Food flavoring compound N-phenylacryloyl amino acid as well as preparation method and application thereof

Abstract

The invention discloses a food flavoring compound N-phenylacryloyl amino acid as well as a preparation method and application thereof. The N-phenylacryloyl amino acid comprises a compound of # imgabs0 # or an edible salt and an edible ester thereof. The preparation method comprises a chemical synthesis method and an enzymatic synthesis method. The N-phenylacryloyl amino acid prepared by the invention can obviously enhance the taste of food and reduce the bitter taste of the food under the addition amount of 1-100mg / L, and can be used as a food additive or flavoring agent for endowing, modifying and / or enhancing one, two or all flavors of sour taste, salty taste, palatable taste and strong taste.

Description

Technical Field

[0001] The invention belongs to the field of flavor substance synthesis, and specifically relates to an N-phenylacryloylamino acid for imparting, modifying and / or enhancing one, two or all of the flavors of sourness, saltiness, umami and richness, and a chemical and enzymatic preparation method thereof. Background Art

[0002] N-phenylacryloyl amino acid compounds are acylated products formed by cinnamic acid and its derivatives connected with amino acids through amide bonds. They are secondary metabolites naturally present in plants. Caffeic acid, ferulic acid, cinnamic acid, p-coumaric acid and amino acid-bound compounds are found in plants such as Dalbergiamelanoxylon, Theobroma cacao, green coffee beans, Abrus mollis Hance, and Althaea officinalis. Cinnamic acid compounds are safe and non-toxic, and have multiple biological activities such as antioxidant, antimicrobial, tyrosinase inhibition, α-glucosidase inhibition, and anti-neuroinflammation. They have broad application prospects in the fields of medicine, food, and cosmetics.

[0003] Amide derivatives can produce many tastes and / or chemical stimulations in the oral cavity. Various cinnamic acid-derived amides naturally present in plants have a variety of taste activities. Vanillin amides (such as capsaicin or piperine) present in pepper or chili have a spicy taste. Hydroxybenzoic acid benzylamine amide synthesized from the herb santa and evaluated as a masking agent for the bitterness of caffeine by sensory methods. N-phenylacryloyl amino acids have also been confirmed to be the key component of cocoa astringency and are considered to be the key flavor compound for the persistent shrinkage, astringency, and dry taste in the oral cavity, which can enhance the complexity and mouthfeel of cocoa drinks and chocolate candies. Patent CN102940224 A discloses a cinnamic acid amide substance that enhances umami, salty and kochi taste, which can be used as a taste imparting or modifying component, and can impart, enhance, improve or modify the umami, salty and kochi taste of condiments. Cinnamic acid-derived amides can also enhance the persistence of sweetness, fat, butter aroma and flavor. Despite this, the role of NPA in enhancing taste perception, including umami, saltiness, and kokumi, remains largely unstudied.

[0004] In recent years, with the booming market of flavor enhancing compounds, the attention and demand for new flavor enhancing compounds have risen sharply. As a naturally occurring taste-active substance, it has become an urgent task to explore efficient and safe synthesis methods for N-phenylacryloylamino acids. At present, the synthesis of these compounds mainly relies on chemical synthesis or enzymatic reactions. The key to the synthesis of N-phenylacryloylamino acids lies in the formation of amide bonds. The Schotten-Baumann reaction is a traditional method for synthesizing amide compounds. Products are obtained by nucleophilic substitution reactions between cinnamic acid chlorides and various amino acids under alkaline conditions. Chemical synthesis is simple to operate and has high efficiency in producing target compounds, but it often involves the use of harmful chemical reagents, which may bring environmental pollution and safety risks. In contrast, enzymatic synthesis methods have become a hot topic of research due to their environmental protection and high efficiency. However, the enzymatic synthesis technology of phenylacryloylamino acids has not been systematically studied. With the development of green synthesis, efficient and highly selective enzyme-catalyzed synthesis methods are the current challenges and future development trends. Some naturally occurring flavor enhancers are widely favored because of their absolute safety. Adding these flavor compounds to food can not only enhance the taste intensity and persistence of food, but also improve its fullness and richness, which is particularly important for the high-end seasoning market. Therefore, how to efficiently, environmentally friendly and safely produce such natural flavor enhancers in the food seasoning industry has become a major challenge that needs to be solved in the field of food science. The synthesis and flavoring research of N-phenylacryloyl amino acids has pointed out an important research direction for its in-depth development and expansion in the field of food applications, and laid a solid theoretical foundation for its future industrial production. Summary of the invention

[0005] Based on the above content, the main purpose of the present invention is to provide a new food flavor enhancing compound N-phenylpropenoyl-L-amino acids (NPAs) and a preparation method thereof, so as to solve the technical problem that the prior art cannot safely produce N-phenylpropenoyl-L-amino acids that can be used in the food field and have enhanced or improved food flavor properties.

[0006] Another object of the present invention is to provide the taste characteristics of the N-phenylacryloylamino acid compound and an evaluation method thereof.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A chemical preparation method of a novel food flavor enhancing compound N-phenylacryloylamino acid comprises the following steps:

[0009] Dissolve cinnamic acid containing phenolic hydroxyl groups in an aqueous sodium hydroxide solution under an ice bath, stir until completely dissolved, and slowly drop acetic anhydride into the solution. After the dropwise addition is completed, naturally warm up to room temperature and stir to react. After the reaction is completed, adjust the pH of the reaction solution to acidity with concentrated hydrochloric acid or concentrated sulfuric acid, precipitate a white solid, filter under reduced pressure, wash with water several times, then recrystallize with anhydrous ethanol, and dry to obtain acetylated hydroxycinnamic acid.

[0010] Cinnamic acid carboxylic acid or acetylated hydroxycinnamic acid, thionyl chloride and chloroform are mixed, DMF is added dropwise, and the mixture is heated and stirred for reaction. After the reaction is completed, the mixture is cooled to room temperature, and the solvent and excess thionyl chloride are removed by rotary evaporation under reduced pressure to obtain the corresponding acyl chloride.

[0011] The amino acid is dissolved in an aqueous solution of sodium hydroxide, stirred until completely dissolved, the prepared acid chloride is dissolved in acetone under an ice bath and then slowly added to the amino acid sodium solution, and the sodium hydroxide aqueous solution is added to keep the pH of the reaction solution alkaline. After the addition is complete, the temperature is naturally raised to room temperature and stirred for reaction. For acetylated hydroxycinnamic acid, sodium hydroxide is added to the reaction solution after the reaction is completed to hydrolyze the acetyl group. After the reaction is completed, acetone is removed by vacuum rotary evaporation, and the pH of the remaining aqueous solution is adjusted to acidic with concentrated hydrochloric acid or concentrated sulfuric acid to precipitate a yellow oily liquid or a white solid. Extract with ethyl acetate and wash with water several times. Finally, the obtained organic layer solution is subjected to rotary evaporation to remove ethyl acetate, and purified N-phenylacryloylamino acid is obtained after drying.

[0012] Furthermore, the cinnamic acid carboxylic acid includes, but is not limited to: cinnamic acid, o-hydroxycinnamic acid, p-hydroxycinnamic acid (p-coumaric acid), m-hydroxycinnamic acid, o-methoxycinnamic acid, m-methoxycinnamic acid, p-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 3,4-dihydroxycinnamic acid (caffeic acid), 3-methoxy-4-hydroxycinnamic acid (ferulic acid), and 4-hydroxy-3,5-dimethoxycinnamic acid (sinapic acid).

[0013] Furthermore, the amino acids include, but are not limited to, any one of alanine, cysteine, aspartic acid, phenylalanine, glutamic acid, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, proline, glycine, ornithine, theanine, sarcosine, γ-aminobutyric acid or β-alanine.

[0014] Furthermore, the molar mass ratio of the hydroxycinnamic acid to the acetic anhydride is related to the number of phenolic hydroxyl groups contained in the cinnamic acid, (molar mass of hydroxycinnamic acid×number of phenolic hydroxyl groups): molar mass of acetic anhydride=0.5-0.8.

[0015] Furthermore, the reaction time of the acetylation of hydroxycinnamic acid is 30 min-300 h.

[0016] Furthermore, after the acetylation reaction of the hydroxycinnamic acid, the pH of the remaining aqueous solution is adjusted to 1-3 with concentrated hydrochloric acid or concentrated sulfuric acid.

[0017] Furthermore, the molar mass ratio of the hydroxycinnamic acid carboxylic acid or acetylated hydroxycinnamic acid to thionyl chloride is 1.2-2.0.

[0018] Furthermore, the reaction time of the acyl chloride is 5-24h.

[0019] A method for preparing a novel food flavor enhancing compound N-phenylacryloylamino acid by enzymatic method comprises the following steps:

[0020] Cinnamic acid carboxylic acid or its sodium / potassium salt, amino acid or its sodium salt are mixed with water, pH is adjusted, food-grade enzyme is then added and placed in a constant temperature shaker for reaction, enzyme is inactivated after the reaction is completed, the obtained reaction mixture is acidified, extracted with ethyl acetate to obtain an organic layer, and washed with water for multiple times, and finally the obtained organic layer solution is subjected to rotary evaporation to remove ethyl acetate, and purified N-phenylacryloyl amino acid is obtained after drying.

[0021] Furthermore, the cinnamic acid carboxylic acid or its sodium / potassium salt includes, but is not limited to: cinnamic acid, o-hydroxycinnamic acid, p-hydroxycinnamic acid (p-coumaric acid), m-hydroxycinnamic acid, o-methoxycinnamic acid, m-methoxycinnamic acid, p-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 3,4-dihydroxycinnamic acid (caffeic acid), 3-methoxy-4-hydroxycinnamic acid (ferulic acid), 4-hydroxy-3,5-dimethoxycinnamic acid (sinapic acid) and their sodium salt or potassium salt.

[0022] Furthermore, the amino acid or its sodium salt includes, but is not limited to, any one of alanine, cysteine, aspartic acid, phenylalanine, glutamic acid, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, proline, glycine, ornithine, theanine, sarcosine, γ-aminobutyric acid or β-alanine.

[0023] Furthermore, the molar mass ratio of the hydroxycinnamic acid to the acetic anhydride is related to the number of phenolic hydroxyl groups contained in the cinnamic acid, (molar mass of hydroxycinnamic acid×number of phenolic hydroxyl groups): molar mass of acetic anhydride=0.5-0.8.

[0024] Furthermore, the molar mass ratio of the cinnamic acid carboxylic acid to the amino acid is 0.1-20.

[0025] Furthermore, the food-grade enzyme is any one of transglutaminase, transglutaminase, pancreatin, aminoacylase, glutaminase Sumizyme GT, protease Sumizyme FLAP-G, dipeptidase Sumizyme DPP-G or protease Sumizyme FP-G, or a combination thereof.

[0026] Furthermore, the enzymatic hydrolysis pH is 6.0-10.0.

[0027] Furthermore, the dosage of the food-grade enzyme is 0.2% to 5.0% w / v.

[0028] Furthermore, the enzymolysis temperature is 25 to 55° C., and the enzymolysis time is 6 to 240 hours.

[0029] Furthermore, the enzyme inactivation temperature is between 80 and 100° C., and the time is between 15 and 25 minutes.

[0030] The novel food flavor enhancing compound N-phenylacryloylamino acid obtained by the preparation method can significantly enhance the umami, salty and thick taste of food and reduce the bitterness of food at an addition amount of 1-100 mg / L.

[0031] A novel food flavor enhancing compound N-phenylacryloylamino acid for imparting, modifying and / or enhancing sourness, saltiness, umami and richness is prepared by one of the above methods, and the structural formula is as follows:

[0032]

[0033] R 1 , R 2 , R 3 , R 4 each independently represents hydrogen, hydroxyl or methoxy,

[0034] - R5 represents an amino acid residue, in particular a residue of a proteinogenic amino acid, ornithine, theanine, sarcosine, γ-aminobutyric acid or β-alanine.

[0035] The present invention also provides a sensory evaluation method for the taste characteristics of the N-phenylacryloylamino acid, wherein the sensory evaluation flavors involved include but are not limited to umami, salty, thick and bitter.

[0036] The N-phenylacryloylamino acid prepared by the present invention can significantly enhance the taste of food (including sour taste, umami taste, salty taste, and rich taste) and reduce the bitterness of food at an addition amount of 1 to 100 mg / L, and can be used as a food additive or flavor enhancer for imparting, modifying and / or enhancing one, two or all of the flavors of sour taste, salty taste, umami taste and rich taste. Suitable examples of these substances include N-cinnamoyltryptophan, N-cinnamoyltyrosine, N-cinnamoylphenylalanine, N-p-coumaroyltyrosine, N-p-coumaroyltryptophan, N-p-coumaroylphenylalanine, N-p-methoxycinnamoyltryptophan, N-p-methoxycinnamoylhistidine, N-p-methoxycinnamoyltyrosine, N-p-methoxycinnamoylphenylalanine, N-caffeoyltyrosine, N-caffeoylphenylalanine, N-caffeoyltryptophan, N-3,4-dimethoxycinnamoyltyrosine, N-3,4-dimethoxycinnamoyl phenylalanine, N-3,4-dimethoxycinnamoyl proline, N-3,4-dimethoxycinnamoyl histidine, N-3,4-dimethoxycinnamoyl arginine, N-3,4-dimethoxycinnamoyl tryptophan, N-feruloyl histidine, N-feruloyl tryptophan, N-feruloyl tyrosine, N-feruloyl phenylalanine, N-sinapoyl tryptophan, N-sinapoyl tyrosine, N-sinapoyl phenylalanine, edible salts thereof and edible esters thereof.

[0037] The present invention synthesizes N-phenylacryloylamino acid with enhanced food taste characteristics by using specific food-grade enzymes under specific enzymatic reaction conditions, and evaluates its taste characteristics by using time-intensity sensory evaluation method (TI), temporary sensory dominance method (TDS) and time-suitability item selection method (TCATA). The N-phenylacryloylamino acid prepared by the present invention has good food taste enhancement characteristics, and can significantly enhance the umami, salty and thick taste of food and reduce bitterness at an extremely low addition amount of 1 to 100 mg / L. At the same time, the preparation method of the present invention is simple and efficient, and solves the technical problem that the prior art cannot safely prepare food-grade N-phenylacryloylamino acid.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] (1) The food-grade enzyme used in the N-phenylacryloylamino acid synthesis process of the present invention complies with relevant food safety standards, poses no risk to human health, and has the characteristic of enhancing the flavor of food.

[0040] (2) The N-phenylacryloyl amino acid prepared by the present invention has the characteristic of enhancing the taste of food, can be used as a food additive or flavor enhancer, and has a good application prospect in the food field.

[0041] (3) The present invention has the advantages of simple process and low production cost.

[0042] (4) The N-cinnamoyltryptophan prepared by the present invention can significantly enhance the umami, salty and kochi taste of food and reduce bitterness at 1-100 mg / L; 1-100 mg / L N-cinnamoyltyrosine can significantly increase the umami and salty taste of food and prolong the duration of umami; 1-100 mg / L N-cinnamoylphenylalanine can significantly enhance the umami intensity and the perception frequency of umami in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the HPLC-MS / MS of cinnamoyltyrosine synthesized by chemical method in Example 1.

[0044] Figure 2 HPLC chromatograms of cinnamic acid, tyrosine and cinnamoyltyrosine.

[0045] Figure 3 HPLC chromatogram of p-coumaric acid, tryptophan and p-coumaryltryptophan in the chemical synthesis system in Example 2.

[0046] Figure 4 This is the HPLC-MS / MS of cinnamoyltryptophan after enzymatic synthesis and purification in Example 8.

[0047] Figure 5 HPLC chromatograms of cinnamic acid, tryptophan and cinnamoyltryptophan.

[0048] Figure 6 This is the HPLC-MS / MS of cinnamoylphenylalanine after enzymatic synthesis and purification in Example 9.

[0049] Figure 7 HPLC chromatograms of cinnamic acid, phenylalanine and cinnamoylphenylalanine.

[0050] Figure 8 HPLC chromatogram of p-coumaric acid, tryptophan, and p-coumaryltryptophan in the enzymatic synthesis system in Example 10.

[0051] Fig. 9 This is the HPLC chromatogram of ferulic acid, phenylalanine, and feruloylphenylalanine in the enzymatic synthesis system in Example 12.

[0052] Fig.10 This is a diagram showing the flavor enhancement effect of cinnamoyl tyrosine measured using the quantitative descriptive analysis (QDA) method in Example 17.

[0053] Fig.11 This is the saltiness intensity TI curve of p-coumaryltryptophan in Example 18 in the simulated chicken soup system.

[0054] Fig.12 This is the TI curve of the umami intensity of p-coumaryltryptophan in Example 18 in the simulated chicken soup system.

[0055] Fig.13 This is the sourness intensity TI curve of cinnamoyl phenylalanine in Example 19 in the sour solution.

[0056] Fig.14 and Fig.15 This is the TDS curve of the feruloylphenylalanine in Example 20 in the model solution.

[0057] Fig.16 This is the TCATA curve of feruloylphenylalanine in Example 20 in the model solution.

[0058] Fig.17 This is the TCATA curve of feruloylphenylalanine in Example 20 in the model solution. DETAILED DESCRIPTION

[0059] The technical scheme of the present invention is further described below by specific embodiments. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included within the scope of the present invention. The method and application of the present invention have been described by preferred embodiments, and those skilled in the art can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0060] Example 1 Chemical Synthesis of Cinnamoyl Tyrosine

[0061] Dissolve 0.0100 mol of cinnamoyl chloride in 5.0 mL of acetone for later use. Weigh 0.0150 mol of tyrosine, 20.0 mL of sodium hydroxide solution (1.5 mol / L) and 30.0 mL of acetone and mix them separately. Slowly drop cinnamoyl chloride and sodium hydroxide solution (0.5 mol / L) in an ice bath to adjust the solution to alkaline. After the addition is complete, warm to room temperature and continue to stir and react for 4 hours. Evaporate the acetone under reduced pressure, cool, and drop dilute hydrochloric acid to adjust the pH of the solution to 2.0. Solids precipitate, filter, wash, and dry to obtain a yellow oily liquid. After extracting with ethyl acetate three times, combine the organic phases (15 mL×3), wash the organic phases with water, dry them with anhydrous magnesium sulfate, filter, and remove ethyl acetate by rotary evaporation. After drying, cinnamoyl tyrosine is obtained.

[0062] UPLC-ESI-MS / MS detection conditions: mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, and the gradient elution program was as follows: 0-1 min, 80% A; 1-4.5 min, 80%-45% A; 4.5-5.5 min, 45% A; 5.5-7.5 min, 45%-80% A; 7.5-8.5 min, 80% A. The flow rate was 0.4 mL / min; the column temperature was 30 °C; the injection volume was 10 μL; and the initial mobile phase ratio was used for 5 min before injection. Mass spectrometry conditions: electrospray ion source was used, the mass spectrometry scanning mode was positive ion scanning mode, the capillary voltage was 3200 V, the desolvation gas (nitrogen), the drying temperature was 220°C, the desolvation gas flow rate was 8 L / min, the dryer temperature was 220°C, the scanning range was m / z 50-1300, the collision gas was argon, and the chromatographic column was ACQUITYUPLC BEH C18 column (2.1 mm×100 mm, 1.7 μm, Waters).

[0063] HPLC detection conditions: The mobile phases were 0.1% (v / v) trifluoroacetic acid-acetonitrile solution (A) and 0.1% (v / v) trifluoroacetic acid-water solution (B), respectively, and the gradient elution method was adopted, and the elution program was: 0min: 85% B+15% A; 25min: 65% B+35% A; 30min: 65% B+35% A; 35min: 85% B+15% A; end elution. Detection was performed using an LC-UV100 ultraviolet detector (Wufeng, Guangzhou), with a detection wavelength of 220nm, a column temperature of 30°C, an injection volume of 10μL, and a flow rate of 1.0mL / min.

[0064] Chromatographic column: Shodex C18-120-5 4E, length: 250 mm, inner diameter: 4.6 mm.

[0065] The purified cinnamoyltyrosine was qualitatively analyzed by UPLC-ESI-MS / MS, and the peak times of cinnamoyltyrosine, cinnamic acid and tyrosine were compared by HPLC.

[0066] Depend on Figure 1 and Figure 2 It can be seen that cinnamoyl tyrosine was successfully synthesized by chemical method.

[0067] Example 2 Chemical Synthesis of Coumaroyl Tryptophan

[0068] (1) Preparation of acetyl p-coumaric acid: Add 64 mmol of sodium hydroxide, 20 mmol of p-coumaric acid and 20 mL of water to a 150 mL single-necked flask under an ice bath and stir. After the solution is completely clarified, add 26 mmol of acetic anhydride to the solution using a constant pressure dropping funnel. Control the dropping speed and temperature. After the addition is completed, naturally warm to room temperature and stir to react for 1 hour. After the reaction is completed, add concentrated hydrochloric acid and adjust the pH of the solution to 2.0 to precipitate a large amount of white solid. After the solution is allowed to stand for 30 minutes, reduce the pressure and filter. The filter cake is washed with water twice and then recrystallized with anhydrous ethanol. After drying, a white powder, namely acetyl p-coumaric acid, is obtained.

[0069] (2) Preparation of acetyl-p-coumaroyl chloride: Add 10 mmol of prepared acetyl-p-coumaric acid, 2 mL of dithionyl chloride and 20 mL of chloroform to a 100 mL two-necked flask equipped with a reflux and tail gas absorption device, add 1-2 drops of DMF and heat to 50°C for stirring and reaction. The white solid gradually dissolves and the reaction is continued for 7-8 hours. After the reaction is completed, cool to room temperature and remove the solvent and excess dithionyl chloride by vacuum rotary evaporation to obtain white crystals, namely acetyl-p-coumaroyl chloride.

[0070] (3) Preparation of p-coumaryltryptophan: Add 10 mmol tryptophan, 10 mmol sodium hydroxide, and 15 mL of a mixture of acetone and water (volume ratio of 1:1) to a 100 mL two-necked flask. Add a magnetic stirrer until the solid is completely dissolved. Dissolve the prepared acetyl p-coumaryl chloride in 15 mL acetone and add it to a constant pressure dropping funnel. Add 15 mL of sodium hydroxide aqueous solution (10 mM) to another constant pressure dropping funnel. Lower the temperature to 0-5 °C under an ice bath. At the same time, slowly add the solutions in the two dropping funnels to the amino acid sodium solution, and control the dropping speed of the two to be equal. After the addition is completed, the temperature is maintained at 10-15 °C and the reaction is continued with stirring for 5 h. Add 40 mmol of sodium hydroxide solid to the reaction solution to hydrolyze the acetyl group, and raise the temperature to 30 °C for 2 h of hydrolysis reaction. After the reaction is completed, remove the acetone by vacuum rotary evaporation. Adjust the pH of the remaining aqueous solution to 1-2 with concentrated hydrochloric acid to precipitate a white solid. After extraction with ethyl acetate three times, the organic phases were combined (15 mL×3), washed with water, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was removed by rotary evaporation to obtain p-coumaryltryptophan.

[0071] The qualitative method is consistent with that in Example 1.

[0072] Depend on Figure 3 It can be seen that the reaction successfully synthesized p-coumaryltryptophan.

[0073] Example 3 Chemical Synthesis of Caffeoylleucine

[0074] (1) Preparation of acetyl caffeic acid: Add 64 mmol sodium hydroxide, 10 mmol caffeic acid and 20 mL water to a 150 mL single-necked flask under an ice bath and stir. After the solution is completely clarified, add 30 mmol acetic anhydride to the solution using a constant pressure dropping funnel. Control the dropping speed and temperature. After the addition is completed, naturally warm up to room temperature and stir to react for 6 hours. After the reaction is completed, add concentrated hydrochloric acid and adjust the pH of the solution to 1-2 to precipitate a large amount of solid. After the solution is allowed to stand for 30 minutes, reduce the pressure and filter. The filter cake is washed twice with an appropriate amount of water and then recrystallized with anhydrous ethanol. After drying, a light brown powder, namely acetyl caffeic acid, is obtained.

[0075] (2) Preparation of acetyl caffeoyl chloride: 10 mmol of prepared acetyl caffeic acid, 2 mL of dichlorothionyl and 25 mL of chloroform were added to a 100 mL two-necked flask equipped with a reflux and tail gas absorption device, and 1-2 drops of DMF were added and the temperature was raised to 60°C for stirring and reaction. The brown solid gradually dissolved and the reaction was continued for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent and excess dichlorothionyl were removed by vacuum rotary evaporation to obtain brown crystals, namely acetyl caffeoyl chloride.

[0076] (3) Preparation of caffeoylleucine: Add 10 mmol leucine, 10 mmol sodium hydroxide and 15 mL of a mixture of acetone and water (volume ratio of 1:1) to a 100 mL two-necked flask. Add a magnetic stirrer until the solid is completely dissolved. Dissolve the prepared acetyl caffeoyl chloride in 15 mL acetone and add it to a constant pressure dropping funnel. Add 15 mL of sodium hydroxide aqueous solution (10 mM) to another constant pressure dropping funnel. Under an ice bath, slowly add the solutions in the two dropping funnels to the amino acid sodium solution at the same time, and control the dropping speed of the two to be equal. After the addition is completed, the temperature is maintained at 10-15°C and the reaction is continued with stirring for 12 hours. Add 30 mmol of sodium hydroxide solid to the reaction solution, raise the temperature to 40°C and react for 5 hours to hydrolyze the acetyl group. After the reaction is completed, remove the acetone by vacuum rotary evaporation. Adjust the pH of the remaining aqueous solution to 1-2 with concentrated hydrochloric acid, and a brown-yellow oily liquid is precipitated. After extraction with ethyl acetate three times, the organic phases were combined (15 mL×3), washed with water, dried over anhydrous magnesium sulfate, filtered, and ethyl acetate was removed by rotary evaporation to obtain caffeoylleucine.

[0077] The qualitative method is consistent with that in Example 1.

[0078] Example 4 Chemical Synthesis of Feruloyl Histidine

[0079] (1) Preparation of acetylferulic acid: Add 64 mmol of sodium hydroxide, 20 mmol of ferulic acid and 20 mL of water to a 150 mL single-necked flask under an ice bath and stir. After the solution is completely clarified, add 26 mmol of acetic anhydride to the solution using a constant pressure dropping funnel. Control the dropping speed and temperature. After the addition is completed, naturally warm up to room temperature and stir to react for 2 h. After the reaction is completed, add concentrated hydrochloric acid and adjust the pH of the solution to 1-2 to precipitate a large amount of white solid. After the solution is allowed to stand for 30 minutes, reduce the pressure and filter. The filter cake is washed twice with an appropriate amount of water and then recrystallized with anhydrous ethanol. After drying, a white powder, namely acetylferulic acid, is obtained.

[0080] (2) Preparation of acetylferuloyl chloride: 10 mmol of the prepared acetylferuloyl acid, 2 mL of dithionyl chloride and 20 mL of chloroform were added to a 100 mL two-necked flask equipped with a reflux and tail gas absorption device, and 1-2 drops of DMF were added and the temperature was raised to 55°C and stirred for reaction. The white solid gradually dissolved and the reaction was continued for 7-8 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent and excess dithionyl chloride were removed by vacuum rotary evaporation to obtain yellow crystals, namely acetylferuloyl chloride.

[0081] (3) Preparation of feruloyl histidine: 10 mmol histidine, 10 mmol sodium hydroxide and 20 mL of a mixture of acetone and water (volume ratio of 1:1) were added to a 100 mL two-necked flask. A magnetic stirrer was added until the solid was completely dissolved. 10 mmol acetylferuloyl chloride was dissolved in 15 mL acetone and added to a constant pressure dropping funnel. 15 mL of sodium hydroxide aqueous solution (10 mM) was added to another constant pressure dropping funnel. Under an ice bath, the solutions in the two dropping funnels were slowly added dropwise to the amino acid sodium solution at the same time, and the dropping speeds of the two were controlled to be equal. After the addition was completed, the temperature was maintained at 10-15°C and the reaction was continued with stirring for 2 h. 50 mmol sodium hydroxide solid was added to the reaction solution, and the temperature was raised to 37°C for hydrolysis reaction to hydrolyze the acetyl group. After the reaction was completed, the acetone was removed by vacuum rotary evaporation. The remaining aqueous solution was adjusted to pH 1-2 with concentrated hydrochloric acid to precipitate a solid. After extraction with ethyl acetate three times, the organic phases were combined (15 mL×3), washed with water, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was removed by rotary evaporation to obtain feruloyl histidine.

[0082] The qualitative method is consistent with that in Example 1.

[0083] Example 5 Chemical Synthesis of 3,4-Dimethoxycinnamoylproline

[0084] (1) Preparation of 3,4-dimethoxycinnamoyl chloride: 20 mmol 3,4-dimethoxycinnamoyl chloride, 5 mL thionyl chloride and 50 mL chloroform were added to a 100 mL two-necked flask equipped with a reflux and tail gas absorption device. After adding 3-5 drops of DMF, the temperature was raised to 60°C and stirred for reaction. The white solid gradually dissolved and the reaction was continued for 12 hours. After the reaction was completed, the solvent and excess thionyl chloride were removed by vacuum rotary evaporation to obtain light yellow crystals, namely 3,4-dimethoxycinnamoyl chloride.

[0085] (2) Preparation of 3,4-dimethoxycinnamoylproline: Add 20 mmol of proline, 20 mmol of sodium hydroxide, and 50 mL of a mixture of acetone and water (volume ratio of 1:1) to a 250 mL two-necked flask. Add a magnetic stirrer until the solid is completely dissolved. Dissolve the prepared 3,4-dimethoxycinnamoyl chloride in 30 mL of acetone and add it to a constant pressure dropping funnel. Add 30 mL of sodium hydroxide aqueous solution (10 mM) to another constant pressure dropping funnel. Under an ice bath, slowly add the solutions in the two dropping funnels to the amino acid sodium solution at the same time, and control the dropping speeds of the two to be equal. After the addition is completed, the temperature rises to room temperature and the reaction is continued with stirring for 6 hours. After the reaction is completed, remove the acetone by vacuum rotary evaporation. Adjust the pH of the remaining aqueous solution to 1-2 with concentrated hydrochloric acid to precipitate a solid. After extraction with ethyl acetate three times, the organic phases were combined (15 mL×3), washed with water, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was removed by rotary evaporation to obtain 3,4-dimethoxycinnamoylproline.

[0086] The qualitative method is consistent with that in Example 1.

[0087] Example 6 Chemical Synthesis of p-Methoxycinnamoylglycine

[0088] (1) Preparation of p-methoxycinnamoyl chloride: 20 mmol p-methoxycinnamoyl chloride, 4 mL thionyl chloride and 40 mL chloroform were added to a 100 mL two-necked flask equipped with a reflux and tail gas absorption device. After adding 3-5 drops of DMF, the temperature was raised to 55°C and stirred for reaction. The white solid gradually dissolved and the reaction was continued for 10 hours. After the reaction was completed, the solvent and excess thionyl chloride were removed by vacuum rotary evaporation to obtain light yellow crystals, namely p-methoxycinnamoyl chloride.

[0089] (2) Preparation of p-methoxycinnamoyl glycine: Add 20 mmol of glycine, 20 mmol of sodium hydroxide, and 30 mL of a mixture of acetone and water (volume ratio of 1:1) to a 200 mL two-necked flask. Add a magnetic stirrer until the solid is completely dissolved. Dissolve the prepared p-methoxycinnamoyl chloride in 30 mL of acetone and add it to a constant pressure dropping funnel. Add 30 mL of sodium hydroxide aqueous solution (10 mM) to another constant pressure dropping funnel. Under an ice bath, slowly drop the solutions in the two dropping funnels into the amino acid sodium solution at the same time, and control the dropping speed of the two to be equal. After the addition is completed, the temperature is maintained at 25°C and the reaction is continued with stirring for 4 hours. After the reaction is completed, the acetone is removed by rotary evaporation under reduced pressure. The remaining aqueous solution is adjusted to pH 1-2 with concentrated hydrochloric acid to precipitate a light yellow solid. Extract with ethyl acetate three times and combine the organic phases (15 mL×3). After the organic phase is washed with water, it is dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate is removed by rotary evaporation to obtain p-methoxycinnamoyl glycine.

[0090] The qualitative method is consistent with that in Example 1.

[0091] Example 7 Chemical Synthesis of Sinapoylglutamic Acid

[0092] (1) Preparation of acetylated sinapinic acid: Add 35 mmol sodium hydroxide, 10 mmol sinapinic acid and 15 mL water to a 150 mL single-necked flask under an ice bath and stir. After the solution is completely clarified, add 13 mmol acetic anhydride to the solution using a constant pressure dropping funnel. Control the dropping speed and temperature. After the addition is completed, the temperature is naturally raised to room temperature and stirred for 3 hours. After the reaction is completed, concentrated hydrochloric acid is added dropwise to adjust the pH of the solution to 1-2 to precipitate a large amount of white solid. After the solution is allowed to stand for 30 minutes, it is filtered under reduced pressure. The filter cake is washed twice with an appropriate amount of water and then recrystallized with anhydrous ethanol. After drying, a yellow-brown powder, namely, acetylated sinapinic acid, is obtained.

[0093] (2) Preparation of acetyl sinapinic acid chloride: 10 mmol of the prepared acetyl sinapinic acid, 2 mL of dithionyl chloride and 20 mL of chloroform were added to a 100 mL two-necked flask equipped with a reflux and tail gas absorption device. After adding 1-2 drops of DMF, the temperature was raised to 55°C and stirred for reaction. The white solid gradually dissolved and the reaction was continued for 8 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent and excess dithionyl chloride were removed by vacuum rotary evaporation to obtain yellow crystals, namely acetyl sinapinic acid chloride.

[0094] (3) Preparation of sinapinic acid: Add 10 mmol glutamic acid, 20 mmol sodium hydroxide and 20 mL of a mixture of acetone and water (volume ratio of 1:1) to a 100 mL two-necked flask. Add a magnetic stirrer until the solid is completely dissolved. Dissolve the prepared acetyl sinapinic acid chloride in 15 mL acetone and add it to a constant pressure dropping funnel. Add 15 mL of sodium hydroxide aqueous solution (10 mM) to another constant pressure dropping funnel. Slowly add the solutions in the two dropping funnels to the amino acid sodium solution under an ice bath, and control the dropping speeds of the two to be equal. After the addition is completed, the temperature is maintained at 20 ° C and the reaction is continued with stirring for 8 h. Add 40 mmol of sodium hydroxide solid to the reaction solution and heat it to 37 ° C for 3 h to hydrolyze the acetyl group. After the reaction is completed, remove the acetone by vacuum rotary evaporation. Adjust the pH of the remaining aqueous solution to 1-2 with concentrated hydrochloric acid to precipitate a yellow oily liquid. After extraction with ethyl acetate three times, the organic phases were combined (15 mL×3), washed with water, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was removed by rotary evaporation to obtain sinapinic acid.

[0095] The qualitative method is consistent with that in Example 1.

[0096] Example 8 Enzymatic Preparation of Cinnamoyl Tryptophan

[0097] (1) Mixing tryptophan and water to a concentration of 160 mM, adding cinnamic acid to a molar mass ratio of acid to amino acid of 10, adjusting the pH to 7.0, adding 1% (w / v) solution of transglutaminase, reacting in a water bath shaker at 37° C. for 24 h, and inactivating the enzyme at 90° C. for 15 min to obtain a reaction mixture;

[0098] (2) The reaction mixture obtained in step (1) is adjusted to pH 2.0 with concentrated hydrochloric acid; extracted with an equal volume of ethyl acetate, then washed multiple times with an equal volume of water, and finally the obtained ethyl acetate layer solution is removed by rotary evaporation (55° C., 15 Bar), then 15 mL of deionized water is added for re-dissolution and freeze-dried (-70° C., 40 Pa, 48 h) to obtain purified cinnamoyl tryptophan.

[0099] The qualitative method is consistent with that in Example 1.

[0100] Depend on Figure 4 and Figure 5 It can be seen that cinnamoyltryptophan was successfully synthesized by enzymatic hydrolysis.

[0101] Example 9 Enzymatic Preparation of Cinnamoyl Phenylalanine

[0102] (1) Phenylalanine and water were mixed to a concentration of 40 mM, cinnamic acid was added to make the molar mass ratio of acid to amino acid 5, the pH was adjusted to 6.0, a 2% (w / v) solution of protease Sumizyme FP-G was added, the mixture was reacted in a water bath shaker at 45° C. for 30 h, and the enzyme was inactivated at 80° C. for 25 min to obtain a reaction mixture;

[0103] (2) The reaction mixture obtained in step (1) is adjusted to pH 2.0 with concentrated hydrochloric acid; extracted with an equal volume of ethyl acetate, then washed multiple times with an equal volume of water, and finally the obtained ethyl acetate layer solution is removed by rotary evaporation (55° C., 15 Bar), then 15 mL of deionized water is added for re-dissolution and freeze-dried (-70° C., 40 Pa, 24 h) to obtain purified cinnamoyl phenylalanine.

[0104] The qualitative method is consistent with that in Example 1.

[0105] Depend on Figure 6 and Figure 7 It can be seen that cinnamoylphenylalanine was successfully synthesized by enzymatic hydrolysis.

[0106] Example 10 Enzymatic Preparation of p-Coumarylphenylalanine

[0107] (1) Phenylalanine and water were mixed to a concentration of 50 mM, p-coumaric acid was added to make the molar mass ratio of acid to amino acid 15, the pH was adjusted to 8.0, a 1% (w / v) solution of dipeptidase Sumizyme DPP-G was added, the mixture was reacted in a water bath shaker at 37° C. for 48 h, and the enzyme was inactivated at 100° C. for 20 min to obtain a reaction mixture;

[0108] (2) The pH of the reaction mixture obtained in step (1) is adjusted to 2.0 with concentrated hydrochloric acid; extracted with an equal volume of ethyl acetate, then washed multiple times with an equal volume of water, and finally the obtained ethyl acetate layer solution is removed by rotary evaporation (55° C., 15 Bar), and then 15 mL of deionized water is added for re-dissolution and freeze-dried (-70° C., 40 Pa, 48 h) to obtain purified p-coumarylphenylalanine.

[0109] The qualitative method is consistent with that in Example 1.

[0110] Depend on Figure 8 It can be seen that the enzymatic hydrolysis reaction successfully synthesized p-coumarylphenylalanine.

[0111] Example 11 Enzymatic Preparation of Caffeoylmethionine

[0112] (1) mixing methionine and water to a concentration of 100 mM, adding caffeic acid to a molar mass ratio of acid to amino acid of 8, adjusting the pH to 7.0, adding 2.5% (w / v) pancreatic enzyme solution, reacting in a water bath shaker at 40° C. for 36 h, and inactivating the enzyme at 90° C. for 20 min to obtain a reaction mixture;

[0113] (2) The reaction mixture obtained in step (1) is adjusted to pH 2.0 with concentrated hydrochloric acid; extracted with an equal volume of ethyl acetate, then washed multiple times with an equal volume of water, and finally the obtained ethyl acetate layer solution is removed by rotary evaporation (55° C., 15 Bar), then 15 mL of deionized water is added for re-dissolution and freeze-dried (-70° C., 40 Pa, 24 h) to obtain purified caffeoylmethionine.

[0114] The qualitative method is consistent with that in Example 1.

[0115] Example 12 Enzymatic Preparation of Feruloyl Phenylalanine

[0116] (1) Phenylalanine and water were mixed to a concentration of 120 mM, ferulic acid was added to make the molar mass ratio of acid to amino acid 6, the pH was adjusted to 8.0, a 1.5% (w / v) solution of protease Sumizyme FLAP-G was added, the mixture was reacted in a water bath shaker at 50° C. for 96 h, and the enzyme was inactivated at 90° C. for 20 min to obtain a reaction mixture;

[0117] (2) The reaction mixture obtained in step (1) is adjusted to pH 2.0 with concentrated hydrochloric acid; extracted with an equal volume of ethyl acetate, then washed multiple times with an equal volume of water, and finally the obtained ethyl acetate layer solution is removed by rotary evaporation (55° C., 15 Bar), then 15 mL of deionized water is added for re-dissolution and freeze-dried (-70° C., 40 Pa, 24 h) to obtain purified feruloylphenylalanine.

[0118] The qualitative method is consistent with that in Example 1.

[0119] Depend on Fig. 9 It can be seen that the enzymatic hydrolysis reaction successfully synthesized feruloylphenylalanine.

[0120] Example 13 Enzymatic Preparation of p-Methoxycinnamoylarginine

[0121] (1) arginine and water were mixed to a concentration of 80 mM, p-methoxycinnamic acid was added to make the molar mass ratio of acid to amino acid 10, the pH was adjusted to 6.0, a 1% (w / v) solution of flavor protease was added, the mixture was reacted in a water bath shaker at 50° C. for 30 h, and the enzyme was inactivated at 80° C. for 25 min to obtain a reaction mixture;

[0122] (2) The reaction mixture obtained in step (1) is adjusted to pH 2.0 with concentrated hydrochloric acid; extracted with an equal volume of ethyl acetate, then washed multiple times with an equal volume of water, and finally the obtained ethyl acetate layer solution is removed by rotary evaporation (55° C., 15 Bar), then 15 mL of deionized water is added for re-dissolution and freeze-dried (-70° C., 40 Pa, 24 h) to obtain purified p-methoxycinnamoyl arginine.

[0123] The qualitative method is consistent with that in Example 1.

[0124] Example 14 Enzymatic Preparation of 3,4-Dimethoxycinnamoylvaline

[0125] (1) mixing valine with water to a concentration of 40 mM, adding p-methoxycinnamic acid to a molar mass ratio of acid to amino acid of 20, adjusting the pH to 8.0, adding 2% (w / v) papain solution, reacting in a water bath shaker at 37° C. for 48 h, and inactivating the enzyme at 90° C. for 20 min to obtain a reaction mixture;

[0126] (2) The reaction mixture obtained in step (1) is adjusted to pH 2.0 with concentrated hydrochloric acid; extracted with an equal volume of ethyl acetate, then washed multiple times with an equal volume of water, and finally the obtained ethyl acetate layer solution is removed by rotary evaporation (55° C., 15 Bar), then 15 mL of deionized water is added for re-dissolution and freeze-dried (-70° C., 40 Pa, 24 h) to obtain purified 3,4-dimethoxycinnamoyl valine.

[0127] The qualitative method is consistent with that in Example 1.

[0128] Example 15 Enzymatic Preparation of Sinapoyl Lysine

[0129] (1) Lysine and water were mixed to a concentration of 50 mM, p-methoxycinnamic acid was added to make the molar mass ratio of acid to amino acid 15, the pH was adjusted to 7.0, a 1% (w / v) solution of protease Debitrase HYW was added, the mixture was reacted in a water bath shaker at 45° C. for 48 h, and the enzyme was inactivated at 90° C. for 20 min to obtain a reaction mixture;

[0130] (2) The pH of the reaction mixture obtained in step (1) is adjusted to 2.0 with concentrated hydrochloric acid; extracted with an equal volume of ethyl acetate, then washed multiple times with an equal volume of water, and finally the ethyl acetate layer solution obtained is removed by rotary evaporation (55° C., 15 Bar), and then 15 mL of deionized water is added for re-dissolution and freeze-dried (-70° C., 40 Pa, 24 h) to obtain purified sinapinic lysine.

[0131] The qualitative method is consistent with that in Example 1.

[0132] The above embodiments 1-15 illustrate that the method of the present invention can synthesize a series of N-phenylacryloyl amino acids.

[0133] Example 16: Evaluation of the flavor enhancement effect of N-phenylacryloylamino acid using a qualitative descriptive analysis method (diluted soy sauce system):

[0134] The diluted soy sauce system was obtained by diluting 1.5 mL of soy sauce with ultrapure water to 100 mL. Different concentrations of N-phenylacryloyl amino acid (10 and 100 mg / L) were added to evaluate the flavor enhancement effect of N-phenylacryloyl amino acid. The evaluation scheme was as follows:

[0135] Sensory evaluator training: The sensory evaluation panel consisted of 12 sensory evaluators, including 6 males and 6 females, aged between 20 and 28 years old. The panelists had undergone 4 to 5 rounds of sensory training and had no known history of taste disorders. We used 5 mg / mL sucrose for sweetness, 0.8 mg / mL citric acid for sourness, 1 mg / mL NaCl for saltiness, 5 mg / L quinine for bitterness, and 1 mg / mL monosodium L-glutamate (MSG) for umami. In addition, 1 mg / mL glutathione was used to evaluate the kokumi taste and 1 g / L gallic acid was used to evaluate the astringency.

[0136] Qualitative descriptive analysis: In a tasting room under normal lighting and room temperature conditions, 12 sensory assessors (6 males and 6 females, aged between 20 and 28 years) evaluated the kokumi characteristics of diluted soy sauce sample solutions of different concentrations. Diluted soy sauce solutions with different concentrations of N-phenylacryloylamino acid (10 and 100 mg / L) were prepared. A blank diluted soy sauce solution was used as a standard reference sample.

[0137] The evaluation results are shown in Table 1. N-phenylacryloylamino acid can enhance the kokumi characteristics of the diluted soy sauce system at an addition amount of 10-100 ppm.

[0138] Table 1 Evaluation of the kokumi enhancement effect of N-phenylacryloyl amino acids

[0139]

[0140]

[0141] “√” indicates that N-phenylacryloyl amino acid has a kokumi effect on the diluted soy sauce system.

[0142] Example 17: Determination of the flavor enhancement effect of cinnamoyltyrosine using quantitative descriptive analysis (QDA)

[0143] The simulated sour, bitter and umami systems were simulated by aqueous solutions of citric acid (0.4 mg / mL), quinine (7.5 mg / L) and monosodium glutamate (1 mg / mL), respectively. Different concentrations of cinnamoyltyrosine (1, 10 and 100 mg / L) were added to evaluate the flavor enhancement effect of cinnamoyltyrosine. The evaluation scheme is as follows:

[0144] Sensory evaluator training: The sensory evaluation panel consisted of 12 sensory evaluators, including 6 males and 6 females, aged between 20 and 28 years old. The panelists had undergone 4 to 5 rounds of sensory training and had no known history of taste disorders. We used 5 mg / mL sucrose for sweetness, 0.8 mg / mL citric acid for sourness, 1 mg / mL NaCl for saltiness, 5 mg / L quinine for bitterness, and 1 mg / mL monosodium L-glutamate (MSG) for umami. In addition, 1 mg / mL glutathione was used to evaluate the kokumi taste and 1 g / L gallic acid was used to evaluate the astringency.

[0145] Quantitative descriptive analysis: In a tasting room under normal lighting and room temperature, 12 sensory assessors (6 males and 6 females, aged between 20 and 28 years old), all familiar with the 15-point scale, used 0-15 points to evaluate saltiness, umami and kochiness, with 0 points indicating that the sample had no taste and 15 points indicating that the sample had a strong taste. Solutions consisting of citric acid (0.4 mg / mL), quinine (7.5 mg / L) and monosodium glutamate (1 mg / mL) and different concentrations of cinnamoyltyrosine (1, 10 and 100 mg / L) were prepared. 0.4, 0.8, 1.2 mg / mL citric acid, 2.5, 5, 7.5 mg / mL quinine and 1, 2, 3 mg / mL monosodium glutamate solutions were used as standard reference samples.

[0146] Evaluation results such as Fig.10 As shown in the results, cinnamoyltyrosine can significantly enhance the sourness of citric acid solution, reduce the bitterness of quinine and increase the intensity of umami taste. With the increase of its addition amount, its enhancement of sourness and inhibition of bitterness also increase.

[0147] Example 18: Using the time intensity sensory evaluation method (TI) to evaluate the effect of p-coumaryl tryptophan on the umami intensity and saltiness intensity of the simulated chicken soup solution:

[0148] Sensory evaluation training: A total of 10 assessors (half male and half female) participated in the time intensity (TI) evaluation, with ages ranging from 20 to 28 years old. These assessors have undergone professional sensory training and have participated in the evaluation process of umami and saltiness many times. All participants are highly proficient in using the 15-point scoring standard to evaluate the intensity changes of food taste and are sensitive to the intensity changes of umami and saltiness.

[0149] Formal evaluation: Before the evaluation test, each sensory evaluation member was required to clean their mouth with drinking water. The simulated chicken soup composition included monosodium glutamate (1 mg / mL), sodium chloride (1 mg / mL), disodium flavor nucleotides (0.1 mg / mL), and additional different concentrations of p-coumaryl tryptophan (1, 10, and 100 mg / L, respectively). The evaluator placed 5 mL of the sample in the mouth and, according to the instructions, spit it out at the 5th second and recorded the perceived taste intensity at the 5th, 10th, 20th, 30th, 45th, 60th, 75th, 90th, 120th, 150th, 180th, and 210th seconds until no salty or umami taste could be perceived. After each sample was evaluated, the evaluator had a 10-minute interval during which the mouth was cleaned with 3% sucrose water, and the samples were evaluated in a random order. Finally, the sensory evaluation results were evaluated by fitting the time-intensity (TI) curve.

[0150] Depend on Fig.11 It can be seen that the CK curve of 0 mg / L is completely covered by the p-coumaryl tryptophan curve. The saltiness intensity increases rapidly in a short period of time and decreases slowly. The umami intensity of p-coumaryl tryptophan is significantly increased and stays in the mouth longer, proving that p-coumaryl tryptophan has the taste property of enhancing saltiness.

[0151] Depend on Fig.12 It can be seen that the p-coumaryl tryptophan curve has similar umami time characteristics, and the 0 mg / L curve is completely covered by the p-coumaryl tryptophan curve. The umami intensity increases rapidly in a short period of time and decreases slowly. The sample with 10 mg / L p-coumaryl tryptophan added has the most obvious increase in umami and stays in the mouth the longest, proving that p-coumaryl tryptophan has the taste characteristics of enhancing umami.

[0152] Example 19: Evaluation of the effect of cinnamoyl phenylalanine on sourness intensity using time intensity sensory evaluation (TI):

[0153] Sensory evaluation training: A total of 8 assessors (half male and half female) participated in the time intensity (TI) evaluation, with an age distribution between 20 and 28 years old. These assessors have undergone one year of professional sensory training and participated in the bitterness evaluation process. All participants have a high degree of proficiency in evaluating the intensity changes of food taste using a 15-point scoring scale and are sensitive to changes in bitterness intensity. Before the evaluation activities began, each assessor signed an informed consent form.

[0154] Formal evaluation: For 0.4, 0.8, and 1.2 mg / mL citric acid solutions, we used a 0 to 15-point scoring system to evaluate the intensity of sour taste, where 0 means the sample has no taste and 15 means the sample has a strong taste. The composition system of the sour solution was composed of 0.4 mg / mL citric acid and an additional 100 mg / L cinnamoylphenylalanine. The assessors placed 5 mL of the sample in the mouth and spit it out at 5 seconds after receiving the instruction. They were asked to evaluate the perceived taste intensity at 5, 10, 20, 30, 45, 60, 75, and 90 seconds until the sour taste could not be perceived. The evaluation interval between each sample was 10 minutes, during which the mouth was cleaned with 3% sucrose water, and the sample evaluation followed a random order. Finally, the sensory evaluation results were evaluated by fitting the time-intensity (TI) curve.

[0155] Depend on Fig.13 It can be seen that cinnamoyl phenylalanine can significantly enhance the sourness intensity of citric acid solution and prolong its duration. The 0 mg / L CK curve is completely covered by the 100 mg / L cinnamoyl phenylalanine TI curve. The sourness intensity increases rapidly in a short period of time and decreases slowly. The sourness intensity of the sample with cinnamoyl phenylalanine added is significantly enhanced, and it stays in the mouth for a longer time, proving that cinnamoyl phenylalanine has the taste property of enhancing sourness.

[0156] Example 20: Evaluation of the effect of feruloylphenylalanine on the taste attributes of model solutions using the transient sensory dominance method (TDS) and the time-appropriate item ticking method (TCATA):

[0157] Sensory evaluation staff training: 12 assessors aged 20-28 years old (6 males and 6 females). All assessors have no aversion to sourness, sweetness, bitterness, saltiness, and umami, and each assessor signed an informed consent form before the test.

[0158] All assessors had no experience with TDS and TCATA, and selected five taste attributes (sour, sweet, bitter, salty, and umami) for TDS and TCATA testing. In order to control the assessors' preference for selecting attributes based on their position in the list, the order of the attributes was randomized among assessors. During product training, assessors needed to be familiar with the following task details: first, assessors needed to be familiar with the position of each attribute on the screen; second, when tasting the sample, assessors had to taste the sample at the same time as clicking the start button; third, assessors had to taste the sample in the same way and had to spit out the sample in the same way; finally, assessors needed to be familiar with the approximate time when the attributes appeared and disappeared.

[0159] Formal evaluation: Evaluators were asked not to eat spicy food for at least 2 hours before any experiment and not to eat or drink any liquid except water within 30 minutes before the evaluation. At the beginning of the test, the evaluators were asked to familiarize themselves with the location of the 5 attributes on the screen again. According to the results of product training, the total evaluation time was set to 240 seconds. Data were collected using a sensory system (http: / / www.cloudsensorylab.com) and recorded every 1 second. Evaluators were instructed to press the start button after the sample was placed in the mouth, fill the mouth and immediately select the attribute. After 5 seconds, the evaluators received a voice instruction to spit out the sample. During the TDS test, the evaluators only needed one applicable attribute (the attribute that attracted the most attention, the most prominent attribute, but not necessarily the strongest attribute at the moment) and immediately select another attribute once it was no longer applicable. Therefore, only one attribute could be selected at a given time point. At 5, 10, 20, 40, 60, 90, 120, 150, 180, 210 and 240 seconds, the evaluators received voice prompts asking them to pay attention to the changes in the sensory attributes of the sample. Drinking water was provided for palate cleansing between assessments. During the TCATA test, assessors were asked to check any applicable attribute and uncheck it once it was no longer applicable. Therefore, multiple attributes could be selected or unchecked at a given time point. At 5, 10, 20, 40, 60, 90, 120, 150, 180, 210, and 240 seconds, assessors received voice prompts asking them to pay attention to changes in the sensory attributes of the samples. Drinking water was provided for palate cleansing between assessments.

[0160] The model solution consisted of monosodium glutamate (2 mg / mL), sodium chloride (2 mg / mL), sucrose (5 mg / mL), citric acid (0.8 mg / mL), quinine (5 mg / L) and feruloylphenylalanine (100 mg / L). Each sensory evaluator put 5 mL of sample in the mouth at the same time and spit it out after receiving the instruction at the 5th second. There was a 10-minute interval between each sample, and the mouth was rinsed with drinking water. The samples were evaluated in random order. Finally, the sensory evaluation results were evaluated by TDS curve fitting.

[0161] Depend on Fig.14 and Fig.15 It can be seen that compared with the control group, the addition of 100 mg / L feruloylphenylalanine can significantly increase the citation ratio of salty and umami in the model solution, with umami leading the dominant rate, followed by salty, indicating that feruloylphenylalanine has a good effect in enhancing salty and umami.

[0162] The model solution consisted of monosodium glutamate (2 mg / mL), sodium chloride (2 mg / mL), sucrose (5 mg / mL), citric acid (0.8 mg / mL), quinine (5 mg / L) and feruloylphenylalanine (100 mg / L). Each sensory evaluator put 5 mL of sample in the mouth at the same time and spit it out after receiving the instruction at the 5th second. There was a 10-minute interval between each sample, and the mouth was rinsed with drinking water. The samples were evaluated in random order. Finally, the sensory evaluation results were evaluated by TCATA curve fitting.

[0163] Depend on Fig.16 and 17 It can be seen that sourness, saltiness and umami are the main taste attributes in the model solution. The addition of 100 mg / L feruloylphenylalanine can significantly increase the proportion of saltiness and umami in the model solution and the duration is significantly higher than that of the blank control. In addition, the duration of sourness is also increased after the addition of 100 mg / L feruloylphenylalanine, which also shows that feruloylphenylalanine has a good effect in enhancing the taste characteristics of food.

Claims

1. A food flavor enhancing compound N-phenylacryloylamino acid, characterized in that The structural formula is as follows: R1, R2, R3, R4 each independently represent hydrogen, hydroxyl or methoxy; R5 represents an amino acid residue, which is a residue of a proteinogenic amino acid, ornithine, theanine, sarcosine, γ-aminobutyric acid or β-alanine.

2. The method for preparing the food flavor enhancing compound N-phenylacryloylamino acid according to claim 1, characterized in that: The chemical organic synthesis preparation method of N-phenylacryloylamino acid comprises the following steps: (1) For cinnamic acid containing phenolic hydroxyl groups, the phenolic hydroxyl groups must first be protected; hydroxycinnamic acid is dissolved in a sodium hydroxide aqueous solution under an ice bath, stirred until completely dissolved, and acetic anhydride is slowly added dropwise to the solution; after the addition is completed, the temperature is naturally raised to room temperature and stirred for reaction; after the reaction is completed, the pH of the reaction solution is adjusted to acidic, a white solid is precipitated, filtered under reduced pressure, washed with water for multiple times, and then recrystallized with anhydrous ethanol, and dried to obtain acetylated hydroxycinnamic acid; (2) mixing cinnamic acid carboxylic acid or acetylated hydroxycinnamic acid, thionyl chloride and chloroform, adding DMF dropwise, heating and stirring to react, cooling to room temperature after the reaction, and removing the solvent and excess thionyl chloride by vacuum rotary evaporation to obtain the corresponding acid chloride; (3) dissolving the amino acid in an aqueous solution of sodium hydroxide and stirring until completely dissolved; dissolving the prepared acid chloride in acetone under an ice bath and then slowly dropping it into the sodium amino acid solution; at the same time, dropping an aqueous solution of sodium hydroxide to keep the pH of the reaction solution alkaline; after the addition is completed, naturally warming to room temperature and stirring the reaction; for acetylated hydroxycinnamic acid, after the reaction is completed, adding sodium hydroxide to the reaction solution to hydrolyze the acetyl group; after the reaction is completed, removing the acetone by rotary evaporation under reduced pressure, adjusting the pH of the remaining aqueous solution to acidic, and precipitating a yellow oily liquid or a white solid; extracting with ethyl acetate and washing with water for multiple times; finally, removing the ethyl acetate from the obtained organic layer solution by rotary evaporation, and drying to obtain the purified N-phenylacryloylamino acid.

3. The method for preparing the food flavor enhancing compound N-phenylacryloylamino acid according to claim 1, characterized in that: The enzymatic synthesis preparation method of N-phenylacryloylamino acid comprises the following steps: Cinnamic acid carboxylic acid or its sodium / potassium salt, amino acid or its sodium salt are mixed with water, pH is adjusted, food-grade enzyme is then added and placed in a constant temperature shaker for reaction, enzyme is inactivated after the reaction is completed, the obtained reaction mixture is acidified, extracted with ethyl acetate to obtain an organic layer, and washed with water for multiple times, and finally the obtained organic layer solution is subjected to rotary evaporation to remove ethyl acetate, and purified N-phenylacryloyl amino acid is obtained after drying.

4. The preparation method according to claim 2, characterized in that: The molar mass ratio of the hydroxycinnamic acid to the acetic anhydride is related to the number of phenolic hydroxyl groups contained in the cinnamic acid, the molar mass of the hydroxycinnamic acid × the number of phenolic hydroxyl groups: the molar mass of the acetic anhydride = 0.5-0.8; The reaction time for acetylation of hydroxycinnamic acid is 1-6h; After the acetylation reaction of hydroxycinnamic acid, the pH of the remaining aqueous solution is adjusted to 1-3 with concentrated hydrochloric acid or concentrated sulfuric acid; The molar mass ratio of the hydroxycinnamic acid carboxylic acid or acetylated hydroxycinnamic acid to thionyl chloride is 1.2-2.

0.

5. The preparation method according to claim 2, characterized in that: The reaction temperature for preparing the acyl chloride is 50-60°C and the reaction time is 5-24h; The molar mass ratio of the acyl chloride to the amino acid is 0.8-1.2; The reaction time after adding the acyl chloride is 2-12h; For acetylated hydroxycinnamic acid, after the reaction is completed, 3-6 times the molar mass of sodium hydroxide is added to the reaction solution to hydrolyze the acetyl group. The hydrolysis temperature is 30-40° C. and the hydrolysis time is 1-5 hours.

6. The preparation method according to claim 3, characterized in that: The cinnamic acid carboxylic acid or its sodium / potassium salt includes cinnamic acid, o-hydroxycinnamic acid, p-hydroxycinnamic acid, m-hydroxycinnamic acid, o-methoxycinnamic acid, m-methoxycinnamic acid, p-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 3,4-dihydroxycinnamic acid, 3-methoxy-4-hydroxycinnamic acid, 4-hydroxy-3,5-dimethoxycinnamic acid and their sodium salt or potassium salt; The amino acid or its sodium salt includes any one of alanine, cysteine, aspartic acid, phenylalanine, glutamic acid, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, proline, glycine, ornithine, theanine, sarcosine, γ-aminobutyric acid or β-alanine.

7. The preparation method according to claim 3, characterized in that: The molar mass ratio of the cinnamic acid carboxylic acid to the amino acid is 0.1 to 20; The food-grade enzyme is any one or more of transglutaminase, transglutaminase, pancreatin, aminoacylase, glutaminase Sumizyme GT, protease Sumizyme FLAP-G, dipeptidase Sumizyme DPP-G or protease Sumizyme FP-G.

8. The preparation method according to claim 3, characterized in that: The enzymatic hydrolysis pH is 5.0-9.0; the dosage of the food-grade enzyme is 1.0%-5.0% w / v; the enzymatic hydrolysis temperature is 25-60° C., and the enzymatic hydrolysis time is 6-240 hours; the enzyme inactivation temperature is 80-100° C., and the time is 15-25 minutes.

9. The N-phenylacryloylamino acid according to claim 1 is used as a food additive or flavor enhancer, characterized in that: To impart, modify and / or enhance one, two or all of the flavors of sourness, saltiness, umami and richness.

10. The use according to claim 9, characterized in that: At an addition amount of 1 to 100 mg / L, one, two or all of the flavors of sourness, saltiness, umami and richness are significantly enhanced.

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