Extraction and application of gastrointestinal digestion resistant octapeptide

Abstract

The invention discloses extraction and application of an octapeptide capable of resisting gastrointestinal digestion, and belongs to the technical field of biology. The octapeptide disclosed by the invention is clear and definite in structure, can be prepared by adopting a solid-phase chemical synthesis method, can also be synthesized by recombinant expression of genetically engineered bacteria, and can also be obtained by separation and purification from fermented scallop skirt hydrolysate. After the octapeptide-ferrous chelate is digested by artificial saliva, artificial gastric juice and artificial intestinal juice, the retention rate of the octapeptide-ferrous chelate is high. The chelate of octapeptide and ferrous iron can improve the serum iron content of mice with iron-deficiency anemia and reduce the total iron binding force of serum. And compared with ferrous sulfate, the iron-rich protein has higher hemoglobin regeneration efficiency and relative biological value, is further suitable for developing novel iron-rich foods and iron supplements, and can also be compounded with other health-care products or food additives for use.

Description

Technical Field

[0001] The present invention relates to the extraction and application of an octapeptide resistant to gastrointestinal digestion, belonging to the field of biotechnology. Background Art

[0002] Iron is one of the essential trace elements for the human body and is crucial for maintaining normal physiological functions, including but not limited to oxygen transport, DNA synthesis, and cellular energy metabolism. Iron deficiency anemia, as a global public health problem, affects a large number of people, especially in developing countries, and is particularly prevalent among children and pregnant women. To improve this situation, researchers have been continuously exploring new products to enhance the absorption and utilization rate of iron in the human body, and bioactive peptides have attracted much attention due to their excellent properties.

[0003] Ferrous chelating peptide is a new type of dietary supplement ingredient, and its advantage lies in that it can form a stable complex with iron ions, thereby increasing the solubility and absorption rate of iron in the intestine. In addition, polypeptides with certain specific structures also exhibit the ability to resist gastrointestinal digestive enzymes, which means that they can maintain structural integrity when passing through the digestive tract, ensuring the delivery of iron to the required sites. However, there is currently a lack of high-efficiency ferrous chelating peptide products with anti-gastrointestinal digestion characteristics on the market.

[0004] Scallop skirt, as a marine resource by-product rich in protein, is an ideal raw material for preparing bioactive peptides. Bacillus altitudinis, as a fermentation strain, performs excellently in protein hydrolysis and can produce various proteases, which helps to release short peptides with specific functions from the substrate. Extracting functional peptide substances from such natural resources using microbial fermentation technology can not only achieve the efficient utilization of waste but also provide new ideas for the green and sustainable development of the food industry.

[0005] The present invention aims to propose an innovative method to extract polypeptides with strong ferrous chelating ability and gastrointestinal digestion stability from fermented scallop skirt hydrolysate to fill the gap in high-efficiency ferrous chelating peptide products with anti-gastrointestinal digestion characteristics. Summary of the Invention

[0006] Aiming at the deficiencies of the above-mentioned existing technologies, the present invention provides the extraction and application of an octapeptide resistant to gastrointestinal digestion, aiming to solve the technical problems such as high price, complex operation, and difficulty in resisting gastrointestinal digestion in the existing enzymatic preparation of ferrous chelating peptides.

[0007] The first technical solution provided by the present invention is a ferrous chelating octapeptide with good digestion stability, and the amino acid sequence of the ferrous chelating octapeptide is DDDHPGIF (Asp-Asp-Asp-His-Pro-Gly-Ile-Phe).

[0008] The second technical solution provided by the present invention is the preparation method of the ferrochelated octapeptide described in the first technical solution, which includes the following steps:

[0009] (1) Sample pretreatment: Wash the scallop skirt, remove impurities, dry it in an oven at 50 °C for 8 hours, crush it, and pass it through a 100-mesh sieve twice.

[0010] (2) Preparation of the sample to be fermented: Mix the scallop skirt powder obtained in step (1) with water and sterilize it under high temperature and high pressure to obtain a sterilized scallop skirt solution.

[0011] (3) Preparation of the fermented scallop skirt crude peptide solution: Add a suspension of Bacillus altitudinis to the cooled sterilized scallop skirt solution, carry out fermentation culture, and freeze-dry the fermentation supernatant to obtain the fermented scallop skirt crude peptide solution.

[0012] (4) Purification and identification of the fermented scallop skirt hydrolysate: First, separate the fermented scallop skirt hydrolysate solution (1 mg / mL) using a BioBasic TM AX HPLC anion exchange chromatography; the eluent is a phosphate gradient buffer (concentration 0.02 mol / L, pH 8.0, containing 0 - 0.5 M NaCl), the flow rate is 0.5 mL / min, monitor the elution peak at 214 nm, and collect the peak with the highest metal chelating activity. Then, further separate it using Sephadex G-25 gel filtration chromatography, the eluent is deionized water, the flow rate is 0.4 mL / min, monitor the elution peak at 214 nm, and collect the fraction with the highest metal chelating activity. Subsequently, further separate it using semi-preparative RP-HPLC-C18 reversed-phase high-performance liquid chromatography, the eluent is a gradient elution of acetonitrile - aqueous solution (volume ratio 0:100 - 30:70), the flow rate is 5 mL / min, and collect the fraction with the highest metal chelating activity. Finally, perform the final separation using analytical RP-HPLC-C18 reversed-phase high-performance liquid chromatography, the eluent is 0 - 10% acetonitrile - aqueous solution (volume ratio 0:100 - 10:90), the flow rate is 1 mL / min, collect each peak and measure the metal chelating activity, collect the elution peak with the highest metal chelating activity, and identify its peptide sequence using mass spectrometry. The result confirms that it contains the octapeptide of the present invention.

[0013] In some embodiments, in step (2), the material-liquid ratio of the scallop skirt powder to deionized water is 1:5 (g / mL); the sterilization condition is sterilization at 90 °C for 20 min.

[0014] In some embodiments, in step (3), the Bacillus altitudinis is Bacillus altitudinis 3*1-3, which was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 10, 2023, with the deposit number CGMCC No. 27846; the fermentation conditions are as follows: the final concentration of the bacterial solution is 1×10 7 CFU / mL, placed in a shaker at 37°C for shaking culture, the shaker speed is 200 rpm, and the culture time is 36 h; the centrifugation conditions are centrifugation at 10000 rpm for 10 min.

[0015] The third technical solution provided by the present invention is a ferrous-octapeptide chelate. The preparation method of the chelate is to adjust the pH of the octapeptide described in the first technical solution to 7.0 and then mix it with ferrous sulfate. The mass ratio of the octapeptide to ferrous sulfate is 5:1 (w / w). After vortex oscillation for 30 min, it is freeze-dried to obtain the ferrous-octapeptide chelate.

[0016] The fourth technical solution provided by the present invention is the application of the octapeptide described in the first technical solution, or the ferrous-octapeptide chelate described in the third technical solution in the preparation of iron-supplementing drugs or health products.

[0017] The fifth technical solution provided by the present invention is the application of the octapeptide described in the first technical solution, or the ferrous-octapeptide chelate described in the third technical solution in the preparation of drugs for relieving and / or treating iron deficiency anemia.

[0018] In some embodiments, the application at least includes one of the following effects:

[0019] (1) Improving the individual's serum iron content and total serum iron-binding capacity;

[0020] (2) Improving the individual's hemoglobin regeneration efficiency.

[0021] The sixth technical solution provided by the present invention is a polynucleotide encoding the octapeptide described in the first technical solution.

[0022] The seventh technical solution provided by the present invention is a recombinant vector carrying the polynucleotide described in the sixth technical solution.

[0023] The eighth technical solution provided by the present invention is a recombinant cell expressing the octapeptide described in the first technical solution, or containing the polynucleotide described in the sixth technical solution, or transformed with the recombinant vector described in the seventh technical solution.

[0024] The ninth technical solution provided by the present invention is a product containing the octapeptide described in the first technical solution.

[0025] In some embodiments, the product includes food, medicine, or health products.

[0026] The technical effects of the present invention are as follows:

[0027] 1. The octapeptide of the present invention has a clear and definite structure, can be prepared by solid-phase chemical synthesis method, can also be recombinantly expressed and synthesized using genetically engineered bacteria, and can also be isolated and purified from fermented scallop skirt hydrolysate.

[0028] 2. The octapeptide of the present invention has good resistance to oral-gastrointestinal digestion. The retention rate of the octapeptide-ferrous chelate of the present invention is high after digestion with artificial saliva, artificial gastric juice, and artificial intestinal juice.

[0029] 3. The octapeptide of the present invention has a significant improvement effect on the symptoms caused by iron deficiency anemia. The experimental results show that this octapeptide can increase the serum iron content of iron-deficient anemia mice and decrease the total iron binding capacity of serum. It has a higher hemoglobin regeneration efficiency and relative biological value compared to ferrous sulfate. The octapeptide in the present invention is suitable for the development of new iron-rich foods and iron supplements, and can also be compounded with other health products or food additives for use.

[0030] Biomaterial Preservation

[0031] Bacillus altitudinis 3*1-3, with the taxonomic name Bacillus altitudinis, was deposited on July 10, 2023, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC No. 27846. Description of the Drawings

[0032] Figure 1 It is the mass spectrometry identification result of the octapeptide.

[0033] Figure 2 It is the peptide sequence identification of the octapeptide and octapeptide-ferrous chelate after digestion. Detailed Embodiments

[0034] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0035] Testing Method:

[0036] 1. Determination of the stability of the octapeptide during digestion

[0037] Prepare an octapeptide-ferrous chelate solution with a concentration of 1 mg / mL, and prepare a ferrous sulfate solution with the same iron content as a control. Add simulated saliva to each solution, stir for 2 minutes at 37 °C to simulate oral digestion, and take 2 mL of samples at 0 and 2 minutes respectively. Then immediately add an equal volume of simulated gastric juice, continue to stir in the dark at 37 °C, take 2 mL of samples every 30 minutes for 2 hours to simulate gastric digestion. Then add an equal amount of simulated intestinal juice to simulate intestinal digestion, and also stir in the dark at 37 °C for 2 hours. The sampling process is the same as that of gastric simulation. Immediately after all samples are collected, place them in an ice bath for 20 minutes to ensure that all enzyme activities are lost, then centrifuge to obtain the supernatant, and determine the retention rate of the octapeptide and the peptide sequence composition after digestion. The method for measuring the retention rate is as follows: the digested samples are filtered through a 0.22 μm organic filter membrane, and the retention rates of the octapeptide and its ferrous chelate during in vitro digestion are measured using an Essentia LC-16 system equipped with a C18 chromatographic column (ZORBAX StableBond C18, 250 mm × 4.6 mm). Mobile phase A is an acetonitrile solution containing 0.1% trifluoroacetic acid, and mobile phase B is an aqueous solution containing 0.1% trifluoroacetic acid. The elution program is that within 0 - 15 min, the ratio of mobile phase A to mobile phase B is 18:82 (V / V). The flow rate is 1 mL / min, the injection volume is 10 μL, and the detection wavelength is 220 nm. Use a liquid chromatography-mass spectrometry (LC-MS) instrument to identify the in vitro simulated digestion products of scallop octapeptide and its ferrous chelate. The sample analysis column is Acclaim PepMap C18 (75 μm × 25 cm). The operating conditions are set as follows: the flow rate of the mobile phase is 300 nL / min, the column temperature is maintained at 40 °C, the injection volume is 2 μL, and the electrospray voltage is set to 2 kV. The liquid chromatography program is that from 0 - 53.6 min, the proportion of mobile phase B increases from 4% to 50%, and from 53.6 - 54 min, the proportion of mobile phase B increases to 95% and is maintained for 5.6 minutes. The tandem mass spectrometry data obtained under the above conditions are analyzed and processed using PEAKS Studio version 10.6 software (Bioinformatics Solutions Inc., Waterloo, Canada). The database search parameters are set as follows: the allowable range of mass deviation of fragment ions is 0.02 Da, and the mass deviation of precursor ions does not exceed 10 ppm. When identifying peptide segments, the -10lgP value is required to be not less than 20 to ensure the reliability of the identification results.

[0038] 2. Establishment of a mouse model of iron deficiency anemia

[0039] Thirty 3-week-old male ICR mice were weaned and acclimated in a barrier system for 1 week. They were randomly divided into 5 groups according to body weight: normal group (Control), positive control (Pos), negative control (Neg), low-dose (Low), and high-dose (High) groups. The normal group was fed with maintenance feed, and the other groups were fed with low-iron feed (5 ppm Fe). All groups had free access to water. The hemoglobin levels of mice in each group were monitored weekly. After the hemoglobin content of mice in each group was lower than 100 g / L, 2 mL / 1000 g bw of the sample was administered by gavage: the normal and negative control groups were gavaged with ultrapure water, the positive control group was gavaged with ferrous sulfate at 4 mg Fe / kg bw, and the low- and high-dose groups were gavaged with octapeptide-ferrous chelate at 1 and 4 mg Fe / kg bw, respectively.

[0040] 3. Determination of hematological indexes of mice

[0041] The collected mouse serum was used to determine the serum iron content and total iron-binding capacity according to the requirements of the test kit instructions. The determination method of the serum iron concentration detection kit (micro method) is as follows: First, prepare Reagent 1 and Reagent 2. Reagent 1 needs to be fully dissolved by adding 7.5 mL of distilled water before use; for Reagent 2, 235 μL of glacial acetic acid needs to be added and dissolved in sequence, and then 7.5 mL of distilled water is added and mixed evenly. Before the experiment, a standard solution of 125 μmol / L needs to be prepared by taking 100 μL of 1000 μmol / L Fe 3+ standard solution and adding 700 μL of distilled water and mixing evenly. Next, adjust the spectrophotometer or microplate reader to a wavelength of 520 nm and preheat for at least 30 minutes, and zero with distilled water. During the sample determination process, corresponding volumes of distilled water, serum (plasma), 125 μmol / L standard solution, Reagent 1, and Reagent 2 are added to the blank tube, determination tube, and standard tube, respectively. After each tube is mixed evenly, it is placed in a boiling water bath and heated for 5 minutes, and then cooled with tap water. Then, 60 μL of chloroform is added to each tube, shaken well, and centrifuged at 10000 rpm for 10 minutes. Finally, carefully aspirate 200 μL of the upper layer liquid into a micro glass cuvette or 96-well plate, and immediately measure the absorbance at a wavelength of 520 nm, recorded as A blank tube, A determination tube, and A standard tube.

[0042] The serum iron content (μmol / L) was calculated according to the following formula: Serum iron content = (A determination tube - A blank tube) / (A standard tube - A blank tube) * 125

[0043] The determination method of the serum total iron-binding capacity (TIBC) detection kit (micro method) is as follows: First, prepare the required reagents. The standard product needs to be dissolved by adding 0.9 mL of distilled water immediately before use to obtain 40 μmol / mL FeSO 4For the solution and the four reagents, the liquid A and liquid B need to be mixed in a ratio of 1:1. At the beginning of the experiment, first prepare a standard solution of 0.5 μmol / mL by taking 20 μL of 40 μmol / mL FeSO4 standard solution and adding 1580 μL of distilled water and mixing well. Then, adjust the spectrophotometer or microplate reader to a wavelength of 562 nm and preheat for at least 30 minutes, and zero with distilled water. At the same time, preheat reagent one at 37 °C for 10 minutes. During the sample determination process, add the corresponding volumes of serum, standard solution, distilled water, reagent one, reagent two, reagent three, and reagent four to the test tube, blank tube, and standard tube in an EP tube respectively. After mixing each tube, take out 200 μL after reacting at 37 °C for 10 minutes and place it in a 96-well plate or microcuvette, and measure the absorbance value at 562 nm, record it as A1 measurement, A1 blank, A1 standard, and calculate ΔA1 measurement and ΔA1 standard. Subsequently, add reagent five to each tube, react again at 37 °C for 5 minutes, take out 200 μL and measure the absorbance value at 562 nm, record it as A2 measurement, A2 blank, A2 standard, and calculate ΔA2 measurement and ΔA2 standard.

[0044] Calculate the total iron binding capacity (μmol / L) according to the following formula: Total iron binding capacity = (ΔA2 measurement / ΔA2 standard - ΔA1 measurement / ΔA1 standard) * 500

[0045] 4. Calculation of mouse hemoglobin regeneration efficiency and relative biological value

[0046] The mouse hemoglobin regeneration efficiency is calculated with reference to the following formula:

[0047] Hemoglobin regeneration efficiency H (%) = (m E - m S ) / m Fc ×100

[0048] The iron content m E (g) of hemoglobin in the mouse at the end of gavage = C E ×V×0.335%

[0049] The iron content m S (g) of hemoglobin in the mouse before gavage = C S ×V×0.335%

[0050] The iron intake m Fc (g) of the mouse = m I ×m F + m G

[0051] Among them, C E is the hemoglobin concentration of the mouse at the end of gavage, C SBefore intragastric administration to mice, Hb is the hemoglobin concentration, V is the blood volume (L), and 0.335% is the content of iron in hemoglobin, which accounts for about 0.335% of the mass of hemoglobin. m I is the food intake of mice, m F is the iron content in the feed, m G is the amount of iron in the intragastric administration of ferrous sulfate or the sample.

[0052] The relative biological utilization rate of the sample is calculated according to the following formula:

[0053] Relative biological utilization rate (%) = Hs - H P × 100

[0054] where Hs is the hemoglobin regeneration efficiency of the mice in the sample group, and Hp is the hemoglobin regeneration efficiency of the mice in the ferrous sulfate group.

[0055] Raw materials used in the examples:

[0056] 1. Bacillus altitudinis 3 * 1 - 3 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 10, 2023, with the deposit number CGMCC N0.27846.

[0057] 2. ICR male mice: Male SPF - level ICR mice (3 - week - old, body weight 12 - 15 g), purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning Experimental Animal Resource Center, license number: SCXK (Liao) 2015 - 0001), and raised in the animal room 401 of the National Engineering and Technology Research Center for Marine Food of Dalian Polytechnic University (license number: SYXK (Liao) 2017 - 0005). The environmental temperature is 22 ± 2 °C, the humidity is 65 ± 5%, and the lights in the animal room alternate between light and dark for 12 hours.

[0058] 3. Simulated saliva: Product number CZ0243 - 500 mL, purchased from Beijing Regene Biotechnology Co., Ltd.

[0059] 4. Simulated gastric juice: Product number CZ0210 - 500 mL, purchased from Beijing Regene Biotechnology Co., Ltd.

[0060] 5. Simulated intestinal juice: Product number CZ0200 - 500 mL, purchased from Beijing Regene Biotechnology Co., Ltd.

[0061] 6. Serum iron concentration detection kit (product number BC1735) and total iron - binding capacity (TIBC) detection kit (product number BC2865) were both purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0062] 7. Liquid Medium Formula: The LB Broth (LB) solid / liquid medium for activating strains is purchased from Qingdao Haibo Biotechnology Co., Ltd. The main components of the liquid medium are (g / L): tryptone 10.0, yeast extract powder 5.0, sodium chloride 10.0. The pH value is 7.0 ± 0.1. The formula of the solid medium contains 15.0 g / L agar in addition to the above components.

[0063] Example 1 Preparation of Ferrous Chelated Octapeptide

[0064] I. Preparation of Fermented Scallop Skirt Hydrolysate

[0065] (1) Sample Pretreatment: Take scallop skirts, wash them, remove impurities, dry them in an oven at 50 °C for 8 hours, crush them, and pass through a 100-mesh sieve twice.

[0066] (2) Preparation of Fermentation Sample: Mix the scallop skirt powder obtained in step (1) with a certain amount of water and sterilize it under high temperature and high pressure to obtain sterilized scallop skirt liquid. The material-liquid ratio of scallop skirt powder to deionized water is 1:5 (g / mL); the sterilization conditions are 90 °C for 20 min.

[0067] (3) Preparation of Fermented Scallop Skirt Crude Peptide Liquid: Inoculate Bacillus altitudinis into LB liquid medium, activate it in a shaking incubator at 37 °C for 18 h, after activation, centrifuge to collect the bacterial cells, and add sterile deionized water to prepare a bacterial suspension. Add the Bacillus altitudinis bacterial suspension to the cooled sterilized scallop skirt liquid, and the final concentration of the bacterial liquid is 10 7 CFU / mL. Place the mixture in a shaking incubator at 37 °C and shake it at a speed of 200 rpm for 36 h. After the cultivation is completed, centrifuge and collect the supernatant, and the centrifugation conditions are centrifugation at 10000 rpm for 10 min. Take the supernatant and freeze-dry it to obtain the fermented scallop skirt hydrolysate.

[0068] II. Isolation and Purification of Ferrous Chelated Octapeptide

[0069] First, use BioBasic TMThe fermented scallop skirt hydrolysate solution (1 mg / mL) was separated by AX HPLC anion exchange chromatography. The eluent was a phosphate gradient buffer (concentration 0.02 mol / L, pH 8.0, containing 0 - 0.5 M NaCl), the flow rate was 0.5 mL / min, the elution peaks were monitored at 214 nm, and the peak with the highest metal chelating activity was collected. Further separation was carried out by Sephadex G-25 gel filtration chromatography. The eluent was deionized water, the flow rate was 0.4 mL / min, the elution peaks were monitored at 214 nm, and the fraction with the highest metal chelating activity was collected. Subsequently, further separation was carried out using semi-preparative RP-HPLC-C18 reversed-phase high performance liquid chromatography. The eluent was a gradient elution of acetonitrile-aqueous solution (volume ratio 0:100 - 30:70), the flow rate was 5 mL / min, and the fraction with the highest metal chelating activity was collected. Finally, final separation was carried out using analytical RP-HPLC-C18 reversed-phase high performance liquid chromatography. The eluent was 0 - 10% acetonitrile-aqueous solution (volume ratio 0:100 - 10:90), the flow rate was 1 mL / min, each peak was collected and the metal chelating activity was measured. The elution peak with the highest metal chelating activity was collected and its peptide sequence was identified by mass spectrometry, and the result confirmed that it contained the octapeptide of the present invention (such as Figure 1 as shown).

[0070] Example 2 Preparation of Ferrous Chelated Octapeptide

[0071] After adjusting the pH of the octapeptide prepared in Example 1 to 7.0, it was mixed with ferrous sulfate. The mass ratio of the octapeptide to ferrous sulfate was 5:1 (w / w). It was vortexed for 30 min and then freeze-dried to obtain the ferrous-octapeptide chelate.

[0072] Example 3 Determination of Digestive Stability of Ferrous Chelated Octapeptide

[0073] The octapeptide obtained in Example 1 and the octapeptide-ferrous chelate obtained in Example 2 were subjected to oral and gastrointestinal digestion, and the products after oral and gastrointestinal digestion were measured as shown in Figure 2 and Table 1. The retention rates of the octapeptide and its ferrous chelate during the entire digestion stage were 95.09% and 93.08% respectively, indicating that these two substances have good digestive stability.

[0074] Table 1 Proportions of Each Peptide Sequence of the Octapeptide and Octapeptide-Ferrous Chelate after Digestion

[0075]

[0076] Example 4 Effects of Octapeptide-Ferrous Chelate on Serum Iron Content and Total Iron Binding Capacity in Mice

[0077] Serum ferritin is a key substance for iron storage in the body, and the serum iron content reflects the availability of iron in the body. Total iron-binding capacity of serum refers to the maximum amount of iron that all proteins capable of binding iron in serum can bind. It is an indicator reflecting the level and function of transferrin in the body and is usually used to evaluate the iron metabolism status in the body. In the case of iron-deficiency anemia, due to the reduction of stored iron in the body, the body will increase the synthesis of transferrin to compensate for this deficiency, so the total iron-binding capacity of serum often increases. As shown in Table 2, the serum iron content of the negative control group was significantly lower than that of the normal group, and the total iron-binding capacity of serum was significantly higher than that of the normal group, indicating that the iron storage in the negative control group of mice was lower, the available iron content decreased, and the iron-deficiency anemia model induced by low-iron diet was successful. After intragastric administration of ferrous sulfate and fermented scallop skirt iron chelate, the serum iron content and total iron-binding capacity of mice were significantly restored, and the serum iron content (49.09±2.37 μmol / L) and total iron-binding capacity of serum (259.68±20.92 μmol / L) in the high-dose group of mice were restored to the level comparable to that of the normal group, which was better than that of the low-dose group and the ferrous sulfate group.

[0078] Table 2 Determination results of serum iron content and total iron-binding capacity of mice

[0079]

[0080] Different lowercase letters indicate significant differences between different groups of the same index (p<0.05).

[0081] Example 5 Hemoglobin regeneration efficiency and relative biological value of octapeptide-iron chelate

[0082] The hemoglobin regeneration efficiency of five groups of mice was measured.

[0083] As shown in Table 3, the hemoglobin regeneration efficiency of mice intragastrically administered with ferrous sulfate component was 12.06±2.65%, and the hemoglobin regeneration efficiencies of mice intragastrically administered with low-dose and high-dose octapeptide-iron chelate were 21.17±3.57% and 14.87±4.11% respectively. The relative biological utilization rates of mice intragastrically administered with low-dose and high-dose octapeptide-iron chelate were 179.32±39.84% and 126.06±30.17% respectively. Mice intragastrically administered with the octapeptide-iron chelate provided by the present invention showed more significant hemoglobin regeneration efficiency and relative biological utilization rate than those intragastrically administered with ferrous sulfate. Compared with the traditional ferrous sulfate supplementation method, the iron supplement provided by the present invention can achieve better improvement effects at lower doses, and at the same time may reduce the risk of side effects caused by unnecessary high doses.

[0084] Table 3 Results of hemoglobin regeneration efficiency and relative biological utilization rate of mice

[0085]

[0086] Different lowercase letters indicate significant differences between different groups of the same index, p < 0.05.

[0087] Determination of the digestive stability of the polypeptide-ferrous chelate extracted from the fermented scallop skirt in Comparative Example 1

[0088] In addition to the octapeptide described in Example 1, another 10 polypeptides identified from the crude peptide of the fermented scallop skirt were selected, and the corresponding polypeptide-ferrous chelates were prepared with reference to the method of Example 2. The peptide sequence retention rates of these 10 polypeptide-ferrous chelates after oral and gastrointestinal digestion were measured, and the results are shown in Table 4. The retention rates of each peptide segment did not exceed 60%, which may be because the octapeptide has a unique amino acid composition and spatial structure, making it have good digestive stability that other peptide segments do not have.

[0089] Table 4. Digestive retention rates of the polypeptide-ferrous chelate extracted from the fermented scallop skirt

[0090]

[0091] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A ferrous chelated octapeptide, characterized in that: The amino acid sequence of the ferrous chelating octapeptide is DDDHPGIF.

2. The method for preparing the ferrous chelated octapeptide according to claim 1, characterized in that: The steps include: (1) preparing a fermented scallop skirt crude peptide liquid: adding a highland Bacillus suspension to the scallop skirt liquid for fermentation, and freeze-drying the fermentation supernatant to obtain a fermented scallop skirt crude peptide liquid; (2) Purification and identification of fermented scallop skirt hydrolysate: First, BioBasic TM The fermented scallop skirt hydrolysate solution was separated by AX HPLC anion exchange chromatography, and the peak with the highest metal chelating activity was collected; Sephadex G-25 gel filtration chromatography is then used for further separation to collect the component with the highest metal chelating activity; subsequently, semi-preparative RP-HPLC-C18 reverse-phase high performance liquid chromatography is used for further separation, each peak is collected and metal chelating activity is determined, and the elution peak with the highest metal chelating activity is collected, and its peptide sequence is identified by mass spectrometry to obtain the ferrous chelating octapeptide.

3. The preparation method according to claim 2, characterized in that: In step (1), the preparation method of scallop skirt liquid is as follows: the scallop skirt is washed, impurities are removed, and it is dried in a 50°C oven for 8 hours, and then crushed and sieved through a 100-mesh sieve twice; the scallop skirt powder and water are mixed according to a solid-liquid ratio of 1g:5mL and then sterilized by high temperature and high pressure to obtain scallop skirt liquid; the highland Bacillus is highland Bacillus (Bacillus altitudinis) 3*1-3, which has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 10, 2023, with a deposit number of CGMCC N0.27846; the fermentation conditions are: the final concentration of the bacterial liquid is 1×10 7 CFU / mL, placed in a 37°C shaker with a shaking speed of 200 rpm for 36 h; In step (2), BioBasic TM The conditions for AX HPLC anion exchange chromatography were as follows: the eluent was a phosphate gradient buffer with a concentration of 0.02 mol / L, pH 8.0, containing 0-0.5 M NaCl, the flow rate was 0.5 mL / min, and the elution peak was monitored at 214 nm; the conditions for Sephadex G-25 gel filtration chromatography were as follows: the eluent was deionized water, the flow rate was 0.4 mL / min, and the elution peak was monitored at 214 nm; the conditions for semi-preparative RP-HPLC-C18 reverse-phase high-performance liquid chromatography were as follows: the eluent was an acetonitrile-water solution with a volume ratio of 0:100-30:70 for gradient elution, and the flow rate was 5 mL / min.

4. A ferrous iron-octapeptide chelate, characterized in that: The preparation method of the chelate is to adjust the pH of the octapeptide of claim 1 to 7.0 and then mix it with ferrous sulfate, the mass ratio of the octapeptide to ferrous sulfate is 5:1 (w / w), vortex oscillation, and freeze-drying to obtain the ferrous-octapeptide chelate.

5. Use of the octapeptide according to claim 1 or the ferrous iron-octapeptide chelate according to claim 4 in the preparation of iron supplement medicines or health products.

6. Use of the octapeptide according to claim 1 or the ferrous iron-octapeptide chelate according to claim 4 in the preparation of a drug for alleviating and / or treating iron deficiency anemia, characterized in that: The application includes at least one of the following functions: (1) Improve individual serum iron content and serum total iron binding capacity; (2) Improve individual hemoglobin regeneration efficiency.

7. A polynucleotide encoding the octapeptide of claim 1.

8. A recombinant vector carrying the polynucleotide according to claim 7.

9. A recombinant cell expressing the octapeptide of claim 1, or containing the polynucleotide of claim 7, or transformed with the recombinant vector of claim 8.

10. A product, characterized in that The product contains the octapeptide according to claim 1.

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