Immunoregulation antiallergic compound powder based on non-active yeast and probiotics

Abstract

The invention relates to the field of anti-allergic compositions, and discloses an immunoregulation anti-allergic compound powder based on non-active yeast and probiotics, and the immunoregulation anti-allergic compound powder comprises the following components in parts by mass: 15-30 parts of non-active yeast powder, 10-25 parts of a probiotic composition, 40-55 parts of a prebiotic composition, 4-6 parts of fucoidin, 0.5-1.5 parts of N-acetylneuraminic acid, and 5-10 parts of an auxiliary material; wherein the prebiotic composition is prepared from resistant dextrin, stachyose, fructo-oligosaccharide and galactooligosaccharide; the auxiliary materials comprise at least two of anhydrous dextrose, acerola cherry powder, elderberry powder, sorbitol and yeast beta-glucan. Through the coupling design of low-temperature mixing and a step-by-step embedding process, the mobility of the powder is maintained, and meanwhile, the activity of thermosensitive components is kept. Compared with a conventional high-temperature mixing or disordered adding process, the problems of component damage and function loss caused by a heat effect or mechanical shearing are solved.

Description

Technical Field

[0001] The invention relates to the technical field of anti-allergic compositions, in particular to an immunomodulatory anti-allergic composite powder based on inactive yeast and probiotics. Background Art

[0002] The immunomodulatory anti-allergic compound powder with inactive yeast and probiotics as the core is mainly aimed at the field of functional food and dietary supplements. This type of product integrates yeast-derived β-glucan, probiotic metabolites and intestinal barrier repair ingredients (L-glutamine) to regulate the intestinal immune microenvironment and relieve allergic reactions. In the existing technology, the inactive yeast wall breaking process, probiotic embedding technology and powder formula design have formed a basic preparation framework. Related products are mostly in the form of granules or capsules, involving conventional processes such as high-pressure homogenization, spray drying, and vacuum packaging.

[0003] In the existing technology, the activity retention and functional synergy of composite powders are limited: the packaging solution relies on a single barrier method (ordinary aluminum foil or vacuum), which cannot simultaneously inhibit oxidation and moisture absorption, resulting in the breakage of the β-glucan molecular chain and the accelerated attenuation of probiotics; the formula design mostly adopts mechanical compounding (simple combination of probiotics and prebiotics), lacks the targeted synergy of multi-component metabolic pathways, and it is difficult to achieve the linkage effect of immune regulation and allergy inhibition; high-temperature mixing or high-pressure shearing in the production process can easily destroy the crystal structure of heat-sensitive ingredients (vitamin C), causing functional loss; the selection of excipients focuses on conventional fillers (maltodextrin), ignoring the dynamic effect of hygroscopicity on powder fluidity and packaging stability, resulting in high agglomeration rate and significant fluctuations in packaging efficiency in industrial production. The above defects jointly restrict the product's activity retention rate, functional accuracy and feasibility of large-scale production. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides an immunomodulatory anti-allergic composite powder based on inactive yeast and probiotics, which solves the problems of insufficient stability of active ingredients, low synergistic efficiency of functional components, lack of protection mechanism for heat-sensitive substances and poor adaptability to industrial production in the prior art.

[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: an immunomodulatory anti-allergic composite powder based on inactive yeast and probiotics, comprising the following components by mass: 15-30 parts of inactive yeast powder, 10-25 parts of probiotic composition, 40-55 parts of prebiotic composition, 4-6 parts of fucoidan, 0.5-1.5 parts of N-acetylneuraminic acid, and 5-10 parts of auxiliary materials; Wherein, the prebiotic composition comprises resistant dextrin, stachyose, oligofructose and oligogalactose; The excipients include at least two of anhydrous glucose, acerola cherry powder, elderberry powder, sorbitol, and yeast β-glucan.

[0006] Immune synergy between inactivated yeast and probiotics: The cell wall component (β-glucan) of inactivated yeast activates pattern recognition receptors (TLR2 / 4) on the surface of intestinal immune cells, enhancing the initiation efficiency of immune responses; while specific probiotics (Bifidobacterium breve M-16V, Lactobacillus rhamnosus GG) directly regulate the Th1 / Th2 balance and inhibit the excessive secretion of allergy-related IgE. The combination of the two forms a "double signal amplification" of immune regulation.

[0007] Functional complementarity between fucoidan and N-acetylneuraminic acid: Fucoidan inhibits mast cell degranulation (reducing histamine release), blocking the effector stage of allergic reactions; N-acetylneuraminic acid, as a sialic acid precursor, modifies the cell surface glycan structure and inhibits the adhesion of allergens to immune cells. The two weaken the allergic cascade reaction from different links.

[0008] Prebiotic-driven metabolic regulation: Prebiotics such as resistant dextrin and stachyose directionally promote the production of butyric acid by Bifidobacterium, enhancing intestinal barrier function, reducing the intestinal leakage and penetration of allergens. At the same time, butyric acid inhibits the inflammatory signaling pathway by activating the GPR109A receptor.

[0009] Preferably, the probiotic composition includes the following strains: Bifidobacterium breve M-16V 8.0×10^9 - 1.2×10^10 CFU / g, Lactobacillus rhamnosus GG 1.8×10^10 - 2.2×10^10 CFU / g, Bifidobacterium animalis subsp. lactis Bb-12 4.5×10^9 - 5.5×10^9 CFU / g, Pediococcus pentosaceus CECT8330 2.5×10^9 - 3.5×10^9 CFU / g.

[0010] Bifidobacterium breve M-16V (8.0×10^9 - 1.2×10^10 CFU / g) as the core strain, its high-activity design (the highest CFU proportion) stems from its unique ability to synthesize extracellular polysaccharides. This strain preferentially colonizes in the proximal colon, and the extracellular polysaccharides (EPS) secreted by it bind to the MUC2 protein in the intestinal mucus layer, forming a physical barrier to reduce the direct contact between allergens (β-lactoglobulin) and intestinal epithelial cells. At the same time, its metabolite butyric acid inhibits the NF-κB-mediated inflammatory response by activating the PPARγ signaling pathway, significantly reducing the levels of Th2 cytokines such as IL-4 and IL-13.

[0011] The activity of Lactobacillus rhamnosus GG (1.8×10^10 - 2.2×10^10 CFU / g) is set higher than the conventional dose (usually ≤1×10^10 CFU / g) to strengthen its regulatory effect on the Th1 / Th2 balance. This strain binds to the TLR2 receptor of intestinal epithelial cells through surface proteins P40 / P75, induces dendritic cells to secrete IL-12, promotes the differentiation of Th1 cells and inhibits Th2-type immune deviation. In addition, the lactic acid it produces reduces the intestinal pH value, inhibits the growth of conditional pathogenic bacteria (Clostridium perfringens), and reduces the systemic allergic reactions caused by endotoxin translocation.

[0012] The activity ratio of Bifidobacterium animalis subsp. lactis Bb-12 (4.5×10^9 - 5.5×10^9 CFU / g) focuses on immunoglobulin regulation. This strain generates acetic acid by metabolizing dietary fiber, stimulates the differentiation of B cells into IgA+ plasma cells in Peyer's patches, and increases the level of secretory IgA (sIgA). sIgA can wrap allergens to form immune complexes, preventing them from passing through the intestinal barrier and entering the blood circulation, thereby reducing systemic allergic responses.

[0013] The introduction of Pediococcus pentosaceus CECT8330 (2.5×10^9 - 3.5×10^9 CFU / g) breaks through the limitations of traditional probiotic combinations. This strain still maintains high metabolic activity in the distal small intestine with low oxygen. The bacteriocin (pediocin) it secretes can specifically inhibit the proliferation of Staphylococcus aureus histamine-producing bacteria, reduce the histamine concentration in the intestine, and reduce the release of allergic mediators from the source. At the same time, the γ-aminobutyric acid (GABA) it produces regulates the stability of mast cells through the vagus nerve-gut-brain axis, further inhibiting histamine release.

[0014] Preferably, the mass ratio of stachyose to galactooligosaccharide in the prebiotic composition is 2.8:1 - 3.2:1, and resistant dextrin accounts for 20 - 30% of the total mass of the prebiotic composition.

[0015] The mass ratio of stachyose to galactooligosaccharide (2.8:1 - 3.2:1) is a core parameter verified by the fermentation kinetics model. As an α-galactoside oligosaccharide, stachyose is preferentially degraded by the α-galactosidase of Bifidobacterium, rapidly ferments in the proximal colon to generate acetic acid and lactic acid, rapidly reduces the intestinal pH value, and inhibits the proliferation of pathogenic bacteria; while the enzymatic hydrolysis rate of galactooligosaccharide (β-1,6 bond type) is slower, and it is mainly metabolized to butyric acid in the middle colon. The control of the ratio of the two ensures: Complementary metabolites: Acetic acid plays an antibacterial role in the proximal colon, and butyric acid activates the PPARγ pathway of intestinal epithelial cells in the middle and distal parts, enhancing the expression of tight junction proteins; Microflora proliferation synergy: After stachyose stimulates the rapid proliferation of Bifidobacterium, galactooligosaccharide serves as a continuous substrate to maintain its metabolic activity and avoid drastic fluctuations in the microflora population.

[0016] The design of the resistant dextrin proportion (20 - 30%) targets the "long-term regulation" of intestinal microflora metabolism. As an indigestible glucan, the β-1,2 / 1,3 glycosidic bond structure of resistant dextrin is slowly fermented by Bifidobacterium in the distal colon, producing a continuous low concentration of butyric acid. This property brings two innovative values: Maintenance of intestinal environmental homeostasis: The butyric acid concentration (0.5 - 1.2 mM) in the distal colon induces the differentiation of regulatory T cells (Treg) by activating the GPR109A receptor, inhibits Th2-type immune responses, and at the same time avoids the toxicity of high-concentration butyric acid to some microflora; Continuation of metabolite signals: The sustained-release effect of resistant dextrin prolongs the inhibitory effect of butyric acid on histone deacetylase (HDAC), enhancing the persistence of the expression of immune tolerance-related genes (Foxp3).

[0017] Preferably, the mass ratio of fucoidan to N-acetylneuraminic acid is 4:1 - 12:1.

[0018] The high proportion design of fucoidan (4 - 12 parts) stems from its direct regulatory ability on immune cells due to its sulfated polysaccharide structure. The sulfate groups of fucoidan can specifically bind to the Siglec-8 receptor on the surface of mast cells, inhibit the phosphorylation of Syk kinase downstream of the FcεRI signaling pathway by activating the SHP-1 phosphatase, and block calcium ion influx and histamine release. At the same time, its branched fucose residues bind to the C-type lectin receptor (DC-SIGN) on the surface of intestinal dendritic cells, inducing the secretion of IL-10 and promoting the differentiation of regulatory T cells (Treg), thus establishing a basis for immune tolerance at the initiation stage (sensitization period) of allergic reactions.

[0019] The low proportion addition of N-acetylneuraminic acid (0.5 - 1.5 parts) focuses on the precise regulation of cell membrane surface modification. As a precursor substance of sialic acid, N-acetylneuraminic acid is integrated into the ends of glycoproteins on the surfaces of intestinal epithelial cells and immune cell membranes through the ST6Gal-I sialyltransferase, forming a negatively charged sialic acid cap structure. This modification can produce two effects: Steric hindrance effect: The electrostatic repulsion of the sialic acid layer hinders the physical contact between allergens (pollen protein Derp1) and the receptors (TLR4) on the surface of immune cells; Epitope masking effect: The sialic acid at the end of the sugar chain masks the antigenic determinant of the allergen, reducing the probability of its recognition by IgE antibodies.

[0020] Preferably, the addition amount of yeast β-glucan in the excipient is 0.5 - 1.5 parts.

[0021] The low-dose design (0.5 - 1.5 parts) of yeast β-glucan stems from the need for its synergistic effect with β-glucan in inactivated yeast powder. The β-glucan in inactivated yeast powder mainly has a short-chain branched structure, which can quickly bind to the Dectin-1 receptor on the surface of macrophages and induce the transient release of pro-inflammatory factors such as IL-1β and IL-6; while the yeast β-glucan in the excipient mainly has a long-chain linear structure, and continuously releases active fragments through slow depolymerization, prolonging the activation time of TLR2 / 4 receptors. The mass ratio of the two (inactivated yeast β-glucan: excipient yeast β-glucan ≈ 10:1 - 20:1) ensures that: Dynamic balance of immune signals: The rapid stimulation of short-chain β-glucan initiates innate immune responses, and the slow-release effect of long-chain β-glucan maintains the continuous secretion of regulatory cytokines (IL-10), preventing excessive immune responses; Complementary receptor activation: The co-activation of Dectin-1 and TLR2 / 4 forms signal cross-talk, amplifying the downstream NFAT signaling pathway and promoting the differentiation of Foxp3+ Treg cells.

[0022] Metabolic competition regulation: Yeast β-glucan is preferentially degraded by Bifidobacterium in the colon into β-1,3 oligosaccharide fragments, which compete with β-glucan from inactivated yeast for binding to immune cell receptors, dynamically regulating the intensity of inflammatory signals; Physical barrier strengthening: The linear structure of yeast β-glucan forms a network scaffold in the intestinal mucus layer, jointly enhancing the mechanical strength of the mucus layer with the short-chain β-glucan of inactivated yeast and reducing the penetration of allergens.

[0023] Compared with the traditional method of using a single-source β-glucan alone or adding doses randomly, the core breakthrough of the present invention lies in: Structure-function adaptation: Through the quantitative compounding of linear and branched β-glucans, multiple binding sites of immune cell receptors are covered (the sugar chain binding domain of Dectin-1 and the lipid binding domain of TLR); Dose threshold control: When the addition amount is less than 0.5 parts, an effective network scaffold structure cannot be formed; when it is higher than 1.5 parts, excessive linear β-glucan causes excessive thickening of the mucus layer, hindering the absorption of nutrients. This range achieves the optimal balance between immune activation and physiological functions; Microbiota-host interaction: Yeast β-glucan, as a metabolic substrate for Bifidobacterium, its degradation product (β-1,3 oligosaccharide) can up-regulate the expression of EPS synthesis genes of Bifidobacterium, indirectly enhancing the colonization advantage of probiotics.

[0024] A preparation method of an anti-allergy composite powder based on the above is provided, including the following steps: S1. Pretreat the probiotics by double-layer embedding with sodium alginate-chitosan, and disrupt the cell walls of inactive yeast powder by high-pressure homogenization; Double-layer embedding with sodium alginate-chitosan: Through the charge complementarity of sodium alginate (outer layer) and chitosan (inner layer) (sodium alginate is negatively charged and chitosan is positively charged), a pH-responsive microcapsule is formed. In the low-pH environment of gastric juice, the sodium alginate layer shrinks to form a dense structure to protect the probiotics from passing through the gastric acid environment; after entering the neutral environment of the intestine, the chitosan layer interacts with the positive charges of the mucus layer, promoting the adhesion of the microcapsule and the slow release of live bacteria, increasing the intestinal colonization efficiency by more than 30%.

[0025] High-pressure homogenization for cell wall disruption: Inactive yeast powder undergoes cavitation effect under ultra-high pressure of 150 - 180 MPa, and the crystalline region of β-1,3 / 1,6-glucan in the cell wall is damaged, releasing soluble linear β-glucan fragments. Compared with the traditional enzymatic method, this process increases the immune activation activity of β-glucan (evaluated by macrophage phagocytosis rate) by 2.3 times and avoids the risk of component degradation caused by enzyme residues.

[0026] S2. Mix the pretreated components with prebiotic composition, fucoidan, and excipients at 4 - 8 °C; Protection of heat-sensitive components: Low temperature conditions (below the probiotic metabolism activation threshold) inhibit the energy consumption of bacterial metabolism, keeping the survival rate of the embedded probiotics above 95%; at the same time, the sulfate groups of fucoidan maintain a stable conformation at low temperature, avoiding desulfation inactivation caused by high temperature.

[0027] Optimization of interfacial interaction: At low temperature, the hydrogen bond binding force between prebiotic (stachyose) and the product of inactivated yeast cell wall disruption is enhanced, forming a uniform blend network structure. This network can delay the moisture absorption and swelling of probiotic microcapsules, ensure the fluidity of the powder (angle of repose ≤ 35°), and lay a physical foundation for subsequent moisture control.

[0028] S3. Subpackage and control the water activity ≤ 0.25; Control of water activity: An environment with Aw ≤ 0.25 puts the probiotics in a dormant state (water molecules are not sufficient to participate in intracellular enzyme reactions), and at the same time inhibits the browning reaction of fucoidan (the Maillard reaction rate is reduced by 80%). This threshold is achieved through the dual mechanisms of freeze-drying and moisture absorption locking by excipients (sorbitol), avoiding local water accumulation.

[0029] Nitrogen filling and subpackaging: Replace oxygen with 99.9% high-purity nitrogen to make the residual oxygen content in the package ≤ 0.5%. This design not only prevents the oxidative degradation of β-glucan (the generation amount of peroxide is reduced by 70%), but also blocks the survival probability of aerobic miscellaneous bacteria (Bacillus), making the attenuation rate of live bacteria number within the shelf life ≤ 0.5 logCFU / month.

[0030] Preferably, the process parameters for embedding probiotics in S1 include: The concentration of sodium alginate solution is 1.5-2.0% (w / v), and the cross-linking time is 30-40 minutes; Chitosan solution concentration 0.5-1.0% (w / v), cross-linking time 20-30 minutes; The particle size of microcapsules is 20-50μm.

[0031] The combination of alginate concentration of 1.5-2.0% (w / v) and cross-linking time of 30-40 minutes is designed to meet the extreme challenges of gastric acid environment. The guluronic acid (G unit) in the sodium alginate chain forms an "egg box" structure under calcium ion cross-linking. When the concentration is lower than 1.5%, the network structure is loose and cannot resist the penetration of gastric acid (pH1.5-3.5); when it is higher than 2.0%, the gel porosity is too low, which hinders the diffusion of probiotic metabolites and leads to a decrease in the viable bacterial rate after embedding. The concentration of 1.5-2.0% combined with 30-40 minutes of cross-linking makes the microcapsule shell form a dense but not completely closed grid (pore size 5-8nm), which can block gastric acid (H + ions with a diameter of about 0.1nm) invade, while allowing small molecule nutrients (glucose, with a diameter of about 0.7nm) to penetrate, maintaining the basal metabolic activity of the bacteria.

[0032] The synergistic effect of chitosan concentration of 0.5-1.0% (w / v) and cross-linking time of 20-30 minutes focuses on intestinal targeted release and mucus layer adhesion. The cationic properties of chitosan cause it to electrostatically adsorb with negatively charged mucin (MUC2) in the intestinal mucus layer, but when the concentration is higher than 1.0%, the excessive positive charge density will cause excessive contraction of the mucus layer, which will reduce the adhesion efficiency. After 20-30 minutes of cross-linking, the 0.5-1.0% chitosan solution forms an inner layer with a thickness of about 2-5μm. Its deacetylation degree (≥85%) ensures slow dissolution in the neutral environment of the intestine. While releasing live bacteria, the chitosan degradation product (low molecular weight chitosan oligosaccharide) can activate the TLR4 receptor of intestinal epithelial cells and enhance the expression of tight junction proteins, forming a dual effect of "release-repair".

[0033] The limitation of microcapsule particle size of 20-50μm is due to the adaptation requirements of the intestinal physiological structure. Microcapsules with a particle size less than 20μm are easily taken up by small intestinal epithelial cells (M cells) and cannot reach the colon colonization site; particles larger than 50μm are easily broken during transportation due to mechanical friction. The particle size range of 20-50μm allows the microcapsules to remain stable during intestinal peristalsis, and through the size-dependent Peyer's patches avoidance mechanism, it ensures that more than 80% of the embedded bacteria safely reach the colon. In addition, the surface area / volume ratio corresponding to this particle size (0.12-0.25μm -1The contact efficiency between the chitosan layer and the intestinal mucosa is optimized, increasing the adhesion strength by 1.8 - 2.5 times.

[0034] Preferably, the parameters for the wall-breaking treatment of the inactive yeast powder in S1 include: High-pressure homogenization pressure of 150 - 180 MPa; Number of cycles of 2 - 4 times; The content of β-glucan after wall-breaking is ≥25%.

[0035] The setting of the high-pressure homogenization pressure of 150 - 180 MPa is based on the mechanical properties of the yeast cell wall. The β-glucan layer of the yeast cell wall cross-links in a triple-helix structure to form nanofibers (with a diameter of about 20 - 50 nm), and its fracture strength threshold is about 130 MPa. Pressures below 150 MPa cannot effectively break the hydrogen bond network between the fibers, resulting in insufficient wall-breaking rate (<60%); while when the pressure is higher than 180 MPa, excessive shear force will break the main chain of β-glucan, generating ineffective fragments with a molecular weight <10 kDa. The pressure range of 150 - 180 MPa, through the synergistic effect of the micro-jet (speed ≥100 m / s) generated by the cavitation effect and the shear force, preferentially destroys the crystalline region of β-glucan (retaining the amorphous region intact), releasing linear β-1,3 glucan with a molecular weight of 50 - 200 kDa (the core immunologically active structure), increasing the yield of water-soluble β-glucan to 2.3 times that of the traditional process.

[0036] The design of the number of cycles of 2 - 4 times focuses on the balance between wall-breaking efficiency and activity retention. The first cycle (pressure of 150 MPa) mainly breaks intact cells, releasing about 60% of the β-glucan inside the wall; the second cycle (pressure increased to 180 MPa) further dissociates the unbroken cell fragments; the third and subsequent cycles break the structure of the released β-glucan. Through experimental verification, 2 - 4 cycles can achieve a wall-breaking rate of 85 - 92%, while ensuring that the immunological activity of β-glucan (evaluated by the activation rate of the macrophage NF-κB pathway) is retained ≥90%. When the number of cycles exceeds 4 times, the helix structure unwinding rate of β-glucan exceeds 30%, resulting in a decrease in its Dectin-1 receptor binding ability by more than 40%.

[0037] The quality standard of β-glucan content ≥25% is the core index of the process effect. This threshold is guaranteed through the following mechanisms: Crystalline structure directional dissociation: High-pressure homogenization preferentially breaks the β-1,6 side-chain connection points of β-glucan, releasing high-purity β-1,3 main-chain fragments (accounting for ≥70% of the total amount), avoiding the interference of mannoprotein impurities; Low-temperature protection: During the homogenization process, the temperature of the feed liquid is controlled at 10 - 15 °C to prevent the oxidative degradation of β-glucan caused by the thermal effect (the residual amount of peroxide ≤0.02%), ensuring the stability of its immunologically active conformation.

[0038] Preferably, the mixing process in S2 includes: Adding L-glutamine microcapsules with an encapsulation efficiency ≥ 90%; The mixing speed is 15 - 25 rpm and the time is 8 - 12 minutes.

[0039] The high encapsulation efficiency (≥ 90%) of L-glutamine microcapsules is the key guarantee for intestinal targeted delivery. As the main energy substrate of intestinal epithelial cells, the free form of L-glutamine is easily degraded into pyroglutamic acid and inactivated in gastric acid. The microcapsule structure (particle size 10 - 30 μm) embedded with sodium alginate-chitosan bilayer can maintain integrity (swelling rate ≤ 5%) at low pH in gastric juice and slowly release L-glutamine through enzymatic hydrolysis of chitosan after entering the intestine. The design with an encapsulation efficiency ≥ 90% ensures: Persistence of intestinal epithelial repair: The concentration of L-glutamine released by the decomposition of microcapsules in the colon is maintained at 2 - 4 mM (optimal repair concentration), which promotes the proliferation of intestinal epithelial cells by activating the mTOR signaling pathway, increasing the expression levels of tight junction proteins (ZO-1, Occludin) by 1.5 - 2 times; Synergy of immunomodulation: The released L-glutamine is metabolized by intestinal flora into precursors of butyric acid, which synergistically enhances the differentiation of regulatory T cells (Treg) with the prebiotic composition and inhibits Th2-type allergic responses.

[0040] The parameter combination of a mixing speed of 15 - 25 rpm and a time of 8 - 12 minutes focuses on the balance between the protection of the microcapsule structure and the dispersion efficiency of components. Low-shear mixing (shear rate ≤ 50 s -1 ) achieves a technological breakthrough through the following mechanisms: Protection of microcapsule integrity: When the rotation speed exceeds 25 rpm, the fluid shear force exceeds the yield strength of the chitosan layer of the microcapsule (about 1.2 kPa), resulting in a decrease in the encapsulation rate to < 85%; while when it is lower than 15 rpm, the powder fluidity is insufficient (repose angle ≥ 40°), causing local caking. The rotation speed range of 15 - 25 rpm controls the shear stress on the microcapsules at 0.8 - 1.1 kPa, ensuring that the loss of encapsulation efficiency ≤ 3%; Maintenance of the stability of active ingredients: The mixing time of 8 - 12 minutes matches the minimum requirement for powder homogenization (mixing uniformity ≥ 95%), and at the same time avoids the temperature rise caused by long-term mechanical action (material temperature ≤ 8°C), preventing the hydrolysis of the sulfate groups of fucoidan or the premature swelling of probiotic microcapsules.

[0041] Preferably, in S3, the sub-packaging uses aluminum foil bags filled with nitrogen, the purity of the filled nitrogen ≥ 99.9%, and the residual oxygen content ≤ 0.5%.

[0042] The multi-layer composite structure of the aluminum foil bag (outer layer polyester / middle layer aluminum foil / inner layer polyethylene) is the core of physical protection. The light reflectivity of the aluminum foil layer (thickness ≥ 7μm) is ≥ 95%, blocking the photolysis of the sulfate group of fucoidan by ultraviolet rays (280 - 400nm); the water vapor transmission rate of the polyethylene layer (thickness ≥ 50μm) is ≤ 0.1g / (m 2 ·day), combined with the control of water activity before packaging (Aw ≤ 0.25), completely isolating the penetration of environmental moisture and preventing the premature activation of probiotics caused by the hygroscopic swelling of microcapsules.

[0043] The chemical protection design with nitrogen filling purity ≥ 99.9% and residual oxygen content ≤ 0.5% focuses on blocking the source of the oxidation chain reaction: Ultra-high purity nitrogen (oxygen content ≤ 0.1%) is used as an inert gas medium to displace the residual oxygen between powder particles, making the initial oxygen concentration in the package ≤ 0.3%. This purity threshold ensures: The oxidation breakage rate of the β-1,3 glycosidic bond of β-glucan is ≤ 1% / year (≥ 5% in traditional packaging); The loss rate of vitamin C (an antioxidant) in acerola cherry powder is ≤ 10% / 24 months, maintaining its function of inhibiting the accumulation of ROS in mast cells; The strict limit of the residual oxygen content ≤ 0.5% is based on the critical oxidation concentration (COC) theory. When the oxygen partial pressure is lower than 0.5%, the propagation rate of the free radical chain reaction (lipid peroxidation) approaches zero, reducing the oxidation crosslinking rate of the chitosan layer of the probiotic microcapsule to ≤ 0.2% / month, ensuring its controllable disintegration performance in the intestine.

[0044] The present invention provides an immunomodulatory anti-allergy composite powder based on inactivated yeast and probiotics. It has the following beneficial effects: 1. The present invention adopts a "physical barrier - chemical inertness" collaborative packaging design. Through the quantitative matching of the aluminum foil multi-layer structure and the high-purity nitrogen filling process, the full-cycle anti-oxidation and moisture-proof protection of active ingredients are achieved. Compared with the single vacuum or ordinary nitrogen filling schemes in the prior art, it solves the stability defects of probiotic inactivation and functional ingredient degradation caused by oxygen residue or moisture penetration.

[0045] 2. Based on the formula system of multi-component functional synergy, the present invention combines probiotic microcapsules, broken-wall yeast β-glucan and fucoidan in a targeted manner to form a multi-target action network of "immunomodulation - intestinal barrier repair - allergy inhibition". Compared with traditional single-component or simple compounding schemes, it solves the technical bottleneck of single efficacy and lack of synergistic effect.

[0046] 3. Through the coupled design of low-temperature mixing and step-by-step embedding processes, the present invention ensures the retention of the activity of heat-sensitive components (vitamin C, L-glutamine) while maintaining the fluidity of the powder. Compared with conventional high-temperature mixing or disordered addition processes, it solves the problems of component damage and function loss caused by thermal effects or mechanical shearing.

[0047] 4. The present invention innovatively introduces a moisture absorption inhibition auxiliary material and a dynamic balance mechanism for the sealed microenvironment. By utilizing the moisture absorption regulation ability of functional auxiliary materials (resistant dextrin) and combining with the low-oxygen and low-humidity conditions inside the packaging, it realizes the efficient and stable operation of industrial packaging. Compared with the existing solutions that rely on single desiccants or conventional auxiliary materials, it solves the problems of low packaging efficiency and insufficient product uniformity caused by moisture absorption caking and poor fluidity. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a schematic flow chart of the method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0049] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0050] Example 1 (basic type) Step S1: Pretreatment Probiotic embedding: Strain: Bifidobacterium animalis subsp. lactis BB-12; Sodium alginate concentration: 1.8% (w / v), cross-linking time 35 minutes (CaCl2 solution concentration 2.5%); Chitosan concentration: 0.8% (w / v, deacetylation degree ≥ 85%), cross-linking time 25 minutes; Microcapsule particle size: 35 ± 5 μm, embedding rate 96.2%.

[0051] Inactivated yeast powder cell wall breaking: High-pressure homogenization pressure: 170 MPa, circulating 3 times; β-glucan content after cell wall breaking: 27.5% (detected by HPLC).

[0052] Step S2: Low-temperature mixing Mixing temperature: 6°C; Mixing speed: 20 rpm, time 10 minutes.

[0053] Added components: Prebiotic composition (stachyose: galactooligosaccharide = 3:1); Fucoidan (purity ≥ 95%); L-glutamine microcapsules (encapsulation efficiency 92%, particle size 25 μm); Excipient: resistant dextrin (proportion 25%); Mixing uniformity: relative standard deviation (RSD) ≤ 4.5%.

[0054] Step S3: Sub-packaging Water activity control: 0.22 (lyophilization + sorbitol adsorption).

[0055] Packaging parameters: Nitrogen filling purity in aluminum foil bag: 99.95%, residual oxygen content 0.3%; Sub-packaging speed: 100 bags / minute.

[0056] Example 2 (high activity type) Step S1: Pretreatment Probiotic embedding: Strain: Bifidobacterium lactis HN019 + Lactobacillus reuteri DSM17938; Sodium alginate concentration: 2.0% (w / v), cross-linking time 40 minutes (CaCl2 solution concentration 3.0%); Chitosan concentration: 1.0% (w / v, deacetylation degree ≥ 90%), cross-linking time 30 minutes; Microcapsule particle size: 45 ± 5 μm, encapsulation efficiency 97.8%.

[0057] Inactive yeast powder cell wall breaking: High-pressure homogenization pressure: 180 MPa, 4 cycles; β-glucan content after cell wall breaking: 30.1% (detected by HPLC).

[0058] Step S2: Low-temperature mixing Mixing temperature: 4°C; Mixing speed: 25 rpm, time 8 minutes.

[0059] Added components: Prebiotic composition (stachyose: galactooligosaccharide = 3.2:1); Fucoidan (purity ≥ 98%); L-glutamine microcapsules (encapsulation efficiency 94%, particle size 15 μm); Excipient: resistant dextrin (proportion 30%) + yeast β-glucan (1.2 parts); Mixing uniformity: RSD ≤ 3.8%.

[0060] Step S3: Sub-packaging Water activity control: 0.18 (spray drying + erythritol).

[0061] Packaging parameters: Nitrogen filling purity in aluminum foil bag: 99.98%, residual oxygen content 0.4%; Sub-packaging speed: 80 bags / minute (thick bag with enhanced sealing).

[0062] Example 3 (quick-acting type) Step S1: Pretreatment Probiotic encapsulation: Strain: Bifidobacterium breve M-16V; Sodium alginate concentration: 1.5% (w / v), cross-linking time 30 minutes (CaCl2 solution concentration 2.0%); Chitosan concentration: 0.5% (w / v, deacetylation degree ≥ 80%), cross-linking time 20 minutes; Microcapsule particle size: 25 ± 5 μm, encapsulation rate 93.5%.

[0063] Inactivated yeast powder cell wall breaking: High-pressure homogenization pressure: 150 MPa, 2 cycles; β-glucan content after cell wall breaking: 25.6% (detected by HPLC).

[0064] Step S2: Low-temperature mixing Mixing temperature: 8°C; Mixing speed: 15 rpm, time 12 minutes.

[0065] Added components: Prebiotic composition (stachyose: galactooligosaccharide = 2.8:1); Fucoidan (purity ≥ 90%); L-glutamine microcapsules (encapsulation rate 90%, particle size 30 μm); Excipients: resistant dextrin (proportion 20%) + acerola cherry powder (vitamin C content 5%); Mixing uniformity: RSD ≤ 5.2%.

[0066] Step S3: Sub-packaging Water activity control: 0.24 (vacuum drying + maltitol).

[0067] Packaging parameters: Nitrogen filling purity in aluminum foil bag: 99.9%, residual oxygen content 0.5%; Sub-packaging speed: 120 bags / minute (thin bag with high-speed sub-packaging).

[0068] Comparative Example 1 (corresponding to Example 1): Compared with Example 1, the difference is that the L-glutamine microcapsules are removed, and the rest are the same.

[0069] Comparative Example 2 (corresponding to Example 1): Compared with Example 1, the difference is that the high-pressure homogenization pressure is adjusted to 130 MPa (circulated 3 times), and the rest are the same.

[0070] Comparative Example 3 (corresponding to Example 2): Compared with Example 2, the difference is that the chitosan concentration is adjusted to 0.3% (w / v), and the rest are the same.

[0071] Comparative Example 4 (corresponding to Example 2): Compared with Example 2, the difference is that the yeast β-glucan excipient is removed, and the rest are the same.

[0072] Comparative Example 5 (corresponding to Example 3): Compared with Example 3, the difference is that the nitrogen filling purity is adjusted to 99.5% (residual oxygen content 1.5%), and the rest are the same.

[0073] Comparative Example 6 (corresponding to Example 3): Compared with Example 3, the difference is that the fucoidan is removed, and the rest are the same.

[0074] Experiment 1: Comprehensive verification of the stability of active ingredients Experiment description and steps Sample preparation: Example group: Take 10 g of the finished powder of Examples 1, 2, and 3 respectively; Comparative example group: Take 10 g of the powders of Comparative Example 1 (without L-glutamine microcapsules), Comparative Example 2 (homogenization pressure 130 MPa), and Comparative Example 5 (nitrogen filling purity 99.5%).

[0075] Simulated gastric juice treatment: Mix each sample with simulated gastric juice (pH 2.0, containing 0.3% pepsin) at a ratio of 1:10, and shake (150 rpm) at 37 °C for 2 hours; Centrifuge to obtain the precipitate, wash it with PBS, and then detect the viable bacteria count (CFU / g).

[0076] Accelerated oxidation experiment: Dispense the powder into transparent glass bottles, place them in a constant temperature incubator at 40 °C / 75% RH for 30 days; Detect the β-glucan content (HPLC method) and vitamin C content (iodometric method).

[0077] Long-term storage test: The powder is stored sealed in an environment of 25°C / 60%RH, and the viable bacteria survival rate and β-glucan retention rate are detected after 12 months.

[0078] The experimental data are shown in Table 1 below: Table 1: Comparison of the stability of active ingredients The viable bacteria survival rate in the examples is significantly higher than that in the comparative examples (the survival rate in Example 1 is 94.5% vs. the survival rate in Comparative Example 2 is 82.3%). The core lies in the synergistic protection effect of the double-layer embedded microcapsules. The dense grid structure (pore size 5-8 nm) formed by the sodium alginate concentration (1.5-2.0%) and the cross-linking time (30-40 minutes) effectively blocks the penetration of gastric acid (H + diameter 0.1 nm), while allowing the diffusion of small molecule nutrients (glucose) to maintain the metabolic activity of the bacteria. In Comparative Example 2, due to insufficient homogenization pressure (130 MPa), β-glucan was not fully released, resulting in a lack of metabolic energy source for probiotics and accelerating their decline.

[0079] The high retention rate of β-glucan (≥98% in Examples 1-2) stems from the directional destruction of the crystal structure by high-pressure homogenization (150-180 MPa). This pressure range preferentially dissociates the β-1,6 side chains through the cavitation effect, releasing the linear β-1,3 main chain. Its molecular weight (50-200 kDa) and low degree of branching (side chain ratio ≤15%) can reduce the exposure of oxidation sites. In contrast, in Comparative Example 5, due to insufficient nitrogen filling purity (99.5%), the residual oxygen content (1.5%) triggered a free radical chain reaction, resulting in the breakage of the β-glucan main chain and the retention rate dropping to 92.2%.

[0080] The retention rate of vitamin C in Example 3 (87.9%) is significantly higher than that in Comparative Example 5 (61.8%), which is directly attributed to the synergistic control of the nitrogen filling purity (≥99.9%) of the aluminum foil bag and the residual oxygen content (≤0.5%). After replacing the oxygen in the powder gap with ultra-high purity nitrogen, the aluminum foil layer (thickness ≥7 μm) further blocks the penetration of external oxygen, reducing the oxidation rate of vitamin C to 0.05% / month (0.23% / month in Comparative Example 5). This result verifies the irreplaceability of the "physical barrier - chemical inertness" dual mechanism in antioxidant protection.

[0081] Experiment 2: Verification of functional synergy Experimental description and procedures Sample grouping: Example group: Example 1 (containing L-glutamine microcapsules), Example 2 (highly active β-glucan), Example 3 (containing fucoidan); Comparative example group: Comparative Example 3 (chitosan concentration 0.3%), Comparative Example 4 (no yeast β-glucan), Comparative Example 6 (no fucoidan).

[0082] Immune regulation effect test: Macrophage culture: RAW264.7 cells were co-incubated with each sample (1 mg / mL) for 24 hours; Detection of NF-κB activation rate: Flow cytometry was used to analyze the nuclear translocation ratio of the p65 subunit.

[0083] Evaluation of intestinal barrier repair function: Caco-2 monolayer model: An intestinal epithelial monolayer was established (TEER ≥ 300 Ω·cm 2 ), and the sample (0.5 mg / mL) was added and treated for 48 hours; Quantification of ZO-1 protein: Immunofluorescence was used to detect the expression level of tight junction proteins.

[0084] Allergy inhibition experiment: Mouse OVA allergy model: Mice were sensitized by intraperitoneal injection of ovalbumin (OVA), and the sample (50 mg / kg) was administered by gavage for 14 consecutive days; Determination of histamine release: ELISA was used to detect the serum histamine concentration.

[0085] The experimental data are shown in Table 2 below: Table 2: Comparison of functional synergy In Examples 1-2, the NF-κB activation rate was significantly higher than that in Comparative Examples 3-4 (the activation rate in Example 2 was 91.8% vs only 47.2% in Comparative Example 4). The core mechanism lies in the molecular conformation regulation of yeast β-glucan. The linear β-1,3-glucan (molecular weight 50-200 kDa) released by high-pressure homogenization (150-180 MPa) activates the Syk signaling pathway through the Dectin-1 receptor, while low homogenization pressure (Comparative Example 2) or excipient deficiency (Comparative Example 4) leads to an increase in the branching degree of β-glucan (the proportion of β-1,6 side chains > 20%), resulting in a decrease in the binding affinity with the receptor by more than 60%.

[0086] In Example 1, the promotion rate of ZO-1 protein (198.7%) was much higher than that in Comparative Example 3 (118.7%), which was attributed to the targeted sustained-release characteristics of L-glutamine microcapsules. The L-glutamine concentration (2-4 mM) released by the sodium alginate-chitosan double-layer microcapsules (particle size 25 μm) in the colon continuously activates the mTORC1 pathway, promoting the migration of intestinal epithelial cells and the assembly of tight junctions. In Comparative Example 3, due to insufficient chitosan concentration (0.3%), the microcapsules disintegrated prematurely in gastric juice (disintegration rate > 50%), and the sudden release of L-glutamine was degraded into ineffective pyroglutamic acid by gastric acid.

[0087] The histamine inhibition rate of Example 3 (73.6%) was significantly different from that of Comparative Example 6 (24.3%), which was due to the charge-mediated effect of the fucose sulfate group (content ≥ 20%). Its sulfated polysaccharide competitively binds to the CD48 receptor on the surface of mast cells, blocks the IgE-FcεRI cross-linking signal, and inhibits calcium ion influx (decrease rate > 70%). After removing the fucose polysaccharide (Comparative Example 6), the allergen directly activates mast cell degranulation, resulting in a three-fold surge in histamine release.

[0088] Experiment 3: Verification of Industrial Applicability Experiment Description and Steps Sample Preparation: Example Group: Example 1 (basic type), Example 2 (high-activity type), Example 3 (quick-acting type); Comparative Example Group: Comparative Example 4 (removing yeast β-glucan), Comparative Example 6 (removing fucose polysaccharide).

[0089] Powder Flowability Test: Angle of Repose Measurement: Using an angle of repose measuring instrument (ASTM D6393 standard), let the powder free fall to form a cone, and measure the inclination angle; Humidity Control: The test environment is 25°C / 60%RH, and the average value is taken after repeating 3 times.

[0090] Sub-packaging Efficiency Test: Fully Automatic Sub-packaging Line: Simulate an industrial production line (nitrogen filling - heat sealing linkage), and record the sub-packaging speed (bags / minute); Sealing Performance Verification: Randomly select 100 bags and detect the residual oxygen content (oxygen analyzer).

[0091] Accelerated Storage Stability Experiment: High Temperature and High Humidity Test: Store for 30 days at 40°C / 75%RH, and detect the viable bacteria attenuation rate (CFU counting method); Powder Caking Rate: Calculate the proportion of caked particles by sieving method (20-mesh sieve).

[0092] The experimental data is shown in Table 3 below: Table 3: Comparison of Industrial Performance In the examples, the angle of repose (32.4 - 34.1°) was significantly lower than that of Comparative Example 4 (44.9°). The core mechanism lies in the moisture absorption regulation of yeast β-glucan. β-glucan preferentially adsorbs environmental moisture through hydrogen bonds (the proportion of bound water ≥ 70%), reducing the liquid bridge force between powder particles. Removing this excipient (Comparative Example 4) led to an increase in the free water activity (Aw) to 0.35, causing particle adhesion and caking. Example 3 further optimized the fluidity (angle of repose 33.8°) through the crystallization inhibition effect of maltitol (the hygroscopicity is only 20% of sorbitol).

[0093] The packaging speed of Example 1 (102 bags / minute) was similar to that of Comparative Example 6 (104 bags / minute) on the surface. However, Example 3 achieved an efficiency breakthrough through thin-bag high-speed packaging (118 bags / minute). The key lies in the precise control of the residual oxygen content (≤0.5%). The low-oxygen microenvironment (oxygen partial pressure ≤ 0.3%) formed by the high purity of nitrogen gas (≥99.9%) in the aluminum foil bag inhibits the electrostatic adsorption of the powder. Removing fucoidan (Comparative Example 6) led to uneven surface charge distribution of the powder. Although the packaging speed reached the standard, the viable bacteria decay rate increased during storage (0.58 log / month).

[0094] The viable bacteria decay rate of Example 2 was the lowest (0.19 log / month), which was due to the dual optimization of water activity (Aw = 0.18) and nitrogen filling purity. The low hygroscopicity of erythritol (moisture adsorption amount ≤ 0.1 g / 100 g) and the hydrophobic modification of chitosan microcapsules (contact angle ≥ 110°) enabled the powder to maintain a glassy state at high temperatures (Tg ≥ 60°C), blocking the microbial metabolic pathway. In Comparative Example 4, due to the lack of excipient, the water activity increased to 0.35 (close to the bacterial growth threshold Aw = 0.6), and the viable bacteria decay rate soared to 0.81 log / month.

[0095] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An immunomodulatory and anti-allergy composite powder based on inactivated yeast and probiotics, characterized in that, It comprises the following components by mass parts: 15 - 30 parts of inactivated yeast powder, 10 - 25 parts of probiotic composition, 40 - 55 parts of prebiotic composition, 4 - 6 parts of fucoidan, 0.5 - 1.5 parts of N-acetylneuraminic acid, 5 - 10 parts of excipients; Among them, the prebiotic composition contains resistant dextrin, stachyose, fructooligosaccharide, galactooligosaccharide; The excipients contain at least two of anhydrous glucose, acerola cherry powder, elderberry powder, sorbitol, yeast β-glucan.

2. The immunomodulatory and anti-allergy composite powder based on inactivated yeast and probiotics according to claim 1, characterized in that, The probiotic composition comprises the following strains: Bifidobacterium breve M-16V 8.0×10^9 - 1.2×10^10 CFU / g, Lactobacillus rhamnosus GG 1.8×10^10 - 2.2×10^10 CFU / g, Bifidobacterium animalis subsp. lactis Bb-12 4.5×10^9 - 5.5×10^9 CFU / g, Pediococcus pentosaceus CECT8330 2.5×10^9 - 3.5×10^9 CFU / g.

3. The immunomodulatory and anti-allergy compound powder based on inactivated yeast and probiotics according to claim 1, wherein The mass ratio of stachyose to galactooligosaccharide in the prebiotic composition is 2.8:1 - 3.2:1, and resistant dextrin accounts for 20 - 30% of the total mass of the prebiotic composition.

4. The immunomodulatory and anti-allergy composite powder based on inactivated yeast and probiotics according to claim 1, wherein The mass ratio of fucoidan to N-acetylneuraminic acid is 4:1 - 12:

1.

5. The immunomodulatory and anti-allergy composite powder based on inactivated yeast and probiotics according to claim 1, wherein The addition amount of yeast β-glucan in the excipients is 0.5 - 1.5 parts.

6. A preparation method of the anti-allergy composite powder according to claim 1, characterized in that, It includes the following steps: S1. Perform sodium alginate-chitosan double-layer embedding pretreatment on probiotics, and perform high-pressure homogenization and cell wall breaking treatment on inactivated yeast powder; S2. Mix the pretreated components with the prebiotic composition, fucoidan, and excipients at 4 - 8°C; S3. Subpackage and control the water activity ≤ 0.

25.

7. An immunomodulatory and anti-allergy composite powder based on inactivated yeast and probiotics according to claim 1, characterized in that, The process parameters for probiotic embedding in S1 include: The concentration of sodium alginate solution is 1.5 - 2.0% (w / v), and the crosslinking time is 30 - 40 minutes; The concentration of chitosan solution is 0.5 - 1.0% (w / v), and the crosslinking time is 20 - 30 minutes; The microcapsule particle size is 20 - 50 μm.

8. An immunomodulatory and anti-allergy composite powder based on inactivated yeast and probiotics according to claim 1, characterized in that, The parameters for cell wall breaking treatment of inactivated yeast powder in S1 include: The high-pressure homogenization pressure is 150 - 180 MPa; The number of cycles is 2 - 4 times; The content of β-glucan after cell wall breaking is ≥ 25%.

9. The immunomodulatory and anti-allergy composite powder based on inactivated yeast and probiotics according to claim 1, characterized in that, The mixing process in S2 includes: Adding L-glutamine microcapsules with an encapsulation efficiency ≥ 90%; The mixing rotation speed is 15 - 25 rpm, and the time is 8 - 12 minutes.

10. The immunomodulatory and anti-allergy composite powder based on inactivated yeast and probiotics according to claim 1, characterized in that, In S3, subpackaging is carried out using nitrogen-filled aluminum foil bags, the nitrogen filling purity is ≥ 99.9%, and the residual oxygen content is ≤ 0.5%.

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