Flavor peptide of termitomyces albuminosus and preparation method and application thereof

Abstract

The present application belongs to the field of food technology, and particularly relates to a fresh taste peptide of Grifola frondosa, a preparation method and application thereof. The fresh taste peptide of Grifola frondosa comprises polypeptide I and / or polypeptide II; the polypeptide I comprises an amino acid sequence as shown in SEQ ID NO. 1; and the polypeptide II comprises an amino acid sequence as shown in SEQ ID NO. 2. The fresh taste peptide of Grifola frondosa is separated from Grifola frondosa, and particularly belongs to a water-soluble component of Grifola frondosa, has fresh taste, can interact with fresh taste receptor T1R1 / T1R3, and enhances the flavor of food.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food technology, and particularly relates to a fresh-taste peptide of Termitomyces albuminosus and a preparation method and application thereof. BACKGROUND

[0002] Taste is an important index for evaluating the quality of food. Small molecule peptides, amino acids, nucleotides and inorganic ions can all contribute to the taste of food. The taste of food is due to the stimulation of small molecule taste substances to the corresponding taste bud cells in the taste organ of the tongue, resulting in a series of taste sensations such as sour, sweet, bitter, salty and umami. Different taste substances bind to different taste bud cells to produce their respective characteristic tastes.

[0003] Peptides in food exhibit different tastes such as sour, sweet, bitter, salty and umami due to the differences in the length of the peptide chain, the composition and arrangement of amino acids. Taste peptides in food can improve or mask the sensory properties of food and play an important role in the flavor of food. Taste peptides are a class of oligopeptides with a molecular weight of less than 3000 Da, which are usually obtained by enzymatic hydrolysis or amino acid synthesis. These peptide components can interact with specific receptors on the taste buds of the tongue to produce characteristic tastes. In addition, some taste peptide components cannot produce taste sensations after binding to these receptors, but only enhance the taste, such as umami peptides and thickening peptides. At present, according to the taste of taste peptides in food, they are divided into two categories: flavor precursor peptides and characteristic taste peptides. Characteristic taste peptides can be divided into sour taste peptides, sweet taste peptides, bitter taste peptides, salty taste peptides, umami taste peptides and thickening peptides according to their taste characteristics. The delicious taste of edible fungi is due to the presence of many taste active substances. Current research on taste substances in edible fungi mainly focuses on taste amino acids, taste nucleotides and carbohydrates, and there is still a lack of research on polypeptides in edible fungi.

[0004] Termitomyces albuminosus belongs to Basidiomycetes, Agaricales, Pleurotaceae and Termitomyces. It is also known as Pleurotus, chicken foot mushroom, chicken mushroom, chicken mushroom, white ant mushroom and ant fungus. Termitomyces albuminosus contains essential proteins, fats, polysaccharides, vitamins, potassium, calcium, phosphorus and 17 kinds of amino acids. Modern research shows that Termitomyces albuminosus has the physiological functions of analgesic and anti-inflammatory, tonifying qi and generating fluid, anti-aging, anti-tumor, anti-fatigue, antioxidant, anti-cancer, hypoglycemic and hypolipidemic. Regular consumption of Termitomyces albuminosus can enhance the body's immunity, improve the anti-viral ability and delay aging. In recent years, research on Termitomyces albuminosus has been conducted in many aspects, such as the culture conditions of Termitomyces albuminosus and the pharmacological research of Termitomyces albuminosus. However, there are few reports on the development and research of umami peptides of Termitomyces albuminosus. Therefore, the development of Termitomyces albuminosus germplasm resources has great practical value. SUMMARY

[0005] The present application aims to provide a tremella fresh taste peptide, a preparation method and application thereof, enrich the types of fresh taste peptide, and improve the flavor of food.

[0006] The present application provides a tremella fresh taste peptide, comprising polypeptide I and / or polypeptide II.

[0007] The polypeptide I comprises an amino acid sequence as shown in SEQ ID NO. 1; and the polypeptide II comprises an amino acid sequence as shown in SEQ ID NO. 2.

[0008] The present application also provides a preparation method of the tremella fresh taste peptide.

[0009] The tremella water-soluble component contains the tremella polypeptide.

[0010] Preferably, the extraction method comprises:

[0011] The fruiting body of tremella is mixed with water at a mass-volume ratio of 1g:(3-10)mL, heated at 80-100℃ for 40-90min, and the extraction liquid is collected to obtain the tremella water-soluble component.

[0012] Preferably, after the extraction, the tremella water-soluble component obtained by extraction is sequentially subjected to concentration and freeze-drying treatment to obtain a tremella water-soluble freeze-dried powder, and then separated and purified to obtain the tremella fresh taste peptide.

[0013] Preferably, the volume ratio of the tremella water-soluble component before and after concentration is (3-5):1.

[0014] Preferably, the separation and purification comprises gel chromatography column separation.

[0015] The present application also provides an application of the tremella fresh taste peptide in food.

[0016] Preferably, the food comprises a fresh taste food.

[0017] The present application also provides a food seasoning, and the effective component comprises the tremella fresh taste peptide.

[0018] Preferably, the concentration of the tremella fresh taste peptide in the food seasoning is 0.5-3mmol / L.

[0019] The present application provides chicken mushroom umami peptides, comprising polypeptide I and / or polypeptide II; the polypeptide I comprises an amino acid sequence as shown in SEQ ID NO. 1; and the polypeptide II comprises an amino acid sequence as shown in SEQ ID NO. 2. The chicken mushroom umami peptides are separated from chicken mushroom, and specifically belong to water-soluble components of chicken mushroom, have umami, can interact with umami receptor T1R1 / T1R3, and enhance the flavor of food. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced.

[0021] Figure 1 A chromatogram of chicken mushroom water-soluble freeze-dried powder after Sephadex G-15 gel chromatography separation;

[0022] Figure 2 Electronic tongue radar fingerprint of chromatographic components A1, A2, A3 and A4;

[0023] Figure 3 Total ion flow chromatogram of A4b component determined by Nano-ESI-LC-MS / MS;

[0024] Figure 4 RP-HPLC separation spectrum of chromatographic component A4;

[0025] Figure 5 Secondary mass spectrum of polypeptide I (DSTDEKFLR);

[0026] Figure 6 Secondary mass spectrum of polypeptide II (AMDDDEADLLLLAM);

[0027] Figure 7 Secondary mass spectrum of polypeptide III (KLNDAQAPK);

[0028] Figure 8 Secondary mass spectrum of polypeptide IV (VGKGAHLSGEH);

[0029] Figure 9 Secondary mass spectrum of polypeptide V (MLKKKKLA);

[0030] Figure 10 Secondary mass spectrum of polypeptide VI (SLGFGGPPGY);

[0031] Figure 11 Secondary mass spectrum of polypeptide VII (TVATFSSSTKPDD);

[0032] Figure 12A bubble chart of umami degree, saltiness and thickness of seven polypeptides of Termitomyces albuminosus, wherein the letter D is polypeptide I, A is polypeptide II, K is polypeptide III, V is polypeptide IV, M is polypeptide V, S is polypeptide VI, and T is polypeptide VII;

[0033] Figure 13 An electronic tongue radar fingerprint chart of the interaction of polypeptide I and polypeptide II with chicken soup;

[0034] Figure 14 A T1R1 / T1R3 umami receptor chart;

[0035] Figure 15 A molecular docking result of polypeptide I and T1R1 / T1R3 umami receptor;

[0036] Figure 16 A molecular docking result of polypeptide II and T1R1 / T1R3 umami receptor;

[0037] Figure 17 A molecular docking result of polypeptide III and T1R1 / T1R3 umami receptor;

[0038] Figure 18 A molecular docking result of polypeptide IV and T1R1 / T1R3 umami receptor;

[0039] Figure 19 A molecular docking result of polypeptide V and T1R1 / T1R3 umami receptor;

[0040] Figure 20 A molecular docking result of polypeptide VI and T1R1 / T1R3 umami receptor;

[0041] Figure 21 A molecular docking result of polypeptide VII and T1R1 / T1R3 umami receptor. DETAILED DESCRIPTION

[0042] The present application provides a Termitomyces albuminosus umami peptide, which comprises polypeptide I and / or polypeptide II.

[0043] The polypeptide I comprises an amino acid sequence as shown in SEQ ID NO. 1; and the polypeptide II comprises an amino acid sequence as shown in SEQ ID NO. 2.

[0044] In the present application, the amino acid sequence shown in SEQ ID NO. 1 is DSTDEKFLR; and the amino acid sequence shown in SEQ ID NO. 2 is AMDDDEADLLLLAM. The Termitomyces albuminosus umami peptide in the present application is isolated from Termitomyces albuminosus, and specifically belongs to a water-soluble component of Termitomyces albuminosus, has umami taste, can interact with umami receptor T1R3, and enhances the flavor of food.

[0045] The chicken mushroom umami peptide of the present application is not strictly required to be obtained from a specific source, and can be artificially synthesized or prepared by oneself. In the specific implementation process of the present application, the chicken mushroom umami peptide is preferably obtained by self-preparation.

[0046] The present application also provides a preparation method of the chicken mushroom umami peptide, comprising the following steps:

[0047] Extracting water-soluble components of chicken mushroom; the chicken mushroom polypeptide is contained in the water-soluble components of chicken mushroom.

[0048] The water-soluble components of chicken mushroom are extracted, and the extraction method preferably comprises the following steps: mixing fruiting bodies of chicken mushroom with water at a mass-volume ratio of 1g:(3-10)mL, heating at 80-100℃ for 40-90min, collecting the extraction liquid to obtain the water-soluble components of chicken mushroom. The mass-volume ratio of the fruiting bodies of chicken mushroom to water is preferably 1g:(5-8)mL, and more preferably 1g:6mL; the heating temperature is preferably 85-95℃, and more preferably 90℃; the heating time is preferably 50-80min, and more preferably 60-70min; and the collection method is preferably centrifugation at 4℃ and 10000r / min for 15min.

[0049] After obtaining the water-soluble components of chicken mushroom, the present application preferably concentrates the extracted water-soluble components of chicken mushroom to obtain a concentrated liquid. The concentration is preferably performed using a vacuum rotary evaporator. The volume ratio of the water-soluble components of chicken mushroom to the concentrated liquid is preferably (3-5):1, and more preferably 4.5:1. For example, after obtaining 1.8L of extraction liquid (i.e. water-soluble components of chicken mushroom) by extraction, the volume is preferably concentrated to 0.4L.

[0050] After obtaining the concentrated liquid, the present application preferably performs freeze-drying treatment on the concentrated liquid to obtain water-soluble freeze-dried powder of chicken mushroom. The freeze-drying treatment is preferably performed for 48h at a temperature of-50℃. The freeze-drying treatment is preferably performed using a vacuum freeze-drying machine.

[0051] After obtaining the water-soluble freeze-dried powder of chicken mushroom, the present application preferably performs separation and purification on the water-soluble freeze-dried powder of chicken mushroom to obtain the chicken mushroom umami peptide. The separation and purification preferably comprises gel chromatography column separation, and more preferably Sephadex G-15 gel chromatography separation and RP-HPLC separation.

[0052] The application also provides application of the chicken mushroom umami peptide in food. The food preferably includes umami food. The chicken mushroom umami peptide is separated from chicken mushroom and specifically belongs to water-soluble components of chicken mushroom, has umami, and can interact with umami receptor T1R3 to enhance the flavor of food.

[0053] The application also provides a food seasoning, and the effective component includes the chicken mushroom umami peptide. The concentration of the chicken mushroom umami peptide in the food seasoning is preferably 0.5-3 mmol / L, and is further preferably 1.24 mmol / L.

[0054] In order to further illustrate the application, the chicken mushroom umami peptide, the preparation method and the application thereof provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0055] Example 1

[0056] 1. Obtain water-soluble extract of chicken mushroom

[0057] Raw material: 200 g of chicken mushroom (Termitomyces albuminosus, purchased from the wild mushroom trading market in Wuding County, Chuxiong Prefecture, Yunnan Province) is cut into pieces by a tissue grinder. The cut sample is transferred into a round-bottom flask, 1:3 (w / w) of ultrapure water is added, and heating is performed on a 100℃ electric heating jacket for 60 min, repeated three times; then the sample is filtered, centrifuged at 10000 r / min for 15 min at 4℃, and the centrifugal liquid of three repeated times is collected to obtain 1.8 L of water-soluble extract of chicken mushroom; the water-soluble extract of chicken mushroom is concentrated to 0.4 L by a vacuum rotary evaporator, and then freeze-dried by a vacuum freeze-drying machine. The freeze-dried powder of chicken mushroom is stored at -8℃ for standby.

[0058] 2. Separation and purification of water-soluble extract of chicken mushroom

[0059] 120 mg of the freeze-dried powder of chicken mushroom obtained in step 1 is dissolved in 2 mL of ultrapure water (60 mg / mL), filtered through a 0.45 μm cellulose membrane, and then separated by a Sephadex G-15 gel chromatography column (eluent: ultrapure water, flow rate: 1 mL / min), and monitored at 220 nm by an HD-3 ultraviolet detector. The chromatogram result is shown in Figure 1 , wherein the horizontal coordinate represents elution time in hours, and the vertical coordinate represents abundance at a detection wavelength of 220 nm. The elution profile is shown in Figure 1Four absorption peaks can be seen, and four elution fractions (i.e. chromatographic fractions, labeled as Al, A2, A3 and A4 in sequence) are collected in sequence during the elution process. The collected elution fractions are subjected to freeze-drying treatment, and the freeze-dried powders of the Al fraction, the freeze-dried powders of the A2 fraction, the freeze-dried powders of the A3 fraction and the freeze-dried powders of the A4 fraction are obtained in sequence, and are stored at -80°C for standby.

[0060] Test Example 1

[0061] Taste property detection - electronic tongue method detection

[0062] The freeze-dried powders of the Al fraction, the freeze-dried powders of the A2 fraction, the freeze-dried powders of the A3 fraction and the freeze-dried powders of the A4 fraction obtained in Example 1 are respectively dissolved in 10 mM KCL, and the concentration is 0.2 mg / mL, which is measured by a TS-5000Z taste sensor system. The taste sensor system is equipped with five sensor probes, and the AAE probe measures umami and richness (umami aftertaste), the CT0 probe measures saltiness, the CA0 probe measures sourness, the C00 probe measures bitterness and bitterness aftertaste, and the AE1 probe measures astringency and astringency aftertaste, and the measurement results are shown in Table 1. Figure 2 Figure 2 It can be seen that the Al, A2, A3 and A4 fractions all have umami, followed by saltiness, and the sourness is the weakest. Among them, the umami intensity of A4 is the highest, so the A4 chromatographic fraction is selected for the next step of RP-HPLC separation and purification.

[0063] Example 2

[0064] The A4 fraction is further separated and purified by RP-HPLC method. 20 mg of the freeze-dried powders of the A4 fraction obtained in Example 1 is dissolved in 2 mL of ultrapure water (20 mg / mL), and filtered through a 0.45 μm cellulose membrane. The chromatographic conditions are as follows: Agilent chromatographic column (ZORBAX SB-C18, 9.4 x 150 mm i.d., 5 μm), sample amount is 10 μL. The mobile phase is 5% methanol and 95% ultrapure water containing 0.05% (v / v) trifluoroacetic acid, the flow rate is 2 mL / min, isocratic elution for 20 min, the column temperature is 35°C, and the detection wavelength is 220 nm. The chromatographic spectrum results are shown in Table 2. Figure 3 Figure 3 It can be seen that the A4 fraction can see one main absorption peak A4b, and the eluent of the separation peak is collected and freeze-dried to obtain the freeze-dried powders of the A4b fraction, which is stored at -80°C.

[0065] Test Example 2

[0066] Structure identification of polypeptide sequence

[0067] ​​1. Sample preparation: The lyophilized powder of A4b component obtained in Example 2 was dissolved in pure water, and a dithiothreitol solution was added to a final concentration of 10 mM, and reduced at 56 °C for 1 h; an iodoacetamide solution was added to a final concentration of 50 mM, and reacted in the dark for 40 min; the extracted polypeptide was lyophilized to near dryness, and dissolved in 2-20 μL of 0.1% formic acid.

[0068] 2. The amino acid sequence and molecular weight of the polypeptide were determined by Nano-ESI-LC-MS / MS, and the determination conditions were as follows:

[0069] Liquid phase conditions: Pre-column: 300 μm i.d. x 5 mm, packed with Acclaim PepMap RPLC C18, 5 μm, Analytical column: 150 μm i.d. x 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, Injection volume: 5 μL; flow rate: 600 nL / min; gradient elution was performed using two eluents as mobile phases, eluent A was 0.1% formic acid aqueous solution, and eluent B was 0.1% formic acid acetonitrile solution; column temperature: 35 °C; analysis time: 66 min; gradient elution conditions were as follows: 0-2 min: 4%-8% B; 2-45 min: 8%-28% B; 45-55 min: 28%-40% B; 55-56 min: 40%-95% B; 56-66 min: 95% B.

[0070] Mass spectrometry parameters: the separated peptide fragments were directly introduced into an electrospray-combined ion trap orbitrap mass spectrometer for online monitoring, and the specific parameters were as follows:

[0071] Primary mass spectrometry parameters: Resolution: 70,000; AGCtarget: 3e6; MaximumIT: 40 ms; Scanrange: 300 to 1800 m / z.

[0072] Secondary mass spectrometry parameters: Resolution: 17,500; AGCtarget: 1e5; MaximumIT: 60 ms; TopN: 20; NCE / steppedNCE: 27.

[0073] Database search: (1) The mass spectrum original file was searched by PEAKS studio against the target protein database, and the search parameters were as follows: Fixed modifications: Carbamidomethyl; (2) Variable modifications: Oxidation (MW); (3) Enzyme: Unspecific; (4) Maximum Missed Cleavages: 2; (5) Peptide Mass Tolerance: 20 ppm; (6) Fragment Mass Tolerance: 0.02 Da; (7) Mass values of peptide and fragment ions: Monoisotopic; (8) Significance threshold: 0.01. The results of the total ion current chromatogram of component A4b determined by Nano-ESI-LC-MS / MS are shown in Fig. 1. Figure 4 After the component A4b was separated and identified by Nano-ESI-LC-MS / MS, the relative molecular weight of the component A4b was directly identified by Biolynx in Masslynx. It was found that seven polypeptides were analyzed from the component A4b; the amino acid sequences in the component A4b were identified by the secondary mass spectrum, and finally seven polypeptide sequences were analyzed after manual verification, and the results are shown in Table 1 and Fig. 2. Figures 5-11 wherein Figures 5-11 the abscissa is the molecular weight (m / z), and the ordinate represents the abundance.

[0074] Table 1 Molecular mass and relative retention time of seven polypeptides

[0075]

[0076] According to Table 1 and Figures 5-11 It can be seen that there are seven polypeptides in the component A4b, and the relative molecular weights are 1090.552 Da, 1109.5352 Da, 983.54 Da, 958.6361 Da, 950.4498 Da, 1354.6252 Da and 1534.6895 Da, respectively, and the sequences are DSTDEKFLR (polypeptide I), AMDDDEADLLLLAM (polypeptide II), KLNDAQAPK (polypeptide III), VGKGAHLSGEH (polypeptide IV), MLKKKKLA (polypeptide V), SLGFGGPPG (polypeptide VI) and TVATFSSSTKPDD (polypeptide VII), respectively.

[0077] Test Example 3

[0078] The polypeptide analyzed in Test Example 1 was synthesized by artificial synthesis.

[0079] (1) Sensory evaluation was used to detect the taste characteristics of the seven synthetic peptides

[0080] Sensory analysis was performed by 10 panelists (5 males and 5 females, aged 23 to 35 years). Through a three-point test, the experimenter was trained to evaluate the taste of the following standard taste compounds: a 1% sucrose solution was used as a sweet standard; a 0.35% sodium chloride solution was used as a salty standard; a 0.08% caffeine solution was used as a bitter standard; a 0.35% monosodium glutamate solution was used as a umami standard; and a 0.08% citric acid solution was used as a sour standard.

[0081] The seven polypeptides were each re-dissolved in deionized water and transferred to sensory evaluation cups. The tasting temperature was 23 ± 2°C, and the evaluators were required to sip the sample, hold it in their mouths briefly, and then spit it out. In order to avoid fatigue and residual effects, the panel members were required to rinse their mouths with 50-60 mL of drinkable water between testing the seven different samples. The umami threshold of the polypeptides was determined by the Taste Dilution Analysis (TDA) method; and a bubble chart of the umami degree, saltiness, and richness values of the seven polypeptides was established. The results of the sensory attributes and umami threshold detection are shown in Table 2, and the bubble chart is shown in Figure 12 wherein the letter D is polypeptide I, A is polypeptide II, K is polypeptide III, V is polypeptide IV, M is polypeptide V, S is polypeptide VI, and T is polypeptide VII.

[0082] Table 2 Sensory attributes and umami threshold of the seven polypeptides

[0083]

[0084] According to Table 2 and Figure 12 It can be seen that only two polypeptides, AMDDDEADLLLLAM and DSTDEKFLR, among the seven polypeptides have umami, and the umami threshold is 1.24 mmol / L, and the umami intensity scores are 5.24 and 4.23, respectively.

[0085] (2) The two polypeptides I and II were added to fresh chicken soup (Cs) at a concentration of 1.24 mmol / L to further test their taste characteristics in the soup. It was found through the electronic tongue radar chart Figure 13 ) that the two polypeptides increased the umami value of the chicken soup by 3.79 (Cs+A) and 2.86 (Cs+D), respectively, wherein Cs+ indicates fresh chicken soup + polypeptide II, and Cs+D indicates fresh chicken soup + polypeptide I.

[0086] (3) To further investigate the molecular mechanism of the interaction between the synthesized peptide and the umami receptors T1R1 / T1R3, CDOCKER was used in Discovery Studio software to analyze the interaction between the Termitomyces albuminosus peptide and the umami receptors T1R1 / T1R3. Figure 14 Docking, the docking result is as follows Figures 15-21 .according to Figures 15-21 It can be seen that the polypeptides with sequences SEQ ID NO.1 to 7 obtained by this invention can all dock with the umami receptor T1R1 / T1R3. However, as mentioned above, only the polypeptides obtained by SEQ ID NO.1 and SEQ ID NO.2 have obvious umami enhancement effects and can be applied in the food field. They can be used as base ingredients and auxiliary ingredients as seasonings to replace saltiness with umami, satisfying sensory needs while ensuring nutritional safety.

[0087] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fresh-taste peptide of Grifola frondosa, characterized in that, The amino acid sequence is shown as SEQ ID NO.

1.

2. Use of the Grifola frondosa umami peptide in claim 1 in food.

3. Use according to claim 2, characterized in that, The food is umami food.

4. A food seasoning, characterized by, The effective component comprises the Grifola frondosa umami peptide in claim 1.

5. The food seasoning according to claim 4, characterized in that, The concentration of the Grifola frondosa umami peptide in the food seasoning is 0.5-3 mmol / L.

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