Pleurotus citrinopileatus peptide and preparation and application thereof
By extracting proteolytics from yam mushrooms and enzymatically purified, the yam mushroom peptide with umami flavor characteristics was successfully obtained, which solved the shortcomings of the research on yam mushroom peptides in the prior art, and achieved efficient separation and umami flavor enhancement effects.
Patent Information
- Application Number
- CN202411916421.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-04-04
- Estimated Expiration
- 2044-12-24
AI Technical Summary
The prior art studies on the extraction and separation and taste characteristics of elm yellow mushroom peptides, and lacks efficient separation methods and in-depth exploration of its umami characteristics.
The proteolytic solution was extracted from the elm yam mushroom, enzymatically dissolved with bromelain, and then ultrafiltration and gel column purification to obtain the umami-flavored yam mushroom peptide.
A variety of elm yelm yelm peptides were successfully extracted and identified, including LLVTVEDELRV, LDDWR, ENLLEK and ELFEPT. These peptides exhibited obvious umami properties, and were weak in bitter and sour taste, with a lower umami threshold than sodium glutamate, which significantly enhanced the umami flavor of simulated broth.
Smart Images
Figure CN119751572A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to citrus luteolin peptide and preparation and application thereof, belonging to the field of food. Background Art
[0002] Professor Kikunae Ikeda of Tokyo Imperial University discovered during his research on kelp soup that its delicious and mellow taste mainly came from the contribution of sodium glutamate, and first proposed the concept of umami in 1908. This discovery opened the prelude to umami research and promoted the exploration of umami substances in food science. Umami research has since become a hot topic. Umami substances mainly show specific umami characteristics by interacting with umami receptors; common umami receptors include heterodimers T1R1 / T1R3, and taste-type metabotropic glutamate receptors mGluR1 and mGluR4; among them, T1R1 / T1R3 heterodimers are generally considered to be the main umami receptors, and the "Venus Fly Trap Domain" (VFTD) in its T1R3 subunit is considered to be the main ligand binding site for umami substances. There are many types of umami ingredients, including organic acids, organic bases, free amino acids and their salts, and small molecule peptides, and each ingredient presents different levels of umami characteristics. Umami peptides refer to small molecule peptides that have an umami taste at a certain concentration. They are widely distributed in meat, fish, shellfish, beans and some fermented foods. Umami peptides have multiple functional properties, such as imparting / enhancing the umami taste of food, reducing food’s dependence on salt, and providing nutritional benefits. They have huge market potential in the field of condiments.
[0003] In recent years, domestic and foreign researchers have isolated a large number of umami peptides from food-derived protein hydrolysates, especially umami peptides from fungal proteins. Li Xiaoming et al. used cellulase and flavor protease to perform mixed enzymatic hydrolysis on white jade mushrooms to obtain white jade mushroom protein hydrolysates with strong umami effects; Chen Daoyou used trypsin to prepare straw mushroom protein hydrolysates and successfully isolated four umami peptides DVELSK, LDELEK, LPDEAR and TTLPDK from straw mushroom protein hydrolysates, among which DVELSK had the lowest umami threshold (0.091 mg / mL), which was close to the umami threshold of sodium glutamate; Song Shiqing et al. obtained 713 peptides from delicious boletus by enzymatic hydrolysis, among which peptides DGF, HHYE and KCGQ showed umami synergistic effects with monosodium glutamate.
[0004] At present, the research on Pleurotus citrinopileatus mainly focuses on antioxidant activity, post-harvest storage and preservation, polysaccharide extraction, etc. There are few reports on the extraction, separation and flavor characteristics of Pleurotus citrinopileatus peptides. Therefore, finding a method for efficient separation and preparation of Pleurotus citrinopileatus peptides, exploring their umami characteristics and freshness mechanism, and developing new flavor-enhancing condiments are the current research focuses. Summary of the invention
[0005] One of the objects of the present invention is to provide a Pleurotus citrinopileatus peptide, the amino acid sequence of which 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 object of the present invention is to provide a method for preparing a citrus citrus peptide. The citrus citrus peptide can be artificially synthesized or extracted from citrus citrus. The specific steps of extracting from citrus citrus include:
[0007] (1) Remove the roots of Pleurotus citrinopileatus, wash and dry to constant weight, crush the fruiting bodies of Pleurotus citrinopileatus, sieve to obtain Pleurotus citrinopileatus powder, dissolve the Pleurotus citrinopileatus powder in water, adjust the pH of the mixed solution after ultrasonic treatment, then add bromelain to the mixed solution and place it in a water bath shaker for enzymolysis, inactivate the system in boiling water after the reaction is completed, centrifuge, and freeze-dry the supernatant to obtain Pleurotus citrinopileatus enzymolysis product powder.
[0008] (2) The protease hydrolysate powder of Pleurotus citrinopileatus was dissolved in deionized water, and the solution was ultrafiltered at 4°C using 3 kDa and 10 kDa filter membranes to obtain ultrafiltration products with molecular weights >10 kDa, 3-10 kDa, and <3 kDa, respectively. The ultrafiltration products were concentrated to obtain concentrated ultrafiltration products.
[0009] (3) The ultrafiltration concentrate with a molecular weight below 3 kDa was prepared into a solution in deionized water, and purified and separated by a Sephadex G-25 gel column. The sample amount on the gel column was 1 mL, and deionized water was used for elution at a flow rate of 1 mL / min. The detection wavelength was 220 nm. Multiple elution peak components were collected and merged, and the component with the strongest umami taste was selected for collection. It was then lyophilized into powder and analyzed by mass spectrometry to obtain the target peptide segment. The sequence of the target peptide segment was 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-to-liquid ratio of Pleurotus citrinopileatus powder to water is 1:15, expressed in 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 Pleurotus citrinopileatus powder.
[0014] Preferably, the temperature of the water bath in step (1) is 55-60°C.
[0015] Preferably, the centrifugation conditions in step (1) are 4°C, 5000-6000 rpm, and 15-20 min.
[0016] Preferably, in step (2), the solid-liquid ratio of the Pleurotus citrinopileatus protein hydrolysate powder to deionized water is 2-3:500, in units of g:mL.
[0017] Preferably, the amount of the ultrafiltration concentrate added to the deionized water in step (3) is 4-5 mg / mL.
[0018] The third object of the present invention is to provide an application of Pleurotus citrinopileatus peptide in the preparation of flavor-enhancing seasonings.
[0019] Beneficial effects of the present invention
[0020] (1) The lindera lutea peptides LLVTVEDELRV, LDDWR, ENLLEK and ELFEPT in the present invention all have obvious umami taste, and the bitterness and sourness are relatively weak. The umami thresholds are 0.0625 mg / mL, 0.1250 mg / mL, 0.0625 mg / mL and 0.2500 mg / mL, respectively, which are all lower than the umami threshold of monosodium glutamate 0.3000 mg / mL.
[0021] (2) The citronella peptide of the present invention has a good flavor enhancement effect on simulated broth. After adding LLVTVEDELRV, LDDWR, ELFEPT or ENLLEK, the umami taste of the simulated broth is increased by 50.0%, 70.0%, 56.0% and 64.0%, respectively. The citronella peptide of the present invention can be used to prepare umami seasonings, and can also be used in combination with other umami products.
[0022] (3) The structure of the Pleurotus citrinopileatus peptide of the present invention is clear and distinct, and it can be prepared by solid phase chemical synthesis method, or by separating and purifying the Pleurotus citrinopileatus protein hydrolysate. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 is the elution curve of Sephadex G-25 gel filtration chromatography.
[0024] Figure 2 This is the sensory radar chart of Pleurotus citrinum peptide.
[0025] Figure 3It is an electronic tongue image of Pleurotus citrinum peptide.
[0026] Figure 4 This is a picture of the freshness enhancement of lindera lutea peptide. DETAILED DESCRIPTION
[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are only 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 shall be subject to the claims.
[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0029] Example 1
[0030] A method for preparing citrus citrus peptide using citrus citrus, comprising the following steps:
[0031] (1) Remove the roots of the Pleurotus citrinum and wash it, place it in a 65° C. forced air drying oven to constant weight, crush the Pleurotus citrinum, select a 40-mesh sieve to sieve, obtain Pleurotus citrinum powder, add water to dissolve the Pleurotus citrinum powder to obtain a mixed solution (the solid-liquid ratio of Pleurotus citrinum powder to water is 1:15, unit is g:mL), ultrasonically treat the mixed solution for 15 min and then adjust the pH to 7.5, add bromelain to the mixed solution (the amount of bromelain added is 4% of the mass of the Pleurotus citrinum powder, and the enzyme activity of bromelain is 300u / mg), place it in a 55° C. water bath shaker for enzymolysis for 4 h, after the reaction is completed, inactivate the system in boiling water for 10 min, centrifuge at 4° C., 5000 rpm for 15 min, and freeze-dry the supernatant to obtain Pleurotus citrinum enzymolysis product powder.
[0032] (2) 2 g of the protease hydrolysate powder of Pleurotus citrinopileatus was dissolved in 500 mL of deionized water, and the solution was subjected to ultrafiltration separation at 4°C. The solution was passed through ultrafiltration membranes with molecular weights of 10 kDa and 3 kDa, respectively, to obtain a U1 component with a molecular weight below 3 kDa, a U2 component with a molecular weight between 3 and 10 kDa, and a U3 component with a molecular weight greater than 10 kDa. The umami taste of U1, U2, and U3 was evaluated by sensory evaluation. Among them, the umami taste of U1 was the most obvious, and that of U2 and U3 was weakened in turn. U1 was concentrated to obtain an ultrafiltration concentrate.
[0033] (3) The ultrafiltration concentrate was dissolved in deionized water to prepare a solution with a mass concentration of 4 mg / mL. The sample volume on the gel column was 1 mL. Deionized water was used for elution at a flow rate of 1 mL / min. All the eluate was collected in one tube every 1-2 minutes, and the wavelength of each tube of eluate at 220 nm was detected. The elution curve was drawn according to the absorbance value, such as Figure 1 As shown, according to the elution curve, two elution peak components, W1 and W2, were obtained. The sensory evaluation results showed that the umami taste of the W2 component was stronger, so the W2 component was selected for mass spectrometry analysis to obtain the amino acid sequence of the citrus ulmoides 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] In order to verify the relevant properties of four citrus erinaceus 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, Jier Biochemical (Shanghai) Co., Ltd. was commissioned to synthesize the four citrus erinaceus peptides. The purity of the synthesized four citrus erinaceus peptides was >95%.
[0036] Example 3
[0037] The sensory evaluation of Pleurotus citrinum peptide includes the following steps:
[0038] (1) The four citrus citrinum peptides synthesized in Example 2 were prepared into a solution with a concentration of 1 mg / mL, and NaCl solution (mass concentration of 0.35%) was used for saltiness, citric acid solution (mass concentration of 0.08%) was used for sourness, monosodium glutamate solution (mass concentration of 0.35%) was used for umami, sucrose solution (mass concentration of 1.00%) was used for sweetness, and L-leucine solution (mass concentration of 0.25%) was used for bitterness as a reference standard solution. The sensory evaluation score of the samples ranged from 0 to 10 points, 0 points indicating no taste, 10 points indicating a more prominent taste, and the taste score of the reference standard solution was 5 points. Ten sensory tasters (5 men and 5 women, aged 20 to 25 years old) who had undergone sensory training were asked to taste the samples, describe the taste characteristics of the samples, and score them with reference to the taste standard. All evaluations were conducted in a sensory evaluation room at a temperature of 24°C. The tasters sipped the samples one by one and kept them in the mouth for 10 seconds before spitting them out. After the evaluation, they rinsed their mouths with pure water. like Figure 2As shown, the sensory evaluation results show that the Pleurotus citrinopileatus peptide of the present invention exhibits complex flavor characteristics, with umami being the main flavor. The umami scores are in the descending order of LLVTVEDELRV (4.143) > LDDWR (3.429) > ELFEPT (3.143) > ENLLEK (2.429).
[0039] (2) Determination of sensory threshold of citrus citrus peptides: The sensory threshold of citrus citrus is determined by the triangle test method. The four citrus citrus peptides synthesized in Example 2 are dissolved in deionized water to obtain a peptide stock solution with a mass concentration of 1 mg / mL. The peptide stock solution is 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. After dilution, the samples are coded in increasing concentrations and submitted to the sensory panel members for evaluation. The panel members are required to distinguish the samples containing synthetic peptides from two cups of purified water and one cup of synthetic peptide samples. The concentration corresponding to the sample and the blank control (purified water) group can be clearly distinguished, and this concentration is taken as the corresponding threshold. The umami threshold is the average of the individual thresholds, and the umami thresholds of the target peptides LLVTVEDELRV, LDDWR, ENLLEK and ELFEPT are 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 Pleurotus citrinum peptides
[0042] The four citrus citrinopileatus peptides synthesized in Example 2 were prepared into a peptide solution with a concentration of 1 mg / mL. The sensor and reference probe of the electronic tongue were inserted into the peptide solution, and the relevant flavor values were responded to by detecting the changes in membrane potential. The results are as follows: Figure 3 As shown, the electronic tongue measurement further confirmed that umami is the main taste of the citrus citrus peptides of the present invention. Among them, LDDWR has the highest umami score, and the umami scores of the other three peptides are similar.
[0043] Example 5
[0044] Molecular docking of Pleurotus citrinum peptide
[0045] Umami taste receptor proteins were constructed by homology modeling. The amino acid sequences of T1R1 / T1R3 were retrieved from the UniProtKB database (https: / / www.UniProt.org / ). The homology model structure with metabolic glutamate (PDBID: 1EWK) as the template was constructed using the online modeling platform SWISS-Model. The constructed initial model was imported into the SYBYL-X 2.1.1 software for dynamic optimization. The optimized model was imported into the open model evaluation platform (https: / / saves.mbi.ucla) to evaluate the applicability of the model to the umami mechanism through the Laplacian graph. The three-dimensional structure of the identified peptide was first constructed using the SYBYL-X 2.1.1 software, and the constructed identified peptide was optimized using the minimized ligand. After obtaining the optimal structure, it was docked with the optimized T1R1 / T1R3 receptor. The score (T-Score) was used to evaluate the rationality of the docking of the peptide with T1R1 / T1R3. The larger the T value, the higher the binding rate of the Pleurotus citrinum peptide to the taste receptor T1R1 / T1R3, and the greater the umami potential. The T-Score values of the target peptides LLVTVEDELRV, LDDWR, ENLLEK and ELFEPT were tested to be 17.75, 10.77, 13.55 and 12.09, respectively, indicating that the Pleurotus citrinum peptide of the present invention has a strong umami potential.
[0046] Example 6
[0047] Analysis on the Freshness Enhancement of Pleurotus citrinum Peptide
[0048] The four citrus citrus peptides prepared in Example 2 were prepared into 5 mg / mL solutions, and 1 mL of the citrus citrus peptide solution was added to 10 mL of simulated broth (the simulated broth contained 2 mg / mL MSG and 2 mg / mL NaCl) after being filtered through a 0.22 μm water membrane. The simulated broth model was scored as 5 points, and the umami taste of the composite aqueous solution of a single citrus citrus peptide and simulated broth was scored on a scale of 0 to 10, with 0 indicating almost no taste and 10 indicating a more prominent taste. The results are as follows: Figure 4 As shown, from Figure 4 It can be seen that after adding LLVTVEDELRV, LDDWR, ELFEPT and ENLLEK, the umami taste of the simulated broth was increased by 50.0%, 70.0%, 56.0% and 64.0%, respectively, indicating that the citronella peptide of the present invention has strong freshness and flavor enhancement properties and can be used to prepare new flavor enhancement condiments.
Claims
1. Pleurotus citrinum peptide, characterized in that: The amino acid sequence of the Pleurotus citrinopileatus peptide 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.
2. The method for preparing the Pleurotus citrinopileatus peptide according to claim 1, characterized in that: The citronella peptide is obtained by separating from citronella enzymatic hydrolysate.
3. Use of the citronella peptide according to claim 1 in the preparation of flavor-enhancing condiments.
Citation Information
Patent Citations
Streptococcus pneumoniae DNA sequences
CA2274311A1
Preparation method of pleurotus citrinopileatus amino acid powder
CN104805162A
Alcoholic beverage with edible-medicinal fungus
CN108034544A
DNA coding for polypeptide participating in biosynthesis of pladienolide
CN1977046A
Novel genes and polypeptides associated with insecticidal activity
WO2008041863A1