A highly active probiotic formulation and a method for its preparation

Abstract

The application provides a high-temperature-resistant and gastric acid-resistant probiotic preparation and a preparation method thereof, and the preparation comprises a probiotic core layer and a shell layer covering the same, and the shell layer comprises succinyl-modified food leaf protein assisted by ultrasonic pretreatment, peach gum polysaccharide and soybean lecithin. The application constructs a single-layer protection system with excellent responsiveness by performing specific functional modification on the food leaf protein and synergizing the food leaf protein with the peach gum polysaccharide and the soybean lecithin. The preparation can significantly improve the survival rate of probiotics during processing, storage and simulated gastrointestinal digestion, and especially realizes excellent gastric acid resistance and intestinal targeting release performance, and the process is simple and suitable for embedding and protecting various probiotics.

Description

Technical Field

[0001] This invention belongs to the field of probiotic products technology, specifically relating to a highly active probiotic preparation and its preparation method. Background Technology

[0002] The commercial value of probiotics is inextricably linked to their ability to maintain high survival rates during processing, storage, and gastrointestinal transit, which constitutes the core driving force behind current research on probiotic delivery technologies. Unprotected probiotics are highly susceptible to irreversible thermal damage and mechanical stress inactivation during common food processing steps such as spray drying and high-temperature sterilization. During their shelf life, fluctuations in oxygen, moisture, and temperature in the environment continuously challenge their stability. The biggest obstacle comes from the physiological environment after oral administration: the synergistic effect of highly acidic gastric juice (pH 1.5-3.0) and high concentrations of bile salts and digestive enzymes can cause most free probiotics to become inactive before reaching the colonization site in the colon, rendering their claimed health benefits impossible to achieve. Therefore, constructing an effective bioactive delivery system is a fundamental technical challenge that urgently needs to be overcome in the field of probiotic functional foods and formulations.

[0003] To address the aforementioned challenges, microencapsulation has become a mainstream strategy for improving probiotic tolerance. The first generation of basic technologies utilizes natural polysaccharides (such as sodium alginate and chitosan) or proteins (such as gelatin and whey protein) as wall materials, employing methods such as extrusion, emulsification gelation, or spray drying for physical encapsulation. While these methods create a simple physical barrier that isolates probiotics from environmental stresses to some extent, their protective mechanism is passive and non-specific. These materials may prematurely swell, disintegrate, or permeate in the acidic environment of the stomach, leading to premature exposure of probiotics. Furthermore, their release in the gut depends on the overall degradation or diffusion of the matrix, failing to achieve precise colonic targeting, thus limiting their protective efficiency. The improvement in survival rate after simulated digestion is limited and unstable.

[0004] To achieve higher targeting and protective efficiency, second-generation intelligent responsive delivery systems have emerged, designed to trigger release by utilizing specific changes in the gastrointestinal environment (such as pH gradients and specific enzymes). For example, they employ enteric polymers (such as certain methacrylic acid copolymers) that are insoluble in gastric acid but soluble at the neutral pH of the intestine, or polysaccharide precursors that can be degraded by enzymes specific to colonic flora (such as pectinase and azoreductase). These systems conceptually represent an advancement from "ordinary protection" to "intelligent navigation," aiming to deliver live bacteria more precisely to the distal intestine. However, excessively increasing the density or cross-linking of the wall material to resist gastric acid often simultaneously hinders the effective release of probiotics into the intestine and even inhibits the metabolic activity of the bacteria during encapsulation, creating a dilemma where "protection" and "release" are difficult to achieve simultaneously.

[0005] In summary, existing technologies have yet to find satisfactory solutions in balancing key dimensions such as processing stability and gut-targeted release efficiency. Therefore, developing a novel probiotic encapsulation system that can synergistically address multiple stresses and is simple and industrially feasible is of significant practical importance for overcoming industry technological bottlenecks. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a highly active probiotic preparation and its preparation method. It utilizes a novel plant protein—leafwort protein—which is modified and then used to encapsulate probiotics, significantly enhancing its pH-responsive protective ability. Finally, it is scientifically compounded with peach gum polysaccharide and soybean lecithin to form a dense and stable monolayer encapsulation system, significantly improving the storage stability and gastric acid resistance of the probiotics, and promoting their release in the intestines. One objective of this invention is to provide a highly active probiotic preparation that can be stably stored, is not easily inactivated in gastric acid, and is released directionally in the intestines. A second objective is to provide a method for preparing the aforementioned highly active probiotic preparation. A third objective is to provide the application of modified leafwort protein in the preparation of highly active probiotic preparations.

[0007] To achieve the above objectives, the present invention employs the following technical solution: On one hand, the present invention provides a probiotic preparation comprising a core layer and a shell layer, wherein the core layer is a probiotic, and the shell layer comprises modified leafy green protein, wherein the modified leafy green protein is succinylated leafy green protein.

[0008] To improve the encapsulation effect of the outer shell on the internal probiotics, this invention abandons traditional and limited protective encapsulation materials such as soy protein and sodium alginate, and creatively selects phytoesophageal protein, an emerging plant protein resource, as the shell material. Phytoesophageal protein differs from common soy and pea proteins in its amino acid composition and molecular structure, possessing a unique molecular basis. However, natural phytoesophageal protein, as an encapsulation material, still suffers from insufficient solubility, and is particularly prone to denaturation and weak protective ability in the acidic environment of the stomach. Therefore, this invention further modifies phytoesophageal protein through succinylation. The core mechanism of this modification lies in the acylation reaction between succinic anhydride and the ε-amino group of lysine residues in the protein molecule, covalently introducing a negatively charged carboxyl group into the protein side chain. This chemical modification fundamentally alters the electrostatic and hydrophobic properties of the protein, transforming it from a common film-forming material into a functional material with intelligent pH-responsive characteristics. In the highly acidic environment of the stomach, the negative charge carried by the modified protein is partially neutralized, the electrostatic repulsion between molecules is weakened, and the hydrophobic interaction is enhanced, thereby driving the protein molecules to aggregate rapidly and actively, forming a dense and insoluble physical barrier that effectively blocks the attack of hydrogen ions and pepsin on the probiotics inside. When the microcapsules enter the neutral to weakly alkaline environment of the intestine, the degree of carboxyl protonation decreases, the electrostatic repulsion is restored, and the aggregated structure is dissociated, thereby realizing the intelligent release of probiotics at the target site.

[0009] Furthermore, the modified leafy green protein is succinylated leafy green protein that has undergone ultrasonic pretreatment, and the succinylated leafy green protein is formed by acylation of leafy green protein with succinic anhydride.

[0010] Considering that the naturally compact higher-order structure of proteins may mask the succinylation reaction sites (amino groups), insufficient or uneven modification could lead to the failure of the protective shell in the acidic environment of the stomach. Therefore, this invention pretreats the succinylation protein with ultrasound before performing the succinylation reaction. Ultrasound treatment can "open up" the globular structure of the succinylation protein, exposing more of the hydrophobic regions and lysine residues originally embedded inside the molecule to the aqueous phase. The protein solution after pretreatment shows significantly improved accessibility, reaction rate, and reaction uniformity in subsequent reactions with succinic anhydride, resulting in a higher and more uniform degree of substitution in the obtained succinylated succinylation protein, with a more regular distribution of negative charges on the molecular chain. This not only further enhances the aggregation ability and barrier density of the modified succinylation protein in gastric acid but also makes the dissociation of aggregates and intestinal release behavior more controllable. The combination of ultrasonic pretreatment and succinylation produces a significant synergistic effect in the composite modification, which fully taps the functional potential of leafy grass protein, thereby obtaining shell materials with performance far exceeding that of conventionally modified materials.

[0011] Furthermore, the shell layer also includes peach gum polysaccharide and soybean lecithin, and the mass ratio of the modified leaf grass protein, peach gum polysaccharide and soybean lecithin is (2.5-4):(0.5-1.5):(0.5-1.5).

[0012] Although modified leafy green protein possesses a smart response core, there is still room for improvement in terms of film-forming mechanical strength and long-term storage physical stability of single protein materials. To address this issue, this invention introduces a shell layer prepared by co-compiling peach gum polysaccharide and soybean lecithin with modified leafy green protein. The peach gum polysaccharide and modified leafy green protein form an interpenetrating network through intermolecular forces, significantly enhancing the mechanical strength and processing tolerance of the shell layer. Simultaneously, their enzymatic hydrolysis properties with colonic flora add a second layer of targeted control to the release process. The addition of soybean lecithin primarily addresses the interfacial compatibility and dispersion stability issues between probiotic cells and the aqueous phase of the shell layer. The surface of probiotic cells is inherently hydrophobic, while the shell material (modified protein, polysaccharide) solution is hydrophilic. Direct mixing of the two easily leads to bacterial aggregation and sedimentation, severely affecting the uniformity and efficiency of encapsulation. Soy lecithin, as an amphiphilic surfactant, spontaneously adsorbs onto the surface of hydrophobic probiotic cells, anchoring its hydrophobic end to the cell wall while its hydrophilic end extends into the surrounding aqueous phase. This adsorption process significantly improves the hydrophilicity of the bacterial surface, greatly enhancing its dispersion stability in the shell solution. This invention experimentally determined that a mass ratio of (2.5-4):(0.5-1.5):(0.5-1.5) represents the optimal balance point for the synergistic effect of these three components. Within this range, protein provides sufficient functional groups, polysaccharides strengthen the structure without interfering with gastric protection, and lecithin ensures process efficiency, thus achieving the best synergistic effect.

[0013] On the other hand, the present invention provides a method for preparing a probiotic preparation, comprising the following steps: (1) Extraction of leafwort protein: The extraction was performed using an enzyme-assisted extraction method; (2) Preparation of modified leafy grass protein: Succinylation of leafy grass protein was assisted by ultrasonic pretreatment; (3) Preparation of core layer solution and shell layer solution: The core layer solution is a probiotic suspension, and the shell layer solution is a mixed solution containing modified leafy grass protein; (4) The probiotic preparation is obtained by mixing, homogenizing and drying the core layer solution and the shell layer solution.

[0014] Furthermore, the enzyme-assisted extraction method employs cellulase to assist in the extraction of leafy grass protein.

[0015] This invention preferably employs a cellulase-assisted extraction method, utilizing the specificity of biological enzymes. Cellulase can efficiently hydrolyze the cellulose components in the cell walls of leafy greens, gently disrupting the cell structure and thus releasing intracellular proteins more completely, while minimizing chemical denaturation or conformational damage to the proteins themselves during extraction. The resulting leafy green protein has higher purity and retains a conformation closer to its natural state and superior initial solubility. This high-quality protein raw material provides a more uniform and reactive substrate for subsequent ultrasonic treatment and succinylation reactions, serving as a crucial starting point for ensuring the superior performance of the final modified product.

[0016] Furthermore, the ultrasonic pretreatment is performed for 5-10 minutes with an ultrasonic power of 100-500 W; the succinylated leafy green protein is obtained by acylation of leafy green protein with succinic anhydride.

[0017] Furthermore, the amount of succinic anhydride added is 1-10% (w / w) of the protein content of the leafy greens.

[0018] Furthermore, the shell solution also contains gum arabic polysaccharide and soybean lecithin, and the mass ratio of the modified leaf grass protein, gum arabic polysaccharide and soybean lecithin is (2.5-4):(0.5-1.5):(0.5-1.5).

[0019] Furthermore, the probiotics are selected from one or more of the genera *Lactobacillus*, *Lactococcus*, *Bifidobacterium*, *Saccharomyces*, and Gram-positive cocci.

[0020] The experimental results of this invention demonstrate that the embedding system provided by this invention has an excellent protective effect on *Lactobacillus plantarum*. Based on similar cell structure and stress tolerance mechanisms, this embedding system can be widely applied to other common probiotic genera that are sensitive to gastric acid and high temperatures, including but not limited to *Lactobacillus* (such as *Lactobacillus acidophilus* and *Lactobacillus casei*), *Lactococcus*, *Bifidobacterium* (such as *Bifidobacterium infantis* and *Bifidobacterium longum*), *Saccharomyces* (such as *Saccharomyces boulardii*), and some Gram-positive cocci (such as *Enterococcus faecalis*).

[0021] In another aspect, the present invention provides the use of modified leafy green protein for preparing probiotic encapsulation formulations, wherein the modified leafy green protein is succinylated leafy green protein that has undergone ultrasonic pretreatment.

[0022] The present invention has the following beneficial effects: 1. Significantly improves the survival rate of probiotics in the gastric acid environment: By succinylation modification, leafy grass protein is given pH responsiveness, which enables it to spontaneously form a dense protective layer in gastric juice, effectively resisting the erosion of low pH and digestive enzymes, increasing the survival rate of probiotics in gastric acid to over 90%, and solving the biggest bottleneck in oral delivery of probiotics.

[0023] 2. It achieves targeted and controllable release of probiotics in the intestine: The shell system has dual environmental response characteristics of "gathering in the stomach and dissociation in the intestine", which ensures that probiotics are stable in the stomach and efficiently released in the intestine, thus improving their colonization and efficacy.

[0024] 3. Significantly enhances the processing and storage stability of probiotic preparations: The synergistic effect of the shell material enables the shell to obtain good mechanical strength and barrier properties, thereby better maintaining the activity of probiotics during production and long-term storage, providing a new and more advantageous encapsulation material. Detailed Implementation

[0025] The preferred embodiments of the present invention will be described in further detail below. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. The raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0026] Example 1: Preparation of the highly active probiotic preparation provided by the present invention This embodiment will describe in detail the complete preparation process of the highly active probiotic preparation provided by the present invention.

[0027] (1) Extraction of leafy grass protein a) Crush the dried leaves of the edible leaf grass and pass them through an 80-mesh sieve. Weigh 100 g of the powder and mix it with 2500 mL of phosphate buffer (0.05 M, pH 7.0) to obtain a 4% (w / v) suspension.

[0028] b) Add cellulase (enzyme activity 33 U / g substrate) to the suspension and react gently in a 47°C water bath for 2 hours.

[0029] c) After the reaction is complete, rapidly heat the mixture to 90°C and maintain the temperature for 10 minutes to inactivate the enzyme. After cooling, centrifuge at 4°C and 8000 rpm for 20 minutes and collect the supernatant.

[0030] d) Slowly adjust the pH of the supernatant to the isoelectric point of the leafminer protein (pH 4.5) with 1M HCl, and observe the formation of a large amount of white precipitate. Centrifuge again (4℃, 8000 rpm, 15 minutes) and collect the precipitate.

[0031] e) The precipitate was washed twice with deionized water, then redispersed in a pH 7.0 buffer solution and adjusted until completely dissolved. Finally, it was freeze-dried to obtain a light yellow leafwort protein powder, which was then stored in a sealed container at -20℃ for later use. The protein extraction rate was determined to be 74.5%, and the purity reached 82.3%.

[0032] (2) Modification of leafy grass protein a) Dissolve the extracted leafwort protein at a concentration of 5% (w / v) in deionized water, and adjust the pH to 8.5 with 0.1 M NaOH solution to obtain a protein solution.

[0033] b) Ultrasonic pretreatment: The protein solution was placed in an ice-water bath and treated using an ultrasonic cell disruptor. The ultrasonic power was set to 300 W, the operating mode was 2 seconds of sonication followed by 3 seconds of intermittent sonication, and the total sonication time was 10 minutes. This step aims to moderately disperse the protein molecules and expose more reaction sites.

[0034] c) Succinylation reaction: Place the pretreated protein solution on a magnetic stirrer and maintain the temperature at 25°C; slowly and in batches add succinic anhydride (SA) equivalent to 8% (w / w) of the protein mass, while continuously adding 0.1 M NaOH dropwise to maintain the pH of the reaction system at a constant 8.5. After all the addition is complete, continue the reaction for 3 hours.

[0035] d) After the reaction is complete, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 8 kDa and dialyzed with flowing deionized water at 4°C for 48 hours to completely remove unreacted succinic anhydride and byproducts.

[0036] e) The dialysate was freeze-dried to obtain succinylated leafwort protein. The degree of succinylation was determined to be 72% using the ninhydrin method.

[0037] (3) Preparation of probiotic preparations a) Preparation of shell solution: Weigh 3 g of the modified leafy grass protein, 1 g of peach gum polysaccharide and 1 g of soybean lecithin, and dissolve them together in 100 mL of phosphate buffer (0.1 M, pH 7.0) at 50 ℃. Stir thoroughly to dissolve completely and obtain a homogeneous shell solution.

[0038] b) Preparation of the core layer solution: Lactobacillus plantarum cultured to the stationary phase ( Lactobacillus plantarum The fermentation broth was centrifuged to collect the bacterial cells, washed twice with sterile physiological saline, and finally resuspended in 10 mL of physiological saline. The viable cell count was approximately 1 × 10⁻⁶. 10 CFU / mL was used to obtain a probiotic suspension.

[0039] c) Dispersion and Homogenization: Under aseptic conditions, 10 mL of probiotic suspension was slowly added to 100 mL of shell solution with continuous stirring. First, the mixture was treated with a high-speed disperser at 10,000 rpm for 2 minutes to initially and uniformly disperse the bacteria in the shell solution. Subsequently, the mixture was homogenized three times using a high-pressure homogenizer at 50 MPa. The core purpose of this process is to further reduce the particle size of the dispersion system using high-pressure shear force and to promote the full adsorption and encapsulation of functional components in the shell material (especially amphiphilic soybean lecithin and modified protein) on and around the surface of the probiotic cells, forming a stable dispersion system.

[0040] d) Spray drying: The homogenized dispersion system described above is immediately subjected to spray drying. The inlet temperature is set to 140℃, the outlet temperature is controlled at 65±2℃, and the feed flow rate is 8 mL / min. The powder collected from the bottom of the drying tower and the cyclone separator yields the probiotic preparation of this invention.

[0041] Example 2: Effects of leafy green protein prepared by different modification methods on probiotic preparations This embodiment aims to investigate the effect of ultrasonic pretreatment-assisted succinylation on the modification effect of leafwort protein. This embodiment follows the method for the probiotic preparation provided in Example 1, wherein the modification of leafwort protein is carried out according to the following four methods: Method 1: No modification treatment required; Method 2: Without ultrasonic pretreatment, the succinylation reaction procedure is the same as in Example 1; Method 3: Perform ultrasonic pretreatment according to the steps of Example 1, without succinylation reaction; Method 4: Follow the steps provided in Example 1 to perform ultrasonic pretreatment before succinylation reaction.

[0042] The encapsulation rate, storage stability, gastric acid survival rate, and enteric release rate of the probiotic preparations in each group were determined. The encapsulation rate was determined by centrifugation of the core and shell solutions, collection of the supernatant, and detection of the free *Lactobacillus plantarum* content. The encapsulation rate was calculated using the formula: Encapsulation rate (%) = (Total *Lactobacillus plantarum* content - Free *Lactobacillus plantarum* content) / Total *Lactobacillus plantarum* content × 100. Storage stability was determined by storing the probiotics at 4 ℃ for 30 days and then detecting the probiotic survival rate. Gastric acid survival rate was determined by placing the probiotics in simulated gastric fluid (containing 0.3% pepsin and 0.2% (w / v) NaCl solution, pH=2.0) and shaking at 37 ℃ (150 rpm) for 2 hours before detecting the probiotic survival rate. Enteric release rate was determined by transferring the probiotics into simulated small intestinal fluid (containing 0.1% trypsin, pH=6.8) for 2 hours before detecting the probiotic release rate. The results are shown in Table 1.

[0043] Table 1. Effects of different modification methods on the encapsulation efficiency of leafy green protein.

[0044] Note: All data in the table are the mean ± standard deviation of at least three independent replicate experiments.

[0045] As shown in Table 1, the protective performance of the prepared probiotic formulations of grass protein treated with different modification processes varied significantly. The ultrasonic pretreatment-assisted succinylation method (Method 4) employed in this invention demonstrated comprehensive and superior advantages. Unmodified grass protein (Method 1) exhibited the most porous encapsulation structure due to its limited solubility and interfacial activity, resulting in the lowest encapsulation rate and virtually no barrier effect against gastric acid, leading to extremely low gastric acid survival. Although it could be rapidly released in intestinal fluid, the significant inactivation in the early stages rendered it unusable. Succinylation modification alone (Method 2) significantly increased the gastric acid survival rate to 78.33%, and also improved the encapsulation rate and storage stability, indicating that this method significantly improved the functional properties of the protein, enabling it to actively aggregate and form a dense barrier in gastric acid. However, the modification may not be sufficiently uniform due to partial masking of the protein's natural structure, resulting in limited effectiveness. Solosonization alone (Method 3) allows protein molecules to expand moderately through physical cavitation, improving their solubility. As a result, the encapsulation rate and gastric acid protection ability are improved to a certain extent compared with the unmodified group. However, due to the lack of fundamental modification by chemical groups, the protective layer formed is not strong enough, and the gastric acid survival rate (45.67%) is much lower than that of the chemically modified group.

[0046] In summary, Method 4, combining ultrasonic pretreatment with succinylation modification, produced a significant synergistic effect. Ultrasonic pretreatment fully exposes the reaction sites of the protein molecules, ensuring a more uniform and efficient subsequent succinylation reaction, resulting in a higher degree of modification. This deep and uniform modification allows the protein to aggregate more rapidly and thoroughly in the acidic environment of the stomach, constructing an exceptionally robust physical barrier, thus significantly improving gastric acid survival rate. Simultaneously, superior solubility leads to the highest encapsulation efficiency and storage stability. Regarding intestinal fluid release, Method 4 is slightly lower than Methods 2 and 3, which precisely demonstrates that the protective structure formed is more stable in the stomach, achieving a controlled and sustained release effect in the intestinal environment, avoiding premature or excessively rapid release, and better meeting the needs of targeted delivery.

[0047] Example 3: Effects of different extraction methods on probiotic preparations of leafy green protein The purpose of this embodiment is to verify the advantages of enzyme-assisted extraction compared to other methods in enhancing the function and final encapsulation effect of leafwort protein. This embodiment follows the method for the probiotic preparation provided in Example 1, wherein the extraction of leafwort protein is performed using the following three methods: Group A: Following the steps provided in Example 1, enzyme-assisted extraction of leafwort protein was performed; Group B: Extraction of leafwort protein using the alkali dissolution and acid precipitation method, the specific steps are as follows: a) Raw material pretreatment: Dry the fresh leaves of the edible grass at 60℃, crush and grind them into powder and pass them through a 100-mesh sieve; b) Alkali extraction: Add 2M sodium hydroxide at a material-to-liquid ratio of 1:40 (w / v) to adjust the pH to 12.0, extract for 2 h at 50℃; after extraction, centrifuge at 5000 r / min for 20 min to obtain the supernatant; c) Acid precipitation: Add 2M hydrochloric acid to the obtained supernatant, adjust the pH to 3.0, precipitate at 4 ℃ for 30 min, and centrifuge at 5000 r / min for 20 min to obtain the precipitate; d) Drying: The precipitate obtained in step S3 is dried to obtain leafy grass protein powder.

[0048] Group C: Extraction of leaf protein from edible grass using salt extraction followed by acid precipitation. The specific steps are as follows: a) Raw material pretreatment: Fresh leaves of edible grass were mixed with 0.5M phosphate buffer at pH 7.5 at a liquid-to-solid ratio of 1:10, heated to boiling, cooled to 25 ℃, homogenized at 14000 r / min for 2 min, and centrifuged at 4000 r / min for 10 min to obtain the supernatant. b) Isoelectric ammonium sulfate precipitation: Adjust the pH of the solution to 5.5, add 8% w / v ammonium sulfate to the supernatant, stir at 4000 r / min for 60 min, and precipitate leaf protein; c) Dry the leaf protein in a vacuum freeze dryer to obtain leafy grass protein powder.

[0049] The extraction rate and solubility (pH 7.0) of the proteins obtained by the three methods were determined respectively. The encapsulation rate, storage stability, gastric acid survival rate and enteric release rate of the probiotic preparations (the protein modification and the preparation of the probiotic preparations were the same as in Example 1) of each group were also tested. The test methods were the same as in Example 2. The test results are shown in Table 2.

[0050] Table 2. Effects of different extraction methods on the protein function and encapsulation efficiency of *Eclipta prostrata* protein.

[0051] Note: All data in the table are the mean ± standard deviation of at least three independent replicate experiments.

[0052] Table 2 clearly shows that the extraction method not only determines the yield and physicochemical properties of leafwort protein, but also affects its encapsulation effect after modification. The alkali-soluble acid precipitation method (Group B), extracted under strongly alkaline conditions, yielded a acceptable leafwort protein. However, the high pH could lead to partial denaturation of the protein, causing molecular aggregation or folding, thus damaging its physicochemical functions and resulting in low solubility. This limited the modification efficiency and uniformity in subsequent modifications, leading to significantly lower encapsulation efficiency, storage stability, and gastric acid survival rate in the final probiotic preparation compared to Group A. The salt extraction combined with ammonium sulfate precipitation method (Group C) caused relatively less damage to the protein's natural structure, but had the lowest extraction rate. Furthermore, the extracted protein may contain more non-protein impurities, resulting in the weakest functional properties (solubility) and the worst final encapsulation effect. In contrast, the enzyme-assisted extraction method (Group A) provided by this invention demonstrated comprehensive superiority, achieving optimal levels in leafwort protein extraction rate, function, and probiotic encapsulation effect. This is because cellulase can specifically and gently degrade plant cell walls, efficiently releasing intracellular proteins while preserving the natural conformation and functional integrity of leafy green proteins to the greatest extent possible, thus achieving the highest extraction rate and solubility. The highly soluble proteins provide excellent substrates for subsequent modification processes, further improving the encapsulation effect of the shell. Therefore, enzyme-assisted extraction has significant advantages in improving the encapsulation of probiotics.

[0053] Example 4: Screening and optimization of shell formulation To optimize the shell formulation, this embodiment investigated the effects of shells composed of different components and the proportions of each component on the final encapsulation effect. First, this embodiment compared the encapsulation effects of shells with different component compositions, following the method for the probiotic preparation provided in Example 1. The shell solutions were prepared according to the following four groups: Group D: Weigh out 3 g of leafy green protein, 1 g of peach gum polysaccharide, and 1 g of soybean lecithin. The subsequent preparation steps are the same as in Example 1. Group E: Weigh 3 g of modified soy protein, 1 g of peach gum polysaccharide and 1 g of soy lecithin. The modified soy protein is prepared according to the method provided in Example 1, and the subsequent preparation steps are the same as in Example 1. Group F: Weigh out 3 g of leafy green protein, 1 g of sodium alginate and 1 g of soybean lecithin, and follow the same preparation steps as in Example 1; Group G: Weigh out 3 g of leafy green protein, 1 g of peach gum polysaccharide, and 1 g of pectin. The subsequent preparation steps are the same as in Example 1.

[0054] The encapsulation rate, storage stability, gastric acid survival rate, and enteric release rate of the four probiotic preparations were tested respectively. The testing methods were the same as in Example 2, and the test results are shown in Table 3.

[0055] Table 3. Effect of different shell formulations on embedding effect

[0056] Note: All data in the table are the mean ± standard deviation of at least three independent replicate experiments.

[0057] As shown in Table 3, the probiotic formulation prepared using the shell formulation (Group D) provided by this invention exhibits the best performance. Group E, using the same modification process but a different protein matrix, still shows significantly lower overall performance than Group D. This result indicates that even with the exact same succinylation and ultrasonic pretreatment processes, physalis protein, as a unique novel plant protein matrix, has superior modification and protective effects compared to traditional soybean protein. This is likely due to the unique amino acid composition, molecular flexibility, or glycosylation pattern of physalis protein itself, allowing it to form a denser and more responsive protective network after modification. Meanwhile, the data from Groups F and G further confirm the irreplaceable role of gum arabic in regulating intestinal release and soybean lecithin in constructing a high-encapsulation-rate initial emulsion system. Therefore, the advantage of this invention lies not only in the innovative modification process but also in the discovery of an unexpected synergistic effect between the specific physalis protein matrix and the aforementioned process and auxiliary components, thereby achieving comprehensively optimal encapsulation and delivery performance.

[0058] Next, this embodiment further investigated the effect of the ratio of physalis protein, gum arabic, and soybean lecithin on the encapsulation effect of the shell layer. The preparation method of the probiotic formulation was also carried out according to the method of Example 1, wherein the modified physalis protein, gum arabic, and soybean lecithin were added to the shell layer solution in the following four proportions: Group H: Modified leafy green protein: peach gum polysaccharide: soybean lecithin = 4:0:1; Group I: Modified leafy green protein: peach gum polysaccharide: soybean lecithin = 3:1:1; Group J: Modified leafy green protein: peach gum polysaccharide: soybean lecithin = 2:2:1; Group K: Modified leafy green protein: peach gum polysaccharide: soybean lecithin = 3:2:0.

[0059] The encapsulation rate, storage stability, gastric acid survival rate, and enteric release rate of the four probiotic preparations were tested respectively. The testing methods were the same as in Example 2, and the test results are shown in Table 4.

[0060] Table 4. Effect of different component ratios on the embedding effect of the shell.

[0061] Note: All data in the table are the mean ± standard deviation of at least three independent replicate experiments.

[0062] As shown in Table 4, the proportion of shell components significantly affects the performance of probiotic preparations. Group I achieved the best balance in terms of encapsulation efficiency, storage stability, gastric acid survival rate, and enteric release rate. Group H lacked gum arabic polysaccharide, resulting in insufficient shell integrity. Although it had a high survival rate in the acidic environment of the stomach, its enteric release rate was low, indicating that the probiotics were difficult to release effectively in the intestines. Group J increased the polysaccharide content, which promoted intestinal release, but the gastric acid survival rate and storage stability decreased significantly, suggesting that excessive polysaccharide may interfere with the formation of a dense protective layer in the stomach. The results of Group K demonstrated the indispensability of soybean lecithin; its absence led to a deterioration in the molding process, and a sharp drop in encapsulation efficiency and storage stability. In summary, the modified leafy green protein, gum arabic polysaccharide, and soybean lecithin ratio (3:1:1) provided by this invention has a significant advantage in improving the encapsulation ability of the shell.

[0063] While the present invention has been disclosed above, it is not limited thereto. Its applications in medicine can be expanded accordingly. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A probiotic preparation, characterized in that, It includes a core layer and a shell layer, wherein the core layer is a probiotic and the shell layer includes modified leafy green protein, wherein the modified leafy green protein is succinylated leafy green protein.

2. The probiotic preparation as described in claim 1, characterized in that, The modified leafy grass protein is succinylated leafy grass protein that has undergone ultrasonic pretreatment. The succinylated leafy grass protein is formed by acylation of leafy grass protein with succinic anhydride.

3. The probiotic preparation as described in claim 2, characterized in that, The shell also includes peach gum polysaccharide and soybean lecithin, and the mass ratio of the modified leaf grass protein, peach gum polysaccharide and soybean lecithin is (2.5-4):(0.5-1.5):(0.5-1.5).

4. A method for preparing a probiotic preparation, characterized in that, Includes the following steps: (1) Extraction of leafwort protein: The extraction was performed using an enzyme-assisted extraction method; (2) Preparation of modified leafy grass protein: Succinylation of leafy grass protein was assisted by ultrasonic pretreatment; (3) Preparation of core layer solution and shell layer solution: The core layer solution is a probiotic suspension, and the shell layer solution is a mixed solution containing modified leafy grass protein; (4) The probiotic preparation is obtained by mixing, homogenizing and drying the core layer solution and the shell layer solution.

5. The preparation method according to claim 4, characterized in that, in, The enzyme-assisted extraction method uses cellulase to assist in the extraction of leafy grass protein.

6. The preparation method according to claim 5, characterized in that, in, The ultrasonic pretreatment takes 5-10 minutes and has an ultrasonic power of 100-500 W; the succinylated leafy grass protein is formed by acylation of leafy grass protein with succinic anhydride.

7. The preparation method according to claim 6, characterized in that, in, The amount of succinic anhydride added is 1-10% (w / w) of the protein content of the leafy greens.

8. The preparation method according to claim 7, characterized in that, in, The shell solution also contains peach gum polysaccharide and soybean lecithin, and the mass ratio of the modified leaf grass protein, peach gum polysaccharide and soybean lecithin is (2.5-4):(0.5-1.5):(0.5-1.5).

9. The preparation method according to claim 8, characterized in that, in, The probiotics are selected from one or more of the following genera: Lactobacillus, Lactococcus, Bifidobacterium, Saccharomyces, and Gram-positive cocci.

10. The use of a modified leafy green grass protein in the preparation of probiotic encapsulation formulations, characterized in that, The modified leafy green protein is succinylated leafy green protein that has undergone ultrasonic pretreatment.

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