A method for preparing a characteristic flavor enhancer of Chinese toon based on an enzymatic reaction

Abstract

The present application relates to a method for preparing a characteristic flavor enhancer of Xanthoceras sorbifolia Bunge based on an enzymatic reaction, which comprises the following steps: 1) fresh or frozen Xanthoceras sorbifolia Bunge sprouts are added with trifluoroacetic acid for enzyme inactivation treatment, ultrasonic water extraction, and then Sevage reagent, petroleum ether and ethanol are sequentially added for impurity removal; 2) preparation of gamma-glutamyl transpeptidase or gamma-glutamyl transpeptidase crude enzyme solution; 3) different sources of gamma-GTP enzyme are reacted with the sulfur-containing flavor precursor substrate of Xanthoceras sorbifolia Bunge in step 1) at a suitable concentration under sealed conditions at 30-50 DEG C and pH 7-8 for 30-60 min, and the characteristic flavor enhancer of Xanthoceras sorbifolia Bunge is obtained. The present application successfully prepares the characteristic flavor of Xanthoceras sorbifolia Bunge in vitro for the first time through an enzymatic reaction, provides a feasible path for the preparation of typical Xanthoceras sorbifolia Bunge flavor spices or flavor enhancers by the biological enzyme method, and is conducive to solving the problems of difficult storage of fresh Xanthoceras sorbifolia Bunge and loss of flavor of Xanthoceras sorbifolia Bunge processing products.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food additives, and particularly relates to a method for preparing a special flavor enhancer of Chinese toon based on an enzymatic reaction. BACKGROUND

[0002] Chinese toon is a traditional vegetable, medicinal plant and wood source in China, and has a long consumption history. Every spring, Chinese toon grows out of tender buds, and due to its unique flavor and nutritional value, the short supply of this seasonal delicacy has been loved by consumers in most areas of China. In recent years, the bud shoots have also become a more and more popular food. However, the tender buds of Chinese toon have typical seasonality, and the supply period is extremely short. A large number of studies have shown that Chinese toon belongs to a heat-sensitive spice plant, and the active components of the characteristic aroma are unstable and easy to escape, decompose or polymerize during the traditional heat processing with the increase of heating temperature, so that the plant characteristic flavor is lost, and even deterioration occurs, which greatly reduces the commodity value. This brings certain difficulties to the identification of the characteristic aroma of Chinese toon, the development of products and the food processing applicability.

[0003] The research on volatile components of Toona sinensis began about 20 years ago. So far, more than 100 volatile components have been identified in Toona sinensis, mainly including sulfides, terpenes, aldehydes, hydrocarbons, acids, alcohols and esters. Liu Changjin et al. analyzed the volatile components of Toona sinensis by GC-MS and GC-O and found that Z / E-2-allyl-3,4-dimethyl-2,3-dihydrothiophene was the main contributor to the characteristic aroma of Toona sinensis (Analysis of volatile compounds and identification of characteristic aroma components of Toona sinensis (A. Juss.) Roem. using GC-MS and GC-O. [J]. Food and Nutrition Sciences, 2013, 04(03)). Sun Baoguo et al. determined the main aroma components in fresh and blanched Toona sinensis by static headspace dilution analysis (SHDA-AEDA) and solvent-assisted flavor evaporation dilution analysis (SAFE-AEDA) combined with GC-MS and GC-O and found that sulfur-containing compounds, especially (E,E)-propenyl-disulfide and (E,Z)-propenyl-disulfide, were the key aroma components and dominated the overall aroma of Toona sinensis, providing a stimulating, sulfur-containing, and garlic-like pungent odor, thus imparting a unique aroma characteristic to Toona sinensis (Characterization of typical potent odorants in raw and cooked Toona sinensis (A. Juss.) M. Roem. by instrumental-sensory analysis techniques. [J]. Food chemistry, 2019, 282). Zhai Xiaoting et al. investigated the aroma differences between red and green Toona sinensis by aroma extract dilution analysis (AEDA) combined with GC-MS and GC-O and found that (E,E)-propenyl-disulfide, (E,Z)-propenyl-disulfide, (Z,Z)-propenyl-disulfide, (E,E)-propenyl-trisulfide, (E,Z)-propenyl-trisulfide, (Z,Z)-propenyl-trisulfide, cis- and trans-2-allyl-3,4-dimethyl-2,3-dihydrothiophene, and dimethyl sulfide were the key aroma components with significant differences in flavor dilution factors, contents, and odor activity values, which resulted in differences in the overall aroma profile and unique sulfur-like odor of each Toona sinensis variety.(Key Odor-Active Compounds in Raw Green and Red Toona sinensis (A.Juss.) Roem. and Their Changes during Blanching.[J].Journal of agricultural and foodchemistry,2020,68(27).). All the above studies indicate that volatile sulfur compounds contribute the most to the characteristic aroma of Toona sinensis and are its main characteristic olfactory components.

[0004] There are few studies on the non-volatile precursors of the above sulfur-containing compounds in Toona sinensis. Only Li Jiaxiao et al. confirmed through the stability isotope tracing method that the sulfur-containing flavor precursors of Toona sinensis are (S,S)-γ-glutamyl-(Z / ES-1-propenyl)thioglycine (structure shown below), γ-glutamyl-(Z / ES-1-propenyl)cysteine, and Z / ES-1-acryloyl-L-cysteine ​​(Identification of (S,S)-γ-Glutamyl-(cis-S-1-propenyl)thioglycine, a Naturally Occurring Norcysteine ​​Derivative, from the Chinese Vegetable Toona sinensis.[J]. Journal of agricultural and food chemistry, 2013, 61(31).).

[0005]

[0006] When the organization is cut or chewed, it will release its typical sulfur-containing irritating smell, combined with the synthesis of similar sulfur-containing volatile flavor substances in Allium plants such as onions, garlic and onions, Li Jiaxiao et al. Research speculates that the flavor precursor substance (γ-glutamyl-propenyl thio glycine) in the Chinese toon forms propenyl mercaptan under the action of peptidase, and the unstable propenyl mercaptan is easy to polymerize or pyrolyze to generate a series of volatile sulfur compounds (dipropyl disulfide, 2-mercapto-2,3-dihydro-3,4-dimethyl thiophene, 3,4-dimethyl thiophene, etc.), thereby producing the characteristic flavor of Chinese toon (synthesis route as shown below). The previous research of the research group found that the freeze-dried powder of fresh Chinese toon obtained by freeze-drying can produce strong fresh Chinese toon inherent sulfur-containing flavor in a few seconds after contacting with a small amount of water, and it is speculated that the typical flavor is the result of enzyme reactivation caused by the contact of freeze-dried powder with water. Similar phenomena have been confirmed in the rehydration of garlic and onion freeze-dried powder, thereby further verifying the enzyme synthesis path of the sulfur-containing flavor of Chinese toon.

[0007]

[0008] Based on this research background, according to the enzymatic formation mechanism of the sulfur-containing characteristic aroma of Chinese toon, the sulfur-containing flavor precursor substrate and γ-glutamyl transpeptidase (γ-GTP) of Chinese toon are separated and purified, and the characteristic flavor of Chinese toon is successfully prepared by constructing an enzymatic reaction system. The enzyme reaction mechanism of the formation of the sulfur-containing characteristic aroma of Chinese toon is verified in vitro for the first time, which provides a feasible path for the preparation of Chinese toon typical flavor enhancer by biological enzyme method. So far, there is no report on the method of separating and purifying Chinese toon flavor precursor substrate and γ-glutamyl transpeptidase (γ-GTP) and preparing Chinese toon characteristic flavor by enzyme reaction. SUMMARY

[0009] The purpose of the present application is to overcome the defects of the prior art and provide a method for preparing Chinese toon characteristic flavor enhancer based on enzyme reaction. Compared with commercialized heterologous γ-GTP enzyme, the extracted and purified Chinese toon γ-GTP enzyme has lower cost, and the prepared Chinese toon characteristic flavor enhancer has the characteristics of high concentration, rich flavor and close to the flavor of fresh Chinese toon, which has good application prospect.

[0010] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0011] A method for preparing Chinese toon characteristic flavor enhancer based on enzyme reaction, comprising the following steps:

[0012] 1) Preparation of Chinese toon sulfur-containing flavor precursor substrate or extract:

[0013] The fresh or frozen tender shoots of Chinese toon are added with 3-8% trifluoroacetic acid aqueous solution, and are crushed and inactivated in a cell breaker for 10-30 min; then deionized water is added for ultrasonic extraction at 30-50℃ for 30-60 min; then the flavor precursor substrate is obtained by centrifugation and concentration; then Sevage reagent and petroleum ether and ethanol are added in sequence for impurity removal; then the flavor precursor substrate extract is obtained by concentration and freeze-drying, and is a yellow-brown powder, which is stored at 4℃ for later use;

[0014] Alternatively, the obtained flavor precursor substrate extract is redissolved in water, and is separated and purified by using a cation exchange resin and a solid-phase extraction column in sequence to obtain the purified flavor precursor substrate.

[0015] 2) Preparation of γ-glutamyl transpeptidase (γ-GTP) or γ-glutamyl transpeptidase crude enzyme solution:

[0016] 3) Preparation of Chinese toon characteristic flavor enhancer based on enzymatic reaction:

[0017] The flavor precursor substrate extract or the flavor precursor substrate is prepared into an aqueous solution with a concentration of 0.1-0.5 g / ml, and γ-glutamyl transpeptidase is prepared into a solution with a concentration of 1-5 mg / ml by using Tris-HCl buffer; then the two solutions are mixed in equal volume and are reacted at 30-50℃ and pH 7-8 in a sealed condition for 30-60 min, and the Chinese toon characteristic flavor enhancer is obtained.

[0018] Alternatively, the flavor precursor substrate extract or the flavor precursor substrate is prepared into an aqueous solution with a concentration of 0.1-0.5 g / ml, and is mixed with γ-glutamyl transpeptidase crude enzyme solution in equal volume and is reacted at 30-50℃ and pH 7-8 in a sealed condition for 30-60 min, and the Chinese toon characteristic flavor enhancer is obtained; the specific enzyme activity of the γ-glutamyl transpeptidase crude enzyme solution is 0.1-0.3 units / mg.

[0019] In the present application, the γ-glutamyl transpeptidase can be a commercial γ-GTP enzyme, an extracted Chinese toon γ-GTP crude enzyme solution or a purified γ-GTP enzyme.

[0020] Specifically, in step 1), the mass ratio of the fresh or frozen tender shoots of Chinese toon to the trifluoroacetic acid aqueous solution is 1:1-2.

[0021] Further, in step 1), the amount of the deionized water added is 10-20 times the mass of the fresh or frozen tender shoots of Chinese toon.

[0022] Specifically, in step 2), the γ-glutamyl transpeptidase crude enzyme solution is prepared by the following steps:

[0023] Fresh, intact Toona sinensis buds are preferably freeze-dried under vacuum conditions at -30 to -40 DEG C and 0.01 KPa for 35-40 hours, crushed and passed through a 40-80 mesh sieve to obtain freeze-dried Toona sinensis powder, and a proper amount of the freeze-dried Toona sinensis powder is added to a buffer solution and rapidly ground for 10-20 minutes, then ultrasonically treated in an ice bath for 30-60 minutes, and centrifuged at 2-8 DEG C for 10-20 minutes to obtain a crude γ-glutamyl transpeptidase solution.

[0024] Further, in the preparation of the crude γ-glutamyl transpeptidase solution, the buffer solution is a Tris-HCl buffer solution having a concentration of 40-60 mM, a pH of 7-9, and containing 8-12% glycerol, 18-22 μM pyridoxal phosphate, 1-2 mM ethylenediaminetetraacetic acid (EDTA), and 4-6 mM β-mercaptoethanol. Preferably, the freeze-dried Toona sinensis powder is added to the buffer solution at a solid-liquid ratio of 1 g: 15-20 ml.

[0025] Further, the crude γ-glutamyl transpeptidase solution is sequentially subjected to ammonium sulfate fractionation, dialysis, and freeze-drying to obtain a purified γ-GTP enzyme sample, and the γ-glutamyl transpeptidase is obtained by the following steps:

[0026] At 2-8 DEG C, ammonium sulfate is added to the crude γ-glutamyl transpeptidase solution to a saturation degree of 40-45%, and left to stand overnight, and then centrifuged at 2-8 DEG C, and the supernatant is collected and ammonium sulfate is added to a saturation degree of 65-70%, and left to stand overnight, and then centrifuged at 2-8 DEG C, and the target protein is collected from the precipitate, dissolved in a Tris-HCl buffer solution, and then dialyzed in a dialysis bag with a molecular weight cut-off of 8-14 KDa, and the dialysis solution is freeze-dried to obtain a purified γ-glutamyl transpeptidase.

[0027] The present application first successfully prepares a characteristic flavor of Toona sinensis by an enzymatic reaction in vitro, and provides a feasible path for preparing a typical Toona sinensis flavoring or flavor enhancer by a biological enzyme method, and is advantageous in solving the problems of difficulty in storage of fresh Toona sinensis and loss of flavor of Toona sinensis processed products.

[0028] 1) The present application constructs an in vitro enzymatic reaction system of Toona sinensis sulfur-containing characteristic aroma, successfully prepares a Toona sinensis typical characteristic flavor enhancer, and first verifies an enzymatic reaction mechanism of formation of Toona sinensis sulfur-containing characteristic aroma in vitro, and provides a feasible path for preparing a Toona sinensis typical characteristic flavor enhancer by a biological enzyme method (results are shown in the examples), which can be applied in the processing of Toona sinensis flavor products and plays a role in flavor enhancement.

[0029] 2) The γ-GTPase extracted and purified from Toona sinensis in this invention has a lower cost compared with commercial heterologous γ-GTPase. Furthermore, the flavor enhancer of Toona sinensis prepared by reacting with the extracted flavor precursor substrate has the characteristics of high concentration, rich flavor, and flavor that is closer to that of fresh Toona sinensis, and has good application prospects. Attached Figure Description

[0030] Fig. 1 This is a GC-MS total ion chromatogram of the volatile components of the crude γ-GTP enzyme solution extracted from Toona sinensis in this invention.

[0031] Fig. 2 This is a GC-MS total ion chromatogram of the volatile components of the flavor precursor substrate extract prepared from Toona sinensis in this invention.

[0032] Fig. 3 This is a GC-MS total ion chromatogram of the volatile components in the enzyme-catalyzed reaction system 1 of this invention;

[0033] Fig. 4 This is a two-dimensional GC-IMS spectrum of the volatile components of the enzyme-catalyzed reaction system 1 and the raw materials in this invention; from left to right: crude enzyme solution of Toona sinensis γ-GTP, aqueous solution of flavor precursor substrate extract, and enzyme-catalyzed reaction system 1;

[0034] Fig. 5 The images show the GC-IMS fingerprints of the volatile components of the enzyme-catalyzed reaction system 1 and the raw materials in this invention; from top to bottom, they are: crude γ-GTP enzyme solution of Toona sinensis, aqueous solution of flavor precursor substrate extract, and enzyme-catalyzed reaction system 1. Detailed Implementation

[0035] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0036] Unless otherwise specified, all raw materials used in the following examples are commercially available products or can be prepared using conventional methods in the art. Room temperature refers to 25±5℃.

[0037] For any procedures or testing methods or steps not mentioned or recorded in detail, conventional techniques in this field may be used.

[0038] Example 1

[0039] A method for preparing a characteristic flavor enhancer of Toona sinensis based on an enzymatic reaction includes the following steps:

[0040] (1) Preparation of flavor precursor substrate extract:

[0041] The fresh Japanese cedrat sprouts were added with 5% trifluoroacetic acid aqueous solution (1:1 in mass), and then were mixed and crushed in a cell breaker for 10 min to inactivate the enzymes. Then, the fresh Japanese cedrat sprouts were ultrasonically extracted for 60 min in deionized water at 40°C and 300W. The crude extract was centrifuged at 4000 rpm / min for 10 min, and was concentrated to 1 / 10 of the volume of the crude extract at 50°C by rotary evaporation to obtain a water solution of the flavor precursor substrate crude extract.

[0042] The water solution of the flavor precursor substrate crude extract was transferred into a separatory funnel, and Sevage reagent was added (volume ratio of the water solution of the crude extract: chloroform: n-butanol = 15:5:1). The mixture was mixed at 150 rpm for 30 min to shake thoroughly to precipitate the proteins. The protein emulsion layer between the upper clear liquid and the lower organic layer was removed, and the supernatant was repeatedly treated by the method for 2-3 times to obtain the protein-removed extract. Petroleum ether was added to the protein-removed extract at a volume ratio of 1:1, and the mixture was shaken in the separatory funnel for 30 min and then was allowed to stand. After the mixture was fully layered, the lower solution was collected, and residual petroleum ether was removed by rotary evaporation at 40°C. An equal volume of anhydrous ethanol was added, and the mixture was mixed and shaken thoroughly in the separatory funnel for 30 min, and was allowed to precipitate at 4°C overnight. The mixture was centrifuged at 4000 rpm / min for 10 min, and the supernatant was collected and was concentrated by rotary evaporation at 50°C to remove ethanol and most of the water. Finally, the mixture was vacuum freeze-dried at -30°C and 0.01 KPa for 36 h to obtain the flavor precursor substrate extract in the form of yellow-brown powder, which was stored at 4°C for use.

[0043] (2) Preparation of the Japanese cedrat γ-GTP crude enzyme solution:

[0044] The fresh and intact Japanese cedrat sprouts were vacuum freeze-dried in a vacuum freeze-drier at -40°C and 0.01 KPa for 40 h, and were crushed through a 40-mesh sieve to obtain freeze-dried Japanese cedrat powder, which was stored in a ultra-low temperature refrigerator at -80°C for use. The freeze-dried Japanese cedrat powder was added to a buffer solution at a solid-liquid ratio of 1 g:20 ml, and was rapidly ground for 10 min. The buffer solution was pre-cooled 50 mM Tris-HCl buffer solution (containing 10% glycerol, 20 μM pyridoxal phosphate, 1 mM EDTA, 5 mM β-mercaptoethanol, pH 8.0). Then, the mixture was ultrasonically treated in an ice bath at 240W for 30 min, and was centrifuged at 10000 rpm / min at 4°C for 10 min to obtain the Japanese cedrat γ-GTP crude enzyme solution (specific enzyme activity: 0.2 units / mg).

[0045] (3) The flavor precursor substrate extract obtained in step (1) was prepared into an aqueous solution with a concentration of 0.3 g / ml, mixed with the γ-GTP crude enzyme solution obtained in step (2) according to a volume ratio of 1:1, and reacted under sealed conditions at 40°C and pH 7.0 for 60 min to construct an enzymatic reaction system 1, i.e. to prepare a Chinese toon characteristic flavor enhancer.

[0046] In this example, the flavors of fresh Chinese toon and the enzymatic reaction system 1 were compared by sensory evaluation, and the volatile components of the Chinese toon γ-GTP crude enzyme solution, the flavor precursor substrate extract aqueous solution, and the enzymatic reaction system 1 were compared and analyzed by GC-IMS and GC-MS, so as to verify the feasibility of the method for preparing the Chinese toon characteristic flavor enhancer by enzymatic reaction.

[0047] Ten sensory evaluation personnel were selected, and the flavors and strengths of fresh Chinese toon and the enzymatic reaction system 1 were described by using a blind evaluation method. The sensory evaluation results are shown in Table 1.

[0048] Table 1 Sensory evaluation table

[0049]

[0050] Note: The flavor intensity of the sample was obtained according to the subjective smell of 10 sensory evaluation personnel, * represents weak, ** represents moderate, *** represents strong, and **** represents rich.

[0051] According to the results in Table 1, the flavor precursor substrate extract aqueous solution extracted from Chinese toon and the Chinese toon γ-GTP crude enzyme solution were reacted in vitro to construct the enzymatic reaction system 1, which had obvious and typical Chinese toon characteristic flavor, and the sulfur irritating odor was stronger. The sensory evaluation results confirmed the feasibility of the method.

[0052] Considering that the sensory evaluation is greatly affected by human subjective factors, the volatile components of the Chinese toon γ-GTP crude enzyme solution, the flavor precursor substrate extract aqueous solution, and the enzymatic reaction system 1 were further determined by GC-MS and GC-IMS in this example. The GC-MS detection results are shown in Figs. 1-3 (total ion chromatogram of volatile components) and Table 2, and the GC-IMS detection results are shown in Fig. 4 .

[0053] Table 2 Comparison and analysis of GC-MS results of sulfur-containing volatile components of the enzymatic reaction system 1 and raw materials

[0054]

[0055] Note: “-” represents not detected.

[0056] The volatile components of the crude γ-GTP enzyme solution of Toona sinensis, the water-soluble flavor precursor substrate extract, and the enzymatic reaction system 1 were detected and analyzed by using the HS-SPME-GC-MS combined technique, and the detection results of the sulfur-containing volatile components are shown in Table 1 and Table 2. Figs. 1 to 3 As shown in Table 1 and Table 2, five sulfur-containing compounds, including propylene sulfide, 2-mercaptoethanol, 3,4-dimethylthiophene, (E)-1-propenyl propyl trisulfide, and 1-(1-propenylthio) propyl disulfide, were detected in the crude γ-GTP enzyme solution of Toona sinensis, with a total content of 0.97 μg / ml; only a trace amount of 3,4-dimethylthiophene (0.03 μg / ml) was detected in the water-soluble flavor precursor substrate extract; and 13 sulfur-containing compounds were detected in the enzymatic reaction system 1, including the characteristic aroma components Z-2-mercapto-3,4-dimethyl-2,3-dihydrothiophene (2.83 μg / ml), E-2-mercapto-3,4-dimethyl-2,3-dihydrothiophene (1.88 μg / ml), (E,Z)-bis-(1-propenyl) disulfide (4.64 μg / ml), and (E,E)-bis-(1-propenyl) trisulfide (0.63 μg / ml), which are unique to fresh Toona sinensis, and the total amount of the sulfur-containing compounds reached 22.97 μg / ml. The results show that the reaction of the crude γ-GTP enzyme solution of Toona sinensis and the water-soluble flavor precursor substrate extract generates the characteristic sulfur-containing aroma components of Toona sinensis, thereby verifying the roles of the γ-GTP enzyme and the flavor precursor substrate in the formation of the characteristic flavor of Toona sinensis.

[0057] The volatile components of the raw materials and products of the enzymatic reaction system 1 were detected and analyzed by using the HS-GC-IMS technique, and the results are shown in Table 3. Fig. 4 As shown in Table 3, the white vertical line at the horizontal coordinate 1.0 is the RIP peak (reaction ion peak), and each point on both sides of the RIP peak represents a volatile organic compound. The brightness of the point represents the concentration of the compound, and the brighter the point, the higher the content. In order to more clearly show the differences in the volatile components of the raw materials and products of the enzymatic reaction system 1, the fingerprint comparison of all the peaks is shown in Table 4. Fig. 5 As shown in Table 4, each row represents all the signal peaks selected from a sample, and each column represents the signal peaks of the same volatile organic compound in different samples. It can be clearly seen that more than 10 new volatile compounds are generated by the enzymatic reaction of the crude γ-GTP enzyme solution and the water-soluble flavor precursor substrate extract.

[0058] Example 2

[0059] A method for preparing a characteristic flavor enhancer of Toona sinensis based on an enzymatic reaction, which specifically comprises the following steps:

[0060] (1) The preparation of the flavor precursor substrate extract is performed according to step (1) of Example 1;

[0061] The obtained flavor precursor substrate extract was dissolved in deionized water to 1 g / ml, first column-purified by 001 x 7 strong acid styrene cation exchange resin, and then gradient eluted by 0.3 M, 0.6 M, 0.9 M, 1.2 M, 1.5 M and 2.0 M NH4OH aqueous solution, respectively, and the eluate corresponding to 0.6 M NH4OH was collected and freeze-dried at -40℃ and 0.01 KPa for 40 h to obtain a brown solid. Then the brown solid was column-purified by ENVI-18 solid phase extraction column, and gradient eluted by 0, 5%, 10%, 25%, 50% and 100% MeCN aqueous solution, respectively, and the eluate corresponding to 0, 5% MeCN was collected and combined, and then freeze-dried at -40℃ and 0.01 KPa for 40 h to obtain a light yellow solid, which was the purified flavor precursor substrate.

[0062] (2) The extraction of the crude γ-GTP enzyme solution of Chinese toon was carried out according to the reference example 1.

[0063] At 4℃, ammonium sulfate was added to the crude γ-GTP enzyme solution of Chinese toon to a saturation degree of 40%, and then the solution was left to stand overnight (12 h), and the precipitated impure proteins were removed by centrifugation at 4℃, and the supernatant was collected and ammonium sulfate was added to the supernatant to a final saturation degree of 70%, and then the solution was left to stand overnight (12 h), and the precipitate was collected by centrifugation at 4℃, and the target protein was dissolved in Tris-HCl buffer (pH 7.0, 0.1 M). Then the target protein was dialyzed in a dialysis bag with a molecular weight cut-off of 8-14 KDa, and the dialysate was freeze-dried at -40℃ and 0.01 KPa for 40 h to obtain the purified γ-GTP enzyme of Chinese toon (specific enzyme activity: 1.5 units / mg).

[0064] (3) The flavor precursor substrate was prepared into a 0.1 g / ml aqueous solution, and the γ-GTP enzyme of Chinese toon obtained in the above step was prepared into a 5 mg / ml solution by using Tris-HCl buffer (pH 7.0, 0.1 M). Then 0.2 ml of the flavor precursor substrate aqueous solution and 0.2 ml of the γ-GTP enzyme solution were precisely measured and added into a 20 mL brown headspace bottle with a silica gel pad, and then the bottle was sealed and reacted at 40℃ in a water bath for 30 min under pH 7 to construct an enzyme reaction system 2, thereby preparing a Chinese toon characteristic flavor enhancer.

[0065] In this example, the sulfur-containing volatile components in the flavor precursor substrate aqueous solution, the purified γ-GTP enzyme solution and the enzyme reaction system 2 were detected and analyzed by GC-MS, and the results are shown in Table 3.

[0066] Table 3 Comparison and analysis of GC-MS results of sulfur-containing volatile components in the enzyme reaction system 2 and raw materials

[0067]

[0068]

[0069] Note: "-" represents not detected.

[0070] The volatile components of the γ-GTP purified enzyme solution, the flavor precursor substrate aqueous solution, and the enzymatic reaction system 2 were detected and analyzed by using the HS-SPME-GC-MS combined technology. The detection results of sulfur-containing volatile components are shown in Table 2: only 2,4-dimethylthiophene (4.24 μg / g) was detected in the γ-GTP purified enzyme solution sample, and only 2,4-dimethylthiophene (0.34 μg / g) and 3,4-dimethylthiophene (0.34 μg / g) were detected in the flavor precursor substrate aqueous solution; and 17 sulfur-containing compounds were detected in the enzymatic reaction system 2, including the characteristic aroma components Z-2-mercapto-3,4-dimethyl-2,3-dihydrothiophene (63.57 μg / g), E-2-mercapto-3,4-dimethyl-2,3-dihydrothiophene (47.14 μg / g), (E,Z)-bis-(1-propenyl) disulfide (0.66 μg / g), (E,E)-bis-(1-propenyl) trisulfide (11.62 μg / g), and the thermal cracking products of the characteristic sulfur-containing compounds of Chinese toon, such as 3,4-dimethylthiophene (156.42 μg / g), and the total amount of sulfur-containing compounds was as high as 334.24 μg / g. The results show that the reaction of the γ-GTP purified enzyme solution and the flavor precursor substrate aqueous solution can synthesize high-content characteristic aroma components of Chinese toon containing sulfur, which confirms that the method is feasible and can prepare the characteristic flavor enhancer of Chinese toon.

[0071] Comparative Example 1

[0072] A method for preparing characteristic flavor substances of Chinese toon based on an enzymatic reaction, which specifically comprises the following steps:

[0073] (1) The preparation step of the purified flavor precursor substrate refers to step (1) of Example 2;

[0074] (2) The commercial γ-glutamyl transpeptidase (sigma company, 5-12 units / mg, derived from horse kidney) was prepared into an enzyme solution of 1 mg / ml by using a Tris-HCl buffer of pH 7.0 and 0.1 M.

[0075] (3) The flavor precursor substrate separated and purified in the above step was prepared into an aqueous solution of 0.1 g / ml, then 0.2 ml of the flavor precursor substrate aqueous solution and 0.2 ml of the enzyme solution were precisely measured and added into a 20 mL brown headspace bottle with a silica gel pad, which was sealed and reacted at 40°C in a water bath for 30 min under the condition of pH 7 to construct an enzymatic reaction system 3, thereby preparing the characteristic flavor substances of Chinese toon.

[0076] The GC-MS was used to detect and analyze the sulfur-containing volatile components in the enzymatic reaction system 3, and the results are shown in Table 4.

[0077] Table 4 GC-MS results of volatile components in the enzymatic reaction system 3

[0078]

[0079] The HS-SPME-GC-MS combined technology was used to detect and analyze the volatile components in the enzymatic reaction system 3, and the detection results are shown in Table 4: four volatile components were detected in the enzymatic reaction system 3, including the characteristic aroma components Z-2-mercapto-3,4-dimethyl-2,3-dihydrothiophene (23.77 μg / g) and E-2-mercapto-3,4-dimethyl-2,3-dihydrothiophene (17.55 μg / g) unique to fresh Chinese toon, the thermal cracking product 3,4-dimethylthiophene (49.03 μg / g) of the characteristic sulfur-containing compound of Chinese toon, and 3,5-dimethylbenzaldehyde (0.68 μg / g), and the total amount of sulfur-containing compounds was 90.35 μg / g. The results show that the commercial γ-GTP enzymes of different species and the purified Chinese toon flavor precursor substrate can still undergo enzymatic reaction to synthesize the sulfur-containing key characteristic aroma components of Chinese toon, which confirms that the method for preparing the characteristic flavor of Chinese toon based on the enzymatic reaction has high feasibility and can successfully prepare the characteristic flavor of Chinese toon.

Claims

1. A method for preparing a characteristic flavor enhancer of Chinese toon based on an enzymatic reaction, characterized by, Comprising the following steps: 1) Preparation of flavor precursor substrate or extract: Add 3-8% trifluoroacetic acid aqueous solution to fresh or frozen Chinese toon shoots, crush and inactivate in a cell disrupter for 10-30 min, add deionized water at 30-50℃ and ultrasonically extract for 30-60 min, then centrifuge and concentrate to obtain a crude flavor precursor substrate aqueous solution, successively add Sevage reagent, petroleum ether and ethanol to remove impurities, concentrate, freeze-dry to obtain a flavor precursor substrate extract; Or, redissolve the obtained flavor precursor substrate extract in water, successively separate and purify with cation exchange resin and solid phase extraction column to obtain a purified flavor precursor substrate; 2) Preparation of purified γ-glutamyl transpeptidase: Under the condition of 2-8℃, add ammonium sulfate to the γ-glutamyl transpeptidase crude enzyme solution to a saturation degree of 40-45%, stand overnight, centrifuge at 2-8℃, take the supernatant and add ammonium sulfate to a saturation degree of 65-70%, stand overnight, centrifuge at 2-8℃, collect the precipitate target protein, dissolve with Tris-HCl buffer, and then dialyze with a dialysis bag with a molecular weight cut-off of 8-14 KDa, freeze-dry the dialysate to obtain purified γ-glutamyl transpeptidase; The γ-glutamyl transpeptidase crude enzyme solution is prepared by the following steps: vacuum freeze-drying fresh and intact Chinese toon shoots, crushing and passing through a 40-80 mesh sieve to obtain freeze-dried Chinese toon powder, adding an appropriate amount of freeze-dried Chinese toon powder to a buffer and grinding for 10-20 min, then ice-bath ultrasonic treatment for 30-60 min, and centrifuging at 2-8℃ to obtain the γ-glutamyl transpeptidase crude enzyme solution; The buffer is a Tris-HCl buffer with a concentration of 40-60 mM, pH 7-9, and containing 8-12% glycerol, 18-22 μM pyridoxal phosphate, 1-2 mM EDTA and 4-6 mM β-mercaptoethanol; 3) Preparation of Chinese toon characteristic flavor enhancer based on enzymatic reaction: prepare the flavor precursor substrate extract or the flavor precursor substrate into an aqueous solution with a concentration of 0.1-0.5 g / ml, prepare the purified γ-glutamyl transpeptidase into a solution with a concentration of 1-5 mg / ml with Tris-HCl buffer, then mix equal volumes and react under sealed conditions at 30-50℃ and pH 7-8 for 30-60 min.

2. The method of claim 1, wherein the preparation of the characteristic flavor enhancer of Prinsepia utilis based on an enzymatic reaction is characterized by, In step 1), the mass ratio of fresh or frozen Chinese toon shoots to trifluoroacetic acid aqueous solution is 1:1-2.

3. The method of claim 1, wherein the preparation of the characteristic flavor enhancer of Prinsepia utilis based on enzymatic reaction is characterized by, In step 1), the amount of deionized water added is 10-20 times the mass of fresh or frozen Chinese toon shoots.

4. The method of claim 1, wherein the preparation of the characteristic flavor enhancer of Prinsepia utilis based on enzymatic reaction is characterized by, The freeze-dried Chinese toon powder and the buffer are added in a solid-liquid ratio of 1 g:15-20 ml.

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