Hypoallergenic tenebrio molitor protein and preparation method thereof

Abstract

The invention provides a low-sensitization tenebrio molitor protein and a preparation method thereof, and the preparation method comprises the following steps: mixing degreased tenebrio molitor powder and alkaline electrolyzed water with the pH value of 9-12 according to a material-liquid ratio of 1: (10-20), adding ferric ammonium citrate and epigallocatechin gallate (EGCG), stirring and reacting at 55-65 DEG C for 30-90 minutes, centrifuging, adjusting the pH value to isoelectric point precipitation protein, filtering, washing, and drying to obtain the low-sensitization tenebrio molitor protein. And washing and drying to obtain the tenebrio molitor protein powder. According to the method, the special property of the alkaline electrolyzed water is utilized, the alkaline electrolyzed water, the ferric ammonium citrate and the EGCG generate a synergistic effect, particle size refinement, foamability, emulsibility and digestibility of the tenebrio molitor protein are synchronously achieved under the mild condition, and the content of the main allergen tropomyosin is effectively reduced. The method is green, safe, controllable in cost and suitable for the field of food processing.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a low-allergenic yellow mealworm protein and its preparation method. Background Technology

[0002] In the global food health and safety field, "alternative proteins" have become a core research hotspot by 2025, driving profound changes in the food industry towards green, efficient, and sustainable development. Among these, insect protein, as a promising emerging alternative protein resource, has attracted significant attention due to its outstanding nutritional value and environmental benefits. Yellow mealworm protein, with its high protein content of 50-60%, has become a key development target in this field. Currently, yellow mealworm protein is widely used in animal feed, but its high-value application in food processing faces two major bottlenecks: firstly, insufficient processability—compared to well-developed plant proteins, its functional property optimization technology still needs breakthroughs; secondly, significant allergenic risks—yellow mealworm protein contains allergens such as tropomyosin and arginine kinase, which can easily trigger allergic reactions, limiting its application in general foods and foods for specific populations.

[0003] Existing methods for improving the functional properties of proteins mainly include physical modification, enzymatic modification, and chemical modification, but all have significant drawbacks: physical modification can only achieve limited functional enhancement and cannot control allergenicity; enzymatic modification is not only costly but also has stringent requirements for reaction temperature, pH, and other conditions, which is not conducive to large-scale production; chemical modification often relies on toxic reagents or poses a safety risk of excessive additives, and may even exacerbate allergenicity due to abnormal changes in protein structure, which contradicts the concept of healthy food. Meanwhile, current insect protein extraction methods mostly use buffer solutions or sodium hydroxide solutions. Although protein extraction can be achieved, it cannot effectively improve functional properties or reduce the risk of allergenicity, making it difficult to reconcile the core requirements of "safe and non-toxic, functionally optimized, and low-allergenicity," thus becoming a key obstacle restricting the expansion of mealworm protein into the food industry.

[0004] Against this backdrop, developing a green, safe, effective, cost-controllable method for modifying mealworm protein that can simultaneously improve functional properties and reduce allergenicity is of great significance for promoting the high-value application of insect protein in the food processing field and improving the alternative protein industry system. This is also the core starting point of this invention. Summary of the Invention

[0005] Technical problem to be solved: In view of the above-mentioned technical problems, the purpose of this invention is to provide a low-allergenic mealworm protein and its preparation method. By utilizing the special properties of alkaline electrolyzed water, it produces a synergistic effect with ferric ammonium citrate and EGCG, and under mild conditions, it simultaneously achieves significant improvement in the particle size, foaming properties, emulsifying properties and digestibility of mealworm protein, and effectively reduces the content of the main allergen tropomyosin.

[0006] Technical solution: A method for preparing low-allergenic mealworm protein, comprising the following steps: (1) Mix defatted mealworm powder with alkaline electrolyzed water to obtain a mixture; (2) Add ferric ammonium citrate and epigallocatechin gallate to the mixture, stir at 55-65℃ for 30-90 min to obtain the reaction solution; (3) Centrifuge the reaction solution, collect the supernatant, adjust the pH of the supernatant to the isoelectric point of yellow mealworm protein 4.5, collect the precipitate, wash and dry it to obtain modified yellow mealworm protein powder.

[0007] Furthermore, the defatted mealworm powder is prepared as follows: mealworms are crushed and mixed with food-grade n-hexane at a mass-volume ratio of 1g:2-4mL, defatted for 4-8 hours, repeated 3 times, and dried to obtain defatted mealworm powder.

[0008] Furthermore, the alkaline electrolyzed water is prepared by electrolyzing a saturated sodium chloride solution at the cathode, and has a pH value of 9-12.

[0009] Furthermore, the ratio of defatted yellow mealworm powder to alkaline electrolyzed water is 1:(10-20).

[0010] Furthermore, the ratio of defatted yellow mealworm powder to alkaline electrolyzed water is 1:15.

[0011] Furthermore, the amount of ferric ammonium citrate added is 0.5%-3% of the mass of defatted mealworm powder, and the amount of epigallocatechin gallate (EGCG) added is 1%-3% of the mass of defatted mealworm powder.

[0012] Furthermore, the amount of ferric ammonium citrate added is 1% of the mass of defatted mealworm powder, and the amount of EGCG added is 1.5% of the mass of defatted mealworm powder.

[0013] Furthermore, in step (2), the stirring reaction is supplemented with physical treatment, which is ultrasonic treatment or high-speed shearing treatment.

[0014] Furthermore, the parameters of the ultrasonic treatment are: power 320 W, working cycle of 6 seconds of ultrasound and 3 seconds of interval; the rotation speed of the high-speed shearing treatment is 10,000 rpm.

[0015] The low-allergenicity mealworm protein prepared by the above preparation method. Beneficial effects

[0016] This invention uses alkaline electrolyzed water as the core extractant, which is obtained by electrolyzing saturated sodium chloride solution. It is safe and non-toxic, and significantly improves product safety compared to the toxic reagents or high-risk sodium hydroxide solutions used in traditional chemical modification. At the same time, the n-hexane used in the process is food grade, and ferric ammonium citrate and EGCG are commonly used additives in the food industry. There are no harmful residues throughout the process, which meets the requirements of food health and safety.

[0017] This invention effectively regulates the particle size distribution of mealworm protein (reducing aggregation) through the negative oxidation-reduction capacity of alkaline electrolyzed water and the synergistic effect of ferric ammonium citrate and EGCG, significantly improving the protein's foaming ability, foaming stability, emulsifying ability, and emulsification stability. In addition, the in vitro digestibility of the protein is improved, solving the problem of poor processability of natural mealworm protein and expanding its application scenarios in food processing (such as dairy products, meat products, baked goods, etc.).

[0018] The process conditions of this invention are mild and the operation steps are simple. It can be flexibly adapted to production needs by adjusting parameters such as the pH of the extract, the material-liquid ratio, and the amount of additives. Compared with the costly and demanding enzymatic modification and the limited effect of physical modification, this invention reduces production energy consumption and cost while ensuring the modification effect. Attached Figure Description

[0019] Figure 1 The average particle size diagram shows the mealworm protein prepared in different embodiments and comparative examples of the present invention. Figure 2 Digestive properties of mealworm proteins prepared in different embodiments and comparative examples of the present invention; Figure 3 The foaming ability and foaming stability of mealworm protein prepared in different embodiments and comparative examples of the present invention are shown, wherein A represents foaming ability and B represents foaming stability. Figure 4 The emulsifying ability and emulsifying stability of mealworm protein prepared in different embodiments and comparative examples of the present invention are shown, wherein A represents emulsifying ability and B represents emulsifying stability. Figure 5 The content of tropomyosin in the mealworm protein prepared in different embodiments and comparative examples of the present invention. Detailed Implementation

[0020] This invention proposes a low-allergenic mealworm protein and its preparation method. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific examples. It should be understood that the specific examples described herein are only for explaining the invention and are not intended to limit the invention.

[0021] Example 1 A method for preparing low-allergenic mealworm protein includes the following steps: (1) Take yellow mealworms, crush them, mix them with food-grade n-hexane at a mass-volume ratio of 1:3 (w / v), defatt them for 6 hours, repeat 3 times, and dry them to obtain defatted yellow mealworm powder. (2) Weigh 10g of defatted yellow mealworm powder and mix it with 150mL of pH10 alkaline electrolyzed water. Under the conditions of 60℃ water bath, add 1% ferric ammonium citrate and 1.5% epigallocatechin gallate in sequence and stir for 1h. (3) Centrifuge, collect the supernatant, adjust the pH of the supernatant to the isoelectric point of the mealworm protein (pI=4.5), collect the precipitate, and freeze-dry it in a -40℃ freeze-dryer for 24 hours to obtain low-allergenic mealworm protein powder.

[0022] Example 2 Except for the pH of the alkaline electrolyzed water, everything else is the same as in Example 1. In this example, alkaline electrolyzed water with pH 12 is used.

[0023] Example 3 Except for the introduction of physical assistance, the rest is the same as in Example 1. In this example, ultrasonic physical assistance is introduced during the 60°C water bath extraction process, with the parameters set to 320W, on for 6 seconds and off for 3 seconds.

[0024] Example 4 Except for the introduction of physical auxiliary operations, the rest is the same as in Example 1. In this example, a shearing physical auxiliary operation is introduced during the 60°C water bath extraction process, and the parameter is set to 10000 rpm.

[0025] Example 5 Except for the pH of the alkaline electrolyzed water, everything else is the same as in Example 3. In this example, alkaline electrolyzed water with pH 12 is used.

[0026] Example 6 Except for the pH of the alkaline electrolyzed water, everything else is the same as in Example 4. In this example, alkaline electrolyzed water with pH 12 is used.

[0027] Example 7 Except for the difference in the amount of ferric ammonium citrate and EGCG added, the rest is the same as in Example 1. In this example, 0.5% ferric ammonium citrate and 1.0% EGCG are added respectively.

[0028] Example 8 Except for the difference in the amount of ferric ammonium citrate and EGCG added, the rest is the same as in Example 1. In this example, 2.0% ferric ammonium citrate and 2.0% EGCG are added respectively.

[0029] Example 9 Except for the difference in the amount of ferric ammonium citrate and EGCG added, the rest is the same as in Example 1. In this example, 3.0% ferric ammonium citrate and 3.0% EGCG are added respectively.

[0030] Comparative Example 1 Except for the change in the extract, everything else is the same as in Example 1. In this example, the extract is a sodium hydroxide solution with a pH of 10.

[0031] Comparative Example 2 Except for the change in the extract, everything else is the same as in Example 1. In this example, the extract is a sodium hydroxide solution with a pH of 12.

[0032] Comparative Example 3 Except for the pH of the alkaline electrolyzed water, everything else is the same as in Example 1. In this example, alkaline electrolyzed water with pH 9 is used.

[0033] Comparative Example 4 Except for the absence of ferric ammonium citrate, it is identical to Example 1.

[0034] Comparative Example 5 Except for the absence of EGCG, it is identical to Example 1.

[0035] The proteins obtained above were subjected to performance characterization and sensitization test results: Particle size determination: Prepare a 1% protein solution and measure the particle size using a Zeta potentiometer. Take the average value of three measurements.

[0036] Determination of in vitro digestibility: The digestion process was divided into two distinct stages. The first stage simulated gastric digestion, with a 1% protein solution prepared at pH 2.0 and pepsin introduced at an enzyme-to-substrate ratio of 1:100 (w / w). The mixture was incubated in a constant temperature water bath at 37°C with a shaking speed of 100 rpm for 90 minutes. The digestion process was then terminated by adjusting the pH to 8.0 using 0.5 M NaOH. The second stage simulated intestinal digestion, with trypsin added at a ratio of 1:50 (w / w). The mixture was incubated in a constant temperature water bath at 37°C with a shaking speed of 100 rpm for 120 minutes. Protein content was determined using a commercial kit (BCA method, Abbkine), and the digestibility of mealworm protein was calculated according to equation (1).

[0037]

[0038] Where A represents the residual protein content after digestion, and B represents the total protein content of the sample before digestion.

[0039] Foaming ability and foam stability: 30 ml of protein suspension was homogenized at 15,000 rpm for 2 minutes at a concentration of 1% (w / v). Foaming ability (FC) was calculated as the percentage increase in suspension volume after homogenization compared to the original volume, while foam stability (FS) was the percentage of foam volume retained after 10 minutes compared to the initial volume.

[0040] Determination of emulsifying ability and emulsifying stability: 15 ml of 1% protein suspension was mixed with 5 ml of corn oil and homogenized at 10,000 rpm / min for 2 minutes. Immediately after homogenization, 50 μl of the emulsion was sampled from the bottom and mixed with 5 ml of 0.1% sodium dodecyl sulfate solution. The absorbance at 500 nm was measured by an ELISA reader, and the emulsification index (EAI) was calculated according to equation (2).

[0041]

[0042] Where A is the absorbance of the diluted emulsion; D is the dilution factor (100); C is the sample concentration (mg / mL); and φ is the oil volume fraction (0.25).

[0043] Emulsion stability (ESI) was measured again after 30 minutes and the ratio of the EAI to the initial value was taken.

[0044] Determination of tropomyosin content: The tropomyosin (Tm) content of the mealworm protein was determined using an insect tropomyosin (Tm) ELISA kit (Shanghai Zhuoeryou Biotechnology Co., Ltd.).

[0045] like Figure 1 As shown, Example 1 exhibits the smallest average particle size. Reducing protein particle size helps increase specific surface area, improves solubility and dispersibility in complex food systems, and facilitates the binding and action of digestive enzymes, laying a physical foundation for subsequent functional expression. The mild alkaline environment provided by alkaline electrolyzed water, combined with the neutralization of surface charge by ferric ammonium citrate and the antioxidant and charge-regulating effects of EGCG, effectively inhibits excessive aggregation between protein molecules. Simultaneously, the introduction of physical-assisted methods increases the protein particle size to some extent compared to Examples 1-2. This phenomenon may be due to the generation of reactive free radicals induced by the physical-assisted process, which promotes covalent cross-linking reactions between protein molecules, accelerates aggregate formation and growth, and ultimately reduces the particle size reduction efficiency of alkaline electrolyzed water. In contrast, the use of strongly alkaline sodium hydroxide in Comparative Examples 1-2 disrupts the protein charge balance, enhances hydrophobic interactions, and induces irreversible aggregation, resulting in a significant increase in particle size.

[0046] like Figure 2As shown, the in vitro digestibility of Examples (1, 3, 4) was significantly better than that of Comparative Examples 1-5, with Examples 3-4 exhibiting the best digestibility. High digestibility implies stronger protein nutrient absorption efficiency, making it better suited for infant formula, health foods for the elderly, and other applications. This difference in digestibility is likely attributed to significant structural modifications caused by the extraction method. Structural analysis revealed that alkaline electrolysis of water at pH 10 effectively reduced protein particle size while better maintaining structural order by reducing random coil content. The reduction in particle size may increase the enzyme-accessible surface area, while the more ordered conformation may promote the exposure and recognition of cleavage sites. These combined effects may synergistically enhance the hydrolytic efficiency of digestive enzymes (pepsin and trypsin). Furthermore, the addition of ferric ammonium citrate during extraction can open protein aggregation structures, eliminating digestive barriers. Simultaneously, EGCG prevents excessive protein aggregation without affecting digestive enzyme activity, further improving protein digestibility. In contrast, under strongly alkaline conditions at pH 12, significant protein denaturation and aggregation may have formed dense aggregates. These aggregates may physically hinder enzyme-substrate contact, thus affecting hydrolysis efficiency. In the comparative example, sodium hydroxide caused excessive protein denaturation and aggregation, forming a dense system that hindered digestive enzyme penetration and reduced digestibility.

[0047] like Figure 3 As shown, foaming capacity (FC) and foam stability (FS) are key functional indicators for proteins used in baking, beverages, and other fields. Examples (2, 5, and 6) are significantly superior to Comparative Examples 1-5 in both aspects. Among them, Example 2 exhibits the most outstanding foam stability. This may be because EGCG can co-adsorb with the protein at the interface, forming a "protein-polyphenol" composite film with higher mechanical strength and greater toughness, effectively delaying liquid film drainage and bubble rupture; ferric ammonium citrate may further strengthen the interfacial film structure by forming cross-linking bridges between protein molecules through metal ions. The two work synergistically to significantly enhance foam persistence while increasing foaming capacity. Comparative Examples 1-2 lack such interfacial reinforcement mechanisms and have damaged protein conformations, resulting in poor foaming performance.

[0048] like Figure 4As shown, emulsifying ability (EAI) and emulsifying stability (ESI) are crucial for the application of proteins in meat products, sauces, and dairy products. Example 4 exhibits significantly superior emulsifying properties compared to the comparative example. This is primarily because EGCG effectively reduces oil-water interfacial tension, promotes protein adsorption, and forms an anti-agglomeration composite film with steric hindrance and electrostatic repulsion on the oil droplet surface; the addition of ferric ammonium citrate enhances the mechanical strength and viscoelasticity of this interfacial film. The synergistic effect of both ensures the stability of the emulsion during preparation and storage. In the comparative example, the proteins, due to denaturation and aggregation, exhibit weak interfacial adsorption and film-forming abilities, making the emulsion system prone to flocculation and stratification. While the physical assistance process may promote initial contact between the protein and oil droplets and improve emulsification efficiency, it may also slightly perturb the integrity of the interfacial film, resulting in slightly lower long-term emulsifying stability compared to the optimal example without assistance.

[0049] Multiple studies have shown that the main allergen of mealworm protein is tropomyosin, whose highly conserved conformation and linear epitopes form the structural basis for triggering cross-sensitivity reactions. Therefore, changes in its extractable content are a direct biochemical indicator for assessing reduced sensitization. Figure 5 As shown, the levels in Examples 1-6 were significantly lower than those in Comparative Examples 1-5. This indicates that the method of the present invention can effectively reduce the risk of protein sensitization. The desensitization mechanism may involve: ① conformational disruption: the special physicochemical properties of alkaline electrolyzed water may selectively disrupt the native conformation of tropomyosin, altering its spatial epitopes; ② epitope masking / degradation: ferric ammonium citrate may bind to or catalyze the degradation of sensitizing epitopes, while EGCG can cover remaining potential epitopes through covalent or non-covalent binding. In the comparative examples, ordinary sodium hydroxide treatment does not possess this selective or synergistic desensitization ability, and may even expose more hidden epitopes due to protein aggregation, failing to reduce the risk of sensitization. The effective reduction in tropomyosin content provides an important safety basis for the application of mealworm protein in a wider range of consumer groups (including people with allergies).

[0050] Different ratios of ferric citrate and EGCG showed significant ratio-dependent differences in their modification effect on mealworm protein in a synergistic system formed with alkaline electrolyzed water. When the amount of ferric ammonium citrate added was controlled within the range of 0.5%-3% of the defatted mealworm powder mass and the amount of EGCG added was controlled within the range of 1%-3%, the combination of the two directly affected the particle size refinement, functional properties (foaming ability and stability, emulsifying ability and stability), in vitro digestibility, and efficiency in reducing tropomyosin content of mealworm protein. Among the selected examples, the combination of 1% ferric ammonium citrate and 1.5% EGCG performed optimally, achieving efficient refinement and uniform distribution of protein particle size, significantly enhancing the foaming-foam stabilization system and the emulsification-emulsification stabilization system of the protein, improving the efficiency of in vitro digestion and absorption of the protein, and minimizing the content of the main allergen, tropomyosin.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing low-allergenic mealworm protein, characterized in that, Includes the following steps: (1) Mix defatted mealworm powder with alkaline electrolyzed water to obtain a mixture; (2) Add ferric ammonium citrate and epigallocatechin gallate to the mixture, stir at 55-65℃ for 30-90 min to obtain the reaction solution; (3) Centrifuge the reaction solution, collect the supernatant, adjust the pH of the supernatant to the isoelectric point of yellow mealworm protein 4.5, collect the precipitate, wash and dry it to obtain modified yellow mealworm protein powder.

2. The method for preparing a low-allergenic mealworm protein according to claim 1, characterized in that, The defatted mealworm powder is prepared as follows: mealworms are crushed and mixed with food-grade n-hexane at a mass-volume ratio of 1g:2-4mL. The mixture is defatted for 4-8 hours, repeated 3 times, and then dried to obtain defatted mealworm powder.

3. The method for preparing a low-allergenic mealworm protein according to claim 1, characterized in that, The alkaline electrolyzed water is prepared by electrolyzing a saturated sodium chloride solution at the cathode, and has a pH value of 9-12.

4. The method for preparing a low-allergenic mealworm protein according to claim 1, characterized in that, The ratio of defatted yellow mealworm powder to alkaline electrolyzed water is 1:(10-20).

5. The method for preparing a low-allergenic mealworm protein according to claim 4, characterized in that, The ratio of defatted yellow mealworm powder to alkaline electrolyzed water is 1:

15.

6. The method for preparing a low-allergenic mealworm protein according to claim 1, characterized in that, The amount of ferric ammonium citrate added is 0.5%-3% of the mass of defatted mealworm powder, and the amount of epigallocatechin gallate added is 1%-3% of the mass of defatted mealworm powder.

7. The method for preparing a low-allergenic mealworm protein according to claim 6, characterized in that, The amount of ferric ammonium citrate added is 1% of the mass of defatted mealworm powder, and the amount of EGCG added is 1.5% of the mass of defatted mealworm powder.

8. The method for preparing a low-allergenic mealworm protein according to claim 1, characterized in that, In step (2), the stirring reaction is supplemented by physical treatment, which is ultrasonic treatment or high-speed shearing treatment.

9. The method for preparing a low-allergenic mealworm protein according to claim 8, characterized in that, The parameters for the ultrasonic treatment are: power 320 W, working cycle of 6 seconds of ultrasound and 3 seconds of interval; the rotation speed for the high-speed shearing treatment is 10,000 rpm.

10. The low-allergenicity mealworm protein prepared by the preparation method according to any one of claims 1-9.

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