Silkworm chrysalis protein gel and preparation method thereof

Abstract

The invention belongs to the technical field of protein gel, and particularly relates to silkworm chrysalis protein gel and a preparation method thereof.The preparation method comprises the following steps that degreased silkworm chrysalis, water, kappa-carrageenan and TG enzyme are mixed at the temperature of 20-50 DEG C, and the silkworm chrysalis protein gel is obtained after standing; according to the mass of the degreased silkworm chrysalis, 10-50U of TG enzyme with catalytic activity is correspondingly added into each gram of the degreased silkworm chrysalis; the mass volume ratio of the kappa-carrageenan to the water is (1.5-3g): 200mL. According to the invention, the synergistic effect of TG enzyme and kappa-carrageenan is utilized to construct a covalent cross-linked network and polysaccharide physical filling dual gel framework, so that silkworm pupa protein can be gelled at normal temperature, and the bottleneck of traditionally depending on heating is broken through.

Description

Technical Field

[0001] This invention belongs to the field of protein gel technology, specifically relating to a silkworm pupa protein gel and its preparation method. Background Technology

[0002] Insects are consumed by nearly 2 billion people worldwide, particularly in some Asian and African countries. Edible insects are rich in various nutrients and can serve as a good protein source to replace other animal protein sources, offering advantages such as higher feed conversion efficiency and sustainable development. Among them, silkworm pupae have attracted considerable attention as a new protein source and a substitute or supplement for traditional protein sources. The protein content of silkworm pupae accounts for more than 50% of their dry weight, higher than that of beef and eggs. The amino acid composition is well-balanced, containing all eight essential amino acids required by the human body. Furthermore, silkworm pupa protein possesses immunomodulatory, anti-tumor, and antioxidant bioactivities, and is considered a high-quality protein source with significant application potential.

[0003] However, silkworm pupa proteins exhibit very poor gelling properties, severely limiting their application in the food industry. Gel formation, a three-dimensional network structure formed through intermolecular interactions of proteins, can encapsulate water, flavor compounds, and lipids, thereby influencing the texture and mouthfeel of food. Furthermore, due to the protective effect of the gel network, protein gels have the potential to serve as delivery carriers for nutrients or bioactive compounds, applicable to functional food processing and biomedical fields. Therefore, improving the gelling properties of silkworm pupa proteins is of great significance in promoting the development and application of insect foods such as silkworm pupae. In one known method, heating silkworm pupa proteins above 85°C only produces flocculation and precipitation, failing to form a continuous gel. Currently, there is no method to achieve high water-holding capacity gelation of silkworm pupa proteins without heating during the gelation stage. Summary of the Invention

[0004] The purpose of this invention is to provide a silkworm pupa protein gel and its preparation method, which achieves the gelation of silkworm pupa protein under heat-free conditions through the synergistic effect of κ-carrageenan and TG enzyme (glutamine transferase).

[0005] This invention provides a method for preparing silkworm pupa protein gel, comprising the following steps: defatted silkworm pupae, water, κ-carrageenan and TG enzyme are mixed at 20~50℃, and the mixture is allowed to stand to obtain silkworm pupa protein gel; Based on the mass of the defatted silkworm pupae, each gram of the defatted silkworm pupae corresponds to the addition of TG enzyme with 10-50U catalytic activity; the mass-volume ratio of κ-carrageenan to water is 1.5-3g:200mL.

[0006] As a preferred embodiment, the mass ratio of κ-carrageenan to defatted silkworm pupae is 1.5~3:10.

[0007] As a preferred embodiment, the mixing method includes: first mixing defatted silkworm pupae and water to obtain a soluble protein solution; second mixing κ-carrageenan and water to obtain a κ-carrageenan solution; third mixing the soluble protein solution and the κ-carrageenan solution to obtain a mixture; and fourth mixing the mixture and TG enzyme to obtain a pregel solution.

[0008] As a preferred embodiment, the first mixing time is 20-40 minutes; the first mixing temperature is 20-30°C. The temperature of the second mixture is 40~50℃; The third mixing time is 20-40 minutes; the temperature of the third mixing is 40-50°C. The fourth mixing time is 60~180 min; the fourth mixing temperature is 30~40℃.

[0009] As a preferred embodiment, the settling temperature is 20~30℃; the settling time is 20~30min.

[0010] As a preferred embodiment, the method for preparing defatted silkworm pupae includes: quick-freezing, vacuum freeze-drying, and pulverizing the silkworm pupae to obtain silkworm pupae powder; mixing the silkworm pupae powder with an organic solvent and defatting to obtain defatted silkworm pupae.

[0011] As a preferred embodiment, the quick-freezing temperature is -200 to -190°C; the quick-freezing time is 10 to 20 minutes.

[0012] As a preferred embodiment, the temperature of the vacuum freeze-drying is ≤-40℃; the time of the vacuum freeze-drying is 55~65h; and the vacuum degree of the vacuum freeze-drying is 10~30Pa.

[0013] As a preferred embodiment, the mass-to-volume ratio of the silkworm pupa powder to the organic solvent is 1g:5~7mL; the organic solvent includes: petroleum ether and / or n-hexane; the degreasing temperature is 35~40℃; and the degreasing time is 4~6h.

[0014] The present invention provides a silkworm pupa protein gel prepared by the above preparation method, wherein the water holding capacity of the silkworm pupa protein gel is ≥99% and the gel strength of the silkworm pupa protein gel is ≥460g.

[0015] Beneficial Effects: This invention provides a method for preparing silkworm pupa protein gel, comprising the following steps: defatted silkworm pupae, water, κ-carrageenan, and TG enzyme are mixed at 20-50°C, and after standing, a silkworm pupa protein gel is obtained; based on the mass of the defatted silkworm pupae, TG enzyme with 10-50 U of catalytic activity is added per gram of defatted silkworm pupae; the mass-to-volume ratio of κ-carrageenan to water is 1.5-3 g: 200 mL. This invention utilizes the synergistic effect of TG enzyme and κ-carrageenan to construct a dual gel framework of covalent cross-linked network and polysaccharide physical filling, enabling silkworm pupa protein to achieve room temperature gelation, breaking through the bottleneck of traditional heating-dependent methods. TG enzyme recognizes glutamine and lysine in protein molecules, catalyzing the formation of very strong covalent bonds, namely ε-(γ-glutamyl)lysine isopeptide bonds, within or between protein molecules. Through this covalent cross-linking, the originally dispersed proteins are woven into a tight and stable three-dimensional network structure. κ-carrageenan is a hydrophilic polysaccharide that can interact with silkworm pupa proteins through hydrophobic interactions and fill into the ordered cross-linked protein network structure. The protein network formed by TG enzyme catalysis and the polysaccharide network formed by κ-carrageenan interpenetrate and intertwine to form an interpenetrating polymer network, making the entire gel structure denser, thicker, and more robust. This invention achieves room-temperature self-gelation of a complex system of silkworm pupa protein, κ-carrageenan, and TG enzyme, eliminating the need for high-temperature heating steps, reducing energy consumption and the loss of nutrients and functions (such as bioactive peptides) due to protein thermal denaturation, and promoting the high-value utilization of insect proteins.

[0016] This invention provides a silkworm pupa protein gel prepared by the above-described method, wherein the water-holding capacity of the silkworm pupa protein gel is ≥99% and the gel strength is ≥460g. The silkworm pupa protein gel of this invention exhibits good freeze-thaw stability and excellent resistance to freezing. It requires no heat treatment during gelation, reducing energy consumption, and also minimizes the loss of protein nutritional value and function caused by high-temperature treatment, thus possessing broad application prospects. Attached Figure Description

[0017] Figure 1 The images show the typical morphology of the silkworm pupa protein gels prepared in Example 1 and Comparative Examples 1-5. Figure 2 The image shows the freeze-thaw stability results of the silkworm pupa protein gels prepared in Example 1 and Comparative Examples 1-2. Figure 3 The images show the microstructure of the silkworm pupa protein gels prepared in Example 1 and Comparative Examples 1-2. Detailed Implementation

[0018] This invention provides a method for preparing silkworm pupa protein gel, comprising the following steps: defatted silkworm pupae, water, κ-carrageenan and TG enzyme are mixed at 20~50℃, and the mixture is allowed to stand to obtain silkworm pupa protein gel; Based on the mass of the defatted silkworm pupae, each gram of the defatted silkworm pupae corresponds to the addition of TG enzyme with 10-50U catalytic activity; the mass-volume ratio of κ-carrageenan to water is 1.5-3g:200mL.

[0019] Unless otherwise specified, the present invention does not have special requirements for the raw materials used, and commercially available products known to those skilled in the art can be used.

[0020] As an optional implementation, the method for preparing defatted silkworm pupae includes: quick-freezing, vacuum freeze-drying, and pulverizing the silkworm pupae to obtain silkworm pupae powder; mixing the silkworm pupae powder with an organic solvent and defatting to obtain defatted silkworm pupae. Preferably, the silkworm pupae are quick-frozen, vacuum freeze-dried, and pulverized to obtain silkworm pupae powder. The silkworm pupae used in this invention are preferably fresh silkworm pupae. The quick-freezing temperature can be any value within the range of -200 to -190℃, for example, -200, -198, -194, -196, -192, or -190℃; the quick-freezing time can be any value within the range of 10 to 20 minutes, for example, 10, 13, 15, 18, or 20 minutes. Conventional freezing forms ice crystals that puncture cell membranes and organelles, leading to protein denaturation and aggregation, and leakage of subcellular components. However, the quick-freezing method used in this invention allows the sample to drop from room temperature to the glass transition temperature in less than 2 seconds. This ultra-rapid freezing causes water molecules to form amorphous ice (glassy state), which eliminates the mechanical damage caused by ice crystals and facilitates subsequent gel formation. Liquid nitrogen is preferably used for quick freezing in this invention. Liquid nitrogen quick freezing can inhibit protease activity and prevent the degradation and oxidation of silkworm pupa proteins. The vacuum freeze-drying temperature of this invention can be any value within the range of ≤-40℃, for example, -60, -55, -50, -45, or -40℃; the vacuum freeze-drying time can be any value within the range of 55~65h, for example, 55, 58, 60, 62, or 65h; the vacuum degree of the vacuum freeze-drying can be any value within the range of 5~15Pa, for example, 5, 10, or 15Pa. The vacuum freeze-drying of this invention can maximize the preservation of the biological activity of silkworm pupae. Vitrification fixation of silkworm pupae tissue at a low temperature of ≤-40℃ can permanently inactivate proteases / lipases, blocking enzyme reactions and preventing oxidative denaturation of silkworm pupa proteins. Direct removal of ice crystals through vacuum sublimation avoids thermal denaturation of proteins, achieving dehydration without damaging the protein structure. Simultaneously, the in-situ sublimation of ice crystals forms honeycomb-like micropores, facilitating efficient diffusion of n-hexane to lipids and improving the rehydration rate of proteins. The pulverizing temperature described in this invention is room temperature, specifically any value within the range of 20~25℃, such as 20, 22, 23, or 25℃; the pulverizing speed is any value within the range of 20000~30000 r / min, such as 20000, 25000, or 30000 r / min. In a specific embodiment of this invention, a high-speed pulverizer is used for pulverization. The pulverizing time (excluding pause time) is any value within the range of 0.5~2 min, such as 0.5, 1, 1.5, or 2 min; pulverization can be paused every 0.5 min. The pauses during pulverization are to prevent the machine from overheating, thus avoiding nutrient loss and denaturation of the silkworm pupa protein. This invention does not have a specific limitation on the pause time, which can be any value within the range of 1~3 min, such as 1, 2, or 3 min. A high-speed pulverizer can be used for pulverization in this invention.The powder described in this invention can pass through a 30-50 mesh sieve, for example, a 30, 40, or 50 mesh sieve.

[0021] In a preferred embodiment, the present invention mixes the silkworm pupa powder obtained above with an organic solvent, defatts it, centrifuges, collects the precipitate, and air-dries it to obtain defatted silkworm pupae. The mass-to-volume ratio of the silkworm pupa powder to the organic solvent in the present invention can be any value within the range of 1g:5~7mL, for example, 1g:5mL, 1g:6mL, or 1g:7mL. The organic solvent in the present invention may include: petroleum ether and / or n-hexane. The preferred solvent in the present invention is n-hexane, which has a single component, good defatting effect, better retention of nutrients, lower risk of harmful impurities, and n-hexane can be recycled for defatting, reducing costs and further lowering the risk of harmful impurities. The defatting temperature in the present invention can be any value within the range of 35~40℃, for example, 35, 36, 38, or 40℃; the defatting time can be any value within the range of 4~6h, for example, 4, 5, or 6h. These defatting temperature and time conditions can maximize the retention of protein nutrients without damaging the protein. The defatting process in the present invention may further include centrifugation, discarding the supernatant, and air-drying to obtain defatted silkworm pupae. The centrifugation speed described in this invention can be any value within the range of 7000~9000×g, for example, 7000, 8000, or 9000×g; the centrifugation time can be any value within the range of 5~15 min, for example, 5, 10, or 15 min; and the centrifugation temperature can be any value within the range of 2~6℃, for example, 2, 4, or 6℃. These centrifugation conditions can satisfy the separation of fat and protein without damaging the nutritional components of the protein. The drying temperature described in this invention can be any value within the range of 25~30℃, for example, 25, 27, or 30℃; and the drying time can be any value within the range of 20~28 h, for example, 20, 24, or 28 h.

[0022] This invention involves mixing defatted silkworm pupae, water, κ-carrageenan, and TG enzyme at 20-50°C, followed by standing to obtain a silkworm pupa protein gel. The mixing temperature described in this invention is any value within the range of 20-50°C, for example, 20, 25, 30, 35, 40, 45, or 50°C. This invention achieves room-temperature gelation, omitting the high-temperature heating step, reducing energy consumption and the loss of nutrients and functions (such as bioactive peptides) due to protein denaturation, and promoting the high-value utilization of insect protein. Based on the mass of the defatted silkworm pupae, each gram of defatted silkworm pupae corresponds to the addition of TG enzyme with 10-50 U of catalytic activity. The catalytic activity of the TG enzyme described in this invention is any value within the range of 10-50 U, for example, 10, 15, 20, 24, 26, 30, 32, 35, 38, 40, 44, 48, or 50 U. The protein network catalyzed by TG enzyme and the polysaccharide network formed by κ-carrageenan interpenetrate and intertwine, forming an interpenetrating polymer network that makes the entire gel structure denser, thicker, and more robust. The mass-to-volume ratio of κ-carrageenan to water in this invention is any value within the range of 1.5~3g:200mL, for example, 1.5g:200mL, 2g:200mL, 2.5g:200mL, or 3g:200mL. κ-carrageenan is a hydrophilic polysaccharide that can interact with silkworm pupa proteins through hydrophobic interactions and other means, filling the ordered cross-linked protein network structure. This invention utilizes the synergistic effect of TG enzyme and κ-carrageenan to construct a dual gel framework of covalently cross-linked network and polysaccharide physical filling, enabling silkworm pupa proteins to gel at room temperature, overcoming the bottleneck of traditional heating-dependent methods.

[0023] In a preferred embodiment, the mixing method includes: first mixing defatted silkworm pupae and water to obtain a soluble protein solution; second mixing κ-carrageenan and water to obtain a κ-carrageenan solution; third mixing the soluble protein solution and the κ-carrageenan solution to obtain a mixed solution; and fourth mixing the mixed solution and TG enzyme to obtain a pre-gelled solution. Preferably, the defatted silkworm pupae and water are first mixed, centrifuged, and the supernatant is collected to obtain the soluble protein solution. Based on the mass of water mixed with the defatted silkworm pupae, the added mass of defatted silkworm pupae in the water can be any value within the range of 8% to 12% (w / v), for example, 8%, 10%, or 12%. The first mixing time can be any value within the range of 20 to 40 minutes, for example, 20, 25, 30, 35, or 40 minutes; the first mixing temperature can be any value within the range of 20 to 30°C, for example, 20, 22, 24, 26, 28, or 30°C. During the mixing process, sufficient dissolution can be achieved by stirring; the stirring can be done using magnetic stirring or manual stirring. The centrifugation speed described in this invention can be any value within the range of 8000~10000×g, for example, 8000, 9000 or 10000×g; the centrifugation time can be any value within the range of 5~15min, for example, 5, 10 or 15min. Centrifugation can remove insoluble proteins.

[0024] In this invention, κ-carrageenan and water are mixed in a second step to obtain a κ-carrageenan solution. The temperature of the water used in this invention can be any value within the range of 40~50℃, for example, 40, 45 or 50℃. The mass concentration of the κ-carrageenan solution used in this invention is any value within the range of 1.5%~3%, for example, 1.5%, 2%, 2.5% or 3%.

[0025] In a preferred embodiment, the present invention involves a third mixing of the obtained soluble protein solution with κ-carrageenan to obtain a mixed solution. The mass ratio of κ-carrageenan to defatted silkworm pupae in the present invention can be any value within the range of 1.5 to 3:10, for example, 1.5:10, 2:10, 2.5:10, or 3:10. The third mixing time in the present invention can be any value within the range of 20 to 40 minutes, for example, 20, 25, 30, 35, or 40 minutes; the third mixing temperature can be any value within the range of 40 to 50°C, for example, 40, 42, 45, 48, or 50°C. The mixing in the present invention can be achieved by stirring to ensure complete dissolution; the stirring can be done using magnetic stirring or manual stirring; stirring ensures more complete dissolution of the carrageenan.

[0026] In a preferred embodiment, the present invention involves a third mixing of the above-obtained mixture and TG enzyme to obtain a pregel solution. TG enzyme recognizes glutamine and lysine in protein molecules, catalyzing the formation of very strong covalent bonds, namely ε-(γ-glutamyl)lysine isopeptide bonds, within or between protein molecules. Through this covalent cross-linking, the originally dispersed proteins are woven into a tight, stable three-dimensional network structure. The third mixing time can be any value within the range of 60-180 min, for example, 60, 90, 120, 160, or 180 min; the third mixing temperature can be any value within the range of 30-40 °C, for example, 30, 32, 35, 37, or 40 °C. Based on the mass of the defatted silkworm pupae, each gram of defatted silkworm pupae corresponds to the addition of TG enzyme with 10-50 U of catalytic activity. The catalytic activity of the TG enzyme described in this invention is any value in the range of 10 to 50 U, for example, 10, 12, 18, 20, 24, 26, 30, 32, 35, 38, 40, 44, 48 or 50 U.

[0027] In a preferred embodiment, the pregel solution obtained above is allowed to stand to obtain silkworm pupa protein gel. The standing temperature described in this invention can be any value within the range of 20~30℃, for example, 20, 22, 23, 25, 27 or 30℃; the standing time can be any value within the range of 20~30 min, for example, 20, 22, 24, 26, 28 or 30 min. The silkworm pupa protein gel is formed by standing.

[0028] This invention provides a silkworm pupa protein gel prepared by the above-described method, wherein the water-holding capacity of the silkworm pupa protein gel is ≥99% and the gel strength is ≥460g. The silkworm pupa protein gel of this invention also exhibits good freeze-thaw stability and excellent anti-freezing properties, making it suitable for developing energy-saving room-temperature ready-to-eat protein gel products, as well as temperature-sensitive active ingredient carriers, such as room-temperature ready-to-eat protein jellies, artificial meat development, sustained-release carriers, or medical dressings.

[0029] To further illustrate the present invention, the following detailed description of a silkworm pupa protein gel and its preparation method provided by the present invention is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 A method for preparing a room-temperature gel of silkworm pupa protein based on κ-carrageenan and TG enzyme (κ-carrageenan accounts for 1% w / v of the total water). (1) Preparation of silkworm pupa protein solution: Fresh silkworm pupae were quick-frozen in liquid nitrogen at -196℃ for 15 min, and then vacuum-frozen (≤-40℃) for 60 h with a vacuum degree of 10 Pa. The freeze-dried silkworm pupae were pulverized at room temperature (23±2℃) using a high-speed pulverizer at a speed of 25000 r / min for a total pulverization time (excluding pause time) of 1 min, with a pause every 30 s for 2 min, to obtain powder; then the powder was passed through a 40-mesh sieve to obtain silkworm pupa powder. The silkworm pupa powder was dissolved in n-hexane at a material-to-liquid ratio of 1 g: 6 mL (w / v), stirred at 38℃ for 5 h for defatting treatment, and then centrifuged at 8000×g for 10 min. The precipitate was collected, dried, and defatted silkworm pupa powder was obtained.

[0031] (2) Take 1g of defatted silkworm pupa powder and add it to 10mL of pure water. Stir magnetically at room temperature (23±2℃) for 30min to fully dissolve it. Centrifuge at 10000×g for 5min and take the supernatant to obtain a soluble protein solution.

[0032] (3) Weigh 0.2g of κ-carrageenan powder and dissolve it completely in 10mL of hot water at 40℃ to obtain a 2% (w / v) κ-carrageenan solution.

[0033] (4) Add 10 mL of the κ-carrageenan solution obtained in step (3) to the soluble protein solution obtained in step (2), and stir magnetically for 30 min under a water bath at 40°C to obtain a mixture; the total volume of pure water in step (2) and hot water in step (3) used to prepare the mixture is 200 mL: 2 g of carrageenan.

[0034] (5) Add 0.2g of TG enzyme to the mixture in step (4). The enzyme activity is 120U / g, that is, the total amount of enzyme added is 24U / g defatted silkworm pupa powder. Stir evenly, and then perform enzymatic reaction by magnetic stirring in a water bath at 37℃ for 2h to obtain a pregel solution.

[0035] (6) Place the pregel solution from step (5) at 25°C and let it stand for 25 minutes to obtain silkworm pupa protein gel.

[0036] Example 2 A method for preparing a room-temperature gel of silkworm pupa protein based on κ-carrageenan and TG enzyme (total enzyme addition: 12 U / g defatted silkworm pupa powder). The process is carried out in accordance with Example 1, except that: (5) 0.1g of TG enzyme is added to the mixture in step (4), the enzyme activity of which is 120U / g, that is, the total amount of enzyme added is 12U / g defatted silkworm pupa powder.

[0037] Example 3 A method for preparing a room-temperature gel of silkworm pupa protein based on κ-carrageenan and TG enzyme (total enzyme addition: 48 U / g defatted silkworm pupa powder). The process was carried out in accordance with Example 1, except that: (5) 0.4g of TG enzyme was added to the mixture in step (4), the enzyme activity of which was 120U / g, that is, the total amount of enzyme added was 48U / g of defatted silkworm pupa powder.

[0038] Comparative Example 1: No κ-carrageenan added and no TG enzyme added (heated) The procedure is carried out in accordance with Example 1, except that: (4) 10 mL of pure water is added to the soluble protein solution obtained in step (2), i.e., κ-carrageenan solution is not added.

[0039] (5) Do not add TG enzyme to the mixture in step (4).

[0040] (6) Heat the pregel solution from step (5) in an 85°C water bath for 10 minutes to generate flocculants.

[0041] Comparative Example 2 without TG enzyme The procedure was carried out in accordance with Example 1, except that: (5) no TG enzyme was added to the mixture in step (4).

[0042] Comparative Example 3 without κ-carrageenan The procedure is carried out in accordance with Example 1, except that: (4) 10 mL of pure water is added to the soluble protein solution obtained in step (2), i.e., κ-carrageenan solution is not added.

[0043] Comparative Example: 4κ-carrageenan at 0.5% of total water (w / v) The procedure was carried out in accordance with Example 1, except that: (3) 0.1 g of κ-carrageenan powder was weighed and dissolved in 10 mL of hot water at 40 °C to obtain a 1% (w / v) κ-carrageenan solution.

[0044] (4) Add 10 mL of the κ-carrageenan solution obtained in step (3) to the soluble protein solution obtained in step (2), and stir magnetically for 30 min under a water bath at 40°C to obtain a mixture; the total volume of pure water in step (2) and hot water in step (3) used to prepare the mixture is 200 mL: 1 g of carrageenan.

[0045] Comparative Example: 5κ-carrageenan accounted for 0.25% of the total water content, w / v The procedure was carried out in accordance with Example 1, except that: (3) 0.05 g of κ-carrageenan powder was weighed and dissolved in 10 mL of hot water at 80 °C to obtain a 0.5% (w / v) κ-carrageenan solution.

[0046] (4) Add 10 mL of 0.5% (w / v) κ-carrageenan solution to the soluble protein solution obtained in step (3), and stir magnetically for 30 min under 40℃ water bath conditions to obtain a mixture; the total volume of pure water in step (2) and hot water in step (3) used to prepare the mixture is 200 mL: 0.5 g of carrageenan.

[0047] Comparative Example 6: No κ-carrageenan added and no TG enzyme added (unheated) The procedure is carried out in accordance with Example 1, except that: (4) 10 mL of pure water is added to the soluble protein solution obtained in step (2), i.e., κ-carrageenan solution is not added.

[0048] (5) Do not add TG enzyme to the mixture in step (4).

[0049] Comparative Example 7: Sodium alginate instead of κ-carrageenan The procedure was carried out in accordance with Example 1, except that in (3) and (4), κ-carrageenan was replaced with sodium alginate.

[0050] Comparative Example 8: Pectin instead of κ-carrageenan The procedure was carried out in accordance with Example 1, except that in (3) and (4), κ-carrageenan was replaced with pectin.

[0051] Comparative Example 9: Chitosan instead of κ-carrageenan The procedure was carried out in accordance with Example 1, except that κ-carrageenan was replaced with chitosan in (3) and (4).

[0052] Experimental Example 1 The appearance and morphology of the silkworm pupa protein products obtained in Examples 1-3 and Comparative Examples 1-9 were observed. The physical results for Examples 1 and Comparative Examples 1-6 are shown below. Figure 1 .

[0053] Depend on Figure 1It can be seen that when κ-carrageenan and TG enzyme are added simultaneously, and the final concentration of κ-carrageenan is 1% (w / v) (Example 1), silkworm pupa protein can form a continuous gel with a smooth and delicate surface, uniform morphology, and good texture. When κ-carrageenan and TG enzyme are added simultaneously, and the final concentration of κ-carrageenan is 0.5% (w / v) (Comparative Example 4), a gel can also be formed, and its appearance is not significantly different from that of Example 1. When κ-carrageenan and TG enzyme are added simultaneously, and the final concentration of κ-carrageenan is 0.25% (w / v) (Comparative Example 5), only a weak gel can be formed, but it is not well-formed and easily breaks, indicating that the concentration of κ-carrageenan added is too low to form a stable gel. When neither κ-carrageenan nor TG enzyme is added (Comparative Example 1), the silkworm pupa protein solution is in a fluid state and no gel is formed. After further heating in an 85°C water bath for 10 minutes, protein aggregation and precipitation occurred, and no continuous gel was formed. When only κ-carrageenan was added without TG enzyme (Comparative Example 2), the silkworm pupa protein formed a continuous gel, with little difference in appearance from Example 1. When only TG enzyme was added without κ-carrageenan (Comparative Example 3), the silkworm pupa protein solution could only form a sol with high viscosity and could not form a gel.

[0054] Observation of the remaining examples and comparative examples showed that the addition of 0.1 g of TG enzyme (Example 2) and 0.4 g of TG enzyme (Example 3) both resulted in gel formation. Without the addition of κ-carrageenan and TG enzyme, and without heating (Comparative Example 6), the gel remained in a fluid state and did not form any precipitate. When carrageenan was replaced with sodium alginate (Comparative Example 7), pectin (Comparative Example 8), and chitosan (Comparative Example 9), none of these methods resulted in the formation of a continuous and stable gel from the silkworm pupa protein; the gel was unstable and easily crumbled.

[0055] The above results collectively demonstrate that the silkworm pupa protein gels of Examples 1-3, utilizing the synergistic effect of κ-carrageenan and TG enzyme, can all form continuous gels at room temperature; while the silkworm pupa protein gels of Comparative Examples 1-9 either fail to form or have very poor gel properties, with some only forming partial flocculent precipitates even under high-temperature heating conditions, and others failing to form continuous gels.

[0056] Experimental Example 2 The performance of the silkworm pupa protein gels prepared in Example 1 and Comparative Examples 1-5 was compared and tested. The results are shown in Tables 1-3.

[0057] Specific methods for performance index testing: (1) The texture properties of the gel were determined using a texture analyzer (TA-XT Plus). Measurement mode: the pre-test rate was 1.5 mm / s; the mid-test rate was 1 mm / s; the post-test rate was 1 mm / s; the probe model was P / 0.5. The sample puncture depth was 90%, and a contact triggering force of 5 g was used. (2) Water retention test: 1 g of fresh gel sample was weighed, centrifuged at 8500 × g for 15 min, and the percentage of the gel mass after centrifugation and removal of the supernatant relative to the initial gel mass was the water retention of the gel. (3) Free amino content test. The content of free amino groups can reflect the degree of covalent cross-linking of proteins catalyzed by TG enzyme. The lower the content of free amino groups in the gel, the higher the degree of cross-linking. The modified O-phthalaldehyde (OPA) method was used for the determination. Dissolve 0.1 g of the lyophilized gel sample thoroughly in 1 mL of phosphate buffer (0.1 M, pH 8.0) containing 2% (w / v) SDS. Incubate at room temperature for 1 h, centrifuge at 4000 × g for 10 min, and take 50 μL of the supernatant. Add 2 mL of OPA reagent and vortex to mix. After reacting at room temperature for 2 min, measure the absorbance at 340 nm using a microplate reader. Construct a standard curve using L-lysine standards. The formula for the standard curve is: y = 0.2132x + 0.2804, R0 2 =0.9984.

[0058] Table 1. Gel texture properties detection

[0059] Note: The data in the table represent the mean ± standard error (n=3). Different superscript letters in the same row indicate significant differences. p <0.05).

[0060] Table 2. Gel water retention test

[0061] Note: The data in the table represent the mean ± standard error (n=3). Different superscript letters in the same row indicate significant differences. p <0.05).

[0062] Table 3. Detection of free amino content in gel.

[0063] Note: The data in the table represent the mean ± standard error (n=3). Different superscript letters in the same row indicate significant differences. p <0.05).

[0064] As shown in Table 1, regarding textural properties, the concentration of κ-carrageenan significantly affects gel strength when preparing silkworm pupa protein gels at room temperature using the synergistic effect of κ-carrageenan and TG enzyme. When the final carrageenan concentration was 1% (w / v) (Example 1), its gel strength (468.24 g) was significantly higher than that formed when the final carrageenan concentrations were 0.5% (w / v) (Comparative Example 4, 103.9 g) and 0.25% (w / v) (Comparative Example 5, 36.14 g), while there was no significant difference in gel strength compared to gels prepared using 1% (w / v) carrageenan alone (Comparative Example 1, 448.52 g).

[0065] Table 2 shows that, in terms of water retention, the room-temperature gel prepared by the synergistic action of κ-carrageenan and TG enzyme has a water retention of 99.18% (Example 1), which is significantly higher than the water retention of the gel prepared using carrageenan alone (Comparative Example 2, 88.79%). However, the gels without κ-carrageenan and without TG enzyme (Comparative Example 1) only showed protein precipitation rather than a continuous gel even after high-temperature treatment, with a very low water retention (46.12%). This means that more than half of the water was lost under the test conditions (such as centrifugation), indicating that the precipitate has a loose structure, lacks a continuous network, and is easily dehydrated under external forces. Water retention refers to the ability of a gel to retain water under external pressure (such as centrifugation, extrusion, and heating), essentially reflecting the density, degree of cross-linking, and binding ability of the gel network to water molecules. Products with good water retention can avoid water separation and deterioration in taste, while poor water retention can lead to water separation, a dry and hard texture, and even stratification during shelf life (such as supernatant separation). Meanwhile, in food processing (such as steaming and refrigeration), gels with good water retention reduce moisture loss and decrease raw material waste.

[0066] As shown in Table 3, regarding the free amino content, the free amino content of the gel in Example 1 of this invention was significantly reduced by 24% compared to Comparative Example 1. This indicates that the addition of TG enzyme caused covalent cross-linking between or within silkworm pupa protein molecules, consuming a large amount of lysine ε-amino groups, thus significantly reducing the free amino content. A highly dense network structure was constructed through extensive covalent cross-linking, which can lock in a large amount of water, thus exhibiting extremely high water retention (99.18%). In contrast, the free amino content of the group with only κ-carrageenan added (Comparative Example 2) was not significantly different from that of Comparative Example 1, indicating that the interaction between κ-carrageenan and silkworm pupa proteins is mainly through physical interaction. When κ-carrageenan is heated and then cooled, it forms a double helix structure and further aggregates to form a three-dimensional network, "capturing" or "bridging" protein aggregates within its network structure.

[0067] Experimental Example 3 Freeze-thaw stability tests were conducted on the silkworm pupa protein gels of Example 1 and Comparative Examples 1 and 2. The results are shown in Table 4 and 2. Figure 2 .

[0068] Freeze-thaw stability refers to the ability of a gel to maintain its structural integrity and physicochemical properties (such as hardness and water retention) without significant deterioration after freezing-thawing cycles. Gels with poor freeze-thaw stability will exhibit severe water separation and structural collapse, completely losing their commercial value. Testing freeze-thaw stability can help screen for freeze-resistant gel formulations. Freeze-thaw stability is considered a key parameter for evaluating the potential application of gels in frozen products. Its stability is quantitatively evaluated by measuring the amount of water separated from the gel after multiple freeze-thaw cycles (water separation rate). The lower the water separation rate, the stronger the water-locking ability of the gel network and the better the freeze-thaw stability. Specific method: Accurately weigh the freshly prepared gel and place it in a centrifuge tube (10 mL). Store it in a freezer at -20°C for 24 hours, then thaw it in a water bath at 30°C for 2 hours. This is considered one freeze-thaw cycle, and this process is repeated five times. After each freeze-thaw cycle, centrifuge the gel sample at 3000×g for 10 minutes to remove the separated water. The formula for calculating the water separation rate is as follows: In the formula, W0 The weight of the centrifuge tube. Wn The weights of the centrifuge tubes and the fresh gel are given. Wn' denoted as , where is the weight of the centrifuge tube and the weight of the remaining gel after centrifugation to remove moisture, and n is the number of freeze-thaw cycles.

[0069] Table 4. Freeze-thaw stability test of gels

[0070] Note: The data in the table represent the mean ± standard error (n=3). Different superscript letters in the same row indicate significant differences. p <0.05).

[0071] From Table 4 and Figure 2 It can be seen that Comparative Example 1 (pure silkworm pupa protein flocculent precipitate) exhibited an extremely high water separation rate, with a large amount of water separating after the first freeze-thaw cycle, followed by near-complete dehydration. This indicates that the flocculent precipitate structure of pure silkworm pupa protein is extremely unstable and completely unable to resist the damage caused by the formation and growth of ice crystals during freezing. Example 1 (silkworm pupa protein gel with simultaneous addition of κ-carrageenan and TG enzyme) maintained a consistently low water separation rate, indicating that this gel system possesses excellent freeze-thaw stability, and its network structure can effectively resist the damage caused by freeze-thaw cycles. Comparative Example 2 (silkworm pupa protein gel with only κ-carrageenan added) showed a significant improvement in water separation rate compared to Comparative Example 1, but the effect was significantly less than that of Example 1. This indicates that although the gel network formed by κ-carrageenan and protein has a certain water-locking capacity, its structure is damaged under freeze-thaw stress, leading to a decrease in water-holding capacity.

[0072] Test Example 4 The silkworm pupa protein gels prepared in Example 1 and Comparative Examples 1 and 2 were observed under a scanning electron microscope (SEM) to determine their microstructure. The results are shown in the figure. Figure 3 .

[0073] The sample was flash-frozen in liquid nitrogen at -196℃ for 15 min, then freeze-dried under vacuum at ≤-40℃ for 48 h at a vacuum degree of 10 Pa. The dried powder was then sputter-coated with gold and its morphology was observed under a scanning electron microscope (SU3500, Hitachi, Tokyo, Japan) with an accelerating voltage of 10 kV.

[0074] Depend on Figure 3 It was found that the SEM structures of pure silkworm pupa protein precipitates without TG enzyme and without κ-carrageenan (Comparative Example 1) showed disordered, loose, flocculent aggregation, exhibiting irregular clumps without a continuous network. The silkworm pupa protein gel with only κ-carrageenan (Comparative Example 2) formed a rough, porous, sponge-like network. Although the structure was continuous, the pores were large, the network walls were thin, and phase separation existed between κ-carrageenan and the silkworm pupa protein. The silkworm pupa protein gel with both κ-carrageenan and TG enzyme (Example 1) showed that the protein network and carrageenan network interpenetrated and intertwined, forming a highly uniformly distributed honeycomb pore and a solid, dense, continuous three-dimensional gel network structure. The pores were small, and the network walls were thick, demonstrating that the covalent cross-linking formed by TG enzyme between protein molecules acted as an anchoring point, fixing the protein conformation, while κ-carrageenan filled the orderly cross-linked protein molecules, effectively inhibiting macroscopic phase separation. The dense, fine pores can firmly lock in a large amount of water, and the thick, strong network can resist the mechanical stress generated when ice crystals form, preventing the structure from collapsing. Thus, it can still maintain its original shape and moisture after thawing, which is consistent with the high water retention and excellent freeze-thaw stability of the protein-polysaccharide composite gel.

[0075] In summary, although adding only carrageenan without enzymes can form a gel at room temperature, this process involves only physical processes, lacking chemical interactions. Therefore, the gel has larger pores, poorer water retention, and lower freeze-thaw stability. In contrast, the addition of both TG enzyme and carrageenan results in gels with better water retention and freeze-thaw stability. Testing water retention and freeze-thaw stability reveals the structural characteristics of the gel network at the molecular level (crosslinking, porosity, intermolecular interactions) and ensures the quality stability of the gel during processing, storage, consumption, and use.

[0076] Therefore, it can be seen that the synergistic treatment of adding κ-carrageenan and TG enzyme can enable silkworm pupa protein to gel at room temperature, breaking through the bottleneck of traditional heating-dependent processes and solving the pain point of traditional processes requiring high-temperature heating.

[0077] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing silkworm pupa protein gel, characterized in that, Includes the following steps: Defatted silkworm pupae, water, κ-carrageenan and TG enzyme were mixed at 20-50℃ and allowed to stand to obtain silkworm pupa protein gel. Based on the mass of the defatted silkworm pupae, each gram of the defatted silkworm pupae corresponds to the addition of TG enzyme with 10-50U catalytic activity; the mass-volume ratio of κ-carrageenan to water is 1.5-3g:200mL.

2. The preparation method according to claim 1, characterized in that, The mass ratio of κ-carrageenan to defatted silkworm pupae is 1.5~3:

10.

3. The preparation method according to claim 1, characterized in that, The mixing method includes: first mixing defatted silkworm pupae and water to obtain a soluble protein solution; second mixing κ-carrageenan and water to obtain a κ-carrageenan solution; third mixing the soluble protein solution and the κ-carrageenan solution to obtain a mixed solution; and fourth mixing the mixed solution and TG enzyme to obtain a pregel solution.

4. The preparation method according to claim 3, characterized in that, The first mixing time is 20-40 minutes; the first mixing temperature is 20-30°C. The temperature of the second mixture is 40~50℃; The third mixing time is 20-40 minutes; the temperature of the third mixing is 40-50°C. The fourth mixing time is 60~180 min; the fourth mixing temperature is 30~40℃.

5. The preparation method according to claim 1, characterized in that, The settling temperature is 20~30℃; the settling time is 20~30min.

6. The preparation method according to claim 1, characterized in that, The method for preparing defatted silkworm pupae includes: quick-freezing, vacuum freeze-drying and pulverizing the silkworm pupae to obtain silkworm pupae powder; mixing the silkworm pupae powder with an organic solvent and defatting to obtain defatted silkworm pupae.

7. The preparation method according to claim 6, characterized in that, The quick-freezing temperature is -200 to -190°C; the quick-freezing time is 10 to 20 minutes.

8. The preparation method according to claim 6, characterized in that, The vacuum freeze-drying temperature is ≤-40℃; the vacuum freeze-drying time is 55~65h; and the vacuum degree of the vacuum freeze-drying is 10~30Pa.

9. The preparation method according to claim 6, characterized in that, The mass-to-volume ratio of the silkworm pupa powder to the organic solvent is 1g:5~7mL; the organic solvent includes: petroleum ether and / or n-hexane; the degreasing temperature is 35~40℃; and the degreasing time is 4~6h.

10. The silkworm pupa protein gel prepared by the preparation method according to any one of claims 1 to 9, characterized in that, The water-holding capacity of the silkworm pupa protein gel is ≥99%; the gel strength of the silkworm pupa protein gel is ≥460g.

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