Method for preparing polypeptides in a salty taste enhancing peptide complex composition

Abstract

The application provides a preparation method of polypeptides in a salty taste enhancing peptide compound composition, comprising the following steps: taking at least two polypeptides as target polypeptides, artificially synthesizing DNA sequences coding the target polypeptides, wherein the DNA sequences of the at least two polypeptides have a predetermined ratio; optimizing according to the codon bias of industrial microorganisms, cloning the optimized sequences into an expression vector to obtain a recombinant plasmid; using an engineering strain as a host, introducing the recombinant plasmid into host cells to obtain a recombinant strain; after expression of the recombinant strain, processing the product after fermentation to obtain a crude solution; and performing protease treatment and purification treatment on the crude solution to obtain a target polypeptide product with a predetermined ratio. According to the preparation method, the hidden danger of heavy metal residues is eliminated from the source, the polypeptide product meets the relevant international food additive specification requirements, and the method of enzymatic treatment after expression realizes high-yield expression of very short polypeptides, and the production efficiency is greatly improved.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and more specifically, to a method for preparing polypeptides in a salty-enhancing peptide compound composition. Background Technology

[0003] In recent years, naturally derived salt-enhancing peptides have become a research hotspot in salt reduction strategies due to their high safety, natural flavor, and well-defined mechanisms of action. To date, several short peptides with salt- or umami-enhancing activities have been reported. However, although these peptides exhibit certain salt- or umami-enhancing abilities in simplified systems, most existing peptide studies rely on His-tagged prokaryotic or eukaryotic expression systems, requiring purification using metal affinity chromatography such as nickel columns. This poses a risk of heavy metal residues and makes it difficult to meet food-grade production requirements. Summary of the Invention

[0004] One objective of this application is to provide a method for preparing peptides in a salty-enhanced peptide compound composition, which can at least solve the problem of heavy metal residue risk in peptide expression in the prior art.

[0005] To achieve the above objectives, this application provides the following technical solutions.

[0006] According to the embodiments of this application, a method for preparing polypeptides in a salty-enhancing peptide compound composition, wherein the polypeptides are polypeptides that can be cleaved by trypsin and chymotrypsin, the preparation method includes the following steps: using at least two polypeptides as target polypeptides, artificially synthesizing DNA sequences encoding the target polypeptides, wherein the DNA sequences of at least two polypeptides have a predetermined ratio; optimizing according to the codon preference of industrial microorganisms, cloning the optimized sequence into an expression vector to obtain a recombinant plasmid; using an engineered strain as a host, introducing the recombinant plasmid into a host cell to obtain a recombinant strain; after the expression of the recombinant strain is completed, processing the fermented product to obtain a crude solution; subjecting the crude solution to protease treatment and purification to obtain a target polypeptide product with the predetermined ratio.

[0007] According to one embodiment of this application, the industrial microorganism is Pichia pastoris.

[0008] According to one embodiment of this application, the expression vector is the ACC signal peptide of pHKA, wherein the ACC signal peptide is a mutated α-factor signal peptide.

[0009] According to one embodiment of this application, the expression vector uses pHKA as a backbone vector and includes the following elements: an AOX1 promoter, an ACC signal peptide coding sequence, a target polypeptide coding sequence, an AOX1 terminator, and a selection marker gene.

[0010] According to one embodiment of this application, the expression vector includes a monocistronic expression unit and a polycistronic expression unit. When the expression vector is a monocistronic expression unit, each expression vector carries only the coding sequence of one target polypeptide. When the expression vector is a polycistronic expression unit, two or more coding sequences of the target polypeptide are inserted tandemly into the same expression vector.

[0011] According to one embodiment of this application, the engineered strain is selected from any one of CBS7435 (NRRL Y-11430), CBS704 (NRRL Y-1603), GS115, MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169.

[0012] According to one embodiment of this application, the purification process of the crude product solution includes: adjusting the pH of the crude product solution with acid or alkali, removing the precipitate by centrifugation or microfiltration to obtain a clear solution; performing chromatography, equilibrating the column bed with a suitable pH-buffer system, eluting with a linear gradient of 0-2M salt solution, and collecting the target elution peak; and desalting the target component by passing it through a nanofiltration membrane with a molecular weight cutoff of less than 5000 Da to remove residual salts and small molecule impurities.

[0013] According to one embodiment of this application, the preparation method further includes: determining the purity of the target polypeptide product to be 0-99% by reversed-phase high-performance liquid chromatography (RP-HPLC), and confirming the purity by liquid chromatography-mass spectrometry (LC-MS) [M+H]. + The deviation from the theoretical molecular weight is ≤±100 Da, and the product has no external labels, no endotoxins, and meets the food safety standards for edible food or pet food.

[0014] According to one embodiment of this application, the target polypeptide product is a tandem polypeptide substrate, and the protease treatment of the crude product solution includes the following steps: dissolving the tandem polypeptide substrate in a buffer solution to obtain a substrate solution; adding an enzyme solution to the substrate solution for mixing to complete the enzymatic digestion reaction and obtain the target polypeptide product having the predetermined ratio.

[0015] According to one embodiment of this application, the mass ratio of the enzyme solution to the substrate solution is 1:10 to 1:1000. The mixed reaction system is placed at 37°C and magnetically stirred at 50 to 100 rpm for 2 to 24 hours. During this period, samples are taken every 2 to 4 hours, and the cleavage progress is detected by HPLC or TLC until the tandem polypeptide substrate peak disappears. After the tandem polypeptide substrate peak disappears, trifluoroacetic acid or HCl is added to the reaction system to make the final concentration of trifluoroacetic acid 0.1% to 1% or the pH of the reaction system is adjusted to 2.0 to 3.0 to complete the enzymatic cleavage reaction.

[0016] The method for preparing peptides in the salty-enhancing peptide compound composition according to the embodiments of this application adopts tag-free gene design throughout the process, eliminating the risk of heavy metal residues from the source. The resulting peptide products meet the requirements of GB 16740 "National Food Safety Standard for the Use of Food Fortifiers" and related international food additive regulations. Furthermore, when designing the DNA sequence of the target peptide, the predetermined proportion of the target peptide product is designed. The predetermined proportion of peptide expression is achieved through tandem expression of the peptide. Moreover, the enzymatic hydrolysis method after expression enables high-yield expression of very short peptides, which greatly improves production efficiency.

[0017] Other features and advantages of this application will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0018] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the present application and, together with their description, serve to explain the principles of the present application.

[0019] Figure 1 This is a flowchart illustrating the preparation method of the polypeptide in the salty-enhanced peptide compound composition according to the embodiments of this application; Figure 2 This is a flowchart of the peptide purification process in the preparation method of the peptide in the salty-enhanced peptide compound composition according to the embodiments of this application; Figures 3-1 to 3-8 This is a schematic diagram of the structure of each recombinant expression plasmid of the polypeptide in the preparation method of the savory-enhancing peptide compound composition according to the embodiments of this application, wherein, Figure 3-1 This is a schematic diagram of the structure of the PHKA-SH428 recombinant expression plasmid. Figure 3-2 This is a schematic diagram of the structure of the PHKA-SH156 recombinant expression plasmid. Figure 3-3 This is a schematic diagram of the structure of the PHKA-SH219 recombinant expression plasmid. Figure 3-4 This is a schematic diagram of the structure of the PHKA-SH305 recombinant expression plasmid. Figure 3-5This is a schematic diagram of the structure of the PHKA-SH791 recombinant expression plasmid. Figure 3-6 This is a schematic diagram of the structure of the PHKA-SH864 recombinant expression plasmid. Figure 3-7 This is a schematic diagram of the structure of the PHKA-SH573 recombinant expression plasmid. Figure 3-8 This is a schematic diagram of the structure of the PHKA-SH428-SH864 recombinant expression plasmid; Figures 4-1 to 4-7 The reversed-phase high-performance liquid chromatograms of each polypeptide in the preparation method of the savory-enhancing peptide compound composition according to the embodiments of this application are shown, with a detection wavelength of 220 nm. Figure 4-1 The image shows the reversed-phase high-performance liquid chromatogram of the SH428 peptide. Figure 4-2 The image shows the reversed-phase high-performance liquid chromatogram of the SH219 peptide. Figure 4-3 This is the reversed-phase high-performance liquid chromatogram of the SH305 peptide. Figure 4-4 This is the reversed-phase high-performance liquid chromatogram of the SH791 peptide. Figure 4-5 The image shows the reversed-phase high-performance liquid chromatogram of the SH864 peptide. Figure 4-6 The image shows the reversed-phase high-performance liquid chromatogram of the SH156 peptide. Figure 4-7 This is the reversed-phase high-performance liquid chromatogram of the SH573 peptide. Detailed Implementation

[0020] The embodiments of this application will now be described in detail. Examples of these embodiments are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0021] The terms "first" and "second" in the specification and claims of this application may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise stated, "multiple" means two or more. Furthermore, "and / or" in the specification and claims indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0022] The following is a detailed description of the salty-enhanced peptide compound composition of the present application.

[0023] According to the embodiments of this application, the salty flavor-enhancing peptide compound composition contains a plurality of polypeptides that can be cleaved by trypsin and chymotrypsin. The C-terminus (carboxyl terminus) of each polypeptide contains a specific amino acid that is specifically recognized by the corresponding enzyme. Specifically, trypsin can specifically recognize arginine (Arg, R) or lysine (Lys, K) in the polypeptide chain, and chymotrypsin can specifically recognize aromatic amino acids in the polypeptide chain, such as phenylalanine Phe / F, tyrosine Tyr / Y, tryptophan Trp / W, etc.

[0024] In this application, the salty-enhancing peptide compound composition is described as containing at least two of the seven peptides with the amino acid sequences DPSPR, DWTDDVEAR, STELFK, MMR, AHSVRFY, YDPNDPEK, and VNVDEVGGEALGR.

[0025] It should be noted that in this application, seven polypeptides with amino acid sequences DPSPR, DWTDDVEAR, STELFK, MMR, AHSVRFY, YDPNDPEK, and VNVDEVGGEALGR are named separately. Unless otherwise specified, the names of each polypeptide in this application correspond one-to-one as follows: SH428 (amino acid sequence DPSPR), SH156 (amino acid sequence DWTDDVEAR), SH219 (amino acid sequence STELFK), SH305 (amino acid sequence MMR), SH791 (amino acid sequence AHSVRFY), SH864 (amino acid sequence YDPNDPEK), and SH573 (amino acid sequence VNVDEVGGEALGR).

[0026] The seven polypeptides are derived from peanut protein, SH156 from zein, SH219 and SH305 from tilapia jaw protein, SH791 from clam protein, SH864 from button mushroom protein, and SH573 from Xuanwei ham protein.

[0027] The following is a brief description of the existing research on the above seven polypeptides.

[0028] SH428 (DPSPR): A pentapeptide derived from peanut protein hydrolysate, first disclosed in the study by Peng Wang et al.: "Identification of a novel salt-enhancing peptide from peanut protein: Salt-enhancing effect and antioxidant properties", Food & Function, 2025, DOI:10.1039 / d4fo05274a. This study confirmed that DPSPR can enhance the saltiness of aqueous solutions in low-salt environments, but this was only a preliminary validation of a single peptide in a simplified model.

[0029] SH156 (DWTDDVEAR): A nonapeptide derived from the hydrolysate of corn alcoholase. It was reported in a study by Zhang et al. published in the Journal of Agricultural and Food Chemistry (2023, DOI: 10.1021 / acs.jafc.3c05395) and was identified as having significant saltiness-enhancing activity, but its application effect in complex food systems has not been verified.

[0030] SH219 (STELFK) and SH305 (MMR) are derived from the lower jaw proteins of tilapia (Oreochromis niloticus), and were first identified by Liu et al. in "Novel umami peptides from tilapia lower jaw and molecular docking to the taste receptor T1R1 / T1R3" (J. Agric. Food Chem., 2023, DOI:10.1021 / acs.jafc.2c08687). While both primarily contribute to umami flavor, they also exhibit the ability to enhance saltiness perception in low-salt environments; however, the study has not evaluated their actual salt-reducing efficacy in condiments.

[0031] SH791 (AHSVRFY): A heptapeptide derived from the protein hydrolysate of the hard clam (Ruditapes philippinarum), reported by Wu et al. in "The enhancement and mechanism of the perception of saltiness byumami peptide from Ruditapes philippinarum and ham" (Food Chemistry, 2023, DOI: 10.1016 / j.foodchem.2023.135789). This peptide can indirectly enhance the perception of saltiness by activating taste pathways; however, the experiment was conducted only in a buffer system, and its stability and flavor compatibility in a real soy sauce matrix were not investigated.

[0032] SH864 / SH512 (YDPNDPEK): An octapeptide derived from the enzymatic hydrolysate of Agaricus bisporus, first discovered by Wang et al. in "Discovery of salt-enhancing peptides from enzymatichydrolysate of Agaricus bisporus protein and evaluation of their salt-reducing performance" (Food Research International, 2023, DOI: 10.1016 / j.foodres.2023.113917). This peptide has been shown to enhance saltiness in a low-sodium soy sauce model, but the study focused only on the effect of a single peptide and did not explore its potential for combination with other salty peptides.

[0033] SH573: A salty taste-enhancing peptide derived from the enzymatic hydrolysate of Xuanwei ham, reported in the study "Identification, flavor characteristics and molecular docking of umami taste peptides of Xuanwei ham" (Food Research International, 2023, DOI: 10.1016 / j.foodres.2023.113211). It was identified as having significant salty taste-enhancing activity, but its application effect in complex food systems has not been verified.

[0034] The sequences, sources, and original literature of each polypeptide are shown in Table 1 below.

[0035] Table 1. Sequences and sources of each polypeptide and original literature. It is evident that, although the above seven polypeptides have all been reported in relevant literature, each polypeptide still has the technical problems pointed out in the background of this application.

[0036] To address the aforementioned technological gap, this application proposes for the first time a method for preparing polypeptides in a salty-enhancing peptide compound composition.

[0037] According to the method for preparing the peptides in the salty-enhancing peptide compound composition according to the embodiments of this application, the salty-enhancing peptide compound composition contains at least two of the following seven peptides with the amino acid sequences DPSPR, DWTDDVEAR, STELFK, MMR, AHSVRFY, YDPNDPEK, and VNVDEVGGEALGR: Figure 1 As shown, the preparation method includes the following steps: Gene design and vector construction: Using at least two peptides as target peptides, DNA sequences encoding the target peptides are artificially synthesized, wherein the DNA sequences of the at least two peptides have a predetermined ratio, and are optimized according to the codon preference of industrial microorganisms, such as Pichia pastoris, without introducing any affinity tag sequences (such as His-tag, GST, FLAG, etc.) to ensure that the final product is free of foreign protein residues. The optimized sequences are cloned into expression vectors to obtain recombinant plasmids. Host strain and transformation: Using an engineered strain, such as Pichia pastoris engineered strain, as the host, the recombinant plasmid was introduced into the host cell to obtain the recombinant strain; Induced expression: The recombinant strain was inoculated into a culture medium for expression, causing the target polypeptide to be secreted into the culture supernatant; Fermentation broth pretreatment: After expression, the fermented product is processed, for example, the fermentation broth is centrifuged to remove the cells, the supernatant is taken, and filtered to obtain a clear and sterile crude solution; Green purification process: The crude product solution is subjected to protease treatment and purification to obtain the target polypeptide product with the predetermined ratio.

[0038] Therefore, the preparation method of the peptides in the salty-enhancing peptide compound composition according to the embodiments of this application adopts tag-free gene design throughout the process, eliminating the risk of heavy metal residues from the source. The obtained peptide products meet the requirements of GB16740 "National Food Safety Standard for the Use of Food Fortifiers" and relevant international food additive regulations. Furthermore, when designing the DNA sequence of the target peptide, the predetermined proportion of the target peptide product is designed. Through tandem expression of the peptides, the predetermined proportion of peptide expression is achieved. Additionally, the enzymatic digestion method after expression enables high-yield expression of very short peptides, significantly improving production efficiency.

[0039] According to one embodiment of this application, the expression vector is the ACC signal peptide of pHKA, wherein the ACC signal peptide is a mutated α-factor signal peptide.

[0040] Optionally, the expression vector uses pHKA as its backbone vector and includes the following elements: an AOX1 promoter, an ACC signal peptide coding sequence, a target polypeptide coding sequence, an AOX1 terminator, and a selection marker gene.

[0041] Therefore, after transforming the Pichia pastoris host strain with the expression vector, the target polypeptide can be efficiently secreted and expressed, and the expression product can be purified by cation exchange chromatography without affinity tagging, which meets the requirements of food-grade production.

[0042] In some specific embodiments of this application, the expression vector includes a monocistronic expression unit and a polycistronic expression unit. When the expression vector is a monocistronic expression unit, each expression vector carries only the coding sequence of one target polypeptide. When the expression vector is a polycistronic expression unit, two or more coding sequences of the target polypeptide are inserted in series on the same expression vector.

[0043] In other words, the expression vector can be selected according to application requirements to adopt a single peptide expression or peptide co-expression strategy: in single peptide expression, each vector carries only the coding sequence of one target peptide, which is suitable for scenarios where a single peptide needs to be prepared separately and then compounded; in peptide co-expression, two or more peptide coding sequences are inserted in series in the same vector, which is suitable for scenarios where multiple target peptides need to be secreted simultaneously in a single fermentation process to directly obtain compound precursors, which can simplify the downstream compounding process and improve the batch consistency of products.

[0044] According to one embodiment of this application, the engineered strain is selected from any one of CBS7435 (NRRL Y-11430), CBS704 (NRRL Y-1603), GS115, MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169.

[0045] The above-mentioned strains can be obtained by referring to published patents, such as: Chinese invention patent application No. 202411671629.9 (publication No. CN119552845A); Chinese invention patent application No. 202411877517.9 (publication No. CN119662696A), etc.

[0046] According to one embodiment of this application, the recombinant plasmid is introduced into a host cell via electroporation to obtain the recombinant bacterial strain. This method can yield high-yield recombinant bacterial strains.

[0047] Optionally, the culture medium is BMMY medium or inorganic basal medium, cultured at 28℃ and 200 rpm, with 0.5%~2% (v / v) methanol as the inducer, to induce expression for 96 hours, causing the target polypeptide to be secreted into the culture supernatant. After expression, the fermentation broth is centrifuged at 8000×g for 10 minutes, the supernatant is collected, and filtered through a 0.22μm microporous membrane to obtain a clear and sterile crude solution.

[0048] In some specific embodiments of this application, such as Figure 2 As shown, the purification steps for the crude solution include: The crude solution is adjusted to pH 3.0 with acid or alkali, and the precipitate is removed by centrifugation or microfiltration to obtain a clear solution; Ion exchange chromatography (SP Sepharose Fast Flow packing material) was used to process the column bed with a suitable pH buffer system, such as a pH 3.0 buffer system, and linear gradient elution was performed using 0-2M salt solution to collect the target elution peak. The target component is desalted by passing it through a nanofiltration membrane with a molecular weight cutoff of less than 5000 Da at an operating pressure of 0.2~0.4 MPa to remove residual salts and small molecule impurities. The resulting desalted solution was freeze-dried in a freeze dryer to obtain a white powdery high-purity polypeptide product.

[0049] The resulting product has a purity of ≥95% and is suitable for salt reduction and salt enhancement applications in food systems such as low-salt soy sauce.

[0050] According to one embodiment of this application, the preparation method further includes: determining the purity of the target polypeptide product to be 0-99% by reversed-phase high-performance liquid chromatography (RP-HPLC), and confirming the purity by liquid chromatography-mass spectrometry (LC-MS) [M+H]. + The deviation from the theoretical molecular weight is ≤±100 Da, and the product is free of heavy metal residues, external labels, and endotoxins, meeting the food safety standards for edible or pet food. The reversed-phase high-performance liquid chromatography (RP-HPLC) detection parameters are as follows: a C4 column is used, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is 0.1% trifluoroacetic acid + 70% acetonitrile aqueous solution, gradient elution program is 0~0.1 min holding 80%A, 20%B, 0.1~7.5 min linearly decreasing to 50%A, 50%B, 7.5~7.6 min rising back to 80%A, 20%B, 7.6~15 min holding 80%A, 20%B, detection wavelength is 220 nm, column temperature is 50℃, injection volume is 5 μL, and flow rate is 1 mL / min.

[0051] By conducting quality control on the prepared product, it can be found that there are no heavy metal residues, no exogenous labels, and no endotoxins in the product. It fully meets the food-grade application standards and can be directly used in soy sauce and other condiments to achieve safe and efficient salt reduction and salt enhancement functions.

[0052] Considering the yield issue, according to the preparation method of this application, when designing the target polypeptide, multiple polypeptides can be used as the target polypeptide. That is, when designing the DNA sequence, a long-chain polypeptide can be designed, and after obtaining the target polypeptide product, it can be digested with enzymes to obtain a short peptide, thus obtaining a single polypeptide product.

[0053] In some specific embodiments of the present invention, the target polypeptide product is a tandem polypeptide substrate, and the protease treatment of the crude product solution includes the following steps: dissolving the tandem polypeptide substrate in a buffer solution to obtain a substrate solution; adding an enzyme solution to the substrate solution for mixing to complete the enzymatic digestion reaction and obtain the target polypeptide product with the predetermined ratio.

[0054] The enzyme solution and the substrate solution are in a mass ratio of 1:10 to 1:1000. The mixed reaction system is placed at 37°C and magnetically stirred at 50 to 100 rpm for 2 to 24 hours. Samples are taken every 2 to 4 hours during the reaction, and the cleavage progress is detected by HPLC or TLC until the tandem polypeptide substrate peak disappears. After the tandem polypeptide substrate peak disappears, trifluoroacetic acid or HCl is added to the reaction system to make the final concentration of trifluoroacetic acid 0.1% to 1% or the pH of the reaction system is adjusted to 2.0 to 3.0 to complete the enzymatic cleavage reaction.

[0055] In other words, the protease treatment of the crude solution in this application includes the following steps: dissolving the tandem polypeptide substrate in a buffer solution to obtain a substrate solution with a concentration of 1-10 mg / mL; adding the enzyme solution to the substrate solution for mixing, wherein the mass ratio of the enzyme solution to the substrate solution is 1:10 to 1:1000; placing the mixed reaction system at 37°C and magnetically stirring at 50-100 rpm for 2-24 hours, taking samples every 2-4 hours during the reaction, and detecting the cleavage progress by HPLC or TLC until the tandem polypeptide substrate peak disappears; after detecting the disappearance of the tandem polypeptide substrate peak, adding trifluoroacetic acid or HCl to the reaction system to make the final concentration of trifluoroacetic acid 0.1%-1% or adjusting the pH of the reaction system to 2.0-3.0 to complete the enzymatic cleavage reaction and obtain a single polypeptide product.

[0056] In addition, the present invention also provides an enzymatic digestion method for depolymerization of tandem polypeptides, comprising the following steps: Preparation of the reaction system: The tandem polypeptide substrate is dissolved in a buffer solution to obtain a substrate solution with a concentration of 1-10 mg / mL; the buffer solution is selected according to the type of enzyme used. If chymotrypsin is used, the buffer solution is 50 mM ammonium acetate buffer or 50 mM sodium phosphate buffer with a pH of 6.0-8.0; if trypsin is used, the buffer solution is 50 mM Tris-HCl buffer with a pH of 7.5-8.5; Ca²⁺ is added to the substrate solution. + , making Ca² + The final concentration is 1~5 mM; Enzymatic digestion reaction: Add the enzyme solution to the substrate solution prepared in the above steps. The mass ratio of the enzyme solution to the substrate solution is 1:10 to 1:100. The chymotrypsin solution is a 1-10 mg / mL chymotrypsin solution prepared with 50 mM ammonium acetate buffer (pH 7.0), and the trypsin solution is a 1-10 mg / mL trypsin solution prepared with 50 mM Tris-HCl buffer (pH 8.0). Place the mixed reaction system at 37°C and magnetically stir at 50-100 rpm for 2-24 hours. During this period, take samples every 2-4 hours and detect the digestion progress by HPLC or TLC until the tandem peptide substrate peak disappears. Reaction termination: After the disappearance of the tandem polypeptide substrate peak is detected, add 10% trifluoroacetic acid (TFA) or 1 M HCl to the reaction system to make the final concentration of 10% trifluoroacetic acid 0.1%~1% or adjust the pH of the reaction system to 2.0~3.0 to complete the termination of the enzymatic digestion reaction; store the terminated reaction system at 4℃.

[0057] Therefore, by designing long-chain polypeptides when designing DNA sequences, and then performing enzymatic digestion to obtain individual polypeptide products after obtaining the target polypeptide product, production efficiency and product yield can be significantly improved.

[0058] In other words, the salty-enhancing peptides described in this application (including SH428 (DPSPR), SH156 (DWTDDVEAR), SH219 (STELFK), SH305 (MMR), SH791 (AHSVRFY), SH864 (YDPNDPEK), and SH573 (VNVDEVGGEALGR)) are all prepared using a uniform, label-free, food-compatible biomanufacturing process, specifically including the following steps: (1) Gene design and vector construction The DNA sequence encoding the target polypeptide is artificially synthesized and codons are optimized based on the host Pichia pastoris (Komagataella phaffii). No affinity tag sequences (such as His-tag, GST, FLAG, etc.) are introduced to ensure that the final product is free of foreign protein residues. The optimized sequence was cloned downstream of the ACC signal peptide in the expression vector pHKA (where ACC is the mutated α-factor signal peptide) to construct a secretory expression unit. Schematic diagrams of the recombinant expression plasmids for each target polypeptide are shown below. Figures 3-1 to 3-8 As shown.

[0059] Depending on the actual needs, any of the following expression strategies can be selected: Single peptide expression: Each vector carries the coding sequence of only one target polypeptide; Co-expression of peptides: Two or more coding sequences of different salty peptides are inserted in series into the same vector. Each sequence is linked to an independent ACC signal peptide, or polycistronic expression is achieved through self-cleaving elements (such as F2A and P2A peptides), thereby simultaneously secreting multiple target peptides during a single fermentation process, which facilitates subsequent compounding.

[0060] (2) Host strain and transformation The host used is *Pichia pastoris* (Komagataella phaffii), preferably including but not limited to the following engineered strains: GS115, MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256 and MF001-169.

[0061] The above-mentioned strains can be obtained by referring to published patents, for example: Chinese invention patent application number 202411671629.9 (publication number CN119552845A); Chinese invention patent application number 202411877517.9 (publication number CN119662696A).

[0062] Recombinant plasmids were introduced into host cells using electroporation to obtain high-yield recombinant strains.

[0063] (3) Induced expression The recombinant strain was inoculated into BMMY medium or inorganic basal medium and cultured at 28°C and 200 rpm. Expression was induced for 96 hours using 0.5%–2% (v / v) methanol as an inducer, resulting in efficient secretion of the target peptide into the culture supernatant.

[0064] (4) Pretreatment of fermentation broth After expression, the fermentation broth was centrifuged at 8000 × g for 10 minutes, and the supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain a clear and sterile crude solution.

[0065] (5) Green purification process This application employs a metal affinity chromatography-free purification route, the specific steps of which are as follows: The pH of the crude solution was adjusted to 3.0 with acid, and a clear solution was obtained by centrifugation or microfiltration. Strong cation exchange chromatography (SP Sepharose Fast Flow packing material) was used, with the column bed equilibrated in a pH 3.0 buffer system, and elution was performed using a linear gradient of 0–500 mM NaCl to collect the target elution peaks. The target component is desalinated by passing it through a nanofiltration membrane with a molecular weight cutoff of 500 Da, which effectively removes residual salts and small molecule impurities. The resulting desalting solution was freeze-dried to obtain a white powdery high-purity polypeptide product.

[0066] (6) Product quality control The obtained polypeptide product was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and its purity was ≥95%; the molecular ion peak [M+H] was confirmed by liquid chromatography-mass spectrometry (LC-MS). + Consistent with theoretical values ​​(e.g., SH156 is 1106.0, SH428 is 567.6).

[0067] The product contains no heavy metal residues, no exogenous labels, and no endotoxins. It fully complies with food-grade application standards and can be directly used in condiments such as soy sauce to achieve safe and efficient salt reduction and salt enhancement functions.

[0068] To ensure the accuracy of the identity, sequence integrity, and high purity of each salty-enhancing peptide in the compound composition prepared in this application, all peptide products were subjected to strict quality control using standardized liquid chromatography-mass spectrometry (LC-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC) methods.

[0069] (1) LC-MS molecular identification Analysis was performed using a high-resolution Q-TOF mass spectrometer equipped with an electrospray ionization (ESI) source in Data Dependent Acquisition (DDA) mode. Specific parameters are as follows: Mass spectrometry (MS¹) scanning range: 350–1500 m / z; Resolution: 60,000 (at m / z 200); Maximum ion implantation time: 118 ms; DDA cycle time: 2 seconds; Secondary mass spectrometry (MS²) employs high-energy collision-induced dissociation (HCD), with the normalized collision energy set to 30%. Maximum injection time for level 2: 22 ms; Dynamic exclusion time: 35 s.

[0070] Under the above conditions, all seven salty peptides covered in this application were tested, and the results are shown in Table 2 below: Table 2. Results of peptide detection The measured mass-to-charge ratios of all peptides and their theoretical [M+H] ratios. + The value deviation is less than ±0.1 Da, which fully meets the theoretical expectation, confirming that the target sequence is correctly expressed and there is no interference from oxidation, deamidation or other side reaction products.

[0071] (2) HPLC purity analysis a) HPLC instrument model: Thermo Fisher Scientific Vanquish Core or Agilent 1260 standalone liquid chromatograph b) Column: C4 250×4.6 mm, 5µm c) Mobile phases: Mobile phase A: 0.1% trifluoroacetic acid; Mobile phase B: 0.1% trifluoroacetic acid + 70% acetonitrile d) Detection wavelength: UV 220 nm e) Column temperature: 50℃ f) Injection volume: 5 μL g) Flow rate: 1 ml / min The gradient elution procedure parameters are shown in Table 3.

[0072] Table 3 Gradient elution program parameters SH428 (DPSPR), SH156 (DWTDDVEAR), SH219 (STELFK), SH305 (MMR), SH791 (AHSVRFY), SH864 (YDPNDPEK) and SH573 (VNVDEVGGEALGR) peptides, ≥95% by HPLC area normalization method.

[0073] The mass spectrometry identification results of each polypeptide are shown in Table 4 and Figures 4-1 to 4-7 As shown.

[0074] Table 4. Mass spectrometry identification results of each polypeptide This invention employs LC-MS / MS technology to analyze purified peptide samples and uses MaxQuant software (v2.0+) for peptide identification and quantification. Mass spectrometry results show that all seven salty-enhancing peptides covered in this invention—SH428 (DPSPR), SH156 (DWTDDVEAR), SH219 (STELFK), SH305 (MMR), SH791 (AHSVRFY), SH864 (YDPNDPEK), and SH573 (VNVDEVGGEALGR)—were successfully detected, with 100% sequence coverage.

[0075] The theoretical molecular weight of SH428 is 566.55 Da, and the measured [M+H] molecular weight is... + The molecular weight is 571.25, and the emPAI value is 128.6; the theoretical molecular weight of SH156 is 1105.00 Da, and the measured [M+H] value is [missing value]. + The emPAI value was 1106.35, with a high emPAI of 185.3. The emPAI values ​​of the remaining peptides were also in the high range of 98.7–136.8. These high emPAI values ​​clearly indicate that all target peptides existed in the sample in a high-abundance, high-purity form, without significant degradation or side reactions. This further verifies their effectiveness and reliability as active components for enhancing saltiness, providing a high-quality raw material guarantee for subsequent compound applications.

[0076] In summary, compared with the prior art, the polypeptide expression and purification process provided by this application has the following significant advantages in the preparation method of the salty-enhanced peptide compound composition according to the embodiments of this application: (1) Supports flexible expression modes: By constructing monocistronic or polycistronic expression units in the pHKA vector, the efficient production of a single salty peptide can be selectively achieved, or two or more target peptides (such as SH428 and SH156, SH219 and SH791, etc.) can be secreted simultaneously in the same fermentation batch, directly obtaining the compound precursor, significantly simplifying the downstream ratio control process, and improving product consistency and process robustness. (2) Completely avoid food safety risks: The entire process adopts tag-free gene design, combined with a purification route that does not use metal chelating media (i.e., does not use Ni²). + Co² + (e.g., IMAC chromatography) eliminates the risk of heavy metal residues at the source, and the resulting peptide products meet the requirements of GB 16740 "National Food Safety Standard for the Use of Food Fortifiers" and relevant international food additive regulations. (3) The process is green and scalable: The combination of strong cation exchange chromatography (SP Sepharose Fast Flow) and 500 Da nanofiltration desalting has mild operating conditions (pH 3.0, room temperature) and a simple solvent system (containing only NaCl and aqueous buffer). It does not require organic solvents or toxic reagents, has high yield and controllable cost, and is easy to scale up to fermentation scale of hundreds of liters or even tons. It is suitable for industrial salt reduction applications in bulk condiments such as soy sauce.

[0077] In summary, the method for preparing peptides in the salty-enhancing peptide compound composition according to the embodiments of this application not only solves the technical bottleneck of the difficulty in mass-producing natural salty peptides for food, but also provides a reliable technical platform for building a safe, efficient, and sustainable next-generation salt reduction solution.

[0078] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments without departing from the scope and spirit of this application. The scope of this application is defined by the appended claims.

Claims

1. A method for preparing polypeptides in a salty-enhancing peptide compound composition, characterized in that, The polypeptide is a polypeptide that can be cleaved by trypsin and chymotrypsin, and the preparation method includes the following steps: Using at least two polypeptides as target polypeptides, a DNA sequence encoding the target polypeptide is artificially synthesized, wherein the DNA sequences of the at least two polypeptides have a predetermined ratio; The codon preferences of industrial microorganisms were optimized, and the optimized sequences were cloned into expression vectors to obtain recombinant plasmids. Using an engineered bacterial strain as a host, the recombinant plasmid was introduced into the host cell to obtain a recombinant bacterial strain; After the recombinant strain expression is completed, the fermented product is processed to obtain a crude solution; The crude solution is subjected to protease treatment and purification to obtain the target polypeptide product with the predetermined ratio.

2. The preparation method according to claim 1, characterized in that, The industrial microorganism is Pichia pastoris.

3. The preparation method according to claim 2, characterized in that, The expression vector is the ACC signal peptide of pHKA, wherein the ACC signal peptide is the mutated α-factor signal peptide.

4. The preparation method according to claim 3, characterized in that, The expression vector uses pHKA as its backbone and contains the following elements: an AOX1 promoter, an ACC signal peptide coding sequence, a target polypeptide coding sequence, an AOX1 terminator, and a selection marker gene.

5. The preparation method according to claim 4, characterized in that, The expression vector includes a monocistronic expression unit and a polycistronic expression unit. When the expression vector is a monocistronic expression unit, each expression vector carries only the coding sequence of the target polypeptide. When the expression vector is a polycistronic expression unit, the coding sequences of two or more target polypeptides are inserted tandemly into the same expression vector.

6. The preparation method according to claim 2, characterized in that, The engineered strain is selected from any one of CBS7435 (NRRLY-11430), CBS704 (NRRL Y-1603), GS115, MF001-29, MF001-94, MF001-136, MF001-143, MF001-249, MF001-251, MF001-256, and MF001-169.

7. The preparation method according to claim 1, characterized in that, The purification process for the crude solution includes: The pH of the crude solution is adjusted with acid or alkali, and the precipitate is removed by centrifugation or microfiltration to obtain a clear solution. Chromatographic processing was employed, the column bed was equilibrated with a suitable pH buffer system, and linear gradient elution was performed using 0-2M salt solution to collect the target elution peak; The target component is desalinated by passing it through a nanofiltration membrane with a molecular weight cutoff of less than 5000 Da to remove residual salts and small molecule impurities.

8. The preparation method according to claim 1, characterized in that, Also includes: The purity of the target peptide product was determined to be 0-99% by reversed-phase high-performance liquid chromatography (RP-HPLC) and confirmed by liquid chromatography-mass spectrometry (LC-MS) [M+H]. + The deviation from the theoretical molecular weight is ≤±100 Da, and the product has no external labels, no endotoxins, and meets the food safety standards for edible food or pet food.

9. The preparation method according to claim 1, characterized in that, The target polypeptide product is a tandem polypeptide substrate, and the protease treatment of the crude product solution includes the following steps: The tandem polypeptide substrate was dissolved in a buffer solution to obtain a substrate solution; The enzyme solution is added to the substrate solution and mixed to complete the enzymatic digestion reaction, thereby obtaining the target polypeptide product with the predetermined ratio.

10. The preparation method according to claim 9, characterized in that, The mass ratio of the enzyme solution to the substrate solution is 1:10 to 1:1000. The mixed reaction system is placed at 37°C and magnetically stirred at 50 to 100 rpm for 2 to 24 hours. Samples are taken every 2 to 4 hours during the reaction, and the cleavage progress is detected by HPLC or TLC until the tandem peptide substrate peak disappears. Once the tandem polypeptide substrate peak disappears, add trifluoroacetic acid or HCl to the reaction system to bring the final concentration of trifluoroacetic acid to 0.1%–1% or adjust the pH of the reaction system to 2.0–3.0 to complete the enzymatic digestion reaction.

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