Method for reducing allergenicity of tenebrio molitor protein by ultrasonic and enzymatic hydrolysis

Abstract

The application discloses a method for reducing the allergenicity of tenebrio molitor by ultrasonic and enzymatic hydrolysis, and relates to the technical field of food processing and food safety. First, the tenebrio molitor protein is extracted by an alkali solution method, and then the tenebrio molitor protein solution is treated by ultrasonic and enzymatic hydrolysis to change the conformational epitope and linear epitope of the tenebrio molitor protein, and finally, the treated tenebrio molitor protein solution is freeze-dried to obtain tenebrio molitor protein powder with low allergenicity. The ultrasonic cavitation effect and the specific hydrolysis of enzymatic hydrolysis are used to produce a synergistic effect, and under the combined treatment of 300 W ultrasonic power and alkaline protease, the allergenicity reduction rate can reach more than 70%, which is significantly better than single processing mode, and provides technical support for the development of tenebrio molitor low allergenic food.

Description

Technical Field

[0001] This invention relates to the fields of food processing and food safety technology, and more specifically to a method for synergistically reducing the allergenicity of yellow mealworm protein through ultrasound and enzymatic hydrolysis. Background Technology

[0002] Yellow mealworms (Tenebrio molitor), as an emerging edible insect resource, have advantages such as high protein content, balanced essential amino acid composition, and sustainable farming, and are considered an important source to replace traditional animal protein.

[0003] However, the food safety risks posed by its endogenous allergenic proteins, especially cross-allergic reactions with crustaceans, severely limit its widespread application in the food industry. Despite the variety of current food processing methods, the desensitization effect on mealworm proteins is generally limited. Conventional heat treatment can only alter the higher-order structure of proteins, lacking sufficient ability to destroy linear epitopes that maintain allergenicity; it may even lead to excessive denaturation, exposing new antigenic determinants and thus increasing allergenicity. While physical processing (such as ultra-high pressure and ultrasound) and enzymatic hydrolysis each have their advantages, a single technique often fails to achieve the desired thorough desensitization effect.

[0004] Therefore, there is an urgent need to develop a method for reducing the allergenicity of mealworm protein that can significantly reduce its allergenicity while maintaining its nutritional value, and that is simple and controllable in terms of process. Summary of the Invention

[0005] In view of this, the present invention provides a highly efficient desensitization method based on ultrasound combined with enzymatic hydrolysis to solve the problem of poor desensitization effect of existing single processing techniques. The present invention uses an alkali dissolution method to extract mealworm protein, and then subjectes the mealworm protein solution to combined ultrasound and enzymatic hydrolysis to change the conformational and linear epitopes of the mealworm protein. Finally, the treated mealworm protein solution is freeze-dried to obtain low-allergenic mealworm protein powder.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for synergistically reducing the sensitizing effect of yellow mealworm protein through ultrasound and enzymatic hydrolysis includes the following steps: (1) Protein extraction: The freeze-dried mealworms were crushed and defatted, and then crude protein was extracted from the mealworms by alkali dissolution method; (2) Combined desensitization treatment: Dissolve yellow mealworm protein in ultrapure water to prepare yellow mealworm protein aqueous solution, sonicate the yellow mealworm protein aqueous solution, and then add alkaline protease to carry out enzymatic hydrolysis reaction; (3) Enzyme inactivation and recovery: After the reaction is completed, the enzyme is inactivated and then freeze-dried to obtain low-allergenic yellow mealworm protein powder.

[0008] Preferably, in step (1), the pulverization is performed by grinding freeze-dried mealworms into powder using a traditional Chinese medicine pulverizer; The defatting process involves mixing mealworm powder with petroleum ether at a ratio of 1:6, magnetically stirring at 4°C, changing the petroleum ether every 3 hours, extracting 3 times, placing the extract in a fume hood and letting it stand overnight until the petroleum ether has completely evaporated, and then obtaining defatted mealworm powder for later use.

[0009] Preferably, in step (1), the alkaline extraction method involves mixing defatted mealworm powder with water at a mass ratio of 1:15, extracting at a pH of 9, at an extraction temperature of 37°C, and for an extraction time of 2 hours.

[0010] Preferably, the concentration of the mealworm protein aqueous solution in step (2) is 10 mg / mL.

[0011] Preferably, the ultrasonic treatment in step (2) is carried out in an ice bath with an ultrasonic intensity of 300W. Intermittent ultrasonic treatment is used, with each ultrasonic treatment lasting 2 seconds and followed by a 2-second pause, for a total of 10 minutes.

[0012] Preferably, the temperature of the enzymatic hydrolysis reaction in step (2) is 50°C, the time is 3 hours, and the amount of enzyme added is 6000 U / g.

[0013] Preferably, the enzyme inactivation in step (3) is achieved by boiling in boiling water for 10 minutes.

[0014] Preferably, the freeze-drying in step (3) involves pre-freezing at -20°C and then freeze-drying at -40~-50°C until dry.

[0015] As can be seen from the above technical solution, compared with the prior art, the present invention has the following technical effects: 1. The ultrasonic combined enzymatic hydrolysis technology provided by this invention significantly improves the desensitization efficiency and increases the reduction rate to 75.65% compared with single cooking (maximum reduction rate 45.24%), microwave (maximum reduction rate 31.16%), ultra-high pressure (maximum reduction rate 53.97%) or single enzymatic hydrolysis (maximum reduction rate 61.64%).

[0016] 2. This invention utilizes the "cavitation effect" of ultrasound to disrupt protein conformational epitopes, combined with the deep hydrolysis of linear peptide epitopes by alkaline proteases, to achieve a synergistic effect. It is a highly efficient and controllable food deep processing technology. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The images show the SDS-PAGE electrophoresis (left) and Western blot immunoblotting (right) of the mealworm protein in Example 1 of this invention.

[0019] Figure 2 This is a comparison chart showing the effects of different single treatments (cooking, microwave, ultra-high pressure, ultrasound, and enzymatic hydrolysis) on sensitization in Example 2 of the present invention.

[0020] Figure 3 This is a graph showing the effect of ultrasonic combined with enzymatic hydrolysis treatment on the reduction rate of protein sensitization in yellow mealworms in Example 3 of the present invention.

[0021] Figure 4 The Western Blot analysis comparison diagram shows the effect of ultrasound combined with enzymatic hydrolysis on the reduction of sensitization of yellow mealworm protein in Example 3 of the present invention. Bands 1-4 represent untreated protein, ultrasound treatment alone, enzymatic hydrolysis treatment alone, and ultrasound and enzymatic hydrolysis combined treatment, respectively. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0023] Example 1: Extraction of protein from yellow mealworms (1) Crushing and defatting of mealworms Weigh an appropriate amount of freeze-dried mealworms, grind them into powder, and place 20 g of the powder in a beaker. Measure 120 mL of petroleum ether and add it to the beaker. Seal the beaker and magnetically stir at 4°C. Replace the petroleum ether every 3 hours, and repeat the extraction three times until the supernatant is clear, indicating complete defatting. Place the extract in a fume hood and let it stand overnight until the petroleum ether has completely evaporated, obtaining defatted mealworm powder for later use.

[0024] (2) Extraction of protein from yellow mealworms The protein from mealworms was extracted using an alkaline dissolution method. Defatted mealworm powder was dispersed in water (37℃) at a ratio of 1:15 (solid-to-liquid ratio). The pH was adjusted to 9.0 using 2M NaOH and maintained at 37℃ for 120 min. The slurry was allowed to stand at room temperature for 15 min, then centrifuged at 8000 r / min for 30 min below 4℃, and the supernatant was collected. The above steps were repeated, and the supernatants from both extractions were combined.

[0025] The obtained mealworm protein was subjected to SDS-PAGE and WB, and the results are as follows: Figure 1 As shown, the protein band with a molecular weight of approximately 35 kDa exhibits significant immunoreactivity, and this protein has been preliminarily identified as a potential allergen of mealworm protein.

[0026] Example 2: Effects of different single processing methods on sensitization (1) Cooking treatment of mealworm protein A 10 mg / mL solution of mealworm protein was prepared and placed in a 50 mL centrifuge tube, followed by boiling treatment. Treatment times were 10 min, 15 min, 20 min, 25 min, and 30 min. The treated samples were immediately placed in an ice bath to terminate the reaction. An untreated protein solution was used as a control to investigate the effect of different boiling times on the sensitization of mealworm protein.

[0027] (2) Ultra-high pressure treatment of mealworm protein A 10 mg / mL solution of mealworm protein was prepared, and an appropriate amount of the solution was dispensed into specially designed packaging bags and vacuum-packed. The bags were then placed in an ultra-high pressure chamber for the experiment. Different pressures (100, 200, 300, 400, and 500 MPa) were applied sequentially to treat the mealworm protein for 20 minutes, with an untreated protein solution serving as a control. The effect of different ultra-high pressure intensities on the sensitization of mealworm protein was investigated.

[0028] (3) Ultrasonic treatment of mealworm protein A 10 mg / mL solution of mealworm protein was prepared and added to a 50 mL centrifuge tube for the experiment. The protein was treated with ultrasonic power of 100, 200, 300, 400, and 500 W for 10 min sequentially. The ultrasonic probe diameter was 6 mm, the frequency was 21-22.5 kHz, and the pulse duration was 2 s on and 2 s off. An untreated protein solution was used as a control to investigate the effect of different ultrasonic powers on the sensitization of mealworm protein.

[0029] (4) Microwave treatment of mealworm protein A 10 mg / mL solution of mealworm protein was prepared in a 50 mL beaker and then microwaved. Treatment times were 30 s, 60 s, 90 s, 120 s, and 150 s. The treated samples were immediately placed in an ice bath to terminate the reaction, and the microwave oven was cooled to room temperature after each treatment. An untreated protein solution was used as a control to investigate the effect of different microwave times on the sensitization of mealworm protein.

[0030] (5) Enzymatic hydrolysis of mealworm protein A 10 mg / mL solution of mealworm protein was prepared in a 50 mL beaker and then enzymatically hydrolyzed. Alkaline protease (ALP), neutral protease (NP), papain (PAP), flavor protease (FP), bromelain (BRO), and complex protease (CP) were used to hydrolyze the mealworm protein at their respective optimal temperatures and pH values, with an enzyme dosage of 6000 U / g for 3 hours. The effects of different enzyme types on the sensitization of mealworm protein were investigated.

[0031] Example 3: Highly efficient desensitization using ultrasound combined with enzymatic hydrolysis Based on the results of Example 2, a combined treatment with ultrasound and alkaline protease was selected. The mealworm protein solution was first treated with ultrasound, followed by the addition of alkaline protease for enzymatic hydrolysis.

[0032] The results are as follows Figure 3 As shown: the reduction rate of alkaline protease treatment alone was 58.92% (control group). When 200 W of ultrasound was introduced, there was no significant difference compared with enzymatic digestion alone. When the ultrasound power was increased to 300 W, the sensitization reduction rate of combined treatment with alkaline protease reached its peak at 75.65%. When the ultrasound power exceeded 300 W, the sensitization reduction rate actually decreased, possibly due to overtreatment leading to protein aggregation or exposure of new epitopes.

[0033] Figure 4 Western blot results showed that sonication alone mainly disrupts the higher-order structure of proteins through physical action, causing denaturation or spatial conformational changes in conformational epitopes, which reduces sensitization but some linear epitopes are still preserved. Enzymatic digestion alone and combined treatment directly destroy the linear sequence of proteins by specifically cleaving peptide bonds, which greatly reduces the binding ability of antibodies that recognize linear epitopes.

[0034] Conclusion: This invention utilizes a combination of 300 W ultrasound and alkaline protease treatment, fully leveraging the cavitation effect of ultrasound to induce moderate conformational stretching of the protein, thereby significantly improving the substrate's accessibility to the alkaline protease and achieving efficient destruction of linear epitopes of allergens. Experimental data confirm that this treatment method exhibits a significant synergistic effect, with the allergenicity of mealworm protein decreasing significantly by 75.65%, far exceeding the effects of ultrasound treatment or enzymatic hydrolysis alone. This method is simple to operate, highly efficient, safe, and low-cost, suitable for industrial application, and provides new ideas and technical support for the development of high-quality, low-allergenic insect protein foods.

[0035] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0036] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for synergistically reducing the sensitizing effect of yellow mealworm protein through ultrasound and enzymatic hydrolysis, characterized in that, Includes the following steps: (1) Protein extraction: The freeze-dried mealworms were crushed and defatted, and then crude protein was extracted from the mealworms by alkali dissolution method; (2) Combined desensitization treatment: Dissolve yellow mealworm protein in ultrapure water to prepare yellow mealworm protein aqueous solution, sonicate the yellow mealworm protein aqueous solution, and then add alkaline protease to carry out enzymatic hydrolysis reaction; (3) Enzyme inactivation and recovery: After the reaction is completed, the enzyme is inactivated and then freeze-dried to obtain low-allergenic mealworm protein powder.

2. The method for synergistically reducing the sensitizing effect of mealworm protein by ultrasound and enzymatic hydrolysis according to claim 1, characterized in that, In step (1), the pulverization is to grind freeze-dried mealworms into powder using a traditional Chinese medicine pulverizer; The defatting process involves mixing mealworm powder with petroleum ether at a ratio of 1:6, magnetically stirring at 4°C, changing the petroleum ether every 3 hours, extracting 3 times, placing the extract in a fume hood and letting it stand overnight until the petroleum ether has completely evaporated, and then obtaining defatted mealworm powder for later use.

3. The method for synergistically reducing the sensitizing effect of yellow mealworm protein by ultrasound and enzymatic hydrolysis according to claim 2, characterized in that, In step (1), the alkaline extraction method involves mixing defatted mealworm powder with water at a mass ratio of 1:15, extracting at a pH of 9, at a temperature of 37°C, and for 2 hours.

4. The method for synergistically reducing the sensitizing effect of mealworm protein by ultrasound and enzymatic hydrolysis according to claim 3, characterized in that, The concentration of the yellow mealworm protein aqueous solution in step (2) is 10 mg / mL.

5. The method for synergistically reducing the sensitizing effect of yellow mealworm protein by ultrasound and enzymatic hydrolysis according to claim 1, characterized in that, The ultrasonic treatment described in step (2) is carried out in an ice bath with an ultrasonic intensity of 200~500W. Intermittent ultrasonic treatment is used, with each ultrasonic treatment lasting 2 seconds and followed by a 2-second pause, for a total of 10 minutes.

6. The method for synergistically reducing the sensitizing effect of mealworm protein by ultrasound and enzymatic hydrolysis according to claim 1, characterized in that, The enzymatic hydrolysis reaction in step (2) is carried out at a temperature of 50°C for 3 hours and with an enzyme dosage of 6000 U / g.

7. The method for synergistically reducing the sensitizing effect of yellow mealworm protein by ultrasound and enzymatic hydrolysis according to claim 1, characterized in that, The enzyme inactivation mentioned in step (3) is to boil in boiling water for 10 minutes.

8. The method for synergistically reducing the sensitizing effect of yellow mealworm protein by ultrasound and enzymatic hydrolysis according to claim 1, characterized in that, The freeze-drying described in step (3) involves first pre-freezing at -20°C, and then freeze-drying at -40~-50°C until dry.

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