CN111560051B - Shrimp-derived nonapeptide with iron absorption promoting activity and application thereof - Google Patents

Abstract

The invention discloses a shrimp-derived nonapeptide and application thereof, wherein the amino acid sequence of the shrimp-derived nonapeptide is as follows: asp-Thr-Asp-Ser-Glu-Glu-Glu-Ile-Arg, molecular weight 1093.05Da. The shrimp-derived nonapeptide is obtained by using a polypeptide synthesizer, synthesizing by a solid phase synthesis method, purifying by high-phase liquid chromatography and freeze-drying. The shrimp-derived nonapeptide can resist gastrointestinal tract digestion, and Fe is transported for 90min by a rat exocarpium-turning model ²⁺ The transport volume is 65.6 +/-12.6 mu g/mL; can enhance the delivery capacity of iron, and can be used in the fields of functional health products, nutrition-enriched foods, etc.

Description

A shrimp source nonapeptide with iron absorption promoting activity and its application

technical field

The invention relates to the fields of functional food, nutritionally fortified food and the like, in particular to a shrimp source nonapeptide and its application.

Background technique

Iron is the trace element with the most content in the human body, and it is widely distributed in the human body. The content in the body of an adult male is 50 mg, and that in an adult woman is 35 mg. The human body regulates iron mainly through the intestinal absorption of iron. The human body loses 1 to 2 mg of iron per day, which is usually obtained through dietary supplementation. Iron in the diet enters the human body and is mainly absorbed in the duodenum and the upper part of the jejunum. absorb. Iron in food is mainly divided into heme iron and non-heme iron. Heme iron exists in animal foods, and its absorption rate is relatively high, ranging from 15% to 35%. However, the diet of Chinese residents is mostly based on plant foods, and the iron in plant foods is non-heme iron. After non-heme iron is ingested, it will be affected by the pH in the human intestinal environment to form insoluble iron hydroxide precipitates, resulting in a low absorption rate, and various dietary factors such as polyphenols and oxalic acid and phytic acid in the diet are also affected. It will affect its absorption and utilization, leading to the appearance of iron deficiency. Therefore, how to improve the absorption and utilization of non-heme iron has attracted extensive attention.

Studies have shown that some "meat factors" have a positive effect on improving the absorption and utilization of iron. Food-derived iron absorption-promoting active peptides can promote iron absorption and enhance iron bioavailability. Food-derived iron absorption-promoting active peptides come from a wide range of sources, usually with smaller molecular weights, and can improve iron transport in the gut.

Contents of the invention

The purpose of the present invention is to provide a shrimp-derived nonapeptide obtained from Antarctic krill trypsin hydrolyzate identified by mass spectrometry and capable of promoting the absorption of iron ions, which can be applied to the fields of functional health products, nutritionally fortified foods and the like. The present invention evaluates the function of the shrimp source nonapeptide in delivering iron ions by using a rat everted intestinal sac model.

A shrimp-derived nonapeptide, the amino acid sequence of which is shown in SEQ ID NO: 1, is Asp-Thr-Asp-Ser-Glu-Glu-Glu-Glu-Ile-Arg, abbreviated as DTDSEEEIR, and has a molecular weight of 1093.05Da; wherein,

Asp means that the English name is Aspartic acid, and the Chinese name is the corresponding amino acid residue of aspartic acid;

Thr means the corresponding residue of the amino acid whose English name is Threonine and whose Chinese name is threonine;

Ser means that the English name is Serine, and the Chinese name is the corresponding amino acid residue of serine;

Glu means that the English name is Glutamic acid, and the Chinese name is the corresponding amino acid residue of glutamic acid;

Ile means that the English name is Isoleucine, and the Chinese name is the corresponding amino acid residue of isoleucine;

Arg represents the corresponding residue of the amino acid whose English name is Arginine and whose Chinese name is arginine.

The amino acid sequence of the present invention adopts the standard Fmoc scheme, through resin screening, and a reasonable polypeptide synthesis method. Connect the C-terminal carboxyl group of the target polypeptide to an insoluble polymer resin in the form of a covalent bond, start from the amino group of this amino acid, react with the carboxyl group of another amino acid to form a peptide bond, repeat this process to form the target polypeptide sequence, and then The peptide chain is separated from the resin to obtain the target polypeptide product. Peptide synthesis is a process of repeated addition of amino acids, and the solid-phase synthesis sequence is synthesized from the C-terminus to the N-terminus. After the synthesis is completed, it is purified by high-performance liquid chromatography, quick-frozen in liquid nitrogen, and vacuum freeze-dried to obtain the finished polypeptide.

The shrimp-derived nonapeptide DTDSEEEIR prepared by the invention has a certain ability to resist digestion by proteases in the gastrointestinal tract, and has the ability to deliver iron ions through the everted intestinal sac model of rats. When the Fe ²⁺ content was 60mg/mL, and the molar ratio of shrimp-derived nonapeptide:Fe ²⁺ was 1:2, the Fe ²⁺ transport amount was 65.6±12.6μg/ mL; while the FeSO ₄ solution with the same concentration of Fe ²⁺ , the Fe ²⁺ transport amount was only 44.7±6.0 μg/mL when the transport time was 90 minutes in the everted intestinal sac model of rats.

An application of shrimp source nonapeptide in delivering Fe ²⁺ , the amino acid sequence of the shrimp source nonapeptide is shown in SEQ ID NO:1.

An application of a shrimp source nonapeptide in the preparation of iron-supplementing medicines, food and/or health products, the amino acid sequence of the shrimp source nonapeptide is shown in SEQ ID NO:1.

An iron supplement, which is prepared by adding shrimp nonapeptide and Fe ²⁺ , contains shrimp nonapeptide and Fe ²⁺ , and the amino acid sequence of the shrimp nonapeptide is shown in SEQ ID NO:1.

In a preferred manner, the molar ratio of shrimp source nonapeptide to Fe ²⁺ in the iron supplement is 1:2.

Compared with the prior art, the present invention has the following advantages and technical effects:

The present invention synthesized the nonapeptide from shrimp for the first time, and the nonapeptide from shrimp can better maintain its integrity after being digested by the gastrointestinal tract, and can deliver iron ions through the everted intestinal sac model of rats, which has the effect of promoting iron absorption , can be applied in fields such as iron supplements and nutritionally fortified foods.

Description of drawings

Fig. 1 is the HPLC chart of the synthetic shrimp source nonapeptide Asp-Thr-Asp-Ser-Glu-Glu-Glu-Ile-Arg (abbreviation DTDSEEEIR) of the present invention.

Fig. 2 is the ESI-MS spectrum of the synthetic shrimp source nonapeptide Asp-Thr-Asp-Ser-Glu-Glu-Glu-Ile-Arg (abbreviation DTDSEEEIR) of the present invention.

Fig. 3 is the HPLC spectrum of the shrimp source nonapeptide DTDSEEEIR of the present invention after simulated digestion in the gastrointestinal tract.

Fig. 4 is the HPLC spectrum after simulated digestion in the gastrointestinal tract when the molar ratio of shrimp source nonapeptide DTDSEEEIR to Fe ²⁺ of the present invention is 1:2.

Fig. 5 is the HPLC spectrum after simulated digestion in the gastrointestinal tract when the molar ratio of shrimp source nonapeptide DTDSEEEIR to Fe ²⁺ of the present invention is 1:1.

Fig. 6 is the HPLC spectrum after simulated digestion in the gastrointestinal tract when the molar ratio of shrimp source nonapeptide DTDSEEEIR to Fe ²⁺ of the present invention is 2:1.

Fig. 7 is a graph of iron transport results when the Fe ²⁺ concentration of the present invention is 60 mg/mL. Different letters indicate significant differences among groups (P<0.05).

Fig. 8 is the HPLC spectrum of the shrimp-derived nonapeptide DTDSEEEIR and Fe ²⁺ in the molar ratio of 1:2 after being absorbed by a rat everted intestinal sac model.

Fig. 9 is the HPLC spectrum of the shrimp-derived nonapeptide DTDSEEEIR and Fe ²⁺ at a molar ratio of 1:1 after being absorbed by a rat everted intestinal sac model.

Fig. 10 is the HPLC spectrum of the shrimp-derived nonapeptide DTDSEEEIR and Fe ²⁺ molar ratio of 2:1 after being absorbed by a rat everted intestinal sac model.

Fig. 11 shows the transfer amount of shrimp-derived nonapeptide DTDSEEEIR absorbed by rat everted intestinal sac model of the present invention. Different letters indicate significant differences among groups (P<0.05).

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the implementation and protection scope of the present invention are not limited thereto. For the process parameters not specified in particular, it can be carried out with reference to conventional techniques.

The abbreviation of shrimp source nonapeptide in the present invention is DTDSEEEIR, and its molecular weight is 1093.05Da. The sequence is: Asp-Thr-Asp-Ser-Glu-Glu-Glu-Ile-Arg; Wherein,

Asp means that the English name is Aspartic acid, and the Chinese name is the corresponding amino acid residue of aspartic acid;

Thr means the corresponding residue of the amino acid whose English name is Threonine and whose Chinese name is threonine;

Ser means that the English name is Serine, and the Chinese name is the corresponding amino acid residue of serine;

Glu means that the English name is Glutamic acid, and the Chinese name is the corresponding amino acid residue of glutamic acid;

Ile means that the English name is Isoleucine, and the Chinese name is the corresponding amino acid residue of isoleucine;

Arg represents the corresponding residue of the amino acid whose English name is Arginine and whose Chinese name is arginine.

The amino acid sequence of the present invention adopts the standard Fmoc scheme, through resin screening, and a reasonable polypeptide synthesis method. Link the C-terminal carboxyl group of the target polypeptide to an insoluble polymer resin in the form of a covalent bond, and then use the amino group of this amino acid as a starting point to form a peptide bond with the carboxyl group of another molecule of amino acid. By repeating this process continuously, the target polypeptide sequence can be obtained. After the synthesis reaction is completed, the protecting group is removed, and the peptide chain is separated from the resin to obtain the target product. Peptide synthesis is a process of repeated addition of amino acids, and the solid-phase synthesis sequence is synthesized from the C-terminus to the N-terminus. After the synthesis is completed, it is purified by high-performance liquid chromatography, quick-frozen in liquid nitrogen, and vacuum freeze-dried to obtain the finished polypeptide.

The shrimp-derived nonapeptide DTDSEEEIR prepared by the invention can resist digestion in the gastrointestinal tract, and has the ability to deliver iron ions through a rat everted intestinal sac model.

In the present invention, when the iron content is 60 mg/mL, and the shrimp source nonapeptide: Fe ²⁺ molar ratio is 1:2, when the transit time of the rat valgus intestinal sac model is 90 minutes, the Fe ²⁺ transport amount is 65.6 ± 12.6 μg /mL; while the FeSO ₄ solution with the same iron concentration, when the transit time was 90min in the rat everted intestinal sac model, the Fe ²⁺ transport amount was only 44.7±6.0μg/mL.

Example 1: Acquisition of shrimp-derived nonapeptide DTDSEEEIR

S1. Preparation of Antarctic krill defatted powder: Add n-hexane/absolute ethanol mixture to Antarctic krill (Euphausia superba) powder, stir at 50°C for 6 hours; then filter with suction to remove the organic solvent, then add an equal volume of Mixed solution of n-hexane/absolute ethanol was stirred at 50°C for 6 hours, the organic solvent was removed by suction filtration, and the filter cake was naturally air-dried and crushed to obtain defatted Antarctic krill powder; wherein, the Antarctic krill powder was mixed with n-hexane/anhydrous The solid-liquid ratio of the ethanol mixed solution is 1:10g/mL (w/v); the n-hexane/dehydrated ethanol mixed solution is formed by mixing n-hexane and absolute ethanol in a volume ratio of 3:1;

S2. Preparation of Antarctic krill enzymatic hydrolyzate: add water to the Antarctic krill defatted powder described in step S1 until the substrate protein concentration is 2g/100mL to prepare an enzymolysis reaction liquid, adjust the pH to 8.0, and use 3000U/g substrate protein Add trypsin to the enzymolysis reaction solution, and react for 3 hours; adjust the pH to 7.0, bathe in 100°C for 10 minutes, centrifuge at 10,000 r/min for 20 minutes, take the supernatant, and freeze-dry to obtain the enzymatic hydrolyzate of Antarctic krill;

S3. Separation and purification of active peptides for promoting iron absorption: fill Sepharose 6Fast flow packing into the chromatography column, install 0.2M FeCl ₃ to the chromatography column at a flow rate of 1mL/min; after incubation for 30min, rinse with ultrapure water to remove Unbound iron ions; equilibrate the chromatographic column with an equilibration buffer; then, dissolve the Antarctic krill hydrolyzate described in step S2 in the equilibration buffer at a concentration of 10 mg/mL, load the sample and incubate for 30 min; After washing away the peptides that are not bound to iron, elute with eluent to obtain iron-binding peptides, collect and freeze-dry them as iron-absorbing active peptides; the equilibrium buffer is pH 5.5 containing 0.05M sodium acetate and 0.1 M NaCl solution; the eluent is pH5.5 containing 0.02M Na ₂ HPO ₄ , 0.1M NaCl, 0.01mg/mL ammonium acetate solution;

S4. HPLC-MS/MS identification of the iron absorption-promoting active peptide: use NanoLC-MS/MS mass spectrometer to identify the peptide sequence of the iron-absorbing active peptide described in step S3, and retrieve the peptide from the established database through Peaks Studio software sequence, a shrimp-derived nonapeptide was identified.

Experimental results: A shrimp-derived nonapeptide with a molecular weight of 1093.05 Da containing two Asp, three Glu, one Ser and one Thr residues was identified. Its amino acid sequence is shown in SEQ ID NO: 1, which is Asp-Thr- Asp-Ser-Glu-Glu-Glu-Ile-Arg (DTDSEEEIR), which contains a potential binding site for iron ions.

Example 2: Solid phase synthesis of shrimp source nonapeptide DTDSEEEIR

Select macromolecule resin (Hefei Saimanuo Biotechnology Co., Ltd.), according to the feature of amino acid sequence Asp-Thr-Asp-Ser-Glu-Glu-Glu-Glu-Ile-Arg (i.e. SEQ ID NO: 1), the Arg The carboxyl group is covalently linked to the resin, then the amino group of Arg and the carboxyl group of Ile are condensed, then Glu is added, amino acids are added from right to left in turn, until the last amino acid Asp is connected, the resin is excised to obtain the target polypeptide. Purify by high performance liquid chromatography, the chromatographic column model is VYDAC-C18, size 4.6*250mm, mobile phase A is acetonitrile containing 0.1% (v/v) trifluoroacetic acid; mobile phase B is acetonitrile containing 0.1% (v/v) ) water of trifluoroacetic acid; elution conditions are: 0～20.0min: mobile phase A rises from 22.0% to 32.0%; 20.0～20.1min: mobile phase A rises from 32.0% to 100.0%; flow rate 1.0mL/min, The detection wavelength is 220nm. Quick-frozen in liquid nitrogen and freeze-dried to obtain nonapeptide from shrimp, the required purity is over 98%, the actual purity is 98.49%, and the structure is identified by ESI-MS. Figure 1 is the HPLC chart of shrimp source nonapeptide Asp-Thr-Asp-Ser-Glu-Glu-Glu-Glu-Ile-Arg. Fig. 2 is an ESI-MS image of shrimp-derived nonapeptide Asp-Thr-Asp-Ser-Glu-Glu-Glu-Glu-Ile-Arg.

Example 3: Digestive properties of shrimp source nonapeptide

S1. Preparation method of simulated gastric digestive juice and intestinal digestive juice: Dissolve 40mg pepsin in 1mL 0.1N HCl to prepare simulated gastric digestive juice; dissolve 120mg sodium taurocholate and 20mg trypsin in 10mL 0.1M NaHCO ₃ solution In the preparation of simulated intestinal digestive juice; the enzyme activity of the pepsin is 3000U/mg, and the enzyme activity of the trypsin is 2500U/mg;

S2. Simulated gastric digestion: prepare 30 mL of 1 mM FeSO ₄ solution, add the shrimp source nonapeptide prepared in Example 2 of the present invention, the molar ratio of the concentration of the shrimp source nonapeptide to the Fe ²⁺ concentration is 1:2, 1, respectively. :1, 2:1, without adding FeSO _1mM shrimp source nonapeptide aqueous solution as a control, incubate at 37°C for 30min, adjust the pH to 2.0 with 1M HCl, add simulated gastric digestion solution (pepsin: shrimp source nonapeptide = 1:100w/w), stirred at 140rpm for 90min, simulated gastric digestion process, obtained simulated gastric digestate, took 2mL simulated gastric digestate, heated in 100°C water bath for 5min, centrifuged at 10000r/min for 10min, and took the supernatant, namely gastric digestate;

S3. Simulated intestinal digestion: adjust the pH of the simulated gastric digest described in step S3 to 6.5, add simulated intestinal digestive juice (trypsin: shrimp source nonapeptide = 1:80w/w), stir at 140rpm, and continue intestinal digestion for 150min Afterwards, heat in a water bath at 100°C for 5 minutes to inactivate the enzyme, and centrifuge at 10,000 rpm for 10 minutes to obtain intestinal digestion products;

S4. Identification of the retention rate of digested products: After filtering the gastric digest and intestinal digest obtained in steps S2 and S3, the retention rate of the shrimp source nonapeptide was determined by HPLC. The determination conditions were as follows: mobile phase A contained 0.1% ( v/v) acetonitrile solution of trifluoroacetic acid; mobile phase B is an aqueous solution containing 0.1% (v/v) trifluoroacetic acid; elution conditions are: 0.0～20.0min, 12%～32%A, 20.0～20.1min , 32% to 100% A, 20.1 to 25 min, 100% to 12% A, the flow rate is 1.0 mL/min, the detection wavelength is 220 nm, and the injection volume is 10 μL.

Experimental results: The HPLC of simulated digestion of shrimp-derived nonapeptide is shown in Figures 3 to 6. It was observed that the peak at about 6.5 minutes was shrimp-derived nonapeptide, and no obvious degradation occurred. The simple shrimp-derived nonapeptide showed digestion resistance after being digested by the simulated gastrointestinal tract. As shown in Table 1, the retention rate of shrimp-derived nonapeptide was 80%. After adding different molar ratios of Fe ²⁺ , the shrimp-derived nonapeptide The ability to resist digestion is improved, and the retention rate is increased to over 90% after simulating gastric digestion. After continuing to simulate intestinal digestion, the retention rate reaches up to 93% when the molar ratio of shrimp source nonapeptide to Fe ²⁺ is 1:2. Therefore, when shrimp-derived nonapeptide was mixed with Fe ²⁺ at a molar ratio of 1:2, shrimp-derived nonapeptide exhibited the highest digestion resistance and was more stable in gastrointestinal digestion.

Table 1: Peptide retention after simulated gastrointestinal digestion

Note: Different letters indicate significant differences among groups (P<0.05).

Example 4: Study on the Iron Absorption-Promoting Properties of Shrimp-Source Nonapeptide

S1. Establishment of the everted intestinal sac model in rats: Before the experiment, SD rats weighing 180-220 g were fasted for 12-16 hours. Inject 4% (w/vg/mL) chloral hydrate intraperitoneally to anesthetize the experimental mice after fasting. After the rats lose consciousness, open the laparotomy and take out the small intestine of about 7 cm, rinse the intestinal contents, and place them in a ventilator. One end of the intestinal segment was ligated in a 4°C buffer solution of oxygen (95% O ₂ ), and then carefully turned out. buffer solution; wherein, the buffer solution is: a solution containing 136mM NaCl, 8.17mM KCl, 1.0mM MgCl ₂ , 11.1mM glucose and 20mM HEPES.

S2. Shrimp-derived nonapeptide-promoting iron absorption experiment: the intestinal segment described in step S1 was placed in a 37°C sample buffer and FeSO ₄ buffer, continuously fed with oxygen, and incubated for 90 minutes, and then the intestinal solution was collected and analyzed by atomic absorption spectrometry. Photometer measures the iron content of the intestinal solution; wherein, the configuration of the sample buffer is: the shrimp source nonapeptide and _FeSO prepared in Example 2 are dissolved in the buffer described in step S1, and the shrimp source nonapeptide and FeSO ²⁺ is dissolved in the buffer solution described in step S1 with a molar ratio of 1:2, 1:1, and ₂ :1, and the iron ion concentration is _60mg /mL; In the buffer solution described in S1, the final concentration of Fe ²⁺ is 60mg/mL.

S3. Stability analysis of shrimp-derived nonapeptide: after the intestinal solution described in step S2 was filtered through a 0.22 μm filter membrane, the content of shrimp-derived nonapeptide was determined by HPLC. The detection conditions are as follows: mobile phase A is an acetonitrile solution containing 0.1% (v/v) trifluoroacetic acid; mobile phase B is an aqueous solution containing 0.1% (v/v) trifluoroacetic acid; the elution conditions are: 0.0～20.0min , 12%～32%A, 20.0～20.1min, 32%～100%A, 20.1～25min, 100%～12%A, the flow rate is 1.0mL/min, the detection wavelength is 220nm, and the injection volume is 10μL.

Experimental results: In the present invention, taking FeSO ₄ as a control, the rat valgus intestinal sac model was used to analyze the activity of promoting iron absorption of shrimp-derived nonapeptide through intestinal cells. As shown in Figure 7, when Fe ²⁺ content is 60mg/mL, shrimp source nonapeptide: ^Fe mol ratio is 1:2, when described shrimp source nonapeptide is transported through rat valgus intestinal sac model for 90min, Fe ²⁺ transport amount was 65.6±12.6μg/mL, significantly higher than FeSO ₄ (44.7±6.0μg/mL) (P<0.05), indicating that shrimp-derived nonapeptide has a good function of promoting iron absorption. The HPLC chromatograms of shrimp-derived nonapeptides with different molar ratios to Fe ²⁺ after transport are shown in Figures 8-10, and shrimp-derived nonapeptides with different molar ratios to Fe ²⁺ were absorbed by the rat everted intestinal sac model. degradation. Figure 11 shows the translocation results of shrimp-derived nonapeptide after being absorbed by the rat eversion intestinal sac model, and the translocation amount is represented by the ratio of the translocated shrimp-derived nonapeptide to the total amount of added shrimp-derived nonapeptide. It can be seen from Figure 11 that when the molar ratio of shrimp-derived nonapeptide to Fe ²⁺ is 1:2, the translocation amount of shrimp-derived nonapeptide is the highest. The peptide may be degraded during the transport process, resulting in a decrease in its transport volume, which has the same trend as the Fe ²⁺ transport volume, so the ability of shrimp-derived nonapeptide to promote iron absorption is related to its integrity during the absorption process.

Conclusion: The present invention synthesized the nonapeptide from shrimp for the first time. The nonapeptide from shrimp can resist simulated digestion in the gastrointestinal tract, can deliver iron ions through the everted intestinal sac model of rats, and has the effect of promoting iron absorption.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone familiar with the technical field within the technical scope disclosed in the present invention, according to the technical solution of the present invention Any equivalent replacement or change of the inventive concepts thereof shall fall within the protection scope of the present invention.

sequence listing

<110> Dalian University of Technology

<120> A nonapeptide derived from shrimp with the activity of promoting iron absorption and its application

<130> ZR201109LQ

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 1

Asp Thr Asp Ser Glu Glu Glu Ile Arg

1 5

Claims (3)

1. The application of a shrimp source nonapeptide in the preparation of iron supplement medicine, characterized in that, the amino acid sequence of the shrimp source nonapeptide is as shown in SEQ ID NO:1. 2. An iron supplement, characterized in that it contains shrimp-derived nonapeptide and Fe ²⁺ , and the amino acid sequence of said shrimp-derived nonapeptide is shown in SEQ ID NO:1. 3. iron supplement according to claim 2, is characterized in that, described shrimp source ^nonapeptide and Fe mol ratio is 1:2.

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