A method for constructing a probiotic gastric acid resistant gel bead using polyphenol cross-linked protein

Abstract

The application discloses a method for constructing a gastric acid-resistant gel bead of probiotics by using polyphenol cross-linked proteins, and belongs to the technical field of microencapsulation of probiotics. In the method, probiotics, whey protein isolate and low-methoxyl pectin are mixed, and then extruded into a calcium ion bath to form ion cross-linked network gel beads; then the gel beads are placed in a solution containing 5-O-caffeoylquinic acid, and catalytic oxidation is generated under the action of laccase and dissolved oxygen to form orthoquinone, which is reacted with a protein side chain to form a covalent cross-linked network, thereby forming an ion-covalent double network interpenetrating structure. The survival rate of the obtained gel bead in simulated gastric juice is greater than 90% for 2 hours, and the complete disintegration release rate of the gel bead in simulated intestinal juice is greater than 95% for 30 minutes.

Description

Technical Field

[0001] This invention relates to the field of microencapsulation and probiotic activity protection technology, and in particular to a method for constructing probiotic acid-resistant gel beads using polyphenol cross-linked proteins. Background Technology

[0002] The health benefits of oral probiotics highly depend on a sufficient quantity of live bacteria successfully crossing the gastric acid barrier and reaching the intestinal target site for colonization. Human gastric juice, when empty, can have a pH as low as 1.2-1.5 and contains pepsin, posing a primary challenge to probiotic survival. Among existing protection strategies, the classic extrusion-based exogenous gelation method utilizes sodium alginate and calcium ions to form gel beads. However, calcium alginate gel networks have large pore sizes and limited pH sensitivity; in a strongly acidic environment, calcium ions are easily replaced by hydrogen ions, leading to rapid gel structure disintegration. This fails to effectively block proton influx, resulting in limited live bacteria protection; after 2 hours of treatment in simulated gastric acid, the survival rate is often below 30%.

[0003] To address this issue, researchers introduced chitosan to form a polyelectrolyte complex coating via electrostatic complexation. However, at pH < 3.0, the highly protonated amino groups of chitosan caused severe swelling and even disintegration, exacerbating core material exposure. While constructing gels using transglutaminase or genipin to cross-link proteins could form stable covalent networks, the cross-linking process was time-consuming and difficult to control precisely. Over-crosslinked networks were also difficult for trypsin to degrade in the neutral intestinal environment, resulting in a low viable cell release rate. Direct assembly of polyphenols such as tannins with proteins through hydrogen bonds and hydrophobic interactions could rapidly form dense networks under mild conditions. However, the single hydrogen bond-driven network lacked stability under the strong ionic strength and low pH environment of gastric acid, making it difficult to resist continuous acid erosion.

[0004] Therefore, existing technologies struggle to simultaneously ensure high gastric acid tolerance and high intestinal release of gel beads while maintaining a mild, efficient, and food-grade preparation process. There is an urgent need to develop a novel gel system that, through the synergistic effect of multiple driving forces at the molecular scale, overcomes the inherent limitations of a single cross-linking mechanism, forming a reversible, dynamically responsive, and highly dense proton barrier in the extremely harsh gastric acid environment, while ensuring rapid disintegration and release in the intestinal environment. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides a method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins, comprising the following steps: Step 1: Mix whey protein isolate solution and low-methoxy pectin solution at a volume ratio of 4:1 to obtain whey protein isolate-low-methoxy pectin composite solution; mix probiotic pre-suspension solution and whey protein isolate-low-methoxy pectin composite solution at a volume ratio of 1:9 to obtain probiotic-polymer mixture. Step 2: The probiotic-polymer mixture is extruded through a nozzle with an inner diameter of 0.5 mm into a calcium chloride crosslinking bath with a pH of 4.0 and a mass-volume concentration of 3%. The mixture is allowed to stand for crosslinking, and the wet gel beads are collected and washed to obtain a probiotic-dispersed single ion crosslinked network gel bead precursor. Step 3: Mix the probiotic-dispersed single-ion cross-linked network gel bead precursor with a 5-O-caffeoylquinic acid solution with a mass / volume concentration of 0.8% and a pH of 6.0. Under light-protected conditions at 10°C, add laccase to a final concentration of [missing information]. The enzyme activity of the laccase is defined as the amount of enzyme required to catalyze the oxidation of 1 micromole of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid per minute under conditions of 30°C and pH 6.0, while maintaining a dissolved oxygen concentration of [missing value - likely a specific concentration]. An enzymatic oxidative cross-linking reaction is carried out to oxidize 5-O-caffeoylquinic acid to generate an ortho-quinone intermediate. This ortho-quinone intermediate undergoes Michael addition and Schiff base reaction with the lysine ε-amino and cysteine ​​sulfhydryl groups of whey protein isolate to form a covalent cross-linked network. This network, together with the ionic cross-linked network in the probiotic dispersed single-ion cross-linked network gel bead precursor, constitutes an ion-covalent double-network interpenetrating structure, yielding probiotic acid-resistant gel beads. The average particle size of the probiotic acid-resistant gel beads is [missing information]. In response to The Young's modulus in the linear viscoelastic region is .

[0006] Preferably, the preparation of the probiotic suspension in step 1 includes: taking Lactobacillus rhamnosus cultured to the stationary phase, centrifuging at 8000 rpm for 10 minutes at 4°C, discarding the supernatant, and collecting the bacterial precipitate; resuspending the collected bacterial precipitate in phosphate buffer with a pH of 6.8 pre-cooled to 4°C, centrifuging at 8000 rpm for 10 minutes at 4°C, discarding the supernatant, and repeating this washing process twice; resuspending the finally obtained washed bacterial precipitate in phosphate buffer with a pH of 6.8, determining the viable cell concentration of the suspension using the plate count method, and adjusting the viable cell concentration to the required pH using phosphate buffer with a pH of 6.8. The probiotic initial suspension was obtained.

[0007] Preferably, the whey protein isolate-low methoxylated pectin composite solution in step 1 is prepared as follows: Under constant temperature of 25°C, a phosphate buffer solution with a pH of 6.8 is stirred at 300 rpm. Whey protein isolate powder is added in batches of 0.5 g at a time, and stirring is continued until the powder is completely dissolved, obtaining a whey protein isolate solution with a mass-to-volume concentration of 12%. Low methoxylated pectin powder is added to another portion of the phosphate buffer solution with a pH of 6.8, and stirred at 400 rpm in a 40°C water bath until completely dissolved. After cooling to 25°C, a low methoxylated pectin solution with a mass-to-volume concentration of 4% is obtained. The methoxylated content of the low methoxylated pectin is 22%-28%, and the weight-average molecular weight is [not specified]. Under stirring conditions of 25°C and 400 rpm, the whey protein isolate solution with a mass-volume concentration of 12% and the low-methoxyl pectin solution with a mass-volume concentration of 4% were mixed at a volume ratio of 4:1, and the mixture was stirred for 30 minutes to obtain the whey protein isolate-low-methoxyl pectin composite solution.

[0008] Preferably, the calcium chloride crosslinking bath in step 2 is prepared by dissolving calcium chloride dihydrate in deionized water to form a calcium chloride solution with a mass-volume concentration of 3%, and then adjusting the pH value to 4.0 with 0.1 mol / L hydrochloric acid. The probiotic-polymer mixture is continuously extruded at 25°C through a nozzle with an inner diameter of 0.5 mm at a flow rate of 1.5 m / s into the calcium chloride crosslinking bath, which is under continuous stirring at 50 rpm. The droplets are allowed to stand in the calcium chloride crosslinking bath for 30 minutes to crosslink, forming wet gel beads. The wet gel beads are collected by filtering with a nylon filter with a pore size of 200 micrometers. The collected wet gel beads are immersed in 20 times their mass of deionized water, stirred at 50 rpm for 1 minute, and then filtered with a nylon filter with a pore size of 200 micrometers. This washing process is repeated 3 times to obtain the probiotic dispersed single ion crosslinked network gel bead precursor.

[0009] Preferably, the 5-O-caffeoylquinic acid solution with a mass-volume concentration of 0.8% and a pH value of 6.0 in step 3 is prepared as follows: At 10°C and under light-protected conditions, 5-O-caffeoylquinic acid powder is dissolved in a 0.05 mol / L phosphate buffer solution with a pH value of 6.0 pre-cooled to 10°C, stirred at 200 rpm to dissolve, and then brought to a final volume with the 0.05 mol / L phosphate buffer solution with a pH value of 6.0; the probiotic-dispersed single-ion cross-linked network gel bead precursor and the 0.8% mass-volume concentration of 5-O-caffeoylquinic acid solution with a pH value of 6.0 are added to a jacketed light-protected reactor at a mass ratio of 1:8, and the reaction is carried out at 10°C and under light-protected conditions with continuous stirring at a rate of 60 rpm for 60 minutes to allow 5-O-caffeoylquinic acid to diffuse into the probiotic-dispersed single-ion cross-linked network gel bead precursor.

[0010] Preferably, the operation of adding laccase and purging sterile air in step 3 for the enzymatic oxidative cross-linking reaction includes: adding a laccase solution pre-prepared with 0.05 mol / L phosphate buffer at pH 6.0 to the reaction system after the diffusion reaction has proceeded for 60 minutes, so that the final concentration of laccase in the reaction system is... Immediately introduce sterile air into the bottom of the reaction solution through a gas distributor with an aperture of 0.22 micrometers, and adjust the aeration rate to maintain a constant dissolved oxygen concentration in the reaction solution. The reaction was carried out at 10°C and in the dark with continuous stirring at 60 rpm for 40 minutes. After the reaction, the gel beads were collected by filtering with a nylon filter with a pore size of 200 micrometers. The gel beads were washed three times with 20 times their mass of deionized water at 10°C and stirred at 50 rpm for 1 minute to obtain the probiotic acid-resistant gel beads.

[0011] Preferably, the dissolved oxygen concentration is monitored in real time by an online dissolved oxygen electrode, and the flow rate of the sterile air is automatically adjusted by a mass flow controller to maintain a constant dissolved oxygen concentration. .

[0012] Preferably, the low-methoxyl pectin in step 1 has a galacturonic acid content of not less than 65%; the whey protein isolate in step 1 has a protein content of not less than 90%, and the β-lactoglobulin content accounts for 55%-65% of the total mass of the whey protein isolate; the phosphate buffer with a pH of 6.8 is obtained by mixing 0.2 mol / L sodium dihydrogen phosphate solution and 0.2 mol / L disodium hydrogen phosphate solution at a volume ratio of 1:2, and then diluting with deionized water to 0.1 mol / L.

[0013] Preferably, in step 1, the probiotic suspension and the whey protein isolate-low methoxy pectin composite solution are mixed at a volume ratio of 1:9, and the mixture is stirred at 300 rpm for 5 minutes at a constant temperature of 25°C; in step 2, the nozzle has an inner diameter tolerance of ±0.02 mm, the driving pressure applied to the probiotic-polymer mixture during extrusion is 0.15 MPa, and the extrusion ambient temperature is maintained at 25°C.

[0014] Preferably, when the probiotic acid-resistant gel beads are tested in the following two systems: after being treated with a pH 1.2 hydrochloric acid solution containing 0.32% pepsin at 37°C and continuously shaken at 50 rpm for 2 hours, the survival rate of Lactobacillus rhamnosus inside the probiotic acid-resistant gel beads is not less than 90%; and after being treated with a pH 6.8 phosphate buffer solution containing 0.5% trypsin at 37°C and continuously shaken at 50 rpm for 30 minutes, the probiotic acid-resistant gel beads completely disintegrate, and the release rate of Lactobacillus rhamnosus is not less than 95%.

[0015] The beneficial effects of this invention are: 1. This invention employs a sequential construction strategy of "first ion shaping, then covalent network locking" to form an ion-covalent dual-network interpenetrating structure that interpenetrates a low-methoxy pectin-calcium ion network and a whey protein isolate-5-O-caffeoylquinic acid quinone covalent network. This structure generates a cascaded synergistic defense effect under simulated gastric acid attack. The ion network sacrificially contracts to consume hydrogen ions, while the covalent network maintains a dense barrier to block proton influx. This results in a survival rate of over 90% for probiotics treated in simulated gastric fluid at pH 1.2 for 2 hours, far exceeding the algebraic sum of the protective effects of a single ion network and a single covalent network, achieving unexpected technical results. 2. The gel beads constructed in this invention exhibit excellent pH and enzyme-responsive disintegration properties. Under the simulated neutral environment of intestinal fluid and the action of trypsin, calcium ions are chelated, leading to the disintegration of the ion network. Protease hydrolyzes the whey protein isolate backbone, causing the covalent network to collapse. This dual mechanism enables the gel beads to completely disintegrate within 30 minutes, with a live bacteria release rate of over 95%, achieving an ideal balance between effective gastric protection and rapid intestinal release. 3. The entire method is implemented under mild conditions, using all food-grade raw materials and laccase-catalyzed cross-linking, avoiding the use of any toxic chemical cross-linking agents, thus ensuring the product's high biocompatibility and food safety. The cross-linking precursor 5-O-caffeoylquinic acid, as a natural dietary polyphenol, can simultaneously exert antioxidant synergistic functions during delivery, further enhancing the product's health value. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0017] Figure 1 This is a flowchart of the steps of the method of the present invention. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should also be noted that, to make the embodiments more comprehensive, the following embodiments are the best and preferred embodiments, and those skilled in the art can use other alternative methods to implement some well-known technologies; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0019] Please see Figure 1 This invention provides a method for constructing probiotic acid-resistant gel beads using polyphenol-crosslinked proteins. The core inventive concept lies in: through strict temporal control of "first ion-based shaping, then covalent network locking," a gel matrix with a "pH-responsive ion-covalent dual-network interpenetrating structure" is constructed at the microscale, thereby forming an intelligent barrier around the probiotics that can cascade against gastric acid erosion and achieve rapid intestinal disintegration.

[0020] First, in step 2, this invention pre-constructs a single ionic cross-linked network formed by low-methoxyl pectin and calcium ions as a precursor for probiotic dispersion gel beads. This ionic network acts as a sacrificial defense layer in the subsequent acidic gastric environment. When the gel beads enter a strongly acidic environment with a pH of 1.2, hydrogen ions preferentially replace calcium ions in this ionic network, causing the low-methoxyl pectin segments to rapidly shrink and densify. This process is not a passive dissolution, but rather an active consumption and neutralization of the large influx of hydrogen ions, thus gaining crucial buffer time for the inner structure and significantly delaying the pH drop in the core region. Without this pre-constructed ionic network, the acidic environment would directly impact the internal protective layer, leading to a sharp decline in protective efficiency.

[0021] Secondly, this invention introduces a uniquely designed enzymatic oxidative cross-linking reaction in step 3 to construct a second covalent cross-linking network. Specifically, under conditions of laccase and precisely controlled dissolved oxygen concentration, the catechol group of 5-O-caffeoylquinic acid is oxidized to a highly reactive o-quinone intermediate. These o-quinone intermediates can spontaneously undergo a Schiff base reaction with the lysine ε-amino group on the whey isolate molecular chain and a Michael addition reaction with the thiol group of cysteine, forming extremely stable CN and CS covalent bonds. The resulting covalent cross-linking network exhibits extremely high chemical inertness to acid hydrolysis, maintaining its highly dense topology throughout the entire gastric acid retention period, thereby forming extremely tortuous proton diffusion channels and minimizing the rate of proton permeation into the inner core cells. This enzymatic process is carried out at a low temperature of 10°C, completely avoiding the use of any toxic chemical cross-linking agents, and the conditions are mild and highly controllable.

[0022] The two networks described above are not simply physically mixed within the gel beads, but rather form an interpenetrating structure, generating a temporally synergistic cascade defense under gastric acid attack. The outer ionic network responds sacrificially to gastric acid, and the resulting contracted and compacted layer further enhances the proton shielding effectiveness of the inner covalent network. This synergistic effect far exceeds the algebraic sum of a single network: tests show that the bacterial survival rate of gel beads with a single ionic cross-linked network is approximately 15% after 2 hours of treatment in simulated gastric acid, while that of gel beads with a single covalent cross-linked network is approximately 45%, with a simple sum of 60%. However, the dual-network interpenetrating structure gel beads constructed in this invention achieve a survival rate of over 90% under the same conditions.

[0023] Finally, upon entering the neutral pH 6.8 environment of the intestine, on the one hand, components such as phosphate and bile salts in the intestine strongly chelate calcium ions, causing the ion network to rapidly disintegrate; on the other hand, trypsin efficiently enzymatically dissolves the whey protein isolate backbone, causing the covalently cross-linked network backbone to break. The simultaneous triggering of these two disintegration mechanisms ensures the rapid disintegration of the gel beads and the instantaneous and complete release of live bacteria.

[0024] To clearly define the technical solution of this invention, the following provides a detailed description of key terms, parameters, and measurement methods that may cause ambiguity.

[0025] Methoxyl content and degree of esterification of low-methoxyl pectin

[0026] Methoxy group content refers to the percentage of methoxy groups (-OCH3) in the total mass of pectin. Esterification degree refers to the percentage of methoxylated galacturonic acid residues in the pectin molecular chain out of the total number of galacturonic acid residues. The two values ​​are highly correlated but not equivalent. In this invention, methoxy group content is used as the limiting standard, specifically within the range of 22%-28%. This determination is performed using the method specified in the People's Republic of China National Standard GB 25533-2010, "National Food Safety Standard for Food Additives: Pectin". Simultaneously, to ensure its cross-linking ability, the galacturonic acid content is limited to no less than 65%.

[0027] Definition of laccase activity

[0028] The final concentration of laccase is expressed in U / mL. The definition of an enzyme activity unit (U) follows the standards of the International Union of Pure and Applied Chemistry (IUPAC): One unit of enzyme activity (1 U) is defined as the amount of enzyme required to catalyze the oxidation of 1 micromolar of the substrate 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) per minute under constant conditions of 30°C and pH 6.0. This assay was performed by detecting the rate of increase in absorbance at 420 nm using a spectrophotometer, based on the molar extinction coefficient of the ABTS cationic radical. Perform the calculation.

[0029] Precise control of dissolved oxygen concentration

[0030] The dissolved oxygen concentration in step 3 is maintained constant at The concentration is monitored in real time by an online dissolved oxygen electrode inserted into the reaction solution, and the signal is fed back to the mass flow controller. When the measured value is lower than the set value, the mass flow controller automatically increases the flow rate of sterile air entering the gas distributor with an orifice diameter of 0.22 micrometers; conversely, it decreases the flow rate. This closed-loop control system ensures a constant supply of oxygen—the electron acceptor required for the laccase-catalyzed oxidation reaction—thereby precisely controlling the degree of crosslinking.

[0031] Physical structural characteristics of gel beads

[0032] The final physical morphology of probiotic acid-resistant gel beads must meet specific structural parameters to ensure their performance. The average particle size was determined using a laser particle size analyzer (wet mode), and the range was limited to [specific parameters missing]. In response to The Young's modulus in the linear viscoelastic region was determined by a texture analyzer under single compression mode, with a defined range of [range missing]. This modulus range reflects the rigidity of the gel beads imparted by the interpenetrating double network structure. If the value is too low, it cannot withstand the pressure of gastric peristalsis, while if it is too high, it will affect the rate of intestinal disintegration.

[0033] Test conditions for simulated gastric and intestinal fluids

[0034] To verify the protective effect and release performance, the present invention uses the following two standard test solutions, and the tests are conducted completely independently.

[0035] The simulated gastric fluid was a hydrochloric acid solution containing 0.32% pepsin (enzyme activity ≥3000 U / mg) at a volume concentration of 1.0 mol / L. The pH was precisely adjusted to 1.2 using 1.0 mol / L hydrochloric acid. The survival rate test method was as follows: 1.0 g of wet probiotic acid-resistant gel beads was accurately weighed and immersed in 10 mL of the simulated gastric fluid preheated to 37°C. The mixture was continuously shaken at 50 rpm for 2 hours in a 37°C constant-temperature shaker. Immediately afterwards, the gel beads were removed, rapidly washed with pre-cooled sterile phosphate buffer, and then placed in 9 mL of sterile phosphate buffer. The mixture was homogenized for 1 minute at 10,000 rpm at 4°C using a high-speed homogenizer. The resulting homogenate was serially diluted, and the viable cell count was determined using the plate count method to calculate the survival rate. Survival rate (%) = (Number of viable cells inside the gel beads after simulated gastric fluid treatment / Initial number of viable cells inside the gel beads) × 100%.

[0036] The simulated intestinal fluid was a pH 6.8 phosphate buffer (0.05 mol / L) containing 0.5% trypsin (enzyme activity ≥250 U / mg). The release rate test method was as follows: 1.0 g of wet probiotic acid-resistant gel beads was accurately weighed and immersed in 10 mL of the simulated intestinal fluid preheated to 37°C. The mixture was continuously shaken at 50 rpm for 30 minutes in a 37°C constant-temperature shaker. The disintegration of the gel beads was observed and recorded. The entire mixture was then passed through a standard test sieve with a pore size of 500 μm. The sieve was rinsed with 10 mL of deionized water, and all filtrates were collected and brought to a final volume. The number of viable bacteria in the filtrate was determined using the plate count method, and the release rate was calculated. Release rate (%) = (Total number of viable bacteria in the filtrate / Total number of viable bacteria inside the initial gel beads) × 100%. The total number of viable bacteria inside the initial gel beads was obtained from parallel samples taken during the same period that were not treated with the simulated intestinal fluid, and determined using the same disruption and plate count methods. The quantitative criterion for determining "complete disintegration" of gel beads is: after the residue on the sieve is dried to constant weight at 105℃, its mass does not exceed 2% of the total dry weight of the initial gel beads.

[0037] The present invention will be described in detail below with reference to specific embodiments and comparative examples, but the present invention is not limited to these embodiments.

[0038] Example 1 (Lactobacillus rhamnosus, standard process)

[0039] Step 1.1: Preparation of the probiotic suspension. Take a culture of *Lactobacillus rhamnosus* GG culture that has reached the stationary phase. Centrifuge the culture at 8000 rpm for 10 minutes at 4°C, discard the supernatant, and collect the bacterial precipitate. Resuspend the collected bacterial precipitate in phosphate buffer (pH 6.8) pre-cooled to 4°C, and centrifuge again at 8000 rpm for 10 minutes at 4°C, discarding the supernatant. Repeat this washing process twice. Resuspend the final washed bacterial precipitate in a small amount of phosphate buffer (pH 6.8). Determine the viable cell concentration of the suspension using the plate count method, and precisely adjust the pH using phosphate buffer (pH 6.8) to obtain the final viable cell concentration. The probiotic initial suspension. The phosphate buffer solution with a pH of 6.8 is prepared by mixing 0.2 mol / L sodium dihydrogen phosphate solution and 0.2 mol / L disodium hydrogen phosphate solution at a volume ratio of 1:2, and then diluting with deionized water to 0.1 mol / L.

[0040] Step 1.2: Preparation of whey protein isolate-low methoxylated pectin composite solution. Under constant temperature of 25℃, continuously stir a phosphate buffer solution with a pH of 6.8 at 300 rpm. Add whey protein isolate powder (protein content not less than 90%, β-lactoglobulin content accounting for 55%-65% of the total whey protein isolate mass) in batches of 0.5 g, continuously stirring until the powder is completely dissolved, obtaining a whey protein isolate solution with a mass concentration of 12%. Separately, take a portion of phosphate buffer solution with a pH of 6.8, add low methoxylated pectin powder, and stir at 400 rpm in a 40℃ water bath until completely dissolved. After cooling to 25℃, obtain a low methoxylated pectin solution with a mass concentration of 4%. The methoxylated content of the low methoxylated pectin used is 22%-28%, and the weight average molecular weight is [not specified]. The galacturonic acid content is not less than 65%. Under stirring conditions of 25°C and 400 rpm, the aforementioned 12% whey protein isolate solution and 4% low-methoxyl pectin solution are mixed at a volume ratio of 4:1, and stirring is continued for 30 minutes to obtain a whey protein isolate-low-methoxyl pectin composite solution. Under constant temperature conditions of 25°C and a speed of 300 rpm, the probiotic suspension obtained in step 1.1 is mixed with the whey protein isolate-low-methoxyl pectin composite solution at a volume ratio of 1:9 for 5 minutes to obtain a probiotic-polymer mixture.

[0041] Step 2: Constructing a single-ion crosslinked network gel bead precursor. A calcium chloride crosslinking bath was prepared by dissolving calcium chloride dihydrate in deionized water to a 3% (w / v) calcium chloride solution, and then adjusting the pH to 4.0 with 0.1 mol / L hydrochloric acid. Under extrusion conditions of 25°C, the probiotic-polymer mixture was continuously extruded at a flow rate of 1.5 m / s through a nozzle with an inner diameter of 0.5 mm and an inner diameter tolerance of ±0.02 mm into the aforementioned calcium chloride crosslinking bath, which was under continuous stirring at 50 rpm. The resulting droplets were allowed to crosslink in the crosslinking bath for 30 minutes to form wet gel beads. Wet gel beads were collected by filtering through a nylon filter with a pore size of 200 micrometers. The collected wet gel beads were then immersed in 20 times their weight of deionized water and stirred at 50 rpm for 1 minute. The mixture was then filtered through a nylon filter with a pore size of 200 micrometers. This washing process was repeated 3 times to obtain a probiotic-dispersed single ion cross-linked network gel bead precursor.

[0042] Step 3: Polyphenol-mediated interfacial diffusion and enzymatic covalent cross-linking. First, under light-protected conditions at 10°C, 5-O-caffeoylquinic acid powder was dissolved in a 0.05 mol / L phosphate buffer solution pre-cooled to 10°C with a pH of 6.0. The solution was stirred at 200 rpm and brought to a final volume to obtain a 0.8% (w / v) 5-O-caffeoylquinic acid solution with a pH of 6.0. The probiotic-dispersed single-ion cross-linked network gel bead precursor obtained in Step 2 was added to this 5-O-caffeoylquinic acid solution at a mass ratio of 1:8 into a jacketed, light-protected reactor. Under light-protected conditions at 10°C, the reaction was continuously stirred at 60 rpm for 60 minutes to allow 5-O-caffeoylquinic acid to fully diffuse into the gel bead precursor. After the diffusion reaction proceeded for 60 minutes, a laccase solution prepared in advance with 0.05 mol / L phosphate buffer at pH 6.0 was added to the reaction system to bring the final concentration of laccase in the reaction system to [value missing]. Here, the enzyme activity of laccase is defined as the amount of enzyme required to catalyze the oxidation of 1 micromole of ABTS per minute at 30°C and pH 6.0. Immediately after adding laccase, sterile air is introduced into the bottom of the reaction solution through a gas distributor with a 0.22-micron pore size. The airflow rate is automatically adjusted by a control system connected to an online dissolved oxygen electrode and a mass flow controller to maintain a constant dissolved oxygen concentration in the reaction solution. The reaction was carried out at 10°C and in the dark, with continuous stirring at 60 rpm for 40 minutes. During this process, 5-O-caffeoylquinic acid was oxidized to an ortho-quinone intermediate. This ortho-quinone intermediate underwent Michael addition and Schiff base reaction with the lysine ε-amino and cysteine ​​thiol groups of whey protein isolate to form a covalent cross-linked network, which together with the original ionic cross-linked network constitutes an ion-covalent interpenetrating double network structure. After the reaction, the gel beads were collected by filtering through a 200-micron nylon filter and washed three times with 20 times their mass of deionized water at 10°C, stirring at 50 rpm for 1 minute, to finally obtain probiotic acid-resistant gel beads.

[0043] Example 2 (Bifidobacterium animalis)

[0044] In Example 1, Lactobacillus rhamnosus in step 1.1 was replaced with Bifidobacterium animalis subsp. lactis BB-12, while the remaining steps, raw materials, and process parameters were exactly the same as in Example 1. The resulting probiotic acid-resistant gel beads had an average particle size of 1.98 mm and a Young's modulus of 19.6 kPa in the linear viscoelastic region.

[0045] Example 3 (Lactobacillus acidophilus)

[0046] In Example 1, Lactobacillus rhamnosus in step 1.1 was replaced with Lactobacillus acidophilus NCFM. All other steps, raw materials, and process parameters were identical to those in Example 1. The resulting probiotic acid-resistant gel beads had an average particle size of 2.10 mm and a Young's modulus of 22.8 kPa in the linear viscoelastic region.

[0047] Example 4 (Short-term diffusion)

[0048] The diffusion reaction time in step 3 of Example 1 was changed from "60 minutes" to "30 minutes", while the remaining steps, raw materials and process parameters were exactly the same as in Example 1.

[0049] Example 5 (Long-term diffusion)

[0050] The diffusion reaction time in step 3 of Example 1 was changed from "60 minutes" to "120 minutes", while the remaining steps, raw materials and process parameters were exactly the same as in Example 1.

[0051] Comparative Example 1 (Single Ion Crosslinked Network)

[0052] Gel beads were prepared according to steps 1 to 2 of Example 1, but all operations in step 3 were omitted, i.e. only the probiotic-dispersed single ion cross-linked network gel bead precursor was used as the final product.

[0053] Comparative Example 2 (Single Covalently Crosslinked Network)

[0054] Step 2 of Example 1 is omitted, i.e., the low-methoxyl pectin-calcium ion crosslinking is not performed. The whey protein isolate-low-methoxyl pectin composite solution in step 1.2 is replaced with an equal volume of pure whey protein isolate solution (concentration adjusted accordingly), mixed with the bacterial culture, and then directly added dropwise to the 5-O-caffeoylquinic acid and laccase reaction system in step 3 for covalent crosslinking. Gel beads are collected. Visual observation shows that the gel beads formed under these conditions are intact spheres that can be picked up with tweezers without breaking, possessing workable physical strength.

[0055] Comparative Example 3 (Reverse Crosslinking Timing)

[0056] First, covalent cross-linking is performed, followed by ionic cross-linking. Specifically, the probiotic suspension is mixed with a pure whey protein isolate solution, and enzymatic covalent cross-linking is performed according to the system described in step 3 of Example 1 (but without the gel bead precursor, directly in the protein solution containing bacteria). This forms a covalently cross-linked gel, which is then cut into spherical pieces approximately 2 mm in diameter using a scalpel. These pieces serve as the starting material for subsequent ionic cross-linking. After washing, this starting material is immersed in a 4% (w / v) low-methoxyl pectin solution to adsorb the pectin layer. Subsequently, it is transferred to a calcium chloride cross-linking bath as described in step 2 of Example 1 for outer layer ionic cross-linking. Finally, core-shell gel beads with an inner covalent layer and an outer ionic layer are obtained.

[0057] Comparative Example 4 (Low Laccase Concentration)

[0058] The final concentration of laccase in step 3 of Example 1 was changed from Modified to The remaining steps, raw materials, and process parameters are exactly the same as in Example 1.

[0059] Comparative Example 5 (High Laccase Concentration)

[0060] The final concentration of laccase in step 3 of Example 1 was changed from Modified to To ensure that the crosslinking reaction in this comparative example is not oxygen-limited, the rate of sterile air introduction was simultaneously doubled to maintain a constant dissolved oxygen concentration. The remaining steps, raw materials, and process parameters are exactly the same as in Example 1.

[0061] Performance Testing and Result Comparison

[0062] For all the gel beads obtained in the above embodiments and comparative examples, the simulated gastric acid survival rate, simulated intestinal fluid release rate, average particle size, and Young's modulus were determined according to the methods defined above in this specific embodiment. The results are detailed in Table 1.

[0063] Table 1. Performance test results of each embodiment and comparative example

[0064] Results Analysis

[0065] The following conclusions can be drawn from the data in Table 1.

[0066] Synergistic innovation: The survival rate of Example 1 (92.5%) was much higher than the simple sum of Comparative Example 1 (14.6%) and Comparative Example 2 (44.8%) (approximately 59.4%), which strongly demonstrates that the dual-network interpenetrating structure constructed in this invention produces a synergistic effect.

[0067] The irreplaceable nature of the timing sequence: Although Comparative Example 3 also contains two networks, due to the reversed construction timing, it forms a core-shell structure of "inner covalent layer - outer ion layer," rather than the interpenetrating structure of this invention. Its protective effect (68.2%) is significantly inferior to Example 1, which fully demonstrates that the operational timing of "ion shaping first, then covalent network locking" is key to producing the technical effect of this invention. It is worth noting that although the Young's modulus (16.5 kPa) of Comparative Example 3 falls within the range of this invention... While the Young's modulus range is within the acceptable range, its performance remains unsatisfactory. This indicates that the Young's modulus range is a characteristic accompanying indicator of the dual-network interpenetrating structure gel beads of this invention, rather than a sufficient condition to independently determine performance. Only when the core structural feature of "ion-covalent dual-network interpenetrating structure" is simultaneously present can this modulus range serve as an indicator of excellent performance.

[0068] Strain applicability: Examples 2 and 3 used different genera of probiotics, Bifidobacterium animalis and Lactobacillus acidophilus, respectively. Their gastric acid survival rate was greater than 90%, and their intestinal fluid release rate was greater than 95%, showing performance essentially consistent with Example 1. This indicates that the method of the present invention is applicable to at least three representative probiotic strains: Lactobacillus rhamnosus, Lactobacillus acidophilus, and Bifidobacterium animalis, and can achieve equivalent levels of protection and release effects.

[0069] Process window tolerance: In Examples 4 and 5, the diffusion time was shortened to 30 minutes and extended to 120 minutes, respectively, and the performance indicators of the resulting gel beads were basically the same as those in Example 1. This indicates that 30 minutes to 120 minutes is an effective process window for this technical solution, and fluctuations in diffusion time will not lead to significant deterioration of product performance. The release rate (95.8%) of the 120-minute diffusion group was slightly lower than that of the other examples, possibly because the extended diffusion time caused a small amount of polyphenols to undergo non-enzymatic oxidation side reactions before crosslinking. However, the release rate of 95.8% is still much higher than that of Comparative Examples 1-5, and fully meets the application requirements.

[0070] Optimization of laccase concentration: Comparative Example 4 used a low laccase concentration ( This resulted in insufficient covalent cross-linking, and the gel bead modulus (11.8 kPa) and survival rate (58.7%) both failed to meet the standards. Comparative Example 5 used a high laccase concentration (…). Under conditions where dissolved oxygen supply is ensured to be unrestricted, its survival rate (85.2%) is still lower than that of Example 1 (92.5%), while its Young's modulus (30.5 kPa) far exceeds the scope of this invention. This result indicates that the degree of crosslinking and the protective effect against gastric acid are not monotonically positively correlated; excessive crosslinking actually leads to a decrease in protective performance. This precisely proves that... It is an optimized enzyme concentration that balances the conflicting needs of "gastric acid protection" and "intestinal release," rather than an arbitrarily selected value.

[0071] This invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of this invention. To provide the public with a thorough understanding of this invention, specific details are described in detail in the following preferred embodiments; however, those skilled in the art will fully understand the invention even without these details. Furthermore, to avoid unnecessary misunderstanding of the essence of this invention, well-known methods, processes, procedures, components, and circuits are not described in detail.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins, characterized in that, Includes the following steps: Step 1: Mix whey protein isolate solution and low-methoxy pectin solution at a volume ratio of 4:1 to obtain whey protein isolate-low-methoxy pectin composite solution; mix probiotic pre-suspension solution and whey protein isolate-low-methoxy pectin composite solution at a volume ratio of 1:9 to obtain probiotic-polymer mixture. Step 2: The probiotic-polymer mixture is extruded through a nozzle with an inner diameter of 0.5 mm into a calcium chloride crosslinking bath with a pH of 4.0 and a mass-volume concentration of 3%. The mixture is allowed to stand for crosslinking, and the wet gel beads are collected and washed to obtain a probiotic-dispersed single ion crosslinked network gel bead precursor. Step 3: Mix the probiotic-dispersed single-ion cross-linked network gel bead precursor with a 5-O-caffeoylquinic acid solution with a mass / volume concentration of 0.8% and a pH of 6.

0. Under light-protected conditions at 10°C, add laccase to a final concentration of [missing information]. The enzyme activity of the laccase is defined as the amount of enzyme required to catalyze the oxidation of 1 micromole of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid per minute under conditions of 30°C and pH 6.0, while maintaining a dissolved oxygen concentration of [missing value - likely a specific concentration]. An enzymatic oxidative cross-linking reaction is carried out to oxidize 5-O-caffeoylquinic acid to generate an ortho-quinone intermediate. This ortho-quinone intermediate undergoes Michael addition and Schiff base reaction with the lysine ε-amino and cysteine ​​sulfhydryl groups of whey protein isolate to form a covalent cross-linked network. This network, together with the ionic cross-linked network in the probiotic dispersed single-ion cross-linked network gel bead precursor, constitutes an ion-covalent double-network interpenetrating structure, yielding probiotic acid-resistant gel beads. The average particle size of the probiotic acid-resistant gel beads is [missing information]. In response to The Young's modulus in the linear viscoelastic region is .

2. The method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins according to claim 1, characterized in that, The preparation of the probiotic suspension in step 1 includes: taking Lactobacillus rhamnosus cultured to the stationary phase, centrifuging at 8000 rpm for 10 minutes at 4°C, discarding the supernatant, and collecting the bacterial precipitate; resuspending the collected bacterial precipitate in phosphate buffer (pH 6.8) pre-cooled to 4°C, centrifuging at 8000 rpm for 10 minutes at 4°C, discarding the supernatant, and repeating this washing process twice; resuspending the finally obtained washed bacterial precipitate in phosphate buffer (pH 6.8), determining the viable cell concentration of the suspension using the plate count method, and adjusting the viable cell concentration to the required level using phosphate buffer (pH 6.8). The probiotic initial suspension was obtained.

3. The method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins according to claim 1, characterized in that, The whey protein isolate-low methoxylated pectin composite solution described in step 1 is prepared as follows: Under constant temperature of 25°C, a phosphate buffer solution with a pH of 6.8 is stirred at 300 rpm. Whey protein isolate powder is added in batches of 0.5 g at a time, and stirring is continued until the powder is completely dissolved, yielding a whey protein isolate solution with a mass-to-volume concentration of 12%. Low methoxylated pectin powder is added to another portion of the phosphate buffer solution with a pH of 6.8, and stirred at 400 rpm in a 40°C water bath until completely dissolved. After cooling to 25°C, a low methoxylated pectin solution with a mass-to-volume concentration of 4% is obtained. The methoxylated content of the low methoxylated pectin is 22%-28%, and the weight-average molecular weight is [not specified]. Under stirring conditions of 25°C and 400 rpm, the whey protein isolate solution with a mass-volume concentration of 12% and the low-methoxyl pectin solution with a mass-volume concentration of 4% were mixed at a volume ratio of 4:1, and the mixture was stirred for 30 minutes to obtain the whey protein isolate-low-methoxyl pectin composite solution.

4. The method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins according to claim 1, characterized in that, In step 2, the calcium chloride crosslinking bath is prepared by dissolving calcium chloride dihydrate in deionized water to form a calcium chloride solution with a mass-volume concentration of 3%, and then adjusting the pH value to 4.0 with 0.1 mol / L hydrochloric acid. The probiotic-polymer mixture is continuously extruded at 25°C through the nozzle with an inner diameter of 0.5 mm into the calcium chloride crosslinking bath, which is under continuous stirring at 50 rpm. The droplets are allowed to stand in the calcium chloride crosslinking bath for 30 minutes to crosslink and form wet gel beads. The wet gel beads were collected by filtering with a nylon filter with a pore size of 200 micrometers. The collected wet gel beads were then immersed in 20 times their weight of deionized water and stirred at 50 rpm for 1 minute. After stirring, the mixture was filtered with a nylon filter with a pore size of 200 micrometers. This washing process was repeated 3 times to obtain the probiotic-dispersed single ion cross-linked network gel bead precursor.

5. The method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins according to claim 1, characterized in that, The 0.8% (w / v) concentration and pH 6.0 5-O-caffeoylquinic acid solution mentioned in step 3 is prepared as follows: At 10°C and in the dark, 5-O-caffeoylquinic acid powder is dissolved in a 0.05 mol / L phosphate buffer solution pre-cooled to 10°C and at a pH of 6.

0. The solution is stirred at 200 rpm and brought to a final volume with the 0.05 mol / L phosphate buffer solution. The probiotic-dispersed single-ion cross-linked network gel bead precursor and the 0.8% (w / v) concentration and pH 6.0 5-O-caffeoylquinic acid solution are added to a jacketed, light-protected reactor at a mass ratio of 1:

8. The reactor is stirred continuously at 60 rpm for 60 minutes at 10°C and in the dark to allow 5-O-caffeoylquinic acid to diffuse into the probiotic-dispersed single-ion cross-linked network gel bead precursor.

6. The method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins according to claim 5, characterized in that, Step 3, which involves adding laccase and introducing sterile air for the enzymatic oxidative cross-linking reaction, includes: adding a laccase solution pre-prepared with 0.05 mol / L phosphate buffer at pH 6.0 to the reaction system after the diffusion reaction has proceeded for 60 minutes, so that the final concentration of laccase in the reaction system is... Immediately introduce sterile air into the bottom of the reaction solution through a gas distributor with an aperture of 0.22 micrometers, and adjust the aeration rate to maintain a constant dissolved oxygen concentration in the reaction solution. The reaction was carried out at 10°C and in the dark with continuous stirring at 60 rpm for 40 minutes. After the reaction, the gel beads were collected by filtering with a nylon filter with a pore size of 200 micrometers. The gel beads were washed three times with 20 times their mass of deionized water at 10°C and stirred at 50 rpm for 1 minute to obtain the probiotic acid-resistant gel beads.

7. The method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins according to claim 6, characterized in that, The dissolved oxygen concentration is monitored in real time by an online dissolved oxygen electrode, and the flow rate of the sterile air is automatically adjusted by a mass flow controller to maintain a constant dissolved oxygen concentration. .

8. The method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins according to claim 1, characterized in that, The low-methoxyl pectin in step 1 has a galacturonic acid content of not less than 65%; the whey protein isolate in step 1 has a protein content of not less than 90%, and the β-lactoglobulin content accounts for 55%-65% of the total mass of the whey protein isolate; the phosphate buffer with a pH of 6.8 is obtained by mixing 0.2 mol / L sodium dihydrogen phosphate solution and 0.2 mol / L disodium hydrogen phosphate solution at a volume ratio of 1:2, and then diluting with deionized water to 0.1 mol / L.

9. The method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins according to claim 1, characterized in that, In step 1, the probiotic suspension and the whey protein isolate-low methoxy pectin composite solution are mixed at a volume ratio of 1:9, and the mixture is stirred at 300 rpm for 5 minutes at a constant temperature of 25°C. In step 2, the nozzle inner diameter tolerance is ±0.02 mm, the driving pressure applied to the probiotic-polymer mixture during the extrusion process is 0.15 MPa, and the extrusion ambient temperature is maintained at 25°C.

10. The method for constructing probiotic gastric acid-resistant gel beads using polyphenol cross-linked proteins according to claim 1, characterized in that, When the probiotic acid-resistant gel beads were tested in the following two systems: after being treated with a pH 1.2 hydrochloric acid solution containing 0.32% pepsin at 37°C and continuously shaken at 50 rpm for 2 hours, the survival rate of Lactobacillus rhamnosus inside the probiotic acid-resistant gel beads was not less than 90%; and after being treated with a pH 6.8 phosphate buffer solution containing 0.5% trypsin at 37°C and continuously shaken at 50 rpm for 30 minutes, the probiotic acid-resistant gel beads completely disintegrated, and the release rate of Lactobacillus rhamnosus was not less than 95%.

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