CN115894657A

CN115894657A - Umami peptide and application thereof

Info

Publication number: CN115894657A
Application number: CN202210725995.2A
Authority: CN
Inventors: 于海; 单艳琴; 刘瑞; 沈银涵; 葛庆丰; 吴满刚; 陈春梅
Original assignee: JIANGSU NATURAL FOOD CO Ltd; Yangzhou University
Current assignee: JIANGSU NATURAL FOOD CO Ltd; Yangzhou University
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2023-04-04
Anticipated expiration: 2042-06-23
Also published as: CN115894657B

Abstract

The invention discloses an umami peptide and application thereof, wherein the amino acid sequence of the umami peptide is as follows: arg-Pro-Gly-Ala-Gly-Gln-Pro-Pro-Arg-Arg, asp-Pro-Ile-Ile-Gln-Asp-Arg-His-Gly-Gly, arg-Ile-Glu-Ala-Gln-Asn-Lys-Pro-Phe, gly-Gly-Lys-Ala-Thr-Ile-Ile-His-Ala-Gln or Ile-Thr-Thr-Asp-Thr-Pro-Glu-Leu-Arg. The 5 umami peptides can be obtained by separating and purifying muscle protein, creatine kinase, myosin heavy chain, LIM structural domain synalbumin or actin, can also be synthesized artificially, and the results show that the 5 umami peptides have good umami and richness by using electronic tongue and sensory detection, can replace monosodium glutamate, have great application significance in food, and are small molecular weight peptides which are easy to be absorbed by human bodies.

Description

Umami peptide and application thereof

Technical Field

The invention relates to protein active peptide, in particular to umami peptide and application thereof.

Background

The flavor peptide serving as a novel active peptide flavor enhancer can enhance the food flavor, has good processing characteristics, fresh effect and nutritive value, and thus becomes a focus of research focus of the food flavor enhancer and a key direction of flavor enhancer development in recent years. In 1972, arai et al isolated and purified 3 dipeptides (Glu-Asp, glu-Ser and Glu-Glu) and 1 tripeptide (Glu-Gly-Ser) from a soy protein hydrolysate for the first time, which had a bouillon-like taste. Until 1978 Yamasaki et al treated beef with papain and obtained therefrom an octapeptide (Beeffy food Peptide, BMP), named Umami Peptide, which received much attention since its research.

Li et al, in order to investigate the umami peptides in the pig bone soup, first isolated the pig bone soup by ultrafiltration and then screened the fractions with the highest umami value by sensory evaluation and identified the peptides by HPLC, detected 3 umami peptides in total (FSGLDGAK, FAGDDAPR and FSGLDGSK), of which the umami peptide FSGLDGAK has the highest umami value, and detected 6 umami peptides (AGPSIVH, IKDDPHVD, TPPKID, IKDDPHVD, FAGDDAPR and NALNDITSL) from the chicken soup by the same method; wang et al optimized the process of stewing beef soup by orthogonal method and separated the delicious peptides such as ultrafiltration, sephadexG-15 column chromatography, reversed-phase high performance liquid chromatography on the basis of the above, finally detected 6 delicious peptides (SDEEVEH, AEVPEVH, GVDPGHP, GSDGSVGCVGPVGP, SDGSVGPVGP and DEGPSIVH) in the stewed beef soup with electric cooker, and detected 7 delicious peptides (VAPEEHPT, VVVPVSNDIL, VGGNVDYK, PFGNTHN, EAGPSIVHR, VDFDDIQK and DEGPSIVH) in the stewed beef soup with the optimal process of orthogonal experimental optimization. These umami peptides contribute significantly to the taste, thereby affecting the taste of the whole soup.

So far, there have been many studies on the isolation and purification of umami peptides from pig bone soup, chicken and beef, but no reports on the isolation and purification of umami peptides from sheep broth have been found.

Disclosure of Invention

Aiming at the problems, the invention provides the umami peptide separated and purified from the mutton soup and the application thereof so as to enrich the source of the umami peptide.

In order to achieve the above object, the present invention provides, in one aspect, an umami peptide having an amino acid sequence of: arg-Pro-Gly-Ala-Gly-Gln-Pro-Pro-Arg-Arg, asp-Pro-Ile-Ile-Gln-Asp-Arg-His-Gly-Gly, arg-Ile-Glu-Ala-Gln-Asn-Lys-Pro-Phe, gly-Gly-Lys-Ala-Thr-Ile-Ile-His-Ala-Gln, or Ile-Thr-Thr-Asp-Thr-Pro-Glu-Leu-Arg.

Further, the umami peptide is derived from muscle protein, creatine kinase, myosin heavy chain, LIM domain associated protein or actin in mutton.

The invention also provides application of the above umami peptide in preparing an umami agent.

Through the technical scheme, the invention has the following beneficial effects:

1. the 5 umami peptides can be obtained by purifying muscle protein, creatine kinase, myosin heavy chain, LIM structural domain protein or actin, can be artificially synthesized, and are detected by using electronic tongue and sense organ, and the results show that the 5 umami peptides have good umami taste and richness.

2. The umami peptide is a small molecular weight peptide and is easy to be absorbed by human body.

3. The umami peptide developed by the invention has high umami intensity, can replace monosodium glutamate, and has great application significance in food.

4. The umami peptide obtained by the invention has high purity.

Drawings

FIG. 1 shows the result of purity verification of the artificially synthesized polypeptide RPGAGQPPRR;

FIG. 2 shows the mass spectrum of the synthetic polypeptide RPGAGQPPRR;

FIG. 3 shows the result of purity verification of the artificially synthesized polypeptide DPIIQDRHGG;

FIG. 4 shows the mass spectrum of the artificially synthesized polypeptide DPIIQDRHGG;

FIG. 5 shows the results of purity verification of the artificially synthesized polypeptide RIAAQNKPF;

FIG. 6 shows the mass spectrum of the artificially synthesized polypeptide RIAAQNKPF;

FIG. 7 shows the results of purity verification of artificially synthesized polypeptide GGKATIIHAQ;

FIG. 8 shows the mass spectrum of artificially synthesized polypeptide GGKATIIHAQ;

FIG. 9 shows the results of the purity verification of the artificially synthesized polypeptide ITTDTPEL;

FIG. 10 shows the mass spectrum of the artificially synthesized polypeptide ITTDTPEL;

FIG. 11 is a molecular docking diagram of 5 umami peptides with umami receptors.

Detailed Description

The following examples are provided to explain the present invention in detail. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The following examples include the following main instruments and devices:

THC-1000SF ultrasonic Decoct Pan Jining Tianhua ultrasonic electronics, inc

Waters E2695 high performance liquid chromatography Watts science and technology, inc

BioLogic LP chromatography System Bio-Rad Inc

TS-50000Z gustatory analysis System INSENT, japan

Ultra pure Water Instrument Millipore Inc. USA

Shanghai Qiangyao Biotechnology GmbH of twelve-channel semi-automatic polypeptide synthesizer

The Marin Christ, germany, of the ALPHA1-2LS PLUS Freeze dryer

Example 1 separation, purification and detection of umami peptide

1. Ultrasonic-assisted stewing of mutton soup:

slaughtering sheep, removing hair and skin, trimming sheep hind legs, cutting into uniform meat blocks with length, width and height of about 9cm, 9cm and 5cm, boiling in cold water, blanching for 15min, taking out mutton, washing with cold water, and draining; placing 1.5kg of mutton pieces into an ultrasonic stewing pot, adding 4.5kg of purified water (m/m =1: 3), setting the stewing temperature at 85 ℃ and the ultrasonic power at 800W, and stewing for 2h to obtain the mutton soup.

2. Separating and purifying the umami peptide:

2.1 ultrafiltration centrifugal separation purification:

defatting mutton soup, lyophilizing, concentrating, making into 5mg/mL solution, adding into 3kDa ultrafiltration centrifuge tube, centrifuging at 4000 Xg for 20min, and collecting the lower filtered liquid.

2.2 separating and purifying the cellulose DEAE-52 ion exchange column:

the DEAE-52 cellulose dry powder was poured into a beaker and added into 10 times by volume of ultra-pure water degassed by ultrasound, and soaked sufficiently at normal temperature for swelling for 2 hours, the floating object on the surface was removed, and the column packing (4.5X 40 cm) was carried out after repeating 5 times. Balancing: the column was washed with phosphate buffer pH 7.0 at a flow rate of 4mL/min, and 4 column volumes were equilibrated.

Adjusting the concentration of liquid obtained by ultrafiltration, centrifugal separation and purification to 5mg/mL, passing 4mL of sample through a 0.22-micron filter membrane and carrying out ultrasonic degassing, slowly adding the sample into a chromatographic column by using a dropper, eluting the polypeptide by using PBS (pH 7.0) eluent with the concentration of NaCl being 0.2M, wherein the elution flow rate is 1mL/min, collecting 1 tube every 3min, detecting the polypeptide absorption peak at 245nm, collecting different components, then filling 20-30mL of collected polypeptide solution containing sodium chloride into a dialysis bag, putting the dialysis bag into a beaker containing distilled water, putting the beaker on a magnetic stirrer, continuously stirring, dialyzing for 48h, and changing water every 6 h.

2.3 Sephadex G-50 column purification:

pouring the dry powder of the sephadex (GFC) into a beaker, adding 10 times of volume of ultra-pure water subjected to ultrasonic degassing, expanding for 3 hours at room temperature, removing upper layer broken gum, repeating for 5 times, and filling columns (1.6 multiplied by 60 cm) by a wet method. Balancing: the packing was rinsed 10 column volumes with ultra-pure water degassed by ultrasound until the baseline stabilized.

The concentration of a sample obtained by separation and purification of a cellulose DEAE-52 ion exchange column was adjusted to 5mg/mL, and then filtered using a 0.22 μm filter membrane, 4mL of the sample was loaded, and eluted with ultrapure water at a flow rate of 0.3mL/min, 1 tube was collected every 10min, and 1200min was collected.

3. Identification of umami peptide

GFC is a separation of samples based on molecular weight, where the fraction with a higher molecular weight is separated earlier and the fraction with a lower molecular weight is eluted later. And (3) analyzing and detecting the umami component which is separated from the Sephadex G-50 chromatographic column by using an ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) technology. Wherein the chromatographic conditions are as follows: buffer conditions: solution A: 0.1% aqueous formic acid solution, solution B: 0.1% aqueous acetonitrile formic acid (80% acetonitrile). The column was equilibrated with 95% solution A, and the sample was first subjected to gradient separation using a trap column (100. Mu. M.times.20mm, 5. Mu.m, C18) and then passed through an analytical column (75. Mu. M.times.150mm, 3. Mu.m, C18) at a flow rate of 300nL/min. The liquid phase gradient was set as follows: 0-2min, the linear gradient of the liquid B is from 5-8 percent; 2-90min, wherein the linear gradient of the liquid B is from 8 to 23 percent; 90-100min, the linear gradient of the liquid B is 23-40%;100-108min, the linear gradient of the B liquid is 40-100%;108-120min, and the B liquid is maintained at 100 percent.

The mass spectrum conditions are as follows: after peptide fragment separation, performing data dependence acquisition mass spectrometry by using a Q-active HF-X mass spectrometer, wherein the analysis time is 120min, and the detection mode is as follows: positive ion, parent ion scan range: 300-1800m/z, first-order mass spectrum resolution: 60000 (m/z 200); automatic gain (AGC target): 3X 10 ⁶ Maximum Injection Time (IT): 50ms. Peptide fragment secondary mass spectrometry was collected as follows: triggering acquisition of secondary mass spectra (MS 2 scan) of 20 highest intensity parent ions after each full scan (full scan), secondary mass resolution: 15,000 (m/z 200), automatic gain: 1 x 10 ⁵ Second order maximum Injection Time (IT): 50ms, isolation window: 1.6m/z, using software to perform mass spectrum database retrieval software; using a protein database: uniprot (Uniprot-Ovis aries (sheet) [ 9940)]-63944-20210902) the species is sheep.

Then, analyzing and matching the whole mass spectrum result by using a database uniprot, and detecting 56 polypeptides in the umami component, wherein 41 polypeptides come from myofibrillar protein and account for 84.24 percent; 14 polypeptides are from sarcoplasmic proteins with a content of 6.44%, and 1 polypeptide is from connective tissue and organelle proteins with a content of 9.32%.

Molecular docking analysis was performed with the appetizing receptor by software Pepsite 2 mimetic polypeptides. A model is constructed by adopting an umami receptor PDB of fish at 5x2m, and the intensity analysis of the umami peptide is carried out according to the combination significance (P is less than 0.05) and the structural characteristics of the polypeptide. Of the 56 polypeptides, 5 polypeptides are predicted to have a high specificity for umami receptors, and they are RPGAGQPPRR (Arg-Pro-Gly-Ala-Gly-Gln-Pro-Pro-Arg-Arg), DPIIQDRHGG (Asp-Pro-Ile-Ile-Gln-Asp-Arg-His-Gly-Gly), RIEAQNKPF (Arg-Ile-Glu-Ala-Gln-Asn-Lys-Pro-Phe), GGKAIHAQ (Gly-Gly-Lys-Ala-Thr-Ile-Ile-His-Ala-Gln), ITTDTPELR (Ile-Thr-Thr-Asp-Thr-Pro-Glu-Leu-Arg). The specific information of the polypeptides is shown in table 1, the results of the electronic tongue evaluation are shown in table 2, and the binding pattern to the umami receptor is shown in fig. 11.

TABLE 1 sequences of potential umami peptides and related information

Polypeptide sequence	Length of	Protein of origin	Location of umami peptides in proteins	P value
					RPGAGQPPRR
	10	Small muscle proteins	f(69-79)	0.037
					RIEAQNKPF	9	Myosin heavy chain	f(84-97)	0.041
ITTDTPELR	9	Actin-binding agent	f(548-557)	0.024
					GGKATIIHAQ	10	LIM domain binding proteins	f(2-11)	0.022
DPIIQDRHGG	10	Creatine kinase	f(156-166)	0.013

Among the 5 polypeptides, RPGAGQPPRR is derived from small muscle protein, DPIIQDRHGG is derived from creatine kinase, RIEAQNKPF is derived from myosin heavy chain, GGKATIIHAQ is derived from LIM domain binding protein, ITTDTPELR is derived from actin.

TABLE 2 electronic tongue evaluation of 5 umami peptides

The results show that the 5 umami peptides have good fresh effect.

Example 2 Artificial Synthesis of umami peptides

Artificially synthesizing the umami peptides RPGAGQPPRR, DPIIQDRHGG, RIAAQNKPF, GGKATIIHAQ and ITTDTPELR which have obvious combination effect with the umami receptor through molecular simulation. Synthesizing polypeptide by a twelve-channel semi-automatic polypeptide synthesizer, taking out the synthesized polypeptide, adding ether for precipitation, centrifuging at 1500g for 10min, removing supernatant, collecting precipitate, and freeze-drying. Dissolving 1.5g crude peptide, centrifuging at 10000 Xg for 2min, removing impurities, detecting and purifying with C18 reverse phase column, and detecting with dual wavelength at 280 nm. And taking 1 mu L of the collected target peak for mass spectrum detection. After the molecular weight of the synthesized polypeptide was determined, it was used in the subsequent experiments. The desired peak was collected and lyophilized again.

And analyzing the purity of the target peptide by adopting ultra-high performance liquid chromatography, wherein the chromatographic conditions are the same as above. The elution peaks were automatically collected and the results are shown in fig. 1, fig. 3, fig. 5, fig. 7 and fig. 9. All of the synthesized polypeptides were more than 95% pure.

And identifying the sample by adopting mass spectrometry. The mass spectrometry conditions were as above. The theoretical molecular weights of the umami peptides RPGAGQPPRR, DPIIQDRHG, RIEAQNKPF, GGKATIIHAQ and ITTDTPELR are 1091.22Da, 1107.17Da, 1102.24Da, 995.13Da and 1045.14Da respectively. The mass spectrometry reports of the five samples are shown in fig. 2, fig. 4, fig. 6, fig. 8, and fig. 10, and the obtained molecular mass-to-charge ratios are 1090.61, 1106.55, 1101.59, 994.56, and 1044.55, respectively, and are consistent with the theoretical molecular weights of umami peptides RPGAGQPPRR, DPIIQDRHG, RIEAQNKPF, ggkatihaq, and itttdtpelr, confirming that the samples are the target peptides.

Electronic tongue evaluation of the synthetic flavor peptide: the synthesized umami peptide was adjusted to a uniform concentration of 0.3mg/mL by BCA protein kit, and 40mL samples were subjected to taste analysis using a TS-50000Z taste analysis system, and the results are shown in Table 3.

TABLE 3 electronic tongue evaluation of synthetic umami peptides

Synthetic peptide sequences	Sour taste	Delicate flavour	Salty taste	Richness of the product
					RPGAGQPPRR	-45.55±1.37	25.26±1.62	-13.56±0.15	0.36±0.03
RIEAQNKPF	-43.69±1.79	23.61±1.35	-14.55±0.25	0.34±0.04
					ITTDTPELR	-42.69±1.31	36.35±1.26	-15.63±0.54	0.12±0.04
GGKATIIHAQ	-43.76±1.56	25.36±0.98	-13.69±0.66	0.23±0.02
					DPIIQDRHGG	-44.36±1.86	34.45±1.92	-12.68±0.33	0.45±0.06
Monosodium glutamate (MSG)	-46.28±0.36	26.32±0.26	-17.84±0.21	0.03±0.01

As can be seen from Table 3, the umami value of RPGAGQPPRR sequence is 25.26, the umami value of DPIIQDRHG sequence is 34.45, the umami value of RIEAQNKPF sequence is 23.61, the umami value of GGKATIHOAQ sequence is 26.36, the umami value of ITTDTPELR sequence is 36.35, which is higher than that of 5 umami peptides obtained by separation and purification because the concentration of artificially synthesized umami peptides is higher.

The synthesized polypeptide was formulated into 0.1mg/mL solution for sensory evaluation, and 0, 0.1, 0.2mg/mL sodium glutamate solution was used as 0, 5 and 10 point controls for more accurate description of the taste of the synthesized umami peptide, and the sensory evaluation of the specific synthesized umami peptide is shown in table 4.

TABLE 4 sensory evaluation of synthetic umami peptides

Synthetic peptide sequences	Length of	Umami value
			RPGAGQPPRR
	10	5.47±0.5
			RIEAQNKPF	9	5.4±0.53
ITTDTPELR	9	7.67±0.58
			GGKATIIHAQ	10	5.56±0.57
DPIIQDRHGG	10	7.03±0.95

As can be seen from Table 4, the sequence RPGAGQPPRR has an umami value of 5.47; the sequence is RIAAQNKPF with an umami value of 5.4; the umami value of ITTDTPELR is 7.67; the sequence is GGKATIIHAQ, and the umami value of the sequence is 5.56; the umami value of the sequence DPIIQDRHGG is 7.03. All the umami peptides have higher umami values at the concentration of 0.1mg/mL than the sodium glutamate with the same concentration, wherein the umami values of the ITTDTPELR and the DPIIQDRHGG are particularly prominent.

The preferred embodiments of the present invention have been described in detail with reference to the examples, however, the present invention is not limited to the details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.

Sequence listing

<110> Jiangsu XingYe food Co., ltd

YANGZHOU University

<120> umami peptide and application thereof

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Asp Pro Ile Ile Gln Asp Arg His Gly Gly

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Claims

1. An umami peptide, wherein the amino acid sequence of the umami peptide is: arg-Pro-Gly-Ala-Gly-Gln-Pro-Pro-Arg-Arg, asp-Pro-Ile-Ile-Gln-Asp-Arg-His-Gly-Gly, arg-Ile-Glu-Ala-Gln-Asn-Lys-Pro-Phe, gly-Gly-Lys-Ala-Thr-Ile-Ile-His-Ala-Gln or Ile-Thr-Thr-Asp-Thr-Pro-Glu-Leu-Arg.

2. The umami peptide according to claim 1, characterized in that the umami peptide is derived from muscle protein, creatine kinase, myosin heavy chain, LIM domain associated protein or actin-chaperones in mutton.

3. Use of the umami peptide according to claim 1 or 2 for the preparation of an umami agent.