A milk-derived probiotic specific aptamer polysaccharide targeted symbiotic proliferation system and a preparation method thereof

Abstract

The application provides a milk-derived probiotic specific adaptation polysaccharide target symbiotic proliferation system and a preparation method thereof, and belongs to the technical field of microbial preparations. The system comprises adaptation structured polysaccharide and milk-derived probiotics in a mass ratio of 2-5:1; the adaptation structured polysaccharide has a regular branched structure of a beta-1,3-glucan main chain and a beta-1,6-glucan side chain, a molecular weight of 3-10 kDa, a branching degree DB of 0.05-0.25, an intrinsic viscosity [eta] of 5-20 mL / g, and a beta-1,3 / 1,6-glucan purity of greater than or equal to 85 wt%; and the milk-derived probiotics are selected from one or more of Bifidobacterium lactis, Streptococcus thermophilus and Lactobacillus delbrueckii bulgaricus. The application first realizes two-way synergy of probiotic reverse activation polysaccharide: the milk-derived probiotics have a degradation activation effect on the structured polysaccharide, and the antioxidant, anti-inflammatory and mucosal repair activity of the generated active polysaccharide fragments is increased by more than 40% compared with that before degradation, thereby filling the technical gap in the field of 'probiotic activation polysaccharide'.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system and its preparation method. Background Technology

[0002] Probiotic preparations have become an important means of regulating gut microbiota and improving host health. Among them, milk-derived probiotics (such as Bifidobacterium lactis, Streptococcus thermophilus, and Lactobacillus delbrueckii subsp. bulgaricus) are widely used in dairy products, health products, and pharmaceuticals due to their high safety and well-defined functions. However, existing milk-derived probiotic preparations generally suffer from problems such as low live bacteria survival rate, poor intestinal colonization ability, and limited functionality, which restrict their efficacy.

[0003] To address the aforementioned issues, existing technologies attempt to combine probiotics with prebiotics to promote the proliferation and functionalization of probiotics. However, currently available commercially available probiotic-prebiotic combination systems suffer from the following key technological deficiencies: (1) There is a lack of structural compatibility between prebiotics and milk-derived probiotics, making it impossible to achieve targeted proliferation.

[0004] Currently available prebiotics (such as broad-spectrum oligosaccharides like fructooligosaccharides (FOS) and galactooligosaccharides (GOS)) are general-purpose carbon sources that can be utilized by a variety of gut microbiota. They are not specifically designed to address the cell interface characteristics and metabolic pathway preferences of dairy probiotics. This results in dairy probiotics being unable to gain a competitive advantage in carbon source competition within the complex gut microbiota, leading to low proliferation efficiency. Furthermore, broad-spectrum prebiotics also promote the growth of non-target bacteria, failing to achieve targeted preferential proliferation of dairy probiotics.

[0005] (2) Traditional bacterial-sugar compounding only achieves a single-layer effect of "polysaccharide supplying bacterial growth" - the polysaccharide is consumed by probiotics as a carbon source, and the probiotics produce acid and gas. The functions of the two are independent, and the activity after compounding is a simple algebraic sum. There is no bidirectional synergistic mechanism in the existing technology that reverses the activation of polysaccharide biological activity by the metabolic process of probiotics, and the overall function cannot achieve a synergistic amplification of 1+1>2.

[0006] (3) Commercial microcapsule protection technology only achieves physical encapsulation, with a viable bacteria retention rate of about 82%, but it cannot improve the metabolic activity of probiotics or activate the function of polysaccharides; although the human milk oligosaccharide (HMO)-probiotic system has a good probiotic effect, HMO is extremely expensive and has limited production, and it only has a one-way probiotic effect without a two-way synergistic mechanism. At present, there is no bacterial-glycoside symbiotic system that combines structural adaptation, two-way synergy, and natural stability.

[0007] (4) Traditional polysaccharide structure-activity relationship studies focus on the one-way activity evaluation of "polysaccharide → host gut / cell", completely ignoring the structure-activity relationship between polysaccharide spatial configuration and probiotic extracellular adsorption, interface recognition and metabolic matching, especially lacking research on the reverse structure-activity mechanism of "probiotic metabolic activation of polysaccharides". In addition, it is traditionally believed that the carbon source utilization spectrum of dairy probiotics (such as Bifidobacterium lactis, Streptococcus thermophilus, etc.) is mainly lactose and oligosaccharides, and it is difficult to directly utilize high molecular weight β-glucan. Therefore, existing technologies have never designed structurally compatible compounding of β-glucan with dairy probiotics.

[0008] Therefore, developing a targeted symbiotic proliferation system for dairy probiotics that combines structural adaptability, bidirectional synergistic effect, and natural homeostasis has significant technological value and market prospects. Summary of the Invention

[0009] Technical problems to be solved: In response to the three core technical problems existing in the existing compound system of dairy probiotics and prebiotics, namely the lack of structural adaptability, unidirectional functional superposition, and lack of system stability, the purpose of this invention is to provide a targeted symbiotic proliferation system of dairy probiotics with specific adaptable polysaccharides and its preparation method. This invention achieves for the first time the bidirectional synergistic effect of probiotics in reverse activation of polysaccharides: the metabolic process of dairy probiotics degrades and activates the structured polysaccharides, and the antioxidant, anti-inflammatory, and mucosal repair activities of the generated active polysaccharide fragments are increased by more than 40% compared with those before degradation.

[0010] Technical solution: A targeted symbiotic proliferation system for milk-derived probiotics with specific adaptive polysaccharides, the system comprising adaptive structured polysaccharides and milk-derived probiotics in a mass ratio of 2-5:1; The adapted structured polysaccharide has a regular branched structure with a β-1,3-glucan backbone and β-1,6-glucan side chains, a molecular weight of 3-10 kDa, a branching degree DB of 0.05-0.25, an intrinsic viscosity [η] of 5-20 mL / g (25°C aqueous solution), and a β-1,3 / 1,6-glucan purity ≥85 wt%. The milk-derived probiotics are selected from Bifidobacterium lactis (Bifidobacterium lactis Bifidobacterium animalis subsp. lactis ), Streptococcus thermophilus ( Streptococcus thermophilus Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus One or more of the following.

[0011] Furthermore, the molecular weight of the adapted structured polysaccharide is 5-8 kDa.

[0012] Furthermore, the mass ratio of the adapted structured polysaccharide to the milk-derived probiotics is 3-4:1.

[0013] Furthermore, the adaptable structured polysaccharide is prepared from yeast β-glucan via enzymatic hydrolysis-ultrafiltration fractionation.

[0014] Furthermore, the enzymatic hydrolysis uses β-1,3-glucan endonuclease, the hydrolysis temperature is 45-55°C, the hydrolysis time is 2-6 h, and the amount of enzyme added is 0.5-2 wt% of the substrate.

[0015] Furthermore, the enzymatic hydrolysis temperature is 50°C, the enzymatic hydrolysis time is 4 h, and the amount of enzyme added is 1 wt% of the substrate.

[0016] Furthermore, the ultrafiltration fractionation adopts a two-stage series ultrafiltration membrane fractionation: first, the permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and then the permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to retain the target components of 3-10 kDa.

[0017] This invention also provides a method for preparing the above-mentioned milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system, comprising the following steps: S1. Activation culture of milk-derived probiotics: Activate milk-derived probiotics in basal medium (MRS or M17 medium) until the logarithmic growth phase OD. 600 =0.6-0.8, to obtain a viable bacteria concentration ≥10 8 CFU / mL bacterial suspension; S2. Structure-activity matching compounding: The adapted structured polysaccharide and the milk-derived probiotic liquid are mixed and compounded under aseptic conditions at 2-8°C for 30-60 min to form a symbiotic system; S3. Steady-state molding: The symbiotic system is vacuum dried at a temperature ≤40°C and a vacuum degree ≤50 Pa until the moisture content of the system is ≤5 wt%, to obtain the dry powder product of the symbiotic proliferation system.

[0018] Furthermore, the vacuum drying temperature in step S3 is 35-38°C.

[0019] This invention also provides the application of the above-mentioned milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system in the preparation of foods, health products or drugs for regulating intestinal flora, enhancing immunity, anti-inflammation or mucosal repair.

[0020] Furthermore, the system operates through the following three-level bidirectional collaborative mechanism: (1) Positive adaptation-structure-promoting bacteria The well-organized branched structure of adaptable structured polysaccharides (β-1,3 main chain + β-1,6 side chain, DB = 0.05-0.25, [η] = 5-20 mL / g) can be preferentially recognized and efficiently utilized by lactic probiotics. Specifically: The regular side chain structure matches the geometry of the substrate binding pocket of the lactogenic probiotic sugar transporter, allowing polysaccharides to be preferentially transported to intracellular metabolism. Compact spatial conformation (low [η] value) reduces steric hindrance and improves the accessibility and degradation efficiency of polysaccharides by probiotic extracellular enzymes; The molecular weight range of 3-10 kDa is significantly better than high molecular weight polysaccharides of >10 kDa (which are hindered by high viscosity) and oligosaccharides of <3 kDa (which have insufficient total carbon source and no spatial configuration for identification) in terms of carbon source utilization efficiency.

[0021] (2) Reverse activation - Bacterial polysaccharide During the metabolic utilization of structured polysaccharides, lactic acid bacteria, through their extracellular glycoside hydrolase system and co-metabolism with intestinal symbiotic bacteria, degrade and activate the structured polysaccharides, directionally generating small-molecule active polysaccharide fragments with a molecular weight of 1-3 kDa. The bioactivity of these degraded fragments is significantly amplified compared to the original structured polysaccharides. The principle lies in the presence of "inert long-chain regions" formed by entangled side chains within the structured polysaccharides. In intact polysaccharides, these regions are shielded from active sites due to steric hindrance and interchain hydrogen bonds. The enzymatic hydrolysis during probiotic metabolism specifically opens these inert regions, releasing low-molecular-weight active fragments and exposing more hydroxyl groups and reducing ends, thus precisely and efficiently amplifying the antioxidant, anti-inflammatory, and mucosal repair activities of the polysaccharides.

[0022] (3) Steady-state bacterial retention - structural bacterial fixation Complete, adaptable, structured polysaccharides, with their hydrophilic and well-organized network, spontaneously form a natural protective film on the surface of lactic probiotic cells, achieving a triple bacterial fixation effect: Stress resistance and protection: The hydrophilic polysaccharide membrane blocks the direct attack of gastric acid and bile salts on the bacterial cells; Mucosal anchoring: The hydrophilic groups of the polysaccharide membrane form a hydrogen bond network with the mucosal mucosa, mediating the anchoring of probiotics to the intestinal mucosal surface; Slow-release carbon source: The surface polysaccharide membrane serves as a slow-release carbon source pool, continuously providing nutrients to probiotics during colonization, maintaining their metabolic activity and competitive advantage. Beneficial effects

[0023] (1) Significant targeted proliferation effect of milk probiotics: Under the same culture conditions, the proliferation rate of milk probiotics by the structured polysaccharide of the present invention is 51.6% higher than that of commercial broad-spectrum prebiotic fructooligosaccharide (FOS), and the total yield of short-chain fatty acids is 48.5% higher than that of FOS.

[0024] (2) For the first time, bidirectional synergy of probiotic reverse activation of polysaccharides was achieved: the metabolic process of dairy probiotics degrades and activates the structured polysaccharides. The antioxidant, anti-inflammatory and mucosal repair activities of the generated active polysaccharide fragments are increased by more than 40% compared with those before degradation, filling the technological gap in the field of "probiotic activated polysaccharides".

[0025] (3) Breakthrough the technical problem of microcapsules “only protects without enhancing efficacy”: The system of this invention has a live bacteria retention rate of 85.8%, and at the same time has the effects of probiotic metabolism enhancement and polysaccharide activity secondary amplification, realizing the organic unity of protection and bidirectional functional enhancement.

[0026] (4) It combines the probiotic effect of HMO system with unique bidirectional synergistic mechanism and has significant cost advantage: The present invention uses yeast β-glucan as raw material, the raw material cost is significantly lower than human milk oligosaccharide (HMO), and the unique bidirectional synergistic mechanism enables the system to have the dual functions of probiotic proliferation and polysaccharide activity amplification, realizing the bidirectional gain that HMO system does not have.

[0027] (5) Long-term colonization effect: The hydrophilic protective film formed by the structured polysaccharide on the surface of probiotics extends the effective colonization time of milk probiotics from the conventional 24 h to 72 h.

[0028] (6) Carbon source competitive advantage inhibits miscellaneous bacteria: The structured polysaccharide of the present invention is preferentially utilized by milk-derived probiotics. The miscellaneous bacteria competitive inhibition rate of Example 1 reached 58.3%, which is about 58% higher than that of the FOS group (36.8%).

[0029] (7) All raw materials are of natural origin and are highly safe. Attached Figure Description

[0030] Figure 1 The figures show a comparison of polysaccharide structure characterization in some embodiments and comparative examples; where A represents molecular weight distribution; B represents degree of branching; and C represents intrinsic viscosity. Figure 2 This is a comparison chart of the probiotic proliferation rate and short-chain fatty acid production of Examples 1-8 and Comparative Examples 1-10. Detailed Implementation

[0031] This invention proposes a milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system and its preparation method. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0032] In the following examples, unless otherwise specified, all raw materials used were commercially available analytical grade; Bifidobacterium lactis ( Bifidobacterium animalis subsp. lactis BB-12, Streptococcus thermophilus ( Streptococcus thermophilus ST-21, Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricusLB340 was purchased from Chr. Hansen; crude yeast β-glucan was purchased from Angel Yeast Co., Ltd.; and β-1,3-glucan endopeptidase was purchased from Novozymes.

[0033] Example 1 I. Preparation of adaptable structured polysaccharides: (1) Yeast β-glucan crude product (molecular weight >100 kDa, β-1,3 / 1,6-glucan content about 72%) was used as raw material and enzymatically hydrolyzed with β-1,3-glucan endoglucanase; the hydrolysis conditions were: temperature 50°C, time 4 h, enzyme addition amount was 1 wt% of substrate, and substrate concentration was 5 wt% (prepared with deionized water); (2) After the enzymatic hydrolysis is completed, heat the hydrolysate to 85°C and keep it warm for 10 min to inactivate the enzyme, then cool it to room temperature; (3) Two-stage tandem ultrafiltration membranes were used for classification: First, an ultrafiltration membrane with a molecular weight cutoff of 10 kDa (polyethersulfone material, effective membrane area 0.1 m²) was used. 2 Operating pressure 0.5 MPa, operating temperature 30°C, collect permeate; (4) Use an ultrafiltration membrane with a molecular weight cutoff of 3 kDa (same material and membrane area) to filter the permeate, operate at a pressure of 0.5 MPa and a temperature of 30°C, and collect the retentate; (5) The retentate was freeze-dried for 24 h under vacuum ≤50 Pa and temperature -50°C to obtain the adapted structured polysaccharide.

[0034] The weight-average molecular weight of the obtained polysaccharide was determined to be 7.2 kDa by high-performance gel permeation chromatography (HPGPC); the degree of branching (DB) was 0.15 by methylation analysis combined with GC-MS; the intrinsic viscosity (η) was 12.5 mL / g by Ubbelohde viscometer (25°C aqueous solution); and the purity of β-1,3 / 1,6-glucan was 88.3% by the Megazyme Mixed Linked β-glucan assay kit (enzymatic method, according to AOAC 995.16 standard method).

[0035] II. Activation and Cultivation of Milk-Derived Probiotics Bifidobacterium lactis BB-12 lyophilized powder was inoculated into MRS liquid medium (containing 0.05% L-cysteine ​​hydrochloride) and activated by anaerobic culture at 37°C for 16 h. The inoculum was then transferred to fresh MRS medium at a 2% inoculation rate and cultured until the logarithmic growth phase (OD200). 600 =0.7), at which point the viable bacteria concentration is 2.6×10 8CFU / mL. Centrifuge the bacterial culture at 4°C and 4000 rpm for 15 min, discard the supernatant, and collect the bacterial precipitate.

[0036] III. Structure-Property Matching Combination The above-mentioned adapted structured polysaccharide and Bifidobacterium lactis BB-12 bacterial cells were centrifuged and precipitated at a mass ratio of 3.5:1 (the mass of polysaccharide is based on dry weight, and the mass of bacterial cells is based on wet weight of centrifuged precipitate) under aseptic conditions at 4°C. The mixture was stirred for 45 min to allow the polysaccharide and probiotics to come into full contact and form a symbiotic system.

[0037] IV. Steady-state forming The compounded symbiotic system was vacuum dried at 37°C and ≤50 Pa for 16 h until the moisture content of the system was 4.2 wt%, thus obtaining the symbiotic proliferation system dry powder product.

[0038] Example 2 The difference between this embodiment and Example 1 is as follows: Preparation of adaptable structured polysaccharide: the enzymatic hydrolysis time is extended to 6 h, and after ultrafiltration fractionation, the low molecular weight component in the 3 kDa retentate is collected. The molecular weight is determined by HPGPC to be 3.1 kDa (branching degree DB=0.22, intrinsic viscosity [η]=6.8 mL / g).

[0039] The molecular weight of the adapted structured polysaccharide is 3.1 kDa, the lactogenic probiotic is Streptococcus thermophilus ST-21, and the mass ratio of polysaccharide to bacteria is 2:1.

[0040] The remaining steps are the same as in Example 1.

[0041] Example 3 The difference between this embodiment and Example 1 is as follows: Preparation of adaptable structured polysaccharide: the enzymatic hydrolysis time is shortened to 2 h, and after ultrafiltration fractionation, the 10 kDa retentate is collected and the fraction after 3 kDa ultrafiltration is determined by HPGPC to be 9.8 kDa (branching degree DB=0.06, intrinsic viscosity [η]=18.7 mL / g).

[0042] The molecular weight of the adapted structured polysaccharide is 9.8 kDa, and the milk-derived probiotic is Lactobacillus delbrueckii subsp. bulgaricus LB340, with a polysaccharide to bacteria mass ratio of 5:1.

[0043] The remaining steps are the same as in Example 1.

[0044] Example 4 The difference between this embodiment and Example 1 is that the molecular weight of the adapted structured polysaccharide is 5.1 kDa (branching degree DB=0.18, intrinsic viscosity [η]=11.2 mL / g), and the mass ratio of polysaccharide to bacteria is 3:1.

[0045] The remaining steps are the same as in Example 1.

[0046] Example 5 The difference between this embodiment and Example 1 is that the molecular weight of the adapted structured polysaccharide is 8.2 kDa (branching degree DB=0.08, intrinsic viscosity [η]=16.3 mL / g), and the mass ratio of polysaccharide to bacteria is 4:1.

[0047] The remaining steps are the same as in Example 1.

[0048] Example 6 The difference between this embodiment and Example 1 is that the molecular weight of the adapted structured polysaccharide is 6.5 kDa (branching degree DB=0.12, intrinsic viscosity [η]=13.8 mL / g), the lactogenic probiotic is a compound of Bifidobacterium lactis BB-12 and Streptococcus thermophilus ST-21 in a 1:1 ratio, and the mass ratio of polysaccharide to compound bacteria is 3.5:1.

[0049] The remaining steps are the same as in Example 1.

[0050] Example 7 The difference between this embodiment and Example 1 is that the molecular weight of the adapted structured polysaccharide is 4.5 kDa (branching degree DB=0.20, intrinsic viscosity [η]=9.5 mL / g), and the milk-derived probiotics are a compound of Bifidobacterium lactis BB-12 and Lactobacillus delbrueckii subsp. bulgaricus LB340 mixed in a 1:1 ratio, with a mass ratio of polysaccharide to compound bacteria of 3:1.

[0051] The remaining steps are the same as in Example 1.

[0052] Example 8 The difference between this embodiment and Example 1 is that the enzymatic hydrolysis temperature in the preparation of the adapted structured polysaccharide is 55°C, the amount of enzyme added is 0.5 wt% of the substrate, the molecular weight of the obtained polysaccharide is 3.8 kDa (branching degree DB=0.23, intrinsic viscosity [η]=7.5 mL / g), and the mass ratio of polysaccharide to bacteria is 4:1.

[0053] The remaining steps are the same as in Example 1.

[0054] Comparative Example 1 The difference between this comparative example and Example 1 is that crude, undigested yeast β-glucan (molecular weight approximately 150 kDa, branching degree DB = 0.32, intrinsic viscosity [η] = 45.6 mL / g) was used instead of the adapted structured polysaccharide, and the mass ratio of polysaccharide to bacteria was 3.5:1.

[0055] The remaining steps are the same as in Example 1.

[0056] Comparative Example 2 The difference between this comparative example and Example 1 is that commercially available fructooligosaccharides (FOS, with an average molecular weight of about 450 Da and no branched structure) were used instead of the adapted structured polysaccharides, and the mass ratio of polysaccharides to bacteria was 3.5:1.

[0057] The remaining steps are the same as in Example 1.

[0058] Comparative Example 3 The difference between this comparative example and Example 1 is that commercially available galactooligosaccharide (GOS, with an average molecular weight of about 600 Da and no branched structure) was used instead of the adapted structured polysaccharide, and the mass ratio of polysaccharide to bacteria was 3.5:1.

[0059] The remaining steps are the same as in Example 1.

[0060] Comparative Example 4 The difference between this comparative example and Example 1 is that the molecular weight of the adapted structured polysaccharide is 2.5 kDa (branching degree DB=0.28, intrinsic viscosity [η]=4.2 mL / g), and the mass ratio of polysaccharide to bacteria is 3.5:1.

[0061] Preparation method: The enzymatic hydrolysis time was extended to 8 h, and the fraction with <3 kDa was collected after ultrafiltration.

[0062] The remaining steps are the same as in Example 1.

[0063] Comparative Example 5 The difference between this comparative example and Example 1 is that the molecular weight of the adapted structured polysaccharide is 15 kDa (branching degree DB=0.04, intrinsic viscosity [η]=28.5 mL / g), and the mass ratio of polysaccharide to bacteria is 3.5:1.

[0064] Preparation method: The enzymatic hydrolysis time was shortened to 1 h, and the fraction with >10 kDa was collected after ultrafiltration.

[0065] The remaining steps are the same as in Example 1.

[0066] Comparative Example 6 The difference between this comparative example and Example 1 is that the degree of branching of the adapted structured polysaccharide DB=0.02 (molecular weight 6.8kDa, intrinsic viscosity [η]=14.1 mL / g), and the mass ratio of polysaccharide to bacteria is 3.5:1.

[0067] Preparation method: First, pretreatment with β-1,6-glucanase at 40°C and pH 5.0 for 2 h removes part of the β-1,6-glycosidic side chain. Then, enzymatic hydrolysis with β-1,3-glucan endonuclease and ultrafiltration fractionation are performed to obtain low-branched polysaccharide.

[0068] The remaining steps are the same as in Example 1.

[0069] Comparative Example 7 The difference between this comparative example and Example 1 is that the degree of branching of the adapted structured polysaccharide DB=0.32 (molecular weight 7.1kDa, intrinsic viscosity [η]=11.8 mL / g) and the mass ratio of polysaccharide to bacteria is 3.5:1.

[0070] Preparation method: Epichlorohydrin was used to chemically cross-link yeast β-glucan to increase the branch density, followed by enzymatic hydrolysis and ultrafiltration fractionation, and repeated washing until no epichlorohydrin residue could be detected.

[0071] The remaining steps are the same as in Example 1.

[0072] Comparative Example 8 The difference between this comparative example and Example 1 is that the mass ratio of polysaccharide to bacteria is 1:1.

[0073] The remaining steps are the same as in Example 1.

[0074] Comparative Example 9 The difference between this comparative example and Example 1 is that the mass ratio of polysaccharide to bacteria is 8:1.

[0075] The remaining steps are the same as in Example 1.

[0076] Comparative Example 10 The difference between this comparative example and Example 1 is that no adaptive structured polysaccharide is added to the system, and only Bifidobacterium lactis BB-12 bacterial culture is used.

[0077] The remaining steps are the same as in Example 1 (only activation culture and drying are performed).

[0078] Performance evaluation: I. Determination of Probiotic Proliferation Rate The symbiotic proliferation system dry powder products prepared in Examples 1-8 and Comparative Examples 1-10 were inoculated into MRS liquid medium (M17 medium was used for Streptococcus thermophilus) at a dosage of 1 wt% and cultured anaerobically at 37°C for 24 h. Using a blank control group without any added polysaccharides as a reference, the viable bacterial count before and after culture was determined by plate counting, and the proliferation rate was calculated.

[0079] Proliferation rate (%) = (Number of viable bacteria after culture - Number of viable bacteria at the beginning) / Number of viable bacteria at the beginning × 100%.

[0080] II. Determination of Short-Chain Fatty Acid (SCFA) Yield The fermentation broth after 24 h of culture was used to determine the contents of acetic acid, propionic acid, and butyric acid by gas chromatography-FID. Chromatographic conditions: DB-FFAP capillary column (30 m × 0.25 mm × 0.25 μm), injection port temperature 250°C, detector temperature 280°C, temperature program: initial 80°C, hold for 1 min, increase to 200°C at 10°C / min, hold for 5 min. Quantification was performed using external standard methods with each acid standard, and the total yield of short-chain fatty acids (acetic acid + propionic acid + butyric acid, mmol / L) was calculated.

[0081] III. Determination of Polysaccharide Degradation Fragment Activity Fermentation broths from Examples 1-8 and Comparative Examples 1-10, cultured for 24 h, were centrifuged (10000 rpm, 10 min) to remove bacterial cells. The supernatant was first filtered through a 3 kDa ultrafiltration membrane, and the permeate (containing <3 kDa degradation fragments) was collected. Then, it was filtered through a 1 kDa ultrafiltration membrane, and the retentate (containing 1-3 kDa active polysaccharide fragments) was collected. Simultaneously, permeate <1 kDa was collected for use as a control. Using the original polysaccharide solution that had not undergone probiotic metabolism as a control, the following indicators were measured: (1) DPPH free radical scavenging rate: 2 mL of sample solution (1 mg / mL) was mixed with 2 mL of DPPH solution (0.1 mmol / L, prepared with anhydrous ethanol), and reacted at room temperature in the dark for 30 min. The absorbance was measured at 517 nm and the scavenging rate was calculated.

[0082] (2) ABTS free radical scavenging rate: Take 0.2 mL of sample solution (1 mg / mL) and mix with 3.8 mL of ABTS working solution, react at room temperature for 6 min, measure absorbance at 734 nm, and calculate scavenging rate.

[0083] (3) IL-6 inhibition rate: An LPS-induced RAW264.7 macrophage inflammation model was used. RAW264.7 cells were injected with 1×10⁻⁶ cells. 5 1 sample per well was seeded into a 96-well plate. After culturing for 24 h, LPS (1 μg / mL) and sample (100 μg / mL) were added, and the plate was cultured for another 24 h. The IL-6 content in the supernatant was detected by ELISA, and the inhibition rate was calculated.

[0084] (4) Mucosal repair activity: The migration promotion rate of human colonic epithelial cells (Caco-2) was evaluated by scratch assay. Caco-2 cells were cultured to a confluent monolayer, scratches were made with a pipette tip, and sample (100 μg / mL) was added. After culturing for 24 h, the scratch healing area was measured and the migration promotion rate was calculated.

[0085] Activity enhancement rate (%) = (Activity of sample after metabolism - Activity of sample before metabolism) / Activity of sample before metabolism × 100%.

[0086] IV. Determination of viable bacteria retention rate The symbiotic proliferation system dry powder products prepared in Examples 1-8 and Comparative Examples 1-10 were treated with simulated gastrointestinal fluid: first, they were treated in simulated gastric fluid (containing 0.32% pepsin, pH 2.0) at 37°C for 2 h, and then in simulated intestinal fluid (containing 1% trypsin, pH 6.8) at 37°C for 4 h. The viable bacterial count was determined by plate counting method before and after treatment, and the viable bacterial retention rate was calculated.

[0087] Viable bacteria retention rate (%) = number of viable bacteria after treatment / number of viable bacteria before treatment × 100%.

[0088] V. Measurement of Planting Time The colonization time of probiotics was evaluated using an in vitro mucosal adhesion model. Caco-2 cells were cultured into a confluent monolayer in Transwell chambers, and samples from Examples 1-8 and Comparative Examples 1-10 (1×10⁻⁶) were added. 7 CFU / well were co-cultured under anaerobic conditions at 37°C. Fresh culture medium (excluding samples) was replaced every 12 h. The number of viable bacteria adhering to the Caco-2 cell layer was determined by plate counting. The effective colonization endpoint was defined as the point at which the number of viable bacteria dropped to less than 10% of the initial adhesion amount. The effective colonization duration (h) was recorded.

[0089] VI. Determination of Competition Inhibition Rate by Contaminating Microorganisms Constructing an in vitro mixed culture system: Bifidobacterium lactis BB-12 (1×10⁻⁶) was used to culture the bacteria. 6 CFU / mL) and Escherichia coli ATCC25922 (1×10⁻⁶ CFU / mL) 6 The samples (CFU / mL) were mixed at a 1:1 ratio and inoculated into MRS medium. Samples from Examples 1-8 and Comparative Examples 1-10 (1 wt%) were added separately, and the mixture was anaerobic at 37°C for 24 h. The number of Bifidobacterium lactis and Escherichia coli were counted separately using selective culture media, and the change in the Bifidobacterium lactis / Escherichia coli ratio was calculated.

[0090] Competition inhibition rate of contaminating bacteria (%) = (number of E. coli in the blank group - number of E. coli in the sample group) / number of E. coli in the blank group × 100% (positive value indicates inhibition).

[0091] VII. Polysaccharide structural characterization The molecular weight and molecular weight distribution of polysaccharides were determined by high-performance gel permeation chromatography (HPGPC); the glycosidic bond linkage mode and branching degree were determined by methylation analysis combined with GC-MS; the intrinsic viscosity [η] (25°C aqueous solution) was determined by Ubbelohde viscometer; the content of β-1,3 / 1,6-glucan was quantitatively determined and the purity was calculated using the Megazyme Mixed Linked β-glucan assay kit (enzymatic method, according to AOAC 995.16 standard method); and the structural characteristics of β-glucan were qualitatively identified by Congo red assay and iodine-potassium iodide reaction.

[0092] The results are as follows: Depend on Figure 2 It can be seen that the average proliferation rate of Examples 1-8 was 198.3%, while the average proliferation rate of Comparative Examples 1-10 was only 114.4%. Among them, Example 1 (molecular weight 7.2 kDa) had the highest proliferation rate (218.6%), which was significantly better than Comparative Example 4 (molecular weight 2.5 kDa, proliferation rate 112.3%) and Comparative Example 5 (molecular weight 15 kDa, proliferation rate 98.6%). It is evident that when the molecular weight is too low (<3 kDa), the polysaccharide spatial configuration is incomplete and cannot provide sufficient recognition sites; when the molecular weight is too high (>10 kDa), the polysaccharide chain is too long and the spatial conformation is loose, which is not conducive to the recognition and utilization of probiotics. The proliferation rate of Comparative Example 6 (DB=0.02) was only 105.8%, and the proliferation rate of Comparative Example 7 (DB=0.32) was 108.4%, both significantly lower than that of Example 1 (DB=0.15, proliferation rate 218.6%). This indicates that when branching is too low (<0.05), the side chains are sparse, failing to provide sufficient recognition sites for probiotic sugar transport proteins to bind; when branching is too high (>0.25), the side chains are dense, increasing steric hindrance and hindering the access and degradation of the main chain by probiotic extracellular enzymes. The proliferation rate of Comparative Example 5 ([η]=28.5 mL / g) was only 98.6%, far lower than that of Example 1 ([η]=12.5 mL / g, proliferation rate 218.6%). Intrinsic viscosity reflects the volume occupied by the polysaccharide in solution. [η]>20 mL / g corresponds to a loose and flexible chain conformation, with high steric hindrance and low interfacial contact efficiency; [η]<5 mL / g corresponds to an overly coiled rigid small molecule (Comparative Example 4, [η]=4.2 mL / g, proliferation rate 112.3%), with insufficient branching structure development. [η]=5-20 mL / g corresponds to a low-coil compact conformation, which has the best compatibility with the probiotic cell interface. The proliferation rate of Comparative Example 8 (mass ratio 1:1) was 132.6%, and that of Comparative Example 9 (mass ratio 8:1) was 118.5%, both significantly lower than that of Example 1. When the mass ratio was less than 2:1, the polysaccharides were insufficient to form a complete protective film on the surface of the probiotics; when the mass ratio was greater than 5:1, the excessive polysaccharides created a high-viscosity environment, inhibiting the metabolic activity of the probiotics.

[0093] Table 1. Activity enhancement rate of polysaccharide degradation fragments in Examples 1-8 and Comparative Examples 1-9

[0094] As shown in Table 1, Example 1 exhibited significantly better performance than the comparative examples, with improvements in DPPH scavenging rate (52.3%), ABTS scavenging rate (48.7%), IL-6 inhibition rate (55.2%), and mucosal repair activity (49.6%). The activity improvement rates of Comparative Examples 2 (FOS) and 3 (GOS) were both below 10%, indicating that broad-spectrum oligosaccharides are almost impossible to degrade by probiotics to produce highly active fragments. The activity improvement rates of Comparative Examples 4-9 ranged from 12-26%, far lower than the average of Examples 1-8 (46.6%). This demonstrates that only polysaccharides simultaneously meeting the following criteria—molecular weight 3-10 kDa, DB=0.05-0.25, and [η]=5-20 mL / g—can produce a significant activity amplification effect after being metabolized by probiotics.

[0095] Table 2. Viable bacterial retention rate and colonization time of Examples 1-8 and Comparative Examples 1-10

[0096] As shown in Table 2, Example 1 achieved a viable bacteria retention rate of 85.8% and an effective colonization time of 72 hours, both significantly better than the comparative examples. Comparative Example 10 (without polysaccharides) had a viable bacteria retention rate of only 58.2% and a colonization time of only 12 hours, demonstrating the crucial role of the polysaccharide protective film in the stress protection and mucosal anchoring of probiotics. Comparative Example 1 (without enzymatic hydrolysis of crude polysaccharides) had a viable bacteria retention rate of 68.2% and a colonization time of 28 hours, indicating that polysaccharides lacking regular structures cannot form an effective protective film. The average viable bacteria retention rate of Examples 1-8 was 83.6%, and the average colonization time was 65.1 hours, representing increases of 21.0% and 114.9% respectively compared to the average values ​​of Comparative Examples 1-10 (69.1%, 30.3 hours).

[0097] Table 3. Competition inhibition rates of contaminating bacteria in Examples 1-8 and Comparative Examples 1-10

[0098] As shown in Table 3, the antimicrobial competition inhibition rate of Example 1 reached 58.3%, significantly better than that of Comparative Example 2 (FOS, 36.8%) and Comparative Example 3 (GOS, 34.2%). This indicates that the structured polysaccharide of the present invention is preferentially utilized by lactic probiotics due to its regular branched structure, while non-target intestinal bacteria cannot efficiently utilize this carbon source due to the lack of corresponding recognition mechanisms. The average antimicrobial competition inhibition rate of Examples 1-8 was 52.3%, which is 47.3% higher than the average of Comparative Examples 2 and 3 (35.5%), achieving effective microbial community regulation at the carbon source allocation level.

[0099] comprehensiveFigures 1-2 Based on the data in Tables 1-3, Examples 1-8 significantly outperformed Comparative Examples 1-10 in all indicators. Taking Example 1 as an example, its proliferation rate (218.6%) was 51.6% higher than that of Comparative Example 2 (FOS, 144.2%); SCFA yield (48.7 mmol / L) was 48.5% higher than that of Comparative Example 2 (32.8 mmol / L); polysaccharide activity enhancement rate (average of 51.5% across four items) was 730% higher than that of Comparative Example 2 (6.2%); viable bacteria retention rate (85.8%) was 47.4% higher than that of Comparative Example 10 (58.2%); and colonization time (72 h) was 500% higher than that of Comparative Example 10 (12 h). These data fully demonstrate the real existence and functional significance of the three-level bidirectional synergistic mechanism of this invention, achieving a strong synergistic effect of 1+1>2.

[0100] Application examples Application Example 1: Probiotic Solid Beverage The symbiotic proliferation system dry powder product prepared in Example 1 was mixed with maltodextrin and fruit powder in a ratio of 1:5:4 to obtain a probiotic solid beverage. Each sachet (5 g) contains ≥1×10⁻⁶ live bacteria. 9 CFU. The viable bacteria retention rate of this product is ≥80% after 12 months of storage at room temperature.

[0101] Application Example 2: Functional Yogurt The symbiotic proliferation system dry powder product prepared in Example 4 was added to fermented milk at a ratio of 0.5 wt% to obtain functional yogurt. This yogurt maintained a viable bacterial count ≥1×10⁻⁶ after 21 days of storage at 4°C. 8 The CFU / mL concentration increases the live bacteria retention rate by approximately 35% compared to regular yogurt.

[0102] Application Example 3: Probiotic Capsules The symbiotic proliferation system dry powder product prepared in Example 6 was directly filled into enteric-coated capsules, with each capsule containing ≥5×10⁶ live bacteria. 9 CFU. The capsule retained ≥82% of its live bacteria after treatment in simulated gastrointestinal fluid.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system, characterized in that, The system comprises adaptive structured polysaccharides and milk-derived probiotics in a mass ratio of 2-5:1; The adapted structured polysaccharide has a regular branched structure with a β-1,3-glucan backbone and β-1,6-glucan side chains, a molecular weight of 3-10 kDa, a branching degree DB of 0.05-0.25, an intrinsic viscosity [η] of 5-20 mL / g, and a β-1,3 / 1,6-glucan purity ≥85 wt%. The lactogenic probiotics are selected from one or more of Bifidobacterium lactis, Streptococcus thermophilus, and Lactobacillus delbrueckii subsp. bulgaricus.

2. The milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system according to claim 1, characterized in that, The molecular weight of the adapted structured polysaccharide is 5-8 kDa.

3. The milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system according to claim 1, characterized in that, The mass ratio of the adapted structured polysaccharide to the milk-derived probiotics is 3-4:

1.

4. The milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system according to claim 1, characterized in that, The adapted structured polysaccharide was prepared from yeast β-glucan via enzymatic hydrolysis and ultrafiltration fractionation.

5. The milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system according to claim 4, characterized in that, The enzymatic hydrolysis was performed using β-1,3-glucan endonuclease at a temperature of 45-55°C for 2-6 hours, with the enzyme added at a concentration of 0.5-2 wt% of the substrate.

6. The milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system according to claim 4, characterized in that, The ultrafiltration fractionation adopts a two-stage series ultrafiltration membrane fractionation: first, the permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and then the permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to retain the target components of 3-10 kDa.

7. The method for preparing the milk-derived probiotic-specific adaptive polysaccharide targeted symbiotic proliferation system according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Activation and culture of milk-derived probiotics: Activate and culture milk-derived probiotics in basal culture medium until the logarithmic growth phase (OD). 600 =0.6-0.8, to obtain a viable bacteria concentration ≥10 8 CFU / mL bacterial suspension; S2. Structure-activity matching compounding: The adapted structured polysaccharide and the milk-derived probiotic liquid are mixed and compounded under aseptic conditions at 2-8°C for 30-60 min to form a symbiotic system; S3. Steady-state molding: The symbiotic system is vacuum dried at a temperature ≤40°C and a vacuum degree ≤50 Pa until the moisture content of the system is ≤5 wt%, to obtain the dry powder product of the symbiotic proliferation system.

8. The preparation method according to claim 7, characterized in that, The vacuum drying temperature in step S3 is 35-38°C.

9. The use of the milk-derived probiotic-specific aptamer-targeted symbiotic proliferation system according to any one of claims 1-6 in the preparation of foods, health products or drugs for regulating intestinal flora, enhancing immunity, anti-inflammation or mucosal repair.

10. The application according to claim 9, characterized in that, The system operates through the following three-level bidirectional collaborative mechanism: (1) Positive adaptation-structure-promoting bacteria: The regular branched structure of the adapted structured polysaccharide is preferentially recognized and efficiently utilized by lactic probiotics, promoting the proliferation of probiotics; (2) Reverse activation - Bacterial polysaccharide: During the process of lactic probiotics metabolizing and utilizing structured polysaccharides, the structured polysaccharides are degraded and activated, generating active polysaccharide fragments with a molecular weight of 1-3 kDa, which enhances the antioxidant, anti-inflammatory and mucosal repair activities of polysaccharides. (3) Steady-state bacterial fixation-structured bacterial fixation: The adapted structured polysaccharide forms a protective film on the surface of the probiotic cells of the milk source, achieving a triple bacterial fixation effect of stress protection, mucosal anchoring and slow-release carbon source.

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