Production method of spina date seed protein gel

Abstract

The invention discloses a production method of spina date seed protein gel, and belongs to the technical field of food processing, the spina date seed protein gel is prepared by taking medicinal and edible spina date seed as a raw material, performing degreasing, alkali extraction and acid precipitation to obtain spina date seed protein isolate, and performing precursor solution preparation, compound probiotic fermentation and post-treatment gelling. The compound probiotic leavening agent is formed by compounding lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, lactobacillus plantarum and lactobacillus casei according to the equal proportion, the inoculation amount is 0.1 g / 100 mL precursor solution, constant-temperature fermentation is carried out at 42 DEG C for 10-20 h, and the preferable fermentation time is 15 h. A standardized spina date seed protein isolate extraction process is established, the screened compound probiotic leavening agent is high in adaptability, and cooperative regulation and control of the gel water-holding capacity and strength are achieved by regulating and controlling the fermentation time; the prepared gel is in a non-transparent solid state, does not have dehydration shrinkage, has the water-holding capacity of more than or equal to 70%, has the gel strength of more than or equal to 18g, has a uniform and porous three-dimensional network structure as a microstructure, and has excellent rheological properties; the process is green and safe, the operation is simple, industrial amplification can be realized, and the product has both nutrition and probiotic functions, can be directly developed into products such as plant-based yoghourt and the like, can also be applied as a food gel, realizes high-value utilization of the spina date seed resource, and has a good industrial prospect.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for producing plant protein gel, and more particularly to a method for preparing jujube seed protein gel by probiotic fermentation. This method uses jujube seed, which is both a food and a medicine, as raw material, and induces the separation of jujube seed protein to form a gel through compound probiotic fermentation. The resulting gel can be applied to food fields such as plant-based yogurt, plant-based cheese, functional gel candies, and meal replacement gels. It can also be used as a food thickener and gelling agent to improve meat and dairy products, belonging to the technical direction of high-value utilization of food and medicinal plant resources. Background Technology

[0002] With global population growth and the deepening of the concept of sustainable development, the production and consumption of animal protein face problems such as high resource consumption, high carbon emissions, and intensified supply-demand imbalances. Finding high-quality plant proteins to replace animal proteins has become a research hotspot in the field of food science. Plant proteins not only have advantages such as high production efficiency, environmental friendliness, and wide availability of raw materials, but their diverse functional properties also provide ample room for the development of novel plant-based foods. Among these, the preparation and application of plant protein gels is one of the important directions for the high-value utilization of plant proteins.

[0003] Protein gels refer to solid systems in which protein molecules aggregate from a dispersed molecular state to form a three-dimensional network structure under the influence of physical, chemical, or biological factors. These systems can retain moisture, flavor compounds, and nutrients, and are crucial for constructing the texture and mouthfeel of plant-based foods. Currently, research and applications of plant protein gels are mainly focused on common oilseed crops such as soybeans, peas, and peanuts. The gel preparation methods primarily include physical methods (thermal induction, high pressure, ultrasound), chemical methods (acid-base induction, salt ion induction), and biological methods (enzymatic induction). However, all of these methods have certain limitations: physical methods consume a large amount of energy and have poor controllability of gel properties; chemical methods easily introduce exogenous reagents, affecting product safety and edibility; and enzymatic methods are costly and prone to excessive protein hydrolysis, leading to a decrease in gel strength.

[0004] Probiotic fermentation, as a green and safe biomodification method, has been applied to the gel preparation of proteins such as soybeans and whey. It lowers the pH of the system by producing acid through probiotic metabolism, weakening the electrostatic repulsion between protein molecules and inducing protein aggregation to form a gel. Simultaneously, probiotic fermentation can also enhance the nutritional and functional properties of products, endowing them with prebiotic characteristics. However, current research on the preparation of plant protein gels through probiotic fermentation still faces the problem of limited raw material availability, and research on fermentation gels of medicinal and edible plant proteins is rarely reported.

[0005] Ziziphus jujuba seed, a traditional Chinese medicinal and edible resource, is rich in protein. Ziziphus jujuba seed protein possesses excellent nutritional properties and physiological activity, making it a high-quality raw material for developing functional plant protein gels. However, current research on ziziphus jujuba seed protein primarily focuses on extraction processes and physiological activity analysis, with insufficient development of its functional properties. In particular, the technology of inducing ziziphus jujuba seed protein to form a gel using probiotic fermentation has not yet been systematically developed, leaving the following technological gaps: There is a lack of standardized extraction technology for jujube seed protein isolate, and the purity and physicochemical properties of jujube seed protein suitable for gel preparation are not clearly defined. No suitable probiotic strain combination for the formation of jujube seed protein gel has been screened out, and the current probiotic compounding scheme cannot be adapted to the molecular characteristics of jujube seed protein. The key process parameters for the formation of jujube seed protein gel induced by probiotic fermentation, such as fermentation temperature, fermentation time, and inoculation amount, are not clearly defined, making it impossible to achieve synergistic regulation of gel water-holding capacity, gel strength, microstructure, and other properties. The lack of in-depth research on the formation mechanism of jujube seed protein gel makes it impossible to provide a theoretical basis for process optimization, resulting in the inability of existing technologies to prepare jujube seed protein gel with stable texture and excellent performance.

[0006] Furthermore, existing plant protein gels suffer from the challenge of simultaneously achieving both water-holding capacity and gel strength. Most gels, while increasing strength, exhibit decreased water-holding capacity and dehydration shrinkage, impacting the product's edibility and shelf-life stability. Therefore, developing a standardized, green, safe, and high-performance method for producing jujube seed protein gel, enabling the high-value utilization of the medicinal and edible jujube seed resource, and filling the gap in plant protein gel raw materials and preparation processes, has significant practical importance and market value.

[0007] This invention addresses the shortcomings of existing technologies by using jujube seed as raw material. Through optimizing the extraction process of jujube seed protein isolate, screening suitable compound probiotic fermentation agents, and clarifying the optimal process parameters for fermentation-induced gel formation, it achieves efficient preparation of jujube seed protein gel. The resulting gel has both excellent water-holding capacity and gel strength, and a uniform and stable microstructure, thus solving many pain points of existing technologies. Summary of the Invention

[0008] The purpose of this invention is to provide a production method for preparing jujube seed protein gel by probiotic fermentation, which aims to solve the problems of single raw materials, limited preparation methods, and poor product performance in existing plant protein gel preparation processes, and at the same time fill the gap in jujube seed protein gel preparation technology.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention discloses a method for producing jujube seed protein gel, which uses jujube seed as raw material and involves protein separation, preparation of precursor solution, probiotic fermentation, and post-treatment to form a gel. Specifically, the method includes the following steps: Step 1: Preparation of Jujube Seed Protein Isolate (JPI): Jujube seed is ground and defatted. Water is added to the defatted meal to make a slurry and the protein is extracted with alkali. After acid precipitation, the protein precipitate is collected, reconstituted and freeze-dried to obtain jujube seed protein isolate. Step 2: Preparation of JPI-based gel precursor solution: Mix jujube seed protein isolate, sucrose and distilled water, adjust the pH to 7.0, refrigerate and then sonicate, then sterilize at high temperature and cool to obtain gel precursor solution; Step 3: Probiotic fermentation: Inoculate the compound probiotic fermentation agent into the gel precursor solution and ferment at a constant temperature of 42℃ for 10~20h to obtain the fermentation product; Step 4: Post-processing to form gel: The fermentation product is placed at 4℃ for 24 hours to complete gel solidification and obtain jujube seed protein gel; The compound probiotic starter is composed of Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei.

[0010] Preferably, the defatting in step 1 involves soaking and grinding jujube seed powder in a mixture of hexane and ethanol, with a volume ratio of hexane to ethanol of 10:1 and a material-to-liquid ratio of 1:5 to 1:10. The defatting is carried out at room temperature for 12 to 24 hours, and the defatted meal is obtained after filtration.

[0011] Preferably, the specific operation of alkaline protein extraction in step 1 is as follows: add distilled water to defatted meal, with a material-to-water ratio of 1:12 (by weight), adjust the pH of the system to 8.0 with 1M NaOH, stir and extract at 600 rpm for 2 hours, centrifuge at 6000×g for 30 minutes at 25°C, and collect the supernatant; the specific operation of acid precipitation is as follows: adjust the pH of the supernatant to 4.5 with 1M HCl, centrifuge at 6000×g for 30 minutes at 25°C, and collect the protein precipitate.

[0012] Preferably, in step 2, the mass-to-volume ratio of jujube seed protein isolate, sucrose, and distilled water is 5g:5g:90mL; the refrigeration is performed by standing at 4℃ for 24 hours; the ultrasonic treatment uses a titanium alloy probe with a diameter of 0.636cm, an ultrasonic frequency of 20kHz, and an ultrasonic time of 20min; the high-temperature sterilization is performed by constant temperature sterilization at 90℃ for 15min, followed by cooling to 25℃.

[0013] Preferably, the inoculation amount of the compound probiotic fermentation agent in step 3 is 0.1g / 100mL of gel precursor liquid; the constant temperature fermentation time is 15h, during which the system naturally produces acid and the pH drops to 4.0~4.5.

[0014] Preferably, after the gel solidification in step 4 is completed, the jujube seed protein gel is an opaque solid gel with no dehydration shrinkage, a water holding capacity ≥70%, a gel strength ≥15g, and a storage modulus (G') ≥100Pa.

[0015] Preferably, the reconstitution in step 1 involves adding the protein precipitate to distilled water and stirring until it is evenly dispersed, and the freeze-drying involves using a vacuum freeze dryer to dry the protein at -40°C and a vacuum of 10 Pa for 24 hours to obtain powdered jujube seed protein isolate with a protein purity ≥85%.

[0016] In a second aspect, the present invention provides a jujube seed protein gel prepared by the production method described above, wherein the gel is a medicinal and edible plant-based gel with a uniform porous three-dimensional network structure, a particle size distribution concentrated in the range of 1 to 10 μm, shear-thinning rheological properties, a relative recovery rate of ≥80%, and no protein hydrolysis. The gel formation is achieved by the synergistic effect of intermolecular covalent cross-linking and non-covalent interactions induced by probiotic acid production.

[0017] Compared with existing technologies, this invention uses jujube seed, a food and medicinal ingredient, as raw material and prepares jujube seed protein gel through a compound probiotic fermentation method. The process design is scientific, and the parameter control is precise. Compared with existing technologies, it has significant advantages in terms of raw materials, process, and product, specifically: Raw material innovation enables high-value utilization of medicinal and edible resources: This invention is the first to apply jujube seed protein to the preparation of plant protein gel, breaking through the limitation of single raw materials in existing plant protein gels, realizing the in-depth development and high-value utilization of medicinal and edible jujube seed resources, and providing a new direction for the expansion of raw materials for plant protein gels; The process is green and safe with a high degree of standardization: This invention uses probiotic fermentation to induce gel formation, with no exogenous chemical reagents added throughout the process, which meets the green and safe requirements of modern food industry; at the same time, it establishes a standardized process for the extraction of jujube seed protein and gel preparation, with clear and easy-to-control parameters for each step, no need for high-end equipment, and can be industrialized and scaled up, making it suitable for large-scale application in food enterprises and with good industrial prospects; The product boasts superior performance and addresses industry technical pain points: By controlling the fermentation time (15h), this invention achieves a synergistic improvement in gel water-holding capacity and gel strength. The resulting jujube seed protein gel has a water-holding capacity of ≥70% and a gel strength of ≥18g. Its microstructure is a uniform, porous, three-dimensional network structure with no dehydration shrinkage. It exhibits excellent rheological properties, shear thinning characteristics, and high elastic recovery rate, thus solving the industry pain points of existing plant protein gels where water-holding capacity and strength are difficult to balance and texture is unstable. The product combines nutrition and function, with a wide range of applications: the resulting jujube seed protein gel not only retains the amino acids, minerals, and other nutrients of jujube seed protein, but also incorporates prebiotic properties due to probiotic fermentation, resulting in a probiotic live bacteria count ≥1.0×10⁻⁶. 9 CFU / g, with both gelling and prebiotic functions; this gel can be directly developed into plant-based yogurt, plant-based cheese, functional gel candies, meal replacement gels and other products, and can also be used as a food gelling agent and thickener to improve the texture of meat products, dairy products and condiments, with a wide range of applications; The fermentation mechanism is clearly understood, providing theoretical support for subsequent research: This invention clarifies the mechanism by which probiotic fermentation induces the formation of jujube seed protein gel, namely, the synergistic effect of intermolecular covalent cross-linking (disulfide bonds) and non-covalent interactions (hydrophobic interactions, hydrogen bonds) induced by probiotic acid production. Furthermore, no protein hydrolysis occurs during fermentation. These findings provide a solid theoretical basis and practical guidance for the process optimization and product upgrading of jujube seed protein gel, as well as the preparation of protein gels from other medicinal and edible plants. Attached Figure Description

[0018] Figure 1 Showing the appearance of the gel at different fermentation times; Figure 2 Showing the changes in average gel particle size at different fermentation times; Figure 3 Show the changes in pH value of the system at different fermentation times; Figure 4 This demonstrates the changes in gel water-holding capacity at different fermentation times; Figure 5 This demonstrates the changes in gel strength at different fermentation times; Figure 6 This demonstrates the effect of different fermentation times on gel viscosity; Figure 7 The effect of different fermentation times on the storage modulus of the gel is demonstrated. Figure 8 The effects of different fermentation times on gel creep and recovery are demonstrated. Figure 9 The images show fluorescence microscopy observations of the gel at different fermentation times. Detailed Implementation

[0019] This invention systematically investigates the effects of probiotic fermentation time (0-20 hours) on the physicochemical properties, microstructure, and molecular interactions of jujube seed protein isolate gel, elucidates its gel formation mechanism, and provides a theoretical basis for the application of jujube seed protein as an innovative gelling agent in the food industry. It thus provides a new production method for preparing jujube seed protein gel by probiotic fermentation, solving the problems of limited raw materials, limited preparation methods, and poor product performance in existing plant protein gel preparation processes, while filling the gap in jujube seed protein gel preparation technology.

[0020] Specifically, the solution of the present invention achieves the following objectives: Establish a standardized extraction process for jujube seed protein isolate suitable for gel preparation to obtain high-purity, highly dispersible jujube seed protein isolate, laying the raw material foundation for gel formation; We screened out a compound probiotic fermentation agent that can efficiently induce the formation of jujube seed protein gel, determined the ratio of bacterial strains, and achieved a match between the acid production rate and the protein aggregation rate of probiotics. Optimize key process parameters for probiotic fermentation, determine the optimal fermentation time, and achieve synergistic regulation of the water-holding capacity, gel strength, and microstructure of jujube seed protein gel, thus solving the problem of difficulty in achieving both water-holding capacity and strength. We provide a standardized, industrially scalable process for producing jujube seed protein gel. The process is green, safe, simple to operate, and low in cost. The resulting product is both nutritious and functional, meeting the development needs of the modern food industry.

[0021] The technical solution of the present invention will be described in detail below in exemplary embodiments.

[0022] To achieve the above technical objectives, this invention provides a method for producing jujube seed protein gel by probiotic fermentation. This method uses jujube seed, a food and medicinal herb, as raw material. High-purity jujube seed protein isolate is obtained through defatting, alkali extraction, and acid precipitation. The isolate is then processed through precursor solution preparation, compound probiotic fermentation, and post-treatment to form a gel, yielding the jujube seed protein gel. The core of this method lies in inducing the aggregation of jujube seed protein molecules through acid production during compound probiotic fermentation, forming a uniform, porous three-dimensional network structure. Simultaneously, the gel performance is optimized by controlling the fermentation time. The method specifically includes the following steps: Step 1: Preparation of Jujube Seed Protein Isolate (JPI) Raw material pretreatment: Remove impurities from jujube seed, dry in a 60℃ oven for 4 hours, grind into 80 mesh powder, sieve and set aside; Degreasing treatment: Add jujube seed powder to a hexane / ethanol mixture with a volume ratio of hexane to ethanol of 10:1 and a material-to-liquid ratio of 1:8 (g / mL). Degrease the mixture by magnetic stirring at room temperature for 18 hours. Remove organic solvent by filtration. Place the defatted meal in a fume hood to air dry and remove residual organic solvent. Alkaline extraction of protein: Add distilled water to defatted meal at a material-to-water ratio of 1:12 (g / mL), slowly adjust the pH of the system to 8.0 with 1 mol / L NaOH solution, and extract at a constant temperature and speed of 600 rpm for 2 hours to fully dissolve the protein in water; Centrifugation: Place the above extract in a high-speed centrifuge and centrifuge at 25℃ and 6000×g for 30 min. Collect the supernatant and discard the insoluble residue at the bottom. Acid precipitation of protein: Slowly adjust the pH of the supernatant to 4.5 (isoelectric point of jujube seed protein) with 1 mol / L HCl solution, stir well and let stand for 30 min to allow the protein to precipitate fully. Then centrifuge at 25℃ and 6000×g for 30 min and collect the protein precipitate at the bottom. Reconstitution freeze-drying: Add the protein precipitate to distilled water and stir until uniformly dispersed to obtain a protein dispersion. Place the dispersion in a vacuum freeze dryer and dry for 24 hours at -40℃ and 10Pa vacuum to obtain powdered jujube seed protein isolate. After sealing, store in a 4℃ refrigerator for later use. The purity of the jujube seed protein isolate is ≥85%.

[0023] Step 2: Preparation of JPI-based gel precursor solution Ingredient mixing: Add 5g of jujube seed protein isolate and 5g of sucrose to 90mL of distilled water, place on a magnetic stirrer, and stir continuously at 25℃ for 1 hour to fully dissolve the jujube seed protein isolate and sucrose to form a uniform mixture; pH adjustment: During stirring, the pH of the mixture is precisely adjusted to 7.0 using 1 mol / L NaOH or 1 mol / L HCl to ensure the dispersion stability of the protein. Refrigeration maturation: Place the pH-adjusted mixture in a 4°C refrigerator for 24 hours to allow the protein molecules to fully hydrate and improve the uniformity of the subsequent gel. Ultrasonic treatment: The refrigerated mixture was taken out and ultrasonically treated using a titanium alloy probe ultrasonic instrument with a probe diameter of 0.636 cm, an ultrasonic frequency of 20 kHz, and an ultrasonic time of 20 min. Ultrasonic treatment broke down the protein aggregates and further improved the dispersibility of the proteins. Sterilization and cooling: Place the ultrasonicated mixture in a water bath and sterilize at 90°C for 15 minutes to kill any bacteria in the system and prevent contamination from affecting the fermentation process. After sterilization, cool rapidly to 25°C to obtain the JPI-based gel precursor solution for later use.

[0024] Step 3: Fermentation with compound probiotics Fermentation agent inoculation: Inoculate 0.1g of compound probiotic fermentation agent into 100mL of gel precursor solution. The compound probiotic fermentation agent is composed of Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus casei in equal proportions. After inoculation, stir thoroughly to ensure that the fermentation agent is evenly dispersed in the precursor solution. Isothermal fermentation: The inoculated precursor solution is placed in an isothermal incubator and fermented at 42℃ for 10-20 hours. No additional stirring is performed during fermentation, allowing the probiotics to naturally metabolize and produce acid. The pH of the system gradually decreases with fermentation time, inducing protein molecules to aggregate and form a gel precursor. The optimal fermentation time is 15 hours, at which point the pH of the system drops to around 4.2, the degree of protein aggregation is moderate, and the water-holding capacity and microstructure of the gel are optimal. If the fermentation time is less than 10 hours, protein aggregation is insufficient, and a complete gel structure cannot be formed. If the fermentation time exceeds 20 hours, excessive protein aggregation occurs, the gel network structure becomes dense, the water-holding capacity decreases, and dehydration and shrinkage occur.

[0025] Step 4: Post-processing into adhesive The fermented product was placed in a 4°C refrigerator for 24 hours to further complete the aggregation process of protein molecules and solidify and stabilize the three-dimensional network structure, thus obtaining the final jujube seed protein gel.

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] All experimental procedures were performed three independent replicates. Results are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 25.0 software. p < 0.05 was considered statistically significant.

[0028] Experimental materials and instruments Experimental materials: Ziziphus jujuba seeds were purchased from Anhui Bozhou Huirui Traditional Chinese Medicine Technology Co., Ltd.; compound probiotic fermentation agent (Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei, all of which were freeze-dried bacterial powders with a viable count ≥1.0×10¹¹ CFU / g) was purchased from Kunshan Baishengyou Biotechnology Co., Ltd.; hexane, ethanol, sodium hydroxide, hydrochloric acid, and other chemical reagents were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.; distilled water was prepared in the laboratory.

[0029] Experimental instruments: High-speed pulverizer (FW100, Tianjin Tester Instrument Co., Ltd.); High-speed centrifuge (Allegra 64R, Beckman Coulter, USA); Vacuum freeze dryer (FD-1A-50, Beijing Boyikang Experimental Instrument Co., Ltd.); Magnetic stirrer (RJ-10, Changzhou Jintan Liangyou Instrument Co., Ltd.); pH meter (PH-100, Shanghai Lijing Bangxi Instrument Technology Co., Ltd.); Probe-type ultrasonic instrument (JY92-IIN, Ningbo Xinzhi Biotechnology Co., Ltd.); Constant temperature incubator (BD23, Binder, Germany); Texture analyzer (RapidTA, Shanghai Tengba Instrument Technology Co., Ltd.); Rheometer (HR-1, TA Instruments, UK); Laser particle size analyzer (LS13320, Beckman Coulter, USA); Fluorescence microscope (DM2500, Leica, Germany).

[0030] Example 1: Preparation of jujube seed protein gel after fermentation for 15 hours The jujube seed protein gel was prepared according to the technical solution of the present invention, with a core fermentation time of 15 hours. The specific steps are as follows: Preparation of Ziziphus jujuba seed protein isolate (1) Remove impurities from jujube seed, dry at 60℃ for 4 hours, and pulverize into 80 mesh powder; (2) Take 100g of jujube seed powder, add 800mL of n-hexane / ethanol mixture (volume ratio 10:1), stir at room temperature for 18h to defatt the powder, filter and dry to obtain defatted meal; (3) Add 1200mL of distilled water to the defatted meal, adjust the pH to 8.0 with 1mol / L NaOH, stir and extract for 2h at 600rpm, centrifuge at 25℃ and 6000×g for 30min, and collect the supernatant; (4) Adjust the pH of the supernatant to 4.5 with 1 mol / L HCl, let it stand for 30 min, then centrifuge at 25℃ and 6000×g for 30 min and collect the protein precipitate; (5) The protein precipitate was reconstituted with distilled water and freeze-dried under vacuum at -40℃ and 10Pa for 24h to obtain jujube seed isolated protein with a protein purity of 88.5±2.3%.

[0031] Preparation of JPI-based gel precursor solution (1) Take 5g of jujube seed protein isolate and 5g of sucrose, add 90mL of distilled water, and stir at 25℃ for 1h until completely dissolved; (2) Adjust the pH of the mixed solution to 7.0 with 1 mol / L NaOH / HCl, and refrigerate at 4℃ for 24 h; (3) Use a titanium alloy probe (0.636 cm in diameter) to perform ultrasonic treatment for 20 min at an ultrasonic frequency of 20 kHz; (4) Sterilize in a 90°C water bath for 15 minutes, then rapidly cool to 25°C to obtain the gel precursor solution.

[0032] Compound probiotic fermentation (1) Inoculate 0.1g of compound probiotic starter (a mixture of Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei in equal proportions) into 100mL of gel precursor solution and stir until evenly dispersed; (2) The inoculated precursor solution was placed in a constant temperature incubator at 42℃ and fermented for 15h. After fermentation, the pH of the system was 4.2±0.1.

[0033] Post-processing into adhesive The fermentation product was placed in a 4°C refrigerator for 24 hours to complete gel solidification and obtain jujube seed protein gel.

[0034] Comparative Example 1: Preparation of Ziziphus jujuba seed protein gel after fermentation for 0 h Except for the fermentation time of 0h (i.e., no constant temperature fermentation is carried out after inoculation, and it is directly refrigerated), the other operation steps and process parameters are completely consistent with those in Example 1.

[0035] Comparative Example 2: Preparation of Ziziphus jujuba seed protein gel after fermentation for 5 hours Except for the fermentation time of 5 hours, the other operating steps and process parameters were completely consistent with those in Example 1. After the fermentation was completed, the pH of the system was 5.8 ± 0.2.

[0036] Comparative Example 3: Preparation of Ziziphus jujuba seed protein gel after fermentation for 10 hours Except for the fermentation time of 10 hours, the other operating steps and process parameters were completely consistent with those in Example 1. After the fermentation was completed, the pH of the system was 4.8 ± 0.1.

[0037] Comparative Example 4: Preparation of Ziziphus jujuba seed protein gel after fermentation for 20 hours Except for the fermentation time of 20 hours, the other operating steps and process parameters were completely consistent with those in Example 1. After the fermentation was completed, the pH of the system was 3.8 ± 0.1.

[0038] Product performance testing The samples obtained in Example 1 and Comparative Examples 1-4 were subjected to performance testing. The test indicators included appearance, pH value, particle size distribution, water holding capacity, gel strength, rheological properties, and microstructure. The test methods are as follows: Appearance: Directly observe the appearance of the sample and record its state (liquid / semi-solid / solid), transparency, and whether there is any dehydration shrinkage. pH value: The pH value of the gel samples was directly measured using a pH meter. Each sample was measured 3 times and the average value was taken. Particle size distribution: After homogenizing the gel sample, a 1% (w / v) dispersion was prepared. The particle size distribution was measured using a laser particle size analyzer, and the average particle size was recorded. Water-holding capacity: Centrifuge the gel sample at 10000×g for 10 min, discard the supernatant, and calculate the water-holding capacity as (gel mass after centrifugation - gel dry weight) / gel wet weight × 100%. Gel strength: Using a texture analyzer with a stainless steel cylindrical probe (36mm in diameter), the gel was compressed to 50% of its original height at a speed of 1mm / s, and the maximum force was recorded as the gel strength. Rheological properties: Steady-state shear viscosity, storage modulus (G'), and creep-recovery properties were measured using a rheometer with 60 mm parallel plates and a 100 μm gap. Microstructure: After labeling the gel sample with fluorescein isothiocyanate (FITC), the microstructure was observed using a fluorescence microscope, and the uniformity and porosity of the network structure were recorded.

[0039] Test Results and Analysis Appearance: Figure 1 Showing the appearance of the gel at different fermentation times.

[0040] Comparative Example 1 (0h): The sample was a transparent liquid with no gel formation; Comparative Example 2 (5h): The sample was a semi-solid turbid liquid with no complete gel structure and strong fluidity; Comparative Example 3 (10h): The sample was a soft solid gel, which was opaque, had a loose gel structure, and had slight fluidity. Example 1 (15h): The sample was a hard solid gel, which was opaque, with an intact gel structure, no dehydration shrinkage, and no fluidity. Comparative Example 4 (20h): The sample was a hard solid gel, which was opaque and had a dense gel structure. It showed obvious dehydration and shrinkage, and water was separated from the surface.

[0041] pH value and average particle size: Figure 2 and Figure 3 The changes in pH value and average particle size of the system under different fermentation times are shown.

[0042] The test results showed that as the fermentation time increased, the pH value of the system continued to decrease, and the average particle size continued to increase. Comparative Example 1 (0h): pH = 7.0 ± 0.0, average particle size = 0.5 ± 0.1 μm; Comparative Example 2 (5h): pH = 5.8 ± 0.2, average particle size = 2.3 ± 0.3 μm; Comparative Example 3 (10h): pH = 4.8 ± 0.1, average particle size = 6.8 ± 0.5 μm; Example 1 (15h): pH=4.2±0.1, average particle size=8.5±0.4μm; Comparative Example 4 (20h): pH=3.8±0.1, average particle size=12.3±0.6μm.

[0043] Analysis shows that the acid production during probiotic fermentation is the core driving force for lowering the pH of the system and inducing protein aggregation. The decrease in pH reduces the net charge on the protein surface, weakens the electrostatic repulsion, and causes protein molecules to gradually aggregate and increase in particle size. When the fermentation time reaches 15 hours, the degree of protein aggregation is moderate, forming a complete gel structure. When the fermentation time exceeds 20 hours, the protein aggregates excessively, the particle size becomes too large, resulting in a dense gel network and water separation.

[0044] Water holding capacity: Figure 4 This demonstrates the changes in gel water-holding capacity at different fermentation times.

[0045] The test results are as follows: Comparative Example 2 (5h): Water holding capacity = 35.2 ± 2.1%; Comparative Example 3 (10h): Water holding capacity = 62.5 ± 3.2%; Example 1 (15h): Water holding capacity = 75.8 ± 2.8%; Comparative Example 4 (20h): Water holding capacity = 68.3 ± 3.5%.

[0046] The water-holding capacity first increases and then decreases with the extension of fermentation time, reaching a peak at 15h. This is because at 15h of fermentation, the gel forms a uniform and porous three-dimensional network structure with moderate porosity, which can effectively retain water. When the fermentation time exceeds 20h, the protein aggregates excessively, the gel network structure becomes dense, the porosity decreases, the water-holding space decreases, the water-holding capacity decreases, and dehydration shrinkage occurs.

[0047] Gel strength: Figure 5 This demonstrates the changes in gel strength at different fermentation times.

[0048] The test results are as follows: Comparative Example 2 (5h): Gel strength = 2.1 ± 0.3 g; Comparative Example 3 (10h): Gel strength = 10.5 ± 0.8 g; Example 1 (15h): Gel strength = 18.6 ± 1.2 g; Comparative Example 4 (20h): Gel strength = 22.3 ± 1.5g.

[0049] The gel strength increases continuously with the extension of fermentation time, reaching its maximum at 20 hours of fermentation. However, at this time, the gel undergoes dehydration and shrinkage, resulting in a decrease in edible quality. At 15 hours of fermentation, the gel strength reaches 18.6±1.2g, exhibiting both excellent strength and water-holding capacity, representing the optimal value that balances gel performance and edible quality.

[0050] Rheological properties: Figure 6 , Figure 7 , Figure 8 The effects of different fermentation times on gel viscosity, storage modulus, and gel creep and recovery were demonstrated.

[0051] All samples that formed gels exhibited typical shear-thinning behavior, meaning that the apparent viscosity decreased with increasing shear rate, consistent with the rheological properties of food gels; the storage modulus (G') continuously increased with prolonged fermentation time, reflecting the continuous improvement in the gel's elasticity and network structure strength. Comparative Example 3 (10h): G' = 65 ± 5 Pa, relative recovery rate = 62 ± 3%; Example 1 (15h): G' = 128 ± 6 Pa, relative recovery rate = 85 ± 4%; Comparative Example 4 (20h): G'=185±8Pa, relative recovery rate=88±3%.

[0052] The gel prepared in Example 1 (15h) has a moderate energy storage modulus and a relative recovery rate of ≥85%, indicating that its gel network structure has good elasticity and resistance to deformation, and has a good taste and chewiness when eaten.

[0053] Microstructure: Figure 9 Fluorescence microscopy images of gels fermented at different times are shown.

[0054] Fluorescence microscopy observations showed that: Comparative Example 3 (10h): The gel network structure was loose, the pore size was uneven, there were a large number of micropores, and the degree of protein aggregation was low. Example 1 (15h): The gel formed a uniform and continuous three-dimensional network structure with moderate and uniform pore size and high porosity, which can effectively retain water and is the microscopic basis for excellent water retention and gel strength. Comparative Example 4 (20h): The gel network structure was dense, the number of pores was reduced, the pore size was smaller, and the proteins were excessively aggregated, resulting in decreased water holding capacity and dehydration shrinkage.

[0055] Summary of Implementation Examples Based on the above test results, fermentation time is a key parameter affecting the formation and performance of jujube seed protein gel. If the fermentation time is less than 10 hours, the probiotics produce insufficient acid, the pH of the system is too high, the protein aggregation is insufficient, and a complete gel structure cannot be formed. The gel strength and water holding capacity are both low. When the fermentation time is 15h, the pH of the system drops to about 4.2, the degree of protein aggregation is moderate, a uniform and porous three-dimensional network structure is formed, the gel water holding capacity reaches 75.8±2.8%, the gel strength reaches 18.6±1.2g, the rheological properties and microstructure are both optimal, and there is no dehydration shrinkage phenomenon. This is the optimal fermentation time for preparing jujube seed protein gel. If the fermentation time exceeds 20 hours, the probiotics produce excessive acid, the system pH becomes too low, proteins aggregate excessively, and the gel network structure becomes dense. Although the gel strength is further improved, the water-holding capacity decreases, and dehydration and shrinkage occur, affecting the edible quality and shelf stability of the product.

[0056] The embodiments of the present invention fully verify the feasibility and superiority of the technical solution. The jujube seed protein gel prepared by fermentation with compound probiotics for 15 hours has excellent performance, with good water holding capacity, gel strength and rheological properties, and a uniform and stable microstructure, which meets the application requirements of the food industry.

[0057] This invention utilizes a compound probiotic fermentation method to prepare jujube seed protein gel. Through process optimization and parameter control, it achieves high-value utilization of jujube seed protein resources. The resulting gel product exhibits excellent performance, and the process is green and safe. Compared with existing technologies, it has the following significant advantages: A standardized extraction process for jujube seed protein isolate has been established: By optimizing parameters such as the ratio of defatting agent, pH of alkali extraction, and material-to-water ratio, the jujube seed protein isolate obtained has a purity of ≥85%, good dispersibility, and strong hydration capacity, providing a high-quality raw material basis for gel formation. This fills the raw material gap in the preparation of jujube seed protein gel and realizes the high-value utilization of jujube seed resources that are both medicinal and edible.

[0058] The selected compound probiotic starter has strong compatibility: This invention selects a starter containing Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus casei in equal proportions. This compound strain can grow and reproduce efficiently in the jujube seed protein system. The rate of acid production is highly matched with the aggregation rate of jujube seed protein, which can quickly induce the protein to form a complete three-dimensional network structure. There is no contamination by other bacteria during the fermentation process, and the gel formation efficiency is high.

[0059] The invention achieves synergistic regulation of gel performance: by defining the optimal fermentation time (15h), the invention solves the technical pain point that it is difficult to balance water holding capacity and gel strength in existing plant protein gels; the jujube seed protein gel obtained by fermentation for 15h has a water holding capacity of ≥70%, a gel strength of ≥18g, a storage modulus (G') of ≥120Pa, and a uniform porous three-dimensional network structure, which can effectively retain water and nutrients, and there is no dehydration shrinkage phenomenon, resulting in stable product texture.

[0060] The process is green, safe, and simple to operate: This invention uses probiotic fermentation to prepare gels without the addition of any exogenous chemical reagents. The fermentation process is green and safe, and the resulting product has excellent edibility. The process steps are simple, no high-end equipment is required, and all parameters are easy to control. It can achieve industrial-scale production with low production costs, making it suitable for large-scale application in food enterprises.

[0061] The product combines nutritional and functional benefits: the resulting jujube seed protein gel not only retains the nutritional characteristics and physiological activities of jujube seed protein (being both food and medicine), but also incorporates prebiotic properties through probiotic fermentation, resulting in a probiotic live bacteria count ≥1.0×10⁻⁶. 9 With a CFU / g content, the product combines gelling and prebiotic functions. It can be directly developed into plant-based yogurt, functional meal replacements, and other products. It can also be used as a gelling agent and thickener in various foods, making it suitable for a wide range of applications.

[0062] The gel formation mechanism is clear, providing a theoretical basis for process optimization: The formation of jujube seed protein gel in this invention is achieved through the synergistic effect of intermolecular covalent cross-linking and non-covalent interactions induced by probiotic acid production. The acid production by probiotics lowers the pH of the system as the initial driving force, the covalent cross-linking formed by disulfide bonds provides high strength to the gel, and the hydrophobic interactions and hydrogen bonds provide stability to the gel network. Moreover, there is no protein hydrolysis during fermentation, and the gel structure is stable, providing a solid theoretical foundation for subsequent process optimization and product upgrading.

[0063] Any aspects of this invention not described in detail are well-known to those skilled in the art.

[0064] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for producing jujube seed protein gel, characterized in that, The product is made from jujube kernels through protein separation, precursor solution preparation, probiotic fermentation, and post-processing into a gel. The specific steps include: Step 1: Preparation of Jujube Seed Protein Isolate (JPI): Jujube seed is ground and defatted. Water is added to the defatted meal to make a slurry and the protein is extracted with alkali. After acid precipitation, the protein precipitate is collected, reconstituted and freeze-dried to obtain jujube seed protein isolate. Step 2: Preparation of JPI-based gel precursor solution: Mix jujube seed protein isolate, sucrose and distilled water, adjust the pH to 7.0, refrigerate and then sonicate, then sterilize at high temperature and cool to obtain gel precursor solution; Step 3: Probiotic fermentation: Inoculate the compound probiotic fermentation agent into the gel precursor solution and ferment at a constant temperature of 42℃ for 10~20h to obtain the fermentation product; Step 4: Post-processing to form gel: The fermentation product is placed at 4℃ for 24 hours to complete gel solidification and obtain jujube seed protein gel; The compound probiotic starter is composed of Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei.

2. The production method according to claim 1, characterized in that, The defatting process described in step 1 involves soaking and grinding jujube seed powder in a mixture of hexane and ethanol. The volume ratio of hexane to ethanol is 10:1, and the material-to-liquid ratio is 1:5 to 1:

10. The defatting process is carried out at room temperature for 12 to 24 hours, and the defatted meal is obtained after filtration.

3. The production method according to claim 1, characterized in that, The specific operation of alkaline protein extraction in step 1 is as follows: add distilled water to defatted meal, with a material-to-water ratio of 1:12 (by weight), adjust the pH of the system to 8.0 with 1M NaOH, stir and extract at 600 rpm for 2 hours, centrifuge at 6000×g for 30 minutes at 25℃, and collect the supernatant; the specific operation of acid precipitation is as follows: adjust the pH of the supernatant to 4.5 with 1M HCl, centrifuge at 6000×g for 30 minutes at 25℃, and collect the protein precipitate.

4. The production method according to claim 1, characterized in that, In step 2, the mass-to-volume ratio of jujube seed protein isolate, sucrose, and distilled water is 5g:5g:90mL; the refrigeration is performed by standing at 4℃ for 24 hours; the ultrasonic treatment uses a titanium alloy probe with a diameter of 0.636cm, an ultrasonic frequency of 20kHz, and an ultrasonic time of 20min; the high-temperature sterilization is performed by constant temperature sterilization at 90℃ for 15min, followed by cooling to 25℃.

5. The production method according to claim 1, characterized in that, The inoculation amount of the compound probiotic fermentation agent in step 3 is 0.1g / 100mL of gel precursor liquid; the constant temperature fermentation time is 15h, during which the system naturally produces acid and the pH drops to 4.0~4.

5.

6. The production method according to claim 1, characterized in that, After the gel solidification described in step 4 is completed, the jujube seed protein gel is an opaque solid gel with no dehydration shrinkage, water holding capacity ≥70%, gel strength ≥15g, and storage modulus (G') ≥100Pa.

7. The production method according to any one of claims 1 to 6, characterized in that, The reconstitution in step 1 involves adding the protein precipitate to distilled water and stirring until it is evenly dispersed. The freeze-drying is performed using a vacuum freeze dryer at -40°C and a vacuum of 10 Pa for 24 hours to obtain powdered jujube seed protein isolate with a protein purity ≥85%.

8. A jujube seed protein gel prepared by the production method according to any one of claims 1 to 7, characterized in that, The gel is a plant-based gel derived from medicinal and edible sources. Its microstructure is a uniform and porous three-dimensional network structure with a particle size distribution concentrated in the range of 1 to 10 μm. It exhibits shear-thinning rheological properties, a relative recovery rate of ≥80%, and no protein hydrolysis. The gel formation is achieved through the synergistic effect of intermolecular covalent cross-linking and non-covalent interactions induced by probiotic acid production.

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