CN110074378B - Flavor development peptide separated from oyster enzymolysis liquid and preparation method and application thereof - Google Patents

Flavor development peptide separated from oyster enzymolysis liquid and preparation method and application thereof Download PDF

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CN110074378B
CN110074378B CN201910432364.XA CN201910432364A CN110074378B CN 110074378 B CN110074378 B CN 110074378B CN 201910432364 A CN201910432364 A CN 201910432364A CN 110074378 B CN110074378 B CN 110074378B
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oyster
enzymolysis
ethanol
supernatant
flavor
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崔春
舒丹阳
张典
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South China University of Technology SCUT
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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/50Molluscs
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/65Addition of, or treatment with, microorganisms or enzymes
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20Synthetic spices, flavouring agents or condiments

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Abstract

The invention discloses a flavor development peptide separated from oyster enzymolysis liquid and a preparation method and application thereof. The method comprises the following steps: taking oyster meat as a raw material, cleaning the oyster meat, beating the oyster meat into meat paste, and adding water to prepare oyster homogenate; adding a compound enzyme into the homogenate for enzymolysis, after the reaction is finished, inactivating the enzyme to obtain an enzymolysis product, and performing centrifugal filtration to obtain an enzymolysis liquid; extracting peptides in the enzymolysis liquid by ethanol in a grading manner; UPLC-MS/MS separates and identifies novel taste peptide with amino acid sequence of Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu. The oyster flavor peptide separated and identified by the invention has rich taste and can provide a theoretical basis for industrial synthesis.

Description

Flavor-developing peptide separated from oyster enzymolysis liquid and preparation method and application thereof
Technical Field
The invention relates to the field of flavor development peptides, in particular to a flavor development peptide separated from oyster enzymolysis liquid and a preparation method and application thereof.
Background
The oyster is the largest cultured shellfish in the world and is also the economic shellfish with the largest yield in China. The oyster is delicious in taste, rich in various physiological active ingredients and has important potential value in the aspects of food and medicine, and the oyster is listed as one of the raw materials which are both food and medicinal materials by the ministry of health in China. At present, products developed by taking oysters as raw materials in the market mainly comprise functional health products, seafood seasonings, medicines and the like. The oyster processing products in China are relatively few, most of the oyster processing products are oyster products in the traditional sense, such as dry products, cans and the like, and how to finely and deeply process oyster meat by using modern food technology high and new technologies, such as fermentation technology, biological enzymolysis technology and the like, becomes a research hotspot by fully utilizing oyster resources.
The enzyme technology is a novel marine raw material processing technology, and is concerned about because the reaction conditions are mild, the process is easy to control, and the nutrient substances of the food raw materials can be well reserved. When the enzyme technology is widely applied to the development of various oyster products, the application mainly comprises three aspects of improving the function of protein through enzyme modification, extracting nutrient components in the protein through enzymolysis and generating flavor substances through enzymolysis.
The enzyme method modification can change the structure of oyster protein, thereby improving the functional characteristics of the oyster protein such as emulsibility, solubility, foamability and the like. The oyster protein is hydrolyzed by enzyme technology to generate a plurality of micromolecule peptides and free amino acids, thereby endowing the oyster enzymolysis product with rich nutrition and full taste. When the oyster protein is enzymolyzed by adopting an enzyme technology, the selection of the protease is very critical, and different proteases have unique action modes, so that different enzymolysis effects can be generated. The protease commonly used for enzymolysis of oysters at present comprises Protemax complex enzyme, trypsin, alkaline protease, neutral protease, flavourzyme and the like. The flavor and color of the enzymolysis liquid prepared by the Protemax complex enzyme are better; pancreatin is a mixed enzyme preparation, consists of single enzymes such as trypsin, pancreatic amylase, pancreatic lipase, aminopeptidase and the like, and has the advantages of high hydrolysis efficiency and strong specificity; the alkaline protease has high hydrolysis efficiency and poor specificity, and has good hydrolysis effect on aromatic amino acid, hydrophobic amino acid and alkaline amino acid; neutral protease is an endonuclease, has the advantages of high hydrolysis efficiency and good flavor of hydrolysate, and is widely applied to the industries of functional foods and seasonings; flavourzyme can hydrolyze proteins to generate free amino acids and oligopeptides with good flavor, so the flavourzyme is widely applied to novel seasonings.
The taste-developing peptide is a kind of small molecular peptide, which can affect or improve the taste of food, and the main ways to obtain the taste-developing peptide are enzymatic hydrolysis and biotechnological synthesis. The flavor peptide is rich in variety, and the flavor covers five basic flavors of sour, sweet, bitter, salty and fresh, so the flavor peptide can replace the traditional seasonings such as sucrose, salt, sodium glutamate and the like to be added into food. In recent years, there have been many studies on the separation and identification of taste-developing peptides from foods, and among many means for separation and purification of taste-developing peptides, food-grade ethanol separation methods have been widely used because of their advantages of non-toxicity, innocuity, high throughput, and high separation speed. In the aspect of flavor peptide structure identification, the precision and sensitivity of an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology and a De Novo sequencing (De Novo sequencing) method greatly exceed those of a conventional detection method, and the polypeptide can be quickly and accurately identified and determined.
Disclosure of Invention
The invention aims to separate and identify a novel flavor peptide from an enzymolysis liquid of oyster by complex enzyme enzymolysis, fresh oyster meat is used as a raw material, a complex enzymolysis technology is adopted to obtain the enzymolysis liquid, an ethanol grading method and UPLC-MS/MS separation are used to identify the flavor peptide, the flavor peptide has strong sourness and delicate flavor, and has no obvious fishy and bitter taste, and the flavor peptide can be industrially synthesized to develop the seafood seasoning.
The purpose of the invention is realized by the following technical scheme.
A flavor peptide has an amino acid sequence of Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu.
The method for separating the flavor-developing peptide from the oyster enzymolysis liquid comprises the following steps:
(1) Pretreatment: taking oyster meat as a raw material, cleaning, draining, beating into meat paste, and adding water to prepare oyster homogenate;
(2) Enzymolysis: adding complex enzyme into the oyster homogenate for enzymolysis reaction, and after the reaction is finished, inactivating the enzyme and centrifuging to obtain filtrate which is oyster enzymolysis liquid;
(3) And (3) carrying out ethanol fractional extraction on the peptides in the oyster enzymolysis liquid: adding ethanol into the oyster enzymolysis liquid, stirring uniformly, centrifuging to obtain a precipitate (I) and a supernatant (I), then adding ethanol into the supernatant (I), stirring uniformly, and centrifuging to obtain a precipitate (II) and a supernatant (II); and adding ethanol into the supernatant (II), stirring uniformly, centrifuging to obtain a supernatant (III), and removing the excessive ethanol from the supernatant (III).
Preferably, the ratio of the oyster homogenate in the step (1) is 1g.
Preferably, the complex enzyme in step (2) is two or more of Pancreatin, papain, acid Protease, ns 37071 (alkaline Protease), protamex (complex hydrolase), flavorzyme500MG (flavourzyme) and alcalase2.4l (alkaline Protease).
Further preferably, the complex enzyme is a mixed enzyme of Flavozyme 500MG and Pancreatin.
More preferably, the mass ratio of Flavozyme 500MG and Pancreatin in the mixed enzyme is 1.
Preferably, the addition amount of the complex enzyme in the step (2) is 0.1-0.5wt% of the oyster meat, and further preferably is 0.2wt%.
Preferably, the temperature of the enzymolysis in the step (2) is 45-55 ℃, and further preferably 50 ℃; the enzymolysis time is 4-8h, and more preferably 7h.
Preferably, the enzyme deactivation time in the step (2) is 10-20min.
Preferably, in the step (3), 3-5 times volume of ethanol is added into the oyster enzymolysis liquid; adding ethanol into the supernatant (I) to ensure that the final concentration of the ethanol in the system is 70-90vol%; ethanol was added to the supernatant (II) so that the final concentration of ethanol in the system was 95vol%.
Further preferably, in the step (3), ethanol with the volume 4 times that of the oyster enzymolysis liquid is added into the oyster enzymolysis liquid; adding ethanol into the supernatant (I) to ensure that the final concentration of the ethanol in the system is 90vol%; ethanol was added to the supernatant (II) so that the final concentration of ethanol in the system was 95vol%.
Use of the above described taste peptide in a food product.
Compared with the prior art, the invention has the following advantages:
(1) The invention adopts the compound enzyme to carry out enzymolysis from a plurality of sites, improves the hydrolysis degree of the oyster protein, and simultaneously improves the yield of the polypeptide;
(2) The flavor development peptide is 8 peptide with the molecular weight less than 800Da, is easy to be absorbed by human body, and improves the nutritive value.
(3) The method has the advantages of mild reaction conditions, short reaction time, low price of enzyme adopted in the reaction, high quality, small dosage and effective cost saving.
(4) The combination of the flavor peptide separated and identified by the invention and other seasonings can obviously enhance the delicate flavor of the seasonings and weaken the fishy and bitter taste.
Drawings
FIG. 1 is a flow chart of ethanol fractionation;
FIG. 2 is a sensory evaluation chart of different enzymatic hydrolysis conditions.
Fig. 3a, fig. 3b, fig. 3c and fig. 3d are graphs showing the taste enhancing effect of oyster enzymolysis products with different concentrations on food.
FIG. 4 is a secondary mass spectrum of taste peptide from UPLC-MS/MS.
Detailed Description
The following describes a specific embodiment of the present invention with reference to the examples and the drawings, but the embodiments of the present invention are not limited thereto.
Example 1
Screening oyster enzymolysis liquid with optimal taste
1. Taking oyster meat as a raw material, cleaning, draining, beating into meat paste, and adding water to prepare oyster homogenate with a feed liquid ratio of 1g;
2. adding 0.1-0.5wt% of mixed enzyme of Flavozyme 500MG and Pancretin (the mass ratio of Flavozyme 500MG to Pancretin is 1:0.1wt% of mixed enzyme, and carrying out enzymolysis for 7 hours at 50 ℃; e-2:0.3wt% of mixed enzyme, and carrying out enzymolysis for 7 hours at 50 ℃; e-3:0.5wt% of mixed enzyme, and carrying out enzymolysis for 7h at 50 ℃; e-4:0.3wt% of mixed enzyme, and carrying out enzymolysis for 7 hours at 45 ℃; e-5:0.3wt% of mixed enzyme, and carrying out enzymolysis for 7h at 55 ℃;
3. sensory evaluation of enzymatic hydrolysate
Five of the men and women (between 24 and 30 years) without taste impairment were selected by the sensory panel of this study, and the temperature in the evaluation room was controlled to room temperature (25. + -. 2 ℃). Panelists were trained with reference solutions for the characteristics of the experiment. The reference solutions were as follows: umami (MSG solution, 16mmol/L,8mmol/L,4 mmol/L); sweet taste (sucrose solution, 50mmol/L,30mmol/L,10 mmol/L); sour (citric acid solution, 8mmol/L,4mmol/L,2 mmol/L); bitter taste (L-isoleucine solution, 40mmol/L,20mmol/L,10 mmol/L); the thick taste is relatively complex, and the research adopts chicken soup prepared from old hens boiled in white water as reference solution (chicken soup boiled for 6 hours, chicken soup boiled for 4 hours and chicken soup boiled for 2 hours). Wherein the flavor scores of the reference liquid with 3 concentrations are respectively positioned according to the strength for 10 scores, 5 scores and 1 score. Each flavor score was referenced to a reference solution score similar to its flavor, and the final result was the average of each panelist's scores.
Sensory evaluation results of the oyster enzymolysis solution are shown in fig. 1. As can be seen from FIG. 1, the sensory evaluation of the oyster enzymolysis liquid E-2 is as follows: umami taste 6.67, sour taste 1.96, sweet taste 3.75, thick taste 4.18, bitter taste 1.65. The fresh taste and the sweet taste of the product have higher scores than other enzymolysis liquid, the thick taste has moderate score, and the sour taste and the bitter taste have lower scores. The results show that the addition amount of the enzyme is 0.3wt%, the oyster enzymolysis liquid obtained by enzymolysis at 50 ℃ for 7 hours under natural pH has rich delicate flavor, full mouthfeel and longer aftertaste, moderate sweetness, no obvious sour taste and bitter taste, and better overall flavor.
Example 2
Taste-optimized enzymolysis liquid taste development synergistic evaluation
1. Taking oyster meat as a raw material, cleaning, draining, beating into meat paste, and adding water to prepare oyster homogenate with a feed liquid ratio of 1g;
2. adding 0.3wt% of mixed enzyme of Flavozyme 500MG and Pancretin (the mass ratio of Flavozyme 500MG to Pancretin is 1).
3. Evaluation of flavor development and flavor enhancement
Respectively adding the chicken soup solution, the beef soup solution, the mixed solution of monosodium glutamate and saline and the mixed solution of monosodium glutamate, saline and I + G, wherein a is shown in a figure 3 a: mixing salt and monosodium glutamate; fig. 3b shows: a mixed solution of salt, monosodium glutamate and I + G; fig. 3c shows: making a model chicken soup; FIG. 3d shows: model beef soup and the pH of each solution prepared was adjusted to 6.5 using sodium hydroxide solution and formic acid solution. The prepared solutions are presented to a sensory evaluation panel according to the order of the concentration of the enzymolysis products from small to large, and the taste development and the efficiency improvement of the solutions are evaluated. The sensory characteristics of each solution were evaluated using a "5-point" intensity scale (0, undetectable; 5, strongly detectable), with target sensory characteristics including umami, kokumi (more complex mouthfeel, which assesses food 10 seconds after it is tasted) and sustainability (sensory effect or taste enhancement that can be sustained, which assesses food 25 seconds after it is tasted).
As can be seen from fig. 3a, fig. 3b, fig. 3c, and fig. 3d, the oyster enzymolysis solution has different degrees of enhancing effects on the umami taste, the kokumi taste, and the persistence of the four solutions, and the taste enhancing value of the 0.1% addition amount of the enzymolysis solution to the four solutions is significantly higher than that of the 0.05% addition amount of the enzymolysis solution. The oyster enzymolysis liquid has good effect of enhancing the persistence and the delicate flavor of the mixed solution of the salt and the monosodium glutamate, and the flavor development and enhancement values of the oyster enzymolysis liquid are as follows in sequence: the persistence is more than the delicate flavor and more than the thick flavor, and the flavor development value ranges from 0.52 to 0.81, from 0.58 to 0.71 and from 0.18 to 0.24 respectively; the continuous enhancement effect on the mixed solution of saline water, monosodium glutamate and I + G is better, and the flavor enhancement value is as follows in sequence: the persistence is more than the delicate flavor and more than the thick flavor, and the ranges of the flavor enhancing values are 0.71-1.2, 0.23-0.42 and 0.32-0.51 respectively; the enhancing effect on the delicate flavor and the thick flavor of the chicken soup is better, and the flavor developing and enhancing values are as follows in sequence: the delicate flavor is more than the thick flavor is more than the persistence, and the ranges of the flavor enhancing values are 0.69-0.84, 0.61-0.82 and 0.21-0.39 respectively; the enhancing effect on the delicate flavor and the thick flavor of the beef soup is better, the flavor enhancing values of the beef soup are sequentially thick flavor > delicate flavor > continuous flavor, and the ranges of the flavor enhancing values are 0.73-0.9, 0.41-0.66 and 0.19-0.38 respectively. Therefore, the oyster enzymolysis liquid can effectively improve the flavor of food, and has certain difference on the taste development influence of different foods.
Example 3
1. Pretreatment: taking oyster meat as a raw material, cleaning, draining, beating into meat paste, and adding water to prepare oyster homogenate with a feed liquid ratio of 1g;
2. adding 0.39wt% of mixed enzyme of Flavozyme 500MG and Pancretin (the mass ratio of Flavozyme 500MG to Pancretin is 1).
3. And (3) extracting peptides in the oyster enzymolysis liquid by ethanol grading:
adding 80mL of absolute ethyl alcohol into 20mL of oyster enzymolysis liquid, stirring for 30min at 25 ℃, centrifuging (8000 r/min,4 ℃,20 min) to obtain a precipitate (1) and a supernatant (1), adding absolute ethyl alcohol into the supernatant (1) to enable the final concentration of the ethyl alcohol in the system to be 90vol%, and stirring and centrifuging under the same conditions to obtain a precipitate (2) and a supernatant (2); and then adding 200mL of absolute ethyl alcohol into the supernatant (2) to enable the final concentration of the ethyl alcohol in the system to be 95vol%, stirring and centrifuging under the same conditions to obtain a supernatant (3), removing excess ethyl alcohol from the supernatant (3), and re-dissolving in deionized water to obtain the component to be detected. The specific operation flow is shown in figure 1.
UPLC-MS/MS separation and identification of novel taste-exhibiting peptides in fractions
The polypeptide in the ethanol extraction component is separated and identified by adopting an ultra performance liquid chromatography tandem mass spectrometry method and a de novo sequencing method, a sample is firstly separated by the ultra performance liquid chromatography, then enters a high-resolution mass spectrometer after electrospray ionization (ESI), is broken into fragment ions after being bombarded by a secondary mass spectrometry, and is separated according to different mass-to-charge ratios to form a secondary mass spectrogram (see figure 2). And searching by Peaks software and a database to match 1 novel peptide chain from the ethanol component, wherein the molecular weight of the peptide chain is less than 800Da, the identified peptide chain is Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu, and the peptide chain can be synthesized artificially.
5. Method for measuring taste development characteristics of short peptides by using artificial sensory evaluation method
(1) Enhanced effect of synthetic peptide-salt and monosodium glutamate mixture
The sensory standard substance is 0.35% monosodium glutamate and salt mixed solution, and the score is 5. The synthesized polypeptide was added to the standard solution, the polypeptide concentration was adjusted to 2mg/mL, and each of the prepared solutions was presented to a sensory panel (10 persons: 5 men and 5 women, trained sensory assessors) and evaluated for taste enhancement. Each solution was evaluated for sensory characteristics, including umami, kokumi (more complex mouthfeel, which assesses food 10 seconds later) and sustainability (sensory or flavour enhancement that can be sustained, which assesses food 25 seconds later). The flavor, saltiness and flavor development synergy of the fresh flavor and the saltiness are evaluated.
(2) Enhancing effect of synthetic peptide-salt: sensory standard was 0.35% saline solution and scored 5 points. Adding the synthetic peptide into a standard solution, adjusting the concentration of the polypeptide to be 2mg/mL, and evaluating the salty taste and the taste development synergy of the polypeptide.
TABLE 1
Figure BDA0002069423800000061
As can be seen from Table 1, the synthetic peptide has enhanced umami taste in the salt solution and reduced sour taste, which shows that the salt solution has certain effect of improving the umami taste of the flavor-developing peptide; the flavor enhancing effect is achieved in the mixed solution of the salt and the monosodium glutamate.

Claims (8)

1. The flavor peptide is characterized in that the amino acid sequence of the flavor peptide is Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu.
2. A method for preparing the taste peptide of claim 1 separated from oyster enzymolysis liquid, which comprises the following steps:
(1) Pretreatment: taking oyster meat as a raw material, cleaning, draining, beating into meat paste, and adding water to prepare oyster homogenate;
(2) Enzymolysis: adding a complex enzyme into the oyster homogenate for enzymolysis reaction, and after the reaction is finished, inactivating the enzyme and centrifuging to obtain filtrate which is oyster enzymolysis liquid, wherein the complex enzyme is mixed enzyme of flavourzyme Flavozyme 500MG and Pancreatin;
(3) And (3) extracting peptides in the oyster enzymolysis liquid by ethanol grading: adding ethanol into the oyster enzymolysis liquid, stirring uniformly, centrifuging to obtain a precipitate (I) and a supernatant (I), then adding ethanol into the supernatant (I), stirring uniformly, and centrifuging to obtain a precipitate (II) and a supernatant (II); and adding ethanol into the supernatant (II), stirring uniformly, centrifuging to obtain a supernatant (III), and removing the excessive ethanol from the supernatant (III).
3. The method according to claim 2, wherein the mass ratio of flavourzyme Flavorzyme500MG and Pancreatin in the mixed enzyme is 1.
4. The preparation method of claim 2, wherein the amount of the complex enzyme added in the step (2) is 0.1-0.5wt% of the oyster meat.
5. The preparation method of claim 2, wherein the temperature of the enzymolysis in the step (2) is 45-55 ℃ and the time is 4-8h.
6. The preparation method of claim 2, wherein the temperature of the enzymolysis in the step (2) is 50 ℃ and the time is 7h.
7. The preparation method according to claim 2, wherein in the step (3), 3-5 times of the volume of the oyster enzymolysis solution is added into the oyster enzymolysis solution; adding ethanol into the supernatant (I) to ensure that the final concentration of the ethanol in the system is 70-90vol%; ethanol was added to the supernatant (II) so that the final concentration of ethanol in the system was 95vol%.
8. Use of the taste peptide of claim 1 in a food product.
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