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WO2025161290A1 - Composé aromatique alimentaire acide aminé succinyle et son procédé de préparation - Google Patents

Composé aromatique alimentaire acide aminé succinyle et son procédé de préparation

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Publication number
WO2025161290A1
WO2025161290A1 PCT/CN2024/106966 CN2024106966W WO2025161290A1 WO 2025161290 A1 WO2025161290 A1 WO 2025161290A1 CN 2024106966 W CN2024106966 W CN 2024106966W WO 2025161290 A1 WO2025161290 A1 WO 2025161290A1
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food
amino acid
protease
preparation
water
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Chinese (zh)
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崔春
黄丕苗
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South China University of Technology SCUT
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South China University of Technology SCUT
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/86—Addition of bitterness inhibitors
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/88—Taste or flavour enhancing agents

Definitions

  • the invention belongs to the field of flavoring substance synthesis, and particularly relates to a food flavoring compound succinylamino acid and a preparation method thereof.
  • Umami plays an integral role in food flavor and is highly favored by consumers. This taste experience, often described as pleasurable, plays a crucial role in enhancing the sensory quality of food. Umami characteristics are often closely associated with various flavor enhancers, such as 5'-nucleotidylcholine disodium (I+G), monosodium glutamate, and disodium succinate. Despite this, the preference for umami can lead to excessive sodium intake, increasing the risk of chronic diseases, particularly cardiovascular disease. Therefore, how to maintain the palatability of food while limiting sodium intake has become a complex issue facing the food science community. Furthermore, previous studies have revealed the key properties of umami, particularly its synergistic and interactive effects with other flavors, prompting researchers to explore more effective and safe flavor enhancers, particularly umami and salty enhancers.
  • I+G 5'-nucleotidylcholine disodium
  • monosodium glutamate monosodium glutamate
  • disodium succinate disodium succinate
  • Succinylamino acids a class of compounds formed by the covalent bonding of succinic acid and amino acids through amide bonds, have been shown to be safe natural flavor enhancers. These compounds have been confirmed in condiments such as soy sauce and exhibit excellent flavor-enhancing properties.
  • succinylamino acids have been confirmed in condiments such as soy sauce and exhibit excellent flavor-enhancing properties.
  • patent CN 117337962 A discloses a flavoring base rich in salty peptides and its preparation method. This method uses a high-voltage pulsed electric field to treat tilapia waste, then uses a fermentation and enzymatic hydrolysis process to act on the protein, before separating and purifying the salty peptides. This method requires high equipment requirements and is relatively complex. Furthermore, the flavoring peptides produced do not have a significant flavor-enhancing effect, requiring a high addition amount of the peptides to achieve a flavoring effect.
  • the main purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art, and to provide a food flavor-enhancing compound succinyl amino acid and a preparation method thereof, so as to solve the technical problem in the prior art that succinyl amino acid cannot be safely produced and can be used in the food field and has the property of enhancing food flavor.
  • Another object of the present invention is to provide the taste characteristics of the succinyl amino acid compound and an evaluation method thereof.
  • a method for preparing succinylamino acid, a food flavor-enhancing compound comprises the following steps:
  • Succinic acid, amino acid and water are mixed, the pH is adjusted, and then food-grade enzyme is added and placed in a constant temperature shaker for reaction. After the reaction is completed, the enzyme is inactivated and the supernatant is obtained by centrifugation; the supernatant is extracted with ethyl acetate, and then water is added for multiple washings. Finally, the obtained organic layer solution is subjected to rotary evaporation to remove ethyl acetate, and deionized water is added for re-dissolution and freeze-dried to obtain the purified succinyl amino acid.
  • amino acids include any one of phenylalanine, citrulline, sarcosine, leucine, ⁇ -aminobutyric acid, methionine, proline, tryptophan, serine, tyrosine, ornithine, hydroxylysine, asparagine, glutamine, glycylproline, thiocystine, taurine, ⁇ -aminoadipic acid, arginine, and histidine.
  • the molar mass ratio of succinic acid to amino acid is 0.2-10.
  • the food-grade enzyme is any one of Protamax PW2A1128, flavor protease, papain, pancreatin, protease Sumizyme FP-G, protease FoodPro 51FP, protease Multifect PR50G, transglutaminase, protease Corolase 7089, alkaline protease Foodpro Alkaline Protease, protease Corolase 8000, protease Sumizyme FLAP-G or lipase 435.
  • the food-grade enzyme is used in an amount of 0.025% to 0.1% w/v .
  • the conditions of the enzymatic hydrolysis reaction are pH 3.0-5.0, temperature 37-65° C., and time 6-36 h.
  • the enzyme inactivation conditions in step (1) are 80-100° C. and heating for 15-25 min.
  • freeze-drying conditions are -70 ⁇ -50°C, 40 ⁇ 100 Pa, and drying for 24 ⁇ 48 hours.
  • the food flavor-enhancing compound succinylamino acid obtained by the above preparation method can significantly enhance the umami, salty and kochi taste of food and reduce the bitterness of food at an extremely low addition amount of 0.25-1 mg/L.
  • the present invention also provides a sensory evaluation method for the taste characteristics of the succinyl amino acid, wherein the sensory evaluation involves flavors including but not limited to umami, saltiness, kochiness, and bitterness.
  • This invention synthesizes succinylamino acids with food flavor-enhancing properties using specific food-grade enzymes under specific enzymatic reaction conditions. Its flavor properties were evaluated using time-intensity sensory evaluation (TI), transient sensory dominance (TDS), and time-appropriate item selection (TCATA) methods.
  • TI time-intensity sensory evaluation
  • TDS transient sensory dominance
  • TCATA time-appropriate item selection
  • the succinylamino acids prepared in this invention exhibit excellent food flavor-enhancing properties, significantly enhancing the umami, salty, and kokumi flavors of foods and reducing bitterness at extremely low addition levels of 0.25 to 1 mg/L. Furthermore, the preparation method of this invention is simple and efficient, resolving the technical challenge of existing technologies that prevent the safe preparation of food-grade succinylamino acids.
  • the food-grade enzyme used in the succinylamino 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.
  • the succinyl amino acid prepared by the present invention has the characteristic of enhancing the taste of food and can be used as a food additive or flavor enhancer, and has good application prospects in the food field.
  • the present invention has the advantages of simple process, low production cost, short cycle and high synthesis rate.
  • the succinyltryptophan prepared by the present invention can significantly enhance the umami, salty and kochi taste of food and reduce bitterness at a concentration of 0.25-1.0 mg/L; succinyltyrosine can significantly increase the umami and salty taste of food and prolong the duration of umami; 1.0 mg/L succinylphenylalanine can significantly enhance the umami intensity and the frequency of umami perception in food.
  • FIG1 is a liquid chromatography-mass spectrometry (LC-MS) graph of succinylphenylalanine after purification from the enzymatic reaction mixture in Example 1.
  • LC-MS liquid chromatography-mass spectrometry
  • FIG2 is a HPLC chromatogram of phenylalanine and succinylphenylalanine.
  • FIG3 is a liquid chromatography-mass spectrometry of succinyltryptophan after purification from the enzymatic reaction mixture in Example 2.
  • FIG4 is a HPLC chromatogram of tryptophan and succinyltryptophan.
  • FIG5 is a liquid chromatography-mass spectrometry of succinyltyrosine after purification of the enzymatic reaction mixture in Example 3.
  • FIG6 is a HPLC chromatogram of tyrosine and succinyltyrosine.
  • FIG7 is a liquid chromatography-mass spectrometry of succinylleucine after purification from the enzymatic reaction mixture in Example 4.
  • FIG8 is a HPLC chromatogram of leucine and succinylleucine.
  • FIG9 shows the enhancing effect of succinyltryptophan in Example 17 on the saltiness, umami, and kokumi of the simulated chicken soup system.
  • FIG10 is a TI curve of the umami intensity of succinyltyrosine in Example 18 in a simulated chicken soup system.
  • FIG11 is a saltiness intensity TI curve of succinyltyrosine in Example 18 in a simulated chicken soup system.
  • FIG12 is a bitterness intensity TI curve of succinyltryptophan in a bitter solution system in Example 19.
  • FIG13 is a TDS curve of succinylphenylalanine in a model solution in Example 20.
  • FIG14 is a TCATA curve of succinylphenylalanine in the model solution in Example 20.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, and then washed multiple times with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (50°C, 10 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-60°C, 100 Pa, 48 h) to obtain purified succinylphenylalanine;
  • UPLC-ESI-MS/MS detection conditions Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile.
  • the gradient elution program was as follows: 0-1 min, 80% A; 1-4.5 min, 80% to 45% A; 4.5-5.5 min, 45% A; 5.5-7.5 min, 45% to 80% A; and 7.5-8.5 min, 80% A.
  • the flow rate was 0.4 mL/min
  • the injection volume was 10 ⁇ L.
  • the sample was equilibrated at the initial mobile phase ratio for 5 min before injection.
  • Mass spectrometry conditions an electrospray ion source was used, the mass spectrometry scan mode was positive ion scan mode, the capillary voltage was 3200 V, the desolvation gas was nitrogen, the drying temperature was 220°C, the desolvation gas flow rate was 8 L/min, the dryer temperature was 220°C, the scan range was m/z 50-1300, the collision gas was argon, and the chromatographic column was an ACQUITY UPLC BEH C18 column (2.1 mm ⁇ 100 mm, 1.7 ⁇ m, Waters).
  • HPLC detection conditions The mobile phases consisted of 0.1% ( v / v ) trifluoroacetic acid in acetonitrile (A) and 0.1% ( v / v ) trifluoroacetic acid in water (B), respectively.
  • a gradient elution method was used, with the following elution program: 0 min: 80% B + 20% A; 25 min: 60% B + 40% A; 30 min: 50% B + 50% A; 35 min: 80% B + 20% A; and end of elution.
  • Detection was performed using an LC-UV100 UV detector (Wufeng, Guangzhou) at a wavelength of 210 nm, a column temperature of 30°C, an injection volume of 10 ⁇ L, and a flow rate of 1.0 mL/min.
  • Chromatographic column XSelect HSS T3, length: 250 mm, inner diameter: 4.6 mm.
  • the purified succinylphenylalanine was qualitatively analyzed by UPLC-ESI-MS/MS, and the peak times of succinylphenylalanine and phenylalanine were compared by HPLC.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, followed by multiple washings with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (55°C, 15 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-70°C, 40 Pa, 24 h) to obtain purified succinyltryptophan.
  • Tyrosine was mixed with water to a concentration of 75 mM, succinic acid was added to a molar mass ratio of succinic acid to amino acid of 6, the pH was adjusted to 3, and a 0.1% ( w/v ) solution of protease FoodPro 51FP was added. The mixture was reacted in a water bath shaker at 55°C for 24 h, and the enzyme was inactivated at 100°C for 20 min to obtain a reaction mixture.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, and then washed multiple times with an equal volume of water. Finally, the ethyl acetate layer solution was removed by rotary evaporation (60°C, 15 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-70°C, 100 Pa, 48 h) to obtain purified succinyltyrosine.
  • Leucine was mixed with water to a concentration of 50 mM, succinic acid was added to make the molar mass ratio of succinic acid to amino acid 1, the pH was adjusted to 5, and 0.025% ( w/v ) pancreatic enzyme was added. The mixture was reacted in a shaking water bath at 45°C for 36 h, and the enzyme was inactivated at 80°C for 15 min to obtain a reaction mixture.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, followed by multiple washings with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (55°C, 10 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-50°C, 70 Pa, 36 h) to obtain purified succinylleucine.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, and then washed multiple times with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (60°C, 50 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-60°C, 100 Pa, 36 h) to obtain purified succinylmethionine.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, and then washed multiple times with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (50°C, 10 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-60°C, 40 Pa, 24 h) to obtain purified succinyl hydroxylysine.
  • Proline was mixed with water to a concentration of 100 mM, succinic acid was added to a molar mass ratio of succinic acid to amino acid of 0.5, the pH was adjusted to 4, 0.1% ( w/v ) solution of lipase 435 was added, the mixture was reacted in a water bath shaker at 45°C for 36 h, and the enzyme was inactivated at 100°C for 20 min to obtain a reaction mixture;
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, and then washed multiple times with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (60°C, 20 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-70°C, 100 Pa, 48 h) to obtain purified succinylproline.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, followed by multiple washings with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (60°C, 10 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-70°C, 70 Pa, 36 h) to obtain purified succinyl sarcosine.
  • Ornithine was mixed with water to a concentration of 100 mM, succinic acid was added to make the molar mass ratio of succinic acid to amino acid 6, the pH was adjusted to 3, and 0.025% ( 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 100°C for 15 min to obtain a reaction mixture.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, and then washed multiple times with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (50°C, 30 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-70°C, 100 Pa, 36 h) to obtain purified succinyl ornithine.
  • Serine was mixed with water to a concentration of 50 mM, succinic acid was added to make the molar mass ratio of succinic acid to amino acid 4, the pH was adjusted to 3, and a 0.1% ( w/v ) solution of protease Multifect PR50G was added. The mixture was reacted in a water bath shaker at 55°C for 18 h, and the enzyme was inactivated at 100°C for 25 min to obtain a reaction mixture.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, followed by multiple washings with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (55°C, 20 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-50°C, 40 Pa, 48 h) to obtain purified succinylserine.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, and then washed multiple times with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (60°C, 20 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-70°C, 70 Pa, 48 h) to obtain purified succinylasparagine.
  • Arginine was mixed with water to a concentration of 50 mM, succinic acid was added to make the molar mass ratio of succinic acid to amino acid 7, the pH was adjusted to 3, and 0.07% ( w/v ) pancreatic enzyme was added. The mixture was reacted in a water bath shaker at 55°C for 24 h, and the enzyme was inactivated at 100°C for 15 min to obtain a reaction mixture.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, followed by multiple washings with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (60°C, 10 Bar), and then 15 mL of deionized water was added for re-dissolution and freeze-dried (-70°C, 100 Pa, 24 h) to obtain purified succinyl arginine.
  • Glutamine and water were mixed to a concentration of 100 mM, succinic acid was added to a molar mass ratio of succinic acid to amino acids of 7, the pH was adjusted to 5, and a 2% ( w/v ) solution of pancreatic enzyme was added.
  • the mixture was reacted in a water bath shaker at 55°C for 24 h, and the enzyme was inactivated at 100°C for 15 min to obtain a reaction mixture.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, and then washed multiple times with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (60°C, 10 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-70°C, 100 Pa, 24 h) to obtain purified succinylglutamine.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, followed by multiple washings with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (50°C, 30 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-50°C, 100 Pa, 24 h) to obtain purified succinyltaurine.
  • Glycylproline was mixed with water to a concentration of 100 mM, succinic acid was added to make the molar mass ratio of succinic acid to amino acid 8, the pH was adjusted to 3, 0.025% ( w/v ) solution of glutamine transaminase was added, the mixture was reacted in a water bath shaker at 55°C for 36 h, and the enzyme was inactivated at 80°C for 20 min to obtain a reaction mixture;
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, followed by multiple washings with an equal volume of water. Finally, the obtained ethyl acetate layer solution was removed by rotary evaporation (60°C, 30 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-60°C, 100 Pa, 24 h) to obtain purified succinylglycylproline.
  • step (2) The reaction mixture obtained in step (1) was centrifuged to obtain the supernatant; the supernatant was extracted with an equal volume of ethyl acetate, followed by multiple washings with an equal volume of water, and finally the obtained ethyl acetate layer solution was removed by rotary evaporation (50°C, 15 Bar), and then 15 mL of deionized water was added for redissolution and freeze-dried (-60°C, 40 Pa, 30 h) to obtain purified succinylcitrulline.
  • Example 17 Determination of the Flavor-Enhancing Effect of Succinyltryptophan Using an Artificial Sensory Evaluation Method (Simulated Chicken Soup System)
  • a simulated chicken broth system consisting of MSG (1 mg/mL), sodium chloride (1 mg/mL), and disodium flavor nucleotides (0.1 mg/mL) was prepared. Succinyltryptophan was added at different concentrations (0.25, 0.5, and 1 mg/L) to evaluate its flavor-enhancing effect.
  • the evaluation protocol was as follows:
  • the sensory evaluation panel consisted of 10 panelists, including 5 males and 5 females aged between 20 and 28 years. The panelists underwent a year of sensory training and had no known history of taste disorders. We used 50 mmol/L sucrose for sweetness, 20 mmol/L citric acid for sourness, 20 mmol/L NaCl for saltiness, 10 mmol/L caffeine for bitterness, and 10 mmol/L monosodium L-glutamate (MSG) for umami. In addition, 5 mmol/L glutathione was used to assess kokumi.
  • Quantitative descriptive analysis In a tasting room under normal lighting and room temperature, ten sensory panelists (five men and five women, aged between 20 and 28 years) were familiar with a 15-point scale, rating saltiness, umami, and kokumi (0–15), with 0 indicating no flavor and 15 indicating a strong flavor. Solutions consisting of monosodium glutamate (1 mg/mL), sodium chloride (1 mg/mL), disodium ribonucleotide (0.1 mg/mL), and varying concentrations of succinyltryptophan (0.25, 0.5, and 1 mg/L) were prepared. Solutions of 0.5, 1.0, and 1.5 mg/mL glutathione, 1, 2, and 3 mg/mL sodium chloride, and 1, 2, and 3 mg/mL monosodium glutamate were used as standard reference samples.
  • Example 18 Evaluation of the effect of succinyltyrosine on the umami intensity and salty intensity of simulated chicken soup solution using time-intensity sensory evaluation (TI)
  • Sensory evaluation training A total of eight 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 had undergone one year of professional sensory training and participated in the evaluation process of umami, kokumi, and saltiness. All participants were highly proficient in using a 15-point rating scale to evaluate the intensity changes of food flavors and were sensitive to the intensity changes of umami and saltiness. Before the evaluation activities began, each assessor signed an informed consent form. To ensure the accuracy of the evaluation process, they received a rigorous 8-day training based on the Standard Guide for Time-Intensity Evaluation of Sensory Attributes (ASTM E, 1909-97).
  • the training content involved: (1) applying a 15-point rating scale to judge the intensity of umami and saltiness, where 0 points represent "none" and 15 points represent "extremely strong”; (2) the assessors were given a detailed introduction to the time-intensity (TI) method procedure and multiple rounds of product taste training.
  • TI time-intensity
  • each assessor uses a paper chart with a scale.
  • the vertical axis represents intensity (out of 15 points) and the horizontal axis represents time (in seconds). Time intervals are set at 5, 10, 20, 40, 60, 90, 120, 150, 180, and 210 seconds.
  • assessor performance can be evaluated by analyzing repeated TI curves. Training is considered adequate if the curve remains consistent for at least 40% of the time.
  • Umami intensity was assessed using a 0-15 scoring system for 1, 2, and 3 mg/mL MSG solutions, with 0 representing a tasteless sample and 15 indicating a strong flavor.
  • the simulated chicken broth system consisted of MSG (1 mg/mL), sodium chloride (1 mg/mL), disodium flavor nucleotides (0.1 mg/mL), and various concentrations of succinyltyrosine (0.25, 0.5, and 1 mg/L, respectively).
  • Panelists placed a 5 mL sample in their mouths and expectorated it 5 seconds after being instructed. They rated the perceived flavor intensity at 5, 10, 20, 30, 45, 60, 75, 90, 120, 150, 180, and 210 seconds, until no umami was perceived.
  • Each sample was evaluated for 10 minutes, with the mouth cleansed with 3% sucrose solution. Samples were randomly evaluated. Sensory evaluation results were then evaluated by fitting a time-intensity (TI) curve.
  • TI time-intensity
  • Figure 10 shows that the succinyltyrosine curves exhibit similar umami temporal characteristics, with the 0 mg/L curve completely overlapping the succinyltyrosine curve.
  • Umami intensity increases rapidly within a short period of time and then decreases slowly. Samples with higher succinyltyrosine concentrations experience a faster increase and a slower decrease, significantly enhancing umami and lingering longer in the mouth. This demonstrates the umami-enhancing properties of succinyltyrosine.
  • the saltiness intensity increases rapidly and then gradually decreases.
  • the 0 mg/L curve is completely covered by the succinyltyrosine curve.
  • Example 19 Evaluation of the effect of succinyltryptophan on the bitterness intensity of a bitter solution using the time-intensity sensory evaluation method (TI):
  • Sensory evaluation training A total of eight 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 had undergone one year of professional sensory training and participated in the bitterness evaluation process. All participants were highly proficient in using a 15-point rating scale to evaluate the intensity changes of food flavors and were sensitive to changes in bitterness intensity. Before the evaluation activities began, each assessor signed an informed consent form. To ensure the accuracy of the evaluation process, they received a rigorous 8-day training based on the Standard Guide for Time-Intensity Evaluation of Sensory Attributes (ASTM E, 1909-97).
  • the training content involved: (1) applying a 15-point rating scale to judge the intensity of umami and saltiness, where 0 represents “none” and 15 represents “extremely strong”; (2) the assessors were given a detailed introduction to the time-intensity (TI) method procedure and multiple rounds of product taste training.
  • TI time-intensity
  • each assessor uses a paper chart with a scale.
  • the vertical axis represents intensity (out of 15 points) and the horizontal axis represents time (in seconds). Time intervals are set at 5, 10, 20, 40, 60, 90, 120, 150, 180, and 210 seconds.
  • assessor performance can be evaluated by analyzing repeated TI curves. Training is considered adequate if the curve remains consistent for at least 40% of the time.
  • Umami taste intensity was assessed using a 0-15 scoring system for 2.5, 5.0, and 7.5 mg/mL isoleucine solutions, with 0 representing a tasteless sample and 15 indicating a strong taste.
  • a bitter solution consisted of 5 mg/mL isoleucine supplemented with varying concentrations of succinyltryptophan (0.25, 0.5, and 1 mg/mL, respectively).
  • Panelists placed a 5 mL sample in their mouths and, upon instruction, expectorated it within 5 seconds. They rated the perceived taste intensity at 5, 10, 20, 30, 45, 60, 75, and 90 seconds, until no bitterness was perceived. Each sample was evaluated for 10 minutes, with the mouth cleansed with 3% sucrose solution, and samples were randomly evaluated. Sensory evaluation results were then evaluated by fitting time-intensity (TI) curves.
  • TI time-intensity
  • Figure 12 shows that the succinyltryptophan curves exhibit similar bitterness time characteristics.
  • the TI curves for 0.25-1 mg/L succinyltryptophan are completely overlapped by the 0 mg/L curve.
  • the bitterness intensity increases rapidly within a short period of time and then decreases slowly.
  • Samples with higher succinyltryptophan concentrations experience slower increases and faster decreases in bitterness intensity, significantly reducing the bitterness and shortening the time it remains in the mouth. This demonstrates that succinyltryptophan has the characteristic of reducing bitterness.
  • Example 20 Evaluation of the Effect of Succinylphenylalanine on the Taste Attributes of Model Solutions Using Temporal Sensory Dominance (TDS) and Time-Appropriate Item Tick-Off (TCATA) Methods:
  • Sensory evaluators were trained: 14 evaluators aged 20-28 years (8 males, 6 females). All evaluators had no aversion to sour, sweet, bitter, salty, or umami flavors, and each evaluator signed an informed consent form before the test.
  • evaluators were required to select only one applicable attribute (the most attention-grabbing and prominent attribute, but not necessarily the strongest attribute at the time). Once it was no longer applicable, they immediately selected another attribute. Therefore, only one attribute could be selected at a given time point.
  • evaluators received voice prompts to note changes in the sample's sensory attributes. Drinking water was provided for palate cleansing between assessments.
  • assessors were asked to check any applicable attribute and uncheck it immediately if it no longer applied. Therefore, multiple attributes could be selected or unchecked at any given time.
  • assessors received voice prompts to note changes in the sample's sensory attributes. Drinking water was provided for palate cleansing between assessments.
  • the model solution consisted of monosodium glutamate (2 mg/mL), sodium chloride (2 mg/mL), disodium nucleotide (0.1 mg/mL), sucrose (20 mg/mL), citric acid (0.5 mg/mL), and succinimidyl phenylalanine (3.5 mg/mL).
  • Each sensory assessor simultaneously placed 5 mL of the sample in their mouth and, after receiving the instruction at the 5th second mark, expelled it. A 10-minute interval was allowed between each sample, followed by a mouthwash with drinking water. Samples were then randomly evaluated. Sensory evaluation results were then assessed using TDS curve fitting.
  • the model solution consisted of monosodium glutamate (2 mg/mL), sodium chloride (2 mg/mL), disodium flavor nucleotides (0.1 mg/mL), sucrose (20 mg/mL), citric acid (0.5 mg/mL), and succinylphenylalanine (3.5 mg/mL).
  • Each sensory assessor simultaneously placed 5 mL of the sample in their mouth and, after receiving the instruction at the 5th second mark, expelled it. A 10-minute interval was allowed between each sample, followed by a mouthwash with drinking water. Samples were then randomly evaluated. Sensory evaluation results were then assessed using TCATA curve fitting.
  • Figure 14 shows that umami, saltiness, and sweetness are the primary taste attributes in the model solution.
  • the addition of 1 mg/L succinylphenylalanine significantly increases the umami contribution in the model solution, and the duration is significantly higher than that of 0 mg/L succinylphenylalanine.
  • the duration of sweetness and the proportion of saltiness also increase with the addition of 1 mg/L succinylphenylalanine, demonstrating that succinylphenylalanine has a significant effect on enhancing food flavor properties.
  • the flavor contribution and duration further demonstrate its excellent umami-enhancing effect.

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Abstract

La présente invention concerne le domaine de la synthèse des substances aromatiques. La présente invention concerne plus particulièrement un composé d'arôme alimentaire, l'acide aminé succinyle, et son procédé de préparation. Le procédé consiste à m mélanger de l'acide succinique, un acide aminé et de l'eau, ajuster le pH, puis ajouter une enzyme de qualité alimentaire, placer le mélange dans une table d'agitation à température constante pour faire réagir l'enzyme, procéder à la désactivation de l'enzyme une fois la réaction terminée, et centrifuger le mélange pour obtenir un surnageant, et extraire le surnageant avec de l'acétate d'éthyle, puis y ajouter de l'eau pour plusieurs lavages, enfin éliminer l'acétate d'éthyle de la solution de la couche organique obtenue au moyen d'une évaporation rotative, y ajouter de l'eau désionisée pour la redissolution, et lyophiliser le tout pour obtenir un acide aminé succinyl purifié. L'acide aminé succinyle préparé peut améliorer de manière significative la saveur (y compris le goût umami, le goût salin et le goût fort) des aliments et réduire le goût amer des aliments sous une quantité d'ajout de 0,25 à 1 mg/L, peut être utilisé comme additif alimentaire ou agent aromatisant, et présente de bonnes perspectives d'application.
PCT/CN2024/106966 2024-01-31 2024-07-23 Composé aromatique alimentaire acide aminé succinyle et son procédé de préparation Pending WO2025161290A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004075663A1 (fr) * 2003-02-26 2004-09-10 Firmenich Sa Derives aminoacides d'acides dicarboxyliques utilises comme aromatisants
US20050233058A1 (en) * 2004-01-29 2005-10-20 Eric Frerot Aminoacid derivatives of dicarboxylic acids as flavor ingredients
US20110244530A1 (en) * 2008-05-07 2011-10-06 Toyo Boseki Kabushiki Kaisha L-succinylaminoacylase and process for producing l-amino acid using it
US20110250653A1 (en) * 2008-12-11 2011-10-13 Sekisui Medical Co., Ltd. L-succinylaminoacylase and process for producing l-amino acid using it
WO2018088434A1 (fr) * 2016-11-10 2018-05-17 東洋紡株式会社 Procédé de production d'acide n-succinyl-hydroxy-d-amino et/ou d'acide hydroxy-d-amino
CN116926141A (zh) * 2023-06-27 2023-10-24 华南理工大学 一种食品级酶制剂催化合成乙酰化氨基酸及寡肽的方法
CN116926140A (zh) * 2023-06-27 2023-10-24 华南理工大学 一种具有增强或改善食物呈味特性的脂肪酰化氨基酸及其制备方法
CN118147247A (zh) * 2024-01-31 2024-06-07 华南理工大学 一种食品增味化合物琥珀酰氨基酸及其制备方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004075663A1 (fr) * 2003-02-26 2004-09-10 Firmenich Sa Derives aminoacides d'acides dicarboxyliques utilises comme aromatisants
US20050233058A1 (en) * 2004-01-29 2005-10-20 Eric Frerot Aminoacid derivatives of dicarboxylic acids as flavor ingredients
US20110244530A1 (en) * 2008-05-07 2011-10-06 Toyo Boseki Kabushiki Kaisha L-succinylaminoacylase and process for producing l-amino acid using it
US20110250653A1 (en) * 2008-12-11 2011-10-13 Sekisui Medical Co., Ltd. L-succinylaminoacylase and process for producing l-amino acid using it
WO2018088434A1 (fr) * 2016-11-10 2018-05-17 東洋紡株式会社 Procédé de production d'acide n-succinyl-hydroxy-d-amino et/ou d'acide hydroxy-d-amino
CN116926141A (zh) * 2023-06-27 2023-10-24 华南理工大学 一种食品级酶制剂催化合成乙酰化氨基酸及寡肽的方法
CN116926140A (zh) * 2023-06-27 2023-10-24 华南理工大学 一种具有增强或改善食物呈味特性的脂肪酰化氨基酸及其制备方法
CN118147247A (zh) * 2024-01-31 2024-06-07 华南理工大学 一种食品增味化合物琥珀酰氨基酸及其制备方法

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