Preparation method of hypoallergenic hermetia illucens protein

Abstract

The invention discloses a preparation method of hypoallergenic hermetia illucens protein, which specifically comprises the following steps: firstly adding teuvincenone H diterpene into a hermetia illucens protein solution, and then treating by ultrasonic waves with the power of 300-900W to reduce the binding capacity of the hermetia illucens protein and immunoglobulin E (IgE) so as to obtain the hypoallergenic hermetia illucens protein. The process flow is simple, the treatment time is short, the temperature is low, wide application of the method in the food industry is facilitated, and a brand new method is provided for producing safe and high-quality hypoallergenic hermetia illucens protein food.

Description

Technical Field

[0001] The invention belongs to the technical field of protein modification, and particularly relates to a method for preparing low-allergenic black soldier fly protein. Background Art

[0002] Black soldier flies are insects of the family Smilodon in the order Diptera. They feed on a wide range of food and play an important role in the recycling of resources. They can efficiently convert part of the garbage produced by humans into their own nutrients. Therefore, black soldier flies need further research, development and utilization. The protein in the body of black soldier flies accounts for a small half of its total content. The types and quantities of essential amino acids in the amino acids that make up the protein are complete and appropriate, so they can be developed and applied in animal feed or biodiesel. However, almost all food allergens are proteins, and food allergies are a public health issue of global concern. About 2% of adults and 6% to 8% of children suffer from food allergies. Therefore, how to reduce the allergenicity of proteins in black soldier flies is an urgent problem to be solved.

[0003] Ultrasound is a sound wave with a frequency higher than 20kHz. From the perspective of frequency, ultrasound can be divided into high-frequency and low-frequency. The former can be used as a non-destructive analytical technology to detect information on physical properties of food such as hardness and acidity. The latter is used in various stages of food processing, such as extraction, auxiliary extraction, auxiliary freezing, sterilization, enzyme inactivation and tenderization. Ultrasonic technology uses the alternating sound pressure cycle of positive and negative pressure generated by ultrasound, accompanied by extrusion. When propagating in a liquid medium, ultrasound will cause a series of cavitation effects, that is, liquid molecules diffuse under the rarefaction wave induced by ultrasound to form bubbles. The volume of the bubble gradually expands, and finally violently implodes or collapses, releasing a large amount of energy and forming turbulence. Ultrasonic waves also generate high-speed microcurrents, changing the properties of the medium, and the degree of cavitation is affected by factors such as the concentration, properties and viscosity of the medium. During the propagation process, the temperature of the medium will increase. This is because the energy of ultrasonic vibration is converted into heat energy, which is the thermal effect caused by ultrasound. When ultrasonic stirring takes effect, the conformation of the medium will be changed. The water in the liquid medium can produce highly active free radicals, which can react with other groups

[0004] Ultrasonic technology is easy to operate in experiments, the experimental materials are low-priced, a large number of protein samples can be processed simultaneously in a short period of time, there is little pollution to the environment, and it does not affect human health and food safety. It has certain feasibility in studying the changes in the conformation, physical and chemical properties and functional characteristics of myofibrillar proteins. However, other functions of ultrasonic technology still need to be developed. Summary of the invention

[0005] The first object of the present invention is to provide a method for preparing a hypoallergenic black soldier fly protein in view of the deficiencies in the prior art, comprising the following steps: The black soldier fly protein was treated with ultrasound in combination with teuvincenone H diterpene to reduce the binding ability of the black soldier fly protein with immunoglobulin E (IgE), and then hypoallergenic black soldier fly protein was obtained.

[0006] Furthermore, the structural formula of the teuvincenone H diterpene is as follows: .

[0007] Furthermore, the power of the ultrasound is 300-900W.

[0008] Furthermore, the ultrasonic treatment process was as follows: ultrasonic treatment for 10 s, then stopped for 10 s, and continued for 30 min.

[0009] Furthermore, the added amount of teuvincenone H diterpene was 10 mg / kg.

[0010] The second object of the present invention is to provide a hypoallergenic black soldier fly protein prepared by the above method.

[0011] The third object of the present invention is to provide a food or health product, wherein the food or health product contains the above-mentioned hypoallergenic black soldier fly protein.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention combines ultrasonic technology with teuvincenone H, effectively reduces the binding force between black soldier fly protein and IgE, and thus obtains a low-allergenic black soldier fly protein; the process flow is simple, the processing time is short and the temperature is low, which is conducive to the wide application of the present invention in the food industry, and provides a new method for producing safe, high-quality low-allergenic black soldier fly protein food. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The effect of different ultrasound powers on the binding capacity of IgE.

[0014] Figure 2 The effect of teuvincenone H on the binding ability of IgE.

[0015] Figure 3 The effect of ultrasound and teuvincenone H on the binding ability of IgE.

[0016] Figure 4 This is an intrinsic fluorescence analysis of the black soldier fly protein-teuvincenone H complex. DETAILED DESCRIPTION

[0017] 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.

[0018] In the embodiments of the present invention, if no specific conditions are specified, the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used in the present invention, if no manufacturer is specified, are all conventional products that can be purchased commercially.

[0019] The materials and instruments involved in the specific implementation of the present invention are as follows: Live sample of black soldier fly insect.

[0020] 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.

[0021] Example 1: Extraction of Tenebrio molitor Protein (TMP) The black soldier flies were cleaned in distilled water, frozen in an ultra-low temperature freezer at -80 °C, and immediately placed in a vacuum freeze dryer for 72 hours. The freeze-dried black soldier flies were ground into powder using a high-speed grinder. The obtained powder was screened through a 20-mesh sieve to obtain untreated black soldier fly powder. Lipids were removed by ethanol extraction. The black soldier fly powder was mixed with ethanol at a ratio of 1:5 (weight / volume) and extracted twice at 45 °C for 50 minutes each time. Then, the mixture was centrifuged at 5500×g for 15 minutes, the supernatant was removed, 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 °C, 180 mBar) overnight to obtain dry defatted powder.

[0022] The dried defatted black soldier fly powder was dispersed in a 2 M NaOH solution, and the mixing ratio of NaOH to defatted black soldier fly powder was 20:1 (v:m). The extraction process lasted for 100 minutes at 45 °C with stirring every 10 minutes. This extraction process was repeated twice. Then, the mixture was centrifuged at 4500 rpm for 15 minutes 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 value of the solution was adjusted to 4.8. When the isoelectric point of TMP was reached, the protein precipitated. Subsequently, the mixture was centrifuged at 4500 rpm at 4 °C for 10 minutes to obtain TMP precipitate. After washing with distilled water, it was centrifuged again at 4500 rpm at 4 °C for 10 minutes. The precipitate obtained was the black soldier fly myofibrillar protein, and the obtained black soldier fly myofibrillar protein was placed in a 4 °C refrigerator for later use.

[0023] Example 2: Ultrasonication The extracted black soldier fly myofibrillar protein was diluted to 10 mg / mL with 0.6 M KCl solution, mixed evenly, and then the myofibrillar protein dilution was divided into 4 portions, 25 mL each, and poured into 50 mL glass beakers. Then the glass beaker was placed in an ultrasonic instrument with different ultrasonic powers (300, 500, 700, 900 W) for treatment. The ultrasonic probe was kept 1 cm below the liquid surface during the ultrasonic process. During the treatment process, the real-time temperature of the sample was monitored by a high-precision temperature probe. The ultrasonic treatment was carried out in an ice water bath. The ultrasonic treatment time was 30 minutes, ultrasonic for 10 seconds, and stopped for 10 seconds.

[0024] Example 3: Teuvincenone H diterpene treatment Teuvincenone H diterpenoid was extracted 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.

[0025] A 0.01 mg / mL black soldier fly 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 black soldier fly 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.

[0026] Example 4: Ultrasound synergistic teuvincenone H diterpene treatment A black soldier fly myofibrillar protein solution with a mass concentration of 0.01 mg / mL 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 black soldier fly myofibrillar protein solution to make the final concentration 10 mg / kg protein. The solution was placed in a 25°C constant temperature water bath and stirred thoroughly to mix the reaction. 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 black soldier fly myofibrillar protein solution containing teuvincenone H. Ultrasonic treatment was performed for 30 min, 10 s for 10 s, and then stopped for 10 s to prevent overheating of myofibrillar protein, and a black soldier fly myofibrillar protein solution treated with ultrasound and diterpenes was obtained.

[0027] Test Example 1: 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 black soldier fly myofibril protein solution treated with ultrasound, teuvincenone H diterpene, and ultrasound-co-teuvincenone H diterpene treatment includes the following steps:

[0028] The binding capacity of IgE was analyzed by indirect competition. Microplate A was blocked after overnight coating with black soldier fly 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:

[0029] The results are as follows Figures 1 to 3 shown.

[0030] Depend on Figure 1 and Figure 2 It can be seen that ultrasound alone and teuvincenone H alone have no significant effect on the binding ability of black soldier fly protein to IgE. Figure 3 As shown, the combination of the two has a significant effect on the binding ability of black soldier fly protein to IgE.

[0031] Intrinsic fluorescence analysis of black soldier fly 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 black soldier fly protein participated in the formation process of the black soldier fly protein-teuvincenone H complex, resulting in a decrease in the quantum yield of fluorescence and inducing quenching.

[0032] The above content describes in detail the specific embodiments of the present invention. However, the present invention is not limited to these specific embodiments. Within the scope allowed by the claims of the present invention, technicians in the relevant fields can make various variations or adjustments. It should be appreciated that it is entirely possible for those skilled in the art to modify or replace the present application with equivalents, and all technical solutions and improvements thereof that do not deviate from the core and scope of the present application should be included in the protection of the claims of the present application.

Claims

1. A method for preparing a hypoallergenic black soldier fly protein, characterized in that: The method comprises the following steps: Teuvincenone H diterpene is added to the black soldier fly protein solution, followed by ultrasonic treatment, thereby obtaining low-allergenic black soldier fly protein.

2. The method for preparing the hypoallergenic black soldier fly protein according to claim 1, characterized in that: The structural formula of the teuvincenone H diterpene is as follows: 。 3. The method for preparing the hypoallergenic black soldier fly protein according to claim 1, characterized in that: The power of ultrasound is 300-900W.

4. The method for preparing the hypoallergenic black soldier fly protein according to claim 1 or 3, characterized in that: The power of ultrasound is 500-700W.

5. The method for preparing the hypoallergenic black soldier fly protein according to claim 1, characterized in that: The ultrasonic treatment time was 30 min, with 10 s on and 10 s off.

6. The method for preparing the hypoallergenic black soldier fly protein according to claim 1, characterized in that: The added amount of teuvincenoneH diterpene was 10 mg / kg.

7. A hypoallergenic black soldier fly protein, characterized in that: Prepared by the method according to any one of claims 1 to 6.

8. A food or health product, characterized in that: The food or health product contains the hypoallergenic black soldier fly protein according to claim 7.

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