CN113527465B - Protein-spice conjugates and uses thereof - Google Patents

Abstract

The present invention relates to the structure and use of a protein-spice conjugate. The protein-spice conjugate has a structure shown in formula I. The protein-spice conjugate of the present invention can be used as a spice precursor with a sustained-release function. Formula I is a conjugate formed by Michael addition reaction of a sulfhydryl-containing protein molecule A and an α,β-unsaturated aldehyde-ketone fragrance molecule. In Formula I, A is a protein molecule such as bovine serum albumin or human serum albumin or a reduction product thereof, Partially unsaturated ketone flavor molecule A part of n is an odd number between 7 and 17. The protein-flavor conjugate synthesized by the present invention has good solubility in aqueous solution, and the carbon-sulfur bond can be broken in a neutral or weakly acidic environment to gradually release the flavor molecules and produce a good flavor sustained-release effect, so that it can be used as a flavor precursor for flavor sustained-release.

Description

Protein-spice conjugates and uses thereof

Technical Field

The invention relates to a protein-spice conjugate with a novel structure and application thereof.

Background Art

Flavors and fragrances are widely used in consumer products in our daily lives, such as food, personal care products, wallpapers, and textiles. However, the inherent volatility and instability of flavors and fragrances make it difficult to achieve long-lasting fragrance. In addition, some aromatic aldehydes and ketones and unsaturated fragrance molecules are easily oxidized and decomposed, which to some extent limits their practical application effects.

Traditional physical encapsulation, including microcapsules, nanocapsules, silica and metal organic frameworks, all physically wrap the fragrance to achieve long-lasting fragrance. In particular, microcapsule technology characterized by the formation of a core-shell structure is an effective solution (Science China: Chemistry. 2019, 49, 575-580). Polyurea and other polymers are used to form microcapsules that can encapsulate fragrance molecules, and then soak or spray them on the surfaces of silk, paper and leather for fragrance, achieving a long-lasting fragrance effect. For example, Bayer AG of Germany prepared polyurethane fragrance microcapsules and sprayed them on the leather surface (US20060216509A1); Givaudan AG of Switzerland developed melamine resin fragrance microcapsules for use in textile finishing (CHIMIA International Journal for Chemistry 2011, 65, 177-181).

However, this technology also has certain shortcomings, such as easy detachment of microcapsules, poor fragrance quality and short fragrance retention time (Fragrance and Fragrance Cosmetics, 2008, 3, 22-25). The emergence of fragrance precursors (i.e. latent fragrances) provides another effective solution for achieving controlled and sustained release of fragrance (Molecules 2018, 23, 1204).

The design of latent fragrances is derived from "prodrugs" in drug delivery, but they are very different in the properties of released molecules, control conditions and application objects. Latent fragrances need to connect highly volatile fragrance molecules to the matrix through characteristic chemical bonds (or non-chemical bonds such as hydrogen bonds) to form non-volatile or low-volatile molecules, which break chemical bonds under mild environmental conditions (such as water vapor, light, oxygen and temperature, etc.) to release fragrance molecules to achieve the effect of controlled release of fragrances. In practical applications, it is necessary to consider the loading problem of latent fragrances in different products, which puts forward requirements for the matrix of latent fragrances: latent fragrances can have good dispersibility and persistence of fragrance in the target product, and have good stability, as well as good biocompatibility and certain degradability. Therefore, the development of excellent matrix and fragrance structure, enriching the way of connecting matrix and fragrance to achieve the sustained release of fragrance under mild biomimetic conditions has become the research focus in this field. This technical strategy is still in the initial development stage, but it shows a bright application prospect (CCS Chemistry 2020, 2, 478-487), and is expected to be used in various products in various fields.

The base materials currently used mainly include special organic molecules and polymers (such as carbohydrate molecules). Organic molecules have poor water solubility and insufficient degradability, which limits the application range of the final prepared latent fragrance. Polymers represented by carbohydrates also have the disadvantages of being difficult to degrade or poorly modifiable. Therefore, developing new bio-friendly and easy-to-modify base materials and preparing corresponding latent fragrances can not only enrich the types of latent fragrances, but also be used in the future. Flavoring in a wider range of fields including organisms. Protein, as a natural compound, is one of the main components of organisms. Protein is a class of water-soluble, easily degradable and harmless compounds, and has increasingly wide applications in functional materials. Therefore, using modifiable proteins as base materials is expected to bring unique properties and a wider range of applications to the final fragrance precursor compounds.

Summary of the invention

The present invention provides a class of protein-flavor conjugates, and uses them as flavor precursors (latent flavor bodies). After testing, the series of compounds all show good flavor sustained-release performance.

Therefore, one object of the present invention is to provide a class of protein-flavor conjugate compounds.

The protein-flavor coupling compound of the present invention has a structure shown in Formula I:

In Formula I, A is a protein such as bovine serum albumin (BSA), human serum albumin (HSA) or a reduction product thereof, Unsaturated ketone flavor molecule A part of, n is an odd number of 7-17, wherein the coupled fragrance is 3-octen-2-one or pharaonic ketone or ionone, and the conjugate is the product of BSA or HSA after reduction and coupling with 12 equivalents of the above-listed coupled fragrances, and each A molecule is coupled with 7-27 small fragrance molecules.

In a preferred technical solution of the present invention, A is bovine serum albumin or its reduction product, and the coupled fragrance is 3-octen-2-one, that is, _R1 is n-butyl, _R2 is H, _R3 is methyl, and n is an odd number of 7-15. The conjugate is the product of BSA or HSA being reduced and coupled with 12 equivalents of 3-octen-2-one.

In another preferred technical scheme of the present invention, A is human serum albumin or its reduction product, and the coupled fragrance is 3-ene-2-one, that is, _R1 is n-butyl, _R2 is H, _R3 is methyl, and n is an odd number of 7-17. The conjugate is the product of BSA or HSA being reduced and coupled with 12 equivalents of 3-octen-2-one.

In another preferred embodiment of the present invention, A is a reduction product of bovine serum albumin, and the coupled fragrance is pharaonic acid, that is, _R1 is H, _R2 is _R3 is n is an odd number of 7-15, and the conjugate is the product of reducing BSA or HSA and coupling it with 12 equivalents of pharaonic ketone.

In another preferred embodiment of the present invention, A is a reduction product of human serum albumin, and the coupling fragrance is pharaonic ketone, that is, _R1 is H, _R2 is _R3 is n is an odd number of 9-15, and the conjugate is the product of reducing BSA or HSA and coupling it with 12 equivalents of pharaonic ketone.

In another preferred embodiment of the present invention, A is a reduction product of bovine serum albumin, and the coupled fragrance is α-ionone, that is, _R1 is _R2 is H, _R3 is methyl, n is an odd number of 7-15, and the conjugate is a product obtained by coupling BSA or HSA with 12 equivalents of α-ionone after reduction.

In another preferred embodiment of the present invention, A is a reduction product of human serum albumin; the coupled fragrance is α-ionone, that is, _R1 is _R2 is H, _R3 is methyl; n is an odd number of 9-13, and the conjugate is a product obtained by coupling BSA or HSA with 12 equivalents of α-ionone after reduction.

In another preferred embodiment of the present invention, A is a reduction product of bovine serum albumin, and the coupled fragrance is β-ionone, that is, _R1 is _R2 is H, _R3 is methyl, n is an odd number of 7-15, and the conjugate is a product obtained by coupling BSA or HSA with 12 equivalents of β-ionone after reduction.

In another preferred embodiment of the present invention, A is a reduction product of human serum albumin, and the coupled fragrance is β-ionone, that is, _R1 is _R2 is H, _R3 is methyl, n is an odd number of 9-15, and the conjugate is a product obtained by coupling BSA or HSA with 12 equivalents of β-ionone after reduction.

A series of protein-flavor conjugates shown in formula I can be prepared with reference to existing preparation techniques.

Formula I is a conjugate formed by Michael addition reaction of a sulfhydryl-containing protein molecule A and an α,β-unsaturated aldehyde and ketone fragrance molecule.

Another object of the present invention is to disclose the use of the above-mentioned protein-flavor conjugate (compound shown in Formula I), that is, the protein-flavor conjugate (compound shown in Formula I) provided by the present invention can be used as a fragrance precursor (latent fragrance body) with fragrance sustained-release performance, and can be applied to the surface of cotton fabrics as a material for long-lasting fragrance.

The invention has the advantage that a latent fragrance body with a lasting fragrance-releasing ability is obtained by using a protein with high biocompatibility as a matrix and combining it with fragrance molecules in the form of chemical bonds, thus having a wide range of application prospects.

The protein-flavor conjugate synthesized by the present invention, such Michael addition product as a conjugate, has good solubility in aqueous solution, and the carbon-sulfur bond can be broken in a neutral or weakly acidic environment, gradually releasing the flavor molecules, producing a good flavor sustained-release effect, so that it can be used as a flavor precursor for flavor sustained-release.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1( a ) is a HR-ESI-MS spectrum of the protein-flavor conjugate BSA-1-1 of Example 1 of the present invention;

Figure 1(b) is a HR-ESI-MS spectrum of the protein-flavor conjugate HSA-1-1 of Example 2 of the present invention;

FIG2( a) is a HR-ESI-MS spectrum of the protein-flavor conjugate BSA-2-1 of Example 3 of the present invention;

FIG2( b ) is a HR-ESI-MS spectrum of the protein-flavor conjugate HSA-2-1 of Example 4 of the present invention;

FIG3 (a) is a HR-ESI-MS spectrum of the protein-flavor conjugate BSA-3-1 of Example 5 of the present invention;

FIG3( b) is a HR-ESI-MS spectrum of the protein-flavor conjugate HSA-3-1 of Example 6 of the present invention;

FIG4 is a HR-ESI-MS spectrum of the protein-flavor conjugate BSA-1-2 of Example 7 of the present invention;

FIG5 is a curve showing the change in volatile concentration of 3-octen-2-one and protein-flavor conjugate BSA-1-1 over time according to Example 8 of the present invention;

FIG. 6 is a curve showing the change in volatile concentration of 3-octen-2-one and protein-flavor conjugate HSA-1-1 over time in Example 9 of the present invention.

DETAILED DESCRIPTION

The present invention is further described below by way of examples, the purpose of which is to provide a better understanding of the content of the present invention. The examples given do not limit the protection scope of the present invention.

Example 1

Take BSA (6.64g, 0.1mmol) and dissolve it in 50mL deionized water, add TCEP (tri(2-carboxyethyl)phosphine) aqueous solution (0.5mL, 1mol/L), and stir at room temperature for 30 minutes. Add sodium hydroxide aqueous solution (10μL, 1mol/L), take 3-octen-2-one (0.15g, 1.2mmol) and dissolve it in 1mL ethanol, and slowly add it dropwise to the BSA aqueous solution. After stirring at room temperature for 5 hours, remove excess small molecules through a Zeba desalting column. Collect the protein, lyophilize to remove the solvent, and obtain 6.28g of protein-flavor conjugate BSA-1-1.

Figure 1(a) shows the HR-ESI-MS spectrum of the protein-flavor conjugate.

Analysis of the data shows that the mixture is a coupling product of partially reduced BSA and the fragrance 3-octen-2-one. Under this condition, each BSA molecule can be coupled with 7-15 fragrance small molecules, with an average value of about 11.

Upon testing, the substance was found to be well soluble in water, and its solid did not undergo significant changes in composition and structure after being exposed at room temperature for 20 days.

Example 2

Take HSA (6.63g, 0.1mmol) and dissolve it in 50mL deionized water, add TCEP (tri(2-carboxyethyl)phosphine) aqueous solution (0.5mL, 1mol/L), and stir at room temperature for 30 minutes. Add sodium hydroxide aqueous solution (10μL, 1mol/L), take 3-octen-2-one (0.15g, 1.2mmol) and dissolve it in 1mL ethanol, and slowly add it dropwise to the HSA aqueous solution. After stirring at room temperature for 5 hours, remove excess small molecules through a Zeba desalting column. Collect the protein, freeze-dry to remove the solvent, and obtain 6.13g protein-flavor conjugate HSA-1-1.

See Figure 1(b) showing the HR-ESI-MS spectrum of the protein-flavor conjugate.

Analysis of the data shows that the mixture is a coupling product of partially reduced HSA and the fragrance 3-octen-2-one. Under this condition, each HSA molecule can be coupled with 7-17 fragrance small molecules, with an average value of about 11.

Upon inspection, the substance was found to be well soluble in water and its solid structure showed no significant changes after being exposed at room temperature for 20 days.

Example 3

Take BSA (6.64g, 0.1mmol) and dissolve it in 50mL deionized water, add TCEP (tri(2-carboxyethyl)phosphine) aqueous solution (0.5mL, 1mol/L), and stir at room temperature for 30 minutes. Add sodium hydroxide aqueous solution (10μL, 1mol/L), take pharaonic acid (0.23g, 1.2mmol) and dissolve it in 1mL ethanol, and slowly add it dropwise to the BSA aqueous solution. After stirring at room temperature for 5 hours, remove excess small molecules through a Zeba desalting column. Collect the protein, freeze-dry to remove the solvent, and obtain 6.07g of protein-flavor conjugate BSA-2-1.

Figure 2(a) shows the HR-ESI-MS spectrum of the protein-flavor conjugate.

Analysis of the data shows that the mixture is the coupling product of partially reduced BSA and the fragrance pharaonic acid. Under this condition, each BSA molecule can be coupled with 7-15 fragrance small molecules, with an average value of about 11.

Upon testing, the substance was found to be well soluble in water, and its solid did not undergo significant changes in composition and structure after being exposed at room temperature for 20 days.

Example 4

Take HSA (6.63g, 0.1mmol) and dissolve it in 50mL deionized water, add TCEP (tri(2-carboxyethyl)phosphine) aqueous solution (0.5mL, 1mol/L), and stir at room temperature for 30 minutes. Add sodium hydroxide aqueous solution (10μL, 1mol/L), take pharaonic acid (0.23g, 1.2mmol) and dissolve it in 1mL ethanol, and slowly add it dropwise to the HSA aqueous solution. After stirring at room temperature for 5 hours, remove excess small molecules through a Zeba desalting column. Collect the protein, lyophilize to remove the solvent, and obtain 5.76g of protein-flavor conjugate HSA-2-1.

Figure 2(b) shows the HR-ESI-MS spectrum of the protein-flavor conjugate.

Analysis of the data shows that the mixture is the coupling product of partially reduced HSA and the fragrance pharaonic acid. Under this condition, each HSA molecule can be coupled with 9-15 fragrance small molecules, with an average value of about 11.

Upon testing, the substance was found to be well soluble in water, and its solid showed no significant changes in composition and structure after being exposed at room temperature for 20 days.

Example 5

Take BSA (6.64g, 0.1mmol) and dissolve it in 50mL deionized water, add TCEP (tri(2-carboxyethyl)phosphine) aqueous solution (0.5mL, 1mol/L), and stir at room temperature for 30 minutes. Add sodium hydroxide aqueous solution (10μL, 1mol/L), take α-ionone (0.23g, 1.2mmol) and dissolve it in 1mL ethanol, and slowly add it dropwise to the BSA aqueous solution. After stirring at room temperature for 5 hours, remove excess small molecules through a Zeba desalting column. Collect the protein, freeze-dry to remove the solvent, and obtain 6.11g protein-flavor conjugate BSA-3-1.

Figure 3(a) shows the HR-ESI-MS spectrum of the protein-flavor conjugate.

Analysis of the data shows that the mixture is the coupling product of partially reduced BSA and the fragrance dynasty ketone. Under this condition, each BSA molecule can be coupled with 7-15 fragrance small molecules, with an average value of about 11.

Upon testing, the substance was found to be well soluble in water, and its solid did not undergo significant changes in composition and structure after being exposed at room temperature for 20 days.

Example 6

Take HSA (6.63g, 0.1mmol) and dissolve it in 50mL deionized water, add TCEP (tri(2-carboxyethyl)phosphine) aqueous solution (0.5mL, 1mol/L), and stir at room temperature for 30 minutes. Add sodium hydroxide aqueous solution (10μL, 1mol/L), take α-ionone (0.23g, 1.2mmol) and dissolve it in 1mL ethanol, and slowly add it dropwise to the HSA aqueous solution. After stirring at room temperature for 5 hours, remove excess small molecules through a Zeba desalting column. Collect the protein, freeze-dry to remove the solvent, and obtain 5.87g of protein-flavor conjugate HSA-3-1.

Figure 3(b) shows the HR-ESI-MS spectrum of the protein-flavor conjugate.

Analysis of the data shows that the mixture is the coupling product of partially reduced HSA and the fragrance dynasty ketone. Under this condition, each HSA molecule can be coupled with 9-13 fragrance small molecules, with an average value of about 11.

Upon testing, the substance was found to be well soluble in water, and its solid showed no significant changes in composition and structure after being exposed at room temperature for 20 days.

Example 7

Take BSA (6.64g, 0.1mmol) and dissolve it in 50mL deionized water, add TCEP (tri(2-carboxyethyl)phosphine) aqueous solution (1.0mL, 1mol/L), and stir at room temperature for 30 minutes. Add sodium hydroxide aqueous solution (10μL, 1mol/L), take 3-octen-2-one (0.31g, 2.4mmol) and dissolve it in 1mL ethanol, and slowly add it dropwise to the BSA aqueous solution. After stirring at room temperature for 5 hours, remove excess small molecules through a Zeba desalting column. Collect the protein, freeze-dry to remove the solvent, and obtain 6.31g protein-flavor conjugate BSA-1-2.

FIG4 shows the HR-ESI-MS spectrum of the protein-flavor conjugate.

Analysis of the data shows that the mixture is a coupling product of partially reduced BSA and the fragrance 3-octen-2-one. Under this condition, each BSA molecule can be coupled with 15-27 fragrance small molecules, with an average value of about 21.

Upon testing, the substance was found to be well soluble in water, and its solid showed no significant changes in composition and structure after being exposed at room temperature for 20 days.

Example 8

Solid phase microextraction-gas chromatography-mass spectrometry was used to detect the release of aroma substances:

The specific implementation method is as follows: 678 mg (0.01 mmol) of the conjugate BSA-1-1 and 1.5 μL (1.26 mg, 1.2 mmol) of 3-octen-2-one were weighed and added to two 10 mL headspace bottles respectively and numbered as No. 1 and No. 2. They were left open overnight, and then on the next day, 2 mL of phosphate buffered saline (PBS, pH=3) and 20 μL of 100 ppm (acetone solution) of o-dichlorobenzene as an internal standard were added to both bottles respectively, and then sealed for use in solid phase microextraction.

The SPME extraction fiber head was aged at 250°C for 30 min at the injection port of the gas chromatograph. Then, the aged fiber extraction head was inserted into the headspace of the sample bottle (be careful not to let the extraction head contact the solution to avoid contamination of the extraction head). The extraction was carried out at 40°C for 20 min. The extraction head was taken out and the needle was quickly inserted into the injection port of the gas chromatograph. The fiber head was pushed out safely and quickly. The fiber head was analyzed at the injection port for 5 min. The adsorbed aroma components were thermally analyzed and the gas chromatography-mass spectrometry (GC-MS) was started to collect data.

The solid phase microextraction and gas chromatography-mass spectrometry analysis were performed with the equilibrium time changed to 20min, 50min, 80min, 110min, 140min and 170min, respectively. The graph was plotted with the equilibrium time as the horizontal axis and the concentration of the flavor substance released as the vertical axis, and Figure 5 was obtained.

Among them, the calculation formula for the release concentration of flavor substances is:

Wherein A1 is the gas chromatographic peak area of the released fragrance, 100 means the internal standard concentration is 100 ppm, 20 means the amount of the added internal standard is 20 μL, A2 is the peak area of the internal standard, and 10 means the volume of the headspace bottle is 10 mL.

SPME operating conditions: equilibration at 40 °C for 20 min, adsorption extraction for 20 min, desorption at the GC-MS injection port for 5 min, and 250 °C.

Example 9

The aroma release of other protein-flavor conjugates (HSA-1-1, BSA-2-1, HSA-2-1, BSA-3-1, HAS-3-1, BSA-1-2) and their corresponding flavor molecules was tested. The test conditions and steps were the same as in Example 8. The exemplary results are shown in FIG6 .

From the above data, we can see that the amount of aroma substances released increases with the passage of time. However, no matter when, the volatile concentration of fragrance molecules is lower than that of protein-fragrance conjugates. This is because after being placed overnight, most of the fragrance compounds have volatilized, while protein-fragrance conjugates undergo a reverse Michael addition reaction under weakly acidic conditions to slowly release fragrance molecules. Therefore, when tested the next day, the concentration of fragrance molecules in protein-fragrance conjugates is relatively high. This shows that this type of protein-fragrance conjugate has a delayed effect on the release of fragrance substances.

However, for the conjugate BSA-1-2, there are too many thiol groups in the protein obtained after the release of the fragrance, which affects the overall aroma of the sample, making it difficult to apply to actual products.

Example 10

This embodiment is an application example: the fragrance effect of the thiourea derivative provided by the present invention applied to cotton fabrics.

Specific implementation method: First weigh 0.5g of protein-flavor conjugate BSA-1-1 into 50mL of deionized water, then add 5g of water-soluble polyurethane, 0.1g of sodium dodecylbenzene sulfonate, and 0.05g of fatty alcohol polyoxyethylene ether, stir at high speed for 4h, then immerse 10cm×15cm pure cotton cloth completely in the emulsion for 30min, take it out and wring it dry naturally overnight. Then take 3-octen-2-one in an equal molar amount to the mixture and repeat the above operation.

The obtained cotton cloth has fragrance. After being placed in the room at room temperature for 5 days, we can clearly feel that the cotton cloth with BSA-1-1 still has a certain fragrance, while the cotton cloth with fragrance has almost no fragrance.

Similar results were obtained by repeating the above experiment with other protein-flavor conjugates and corresponding flavor compounds.

As can be seen from Application Example 1, the thiourea derivatives provided by the present invention can not only be applied to cotton clothing, but also can achieve a lasting fragrance effect when combined with spices.

Claims (6)

1. A protein-flavor conjugate, which is represented by the following structural formula I: In formula I, A is bovine serum albumin BSA or human serum albumin HAS or the reduction product of these proteins, Unsaturated ketone flavor molecule A part of, n is an odd number of 7-17, wherein the coupled fragrance is 3-octen-2-one or pharaonic ketone or ionone, and the conjugate is the product of BSA or HSA after reduction and coupling with 12 equivalents of the above-listed coupled fragrances, and each A molecule is coupled with 7-27 small fragrance molecules. 2. The protein-flavor conjugate according to claim 1, wherein A is bovine serum albumin or human serum albumin or a reduced product thereof, and the coupled flavor is 3-octen-2-one, i.e., _R1 is n-butyl, _R2 is H, _R3 is methyl, and n is an odd number of 7-17, and the conjugate is the product of BSA or HSA being reduced and coupled with 12 equivalents of 3-octen-2-one. 3. The protein-flavor conjugate according to claim 1, wherein A is bovine serum albumin or human serum albumin and its reduction product, and the conjugated flavor is pharaonic acid, that is, _R1 is H, _R2 is _R3 is n is an odd number of 7-17, and the conjugate is the product of reducing BSA or HSA and coupling it with 12 equivalents of pharaonic ketone. 4. The protein-flavor conjugate according to claim 1, wherein A is bovine serum albumin or human serum albumin and its reduction product, and the conjugated flavor is ionone, that is, _R1 is _R2 is H, _R3 is methyl, n is an odd number of 7-15, and the conjugate is a product obtained by coupling BSA or HSA with 12 equivalents of ionone after reduction. 5. Use of the protein-flavor conjugate according to claim 1 as a flavor precursor. 6. Use of the protein-flavor conjugate according to claim 5, wherein the flavor precursor is used for sustained release of fragrance and is applied to cotton fabric as a long-lasting fragrance material.