PH-responsive modified protein-polysaccharide-based functional factor delivery dual-network hydrogel as well as preparation method and application of pH-responsive modified protein-polysaccharide-based functional factor delivery dual-network hydrogel

Abstract

The invention provides a pH response type modified protein-polysaccharide-based functional factor delivery dual-network hydrogel as well as a preparation method and application of the pH response type modified protein-polysaccharide-based functional factor delivery dual-network hydrogel. The preparation method comprises the following steps: by taking low-temperature soybean meal as a raw material, carrying out ultrasonic treatment, alkali extraction and acid precipitation on the soybean meal to prepare modified soybean protein isolate, mixing the modified soybean protein isolate with an arabinoxylan solution, and carrying out operations such as compound enzyme gelation and embedding of water-soluble functional factors to prepare the pH-responsive modified protein-based functional factor delivery dual-network hydrogel. The prepared pH-responsive protein-based functional factor delivery dual-network hydrogel has the advantages of simplicity in operation, few production processes, low cost, low energy consumption and the like, and compared with conventional single-network gel, the dual-network hydrogel has good mechanical performance and water binding capacity and higher functional factor loading rate. By simulating in-vitro digestion, the gel slows down the release efficiency of functional factors in the stomach and optimizes the release efficiency in the intestinal tract, so that a potential targeted delivery carrier is provided for the edible gel in the fields of food, drug embedding and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel and a preparation method and application thereof. Background Art

[0002] Soy protein isolate (SPI) is a high-quality plant protein extracted from soybeans. It is rich in nine essential amino acids required by the human body and has a nutritional value comparable to that of animal protein. Therefore, it is widely used in the food industry. At the same time, due to its good bioavailability and functions such as lowering blood lipids and enhancing immunity, it shows broad application prospects in the fields of medicine and cosmetics. However, in the application scenario of the delivery system, the structural characteristics of soy protein and the limitations of the existing processing technology level limit the competitiveness of food delivery products carrying soy protein in the market, and cannot fully meet the needs of stable encapsulation, effective delivery and controlled release of active ingredients under complex physiological environments. In order to optimize the performance of soy protein in the delivery system, soy protein was ultrasonically modified to improve its functionality. Ultrasonic optimization of the structure and functional properties of soy protein isolate, enhanced the gel properties of SPI, and made the structure more dense and uniform. The preparation process is energy-saving and efficient, which is conducive to the research and development of the steady-state delivery system of active ingredients in the soy protein hydrogel system.

[0003] However, there are some limitations in the actual application of the single soybean protein gel delivery system, especially in the gastrointestinal environment, where its stability is low, it is easily affected by strong acid, strong alkali or heating conditions and aggregates, and it is sensitive to digestive enzymes. These factors are not conducive to the stable encapsulation and sustained release of active substances. Given that the sensitivity of active ingredients to environmental factors may lead to a decrease in bioactivity and bioavailability, the food, light industry and pharmaceutical industries are in urgent need of seeking a new delivery solution that can overcome these challenges. Protein-polysaccharide double network hydrogel is an important oral drug delivery carrier, and its release mechanism includes simple diffusion, swelling diffusion, matrix erosion and matrix fragmentation. Compared with simple protein or polysaccharide gels, protein-polysaccharide double network hydrogels exhibit superior mechanical properties. However, the current protein-polysaccharide double network hydrogel carrier still has certain limitations in inhibiting the premature swelling and degradation of the network in the stomach. Further research is still needed to study its response to microenvironments such as different pH, improve its nutrient release efficiency in the small intestine and the accuracy of targeted delivery. Therefore, it is of great value to develop pH-responsive and efficient water-soluble nutrient oral delivery carriers. Summary of the invention

[0004] The purpose of the present invention is to provide a pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel and a preparation method and application thereof.

[0005] The present invention is conceived as follows: ultrasonically modified soy protein isolate and polysaccharide are used to construct a double network hydrogel, which significantly improves the mechanical strength and water holding capacity of the hydrogel, has a high degree of gel crosslinking, improves the gel functional factor embedding rate, avoids its loss rate due to digestion and degradation in the stomach, and enables the delivered functional factors to be fully released in the small intestine. The problems of poor embedding stability and low delivery efficiency of the hydrogel are solved, and the targeted release of delivered nutrients is achieved, thereby improving the bioavailability of the functional factors. At the same time, the raw material cost is low and it is easy to prepare.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a method for preparing a pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel, comprising the following steps: (1) Using low-temperature soybean meal as raw material, modified soy protein isolate was prepared by ultrasonication, alkali extraction and acid precipitation of low-temperature soybean meal; (2) A modified soy protein isolate solution of a certain concentration is prepared with water, polysaccharides and water-soluble functional factors are added, and after dissolution, a complex enzyme is added. The resulting mixed system is gelled at 25-70°C, and finally the enzyme is inactivated and the system is cooled on ice to room temperature to obtain a pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel.

[0007] In the present invention, the polysaccharide is selected from at least one of arabinoxylan, beet pectin, citrus pectin, corn fiber gum, etc. The water-soluble functional factor is at least one of the following water-soluble nutrients: riboflavin, polyphenols, vitamin C, peptides, etc.

[0008] The complex enzyme consists of laccase and mTGase enzymes.

[0009] Furthermore, step (1) comprises: crushing low-temperature soybean meal, mixing and stirring the sieved soybean meal with water in a mass ratio of 1:(8-12), adjusting the pH to 8.0-10.0 (for example, adjusting the pH with a 1 mol / L NaOH solution), stirring for 1-3 hours, ultrasonically treating, and centrifuging to obtain a supernatant; adjusting the pH value of the supernatant to allow protein to precipitate therein (for example, adjusting the pH with a 1 mol / L HCl solution), then centrifuging, re-dissolving the precipitate with water and adjusting the pH to neutral, and freeze-drying to obtain a modified soy protein isolate.

[0010] Furthermore, the ultrasonic power is 200-600 W, the ultrasonic time is 1-20 min, and the pH value of the supernatant is adjusted to 3.5-5.0.

[0011] Furthermore, in the mixed system of step (2), the concentration of modified soy protein isolate is 5-20% w / v, the concentration of polysaccharide is 0.5-5% w / v, the concentration of water-soluble functional factor is 0.1-5% w / v, and the added amounts of mTGase and laccase are 10-100 u / g and 1-50 u / g, respectively.

[0012] Preferably, in the mixed system, the concentration of modified soy protein isolate is 8-15% w / v, the concentration of polysaccharide is 0.5-2% w / v, the concentration of water-soluble functional factor is 0.1-5% w / v, and the added amounts of mTGase and laccase are 10-50 u / g and 1-20 u / g, respectively.

[0013] More preferably, in the mixed system, the concentration of modified soy protein isolate is 8-15% w / v, the concentration of polysaccharide is 0.5-2% w / v, the concentration of water-soluble functional factor is 0.1-5% w / v, and the added amounts of mTGase and laccase are 40 u / g and 10 u / g, respectively.

[0014] Furthermore, the mixed system was subjected to gelation treatment in a water bath at 25-70° C. for more than 10 min, and then the enzyme was inactivated at 90° C. for more than 5 min.

[0015] Preferably, the mixed system is subjected to gelation treatment in a water bath at 30-50° C. for more than 1 hour, and then the enzyme is inactivated at 90° C. for 10 minutes.

[0016] More preferably, the mixed system is subjected to gelation treatment in a water bath at 45° C. for 1.5 h, and then the enzyme is inactivated at 90° C. for 10 min.

[0017] In a second aspect, the present invention provides a pH-responsive modified protein-polysaccharide-based functional factor delivery double-network hydrogel prepared according to the method.

[0018] In a third aspect, the present invention provides application of the pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel in a food functional factor delivery system.

[0019] In a fourth aspect, the present invention provides the use of the pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel in the preparation of a drug delivery carrier.

[0020] The embedding rate of the water-soluble functional factors in the double-network hydrogel of the present invention is 98.9%, and the total release rate after the in vitro digestion model reaches 98.0%.

[0021] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention constructs a modified SPI-AX double network hydrogel delivery system, in which the hydroxyl groups on the AX (arabinoxylan) molecular chain in the hydrogel generate hydrophobic interactions and covalent bonds with the SPI molecules, and the synergistic action of enzymes induces changes in the spatial arrangement and conformation of the hydrogel, thereby forming a high-strength double network hydrogel structure, significantly enhancing the mechanical and functional properties of the SPI-AX double network hydrogel, and increasing the degree of gel cross-linking to the highest level and the densest network structure.

[0022] The double network hydrogel significantly improves the embedding rate and loading rate of water-soluble nutrients, inhibits the swelling or erosion of the double network hydrogel in simulated gastric juice, slows down the release process of water-soluble nutrients in the stomach, optimizes the release efficiency in the intestine, and the total release efficiency reaches 98.0%. It has good responsiveness to the gastrointestinal microenvironment pH and digestive enzymes. The delivered functional factors are fully released in the small intestine segment. It solves the problems of poor embedding stability and low delivery efficiency of hydrogels, realizes the targeted release of delivered nutrients, and improves the bioavailability of functional factors. At the same time, the raw material cost is low, it is easy to prepare, and has excellent industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a double network hydrogel laser-controlled focusing microstructure prepared by combining SPI with beet pectin in the comparative example of the present invention.

[0024] Figure 2 This is a double network hydrogel microstructure prepared by combining SPI with different concentrations of AX in a preferred embodiment of the present invention.

[0025] Figure 3 This is a double-network hydrogel laser-controlled focusing microstructure prepared by combining SPI with different concentrations of AX in a preferred embodiment of the present invention.

[0026] Figure 4 The release efficiency of riboflavin in the SPI-AX double network hydrogel encapsulating riboflavin constructed with different concentrations of AX in the preferred embodiment of the present invention in the simulated oral cavity, stomach and small intestine environment. DETAILED DESCRIPTION

[0027] The invention provides a pH-responsive modified protein-polysaccharide functional factor delivery double-network hydrogel, which comprises a soybean protein and polysaccharide hydrogel matrix and a water-soluble functional factor loaded in the hydrogel matrix.

[0028] The soy protein is ultrasonically extracted modified soy protein isolate, the polysaccharide is arabinoxylan, beet pectin, citrus pectin, corn fiber gum and the like, and the water-soluble functional factors are riboflavin, polyphenols, vitamins, peptides and other water-soluble nutrients.

[0029] The present invention adopts the following technical solution: According to a specific embodiment of the present invention, the mass ratio of soybean protein to polysaccharide is 1-20:1.

[0030] Preferably, the mass ratio of the soy protein to the polysaccharide is 5-20:1

[0031] According to a specific embodiment of the present invention, the modified soy protein isolate is prepared by the following method: crushing soybean meal, mixing the sieved soybean meal with water and stirring, adjusting the pH to 8.0-10.0, stirring for 1-3 hours, ultrasonically treating, and centrifuging to obtain the supernatant; adjusting the pH value of the supernatant to allow protein to precipitate therein, then centrifuging, taking the precipitate, re-dissolving, adjusting the pH to neutral, and freeze-drying to obtain the soy protein isolate.

[0032] According to a specific embodiment of the present invention, the mass ratio of soybean meal to water is 1:8-12, the ultrasonic power is 100-1000W, the ultrasonic time is 1-20min, and the pH of the supernatant is 3.5-5.0.

[0033] The present invention also provides a method for preparing the modified protein-polysaccharide-based functional factor delivery double network hydrogel, comprising: preparing a solution of a certain concentration of modified soy protein isolate, then dissolving polysaccharide in the soy protein isolate solution at a certain concentration, and continuously stirring to ensure that it is fully dissolved. Adding a complex enzyme to the solution, killing the enzyme after water bathing, and cooling it to room temperature in an ice bath to obtain a protein-polysaccharide-based double network hydrogel.

[0034] Soy protein isolate and water-soluble functional factors are prepared into a solution of a certain concentration, and then polysaccharide is dissolved in the soy protein isolate at a certain concentration, and continuously stirred to ensure that it is fully dissolved. Complex enzyme is added to the solution, and the enzyme is inactivated after water bathing to obtain a pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel.

[0035] According to a specific embodiment of the present invention, the concentration of the soy protein isolate solution is 5-20% (w / v), the concentration of the polysaccharide solution is 0.5-5% (w / v), and the mixture is stirred for 2-4 hours until completely dissolved.

[0036] Preferably, the concentration of the soy protein isolate solution is 8-15% (w / v), and the concentration of the polysaccharide solution is 0.5-2% (w / v).

[0037] According to a specific embodiment of the present invention, the complex enzyme is mTGase enzyme and laccase, and the added amounts are 10-100 u / g and 1-50 u / g respectively.

[0038] Preferably, the added amounts of the mTGase enzyme and laccase are 10-50 u / g and 1-20 u / g, respectively.

[0039] More preferably, the added amounts of the mTGase enzyme and laccase are 40 u / g and 10 u / g, respectively.

[0040] According to a specific embodiment of the present invention, the water bath temperature is 25-70°C, the water bath is for more than 10 min, and the enzyme is inactivated at 90°C for more than 5 min.

[0041] Preferably, the water bath temperature is 30-50°C, the time is more than 1 h, and the enzyme is inactivated for 10 min.

[0042] More preferably, the water bath temperature is 45° C. and the time is 1.5 h.

[0043] According to a specific embodiment of the present invention, the concentration of the added functional factor is 0.1-5% (w / v).

[0044] In the present invention, the properties of the double network hydrogel and the embedding rate of the functional factors are greatly improved by modifying the soy protein isolate and optimizing the preparation process of the double network hydrogel.

[0045] The present invention also provides application of the aforementioned delivery double network hydrogel in a food functional factor delivery system.

[0046] In the present invention, the functional factor embedding rate of the double network hydrogel is 98.9%, and the total release rate after the in vitro digestion model reaches 98.0%.

[0047] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0048] In the following examples, soybean meal uses low-temperature defatted soybean meal provided by Shandong Yuwang Ecological Food Co., Ltd. Arabinoxylan was purchased from Shanghai Bevanta Biotechnology Co., Ltd., beet pectin and corn fiber gum were purchased from Nanjing Four Seasons Qianshun Biotechnology Co., Ltd., and citrus pectin (high methylation) was purchased from Herbstreith & Fox KG, Germany. Transglutaminase (mTGase), laccase and riboflavin were purchased from Beijing Solebow Technology Co., Ltd. Artificial oral fluid, artificial gastric juice, artificial small intestinal fluid and salivary amylase, trypsin and pepsin therein were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd. and Sigma-Aldrich, USA. The alkaline solution used to adjust the pH uses a NaOH solution with a concentration of 1 mol / L. The acid solution used to adjust the pH uses HCl with a concentration of 1 mol / L.

[0049] Example 1 Preparation of Ultrasonic Modified Protein

[0050] The soybean meal is crushed, the sieved soybean meal and water are mixed and stirred at a mass ratio of 1:8, the pH is adjusted to 8.0-10.0, and ultrasonic treatment is performed after stirring for 1-3 hours, and the supernatant is obtained by centrifugation; the pH value of the supernatant is adjusted to a value where protein precipitates therein, and then centrifuged, the precipitate is re-dissolved with water, the pH is adjusted to neutral, and freeze-dried to obtain soy protein isolate (SPI).

[0051] Example 2 Preparation of pH-responsive modified protein-polysaccharide based double network hydrogel

[0052] The SPI in Example 1 was dissolved in deionized water to prepare a 10% (w / v) SPI solution. AX (arabinoxylan) and riboflavin powder were then added to the SPI solution and stirred in the dark to prepare AX concentrations of 0%, 0.5%, and 1.0% (w / v), and riboflavin concentrations of 0.4% (w / v). TG enzyme and laccase were added to the solution successively, and the enzymes were inactivated at 90°C for 10 min after being placed in a 45°C water bath for 1.5 h. The gel was placed in an ice bath and cooled to obtain a protein-arabinoxylan double network hydrogel.

[0053] Comparative Example 1 Preparation of pH-responsive modified protein-beet pectin double network hydrogel

[0054] The SPI in Example 1 was dissolved in deionized water to prepare a 10% (w / v) SPI solution. Beet pectin and riboflavin powder were then added to the SPI solution, stirred in the dark, and the AX concentrations were 0%, 0.4%, 0.6%, 0.8%, and 1.0% (w / v), respectively, and the riboflavin concentration was 0.4% (w / v). TG enzyme and laccase were added to the solution successively, and the enzyme was inactivated at 90°C for 10 min after being incubated in a 45°C water bath for 1.5 h. The gel was cooled in an ice bath.

[0055] The gel obtained by the above steps is a composite gel. The double network structure of protein and polysaccharide is not produced by laser confocal microscopy ( Figure 1 ), which may be due to the extraction process and source of the raw materials, resulting in a low content of ferulic acid and other substances in the side chains of beet pectin, resulting in the failure to form a network structure between the pectin network and the protein.

[0056] Comparative Example 2 Preparation of pH-responsive modified protein-citrus pectin double network hydrogel

[0057] The SPI in Example 1 was dissolved in deionized water to prepare a 10% (w / v) SPI solution. Citrus pectin and riboflavin powder were then added to the SPI solution and stirred in the dark to prepare AX concentrations of 0%, 0.4%, 0.6%, 0.8%, and 1.0% (w / v), respectively, and riboflavin concentration of 0.4% (w / v). TG enzyme and laccase were added to the solution successively, and the enzyme was inactivated at 90°C for 10 min after being incubated in a 45°C water bath for 1.5 h. The gel was then cooled in an ice bath.

[0058] The gel obtained by the above steps is a composite gel, and the gel strength is low and the structure is relatively loose. Rheological measurement shows that the elastic modulus is less than the viscous modulus, and no solid elastic gel is produced. This may be because the polysaccharide side chain groups in the raw material do not produce hydrogen bonds or other interactions with the protein side chain groups, resulting in no double network structure formed between pectin and protein.

[0059] Comparative Example 3 Preparation of pH-responsive modified protein-corn fiber gum double network hydrogel

[0060] The SPI in Example 1 was dissolved in deionized water to prepare a 10% (w / v) SPI solution. Corn fiber gum and riboflavin powder were then added to the SPI solution and stirred in the dark to prepare AX concentrations of 0%, 0.4%, 0.6%, 0.8%, and 1.0% (w / v), respectively, and riboflavin concentration of 0.4% (w / v). TG enzyme and laccase were added to the solution successively, and the solution was incubated in a water bath at 45°C for 1.5 h, then inactivated at 90°C for 10 min, and the gel was cooled in an ice bath.

[0061] The gel obtained by the above steps is a composite gel, and the gel strength is low and the structure is relatively loose. Rheological measurement shows that the elastic modulus is less than the viscous modulus, and no solid elastic gel is produced. This may be because the polysaccharide side chain groups in the raw material do not produce covalent bonds and hydrophobic bonds with the protein side chain groups, resulting in no double network structure formed between pectin and protein.

[0062] Example 3 Determination of mechanical and water holding properties of pH-responsive modified protein-polysaccharide based double network hydrogel functional factors

[0063] The texture characteristics of the sample in Example 2 were measured using a P / 36R cylindrical probe equipped with a texture analyzer through a TPA test cycle. The texture measurement parameters were as follows: the trigger point load was 5 g, the test speed was 1.0 mm·s -1 , return speed is 1.0 mm·s -1 , compression set is 50%, pre-test speed is 2.0 mm·s -1 , data frequency is 10 points·s -1The load unit is 1500 g. The sample in Example 2 was placed in a centrifuge tube and centrifuged at 7000 r / min for 20 min at 4°C to remove water. The calculation formula for WHC is: ×100,W r is the mass of water overflowed after centrifugation (g), W t is the total mass of water in the gel sample (g).

[0064] Table 1 Texture and water holding properties of double network hydrogels prepared by combining SPI with different concentrations of AX

[0065] Note: Different lowercase letters indicate significant differences.

[0066] As shown in Table 1, with the increase of AX concentration, the mechanical properties of the double network gel are significantly improved, and the ability to resist damage is increased. Appropriate repulsion between molecules is conducive to the formation of the gel network and increases the uniformity of the gel network. With the increase of total solid content, the formation and performance of the gel are further improved. Compared with the pure SPI gel, the hardness and cohesion of the double network gel are enhanced. The addition of AX also has a positive effect on the elasticity of the gel. With the increase of AX concentration, the elasticity of the gel gradually increases. This is because after AX is added to the SPI solution, an independent network structure is formed, which interpenetrates with the protein network. This double network structure can enhance the elasticity and ductility of the gel. In terms of water holding capacity, when the AX concentration increases, the WHC of the gel shows a trend of first decreasing and then increasing. This is because the introduction of AX may disrupt the construction process of the gel network, resulting in the dissociation or loosening of some gel structures and a decrease in WHC. When the AX concentration is 1.0% (w / v), the WHC of the double network gel is significantly increased to 98.9%. This is due to the enhanced interaction between SPI and AX, which results in a higher cross-linking density of the double network gel, which may cause the gel network to rearrange or strengthen, thereby improving the WHC of the gel. This interaction may involve mechanisms such as hydrogen bonding, electrostatic interaction, or hydration, which helps to form a more compact and stable gel structure.

[0067] Example 4 Microstructure of pH-responsive modified protein-polysaccharide based double network hydrogel functional factors

[0068] The samples in Example 2 were freeze-dried, and each sample was then cut into squares with a diameter of approximately 0.1 cm. Before observation, the samples were plated with gold or palladium and then critical point dried. The microstructure of the protein gel was observed using a scanning electron microscope at a voltage of 10 kV ( Figure 2 ).

[0069] 0.5 g of AX was dissolved in dimethyl sulfoxide at 10% (w / v), and then 50 mg of FITC and 20 mg of dibutyltin dilaurate were added. The mixture was stirred at 65 °C in the dark for 3 h (200 r / min). When the mixture cooled to room temperature, it was washed with ethanol (75%) to remove excess dye, and the precipitate was collected by centrifugation at 8000 r / min for 5 min and washed until the residual free dye was completely removed. After freeze-drying, dry AX was obtained. Nile blue was dissolved in ethanol to prepare a 0.1% (w / v) solution. Then, in the gel preparation process, 40 μL of 0.1% Nile blue solution was mixed into each milliliter of gel sample to stain SPI. After ensuring sufficient mixing, TGase and laccase were added in sequence. Then, 60 μL of the stained gel sample was quickly taken and dropped onto the grooved glass slide, covered with a cover glass and smeared with silicone oil to seal, and then the sample was operated according to the steps of Example 2 for subsequent observation. The stained gel sample was loaded on the glass slide and mounted on the stage of the laser confocal microscope, and the sample was observed using a 20x objective lens, and the dual laser mode was enabled to scan the sample in all directions.

[0070] Pure SPI has a uniform and ordered three-dimensional gel network structure. The double network gel of SPI-AX has both a layered structure and a porous structure. With the increase of AX concentration, the pore size of the double network gel becomes smaller. AX enhances the intermolecular interaction and thus improves the density of the hydrogel network. Figure 3 Laser confocal images showed that the structure of pure SPI gel was denser and had a few irregular aggregates. The microstructure of SPI-AX double network gel showed a continuous three-dimensional network structure, and the concentration of AX had a significant effect on the structural properties of the double network gel. When its concentration was low, the cross-linking of AX was insufficient to form a three-dimensional network, and the double network gel structure was mainly a continuous protein network, and incomplete AX gel particles were wrapped in SPI gel. With the increase of AX concentration, the network structure of AX gel in the double network gel tended to be tighter, and the network structure formed by SPI was further dense. The double network gel showed an interlaced network structure, and the AX gel structure tended to be dominant. When the AX concentration reached 1.0% (w / v), the interpenetrating network was more obvious, forming an interconnected network structure, which helped to improve the mechanical properties of the gel.

[0071] Example 5 Determination of the embedding rate and loading rate of functional factors in pH-responsive modified protein-polysaccharide based double network hydrogel

[0072] The sample in Example 2 was freeze-dried and ground into powder. About 20 mg of the powder sample was accurately measured and added to 20 mL of simulated intestinal fluid containing 0.125% trypsin, and incubated at 37°C with shaking for 6 h. Subsequently, centrifugation was performed at 10,000 r / min for 20 min at room temperature. The riboflavin content in the supernatant was determined by comparing the standard curve at a wavelength of 445 nm. The concentration of riboflavin was calculated from the riboflavin standard curve.

[0073] Table 2 Wet gel yield, embedding efficiency and loading rate of double network hydrogels prepared by SPI combined with different concentrations of AX

[0074] Note: Different lowercase letters indicate significant differences.

[0075] As shown in Table 2, with the increase of AX concentration, the wet gel yield, EE and LC of the double network hydrogel showed a trend of first decreasing and then increasing. Compared with the gel prepared by pure SPI, when the AX concentration was 0.5% (w / v), the wet gel yield, embedding rate and loading rate of the double network hydrogel constructed by SPI-AX were significantly reduced. The low concentration of AX disrupted the formation of the SPI gel network, affected the connectivity of the gel structure, made the gel network uneven, and decreased the hydration capacity, resulting in a decrease in the embedding rate and loading rate of the gel. When the AX concentration increased to 1.0% (w / v), the yield of the wet gel was significant, which was 1.1 times that of the SPI gel. This is because the interaction between AX and SPI was enhanced, forming an interpenetrating network structure, thereby enhancing the ability to retain water and increasing the gel yield. In addition, the stable and dense double network gel structure can mix riboflavin evenly and tightly in the gel network, improving the embedding rate (98.91%) and loading rate (3.96%).

[0076] Example 6 In vitro release assay of functional factors from pH-responsive modified protein-polysaccharide based double network hydrogels

[0077] The sample in Example 2 was cut into small pieces to simulate the chewing process, immersed in simulated saliva containing salivary amylase, and continuously shaken. Then, it was taken out and quickly inactivated in a 95°C hot water bath and cooled to room temperature, and its absorbance at a wavelength of 445 nm was measured; next, the gel sample was added to SGF containing pepsin and continued to be shaken, and the absorbance at 445 nm was measured at multiple predetermined time points until 120 min later, the sample was placed in a 95°C hot water bath again to inactivate the enzyme and cooled to room temperature; finally, the sample was placed in SIF and continued to be shaken, and the absorbance was repeatedly measured at various time periods to monitor the release process of riboflavin, and the digestion was continued in SIF for 240 min. Pepsin and trypsin need to be added to SGF and SIF 1 h in advance, where the concentration of pepsin is set to 0.1%, and the concentration of trypsin is set to 1.0%, and the digestion experiment lasts for 6 h. Riboflavin release rate (%) = m / M×100, m is the mass of released riboflavin, and M is the mass of total embedded riboflavin.

[0078] Figure 4 The results showed that riboflavin in all gel samples was released with the increase of time. For the gel prepared by single SPI, the release efficiency of riboflavin in the simulated stomach was high, reaching 75.2%. Since the network structure of single SPI is relatively simple and easily eroded and dissolved by gastric acid, SPI itself will undergo conformational changes and protein denaturation in an acidic environment, which will reduce the stability of the gel and accelerate the dissolution and release process of the gel. However, for the SPI-AX double network hydrogel, the release efficiency of riboflavin in the stomach decreased significantly with the increase of AX concentration. The AX network remained in a contracted state, firmly locking the bioactive components in the gel and slowing down its rapid release in gastric juice, indicating that the double network constructed by SPI combined with AX has a protective effect on the penetration of pepsin. In simulated intestinal fluid, the release efficiency of riboflavin in the intestine increased with the increase of AX concentration, which was 24.8% higher than the riboflavin release rate of single SPI gel, and this result was better than the delivery effect of the double network gel constructed by SPI combined with other polysaccharides. Therefore, SPI-AX double network hydrogel can inhibit the release of riboflavin in the stomach and promote the effective release of riboflavin in the small intestine.

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a pH-responsive modified protein-polysaccharide functional factor delivery double network hydrogel, characterized in that: The steps include: (1) Using low-temperature soybean meal as raw material, modified soy protein isolate was prepared by ultrasonication, alkali extraction and acid precipitation of low-temperature soybean meal; (2) A modified soy protein isolate solution of a certain concentration is prepared with water, polysaccharides and water-soluble functional factors are added, and after dissolution, a composite enzyme is added. The resulting mixed system is gelled at 25-70°C, and finally the enzyme is inactivated and cooled on ice to room temperature to obtain a pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel; Wherein, the polysaccharide is selected from at least one of arabinoxylan, beet pectin, citrus pectin, and corn fiber gum; The water-soluble functional factor is at least one of the following water-soluble nutrients: riboflavin, polyphenols, vitamin C, peptides; The complex enzyme consists of laccase and mTGase enzymes.

2. The method according to claim 1, characterized in that The step (1) comprises: crushing low-temperature soybean meal, mixing and stirring the sieved soybean meal with water in a mass ratio of 1:(8-12), adjusting the pH to 8.0-10.0, stirring for 1-3 hours, ultrasonically treating, and centrifuging to obtain a supernatant; adjusting the pH value of the supernatant to allow protein to precipitate therein, then centrifuging, re-dissolving the precipitate with water, adjusting the pH to neutral, and freeze-drying to obtain a modified soybean protein isolate.

3. The method according to claim 2, characterized in that The ultrasonic power is 200-600 W, the ultrasonic time is 1-20 min, and the pH value of the supernatant is adjusted to 3.5-5.

0.

4. The method according to claim 1, characterized in that: In the mixed system of step (2), the concentration of modified soy protein isolate is 5-20% w / v, the concentration of polysaccharide is 0.5-5% w / v, the concentration of water-soluble functional factor is 0.1-5% w / v, and the added amounts of mTGase and laccase are 10-100 u / g and 1-50 u / g, respectively.

5. The method according to claim 4, characterized in that In the mixed system of step (2), the concentration of modified soy protein isolate is 8-15% w / v, the concentration of polysaccharide is 0.5-2% w / v, the concentration of water-soluble functional factor is 0.1-5% w / v, and the added amounts of mTGase and laccase are 10-50 u / g and 1-20 u / g, respectively.

6. The method according to claim 5, characterized in that In the mixed system of step (2), the concentration of modified soy protein isolate is 8-15% w / v, the concentration of polysaccharide is 0.5-2% w / v, the concentration of water-soluble functional factor is 0.1-5% w / v, and the added amounts of mTGase and laccase are 40 u / g and 10 u / g, respectively.

7. The method according to any one of claims 4 to 6, characterized in that: The mixed system was gelled in a water bath at 25-70°C for more than 10 min, and then the enzyme was inactivated at 90°C for more than 5 min; Preferably, the mixed system is subjected to gelation treatment in a water bath at 30-50°C for more than 1 hour, and then the enzyme is inactivated at 90°C for 10 minutes; More preferably, the mixed system is subjected to gelation treatment in a water bath at 45° C. for 1.5 h, and then the enzyme is inactivated at 90° C. for 10 min.

8. A pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel prepared according to the method of any one of claims 1 to 7.

9. Use of the pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel according to claim 8 in a food functional factor delivery system.

10. Use of the pH-responsive modified protein-polysaccharide-based functional factor delivery double network hydrogel according to claim 8 in the preparation of a drug delivery carrier.

Images