Odor-free silkworm pupa protein compound as well as deodorization method and application thereof

Abstract

The deodorization method comprises the following steps: step 1, dissolving silkworm chrysalis protein by using a solvent, mixing the dissolved silkworm chrysalis protein with polyphenol, and stirring under an alkaline condition, so that the silkworm chrysalis protein and polyphenol are covalently bound; 2, carrying out heat treatment on the mixture treated in the step 1, and monitoring by using a solid-phase microextraction-gas chromatography-mass spectrometry method until 98% of odor volatile matters are released; and step 3, purifying protein from the mixture treated in the step 2, adjusting the pH value of the mixture to be neutral by using an acid solution, and drying to obtain odorless silkworm chrysalis protein-polyphenol compound powder.

Description

Technical Field

[0001] The invention belongs to the technical field of silkworm pupa protein processing, and in particular relates to an odorless silkworm pupa protein complex and a deodorizing method and application thereof. Background Art

[0002] Silkworm pupae are the only insect food on the list of new food resources under general food management. Dry silkworm pupae contain about 60% crude protein and 18 kinds of amino acids, of which 8 essential amino acids for the human body account for 42% of the total amino acids, which is in line with the ideal amino acid pattern recommended by FAO / WHO (European Food Research and Technology, 2021, 247(2): 343-352.). Therefore, silkworm pupa protein is an insect protein resource with great development prospects.

[0003] However, silkworm pupa protein carries a distinct odor, which greatly reduces consumer acceptance. Therefore, removing silkworm pupa protein is a necessary step in its food development. At present, there have been some studies on the deodorization of silkworm pupa protein. The use of sodium hypochlorite solution, activated carbon adsorption, and composite fiber adsorption of silkworm pupa odor only has a partial deodorization effect, and the odor of silkworm pupa is still retained (Food Science, 2005, 26 (9): 291-294). The strong binding ability between odorous volatiles and silkworm pupa protein makes it difficult for conventional physical means to effectively deodorize. Therefore, some scholars use strong oxidants such as ozone and hydrogen peroxide to deodorize silkworm pupa protein. However, these methods have a great impact on the color and structure of silkworm pupa protein, limiting its subsequent processing (China Food Additives, 2012, 3: 133-136). In addition, microbial fermentation is also used for silkworm pupa deodorization. Silkworm pupa powder is fermented by inoculating tropical Candida and washed with ethanol to obtain odorless silkworm pupa powder. However, microbial fermentation significantly changes the amino acid composition of silkworm pupa protein, destroying its nutritional composition (CN101926406A). Therefore, a milder silkworm pupa protein deodorization process has great application prospects.

[0004] Odor compounds can bind to proteins through ionic bonds, hydrogen bonds, hydrophobic interactions, and covalent bonds, thereby changing their sensory evaluation. Aldehyde volatiles are considered to be the main odor volatile components in food and can present unpleasant odor characteristics. In addition, aldehyde compounds can interact with hydrophobic groups on the surface of proteins, thereby binding more tightly (Flavour and Fragrance Journal, 2017, 32(2): 92-101). Polyphenols can bind to hydrophobic groups on the surface of proteins through free radical mediation, pH drive, etc. to form conjugates, which can improve the functional properties of proteins based on this principle. Kong Fansheng et al. used sugarcane leaf polyphenols to modify zein, which improved the antioxidant capacity of proteins and reduced their surface hydrophobicity (CN117659450A). In addition, polyphenols have the function of attaching to odorous volatiles. Li Jianquan et al. used plant polyphenol deodorizing film to make an odor-removing mask, which effectively solved the problem of oral odor (CN218474159U). So far, there has been no report on the use of polyphenols to remove the odor of silkworm pupae. The present invention utilizes polyphenols to covalently modify silkworm pupa proteins, and is expected to remove the peculiar smell under milder conditions. Summary of the invention

[0005] Technical problem to be solved: In view of the shortcomings of the prior art, the present invention provides an odorless silkworm pupa protein complex and a deodorization method and application thereof, covalently combining silkworm pupa protein with polyphenols to promote the release of odorous volatiles, and removing the odor of silkworm pupae by heat treatment, providing a new green and efficient silkworm pupa protein deodorization method for the food industry.

[0006] Technical solution: A method for deodorizing silkworm pupa protein, comprising the following steps: step 1, using a solvent to dissolve the silkworm pupa protein and mixing it with polyphenols, and stirring under alkaline conditions to covalently bind the two; step 2, heat-treating the mixture treated in step 1, and using solid phase microextraction-gas chromatography-mass spectrometry to monitor until 98% of the odor volatiles are released; step 3, purifying the protein from the mixture treated in step 2, using an acidic solution to adjust the pH of the mixture to neutral, and drying to obtain odorless silkworm pupa protein-polyphenol complex powder.

[0007] Preferably, the polyphenol in step 1 is a flavonoid or a phenolic acid, the flavonoid is rutin, and the phenolic acid is gallic acid.

[0008] Preferably, in step 1, the silkworm pupa protein is mixed with the polyphenol solution, and the mass ratio of the silkworm pupa protein to the polyphenol is 1:(0.1-10).

[0009] Preferably, the solvent in step 1 is pure water or phosphate buffer.

[0010] Preferably, the pH of the alkaline condition in step 1 is 10-12.5.

[0011] Preferably, the stirring time in step 1 is 0.5-12h.

[0012] Preferably, the heat treatment in step 2 is performed for 3-24 hours at a temperature of 30-70°C.

[0013] Preferably, the acidic solution in step 3 is hydrochloric acid, and the pH is adjusted to neutral.

[0014] The silkworm pupa protein-polyphenol complex prepared by the above method.

[0015] Based on the application of the silkworm pupa protein-polyphenol complex as an additive in food, health products and cosmetics.

[0016] Beneficial effects: Compared with the prior art, the present invention uses polyphenols as deodorants to achieve deodorization of silkworm pupa protein with little damage to the protein structure. The reaction system conditions are mild, easy to operate, and the production cost is low, with good industrial application prospects. The present invention removes 98% of silkworm pupa odorous volatiles, the secondary structure of the protein changes little after deodorization, the polyphenol binding rate can reach up to 14%, and the silkworm pupa protein DPPH removal rate reaches 74.59%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flow chart for deodorization of silkworm pupa protein.

[0018] Figure 2 This is the Fourier infrared analysis of silkworm pupa protein and deodorized silkworm pupa protein.

[0019] Figure 3 Solid phase microextraction-gas chromatography analysis of silkworm pupa protein and deodorized silkworm pupa protein. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0021] Example 1

[0022] Combination of silkworm pupa protein and rutin

[0023] Covalently bind silkworm pupa protein to rutin. Take 1g silkworm pupa protein and 1g rutin, dissolve in 1L pure water, adjust pH to 12 with 2M sodium hydroxide, stir magnetically for 1h, and adjust pH to 7 with dilute hydrochloric acid to obtain silkworm pupa protein-rutin complex solution. After purifying the complex with an 8kD dialysis bag, the content of rutin bound to silkworm pupa protein was determined. The concentration of rutin was determined using the Folin phenol method, and the concentration of silkworm pupa protein was determined using the Coomassie Brilliant Blue method. The content of rutin in this complex was 0.14±0.007g / L, indicating that rutin was well bound to silkworm pupa protein.

[0024] The protein structure changes were analyzed by Fourier transform infrared spectroscopy. Figure 2 The α-helix content of silkworm pupa protein-rutin complex was 29.71±0.22%, the β-turn content was 11.95%±0.35%, the β-sheet content was 23.39±0.25%, and the random coil content was 34.95±0.15%, which was not much different from that of silkworm pupa protein, indicating that the protein was not significantly damaged.

[0025] Analysis of the effect of silkworm pupa protein-rutin complex on promoting the release of odor volatiles

[0026] 5g of silkworm pupa protein-rutin complex solution was taken, and the release of each odor volatile was analyzed by solid phase microextraction-gas chromatography-mass spectrometry. The key odor volatiles nonanal and decanal were used as indicators to evaluate the effect of rutin on the odor release of silkworm pupa protein. The results showed that compared with silkworm pupa protein, the release of nonanal in silkworm pupa protein-rutin complex increased to 270±13%, and the release of decanal increased to 256±55%, which was more conducive to further deodorization.

[0027] Deodorization of heat-treated silkworm pupa protein-rutin complex

[0028] The samples were placed in a 60°C hot water bath and treated for 6 hours before the odor volatiles were analyzed using solid phase microextraction-gas chromatography-mass spectrometry. The results are shown in Figure 3 . Before deodorization, the odor volatiles in silkworm pupa protein were complex, and the total odor volatiles concentration could reach 4630ng / g. However, after deodorization, only 5 odor volatiles were detected in silkworm pupa protein, with a total concentration of 118±27ng / g, indicating a good deodorization effect. In addition, the nonanal content was reduced from 1450±320ng / g to 45±7ng / g, and the decanal content was reduced from 140±30ng / g to 16±3ng / g. The calculation of the odor activity value and sniffing proved that the odor of silkworm pupa protein was basically removed.

[0029] Determination of Antioxidant Activity of Silkworm Pupa Protein-Rutin Complex

[0030] DPPH free radical scavenging activity determination method: freeze-dried silkworm pupa protein-rutin complex was prepared into a 1 mg / mL solution with deionized water. 0.5 mL of the solution was added to 0.5 mL of 0.2 mmol / L DPPH solution, reacted in the dark at 37°C for 30 minutes, and the absorbance was measured at 517 nm. The calculation method is as follows:

[0031]

[0032] Where A s is the absorbance value of the sample group, A b is the absorbance value of the 95% ethanol replacement sample, A c The absorbance value of the DPPH solution replaced by 95% ethanol.

[0033] The results showed that the DPPH free radical scavenging rate of the complex was 74.59±0.58%.

[0034] Example 2 Deodorizing effect of gallic acid on silkworm pupa protein

[0035] 1g of gallic acid, quercetin, chlorogenic acid and caffeic acid solution were used to dissolve 1g of silkworm pupa protein in 1L pure water, and 2M sodium hydroxide was used to adjust the pH to 12. After magnetic stirring for 1h, dilute hydrochloric acid was used to adjust the pH to 7 to obtain silkworm pupa protein-polyphenol complex solution. The odor release was analyzed by solid phase microextraction-gas chromatography-mass spectrometry. The results showed that the release of nonanal in silkworm pupa protein-gallic acid increased to 275±18% and the release of decanal increased to 392±24%. After silkworm pupa protein-gallic acid was treated in a 60℃ hot water bath for 6h, only 5 odor volatiles were detected, with a total concentration of 135±15ng / g, nonanal content decreased to 63±8ng / g, and decanal content decreased to 14±5ng / g.

[0036] Table 1 Contents of main volatile substances in polyphenol-treated samples

[0037]

Claims

1. A method for deodorizing silkworm pupa protein, characterized in that: The following steps are involved: Step 1, using a solvent to dissolve silkworm pupa protein and mix it with polyphenols, stirring under alkaline conditions to make the two covalently bonded; Step 2, heat-treating the mixture treated in step 1, and using solid phase microextraction-gas chromatography-mass spectrometry to monitor until 98% of the odor volatiles are released; Step 3, purifying the protein from the mixture treated in step 2, adjusting the pH of the mixture to neutral with an acidic solution, and drying to obtain odorless silkworm pupa protein-polyphenol complex powder.

2. The method for deodorizing silkworm pupa protein according to claim 1, characterized in that: In step 1, the polyphenol is a flavonoid or a phenolic acid, the flavonoid is rutin, and the phenolic acid is gallic acid.

3. The method for deodorizing silkworm pupa protein according to claim 1, characterized in that: The silkworm pupa protein described in step 1 is mixed with the polyphenol solution, and the mass ratio of the silkworm pupa protein to the polyphenol is 1:(0.1-10).

4. The method for deodorizing silkworm pupa protein according to claim 1, characterized in that: The solvent in step 1 is pure water or phosphate buffer.

5. The method for deodorizing silkworm pupa protein according to claim 1, characterized in that: The pH of the alkaline condition in step 1 is 10-12.

5.

6. The method for deodorizing silkworm pupa protein according to claim 1, characterized in that: The stirring time in step 1 is 0.5-12h.

7. The method for deodorizing silkworm pupa protein according to claim 1, characterized in that: The heat treatment in step 2 is performed for 3-24 hours at a temperature of 30-70°C.

8. The method for deodorizing silkworm pupa protein according to claim 1, characterized in that: The acidic solution in step 3 is hydrochloric acid, and the pH is adjusted to neutral.

9. The silkworm pupa protein-polyphenol complex obtained by the method according to any one of claims 1 to 8.

10. Use of the silkworm pupa protein-polyphenol complex according to claim 9 as an additive in food, health products and cosmetics.

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