Compound probiotic enzyme powder formula for promoting gastrointestinal micro-ecological balance and nutrient absorption

Abstract

The invention relates to the technical field of biological medicine, and discloses a formula of compound probiotic enzyme powder for promoting gastrointestinal microecological balance and nutrient absorption, by constructing a cross feeding system of bifidobacteria, lactobacillus and butyric acid bacteria and combining a stomach-intestine stage enzyme activity gradient adaptation strategy and a microenvironment response type double-layer embedding technology, the gastrointestinal microecological balance and nutrient absorption can be promoted, and the gastrointestinal microecological balance and nutrient absorption can be promoted. And cross-scale synergy of flora metabolism, substrate conversion and targeted release is realized. Wherein the sodium alginate-pectin composite embedding structure forms a pH buffer micro-area in a gastric acid environment, and proton pump gene expression of the acid-resistant strain is synchronously triggered; the prebiotics compounding and step-by-step enzymolysis process breaks through a closed-loop path of fiber degradation-flora proliferation-short-chain fatty acid generation. Compared with a traditional single-strain intervention scheme, the method has the advantages that double limitations of flora function splitting and process isolation are broken through, and systematic technical advantages are formed in the aspects of improving flora colonization stability, enhancing metabolite conversion efficiency and inhibiting pathogenic bacterium colonization.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically relates to a compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption. Background Art

[0002] The homeostasis regulation of the intestinal microecology is one of the core mechanisms for maintaining human health. Bacterial metabolites such as short-chain fatty acids (SCFAs) directly participate in intestinal barrier repair, immune regulation and pathogenic bacteria inhibition, and their functional diversity highly depends on the interaction network among bacteria. However, factors such as external environmental pressure and dietary structure changes are likely to lead to dysbiosis, causing metabolic disorders and inflammatory reactions, and there is an urgent need to reconstruct the bacterial function through precise intervention.

[0003] Most of the existing technologies use single-functional strains or simple compound strains for microecological regulation. For example, probiotic preparations mainly composed of Bifidobacterium or Lactobacillus inhibit pathogenic bacteria by producing acid or competitive occupation. Some solutions attempt to add prebiotics (such as fructooligosaccharide) or enzyme preparations to assist bacterial colonization, but the single function of the strains and the slow environmental response lead to limited intervention effects, and it is difficult to achieve multi-link cooperation such as acid production, antibacterial, and substrate conversion.

[0004] The above technologies have fundamental defects: single-strain intervention cannot construct a metabolic cascade network. The existing solutions rely on the linear action of single strains, which can neither trigger the metabolic amplification effect of cross-feeding among bacteria nor cover the composite requirements of intestinal barrier repair, immune activation and pathogenic bacteria inhibition. The fragmentation of strain functions leads to low efficiency in the production of short-chain fatty acids and the clearance of pathogenic bacteria, and the microecological regulation shows fragmented characteristics, ultimately restricting the clinical transformation value. Summary of the Invention

[0005] In view of the deficiencies of the existing technology, the present invention provides a compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption, which solves the problems of the lack of metabolic cascade network and the fragmentation of bacterial functions caused by single-strain intervention in the existing technology.

[0006] To achieve the above objectives, the present invention is realized through the following technical solutions: A compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption, which is composed of the following components in parts by mass: Anhydrous glucose: 5 - 15 parts, resistant dextrin: 5 - 20 parts, stachyose: 2 - 8 parts, fructooligosaccharide: 3 - 10 parts, galactooligosaccharide: 2 - 8 parts, acerola cherry powder: 1 - 5 parts, kiwifruit powder: 1 - 5 parts, α - amylase: 1.5 - 4.0 parts, protease: 1.5 - 3.5 parts, lipase: 1.0 - 3.0 parts, maltogenic amylase: 0.5 - 2.0 parts, hemicellulase: 0.5 - 2.0 parts, xylanase: 0.5 - 2.0 parts, sucrase: 0.5 - 2.0 parts, bromelain: 0.1 - 1.0 parts, papain: 0.1 - 1.0 parts, cellulase: 0.5 - 2.0 parts, Bifidobacterium animalis subsp. lactis Bb - 12: 1.0 - 3.0 parts, Bifidobacterium breve M - 16V: 1.0 - 3.0 parts, Lactobacillus rhamnosus HN001: 1.0 - 3.0 parts, Bifidobacterium animalis subsp. lactis HN019: 1.0 - 3.0 parts.

[0007] Preferably, the total viable count ratio of Bifidobacterium animalis subsp. lactis Bb - 12, Bifidobacterium breve M - 16V, Lactobacillus rhamnosus HN001, and Bifidobacterium animalis subsp. lactis HN019 is ≥ 1.0×10 9 CFU per gram, and the viable ratio of each strain satisfies the following range: Bifidobacterium animalis subsp. lactis Bb - 12: 25 - 35% of the total viable count of probiotics Bifidobacterium breve M - 16V: 20 - 30% of the total viable count of probiotics Lactobacillus rhamnosus HN001: 15 - 25% of the total viable count of probiotics Bifidobacterium animalis subsp. lactis HN019: 20 - 30% of the total viable count of probiotics.

[0008] Preferably, the activity of the α - amylase is 5,000 - 20,000 U / g, the protease activity is 6,000 - 15,000 U / g, the lipase activity is 3,500 - 10,000 U / g, and the activity ratio of each enzyme is as follows: α - amylase: protease: lipase = 1:(1.2 - 1.8):(0.7 - 1.2).

[0009] Preferably, the mass ratio of the resistant dextrin to the fructooligosaccharide is 1:0.5 - 1:2, and the mass ratio of the stachyose to the galactooligosaccharide is 1:1 - 1:3.

[0010] Preferably, the mass ratio of the cellulase to the hemicellulase is 1:1 - 2:1, and the mass ratio of the xylanase to the sucrase is 1:0.8 - 1:1.5.

[0011] Preferably, the total activity of the bromelain and papain is 2,000 - 8,000 U / g, and the mass ratio of the two is 1:1 - 1:2.

[0012] Preferably, the particle sizes of the acerola cherry powder and the kiwifruit powder are 80 - 120 mesh, and their total mass proportion is 2 - 10 parts.

[0013] Preferably, the mass ratio of anhydrous glucose to resistant dextrin in the formula is 1:0.8 - 1:3, and their total proportion is 10 - 35 parts.

[0014] A preparation process for a compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption includes the following steps: Screen the solid components to 80 - 120 mesh and then mix them. The mixing time is 30 - 60 minutes, and the temperature is controlled at 20 - 25°C. During spray drying, the feeding temperature is 20 - 40°C, the discharging temperature is 40 - 70°C, and the nozzle diameter is 0.5 - 1.0 mm. After drying, screen to a particle size of ≤100 μm, and encapsulate the probiotics with sodium alginate. The encapsulation ratio is 2 - 5%.

[0015] Preferably, in the encapsulation, the concentration of sodium alginate is 1.5 - 3.0%, and after encapsulation, it is stored frozen at -20°C to -40°C, and the shelf life is ≤24 months.

[0016] The present invention provides a compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption. It has the following beneficial effects: 1. Through the multi-strain metabolic network reconstruction technology, the present invention realizes precise regulation of the intestinal microecology. The prior art relies on the functional limitations of a single strain and cannot cover complex metabolic requirements. The present invention constructs a bacterial community division of labor system, with multiple links such as acid production, antibacterial, and substrate conversion cooperating synergistically, forming a closed loop between short-chain fatty acid production and pathogenic bacteria inhibition, breaking through the fragmented defects of traditional interventions.

[0017] 2. The enzyme system dynamic adaptation technology of the present invention breaks through the bottleneck of digestion efficiency. Traditional solutions ignore the spatio-temporal matching of enzyme activities, resulting in low substrate utilization rates. The present invention dynamically regulates the action sequence of enzymes in combination with the characteristics of the gastric-intestinal environment, seamlessly connecting macromolecule decomposition and bacterial community metabolism, and simultaneously increasing the fiber conversion rate and nutrient release efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic flow chart of the method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying 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 making creative efforts belong to the scope of protection of the present invention.

[0020] Please refer to the attached Figure 1 , the embodiments of the present invention provide a compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption, including: 1. Probiotic components This formula contains four specific probiotics: Bifidobacterium animalis subsp. lactis Bb-12 (1.0 - 3.0 parts) Bifidobacterium breve M-16V (1.0 - 3.0 parts) Lactobacillus rhamnosus HN001 (1.0 - 3.0 parts) Bifidobacterium animalis subsp. lactis HN019 (1.0 - 3.0 parts) Bb-12: Its metabolites (such as lactic acid and acetic acid) can stabilize the intestinal pH value at 4.5 - 5.5, inhibiting the growth of pathogenic bacteria such as Clostridium perfringens (experimental data: the survival rate of pathogenic bacteria decreases by 90% at pH 5.0). At the same time, its extracellular polysaccharide (EPS) promotes the synthesis of intestinal secretory immunoglobulin A (sIgA) by activating the TLR2 receptor, enhancing the mucosal immune barrier.

[0021] M-16V: Directly kills Salmonella by secreting antibacterial peptides (such as bacteriocin Bifidocin M16), and competitively occupies the receptor sites of intestinal epithelial cells through adhesin proteins (such as Mub protein), blocking the colonization of pathogenic bacteria (adhesion rate ≥ 85%).

[0022] HN001: The butyric acid produced by metabolism can promote the expression of tight junction proteins (such as occludin) in intestinal epithelial cells by activating the GPR43 receptor, reducing intestinal permeability (in vitro model shows that the trans-epithelial resistance value increases by 30%).

[0023] HN019: Its β-galactosidase activity can decompose lactose into glucose and galactose, reducing lactose intolerance symptoms; at the same time, it improves the glucose absorption efficiency by upregulating the expression of SGLT-1 transporter (clinical data shows that the lactose digestibility increases by 40%).

[0024] The ratio design of the four strains (Bb-12:M-16V:HN001:HN019 = 1:0.8:0.6:0.9) is based on the principle of metabolic complementarity: Bb-12 and HN019 preferentially utilize lactose, while M-16V and HN001 prefer to decompose oligosaccharides, avoiding nutritional competition.

[0025] Butyric acid produced by HN001 provides a carbon source for Bb-12, forming cross-feeding and enhancing the stability of the microbial community.

[0026] 2. Enzyme components The formula contains three types of digestive enzymes: Carbohydrate-degrading enzymes: α-amylase (1.5 - 4.0 parts), maltogenic amylase (0.5 - 2.0 parts), xylanase (0.5 - 2.0 parts) Protein-degrading enzymes: protease (1.5 - 3.5 parts), bromelain (0.1 - 1.0 part), papain (0.1 - 1.0 part) Lipid-degrading enzyme: lipase (1.0 - 3.0 parts) α-Amylase: Retains ≥70% activity in gastric acid environment (pH 2.0 - 3.0), hydrolyzes amylose into α-limit dextrin (molecular weight ≤5 kDa) through endo-action, reducing the production of gas by fermentation of undigested starch in the colon (in vitro digestion model shows that the starch decomposition rate ≥90%).

[0027] Protease: Its active center contains metal ions (such as Zn²⁺), can specifically cleave the peptide bonds of hydrophobic amino acids (such as leucine, phenylalanine), decompose allergenic macromolecules such as soy protein into oligopeptides <3 kDa, reducing the risk of IgE-mediated allergic reactions (SDS-PAGE detection shows that the antigen protein degradation rate ≥95%).

[0028] Lipase: Under the emulsification of bile salts, hydrolyzes triglycerides into 2-monoacylglycerol and free fatty acids, and promotes the absorption of fat-soluble vitamins (such as vitamin D3) by forming mixed micelles (in vitro experiments show that the fat digestion rate increases by 50%).

[0029] Optimization of enzyme activity ratio: α-amylase: protease: lipase = 1:1.5:0.8 (mass ratio), matching the enzyme spectrum of human digestive juice (pancreatic juice enzyme ratio 1:1.2:0.7), avoiding substrate competition caused by excessive single enzyme.

[0030] pH adaptability design: Bromelain (optimal pH 3.0 - 6.0) initiates protein predigestion in the stomach; Trypsin-like protease (optimal pH 7.0 - 8.0) continues to act in the intestine, forming relay digestion under a pH gradient.

[0031] 1. Mixing process Mix the dry probiotics, enzymes and excipients at 20 - 25°C for 30 - 60 minutes, and the rotation speed of the mixing equipment is 20 - 40 rpm.

[0032] Requirements for mixing uniformity: The coefficient of variation (CV) of the content of each component ≤ 5%.

[0033] Temperature control: 20 - 25°C can avoid the thermal denaturation and inactivation of enzymes (such as lipase) (TGA analysis shows that the thermal inactivation rate of lipase increases 3 times when > 30°C), and at the same time prevent probiotics (such as HN001) from entering the logarithmic growth phase in advance and consuming stored energy.

[0034] Optimization of rotation speed: Low-speed mixing (20 - 40 rpm) reduces the damage of mechanical shear force to the cell wall of probiotics (flow cytometry shows that the loss rate of viable bacteria < 5%).

[0035] Sequential addition: First mix the prebiotics (resistant dextrin, oligosaccharide) to form a matrix layer, and then gradually add probiotics and enzymes, and use the hygroscopicity of prebiotics to form a protective microenvironment.

[0036] 2. Spray drying process Feeding temperature: 20 - 40°C, discharging temperature: 40 - 70°C Nozzle pressure: 0.5 - 1.5 MPa, rotation speed of the atomizing disk: 15,000 - 25,000 rpm Moisture content of the final product: ≤ 3% (determined by the Karl Fischer method) Low-temperature feeding: The feeding temperature ≤ 40°C ensures that the oxidation loss rate of heat-sensitive components (such as vitamin C) < 10% (the retention rate detected by HPLC ≥ 90%).

[0037] Rapid drying: The diameter of the atomized liquid droplets is controlled at 20 - 50 μm (determined by a laser particle size analyzer), the specific surface area ≥ 300 m 2 / g, so that the water evaporation time ≤ 5 seconds, and avoid exposing probiotics to high-temperature environments (the survival rate of viable bacteria ≥ 80%).

[0038] Addition of cryoprotectant: Add 5% trehalose as a freeze-drying protectant to the feed liquid, and replace water molecules through hydrogen bonds to stabilize the phospholipid bilayer of the cell membrane (DSC analysis shows that the glass transition temperature is increased to 65°C).

[0039] 3. Encapsulation process Encapsulation material: Compound of sodium alginate (1.5 - 3.0% w / v) and chitosan (0.5 - 1.5% w / v) Encapsulation rate: ≥ 95% (determined by fluorescence labeling method) Particle size of the embedded particles: 100 - 300 μm (determined by laser scattering method) Ionic crosslinking: Sodium alginate and Ca 2+Form a "egg box" structure gel that maintains structural integrity in gastric acid (pH 1.2 - 3.0); after entering the intestine (pH 6.8 - 7.4), Ca 2+ is replaced by Na + , and the gel dissolves and releases live bacteria (in vitro simulated release rate ≥ 90%).

[0040] Positive and negative charge adsorption: Chitosan (positively charged) is adsorbed on the surface of sodium alginate microspheres through electrostatic interaction to form a dense outer layer, reducing the erosion of pepsin on probiotics (scanning electron microscopy shows that the surface porosity ≤ 5%).

[0041] Cryoprotection: Immediately freeze at -40 °C for 24 hours after encapsulation to form amorphous ice in the intracellular water, avoiding ice crystals piercing the cell wall (the survival rate of live bacteria after resuscitation ≥ 85%).

[0042] Example 1: Components: anhydrous glucose: 12%, resistant dextrin: 18%, stachyose: 4%, fructooligosaccharide: 7%, galactooligosaccharide: 3%, acerola cherry powder: 2%, kiwi fruit powder: 3%, α - amylase: 3%, protease: 2.5%, lipase: 1.8%, maltogenic amylase: 1%, hemicellulase: 1%, xylanase: 0.8%, sucrase: 0.7%, bromelain: 0.5%, papain: 0.5%, cellulase: 1.2%, Bb - 12 bacteria: 2%, M - 16V bacteria: 2%, HN001 bacteria: 1.5%, HN019 bacteria: 2%; Steps: Screening: Pass the resistant dextrin and acerola cherry powder through a 100 - mesh sieve to remove lumps.

[0043] Premixing: Anhydrous glucose and oligosaccharides (stachyose, fructooligosaccharide, galactooligosaccharide) are premixed at 22 °C for 15 minutes with a rotation speed of 25 rpm.

[0044] Adding enzymes: Add enzymes in three batches, with an interval of 5 minutes each, and control the mixing temperature at 23 °C for a total duration of 40 minutes.

[0045] Adding bacteria: The probiotic powder is added in two batches, with an interval of 10 minutes to avoid electrostatic adsorption.

[0046] Drying: The inlet temperature for spray drying is 35 °C, the outlet temperature is 55 °C, the nozzle diameter is 0.8 mm, and the residual moisture ≤ 3%.

[0047] Encapsulation: Spray - embed with a 2.5% sodium alginate solution, the particle size is 150 μm, and store frozen at -30 °C.

[0048] Example 2: Components: anhydrous glucose: 8%, resistant dextrin: 22%, stachyose: 5%, fructooligosaccharide: 9%, galactooligosaccharide: 4%, acerola cherry powder: 4%, kiwifruit powder: 1%, α-amylase: 3.5%, protease: 3%, lipase: 2.2%, maltogenic amylase: 1.5%, hemicellulase: 0.8%, xylanase: 1.2%, sucrase: 0.5%, bromelain: 0.6%, papain: 0.6%, cellulase: 1.5%, Bb-12 bacteria: 3%, M-16V bacteria: 2.5%, HN001 bacteria: 2%, HN019 bacteria: 2.2%.

[0049] Steps: Low-temperature mixing: All solid components are pre-cooled in an 18°C cold storage for 2 hours, and nitrogen is introduced during mixing to prevent oxidation.

[0050] Gradient feeding: First add resistant dextrin and enzymes, and mix for 30 minutes; then add probiotics in four batches at intervals of 8 minutes each.

[0051] High-pressure atomization: Spray drying uses a two-fluid nozzle, with a pressure of 1.2 MPa, an atomization particle size of 30 μm, and an outlet temperature of 60°C.

[0052] Double-layer embedding: First coat with sodium alginate, and then spray a 0.5% chitosan outer layer, with an encapsulation efficiency of 97%.

[0053] Quick freezing: Immediately put into liquid nitrogen for quick freezing for 10 seconds after embedding, and transfer to -25°C for storage.

[0054] Example 3: Components: anhydrous glucose: 10%, resistant dextrin: 15%, stachyose: 6%, fructooligosaccharide: 5%, galactooligosaccharide: 5%, acerola cherry powder: 3%, kiwifruit powder: 2%, α-amylase: 2.8%, protease: 2.2%, lipase: 1.5%, maltogenic amylase: 1.2%, hemicellulase: 1.5%, xylanase: 1%, sucrase: 0.9%, bromelain: 0.7%, papain: 0.7%, cellulase: 1.8%, Bb-12 bacteria: 1.8%, M-16V bacteria: 2.2%, HN001 bacteria: 1.8%, HN019 bacteria: 2%; Steps: Enzyme activation: Pre-dissolve α-amylase and lipase with a pH 6.0 buffer solution and premix for 10 minutes before spray drying.

[0055] Staged mixing: First stage: Mix prebiotics and anhydrous glucose at 25 rpm for 20 minutes.

[0056] Second stage: Add the enzyme solution, raise the temperature to 28°C, and increase the mixing speed to 40 rpm for 25 minutes.

[0057] Bacterial powder dispersion: The probiotics are premixed with 5% microcrystalline cellulose to reduce caking, and then put into the main mixer.

[0058] Dynamic drying: Fluidized bed drying is adopted, the inlet air temperature is 45°C, the residence time of the material is 15 minutes, and the moisture content ≤ 2.5%.

[0059] Microencapsulation: Electrostatic spraying technology is used, the voltage is 15 kV, the microcapsule particle size is 80 μm, and the encapsulation efficiency ≥ 95%.

[0060] Example 4: Components: Anhydrous glucose: 14%, resistant dextrin: 12%, stachyose: 3%, fructooligosaccharide: 8%, galactooligosaccharide: 3%, acerola cherry powder: 1%, kiwi fruit powder: 4%, α-amylase: 4%, protease: 3.5%, lipase: 2.5%, maltogenic amylase: 0.8%, hemicellulase: 0.6%, xylanase: 1.5%, sucrase: 0.6%, bromelain: 0.4%, papain: 0.4%, cellulase: 0.8%, Bb-12 bacteria: 2.5%, M-16V bacteria: 3%, HN001 bacteria: 2%, HN019 bacteria: 3%.

[0061] Steps: Enzyme protection: The protease and lipase are premixed with 5% maltodextrin to form a protective layer.

[0062] Reverse feeding: First add the probiotics, and then gradually cover the enzymes and prebiotics, the mixing temperature is 20°C, and the duration is 50 minutes.

[0063] Low-temperature drying: The freeze dryer is pre-frozen to -45°C, the pressure in the sublimation stage is 10 Pa, and the temperature is raised to 30°C in the desorption stage.

[0064] Composite encapsulation: Sodium alginate and pectin (ratio 3:1) are compounded, and after encapsulation, it is vacuum packaged, and the water activity ≤ 0.3.

[0065] Activity detection: Sampling and detecting each batch to ensure that the viable bacteria count ≥ 1×10^9 CFU / g and the enzyme activity retention ≥ 85%.

[0066] Example 5 Components: anhydrous glucose: 9%, resistant dextrin: 20%, stachyose: 2%, fructooligosaccharide: 6%, galactooligosaccharide: 4%, acerola cherry powder: 5%, kiwifruit powder: 1%, α-amylase: 2%, protease: 1.8%, lipase: 1.2%, maltogenic amylase: 0.9%, hemicellulase: 1.1%, xylanase: 0.7%, sucrase: 0.8%, bromelain: 0.3%, papain: 0.3%, cellulase: 0.9%, Bb-12 bacteria: 3%, M-16V bacteria: 2.8%, HN001 bacteria: 2.5%, HN019 bacteria: 3%.

[0067] Steps: Ultrasonic dispersion: The probiotic powder is ultrasonically treated at 20 kHz for 5 minutes before mixing to improve dispersion.

[0068] Stepwise temperature control: The prebiotics and enzymes are mixed at 22°C for 30 minutes.

[0069] After adding the probiotics, the temperature is lowered to 18°C and the mixing speed is reduced to 15 rpm.

[0070] Pulse drying: Spray drying uses intermittent pulsed air flow with a pulse cycle of 3 seconds, increasing the drying efficiency by 20%.

[0071] Double-layer coating embedding: The inner layer is sodium alginate and the outer layer is gelatin, with a thickness ratio of 1:2 and a gastric acid resistance time of ≥2 hours.

[0072] Stability test: Accelerated test at 40°C for 3 months, with a viable bacteria survival rate of ≥90% and an enzyme activity decay of ≤10%.

[0073] Control example 1: Adjustment points: Removing strain ratio control: Only a single strain (Bifidobacterium animalis subsp. lactis Bb-12, accounting for 8%) is used, and the other strains are deleted.

[0074] Simplifying the mixing process: The stepwise feeding is cancelled, all components are mixed at once, the temperature is raised to 35°C, and the mixing time is shortened to 15 minutes.

[0075] No encapsulation treatment: Direct screening after drying without sodium alginate embedding.

[0076] Preparation process: According to the component ratio of Example 1, but only Bb-12 bacteria (8%) are retained, and the other strains are replaced with an equal amount of anhydrous glucose.

[0077] All components are put into the mixer at once and mixed at 35°C for 15 minutes.

[0078] The spray drying parameters are the same as those in Example 1, but the encapsulation step is skipped and the powder is directly packaged.

[0079] Comparative Example 2: Adjustment points: Imbalance in enzyme activity ratio: Adjust α - amylase: protease: lipase to 1:0.5:3 (mass ratio) to disrupt the synergistic effect.

[0080] Cancel cryoprotection: The mixing environment is at room temperature (25°C), nitrogen is not introduced, and the mixing time is extended to 60 minutes.

[0081] Single - layer embedding: Only sodium alginate is used for encapsulation, and the chitosan outer layer is omitted.

[0082] Preparation process: According to the component ratio of Example 2, but adjust the enzyme ratio: α - amylase (1.5%), protease (0.75%), lipase (4.5%).

[0083] Keep at 25°C during mixing, do not introduce nitrogen, and mix for 60 minutes.

[0084] Only encapsulate with sodium alginate, do not spray chitosan, and the remaining steps are the same as in Example 2.

[0085] Comparative Example 3 (corresponding to Example 3) Adjustment points: Delete prebiotic synergy: Remove fructooligosaccharide and galactooligosaccharide, and replace them with an equal amount of resistant dextrin.

[0086] Reverse the mixing order: First add probiotics, then mix the enzymes, and raise the mixing temperature to 30°C.

[0087] Cancel dynamic drying: Use an ordinary oven for drying (60°C, 2 hours).

[0088] Preparation process: According to the component ratio of Example 3, but delete fructooligosaccharide and galactooligosaccharide, and increase the proportion of resistant dextrin to 20%.

[0089] Mix probiotics and anhydrous glucose first, stir at 30°C for 10 minutes, and then add the enzymes.

[0090] Dry the wet material in an oven at 60°C for 2 hours, crush and sieve, and do not use a fluidized bed.

[0091] Comparative Example 4 (corresponding to Example 4) Adjustment points: Disrupt strain metabolic complementarity: Only retain HN019 bacteria (accounting for 8%), and delete the remaining strains.

[0092] Pre - mix enzymes at high temperature: Pre - mix the enzymes and maltodextrin at 40°C, resulting in partial inactivation of some enzymes.

[0093] Simplified encapsulation material: Only embedded with gelatin, without using the sodium alginate - pectin composite layer.

[0094] Preparation process: According to the component ratio of Example 4, but only retain HN019 bacteria (8%), and replace the remaining strains with an equal amount of resistant dextrin.

[0095] The protease, lipase and maltodextrin are premixed at 40 °C for 30 minutes.

[0096] Embedded with gelatin solution (5%), without compounding pectin, and the remaining steps are the same as in Example 4.

[0097] Comparative Example 5 (corresponding to Example 5) Adjustment points: Cancel ultrasonic dispersion: The probiotics are directly added to the mixer without pretreatment.

[0098] Mixing temperature fluctuation: Mix at 25 °C throughout the process without stage - controlled temperature.

[0099] Single - pulse drying: Adopt continuous spray drying and cancel the pulsed air flow.

[0100] Preparation process: According to the component ratio of Example 5, but the probiotics are not ultrasonically treated and are directly put into the mixer.

[0101] All components are mixed at 25 °C for 40 minutes without stage - controlled temperature.

[0102] The spray drying is a continuous air flow, without using the pulsed mode, and the rest is the same as in Example 5.

[0103] Experiment 1: Verification of the synergistic effect of strains Experiment description: Use the SHIME® system to simulate the human intestinal environment, which is divided into five segments: stomach, small intestine, ascending colon, transverse colon, and descending colon. The samples of Example 1 and Comparative Example 1 are continuously intervened for 7 days at a dose of 0.5 g / day. The contents of the colon segment are sampled daily and stored frozen at - 80 °C. The specific genes of Bifidobacterium and Escherichia coli are detected by qPCR, and the content of short - chain fatty acids (SCFAs) is analyzed by gas chromatography.

[0104] Experiment steps: Model startup: Inoculate the fecal flora of healthy people into the SHIME colon reactor and operate stably for 14 days.

[0105] Intervention stage: Group A (Example 1): Add the sample to the gastric simulation cavity at 9:00 every day.

[0106] Group B (Comparative Example 1): Add in the same way with the same dose.

[0107] Sample collection: Collect the ascending colon contents on the 3rd, 5th, and 7th days and store them at -80°C.

[0108] Detection method: Proportion of flora: After DNA extraction, qPCR amplification of hsp60 (Bifidobacterium) and uidA (Escherichia coli) was performed, and the ratio was calculated.

[0109] SCFAs: After acidification treatment of the samples, the concentrations of acetic acid, propionic acid, and butyric acid (μg / mg) were determined by GC-MS..

[0110] Table 1: Proportion of flora and content of SCFAs The multi-strain combination in Example 1 significantly inhibited the proliferation of Escherichia coli. The data showed that the ratio of Bifidobacterium to pathogenic bacteria increased from 8.2 on the 3rd day to 14.7 on the 7th day, while this ratio continued to decline to 1.9 under the intervention of the single strain in Comparative Example 1. The difference in short-chain fatty acids was particularly obvious - the butyric acid concentration in Example 1 reached 41.5 μg / mg on the 7th day, nearly 6 times higher than that in Comparative Example 1 (6.3 μg / mg). This result is directly related to the metabolic complementarity of the strains: Bb-12 and HN019 produce organic acids by metabolizing lactose, reducing the intestinal pH value and inhibiting the growth of pathogenic bacteria; M-16V secretes antibacterial peptides to directly inhibit pathogens; HN001 further metabolizes fiber into butyric acid, forming a positive cycle.

[0111] The single-strain strategy in Comparative Example 1 exposed the limitations of the prior art. In a complex intestinal environment, a single strain cannot cover the multi-level regulatory requirements. Although Bb-12 can produce acid, it lacks the adhesion competition ability of M-16V and the butyric acid conversion function of HN001, resulting in limited inhibition effect on pathogenic bacteria. Example 1 achieved "offense and defense integration" through strain division of labor (acid production, antibacterial, energy supply), and the pH value dropped from the initial 6.8 to 5.2, completely destroying the living environment of pathogenic bacteria.

[0112] The core role of butyric acid was further highlighted in this experiment. The increase in its concentration not only provides energy for intestinal epithelial cells, but also can enhance the expression of tight junction proteins by activating the PPAR-γ pathway, reducing intestinal leakage. The lack of butyric acid in Comparative Example 1 led to ineffective repair of the intestinal barrier function. In addition, the propionic acid concentration in Example 1 (16.8 μg / mg) was continuously higher than that in Comparative Example 1 (6.5 μg / mg), suggesting cross-feeding between Bifidobacterium and propionic acid bacteria - the galactose produced by HN019 decomposing lactose becomes the substrate of propionic acid bacteria, and this metabolic network cannot be constructed by a single strain.

[0113] Experimental Example 2: Experimental description An in vitro digestion model was constructed based on the INFOGEST 2.0 standard to simulate the digestion process in the gastric-intestinal stage. The standardized meal contained 20% corn starch, 15% casein, and 10% olive oil. The samples of Example 2 and Comparative Example 2 were added at a ratio of 1%, and the substrate decomposition efficiency was measured after the intervention. The gastric stage was 2 hours, the intestinal stage was 4 hours, and the temperature was kept constant at 37 °C. The sampling interval was 30 minutes, and the supernatant was taken by centrifugation to detect the indicators.

[0114] Experimental procedure Gastric phase simulation: Mix 5 g of the standardized meal with 10 mL of simulated gastric juice (containing 0.3% pepsin, pH 3.0).

[0115] Add the sample of Example 2 or Comparative Example 2, and stir magnetically (200 rpm) for 2 hours.

[0116] Intestinal phase simulation: Adjust the pH to 7.0, and add pancreatic enzymes (100 U / mL trypsin) and bile salts (10 mM).

[0117] Continue stirring for 4 hours, and take 1 mL of sample every 30 minutes.

[0118] Detection treatment: Starch hydrolysis: DNS reagent colorimetric method, measure the absorbance at 540 nm, and convert it to glucose equivalent.

[0119] Protein decomposition: BCA method was used to determine the free amino groups, and the absorbance at 562 nm was compared with the standard curve.

[0120] Fat decomposition: Titration method was used to determine the released free fatty acids, with the unit of mmol / g.

[0121] Table 2. Substrate decomposition rate in the digestion stage The enzyme activity ratio of Example 2 significantly improved the digestion efficiency. The data showed that at 240 minutes, the starch hydrolysis rate of Example 2 reached 96.8%, while that of Comparative Example 2 was only 59.3%. This difference was due to the synergistic effect of α-amylase, protease, and lipase. The proportion of protease activity in Example 2 was higher (1.5 times that of α-amylase), which could rapidly decompose casein in the gastric stage and reduce the residual macromolecular antigens, which was consistent with the "cutting of hydrophobic peptide chains" mentioned in the mechanism. The excessive lipase in Comparative Example 2 led to substrate competition, which was instead unfavorable for lipid decomposition.

[0122] The efficiency of lipase is closely related to the emulsifying effect of bile salts. The lipase ratio in Example 2 (0.8 times that of α-amylase) is adapted to the bile salt concentration, and the free fatty acid release amount (4.13 mmol / g) is much higher than that in Comparative Example 2 (1.55 mmol / g). The imbalance of enzyme activity in Comparative Example 2 leads to insufficient protease action in the gastric phase, and the undigested casein forms a gel in the intestinal phase, hindering the lipase from contacting the substrate. This is directly related to the failure of "pH gradient relay digestion" in the mechanism.

[0123] The synergistic effect is also reflected in the time dimension. The starch hydrolysis rate in Example 2 reached 67.3% at 90 minutes, while that in Comparative Example 2 was only 41.8%. The sufficient decomposition of starch at an early stage creates space for subsequent lipase action, avoiding substrate accumulation. This temporal advantage verifies the design logic of "matching the enzyme activity ratio to the digestion stage requirements" in the claims. The data fluctuation in Comparative Example 2 (such as the fat decomposition amount of 0.12 mmol / g at 30 minutes) further exposes the system disorder caused by excessive single enzyme.

[0124] Experimental Example 3: Experimental description: The BALB / c mouse model was used to evaluate the effects of prebiotic combinations and mixed processes on intestinal flora colonization. Example 3 contained fructooligosaccharide (FOS) and galactooligosaccharide (GOS), and Comparative Example 3 only retained resistant dextrin. The mice were intervened by gavage daily for 4 weeks, and feces were collected weekly to detect the flora abundance and digestion residues.

[0125] Experimental steps: Animal grouping: 40 mice were randomly divided into 2 groups, Group A (Example 3) and Group B (Comparative Example 3), and fed with basal diet.

[0126] Intervention treatment: The mice were gavaged with 200 mg / kg of the sample daily, and the control group was gavaged with an equal volume of normal saline.

[0127] Sample collection: Fresh feces were collected on the 7th day of each week and stored at -80 °C.

[0128] Detection methods: Bifidobacterium abundance: 16S rRNA sequencing (V4 region primers 515F / 806R), and the relative abundance was calculated.

[0129] Fecal pH: 1 g of feces was suspended in 9 mL of pure water, centrifuged to take the supernatant, and directly measured with a pH meter.

[0130] Undigested residue: Dried to a constant weight in an oven at 105 °C, and the proportion of dry matter was calculated.

[0131] Table 3. Dynamics of intestinal flora and digestion indicators The prebiotic combination (fructooligosaccharide + galactooligosaccharide) in Example 3 significantly improved the colonization efficiency of Bifidobacterium. After 4 weeks of intervention, the abundance of Bifidobacterium reached 31.2%, while that in Comparative Example 3 was only 4.8%. This difference stems from the synergistic effect of the two types of prebiotics: fructooligosaccharide is preferentially metabolized by Bifidobacterium, and galactooligosaccharide stimulates its proliferation, and the two together promote the expansion of the flora. Comparative Example 3 only relied on resistant dextrin, and its fermentation rate was slow, unable to effectively reduce the intestinal pH (7.3 vs 5.3), resulting in pathogenic bacteria competitively occupying the ecological niche.

[0132] The difference in the process sequence further amplified the effect. The staged mixing in Example 3 protected the enzyme activity, enabling the cellulase to fully decompose the fiber, and the amount of undigested residue (19.8%) was significantly lower than that in Comparative Example 3 (47.5%). The high-temperature mixing in Comparative Example 3 led to enzyme inactivation, and the resistant dextrin was not effectively degraded, instead forming a physical barrier that hindered the flora from contacting the substrate.

[0133] The generation of short-chain fatty acids and the decrease in pH formed a positive cycle. When the pH dropped to 5.3 in Example 3, the butyric acid concentration increased synchronously, directly enhancing the energy supply of intestinal epithelial cells and inhibiting pathogenic bacteria. The increase in pH (7.3) in Comparative Example 3 disrupted this cycle, and the accumulation of undegraded fiber further exacerbated the deterioration of the intestinal environment. The dual optimization of the process and formula was the key to the success of Example 3.

[0134] Experimental Example 4: Experimental description: The protective effect of the encapsulation process on probiotics was tested by simulating the gastric acid environment. Example 4 used sodium alginate-pectin double-layer embedding, and Comparative Example 4 used only gelatin single-layer embedding. Two groups of samples were exposed to artificial gastric juice (pH 2.0, containing 0.3% pepsin) for 2 hours, and the viable bacteria count was detected by sampling every 30 minutes.

[0135] Experimental steps: Preparation of artificial gastric juice: 0.3% pepsin was dissolved in 0.1 M HCl solution, and the pH was adjusted to 2.0.

[0136] Sample treatment: Group A (Example 4): 1 g of embedded particles was added to 10 mL of gastric juice and shaken at 37°C (120 rpm).

[0137] Group B (Comparative Example 4): The unembedded bacterial powder was treated in the same way.

[0138] Neutralization and counting: 1 mL of the mixed solution was taken every 30 minutes and added to 9 mL of PBS (pH 7.0) to neutralize the gastric acid.

[0139] After gradient dilution, it was spread on MRS medium and anaerobically cultured at 37°C for 48 hours, and the viable bacteria count (CFU / g) was calculated.

[0140] Table 4: Survival rate after simulated gastric acid treatment Experimental Example 5: Experimental description: The protective effect of the encapsulation process on probiotics was tested by simulating the gastric acid environment. In Example 4, sodium alginate-pectin double-layer embedding was used, and in Comparative Example 4, only gelatin single-layer embedding was used. Two groups of samples were exposed to artificial gastric juice (pH 2.0, containing 0.3% pepsin) for 2 hours, and the viable cell count was detected by sampling every 30 minutes.

[0141] Experimental procedure Preparation of artificial gastric juice: 0.3% pepsin was dissolved in 0.1 M HCl solution, and the pH was adjusted to 2.0.

[0142] Sample treatment: Group A (Example 4): 1 g of embedded particles was taken and added to 10 mL of gastric juice, and shaken at 37°C (120 rpm).

[0143] Group B (Comparative Example 4): The unembedded bacterial powder was treated in the same way.

[0144] Neutralization and counting: 1 mL of the mixed solution was taken every 30 minutes, and 9 mL of PBS (pH 7.0) was added to neutralize the gastric acid.

[0145] After gradient dilution, it was spread on MRS medium and anaerobically cultured at 37°C for 48 hours, and the viable cell count (CFU / g) was calculated.

[0146] Table 5. Survival rate after simulated gastric acid treatment The double-layer embedding process of Example 4 protects probiotics through the dual mechanisms of physical barrier and chemical buffering. Sodium alginate rapidly forms a gel layer in gastric acid to block the penetration of pepsin, and the carboxyl groups of pectin neutralize the local pH, raising the microenvironment from 2.0 to 4.5. The data shows that the survival rate of Example 4 (45.8%) after 120 minutes of treatment is more than 18 times higher than that of Comparative Example 4 (2.5%), and the difference in survival rate at 30 minutes (80.7% vs 35.8%) more prominently highlights the early protection advantage of the double-layer structure.

[0147] There is a synergistic effect between the acid resistance of the strain and the embedding process. The F0F1-ATPase gene of HN019 bacteria can still maintain the transmembrane proton gradient at pH 4.5, and the slow-release characteristics of pectin extend its metabolic activity time. The gelatin layer of Comparative Example 4 dissolved within 30 minutes, and the bacterial cells were directly exposed to the pH 2.0 environment, resulting in the interruption of ATP synthesis and the loss of cell membrane integrity, and the survival rate dropped sharply to 9.9% at 60 minutes.

[0148] The embedding effect directly determines the downstream functional performance. The live bacteria in Example 4 were precisely released in the intestine, forming a metabolic complement with the bifidobacteria in Experiment 1 and promoting the continuous production of SCFAs. In contrast, the inactivation of the flora in Comparative Example 4 led to the accumulation of undigested residues (47.5%) in Experiment 3, verifying the positive correlation between the flora survival rate and intestinal function. The chain reaction caused by process differences proves that the embedding technology is the core barrier of the flora preparation.

[0149] 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 in these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption, characterized in that, Composed of the following components in parts by mass: Anhydrous glucose: 5 - 15 parts, resistant dextrin: 5 - 20 parts, stachyose: 2 - 8 parts, fructooligosaccharide: 3 - 10 parts, galactooligosaccharide: 2 - 8 parts, acerola cherry powder: 1 - 5 parts, kiwifruit powder: 1 - 5 parts, α - amylase: 1.5 - 4.0 parts, protease: 1.5 - 3.5 parts, lipase: 1.0 - 3.0 parts, maltogenic amylase: 0.5 - 2.0 parts, hemicellulase: 0.5 - 2.0 parts, xylanase: 0.5 - 2.0 parts, sucrase: 0.5 - 2.0 parts, bromelain: 0.1 - 1.0 parts, papain: 0.1 - 1.0 parts, cellulase: 0.5 - 2.0 parts, Bifidobacterium animalis subsp. lactis Bb - 12: 1.0 - 3.0 parts, Bifidobacterium breve M - 16V: 1.0 - 3.0 parts, Lactobacillus rhamnosus HN001: 1.0 - 3.0 parts, Bifidobacterium animalis subsp. lactis HN019: 1.0 - 3.0 parts.

2. The formulation of a compound probiotic enzyme powder for promoting gastrointestinal microecological balance and nutrient absorption according to claim 1, characterized in that, The total viable count ratio of Bifidobacterium animalis subsp. lactis Bb-12, Bifidobacterium breve M-16V, Lactobacillus rhamnosus HN001, and Bifidobacterium animalis subsp. lactis HN019 is ≥ 1.0 × 10 9 CFU per gram, and the viable count ratio of each strain meets the following range: Bifidobacterium animalis subsp. lactis Bb - 12: 25 - 35% of the total viable probiotic count Bifidobacterium breve M - 16V: 20 - 30% of the total viable probiotic count Lactobacillus rhamnosus HN001: 15 - 25% of the total viable probiotic count Bifidobacterium animalis subsp. lactis HN019: 20 - 30% of the total viable probiotic count.

3. The formulation of a compound probiotic enzyme powder for promoting gastrointestinal microecological balance and nutrient absorption according to claim 1, wherein The activity of the α - amylase is 5,000 - 20,000 U / g, the protease activity is 6,000 - 15,000 U / g, the lipase activity is 3,500 - 10,000 U / g, and the activity ratio of each enzyme is as follows: α - amylase: protease: lipase = 1:(1.2 - 1.8):(0.7 - 1.2).

4. A compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption according to claim 1, characterized in that, The mass ratio of the resistant dextrin to the fructooligosaccharide is 1:0.5 - 1:2, and the mass ratio of the stachyose to the galactooligosaccharide is 1:1 - 1:

3.

5. The formulation of a compound probiotic enzyme powder for promoting gastrointestinal microecological balance and nutrient absorption according to claim 1, characterized in that, The mass ratio of the cellulase to the hemicellulase is 1:1 - 2:1, and the mass ratio of the xylanase to the sucrase is 1:0.8 - 1:1.

5.

6. The formula of a compound probiotic enzyme powder for promoting gastrointestinal microecological balance and nutrient absorption according to claim 1, characterized in that, The total activity of the bromelain and the papain is 2,000 - 8,000 U / g, and the mass ratio of the two is 1:1 - 1:

2.

7. A compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption according to claim 1, characterized in that, The particle size of the acerola cherry powder and the kiwifruit powder is 80 - 120 mesh, and the total mass ratio of the two is 2 - 10 parts.

8. A compound probiotic enzyme powder formulation for promoting gastrointestinal microecological balance and nutrient absorption according to claim 1, characterized in that, The mass ratio of the anhydrous glucose to the resistant dextrin in the formula is 1:0.8 - 1:3, and the total ratio of the two is 10 - 35 parts.

9. A preparation process for a compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption, based on the compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption according to any one of claims 1-8, characterized in that, Including the following steps: Sieve the solid components to 80 - 120 mesh and then mix them. The mixing time is 30 - 60 minutes, and the temperature is controlled at 20 - 25°C; During spray drying, the feeding temperature is 20 - 40°C, the discharging temperature is 40 - 70°C, and the nozzle diameter is 0.5 - 1.0 mm; After drying, sieve to a particle size of ≤100 μm, and encapsulate the probiotics with sodium alginate. The encapsulation ratio is 2 - 5%.

10. The preparation process of a compound probiotic enzyme powder formula for promoting gastrointestinal microecological balance and nutrient absorption according to claim 9, characterized in that, In the encapsulation, the concentration of sodium alginate is 1.5 - 3.0%, and after encapsulation, it is stored frozen at - 20°C to - 40°C, and the shelf life is ≤24 months.

Images