Peptides from pleurotus citrinopileatus and preparation and use thereof

Abstract

This invention discloses *Pleurotus ostreatus* peptides, their preparation, and applications, belonging to the food field. The amino acid sequence of the *Pleurotus ostreatus* peptides of this invention is: Leu-Leu-Val-Thr-Val-Glu-Asp-Glu-Leu-Arg-Val, Leu-Asp-Asp-Trp-Arg, Glu-Asn-Leu-Leu-Glu-Lys, or Glu-Leu-Pro-Glu-Pro-Thr. The *Pleurotus ostreatus* peptides can be obtained by isolation from *Pleurotus ostreatus* protease hydrolysates or by solid-phase chemical synthesis. The *Pleurotus ostreatus* peptides of this invention have a distinct umami flavor, with umami thresholds of 0.063 mg / mL, 0.125 mg / mL, 0.063 mg / mL, and 0.250 mg / mL, respectively, all lower than the umami threshold of monosodium glutamate (MSG) at 0.3 mg / mL. Furthermore, the *Pleurotus ostreatus* peptides of this invention have a significant umami-enhancing effect on simulated meat broth, making them suitable for preparing novel flavor-enhancing seasonings.

Description

Technical Field

[0001] This invention relates to elm oyster mushroom peptides and their preparation and application, belonging to the food field. Background Technology

[0002] Professor Kikunae Ikeda of Tokyo Imperial University discovered during her research on kelp soup that its umami and mellow taste primarily stemmed from monosodium glutamate (MSG). She first proposed the concept of umami in 1908, a discovery that opened the door to umami research and propelled food science's exploration of umami-enhancing substances. Umami research has since become a hot topic. Umami substances primarily exhibit specific umami characteristics through interactions with umami receptors. Common umami receptors include the heterodimer T1R1 / T1R3, as well as the taste-type metabolic glutamate receptors mGluR1 and mGluR4. Among them, the T1R1 / T1R3 heterodimer is generally considered the primary umami receptor, and the "Venus Fly Trap Domain" (VFTD) in its T1R3 subunit is considered the main ligand binding site for umami substances. Umami components are diverse, including organic acids, organic bases, free amino acids and their salts, and small peptides, each exhibiting different levels of umami characteristics. Umami peptides are small molecule peptides that have a umami taste at certain concentrations. They are widely distributed in meat, fish, shellfish, beans, and some fermented foods. Umami peptides have a variety of functional properties, such as imparting / enhancing the umami flavor of food, reducing the dependence of food on salt, and providing nutritional benefits. They have huge market potential in the condiment industry.

[0003] In recent years, researchers both domestically and internationally have isolated a large number of umami peptides from food-derived protease hydrolysates, especially those derived from fungi. Li Xiaoming et al. used a mixture of cellulase and flavor protease to hydrolyze *Mammillaria leucocephala*, obtaining a *Mammillaria leucocephala* protease hydrolysate with a strong umami effect. Chen Daoyou used trypsin to prepare *Mammillaria pulcherrima* protease hydrolysate and successfully isolated four umami peptides from it: DVELSK, LDELEK, LPDEAR, and TTLPDK. Among them, DVELSK had the lowest umami threshold (0.091 mg / mL), close to the umami threshold of monosodium glutamate. Song Shiqing et al. obtained 713 peptide fragments from *Boletus edulis* using enzymatic hydrolysis, among which peptides DGF, HHYE, and KCGQ showed a synergistic effect with monosodium glutamate (MSG) umami.

[0004] Currently, research on *Pleurotus ostreatus* mainly focuses on its antioxidant activity, post-harvest storage and preservation, and polysaccharide extraction. However, there are few reports on the extraction, separation, and flavor characteristics of *Pleurotus ostreatus* peptides. Therefore, finding efficient methods for separating and preparing *Pleurotus ostreatus* peptides, exploring their umami characteristics and flavor mechanisms, and developing novel flavor-enhancing seasonings are the current research priorities. Summary of the Invention

[0005] One of the objectives of this invention is to provide elm oyster mushroom peptides, wherein the amino acid sequence of the elm oyster mushroom peptides is: Leu-Leu-Val-Thr-Val-Glu-Asp-Glu-Leu-Arg-Val (LLVTVEDELRV), Leu-Asp-Asp-Trp-Arg (LDDWR), Glu-Asn-Leu-Leu-Glu-Lys (ENLLEK) or Glu-Leu-Pro-Glu-Pro-Thr (ELFEPT).

[0006] The second objective of this invention is to provide a method for preparing elm-yellow mushroom peptides, which can be synthesized artificially or extracted from elm-yellow mushrooms. The specific steps for extraction from elm-yellow mushrooms include:

[0007] (1) Remove the roots of the elm mushroom, wash it, dry it to constant weight, crush the fruiting body of the elm mushroom, sieve it to obtain elm mushroom powder, dissolve the elm mushroom powder in water, sonicate it to adjust the pH of the mixed solution, then add bromelain to the mixed solution and place it in a water bath shaker for enzymatic hydrolysis. After the reaction is completed, inactivate the system in boiling water, centrifuge it, take the supernatant and freeze dry it to obtain elm mushroom enzyme hydrolysate powder.

[0008] (2) Dissolve the protein hydrolysate powder of elm mushroom in deionized water, and use 3kDa and 10kDa filter membranes to perform ultrafiltration separation at 4℃ to obtain ultrafiltration products with molecular weights >10kDa, 3-10kDa and <3kDa respectively. After concentrating the ultrafiltration, concentrated ultrafiltration is obtained.

[0009] (3) The ultrafiltration concentrate with a molecular weight below 3kDa was prepared into a solution in deionized water and purified and separated by Sephadex G-25 gel column. The sample loading volume of the gel column was 1mL, and deionized water was eluted at a flow rate of 1mL / min. The detection wavelength was 220nm. The elution peaks were collected and combined into multiple elution peaks. The component with the strongest umami flavor was selected for collection and then freeze-dried into powder. The target peptide was obtained by mass spectrometry analysis. The sequence of the target peptide is Leu-Leu-Val-Thr-Val-Glu-Asp-Glu-Leu-Arg-Val, Leu-Asp-Asp-Trp-Arg, Glu-Asn-Leu-Leu-Glu-Lys and Glu-Leu-Pro-Glu-Pro-Thr.

[0010] Preferably, in step (1), the solid-liquid ratio of elm mushroom powder to water is 1:15, with units of g:mL.

[0011] Preferably, the ultrasonic treatment time in step (1) is 15-20 min.

[0012] Preferably, the pH of the mixed solution in step (1) is 7.0-7.5.

[0013] Preferably, the amount of bromelain added in step (1) is 3%-4% of the mass of the mushroom powder.

[0014] Preferably, the temperature of the water bath in step (1) is 55-60℃.

[0015] Preferably, the centrifugation conditions in step (1) are 4℃, 5000-6000rpm for 15-20min.

[0016] Preferably, in step (2), the solid-liquid ratio of the elm bark protein hydrolysate powder to deionized water is 2-3:500, with units of g:mL.

[0017] Preferably, the amount of ultrafiltration concentrate added to the deionized water in step (3) is 4-5 mg / mL.

[0018] The third objective of this invention is to provide an application of elm oyster mushroom peptide in the preparation of flavor-enhancing seasonings.

[0019] Beneficial effects of the present invention

[0020] (1) The elm shiitake peptides LLVTVEDELRV, LDDWR, ENLLEK and ELFEPT in this invention all have obvious umami flavor, and the bitterness and sourness are relatively weak. The umami thresholds are 0.0625mg / mL, 0.1250mg / mL, 0.0625mg / mL and 0.2500mg / mL respectively, all of which are lower than the umami threshold of monosodium glutamate 0.3000mg / mL.

[0021] (2) The elm shiitake peptide of the present invention has a good flavor-enhancing effect on simulated broth. After adding LLVTVEDELRV, LDDWR, ELFEPT or ENLLEK, the umami flavor of simulated broth is increased by 50.0%, 70.0%, 56.0% and 64.0% respectively. The elm shiitake peptide of the present invention can be used to prepare umami seasonings, or can be used in combination with other umami products.

[0022] (3) The peptide structure of the elm yellow mushroom of the present invention is clear and distinct. It can be prepared by solid-phase chemical synthesis or by separating and purifying the enzyme hydrolysate of elm yellow mushroom. Attached Figure Description

[0023] Figure 1 This is the elution curve from Sephadex G-25 gel filtration chromatography.

[0024] Figure 2 This is a sensory radar diagram of elm oyster mushroom peptides.

[0025] Figure 3This is an electronic tongue diagram of elm yellow mushroom peptide.

[0026] Figure 4 This is a picture showing the flavor-enhancing effect of elm shiitake peptides. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following embodiments are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Example 1

[0030] A method for preparing elm oyster mushroom peptides from elm oyster mushrooms specifically includes the following steps:

[0031] (1) Remove the roots of the elm mushroom, wash it, and dry it in a 65℃ forced-air drying oven until constant weight. Crush the elm mushroom and sieve it through a 40-mesh sieve to obtain elm mushroom powder. Dissolve the elm mushroom powder in water to obtain a mixed solution (the solid-liquid ratio of elm mushroom powder to water is 1:15, and the unit is g:mL). After sonicating the mixed solution for 15 min, adjust the pH to 7.5. Add bromelain to the mixed solution (the amount of bromelain added is 4% of the mass of elm mushroom powder, and the enzyme activity of bromelain is 300u / mg). Place it in a 55℃ water bath shaker for 4 h for enzymatic hydrolysis. After the reaction is completed, inactivate the system in boiling water for 10 min, centrifuge at 4℃ and 5000rpm for 15 min, take the supernatant and freeze-dry it to obtain elm mushroom protein hydrolysate powder.

[0032] (2) Take 2g of elm yellow mushroom protein hydrolysate powder and dissolve it in 500mL of deionized water. The solution is separated by ultrafiltration at 4℃. It is passed through ultrafiltration membranes with molecular weights of 10kDa and 3kDa in sequence to obtain U1 component with molecular weight below 3kDa, U2 component with molecular weight between 3-10kDa and U3 component with molecular weight greater than 10kDa. The umami flavor of U1, U2 and U3 is evaluated by sensory evaluation method. U1 has the most obvious umami flavor, while U2 and U3 have the weakest flavor in sequence. After concentrating U1, the ultrafiltration concentrate is obtained.

[0033] (3) Dissolve the ultrafiltration concentrate in deionized water to prepare a solution with a mass concentration of 4 mg / mL. Load 1 mL onto the gel column and elute with deionized water at a flow rate of 1 mL / min. Collect all eluent in one tube every 1-2 minutes, and measure the wavelength at 220 nm for each tube. Plot an elution curve based on the absorbance values. Figure 1 As shown, based on the elution curve, two elution peaks, W1 and W2, were obtained. Sensory evaluation results showed that the W2 component had a stronger umami flavor. Therefore, the W2 component was selected for mass spectrometry analysis to obtain the amino acid sequences of the *Pleurotus ostreatus* peptide, which were Leu-Leu-Val-Thr-Val-Glu-Asp-Glu-Leu-Arg-Val, Leu-Asp-Asp-Trp-Arg, Glu-Asn-Leu-Leu-Glu-Lys, and Glu-Leu-Pro-Glu-Pro-Thr.

[0034] Example 2

[0035] To verify the properties of four elm oyster mushroom peptides—Leu-Leu-Val-Thr-Val-Glu-Asp-Glu-Leu-Arg-Val, Leu-Asp-Asp-Trp-Arg, Glu-Asn-Leu-Leu-Glu-Lys, and Glu-Leu-Pro-Glu-Pro-Thr—these four elm oyster mushroom peptides were synthesized by Jier Biochemical (Shanghai) Co., Ltd. The purity of the synthesized four elm oyster mushroom peptides was >95%.

[0036] Example 3

[0037] Sensory evaluation of elm shiitake peptides includes the following steps:

[0038] (1) The four types of elm shiitake peptides synthesized in Example 2 were prepared into solutions with a concentration of 1 mg / mL. NaCl solution (0.35% by mass) was used as a salty solution, citric acid solution (0.08% by mass) as a sour solution, monosodium glutamate solution (0.35% by mass) as a umami solution, sucrose solution (1.00% by mass) as a sweet solution, and L-leucine solution (0.25% by mass) as a bitter solution as reference standards. Sensory evaluation scores for the samples ranged from 0 to 10, with 0 indicating no taste and 10 indicating a prominent taste. The reference standard solutions were scored out of 5. Ten sensory evaluators (5 men and 5 women, aged 20-25 years) who had received sensory training evaluated the samples, described their taste characteristics, and scored them against the taste standards. All evaluations were conducted in a sensory evaluation room at 24°C. Evaluators sipped the samples repeatedly, holding them in their mouths for 10 seconds before spitting them out, and rinsed their mouths with pure water after the evaluation. like Figure 2As shown, the sensory evaluation results show that the elm shiitake peptide of the present invention exhibits complex flavor characteristics, with umami being the main flavor. The umami scores decrease in the following order: LLVTVEDELRV (4.143) > LDDWR (3.429) > ELFEPT (3.143) > ENLLEK (2.429).

[0039] (2) Sensory Threshold Determination of Peptides from *Pleurotus ostreatus*: The sensory threshold of *Pleurotus ostreatus* was determined using the triangular test method. The four types of *Pleurotus ostreatus* peptides synthesized in Example 2 were dissolved in deionized water to obtain a peptide stock solution with a mass concentration of 1 mg / mL. The stock solution was then diluted with purified water to obtain mass concentrations of 0.5000 mg / mL, 0.2500 mg / mL, 0.1250 mg / mL, 0.0625 mg / mL, and 0.03125 mg / mL. The diluted samples were coded in ascending order of concentration and submitted to the sensory evaluation panel. The panel members were required to distinguish the sample containing the synthetic peptide from two glasses of purified water and one glass of synthetic peptide sample. The concentration that clearly distinguished the sample from the blank control (purified water) group was recorded, and this concentration was taken as its corresponding threshold. The umami threshold is the average value of individual thresholds. The umami thresholds of the target peptides LLVTVEDELRV, LDDWR, ENLLEK and ELFEPT were obtained as 0.0625 mg / mL, 0.1250 mg / mL, 0.0625 mg / mL and 0.2500 mg / mL, respectively.

[0040] Example 4

[0041] Electronic tongue analysis of elm oyster mushroom peptides

[0042] The four types of *Pleurotus eryngii* peptides synthesized in Example 2 were prepared into peptide solutions with a concentration of 1 mg / mL. The sensor and reference probe of an electronic tongue were inserted into the peptide solutions, and the corresponding flavor values ​​were detected by measuring changes in membrane potential. The results are as follows: Figure 3 As shown, electronic tongue assays further confirmed that umami is the primary flavor characteristic of the *Pleurotus ostreatus* peptides of this invention. Among them, LDDWR had the highest umami score, while the other three peptides had similar umami scores.

[0043] Example 5

[0044] Molecular docking of elm mushroom peptides

[0045] Umami receptor proteins were constructed using homology modeling. The amino acid sequences of T1R1 / T1R3 were retrieved from the UniProtKB database (https: / / www.UniProt.org / ). Homology model structures using metabolic glutamate (PDBID: 1EWK) as templates were constructed using the online modeling platform SWISS-Model. The initial model was imported into SYBYL-X 2.1.1 software for dynamic optimization. The optimized model was then imported into an open model evaluation platform (https: / / saves.mbi.ucla) to evaluate its applicability to umami mechanisms using Laplacian plots. The three-dimensional structures of the identification peptides were first constructed using SYBYL-X 2.1.1 software. The constructed identification peptides were optimized using ligand minimization, and the optimal structure was then docked with the optimized T1R1 / T1R3 receptors. The T-score was used to evaluate the rationality of the peptide docking with T1R1 / T1R3. The higher the T value, the higher the binding rate of the elm shiitake peptide to the taste receptors T1R1 / T1R3, and the greater the umami potential. The T-Score values ​​of the target peptides LLVTVEDELRV, LDDWR, ENLLEK and ELFEPT were detected to be 17.75, 10.77, 13.55 and 12.09, respectively, indicating that the elm shiitake peptide of the present invention has a strong umami potential.

[0046] Example 6

[0047] Flavor-enhancing analysis of elm oyster mushroom peptides

[0048] The four types of *Ichthyophthirius multiflorus* peptides synthesized in Example 2 were each prepared into 5 mg / mL solutions. After filtration through a 0.22 μm water membrane, 1 mL of the *Ichthyophthirius multiflorus* peptide solution was added to 10 mL of simulated broth (containing 2 mg / mL MSG and 2 mg / mL NaCl). The simulated broth model was scored out of 5. The umami flavor of the combined aqueous solution of individual *Ichthyophthirius multiflorus* peptides and the simulated broth was scored from 0 to 10, where 0 indicates almost no flavor and 10 indicates a more pronounced flavor. The results are as follows: Figure 4 As shown, from Figure 4 As can be seen from the results, the addition of LLVTVEDELRV, LDDWR, ELFEPT and ENLLEK increased the umami flavor of the simulated broth by 50.0%, 70.0%, 56.0% and 64.0% respectively, indicating that the elm shiitake peptide of the present invention has strong umami-enhancing and flavor-enhancing properties and can be used to prepare novel flavor-enhancing seasonings.

Claims

1. Elm Leaf Peptide, characterized in that: The amino acid sequence of the *Eriocaulon buergerianum* peptide is: Leu-Asp-Asp-Trp-Arg.

2. The method for preparing the *Pleurotus ostreatus* peptide according to claim 1, characterized in that: The elm oyster mushroom peptide was isolated from the bromelain hydrolysate of elm oyster mushroom.

3. The application of the elm shiitake peptide according to claim 1 in the preparation of flavor-enhancing seasonings.

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