Application of ultrasound and teuvincenone H diterpenoid treatment in reduction of protein allergy of tenebrio molitor

Abstract

The invention discloses an application of ultrasound and teuvincenone H diterpenoid treatment in reduction of tenebrio molitor protein allergy, the tenebrio molitor protein is treated by ultrasound and teuvincenone H diterpenoid, so that the binding capacity of the tenebrio molitor protein and immunoglobulin E (IgE) can be reduced, and the allergy of the tenebrio molitor protein is further reduced. The invention discloses other effects of teuvincenone H diterpene except for anti-tumor and anti-inflammatory effects for the first time, and the method has the advantages of simple process steps, short treatment time, low temperature, high efficiency, rapidness and environmental friendliness, is beneficial to wide application of related technologies in the field of food industry, and provides a brand new scheme for developing safe and high-quality insect protein food.

Description

Technical Field

[0001] The invention belongs to the technical field of biological sciences, and particularly relates to application of ultrasound-assisted teuvincenone H diterpene treatment in reducing the allergenicity of yellow mealworm protein. Background Art

[0002] At present, with the continuous increase of the world's population, people's demand for animal protein is also increasing day by day, but the existing animal protein resources cannot meet human needs, and a large amount of gases harmful to the environment will be produced during the animal breeding process. Therefore, humans are urgently seeking a new source of protein as a substitute. As a large number of diverse animal groups on the earth, insects are rich in protein and various amino acids, which can meet the daily protein needs of humans, and have less harm to the environment during the breeding process. Therefore, insect protein can be developed as a protein source for humans.

[0003] However, protein allergy is an issue that cannot be ignored in food consumption. Certain proteins can trigger abnormal reactions in the human immune system, leading to skin, digestive tract or respiratory allergic symptoms. For example, allergenic proteins in milk, peanuts and seafood are the main allergens for many consumers. Similarly, as a new source of protein, insect protein also needs to be given sufficient attention for its potential allergenicity, which is of great significance for ensuring food safety and promoting new protein resources. At present, protein modification through some physical, chemical or biological means can destroy the three-dimensional structure of the protein, thereby changing the allergenicity of the protein, such as heating, glycosylation, enzymatic hydrolysis, fermentation, etc.

[0004] Tenebrio molitor, also known as mealworms and high-protein worms, belongs to the class Insecta, order Coleoptera, and family Tenebrio. It has a high protein content (48% to 66%, dry basis) and is rich in a variety of essential amino acids. It is one of the good edible insect protein resources, and is therefore known as the third largest industrial insect after silkworms and bees, with broad application prospects. Tenebrio molitor has a unique flavor and good taste, and can be processed into protein drinks, refined protein powder and other forms of food. As the world's protein crisis becomes increasingly serious, mealworms, as a food protein resource to be developed, have great potential for development and application.

[0005] So far, most of the research on mealworm protein has focused on how to optimize the extraction process of mealworm and evaluate the effect of structural changes of mealworm protein obtained by different extraction methods on functional properties. There are few studies on the allergenicity of mealworm protein. However, studies have shown that some mealworm proteins have immune reactivity and can trigger specific allergic mechanisms. Therefore, in the food industry, how to reduce the allergenicity of mealworm protein is an urgent problem to be solved. Summary of the invention

[0006] The purpose of the present invention is to provide an application of ultrasound-assisted teuvincenone H diterpene treatment in reducing the allergenicity of yellow mealworm protein in view of the deficiencies of the prior art.

[0007] Furthermore, the structural formula of the teuvincenone H diterpene is as follows: .

[0008] Furthermore, after treatment with ultrasound and teuvincenone H diterpenoid, the binding ability of mealworm protein to immunoglobulin E (IgE) decreased.

[0009] Furthermore, the power of the ultrasound is 300-900W.

[0010] Furthermore, the ultrasonic treatment process is: ultrasonic treatment for 10 s followed by a 10 s break, and lasting for 30 to 45 min, more preferably for 30 min.

[0011] Furthermore, the added amount of teuvincenone H diterpene is 10-12 mg / kg, more preferably 10 mg / kg.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention is the first to use ultrasound technology and diterpenoid compounds to reduce the allergenicity of insect proteins, breaking the limitation of traditional technology that only relies on chemical modification or high-temperature heat treatment. At present, ultrasound technology is mostly used in extraction processes or particle dispersion. As a diterpenoid compound, teuvincenone H is mostly used to exert anti-tumor activity. The present invention combines ultrasound with teuvincenone H diterpenoid to significantly reduce the binding ability of mealworm protein to IgE.

[0013] The process steps of the present invention are simple, the processing time is short and the temperature is low, which is conducive to the wide application of related technologies in the field of food industry and provides a new solution for the development of safe and high-quality insect protein food. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The effect of different ultrasound powers on the binding capacity of IgE.

[0015] Figure 2 The effect of teuvincenone H on the binding ability of IgE.

[0016] Figure 3 The effect of ultrasound and teuvincenone H on the binding ability of IgE.

[0017] Figure 4Intrinsic fluorescence analysis of the mealworm protein-teuvincenone H complex. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific implementation methods. Obviously, the described implementation methods are only part of the implementation methods of the present invention, rather than all of the implementation methods.

[0019] Based on the technical solution and the given implementation mode provided by the present invention, all other technical solutions obtained by those skilled in the art without creative work are within the scope of protection of the present invention. If no specific conditions are specified in the implementation mode, the conventional conditions or the conditions recommended by the manufacturer shall be followed. If the manufacturer is not specified for the reagents or instruments used in the present invention, they are all conventional products that can be purchased commercially.

[0020] The materials and instruments involved in the specific implementation of the present invention are as follows: Live sample of mealworm insect.

[0021] Ultrasonicator (Scientz-IID, 1000 W NingBo Scientz Biotechnology Co. Ltd., China), ultra-low temperature refrigerator, vacuum freeze dryer, high-speed grinder, centrifuge, BUCHI rotary evaporator, vacuum drying oven, refrigerator.

[0022] The teuvincenone H diterpene is obtained by the following method: Zhejiang Daqing was dried and crushed, and then extracted with 75% ethanol at 75°C for 3 times. The extract was vacuum filtered and concentrated by rotary evaporator under reduced pressure to recover the solvent to obtain an extract. Part of the extract was dispersed in water to make it suspended, and extracted with an equal volume of petroleum ether for 3 times. The petroleum ether extract was concentrated by rotary evaporation under reduced pressure to obtain a petroleum ether phase extract. The aqueous phase after extraction was extracted with an equal volume of ethyl acetate for 3 times to obtain an ethyl acetate extract, which was concentrated by rotary evaporation under reduced pressure to obtain an ethyl acetate phase extract. Part of the extract of the ethyl acetate part was subjected to silica gel column chromatography, with petroleum ether, petroleum ether-ethyl acetate with a volume ratio of 20:1, 10:1, 5:1, 1:1, 1:3, and 1:5, and ethyl acetate eluted. The same components were combined by TLC detection to obtain 10 groups of different fractions (Fr1~Fr10). Fr4 was then chromatographed on a silica gel column (200-300 mesh, L 450 mm, ø 25 mm) and eluted with a gradient of petroleum ether-ethyl acetate (6:1-1:1). The fraction was further chromatographed on a silica gel column and Sephadex LH-20 column to obtain the compound teuvincenone H.

[0023] Example 1: Extraction of Tenebrio molitor Protein (TMP) Take the mealworms and wash them with distilled water, freeze them in a -80 ℃ ultra-low temperature refrigerator, and immediately put them into a vacuum freeze dryer for 72 hours. The freeze-dried mealworms were ground into powder using a high-speed grinder and passed through a 20-mesh sieve. The lipids of the mealworm powder were removed by ethanol extraction. The mealworm powder was mixed with ethanol at a ratio of 1:5 (m / V), extracted at 45 ℃, and extracted twice. Then, the mixture was placed in a centrifuge and centrifuged at a speed of 5500g for 15 min. The precipitate was retained and a small amount of ethanol in the precipitate was removed using a BUCHI rotary evaporator. The mixture was placed in a vacuum drying oven (30 ℃, 180 mBar) overnight to obtain dry defatted powder.

[0024] The dried defatted mealworm powder was mixed with 2 M NaOH solution at a ratio of 20:1 (V:m), extracted at 45 °C for 100 min, stirred every 10 min, and the extraction process was repeated twice. Then, the mixture was centrifuged at 4500 rpm for 15 min at 4 °C, the supernatant was collected, and the precipitate was extracted twice more. At room temperature, the extracted supernatant was dispersed in 2M HCl, and the pH of the solution was adjusted to 4.4. When the isoelectric point of TMP was reached, the protein precipitated. Subsequently, the mixture was centrifuged at 4500 rpm at 4 °C for 10 min to obtain TMP precipitate. After washing with distilled water, it was centrifuged again at 4500 rpm at 4 °C for 10 min. The precipitate obtained was the mealworm myofibrillar protein. The above-mentioned mealworm myofibrillar protein was dissolved in water, adjusted to pH 12, and placed in a -80 °C refrigerator after it was completely dissolved. The mealworm myofibrillar protein powder was obtained after freeze-drying with a freeze dryer, and placed in a 4 °C refrigerator for standby use.

[0025] Example 2: Ultrasonication The myofibrillar protein powder obtained by extraction was used to prepare a myofibrillar protein solution with a mass concentration of 0.01 mg / mL in phosphate buffer (0.01 M, pH 7.0), and the ultrasonic power of the ultrasonic instrument was set to 0, 300, 500, 700, and 900 W, respectively, and the probe of the ultrasonic instrument was immersed in the myofibrillar protein solution. The myofibrillar protein was ultrasonically treated for 30 min, with an ultrasonic treatment of 10 s and an interval of 10 s to prevent the myofibrillar protein from overheating, and an ultrasonically treated myofibrillar protein solution was obtained.

[0026] Example 3: Teuvincenone H diterpene treatment A 0.01 mg / mL Tenebrio molitor myofibrillar protein solution was prepared with phosphate buffer (0.01 M, pH 7.0), and a certain amount of teuvincenone H was weighed on a balance and dissolved in the Tenebrio molitor myofibrillar protein solution to make the final concentration 10 mg / kg protein. The mixture was placed in a 25°C constant temperature water bath and stirred thoroughly to mix the reaction.

[0027] Example 4: Ultrasound synergistic teuvincenone H diterpene treatment A 0.01 mg / mL yellow mealworm myofibrillar protein solution was prepared with phosphate buffer (0.01 M, pH 7.0). A certain amount of teuvincenone H was weighed on a balance and dissolved in the yellow mealworm myofibrillar protein solution to a final concentration of 10 mg / kg protein. The solution was placed in a 25°C constant temperature water bath and stirred thoroughly to react. The ultrasonic power of the ultrasonic instrument was set to 0, 300, 500, 700, and 900 W, and the probe of the ultrasonic instrument was immersed in the yellow mealworm myofibrillar protein solution containing teuvincenone H. Ultrasonic treatment was performed for 30 min, 10 s for 10 s, and an interval of 10 s to prevent overheating of the myofibrillar protein, and a yellow mealworm myofibrillar protein solution treated with ultrasound and diterpenes was obtained.

[0028] The present invention adopts a two-tailed TTEST test method to analyze the experimental data, and the experimental data are all from three independent repeated experiments, and the experimental results are expressed as mean ± standard deviation (SD). Determining the IgE binding capacity of the yellow mealworm myofibrillar protein solution treated with ultrasound, teuvincenone H diterpene, and ultrasound-co-teuvincenone H diterpene treatment includes the following steps:

[0029] The binding capacity of IgE was analyzed by indirect competition. Microplate A was blocked after overnight coating with mealworm protein (100 μL / well, 2.0 μg / mL). At the same time, microplate B was blocked with 3% gelatin, and then 60 μL of human serum (dilution ratio 1:20) and 60 μL of sample were added respectively. After incubation at 37°C for 1 hour, 100 μL of the reaction solution was transferred to plate A. After 1 hour of incubation and washing, 100 μL of biotin-labeled goat anti-human IgE (dilution ratio 1:5000) was added to plate A, and then incubated for another 1 hour and washed. Then, 100 μL of HRP-labeled streptavidin (dilution ratio 1:60) was added to each well, incubated at 37°C for 1 hour and then washed. After stopping the color development, the OD value was measured at 450 nm using an enzyme reader. The inhibition rate reflects the binding capacity of IgE, and the calculation formula is as follows:

[0030] The results are as follows Figures 1 to 3 shown.

[0031] Depend on Figure 1 It can be seen that different ultrasonic powers have little effect on the binding ability of mealworm protein and IgE, which is slightly reduced at 300-700W, and almost unchanged at 900W.

[0032] Depend on Figure 2 It can be seen that the addition of teuvincenone H diterpene alone has almost no effect on the binding ability of mealworm protein with IgE.

[0033] Depend on Figure 3 It can be seen that ultrasound synergistically affects teuvincenone H on the binding ability of mealworm protein and IgE, among which the most significant effect is when the ultrasonic power is 500-700W. When the ultrasonic power is 300W and 900W, the synergistic effect of teuvincenone H can also significantly reduce the binding ability of mealworm protein and IgE.

[0034] Intrinsic fluorescence analysis of the mealworm protein-teuvincenone H complex Figure 4 It can be seen that compared with the control group, the fluorescence intensity of the sample group treated with ultrasound and teuvincenone H was significantly reduced. This result may be due to the fact that the fluorescent groups such as Trp and Tyr in the mealworm protein participated in the formation process of the mealworm protein-teuvincenone H complex, which led to a decrease in the quantum yield of fluorescence and caused quenching.

[0035] In summary, the present invention can significantly reduce the allergenicity of mealworm protein by synergizing ultrasound technology with diterpenoid compounds.

[0036] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

[0037] It should be understood by those skilled in the art that the present application can still be modified or replaced by equivalents, and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present application should be included in the scope of the claims of the present application.

Claims

1. Application of ultrasound combined with teuvincenone H diterpenoid treatment in reducing the allergenicity of mealworm protein.

2. The use according to claim 1, characterized in that: The structural formula of the teuvincenone H diterpene is as follows: 。 3. The use according to claim 1, characterized in that: After ultrasound and teuvincenone H diterpenoid treatment, the binding ability of Tenebrio molitor protein to immunoglobulin E decreased.

4. The use according to claim 1, characterized in that: The power of ultrasound is 300-900W.

5. The use according to claim 4, characterized in that: The ultrasonic treatment process was as follows: ultrasonic treatment for 10 s followed by a 10 s break, lasting for 30-45 min.

6. The use according to claim 5, characterized in that: The ultrasonic treatment process was as follows: ultrasonic treatment for 10 s followed by a 10 s break for 30 min.

7. The use according to claim 1, characterized in that: The added amount of teuvincenone H diterpene is 10~12mg / kg.

8. The use according to claim 1, characterized in that: The amount of teuvincenone H diterpenoid added was 10 mg / kg.

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