assessment of stress-induced gastrointestinal injury and motility repair Combined physical stress can easily lead to damage to the gastrointestinal mucosa and impaired motility. Serum / gastric tissue markers were detected using ELISA. Results are as follows: Figure 1 As shown, the pepsinogen ratio (PG I / PG II), motilin (MTL), gastrin (GAS), and prostaglandin E2 (PGE2) in the AO group rats all experienced a sharp decline. After intervention, the Form.3 group showed a highly significant recovery in all gastrointestinal protective and motility indicators, demonstrating a strong anti-stress gastrointestinal targeted protective ability. In contrast, the naked bacteria group (Form.1), lacking matrix protection, suffered a large number of deaths in the stressful gastric acid, and its recovery effect was no different from that of the AO group.

[0073] (2) Assessment of targeted regulation of central tryptophan metabolism Tryptophan is the only precursor for the central synthesis of serotonin (5-HT). The L-tryptophan content in hypothalamic tissue was detected using LC-MS. Results are as follows: Figure 2 As shown, combined AO stress leads to a rapid depletion of tryptophan reserves in the brain (resulting in neurotoxic shift). The main formulation, Form.3, significantly restored tryptophan levels in the hypothalamus through ascending regulation along the gut-brain axis. In contrast, the Form.2 group with altered proportions failed to exert a synergistic metabolic effect, resulting in extremely limited recovery of tryptophan levels.

[0074] (3) Macroscopic assessment of depressive / anxiety behavioral patterns The results of the open field experiment are as follows Figure 3 As shown in Figures A through D: The total distance traveled and the distance traveled in the four corner areas were significantly shortened in the AO group rats, and the resting time was longer, indicating strong anxiety. The Form.3 group showed a remarkably significant recovery of exploratory movement trajectories and eliminated anxiety-like behaviors.

[0075] Results of the tail suspension and sugar water preference experiment are as follows Figure 3 As shown in E and F, the AO group exhibited increased tail suspension immobility time and a sharp decrease in sucrose preference, demonstrating severe despair and anhedonia. Form.3 successfully reversed the aforementioned depressive-like phenotypes, with significantly better results than Form.1 and Form.2.

[0076] Example 6 A probiotic pill for combating shipboard combined stress is made from 25 parts by weight of the probiotic freeze-dried powder described in Example 4, 20 parts by weight of tryptophan metabolic substrate, 5 parts by weight of neurotransmitter synthesis cofactor, and 50 parts by weight of prebiotic carrier; the tryptophan metabolic substrate is composed of L-tryptophan and α-lactalbumin, with a mass ratio of L-tryptophan to α-lactalbumin of 1:3; the neurotransmitter synthesis cofactor is composed of vitamin B6 and magnesium glycine, with a mass ratio of vitamin B6 to magnesium glycine of 1:10; the prebiotic carrier is composed of water-soluble dietary fiber (polydextrose) and microcrystalline cellulose, with a mass ratio of water-soluble dietary fiber to microcrystalline cellulose of 4:1.

[0077] The preparation method of the probiotic pills is as follows: (1) Preparation of spherical pellet cores based on extrusion-spheronization process Premix: Add the tryptophan metabolism substrate, neurotransmitter synthesis cofactor and prebiotic carrier to a high-shear mixing granulator according to the weight ratio, and dry mix for 8 minutes until uniformly mixed.

[0078] To make the soft material: While stirring, spray in a 3% hydroxypropyl methylcellulose (HPMC, viscosity specification E5) aqueous solution as a binder at a uniform speed, stir for 4 minutes, and make a suitable soft material that can be formed into a ball by hand and crumbles when lightly pressed.

[0079] Extrusion: The soft material is added to a swing extruder, an extrusion screen with a 1.0 mm aperture is selected, and the extrusion speed is set to 40 rpm to obtain a smooth cylindrical strip.

[0080] Rounding and Drying: Quickly transfer the strip-shaped material to a centrifugal granulator and rounding machine. Set the toothed disc speed to 800 rpm and the rolling time to 5 minutes to break the strip-shaped material and round it into spherical cores with uniform surface height. Then, transfer the cores to a bottom-spray fluidized bed, set the inlet air temperature to 42℃, and fluidize and dry for 35 minutes until the core moisture content is <3%. Collect and set aside for later use.

[0081] (2) Bottom-spray fluidized bed process for loading probiotic active layer Preparation of coating solution: The probiotic freeze-dried powder obtained in Example 4 was uniformly suspended in an anhydrous ethanol / purified water mixed solvent containing 2% HPMC (as a film-forming binder) (the volume ratio of anhydrous ethanol to purified water was 7:3), and continuously stirred at low speed with magnetic force to prepare the drug-containing coating solution.

[0082] Active layer loading: The spherical pellets obtained in step (1) were placed in a Wurster bottom-spray fluidized bed coating machine. The core process parameters were controlled as follows: the inlet air temperature was strictly controlled at 32℃, and this low-temperature environment ensured that the fragile live bacteria were not inactivated by heat; the material temperature was maintained at 29℃; the atomizing air pressure was set to 0.18MPa; and the spraying rate was 3g / min. The atomized live bacteria coating liquid adhered evenly to the outer surface of the spherical pellets, and the moisture evaporated rapidly, forming a dense microsphere with a beneficial bacteria active layer.

[0083] (3) Coating (forming process) Preparation of enteric coating solution: Select Eudragit-type acrylic resin aqueous dispersion (model Eudragit L30D-55, which dissolves precisely in the intestinal environment with pH>5.5), add 10% by weight of dry polymer triethyl citrate (as plasticizer) and 5% talc (to prevent adhesion), dilute with water to a solid content of about 15%, and stir evenly.

[0084] Enteric coating: Continue spray coating of the microspheres from step (2) in the fluidized bed bottom spray coating machine. Process parameters: The inlet air temperature is slightly increased to 40℃, and the atomization pressure is set to 0.25MPa. Continue spray coating until the physical coating weight gain of the microspheres reaches 12% (too low a weight gain will result in the inability to tolerate extreme gastric acid stress, while too high a weight gain will delay intestinal disintegration and release).

[0085] Curing and Sieving: After coating, continue fluidization (curing) at the original inlet air temperature for 13 minutes to ensure complete fusion and densification of the polymer film. Cool and discharge the material, then screen it through a 20-mesh standard sieve to obtain a finished probiotic pellet product with uniform particle size, smooth and dense surface, and targeted sustained-release function to resist shipboard compound stress.

[0086] Example 7 A probiotic pill for combating shipboard combined stress is made from 35 parts by weight of the probiotic freeze-dried powder described in Example 5, 25 parts by weight of tryptophan metabolic substrate, 8 parts by weight of neurotransmitter synthesis cofactor, and 60 parts by weight of prebiotic carrier; the tryptophan metabolic substrate is composed of L-tryptophan and α-lactalbumin, with a mass ratio of L-tryptophan to α-lactalbumin of 1:3; the neurotransmitter synthesis cofactor is composed of vitamin B6 and magnesium glycine, with a mass ratio of vitamin B6 to magnesium glycine of 1:10; the prebiotic carrier is composed of water-soluble dietary fiber (polydextrose) and microcrystalline cellulose, with a mass ratio of water-soluble dietary fiber to microcrystalline cellulose of 3:1.

[0087] The preparation method of the probiotic pills is as follows: (1) Preparation of spherical pellet cores based on extrusion-spheronization process Premix: Add the tryptophan metabolism substrate, neurotransmitter synthesis cofactor and prebiotic carrier to a high-shear mixing granulator according to the weight ratio, and dry mix for 10 minutes until uniformly mixed.

[0088] To make the soft material: While stirring, spray a 5% hydroxypropyl methylcellulose (HPMC, viscosity specification E5) aqueous solution at a uniform speed as a binder, stir for 5 minutes, and make a suitable soft material that can be formed into a ball by hand and crumbles when lightly pressed.

[0089] Extrusion: The soft material is added to a swing extruder, an extrusion screen with a 1.2mm aperture is selected, and the extrusion speed is set to 50rpm to obtain a smooth cylindrical strip.

[0090] Rounding and Drying: Quickly transfer the strip-shaped material to a centrifugal granulator and rounding machine. Set the toothed disc speed to 1000 rpm and the rolling time to 8 minutes to break the strip-shaped material and round it into spherical cores with uniform surface height. Then, transfer the cores to a bottom-spray fluidized bed, set the inlet air temperature to 45℃, and fluidize and dry for 45 minutes until the moisture content of the cores is <3%. Collect them for later use.

[0091] (2) Bottom-spray fluidized bed process for loading probiotic active layer Preparation of coating solution: The probiotic freeze-dried powder obtained in Example 5 was uniformly suspended in an anhydrous ethanol / purified water mixed solvent containing 2% HPMC (as a film-forming binder) (the volume ratio of anhydrous ethanol to purified water was 7:3), and continuously stirred at low speed with magnetic force to prepare the drug-containing coating solution.

[0092] Active layer loading: The spherical pellets obtained in step (1) were placed in a Wurster bottom-spray fluidized bed coating machine. The core process parameters were controlled as follows: the inlet air temperature was strictly controlled at 35℃, and this low-temperature environment ensured that the fragile live bacteria were not inactivated by heat; the material temperature was maintained at 30℃; the atomizing air pressure was set at 0.25MPa; and the spraying rate was 5g / min. The atomized live bacteria coating liquid adhered evenly to the outer surface of the spherical pellets, and the moisture evaporated rapidly, forming a dense microsphere with a beneficial bacteria active layer.

[0093] (3) Coating (forming process) Preparation of enteric coating solution: Select Eudragit-type acrylic resin aqueous dispersion (model Eudragit L30D-55, which dissolves precisely in the intestinal environment with pH>5.5), add 10% by weight of dry polymer triethyl citrate (as plasticizer) and 5% talc (to prevent adhesion), dilute with water to a solid content of about 15%, and stir evenly.

[0094] Enteric coating: Continue spray coating of the microspheres from step (2) in the fluidized bed bottom spray coating machine. Process parameters: The inlet air temperature is slightly increased to 42℃, and the atomization pressure is set to 0.30MPa. Continue spray coating until the physical coating weight gain of the microspheres reaches 15% (too low a weight gain will result in the inability to tolerate extreme gastric acid stress, while too high a weight gain will delay intestinal disintegration and release).

[0095] Curing and Sieving: After coating, continue fluidization (curing) at the original inlet air temperature for 15 minutes to ensure complete fusion and densification of the polymer film. Cool and discharge the material, then sieve it through a 14-mesh standard sieve to obtain a finished probiotic pellet product with uniform particle size, smooth and dense surface, and targeted sustained-release function to resist shipboard compound stress.

[0096] Experimental Example 2 Experimental model construction (simulating the complex environment of an aircraft carrier) SPF-grade male SD rats were used, and the modeling environment was set as follows: (1) High G exposure: Using an animal centrifuge, set the peak value to +10 Gz for 1 minute, 3 times a day, 5 days a week.

[0097] (2) Exposure to the marine environment: The animal cages were fixed on a six-degree-of-freedom (6-DOF) electric simulation platform. The control system was equipped with measured parameters of sea state 6-8 (pitch ±15°, yaw ±25°, heave amplitude 1.5m, frequency 0.15~0.3Hz). Simultaneously, simulated background noise (85dB) was applied to the cabin, and day and night light reversal (work-rest disorder) was applied. The animals were exposed for 2 hours continuously every day.

[0098] The above composite molding process was carried out continuously for 28 days.

[0099] Animal grouping: High-dose group of probiotic pills obtained in Example 6: During the modeling period, the probiotic pills obtained in Example 6 were mixed into the rat feed at a dose of 250 mg / kg per day.

[0100] Low-dose group of probiotic pills obtained in Example 6: During the modeling period, the probiotic pills obtained in Example 6 were mixed into the rat feed at a dose of 125 mg / kg per day.

[0101] Blank stress group: During the modeling period, they were fed the same amount of ordinary feed every day without any intervention.

[0102] Standard powder group: This group consists of commercially available probiotics, specifically standard lyophilized powder. A daily oral gavage dose of a commercially available mixed lyophilized powder of Bifidobacterium and Lactobacillus was administered in aqueous solution (same as in Example 6).

[0103] Normal healthy control: No modeling was performed, and the animals were fed the same amount of ordinary feed every day.

[0104] On day 28 of the experiment, behavioral tests were conducted (elevated maze to assess anxiety; stool samples were collected to determine water content and assess constipation). The animals were then euthanized for tissue analysis. Metabolites in serum and brain tissue were detected by LC-MS, and hippocampal protein expression was detected by Western blotting. The results are shown in Table 1.

[0105] Table 1 Results after 28 days of experiment

[0106] As shown in Table 1, the combined stress of long-distance voyages not only triggered severe anxiety behavior (very little arm-opening exploration) but also led to severe constipation (a sharp decrease in fecal water content). After intervention with the probiotic pills obtained in Example 6 of this invention, the anxiety-like behavior of rats was significantly reversed, and their defecation function returned to normal.

[0107] The serum Kyn / Trp ratio is the gold standard for measuring the metabolic shift of tryptophan toxicity under stress. In the blank stress group, this ratio surged, indicating the production of a large amount of neurotoxins; however, the probiotic pills obtained in Example 6 of this invention successfully suppressed this ratio to near-normal levels, while simultaneously restoring the concentration of the beneficial brain metabolite IAA. This demonstrates that the probiotic pills of this invention successfully achieved metabolic diversion of tryptophan.

[0108] The ordinary powder group (conventional uncoated powder) had poor efficacy, demonstrating that in the absence of enteric-coated sustained-release protection and specific coenzyme substrates, simply supplementing with ordinary live bacteria is extremely difficult to exert sufficient efficacy in the stress-damaged intestine.

[0109] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A probiotic composition for protection against combined stress of air and sea travel, characterized in that, It is composed of Lactobacillus rhamnosus, Ruminococcus flavomarginata and Broutella wesleyanus, wherein the ratio of the number of viable Lactobacillus rhamnosus, Ruminococcus flavomarginata and Broutella wesleyanus is (3~5):(1~2):

1.

2. A probiotic lyophilized powder against combined stress of carrier, characterized in that, It is composed of the probiotic composition of claim 1 and an anti-stress matrix carrier; the anti-stress matrix carrier is composed of skim milk powder, trehalose and sodium ascorbate.

3. The probiotic lyophilized powder according to claim 2, characterized in that, The mass ratio of the skim milk powder, trehalose, and sodium ascorbate is (9~11):(4~6):(0.8~1.2).

4. The probiotic freeze-dried powder according to claim 2, characterized in that, The mass ratio of the probiotic composition to the stress-resistant matrix carrier is 20~35:65~80.

5. A probiotic pill for resisting shipborne combined stress, characterized in that, It is made from the probiotic freeze-dried powder, tryptophan metabolic substrate, neurotransmitter synthesis cofactor and prebiotic carrier as described in claim 2; the tryptophan metabolic substrate is composed of L-tryptophan and α-lactalbumin; the neurotransmitter synthesis cofactor is composed of vitamin B6 and magnesium glycine; the prebiotic carrier is composed of water-soluble dietary fiber and microcrystalline cellulose.

6. The probiotic pill according to claim 5, characterized in that, The weight proportions of each raw material are as follows: 15-35 parts of probiotic freeze-dried powder, 10-25 parts of tryptophan metabolism substrate, 2-8 parts of neurotransmitter synthesis cofactor, and 30-60 parts of prebiotic carrier.

7. The probiotic pill according to claim 5, characterized in that, The mass ratio of L-tryptophan to α-lactalbumin is 1:3; the mass ratio of vitamin B6 to magnesium glycine is 1:10; and the mass ratio of water-soluble dietary fiber to microcrystalline cellulose is (3~4):

1.

8. The method for preparing the probiotic pills according to any one of claims 5 to 7, characterized in that, The process includes the following steps: *Lactobacillus rhamnosus*, *Ruminococcus flavinatus*, and *Brutella westermani* are cultured separately in a medium containing 0.5%–1.5% L-tryptophan. The bacterial sludge from the three strains is collected and mixed, then mixed with an aqueous solution of an anti-stress matrix carrier, and freeze-dried to obtain a probiotic lyophilized powder. *Tryptophan metabolic substrate, neurotransmitter synthesis cofactor, and prebiotic carrier are mixed, and an aqueous binder solution is added to form spherical pellet cores. *The probiotic lyophilized powder is mixed with anhydrous ethanol binder solution and sprayed onto the surface of the spherical pellet cores to obtain microspheres with a probiotic active layer. *An enteric coating solution is prepared by mixing acrylic resin, triethyl citrate, and talc. *The microspheres with the probiotic active layer are spray-coated and dried to obtain probiotic pellets.

9. The preparation method according to claim 8, characterized in that, The culture medium for culturing Lactobacillus rhamnosus is MRS liquid medium, and the culture medium for culturing Ruminococcus luteus and Broutbacter vegetans is anaerobic BHI medium; the adhesive includes hydroxypropyl methylcellulose, and the mass-volume concentration of the adhesive in the aqueous solution of the adhesive is 3%~5%; the acrylic resin includes Eudragit L30D-55, and the coating weight gain is 8%~15%.

10. The use of the probiotic composition of claim 1, the probiotic freeze-dried powder of any one of claims 2-4, or the probiotic pills of any one of claims 5-7 in the preparation of anti-shipborne compound stress products, characterized in that, The shipboard combined stress includes psychological anxiety, cognitive decline, and / or intestinal constipation.

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