CN111537675A

CN111537675A - Method for determining key taste substances of grifola frondosa

Info

Publication number: CN111537675A
Application number: CN202010406715.2A
Authority: CN
Inventors: 宋诗清; 庄金达; 许锐; 冯涛; 孙敏; 姚凌云
Original assignee: Shanghai Institute of Technology
Current assignee: Shanghai Institute of Technology
Priority date: 2020-05-14
Filing date: 2020-05-14
Publication date: 2020-08-14

Abstract

The invention discloses a method for determining key taste substances of grifola frondosa. The method comprises the following steps: firstly, water-soluble components are extracted from grifola frondosa by cooking, the taste active substances judged in the grifola frondosa crude extract are quantitatively analyzed, then taste recombination, deletion and additional tests are carried out, and finally key taste contributors are identified and the sensory characteristics of the key taste contributors are verified. Dose response analysis was performed in simplified flavor recombinants to assess the flavor contribution of taste-presenting peptides in grifola frondosa.

Description

Method for determining key taste substances of grifola frondosa

Technical Field

The invention relates to a method for determining key taste substances of grifola frondosa, and belongs to the technical field of food science.

Background

Grifola frondosa (Grifola frondosa) belongs to Basidiomycotina, Hymenomycetes, Aphyllophorales, Polyporaceae, Grifola, also called Polyporus frondosus, Zhejiang Qingyuan, etc. commonly called as Clinopodium sinensis, Hebei Qianxi, etc. commonly called as chestnut mushroom, Sichuan called as Chiba, Japanese called as Maitake mushroom. The grifola frondosa is delicious in taste, rich in protein, carbohydrate, polysaccharide, vitamins and various trace elements, and has immune function and anticancer effect.

Due to the interaction between various nutritional components in food products, it is difficult to reveal the effect of each flavor compound by only quantitative analysis and individual flavor profile studies. The deficiency experiment establishes a bridge between physicochemical analysis and sensory data. In addition, complex relationships exist, such as synergistic or masking effects. Deletion experiments can be used to simplify the association between ingredients so that the taste contribution of the food extract under study can be analyzed in a comprehensive manner.

On the basis of the above sensory analysis, it is the purpose of this study to answer a confusing question of which key flavor compounds are responsible for characteristic flavors, and the role of taste-imparting peptides in the flavor of Grifola frondosa. The main research works in this chapter are (i) quantitative analysis of the taste active substances identified in the grifola frondosa crude extract and (ii) taste reorganization, deletion and additional tests, and finally identification of key taste contributors and verification of their organoleptic properties. Dose response analysis was performed in simplified flavor recombinants to assess the flavor contribution of taste-presenting peptides in grifola frondosa.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a method for determining key taste substances of grifola frondosa, and provides a basis for the development and application of grifola frondosa.

In order to solve the problems, the method for determining the key taste substances of the grifola frondosa is characterized by comprising the following steps of:

step 1): pretreating the grifola frondosa by adopting water bath reflux cooking, and cooling for later use;

step 2): measuring the flavor development substance;

step 3): selecting sensory evaluators, familiar with sensory processes, and respectively determining the concentrations of five basic taste and thick sense reference compounds, namely, sour, sweet, bitter, salty and fresh;

step 4): preparing a plurality of groups of samples, and evaluating the samples by a sensory evaluator;

step 5): the TAV and EUC values of the taste-imparting substances were measured.

Preferably, the step 1) is specifically: pretreating Grifola frondosa by adopting water bath reflux cooking, wherein the mass ratio of feed liquid is 1:20, the water bath temperature is 100 ℃, and the time lasts for 1.5 h; then subjecting the crude extract to refrigerated centrifugation at 7000rmp at below 4 deg.C for 15 min; filtering the supernatant with gauze, and filtering with 0.45 μm microporous filter for the second time.

Preferably, the taste substances in step 2) are free amino acids, 5' -nucleotides, organic acids, inorganic ions and taste peptides.

More preferably, the free amino acids are 17, respectively aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, alanine, tyrosine, cysteine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline; the nucleotide comprises 3 kinds, namely 5' -GMP, 5' -IMP and 5' -AMP; two organic acids are provided, namely lactic acid and succinic acid; the inorganic ions are 4 kinds, and are respectively Na⁺、K⁺、PO₄ ^3-And Cl^-。

Preferably, the step 3) is specifically:

step 3.1): selecting the same number of women and men with the age of 20-26 years, and requiring no taste disturbance and having more than half a year of sensory evaluation experience; the use of a nose clip to prevent the effects of other tastes; controlling the temperature of the sample and the mouthwash to be about 25 ℃; during the test, the sensory evaluator drunk 10mL of the sample in the mouth for 10s during the tasting process and then spit it off, the mouthwash was used for mouth rinsing to prevent aftertaste; the sensory evaluator had to rest for at least 30 seconds before tasting the next sample;

step 3.1): all sensory evaluators will learn how to distinguish and standardize five basic flavors, richness and complex flavors perceived according to a series of sensory methods; first, they will evaluate five standard basic flavor aqueous solutions, citric acid, sugar, quinine, salt and MSG corresponding to five basic flavors of sour, sweet, bitter, salty and fresh; then using a three-point test to estimate the sensitivity of the panel to each of the five basic flavors at lower concentrations than the above five basic flavors; training the panellists for a sense of richness according to the Meyer method, using GSH (glutathione) as the standard; finally, sensory evaluation personnel sequence the MSG with different concentrations in delicate flavor; in tests involving samples, standardization of taste intensity for the samples was determined by intra-panel discussions; all sensory evaluators need to use standardized methods and remember consistency of taste intensity; the test cannot be started until the sensory data of the sensory evaluators are stable.

Preferably, in the step 4), a sensory evaluator tastes the prepared sample, and the sensory evaluator evaluates the sample by using a score of 0-4, wherein 0-none, 1-weak, 2-medium, 3-strong and 4-strong; the intensity of the ultrapure water was set to 0 and the citric acid, sugar, quinine, salt, MSG and GSH were all set to 4.

Preferably, in the step 4), the grifola frondosa flavor substances are analyzed respectively from a deletion experiment, an addition experiment and a dose reaction experiment; the deletion experiment was:

comparing a plurality of incomplete flavor reconstitution samples lacking a single set or group of flavor compounds to CTR; selecting a sample different from the other two samples by a sensory tester according to a three-point test method from three samples comprising an incomplete taste recombined sample and two CTR samples, and sequentially preparing all the samples; meanwhile, accurately judged sensory evaluators need to describe how different the sample is from the CTR and score the sample according to the taste characteristic analysis;

the additional experiments described were:

firstly, preparing a sample model according to the concentrations of different flavor active compounds formed by a deletion experiment, then judging compounds which do not contribute to flavor, and adding the compounds into the sample model one by one according to the original concentration; finally, each sample is submitted to a sensory evaluator to be compared with the sample model according to a three-point test method;

the dose response experiment was:

respectively preparing STRs of flavor peptides with the addition concentration of 20.0mg/100mL, and setting the delicate flavor and the thick feeling intensity to be 0; then adding the mixture into STR respectively to make the dosage reach the set concentration; sensory evaluators tasted the samples first and then rated on an 8cm non-structural linear scale for the value of-4 to +4 representing varying degrees of "antagonism" to "contribution" based on the perceived taste contribution, including umami and richness. All sensory evaluation tests were carried out in a constant temperature sensory laboratory equipped with a ventilation system and a white light fixture.

Preferably, the method for measuring the TAV and EUC values in step 5) is as follows:

TAV is generally adopted to evaluate the contribution of a single flavor substance to the flavor of food, and is a relatively objective evaluation method, which is widely applied to flavor research of various foods at present, and the calculation formula is as follows:

when the TAV is more than 1, the substance is indicated to contribute to the flavor of the food, and the contribution degree is in direct proportion to the value; when the TAV is less than 1, it indicates that the substance does not contribute to the flavor of the food;

the flavor nucleotide (such as 5' -GMP, 5' -IMP, 5' -AMP, etc.) has synergistic effect on the flavor amino acid (such as Glu or Asp), and can enhance the flavor; therefore, EUC is used to evaluate the synergistic umami effect of the two, i.e. 100g of synergistic umami effect of the two produces the umami intensity equal to the umami intensity produced by corresponding grams of sodium glutamate (MSG), the unit is g/100g, and the calculation formula is:

EUC/(g/100g)＝∑a_pb_p+1218×∑a_pb_p×∑a_qb_qformula 2;

in formula 2: ap is the mass fraction of Glu or Asp,%; bp is the relative freshness coefficient of Glu or Asp equivalent to MSG (Glu-1; Asp-0.077); aq is the mass fraction of 5' -GMP, 5' -IMP and 5' -AMP,%; bq is 5 '-GMP, 5' -IMP, 5 '-AMP equivalent to MSG relative freshness coefficient (5' -GMP ═ 2.3, 5 '-IMP ═ 1, 5' -AMP ═ 0.18).

The invention also provides a grifola frondosa flavor-presenting peptide which is characterized in that the amino acid sequence is Arg-Ser-Gly-Val (RSGV).

Preferably, the grifola frondosa taste peptide is derived from grifola frondosa extract.

Preferably, the grifola frondosa flavor-developing peptide is artificially synthesized.

The invention has the beneficial effects that:

the invention adopts a series of flavor substance determination and sensory evaluation experiments to establish a set of identification method of key flavor substances of grifola frondosa.

Drawings

FIGS. 1a-e show the scores of Grifola frondosa crude extract after recombination with different flavors; wherein, the solid line is a taste profile of the crude extract, and the dotted line is a taste profile of each group of taste recombinant;

figure 2 is a graph of the effect of adding different concentrations of RSGV in STR on two flavor attributes (umami and richness).

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

Example 1

The embodiment provides a grifola frondosa flavor peptide (the flavor peptide is obtained by artificially synthesizing a grifola frondosa extracting solution after separation, purification and identification to obtain an amino acid sequence) with an amino acid sequence of Arg-Ser-Gly-Val (RSGV) in a dry grifola frondosa product (sold in Qingyuan Zhejiang), and the method comprises the following specific steps:

step 1): determining the composition, concentration, threshold value and TAV value of non-volatile flavor substances in the Grifola frondosa crude extract

In order to elucidate the main flavor active compounds and construct a solution simulating the flavor of natural extracts, it is first necessary to quantify the content of flavor compounds in the crude extract of Grifola frondosa.

TABLE 1 composition, concentration, threshold and TAV value of non-volatile flavor substances in Grifola frondosa crude extract

Taste Activity Value (TAV), ratio of concentration of taste substance to its threshold value. The threshold value (mg/100mL) was verified by reference.

TABLE 2 Grifola frondosa EUC

Step 2): grifola frondosa taste feature analysis

According to quantitative analysis, Complete Taste Reorganization (CTR) was reorganized from the 26 taste substances provided in Table 1, and its taste characteristics were compared with those of fresh Grifola frondosa aqueous extracts by a five-part method. Sensory analysis showed that the grifola frondosa crude extract had the highest umami and richness scores of 2.5 and 2.7, respectively, and the second bitterness and saltiness scores of 1.1 and 1.5, respectively (fig. 1a), compared to very low sour and sweet scores of 0.8 and 0.5, respectively. Therefore, the characteristic taste of the grifola frondosa crude extract is umami and rich, followed by salty and bitter tastes, and sour and sweet tastes are negligible. Meanwhile, the taste profile of CTR (fig. 1b) is similar to that of the grifola frondosa crude extract, the sour and sweet taste scores are consistent, and the umami and rich taste scores of CTR are slightly lower than that of the grifola frondosa crude extract. It can be concluded that these 26 flavors can effectively replicate the characteristic flavor of the Grifola frondosa crude extract, and also illustrate that the key flavors of Grifola frondosa have been successfully identified and quantified.

Step 3): deletion experiments

After the taste analysis was completed, a deletion experiment was performed to explore the taste contribution of the small groups and the single taste substances to the grifola frondosa, and key taste substances were obtained from table 3. The first step is a panel deletion experiment, and when panels one, two, three, four are removed from the CTR, significant flavor differences can be found. By way of example, when a small group one (free amino acids) is knocked out, a significant reduction in umami and richness can be found. Similarly, when small groups of di (nucleotides) were knocked out, a significant reduction in umami and richness occurred. According to previous studies, free amino acids and nucleotides are reported to be major contributors to umami taste and richness. In the same way, it was demonstrated that the absence of panel four (taste-imparting inorganic ion) resulted in a reduction of umami and salty taste. In this study, it was found that the characteristic taste of the grifola frondosa was umami and rich, and sweet taste and sour taste were weak. And, the taste contribution of the third group (organic acid) to the grifola frondosa is basically negligible.

After the experiment of the taste contribution of the small groups of taste substances to the grifola frondosa is completed, the same deletion experiment is used for researching the single taste substances of the four small groups, and specifically, the taste substances are removed from the four small groups one by one to research the taste contribution of each taste substance to the grifola frondosa. According to table 3, in group one, the absence of glutamic acid resulted in a significant decrease in umami taste. In panel two, the lack of GMP alone resulted in a reduction in umami, sour, salty and bitter taste, combining sensory results with TAV to arrive at GMP as one of the key flavors of grifola frondosa. In panel three, the absence of succinic acid resulted in a decrease in umami, sour and tartness. In subgroup four, Cl^-The lack of (b) results in a decrease in umami taste, an increase in sour and bitter taste.

TABLE 3 influence of the subgroup or the deletion of the Single Compound on the complete taste reorganization of Grifola frondosa

So, sour taste; s, sweet taste; b, bitterness; sa, salty taste; u, umami; k, thick feeling; -, no significance; p < 0.05; p < 0.01; p < 0.001

Step 4): additional experiments

10 (glutamic acid, serine, arginine, lysine, proline AMP, GMP, IMP, succinic acid, Cl) combined with the panel and deletion experiments of single flavor substances^-) The taste substances are identified as key taste active substances of the grifola frondosa. To avoid losing potentially critical flavor substances, additional experiments were further conducted to determine if the combination of several substances would have an effect on one or more flavor characteristics. However, none of the substances were identified as significantly different.

Therefore, according to the deletion experiment and the additional experiment, the simple flavor recombination (STR) is composed of the above 10 flavor active substances, and the comparison graph of the STR and the flavor profile of the crude extract of the grifola frondosa is shown in FIG. 1 c. Glutamic acid, IMP, succinic acid, Cl-significantly contribute to the taste reorganization of Grifola frondosa (p < 0.05). According to the analysis of the TAV results, the TAV of 6 flavors was less than 1, but was still identified as the key flavor. This demonstrates that these substances still exhibit the characteristic taste of grifola frondosa at subthreshold levels. Although the TAV value of lactic acid is greater than 1, it does not contribute to the characteristic taste of Grifola frondosa through a series of sensory evaluations. The reason may be that TAV is a useful value to assess a single flavor, while interactions (e.g., synergy or masking) of compounds in a food environment may not be taken into account.

As previously described, the flavor characteristics between CTR and STR were compared by the three-point test and sensory evaluation. Although significant differences in individual taste characteristics were found, there was little discrimination between these two solutions. Umami amino acids (aspartic acid and glutamic acid) and 5' -nucleotides, especially AMP, GMP and IMP, play a major role in umami, and the synergistic effect of these compounds can further enhance umami, such as aspartic acid, glutamic acid and IMP as well as sweet amino acids (serine, glycine and alanine) and IMP. The lower intensity of umami taste in STR compared to CTR may be due to partial deletion of these compounds, resulting in a weaker synergistic effect, while the difference in CTR and STR may have other unknown compounds, such as oligopeptides.

Step 5): the taste peptide contributes to the taste of Grifola frondosa.

By adding taste peptide RSGV to CTR and STR, and adding the peptide to CTR, a significant increase in umami and richness (p <0.05) was detected by three-point assay (table 4). Furthermore, according to the taste profile analysis of the recombinant, it was revealed that the umami taste (2.5 → 2.8) and the rich feeling (2.7 → 2.9) were well matched with the taste profile in the Grifola frondosa crude extract (FIG. 1 d). Similarly, near positive results were also observed in STRs. By adding peptides separately, the scores of umami (2.2 → 2.4) and chunky (2.2 → 2.5) also increased (fig. 1 e). Therefore, the addition of the peptide can compensate for the difference in taste between the Grifola frondosa crude extract and the artificially synthesized Grifola frondosa recombinant. On this basis, deletion experiments were also performed to narrow down the major flavor peptides that contributed to the characteristic taste of Grifola frondosa. As a result, it can be concluded that the taste peptide RSGV can be a key taste active substance of Grifola frondosa.

TABLE 4 Effect of addition of taste peptides on CTR and STR taste characteristics

TABLE 5 Effect of deletion of Single taste peptides on the taste characteristics of CTR and STR

Step 6): dose response analysis

According to FIG. 2, the positive correlation between the concentration of the taste peptide RSGV and the intensity of umami and richness is revealed. The pearson correlation coefficients for the umami and rich taste of RSGV were 0.94 and 0.92, respectively. Thus, this taste peptide RSGV has a clear umami and rich impact in grifola frondosa. The results prove that the taste development peptide RSGV is one of essential components in the characteristic taste of the grifola frondosa. Further studies are necessary to quantify the concentration of the taste peptide RSGV so that the contribution to the taste of grifola frondosa can be accurately elucidated.

The polypeptide extracted from the grifola frondosa has an obvious umami enhancing effect, can be applied to the field of food, can be used as a base material and an auxiliary material as a seasoning, is salty in the fresh generation, is nutritious and safe, and can meet the sensory requirement.

Claims

1. A method for determining key taste substances of grifola frondosa is characterized by comprising the following steps:

step 2): measuring the flavor development substance;

2. The method for determining key flavor substances of grifola frondosa according to claim 1, wherein the step 1) is specifically as follows: pretreating Grifola frondosa by adopting water bath reflux cooking, wherein the mass ratio of feed liquid is 1:20, the water bath temperature is 100 ℃, and the time lasts for 1.5 h; then subjecting the crude extract to refrigerated centrifugation at 7000rmp at below 4 deg.C for 15 min; filtering the supernatant with gauze, and filtering with 0.45 μm microporous filter for the second time.

3. The method of claim 1, wherein the taste substances in step 2) are free amino acids, 5' -nucleotides, organic acids, inorganic ions and taste peptides.

4. The method of claim 3, wherein said 17 kinds of free amino acids are aspartic acid, glutamic acid, serine, histidine,Glycine, threonine, arginine, alanine, tyrosine, cysteine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline; the nucleotide comprises 3 kinds, namely 5' -GMP, 5' -IMP and 5' -AMP; two organic acids are provided, namely lactic acid and succinic acid; the inorganic ions are 4 kinds, and are respectively Na⁺、K⁺、PO₄ ^3-And Cl^-。

5. The method for determining key flavor components of Grifola frondosa according to claim 1, wherein the step 3) is specifically:

step 3.1): all sensory evaluators will learn how to distinguish and standardize five basic flavors, richness and complex flavors perceived according to a series of sensory methods; first, they will evaluate five standard basic flavor aqueous solutions, citric acid, sugar, quinine, salt and MSG corresponding to five basic flavors of sour, sweet, bitter, salty and fresh; then using a three-point test to estimate the sensitivity of the panel to each of the five basic flavors at lower concentrations than the above five basic flavors; training the thick feeling of the panelists according to the Meyer method, wherein the used standard substance is GSH; finally, sensory evaluation personnel sequence the MSG with different concentrations in delicate flavor; in tests involving samples, standardization of taste intensity for the samples was determined by intra-panel discussions; all sensory evaluators need to use standardized methods and remember consistency of taste intensity; the test cannot be started until the sensory data of the sensory evaluators are stable.

6. The method for determining key taste substances of grifola frondosa according to claim 1, wherein in the step 4), sensory evaluators taste the prepared samples, and the sensory evaluators evaluate the samples by using a score of 0-4, wherein 0-none, 1-weak, 2-moderate, 3-strong and 4-strong; the intensity of the ultrapure water was set to 0 and the citric acid, sugar, quinine, salt, MSG and GSH were all set to 4.

7. The method for determining key flavor components of Grifola frondosa according to claim 1, wherein in the step 4), the flavor components of Grifola frondosa are analyzed respectively from a deletion experiment, an addition experiment and a dose reaction experiment; the deletion experiment was:

the additional experiments described were:

the dose response experiment was:

respectively preparing STRs of flavor peptides with the addition concentration of 20.0mg/100mL, and setting the delicate flavor and the thick feeling intensity to be 0; then adding the mixture into STR respectively to make the dosage reach the set concentration; sensory evaluators tasted the samples first and then rated on an 8cm non-structural linear scale for the value of-4 to +4 representing varying degrees of "antagonism" to "contribution" based on the perceived taste contribution, including umami and richness.

8. The method for determining key flavor components of Grifola frondosa according to claim 1, wherein the TAV and EUC values in step 5) are determined by:

the evaluation of the contribution of single flavor substances to the taste of food usually adopts TAV, and the calculation formula is as follows:

the flavor nucleotide has a synergistic effect, so that the delicate flavor can be enhanced; therefore, the EUC is adopted to evaluate the synergistic freshness-enhancing effect of the two, namely the delicious taste intensity generated by 100g of the synergistic freshness-enhancing effect of the two is equal to the delicious taste intensity generated by corresponding grams of sodium glutamate, the unit is g/100g, and the calculation formula is as follows:

EUC/(g/100g)＝∑a_pb_p+1218×∑a_pb_p×∑a_qb_qformula 2;

in formula 2: ap is the mass fraction of Glu or Asp,%; bp is Glu or Asp which is equal to the relative freshness coefficient of MSG; aq is the mass fraction of 5' -GMP, 5' -IMP and 5' -AMP,%; bq is the relative freshness coefficient of 5' -GMP, 5' -IMP, 5' -AMP is equivalent to MSG.