A Bacillus subtilis strain and its applications

Abstract

The present invention discloses a Bacillus subtilis and its application, belonging to the field of modern food processing biotechnology. The present invention provides a Bacillus subtilis that can utilize seafood by-products to prepare ferrous chelated peptides. Through the pretreatment of raw materials, the preparation of fermentation broth, centrifuging the fermentation broth to obtain the supernatant, freeze-drying, re-dissolving, combining with ferrous sulfate, alcohol precipitation, and spray drying, a dry powder of ferrous chelate is obtained. The chelation rate of the fermented ferrous chelated peptides prepared by the present invention reaches 96.50%, and the chelation effect is good. The Bacillus subtilis and its application provided by the present invention provide new ideas for the preparation of ferrous chelated peptides; the fermentation method adopted by the present invention has a simple operation process, does not require the addition of exogenous enzymes throughout the process, and has low costs; at the same time, it makes full use of seafood by-products, laying a foundation for the high-value processing of seafood by-products and enhancing their development value.

Description

Technical Field

[0001] The present invention relates to a Bacillus subtilis strain and its application, belonging to the field of modern food processing biotechnology. Background Art

[0002] The World Health Organization states that iron is an essential trace element for the human body and plays important roles such as oxygen transport, DNA synthesis, and promotion of muscle metabolism in the body. During infancy, adolescence, and pregnancy, the human body's demand for iron increases rapidly, easily leading to iron deficiency. Iron deficiency disorders and iron deficiency anemia are common and important health problems affecting the world.

[0003] Iron-chelated peptides are a new type of dietary supplement, with advantages such as high absorption and utilization rate, low energy consumption during metabolism, fast transport speed, and being not easily saturated. Moreover, polypeptides can enter intestinal mucosal cells through the small peptide transport system as metal element ligands, with high stability, and can reduce metal antagonism, having good application prospects.

[0004] The microbial fermentation method is a method for preparing bioactive peptides by using proteases produced by microbial strains during the production and metabolism process to hydrolyze substrate proteins. Microorganisms can produce a complex enzyme system during metabolism, releasing specific high-concentration bioactive peptides through enzymatic hydrolysis, with higher enzymatic hydrolysis efficiency; at the same time, they can also produce peptidases to remove bitter peptides, making the taste and flavor of bioactive peptide products better; in addition, the metabolites of the bacterial cells can further modify certain functional groups to improve their biological activity. Microorganisms also have lower production costs compared to the enzymatic method. However, existing microbial fermentation methods often still need to be combined with the enzymatic method and cannot use microorganisms alone to achieve the preparation of iron-chelated peptides, and additional carbon sources, etc. need to be added to the fermentation system to promote fermentation. For example, in the invention patent application with the publication number CN102827908A, Bacillus and protease are combined for fermentation and enzymatic hydrolysis; in the invention patent application with the publication number CN110693033B, walnut meal needs to be preliminarily hydrolyzed with alkaline protease before using microorganisms for fermentation. It can be seen that there is almost no research on using microorganisms to hydrolyze and prepare ferrous chelated peptides without enzymatic method assistance, and the existing technology has problems of complex processes and inability to reduce production costs.

[0005] Scallops are known as one of the eight treasures of seafood. The scallop skirt is a by-product generated during the processing process, with a large quantity and high nutritional value. Especially, the protein content accounts for about 78% of the dry weight of the skirt. Therefore, the scallop skirt is a good raw material for polypeptide preparation. Currently, there is little research and development on scallop peptides in China, and no research reports on the preparation method of fermented scallop skirt peptide iron chelate have been seen. Summary of the Invention

[0006] In view of the problems of existing iron supplements, such as low iron content, strong gastrointestinal irritation, and high production costs (enzymatic method), the present invention provides a method for preparing a fermented scallop skirt ferrous chelate. Its main objectives include: First, to realize the recycling of seafood waste and prepare peptide iron chelate from waste scallop skirts; Second, to prepare a peptide iron chelate that is convenient for human absorption; Third, to increase the iron content in the peptide iron chelate; Fourth, to provide a method for preparing ferrous chelated peptides that only uses microorganisms, saving production costs and simplifying the operation process.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions.

[0008] The first objective of the present invention is to provide Bacillus subtilis M17 b7, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 10, 2023, with the deposit number CGMCC No. 27847.

[0009] The second objective of the present invention is to provide the application of the Bacillus subtilis M17 b7 in the preparation of ferrous chelated peptides.

[0010] In one embodiment, the application includes the following steps:

[0011] A. Raw material pretreatment: Take the seafood by-product raw material, remove impurities, dry it, crush it, and sieve it to obtain dry powder.

[0012] B. Preparation of sterilized raw material liquid: Mix the dry powder obtained in step A with a certain amount of water to obtain a mixed liquid, and sterilize it.

[0013] C. Preparation of fermented raw material hydrolysate: After the sterilized raw material liquid obtained in step B is cooled, add the Bacillus subtilis M17 b7 bacterial suspension, and shake and culture the mixed liquid; centrifuge it, collect the supernatant, and freeze-dry the supernatant to obtain the fermented raw material hydrolysate.

[0014] D. Preparation of ferrous chelate: Re-dissolve the fermented raw material hydrolysate obtained in step C into a sample solution with a certain concentration, and add ferrous sulfate for chelation reaction.

[0015] E. Separation of ferrous chelate: Add ethanol to the ferrous chelate obtained in step D, dry the precipitate, and obtain ferrous chelated peptide powder.

[0016] In one embodiment, in step A, the drying conditions are drying at 40 - 60 °C for 4 - 6 hours; the sieve mesh specifications for sieving are 50 - 500 meshes.

[0017] In one embodiment, the mass-volume ratio of the dry powder to water in step B is (1:10)-(1:50) (g / mL); the sterilization condition is sterilization at 121 °C for 10-40 min.

[0018] In one embodiment, in step C, the final concentration of Bacillus subtilis M17 b7 is 10 5 -10 9 CFU / mL.

[0019] In one embodiment, in step C, the culture temperature is 20-45 °C; the shaker speed is 100-300 rpm / min; the culture time is 12-72 h; the centrifugation condition of the fermentation raw material hydrolysate is centrifugation at 4000-8000 rpm for 5-40 min.

[0020] In one embodiment, in step D, the reconstitution concentration of the fermentation raw material hydrolyzate is 1 mg / mL, the concentration of ferrous sulfate added is 15-25 mM, and the mass ratio of the hydrolyzate to ferrous sulfate is 20:1 (w / w); the reaction time is 15-25 min.

[0021] In one embodiment, in step E, the volume ratio of ethanol to ferrous chelate added is 5:1, and the centrifugation condition is 10000 rpm for 10 min.

[0022] In one embodiment, in step E, the drying method is spray drying or freeze drying.

[0023] Compared with the existing methods, the present invention has the following beneficial effects:

[0024] 1. Using seafood by-products, especially scallop skirts as raw materials, high-value utilization of scallop by-products can be achieved, saving resources and increasing economic benefits.

[0025] 2. Using the microbial method to produce bioactive peptides, without the addition of extra enzymes, the production cost is low, the production process is simple, and no complex equipment is required. The chelation rate of the prepared ferrous chelated peptide is as high as 96.5%.

[0026] Biological material preservation

[0027] Bacillus subtilis M17 b7 has been deposited with the China General Microbiological Culture Collection Center (CGMCC) on July 10, 2023, and the deposit number is CGMCC No. 27847. Description of the drawings

[0028] Figure 1It is a streak plate photo of Bacillus subtilis M17 b7 of the present invention.

[0029] Figure 2 It is the ultraviolet spectrogram of the fermented scallop skirt hydrolysate, the scallop skirt ferrous chelate and ferrous sulfate prepared by this method;

[0030] Figure 3 It is the scanning electron microscope (a) and elemental analysis diagram (b) of the fermented scallop skirt hydrolysate, the scallop skirt ferrous chelate and ferrous sulfate prepared by this method;

[0031] Figure 4 It is the X-ray diffraction analysis of the fermented scallop skirt hydrolysate, the scallop skirt ferrous chelate and ferrous sulfate prepared by this method. Specific embodiments

[0032] The present invention will be further described in detail below in conjunction with specific embodiments, but the implementation mode of the present invention is not limited to the scope represented by this embodiment.

[0033] (1) The method for measuring the ferrous chelation rate of the scallop skirt hydrolysate in the following embodiments is as follows (ferrozine colorimetric method):

[0034] The freeze-dried scallop skirt hydrolysate is redissolved into a solution with a concentration of 1 mg / mL. Take 0.5 mL of the above solution, add 50 μL of a ferrous sulfate solution with a concentration of 2 mM, and vortex and oscillate for 20 min. Add 250 μL of ferrozine (2 mM) to the mixture, mix well and react at room temperature for 10 min, and then measure the absorbance at a wavelength of 562 nm. The calculation formula for the chelation rate is:

[0035] Chelation rate (100%) = (A b - A S ) / A b * 100%, A b is the absorbance with pure water instead of the sample as a control, and A S is the absorbance of the sample.

[0036] (2) Cultivation of strains:

[0037] Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus natto: Cultured in LB liquid medium, taken out after culturing in a 37 °C incubator for 18 - 24 hours and adjusted to the appropriate concentration for subsequent fermentation experiments.

[0038] Aspergillus niger: Cultured in PDA solid medium, taken out after culturing in a 30 °C incubator for 4 - 5 days. After the flat plate colonies turn from yellow to black, adjust the spore concentration for subsequent fermentation experiments.

[0039] Yeast: Cultured in YPD liquid medium, taken out after culturing in a 30°C incubator for 18 - 24 hours, and the concentration was adjusted for subsequent fermentation experiments.

[0040] Isolation and Identification of Bacillus subtilis M17 b7 in Example 1

[0041] (1) Collection of strains:

[0042] The sample was collected from local Northeast farmhouse sauce in Dalian. Take 1 g of farmhouse sauce, add it to 10 mL of physiological saline, transfer it into a homogenization bag, beat for 30 min, centrifuge at 500 g for 5 min to remove sauce residue, and aseptically transfer the turbid liquid part containing bacteria to a 50 mL centrifuge tube. After appropriately diluting the culture solution, it was spread on LB solid medium. After the liquid was completely absorbed, it was cultured inverted at 37°C for 24 - 48 h. Then, single colonies were picked and streaked on LB solid medium to isolate single colonies ( Figure 1 ). After continuous purification for three generations, single colonies were picked for microscopic examination. After determining that there was no contamination, they were inoculated into LB liquid medium for enlarged culture. The cultured strains were frozen and preserved, named M17 b7. Another part of the cultured bacterial liquid was used for identification.

[0043] (2) Identification of strain M17 b7:

[0044] Extract the genome of strain M17 b7 for 16s rDNA identification. The genomic extraction method was carried out according to the glass bead method in the "Concise Guide to Molecular Biology Experiments".

[0045] The PCR conditions are as follows:

[0046] The amplification system is: 2×Taq Master Mix 25 μL, primer 27F, 2 μL, primer 1492R, 2 μL, sterilized water 19 μL, template 2 μL. The PCR reaction conditions are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 S; annealing at 55°C for 15 S; extension at 72°C for 1 min; 72°C for 5 min; 30 cycles; 4°C ∞. After PCR, agarose gel (1.0%) electrophoresis was used to detect the PCR products of yeast samples. The samples with bright bands were the successfully amplified genomes, and the successful genomes could be sent for sequencing.

[0047] The 16s rDNA sequence of the strain M17 b7 is as follows:

[0048] GGAGGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGG

[0049] GTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGG

[0050] TTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATT

[0051] AGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACA

[0052] CTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGT

[0053] CTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAA

[0054] GTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGC

[0055] CGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAA

[0056] GTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGA

[0057] GGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGA

[0058] CTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTC

[0059] CACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCA

[0060] CTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGG

[0061] AGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGAT

[0062] AGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG

[0063] GGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTG

[0064] CCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACG

[0065] TGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTT

[0066] CGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTG

[0067] AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGG

[0068] TAACCTTTTAGGAGCCAGCCGCCGAATGGGGG(SEQ ID NO.1).

[0069] The above sequencing results were analyzed by Blastn, and it was found that the bacterium had the highest homology with Bacillus subtilis, reaching 98%. Therefore, this strain was identified as Bacillus subtilis and named Bacillus subtilis M17 b7. The Bacillus subtilis M17 b7 grew well on the LB solid plate. After culturing at 37 °C for 24 h, as Figure 1 shown, the colonies of Bacillus subtilis were milky white, round or irregular in shape, with neat edges, dry surfaces, and slightly sticky characteristics.

[0070] Through morphological and 16S rDNA identification, this strain M17 b7 was tentatively identified as Bacillus subtilis and was deposited in the China General Microbiological Culture Collection Center (CGMCC) on July 10, 2023. The deposit number is: CGMCC No. 27847. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0071] Example 2 Preparation of Ferrous Chelated Peptide

[0072] S1. Raw material pretreatment: Wash the scallop skirts, remove impurities, dry them at 50 °C for 4 h, pulverize them, and pass through a 100-mesh sieve to obtain scallop skirt powder for standby;

[0073] S2. Preparation of sterilized scallop skirt liquid: Mix the scallop skirt powder obtained in step S1 with water at a ratio of 1:20 (g / mL) to obtain a mixed solution; sterilize it at 121 °C for 20 min to obtain a sterilized scallop skirt liquid;

[0074] S3. Preparation of scallop skirt mixed solution: After the sterilized scallop skirt liquid obtained in step S2 is cooled, add a Bacillus subtilis M17 b7 bacterial suspension with a final concentration of 10 7 CFU / mL.

[0075] S4. Preparation of fermented scallop skirt hydrolyzate: Shake and culture the mixed solution in a shaker at 37 °C at a rotation speed of 200 rpm / min for 36 hours;

[0076] S5. Preparation of scallop skirt ferrous chelate: Centrifuge the fermented scallop skirt hydrolyzate obtained in step S4 at 6000 rpm / min for 20 min, collect the supernatant, and freeze-dry the supernatant to obtain the fermented scallop skirt hydrolyzate. Then, re-dissolve the obtained scallop skirt hydrolyzate with water at a re-dissolution concentration of 1 mg / mL, add ferrous sulfate at a concentration of 2 mM, and the mass ratio of the hydrolyzate to ferrous sulfate is 20:1 (w / w); the reaction time is 20 min to obtain the scallop skirt ferrous chelate. The ferrous chelation rate was determined by the ferrozine colorimetric method.

[0077] S6, Freeze-drying: Add ethanol to the scallop skirt iron chelate obtained in S5. The volume ratio of ethanol to the sample solution is 5:1 (V / V). The centrifugation conditions are 10000 rpm for 10 min. Spray-dry to obtain scallop skirt iron chelated peptide powder.

[0078] Preparation of Iron Chelated Peptide in Example 3

[0079] S1, Raw material pretreatment: Take scallop skirts, wash them, remove impurities, dry them at 40 °C for 6 h, pulverize them, and pass through a 200-mesh sieve to obtain scallop skirt powder for standby;

[0080] S2, Preparation of sterilized scallop skirt solution: Mix the scallop skirt powder obtained in step S1 with water at a ratio of 1:10 (g / mL) to obtain a mixed solution; Sterilize at 121 °C for 15 min to obtain a sterilized scallop skirt solution;

[0081] S3, Preparation of scallop skirt mixed solution: After the sterilized scallop skirt solution obtained in step S2 is cooled, add a Bacillus subtilis M17 b7 bacterial suspension with a final concentration of 10 6 CFU / mL.

[0082] S4, Preparation of fermented scallop skirt hydrolyzate: Shake and culture the mixed solution in a shaker at 25 °C at a rotation speed of 150 rpm / min for 24 hours;

[0083] S5, Preparation of scallop skirt iron chelate: Centrifuge the fermented scallop skirt hydrolyzate obtained in step S4 at 5000 rpm / min for 30 min, collect the supernatant, and freeze-dry the supernatant to obtain the fermented scallop skirt hydrolyzate. The preparation of scallop skirt iron chelate and the determination of the chelation rate refer to Example 2.

[0084] S6, Freeze-drying: Add ethanol to the scallop skirt iron chelate obtained in S5. The volume ratio of ethanol to the sample solution is 5:1 (V / V). The centrifugation conditions are 10000 rpm for 10 min. Spray-dry to obtain scallop skirt iron chelated peptide powder.

[0085] Preparation of Iron Chelated Peptide in Example 4

[0086] S1, Raw material pretreatment: Take scallop skirts, wash them, remove impurities, dry them at 60 °C for 4 h, pulverize them, and pass through a 50-mesh sieve to obtain scallop skirt powder for standby;

[0087] S2, Preparation of sterilized scallop skirt solution: Mix the scallop skirt powder obtained in step S1 with water at a ratio of 1:10 (g / mL) to obtain a mixed solution; Sterilize at 121 °C for 10 min to obtain a sterilized scallop skirt solution;

[0088] S3. Preparation of scallop skirt mixed solution: After the sterilized scallop skirt solution obtained in step S2 is cooled, add a Bacillus subtilis M17 b7 bacterial suspension with a final concentration of 10 5 CFU / mL.

[0089] S4. Preparation of fermented scallop skirt hydrolyzate: Shake and culture the mixed solution in a shaker at 20°C at a rotation speed of 100 rpm / min for 12 hours;

[0090] S5. Preparation of scallop skirt ferrous chelate: Centrifuge the fermented scallop skirt hydrolyzate obtained in step S4 at 4000 rpm / min for 40 min, collect the supernatant, and freeze-dry the supernatant to obtain the fermented scallop skirt hydrolyzate. The preparation and chelation rate determination of scallop skirt ferrous chelate refer to Example 2.

[0091] S6. Freeze-drying: Add ethanol to the scallop skirt ferrous chelate prepared in S5, with the volume ratio of ethanol to the sample solution being 5:1 (V / V), and the centrifugation conditions being 10000 rpm for 10 min. Spray-dry to obtain scallop skirt ferrous chelate peptide powder.

[0092] Preparation of Ferrous Chelate Peptide in Example 5

[0093] S1. Raw material pretreatment: Take scallop skirts, wash them, remove impurities, dry them at 50°C for 4 h, pulverize them, and pass through a 300-mesh sieve to obtain scallop skirt powder for standby;

[0094] S2. Preparation of sterilized scallop skirt solution: Mix the scallop skirt powder obtained in step S1 with water at a ratio of 1:30 (g / mL) to obtain a mixed solution; sterilize it at 121°C for 25 min to obtain a sterilized scallop skirt solution;

[0095] S3. Preparation of scallop skirt mixed solution: After the sterilized scallop skirt solution obtained in step S2 is cooled, add a Bacillus subtilis M17 b7 bacterial suspension with a final concentration of 10 8 CFU / mL.

[0096] S4. Preparation of fermented scallop skirt hydrolyzate: Shake and culture the mixed solution in a shaker at 30°C at a rotation speed of 250 rpm / min for 48 hours;

[0097] S5. Preparation of scallop skirt ferrous chelate: Centrifuge the fermented scallop skirt hydrolyzate obtained in step S4 at 7000 rpm / min for 10 min, collect the supernatant, and freeze-dry the supernatant to obtain the fermented scallop skirt hydrolyzate. The preparation and chelation rate determination of scallop skirt ferrous chelate refer to Example 2.

[0098] S6. Freeze-drying: Ethanol was added to the scallop skirt ferrous chelate prepared in S5, with the volume ratio of ethanol to the sample solution being 5:1 (V / V). The centrifugation conditions were 10,000 rpm for 10 min. The obtained scallop skirt ferrous chelate precipitate was spray-dried to obtain scallop skirt ferrous chelated peptide powder.

[0099] Preparation of Ferrous Chelated Peptide in Example 6

[0100] S1. Raw material pretreatment: The scallop skirt was taken, washed, impurities removed, dried at 50 °C for 4 h, pulverized, and passed through a 400-mesh sieve to obtain scallop skirt powder for standby.

[0101] S2. Preparation of sterilized scallop skirt solution: The scallop skirt powder prepared in step S1 was mixed with water at a ratio of 1:40 (g / mL) to obtain a mixed solution, which was sterilized at 121 °C for 30 min to obtain a sterilized scallop skirt solution.

[0102] S3. Preparation of scallop skirt mixed solution: After the sterilized scallop skirt solution prepared in step S2 was cooled, a Bacillus subtilis M17 b7 bacterial suspension with a final concentration of 10 9 CFU / mL was added.

[0103] S4. Preparation of fermented scallop skirt hydrolyzate: The mixed solution was shaken and cultured in a shaker at 40 °C at a rotation speed of 300 rpm / min for 60 hours.

[0104] S5. Preparation of scallop skirt ferrous chelate: The fermented scallop skirt hydrolyzate prepared in step S4 was centrifuged at 8,000 rpm for 5 min, the supernatant was collected, and the supernatant was freeze-dried to obtain fermented scallop skirt hydrolyzate. The preparation and chelation rate determination of scallop skirt ferrous chelate refer to Example 2.

[0105] S6. Freeze-drying: Ethanol was added to the scallop skirt ferrous chelate prepared in S5, with the volume ratio of ethanol to the sample solution being 5:1 (V / V). The centrifugation conditions were 10,000 rpm for 10 min. Spray-drying was carried out to obtain scallop skirt ferrous chelated peptide powder.

[0106] Preparation of Ferrous Chelated Peptide in Example 7

[0107] S1. Raw material pretreatment: The scallop skirt was taken, washed, impurities removed, dried at 50 °C for 4 h, pulverized, and passed through a 500-mesh sieve to obtain scallop skirt powder for standby.

[0108] S2. Preparation of sterilized scallop skirt solution: The scallop skirt powder prepared in step S1 was mixed with water at a ratio of 1:50 (g / mL) to obtain a mixed solution, which was sterilized at 121 °C for 40 min to obtain a sterilized scallop skirt solution.

[0109] S3. Preparation of scallop skirt mixed solution: After the sterilized scallop skirt solution obtained in step S2 is cooled, add Bacillus subtilis M17 b7 bacterial suspension with a final concentration of 10 7 CFU / mL.

[0110] S4. Preparation of fermented scallop skirt hydrolysate: Shake and culture the mixed solution in a shaker at 30 °C at a rotation speed of 200 rpm / min for 72 hours;

[0111] S5. Preparation of scallop skirt ferrous chelate: Centrifuge the fermented scallop skirt hydrolysate obtained in step S4 at 6000 rpm / min for 20 min, collect the supernatant, and freeze-dry the supernatant to obtain the fermented scallop skirt hydrolyzate. The preparation and chelation rate determination of scallop skirt ferrous chelate refer to Example 2.

[0112] S6. Freeze-drying: Add ethanol to the scallop skirt ferrous chelate prepared in S5, with the volume ratio of ethanol to the sample solution being 5:1 (V / V), and the centrifugation conditions being 10000 rpm and 10 min. Spray-dry to obtain scallop skirt ferrous chelated peptide powder.

[0113] Verification of Ferrous Chelated Peptide in Example 8

[0114] Perform spectroscopic analysis on the scallop skirt ferrous chelated peptide prepared in Example 2. As Figure 2 , by comparing the ultraviolet absorption spectra of the fermented scallop skirt hydrolyzate (FSH) prepared in step S4 and the scallop skirt peptide iron chelate (FSH-Fe) prepared in step S5, it can be seen that after chelation, the highest absorption peak moves from 271.2 nm to 268.4 and 268.6 nm (adding 10 and 20 mM FeSO 4 respectively), and the absorption peak undergoes a blue shift. This is because the oxygen atom on the carbonyl group participates in the complexation of Fe 2+ , and the addition of Fe 2+ causes charge transfer transition and ligand field transition, resulting in a change in the charge of the carbonyl group and a blue shift of the absorption band. This shows that the chelate and the unchelated substance are two different substances.

[0115] Verification of Ferrous Chelated Peptide in Example 9

[0116] Perform morphological analysis on the scallop skirt ferrous chelated peptide prepared in Example 2. As Figure 3 a shows the results of scanning electron microscopy of FSH, FSH-Fe, and ferrous sulfate respectively. The structure of ferrous sulfate is different from that of FSH-Fe and FSH, and it has a standard crystal structure. The surface of FSH is smooth and dense, while the surface of FSH-Fe is rough and loose. It can be inferred that FSH-Fe is a new substance. According to elemental analysis, as Figure 3b. The surface elemental composition of FSH includes C, O, S, and N, while that of FSH-Fe includes C, O, S, N, and Fe. This finding indicates that ferrous iron has been successfully chelated with FSH.

[0117] Example 10 Verification of Ferrous Chelated Peptide

[0118] X-ray diffraction analysis was performed on the ferrous chelated peptide from scallop skirt prepared in Example 2. The XRD results are as Figure 4 shown. Ferrous sulfate has a high diffraction peak at a diffraction angle of 28.68°. However, the XRD results of FSH and FSH-Fe are completely different from those of ferrous sulfate. The diffraction peaks of FSH appear at diffraction angles of 31.76° and 45.52°. At these two angles, the peak intensity of the ferrous chelate is significantly weakened and is positively correlated with the addition amount of ferrous sulfate. This shows that ferrous iron has been successfully chelated with FSH.

[0119] Comparative Example 1 Preparation of Ferrous Chelated Peptide

[0120] Control group without adding Bacillus subtilis M17 b7 of the present invention

[0121] S1. Raw material pretreatment: Wash scallop skirt, remove impurities, dry at 50 °C for 4 h, pulverize, and sieve through a 100-mesh sieve to obtain scallop skirt powder for standby;

[0122] S2. Preparation of sterilized scallop skirt liquid: Mix the scallop skirt powder prepared in step S1 with water at a ratio of 1:20 (g / mL) to obtain a mixed liquid; sterilize at 121 °C for 20 min to obtain sterilized scallop skirt liquid;

[0123] S3. Preparation of scallop skirt hydrolyzate: After the sterilized scallop skirt liquid prepared in step S2 is cooled, shake and culture in a shaker at 30 °C at a rotation speed of 200 rpm / min for 36 hours;

[0124] S4. Preparation of scallop skirt ferrous chelate: Centrifuge the scallop skirt hydrolyzate prepared in step S4 at 6000 rpm / min for 20 min, collect the supernatant, and freeze-dry the supernatant to obtain fermented scallop skirt hydrolyzate. The preparation of scallop skirt ferrous chelate refers to Example 2.

[0125] S5. Freeze-drying: Add ethanol to the scallop skirt ferrous chelate prepared in S5, with the volume ratio of ethanol to the sample solution being 5:1 (V / V), and the centrifugation conditions being 10000 rpm and 10 min. Spray-dry to obtain scallop skirt ferrous chelated peptide powder.

[0126] Comparative Example 2 Preparation of Ferrous Chelated Peptide

[0127] S1. Raw material pretreatment: Take the adductor muscle of bay scallop, wash it, remove impurities, dry it at 50 °C for 4 h, pulverize it, and sieve it through a 100-mesh sieve to obtain scallop adductor muscle powder for standby.

[0128] S2. Preparation of scallop adductor muscle hydrolysate: Add deionized water to the scallop adductor muscle powder to make the concentration of the scallop adductor muscle powder reach 6.25% (w / w). Then add bromelain (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The enzymatic hydrolysis conditions are: reaction temperature 68.37 °C, pH 9.35, substrate concentration 6.25%, enzyme dosage 6000 U / g, and reaction time 6 h.

[0129] S3. Preparation of scallop adductor muscle ferrous chelate: Centrifuge the scallop adductor muscle hydrolysate prepared in step S3 at 6000 rpm / min for 20 min, collect the supernatant, and freeze-dry the supernatant to obtain scallop adductor muscle hydrolyzate. The preparation of scallop adductor muscle ferrous chelate refers to Example 2.

[0130] S4. Freeze-drying: Add ethanol to the scallop adductor muscle ferrous chelate prepared in S4, and the volume ratio of ethanol to the sample solution is 5:1 (V / V). The centrifugation conditions are 10000 rpm and 10 min. Spray-dry the obtained scallop adductor muscle ferrous chelate precipitate to obtain scallop adductor muscle ferrous chelated peptide powder.

[0131] Preparation of ferrous chelated peptide in Comparative Example 3

[0132] S1. Raw material pretreatment: Take scallops (from Qinhuangdao sea area), wash them, remove impurities, dry them at 50 °C for 4 h, pulverize them, and sieve them through a 100-mesh sieve to obtain scallop powder for standby.

[0133] S2. Preparation of scallop liquid to be hydrolyzed: Soak the scallop powder and water at a material-liquid mass ratio of 1:10, heat it to 85 °C, and then pour it into a colloid mill to grind it into scallop slurry.

[0134] S3. Preparation of scallop hydrolysate: Add the first protease (4000 U·g -1 ) according to the mass of the total protein measured in the scallop slurry for the first enzymatic hydrolysis, and carry out constant-temperature hydrolysis for 90 min under the appropriate pH and temperature conditions of this enzyme to obtain the first hydrolysate; after inactivating the enzyme in the first hydrolysate, add the second protease (4000 U·g -1 , calculated according to the total protein mass of the scallop slurry) for the second enzymatic hydrolysis, and adjust the temperature and pH for constant-temperature hydrolysis for 70 min. Inactivate the enzyme in the second hydrolysate at 90 °C for 10 min. Different enzyme combinations for scallop hydrolysis are shown in Table 1, and the enzymes used are purchased from Peptide Technology (Xiamen) Co., Ltd.

[0135] Table 1 Different enzyme combinations for scallop hydrolysis

[0136]

[0137] S4. Preparation of scallop ferrous chelate: Centrifuge the scallop hydrolyzate obtained in step S3 at 6000 rpm / min for 20 min, collect the supernatant, and freeze-dry the supernatant to obtain scallop hydrolyzate. The preparation of scallop skirt ferrous chelate refers to Example 2.

[0138] S5. Freeze-drying: Add ethanol to the scallop ferrous chelate prepared in S4, with the volume ratio of ethanol to the sample solution being 5:1 (V / V), and the centrifugation conditions being 10000 rpm for 10 min. Spray-dry the obtained scallop ferrous chelate precipitate to obtain scallop ferrous chelated peptide powder.

[0139] Preparation of ferrous chelated peptide in Comparative Example 4

[0140] S1. Raw material pretreatment: Take scallop skirt (purchased from Qinhuangdao Haidongqing Food Co., Ltd.), wash, remove impurities, dry at 50 °C for 4 h, pulverize, and pass through a 100-mesh sieve to obtain scallop skirt powder for standby;

[0141] S2. Preparation of scallop skirt hydrolyzate: Disperse the scallop skirt powder obtained in step S1 evenly in distilled water at a ratio of 1:25 (w / v), and hydrolyze with neutral protease (the enzyme addition amount is 7%, purchased from Beijing Solarbio Science & Technology Co., Ltd.) at 50 °C and pH 7 for 4 hours. Heat the obtained hydrolyzate in a water bath at 100 °C for 10 min to inactivate the enzyme.

[0142] S3. Preparation of scallop skirt ferrous chelate: Centrifuge the hydrolyzate obtained in step S2 at 4 °C at 10,610×g for 25 min. Filter the supernatant through a 0.22-μm membrane, and the filtrate is the protein hydrolyzate. Freeze-dry the filtrate to obtain scallop skirt hydrolyzate. The preparation of scallop skirt ferrous chelate refers to Example 2.

[0143] S4. Freeze-drying: Add ethanol to the scallop skirt ferrous chelate prepared in S3, with the volume ratio of ethanol to the sample solution being 5:1 (V / V), and the centrifugation conditions being 10000 rpm for 10 min. Spray-dry the obtained scallop skirt ferrous chelate precipitate to obtain scallop skirt ferrous chelated peptide powder.

[0144] Preparation of ferrous chelated peptide in Comparative Example 5

[0145] S1. Raw material pretreatment: Take scallop skirt (purchased from Qinhuangdao Haidongqing Food Co., Ltd.), wash, remove impurities, dry at 50 °C for 4 h, pulverize, and pass through a 100-mesh sieve to obtain scallop skirt powder for standby;

[0146] S2. Preparation of sterilized scallop skirt solution: Mix the scallop skirt powder obtained in step S1 with water at a ratio of 1:20 (g / mL) to obtain a mixed solution; sterilize at 121 °C for 20 min to obtain a sterilized scallop skirt solution;

[0147] S3. Preparation of Fermented Scallop Skirt Hydrolysate: Inoculate 1 mL of bacterial solution (10 6 viable bacteria per mL of culture medium) into 100 mL of sterilized scallop skirt solution (pH 7.0). The inoculated strains are Bacillus subtilis CICC 20030 (5-1), Bacillus subtilis CICC 20076 (5-2), Bacillus licheniformis CICC 20033 (5-3), Bacillus licheniformis CICC 23631 (5-4), and Bacillus amyloliquefaciens CICC 20029 (5-5). Shake and culture the mixture in a shaker at 30 °C at a rotation speed of 150 rpm / min for 48 hours;

[0148] S4. Preparation of Ferrous Chelate of Scallop Skirt: Centrifuge the fermented scallop skirt hydrolysate obtained in step S3 at 10,610×g for 25 min at 4 °C. Filter the supernatant through a 0.22 μm membrane. The filtrate is the protein hydrolysate. Freeze-dry the filtrate to obtain the scallop skirt hydrolyzate. The preparation of the ferrous chelate of scallop skirt refers to Example 2.

[0149] S5. Freeze-drying: Add ethanol to the ferrous chelate of scallop skirt obtained in S4. The volume ratio of ethanol to the sample solution is 5:1 (V / V). The centrifugation conditions are 10000 rpm for 10 min. Spray-dry the obtained precipitate of the ferrous chelate of scallop skirt to obtain the powder of ferrous chelated peptide of scallop skirt.

[0150] Preparation of Ferrous Chelated Peptide in Comparative Example 6

[0151] S1. Raw Material Pretreatment: Wash the scallop skirt, remove impurities, dry it at 50 °C for 4 h, crush it, and pass it through a 100-mesh sieve to obtain the dry powder of scallop skirt for standby;

[0152] S2. Preparation of Sterilized Scallop Skirt Solution: Take 6 g of scallop skirt, add water to 100 mL, add 3 g of sucrose, adjust the pH to 8.2 to obtain a mixed solution; sterilize it at 121 °C for 20 min to obtain the sterilized scallop skirt solution;

[0153] S3. Preparation of Scallop Skirt Hydrolysate: Inoculate 5 mL of natto bacteria suspension (purchased from Ningbo Mingzhou Biotechnology Co., Ltd., numbered BMZ125132) into the above sterilized scallop skirt solution, with a final concentration of 10 7 CFU / mL. Shake and culture the mixture in a shaker at 37 °C at a rotation speed of 180 rpm / min for 60 hours;

[0154] S4. Preparation of scallop skirt iron chelate: Centrifuge the scallop skirt hydrolysate obtained in step S3 at 7000 rpm / min for 20 min to obtain scallop skirt fermentation broth. Take the supernatant and freeze-dry it to obtain scallop skirt hydrolysate. The preparation of scallop skirt iron chelate refers to Example 2.

[0155] S5. Freeze-drying: Add ethanol to the scallop skirt iron chelate prepared in S4, with the volume ratio of ethanol to the sample solution being 5:1 (V / V). The centrifugation conditions are 10000 rpm and 10 min. Spray-dry the obtained scallop skirt iron chelate precipitate to obtain scallop skirt iron chelated peptide powder.

[0156] Preparation of ferrous chelated peptide in Comparative Example 7

[0157] S1. Raw material pretreatment: Take the by-products of Fenneropenaeus chinensis, including heads and shells, and chop them with a cell breaker.

[0158] S2. Preparation of fermentation broth of Fenneropenaeus chinensis by-products: Add the chopped raw materials obtained in step S1 to sterile water at a ratio of 30:100 (w / v) to obtain a fermentation medium.

[0159] S3. Preparation of mixed solution of Fenneropenaeus chinensis by-products: Add 50 mL of the fermentation medium obtained in step S2 to a 250 mL conical flask, and inoculate 2.5 mL of Bacillus subtilis (CICC 10023) bacterial solution (5% of the volume of the fermentation medium, and the inoculation concentration is 1*10 7 CFU / mL).

[0160] S4. Preparation of fermented Fenneropenaeus chinensis by-product hydrolysate: Incubate the mixed solution at 37 °C for 7 days, and the shaking speed of the shaker is 150 rpm.

[0161] S5. Preparation of Fenneropenaeus chinensis iron chelate: Centrifuge the fermented Fenneropenaeus chinensis by-product hydrolysate obtained in step S4 at 4 °C and 4000 g for 20 min, collect the supernatant, and take the supernatant and freeze-dry it to obtain fermented Fenneropenaeus chinensis by-product hydrolysate. The preparation of Fenneropenaeus chinensis iron chelate refers to the preparation of scallop skirt iron chelate in Example 2.

[0162] S6. Freeze-drying: Add ethanol to the Fenneropenaeus chinensis iron chelate prepared in S5, with the volume ratio of ethanol to the sample solution being 5:1 (V / V). The centrifugation conditions are 10000 rpm and 10 min. Spray-dry the obtained fermented Fenneropenaeus chinensis by-product iron chelate precipitate to obtain fermented Fenneropenaeus chinensis by-product iron chelated peptide powder.

[0163] Preparation of ferrous chelated peptide in Comparative Example 8

[0164] S1. Raw material pretreatment: Take the muscles of sardines, zebrafish, gobies, and rays, and then rinse them with cold distilled water to remove salt and other contaminants. Take 500 g of raw muscles of sardines, zebrafish, gobies, and rays, place them in 1000 mL of distilled water, and boil for 20 min at 100 °C. Remove the bones from the cooked fish, collect the fish meat, and dry it in an oven at 80 °C for 18 hours. Then perform a crushing process to obtain fish meat powder.

[0165] S2. Preparation of fish meat fermentation broth: Add the fish meat powder obtained in step S1 to sterile water at a ratio of 30:100 (w / v), adjust the pH to 8, and sterilize at 121 °C for 20 min.

[0166] S3. Preparation of fish meat mixture: Place 100 mL of the fermentation broth obtained in S2 in a 1000 mL conical flask, and inoculate it with a Bacillus subtilis A26 (CTM 50700) bacterial suspension at 10% of the volume of the fermentation broth. The initial inoculation concentration is 1*10 7 CFU / mL.

[0167] S4. Preparation of fermented fish meat hydrolysate: Incubate the mixture obtained in step S3 at 37 °C for 24 h with a shaker speed of 200 rpm.

[0168] S5. Preparation of fish meat ferrous chelate: Centrifuge the fermented fish meat hydrolysate obtained in step S4 at 4 °C and 8500 g for 30 min, collect the supernatant, and freeze-dry the supernatant to obtain the fermented fish meat hydrolysate. The preparation of the fish meat ferrous chelate refers to the preparation of the scallop skirt ferrous chelate in Example 2.

[0169] S6. Freeze-drying: Add ethanol to the fish meat ferrous chelate prepared in S5, with the volume ratio of ethanol to the sample solution being 5:1 (V / V). The centrifugation conditions are 10000 rpm for 10 min. Spray-dry the obtained precipitate of the fermented fish meat ferrous chelate to obtain the powder of the fermented fish meat ferrous chelated peptide.

[0170] Preparation of Ferrous Chelated Peptide in Comparative Example 9

[0171] S1. Raw material pretreatment: Remove the internal organs and fish skin from the by-products of tilapia (purchased from Zhanjiang Asia Aquatic Science and Technology Co., Ltd.), wash the remaining fish heads, fish bones, and the attached fish meat in the by-products, and grind the cleaned remaining materials with a meat grinder.

[0172] S2. Preparation of tilapia by-product fermentation broth: Add a certain amount of distilled water to the ground tilapia by-products, with the solid-liquid ratio of 13.8% (w / v), place them in a 250 mL Erlenmeyer flask, with a loading volume of 48.6 mL / 250 mL, and the glucose addition amount of 0.2% (w / v), and sterilize at 121 °C for 20 min.

[0173] S3. Preparation of tilapia by - product mixture: Inoculate the cooled tilapia by - product fermentation broth obtained in S2 with the seed solution of Bacillus subtilis HL - 1. Adjust the OD 600 of the seed solution to 1.01 - 1.05, and the inoculation amount is 5% (V / V).

[0174] S4. Preparation of tilapia by - product hydrolysate: Incubate the mixture obtained in step S3 at 34 °C and 150 rpm / min for 48 h.

[0175] S5. Preparation of tilapia by - product ferrous chelate: Centrifuge the fermented tilapia by - product obtained in step S4 at 8000 rpm / min for 10 min, collect the supernatant, and finally keep the supernatant at 95 °C for 5 min to inactivate the enzyme, obtaining an aqueous solution of tilapia by - product protein peptides. The preparation of tilapia by - product ferrous chelate refers to the preparation of scallop skirt ferrous chelate in Example 2.

[0176] S6. Freeze - drying: Add ethanol to the scallop skirt ferrous chelate prepared in S5, with the volume ratio of ethanol to the sample solution being 5:1 (V / V), and the centrifugation conditions being 10000 rpm and 10 min. Spray - dry the obtained tilapia by - product ferrous chelate precipitate to obtain a powder of fermented tilapia by - product ferrous chelated peptide.

[0177] Preparation of ferrous chelated peptide in Comparative Example 10

[0178] S1. Raw material pretreatment: Take fish processing by - products (mainly small yellow croaker heads and fish viscera), wash them, add 10 mL of deionized water, and the solid - liquid ratio is 1:2 (m:v) to prepare the fermentation broth.

[0179] S2. Preparation of fish processing by - product mixture: Add the cultured Bacillus subtilis (CICC 10023) to the fermentation broth, with the initial inoculation concentration of 1*10 7 CFU / mL, the strain addition amount is 5.3% (V / V), and the soybean meal addition amount is 10.1% (w / w).

[0180] S3. Preparation of fermented fish processing by - product hydrolysate: Incubate the mixture obtained in step S2 at 28 °C for 4 days, and the shaker speed is 160 rpm.

[0181] S4. Preparation of fish processing by - product ferrous chelate: Centrifuge the fermented fish processing by - product hydrolysate obtained in step S3 at 4 °C and 8500 g for 30 min, collect the supernatant, and freeze - dry the supernatant to obtain the fermented fish processing by - product. The preparation of fish processing by - product ferrous chelate refers to the preparation of scallop skirt ferrous chelate in Example 2.

[0182] S5. Freeze-drying: Ethanol was added to the scallop skirt ferrous chelate prepared in S4, and the volume ratio of ethanol to the sample solution was 5:1 (V / V). The centrifugation conditions were 10,000 rpm for 10 min. The obtained precipitate of the fermented fish processing by-product ferrous chelate was spray-dried to obtain the fermented fish processing by-product ferrous chelated peptide powder.

[0183] Preparation of Ferrous Chelated Peptide in Comparative Example 11

[0184] S1. Raw material pretreatment: Fresh cod fish skin was cleaned, cooked to denature the fish skin, and ground with a tissue grinder. 100 g of cod fish skin was taken, 50 mL of distilled water was added, the pH was natural, and it was autoclaved at 121 °C for 20 min for standby.

[0185] S2. Preparation of cod skin mixed solution: The solid-liquid ratio was set to 1:1.5 (w / v), and the strain ratio of bacteria (Bacillus subtilis BNⅣ-1; Bacillus licheniformis JF0 Y3): mold (Aspergillus niger H-2): yeast (Saccharomyces cerevisiae JQⅡJM) was 2:1:1. The inoculation amount was 8%, and the initial inoculation concentration was 1×10 7 CFU / mL.

[0186] S3. Preparation of fermented cod skin fermentation broth: The mixed solution prepared in step S2 was cultured at 30 °C for 24 h, and the rotation speed of the shaker incubator was 200 rpm.

[0187] S4. Preparation of fish processing by-product ferrous chelate: After the cod skin fermentation broth prepared in step S3 was inactivated and sterilized, it was centrifuged at 4,000 r / min for 20 min, the supernatant was collected, and freeze-dried to obtain fermented fish skin. The preparation of the fish processing by-product ferrous chelate refers to the preparation of the scallop skirt ferrous chelate in Example 2.

[0188] S5. Freeze-drying: Ethanol was added to the fish processing by-product chelate prepared in freeze-drying: S4 to make the ethanol content in the system reach 60%. It was refrigerated and left standing at 4 °C for 12 h, centrifuged at 6,000 r / min to collect the supernatant, the ethanol was evaporated, and freeze-dried to obtain the powdery fish skin polypeptide ferrous chelate.

[0189] Preparation of Ferrous Chelated Peptide in Comparative Example 12

[0190] S1. Raw material pretreatment: Weigh 25 g of the by-product of Solenocera melantho, add distilled water according to the ratio of 1:4 (m / v), adjust the pH to 8.0, and autoclave at 121 °C for 15 min for standby.

[0191] S2. Preparation of the Solenocera melantho by-product mixed solution: Wait for the Solenocera melantho by-product prepared in step S1 to cool to room temperature, inoculate the cultured Bacillus subtilis (CICC 10023), and the inoculation amount is 4% (V / V). The initial inoculation concentration is 1×107 CFU / mL..

[0192] S3. Preparation of the fermented by - product fermentation broth of Solenocera crassicornis: Cultivate the mixed solution obtained in S2 at 35 °C for 3 days, and the rotation speed of the shaker incubator is 180 rpm.

[0193] S4. Preparation of the ferrous chelate of Solenocera crassicornis by - products: Centrifuge the fermented by - product fermentation broth of Solenocera crassicornis obtained in S3 at 4000 r / min for 20 min, collect the supernatant, and freeze - dry to obtain the hydrolysate of Solenocera crassicornis. The preparation of the ferrous chelate of Solenocera crassicornis by - products refers to the preparation of the ferrous chelate of scallop skirt in Example 2.

[0194] S5. Freeze - drying: Add ethanol to the ferrous chelate of Solenocera crassicornis by - products prepared in S4. The volume ratio of ethanol to the sample solution is 5:1 (V / V), and the centrifugation conditions are 10000 rpm and 10 min. Spray - dry the obtained precipitate of the fermented ferrous chelate of Solenocera crassicornis by - products to obtain the powder of the fermented ferrous chelate peptide of Solenocera crassicornis by - products.

[0195] Preparation of Ferrous Chelate Peptide in Comparative Example 13

[0196] S1. Raw material pretreatment: Take Euphausia superba (purchased from Dalian Ocean Fishery Group Corporation, Liaoning Province), and obtain Euphausia superba shells after mincing the meat three times with a meat - mincing machine.

[0197] S2. Preparation of the fermentation solution of Euphausia superba shells to be fermented: Accurately weigh 25 g of Euphausia superba shells, sterilize them at 121 °C, place them in a 250 - mL glass bottle, and add deionized water to make the solid - liquid ratio 1:3 (w / V).

[0198] S3. Preparation of the mixed solution of Euphausia superba shells: Inoculate the cultivated Bacillus subtilis (CICC 10023) into the fermentation broth obtained in S2, and the inoculation amount is 4%, and the inoculation concentration is 1×10 8 CFU / mL.

[0199] S4. Preparation of the fermented Euphausia superba shell fermentation broth: Cultivate the mixed solution obtained in S3 at 37 °C for 72 h, and the rotation speed of the shaker incubator is 120 rpm.

[0200] S5. Preparation of the ferrous chelate of Euphausia superba shells: Centrifuge the fermented Euphausia superba shell fermentation broth obtained in S4 at 4000 r / min for 20 min, collect the supernatant, and freeze - dry to obtain the hydrolysate of Euphausia superba shells. The preparation of the ferrous chelate of Euphausia superba shells refers to the preparation of the ferrous chelate of scallop skirt in Example 2.

[0201] S6. Freeze-drying: Ethanol was added to the scallop skirt iron chelate prepared in S5, and the volume ratio of ethanol to the sample solution was 5:1 (V / V). The centrifugation conditions were 10000 rpm for 10 min. The obtained Antarctic krill shell iron chelate precipitate was spray-dried to obtain Antarctic krill shell iron chelated peptide powder.

[0202] Table 2 Iron Chelation Rates of Samples with Different Components

[0203]

[0204]

[0205] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations 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 strain of Bacillus subtilis M17 b7 has been deposited with the China General Microbiological Culture Collection Center (CGMCC), and the deposit number is CGMCC No. 27847.

2. Use of the Bacillus subtilis M17 b7 described in claim 1 in the preparation of ferrous chelated peptides, characterized in that, using the Bacillus subtilis M17 b7 to ferment a scallop skirt powder solution to obtain a hydrolyzate, and reacting the obtained hydrolyzate with ferrous sulfate, then ferrous chelated peptides can be prepared.

3. The use according to claim 2, characterized in that, comprises the following steps: A. Raw material pretreatment: Take scallop skirts, remove impurities, dry, pulverize, and sieve to obtain dry powder; B. Preparation of sterilized raw material liquid: Mix the dry powder obtained in step A with a certain amount of water to obtain a mixed solution, and sterilize; C. Preparation of fermented raw material hydrolyzate: After the sterilized raw material liquid obtained in step B is cooled, add the Bacillus subtilis M17 b7 bacterial suspension, and shake and culture the mixed solution; centrifuge, collect the supernatant, and freeze-dry the supernatant to obtain fermented scallop skirt hydrolyzate; D. Preparation of ferrous chelate: Re-dissolve the fermented scallop skirt hydrolyzate obtained in step C into a sample solution with a certain concentration, and add ferrous sulfate for chelation reaction; E. Separation of ferrous chelate: Add ethanol to the ferrous chelate obtained in step D, and dry the precipitate to obtain ferrous chelated peptide powder.

4. The use according to claim 3, characterized in that, In step A, the drying conditions are drying at 40 - 60 °C for 4 - 6 hours; the sieve mesh size for sieving is 50 - 500 meshes.

5. The use according to claim 3, characterized in that, In step B, the mass - volume ratio of dry powder to water is (1:10) - (1:50).

6. The use according to claim 3, characterized in that, In step C, the final concentration of the Bacillus subtilis is 10 5 -10 9 CFU / mL.

7. The use according to claim 3, characterized in that, In step C, the culture temperature is 20 - 45 °C; the rotation speed is 100 - 300 rpm / min; the culture time is 12 - 72 h.

8. The use according to claim 3, characterized in that, In step D, the re - dissolution concentration of the fermented scallop skirt hydrolyzate is 1 mg / mL, the concentration of added ferrous sulfate is 15 - 25 mM, the mass ratio of hydrolyzate to ferrous sulfate is 20:1; the reaction time is 15 - 25 min.

9. The use according to claim 3, characterized in that, In step E, the volume ratio of added ethanol to ferrous chelate is 5:

1.

10. The use according to claim 3, characterized in that, In step E, the drying method is spray drying or freeze drying.

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