Preparation method of micromolecule peptide for directional metabolism of saccharomycetes

Abstract

The preparation method comprises the following steps: (1) preparing a seed culture medium, and culturing at the temperature of 30 + / -1 DEG C until the logarithmic phase to obtain a seed solution; (2) adding the seed solution into a fermentation tank filled with a fermentation basal culture medium, and carrying out two-stage pH regulation and control fermentation; stage 1: under the conditions that the pH is 5.5 + / -0.1, the temperature is 30 + / -0.5 DEG C and the dissolved oxygen DO is 50 + / -5%, carrying out enrichment culture on the thalli until the OD600 is 15-20; stage 2, reducing the pH to 3.8 + / -0.1, reducing the temperature to 28 + / -0.5 DEG C, reducing dissolved oxygen (DO) to 20 + / -5%, and carrying out small molecule peptide synthesis, wherein the reaction time is 18-60 hours; 0.1-0.2 mM isopropyl-beta-D-thiogalactoside is added at the beginning of the stage 2 to continuously induce the expression of the exogenous gene, and the concentration of residual sugar is maintained at 2-5g / L in the synthesis process; and 3) separating and purifying the fermentation broth to obtain the peptide. According to the method, the metabolism of amino acid is enhanced, the degradation of peptide is blocked, the yield of small molecule peptide is increased, and the metabolism has antibacterial and antioxidant effects by virtue of precise fermentation control and an efficient purification process.

Description

Technical Field

[0001] The invention belongs to the technical field of feed additives and mainly relates to a method for preparing a yeast-directed metabolic small-molecule peptide. Background Art

[0002] In the field of feed additive technology, there are currently the following industry pain points: 1. Antibiotics cause drug-resistant bacteria, and natural alternatives to antibiotics (such as antimicrobial peptides) are urgently needed; 2. Existing feed additives (such as plant extracts) have defects such as low activity, high cost and poor stability; 3. The traditional fermentation method has low efficiency in producing small molecule peptides (<0.5 g / L) and cannot meet the industrial demand for feed.

[0003] In response to the above-mentioned industry pain points, the key issue is how to make innovative breakthroughs in technology regarding the effects of small peptides and the metabolites of probiotics.

[0004] Based on this, the present application was developed. The present invention enhances amino acid metabolism, blocks peptide degradation, increases small molecule peptide production, and uses precise fermentation control and efficient purification processes to make the metabolites have both antibacterial and antioxidant effects. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art by providing a method for preparing small-molecule peptides through the targeted metabolism of yeast. This method enhances amino acid metabolism, blocks peptide degradation, and increases small-molecule peptide yield. Furthermore, through precise fermentation control and efficient purification, the metabolites possess both antibacterial and antioxidant properties.

[0006] Another object of the present invention is to provide the effects of the product prepared by the above method in terms of physicochemical properties, biological activity and antibacterial activity.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A method for preparing a small molecule peptide through yeast-directed metabolism comprises the following steps: 1) Prepare seed culture medium in a dedicated seed tank, then culture with shaking at 30±1°C until the logarithmic growth phase to obtain seed solution; 2) The seed liquid is pumped through a sterile pipe into a fermentation tank filled with fermentation basal medium (the fermentation tank has been pre-sterilized) for a two-stage pH-controlled fermentation. The inoculum size is usually 5%-10% of the fermentation tank volume. Stage 1: Under the conditions of pH 5.5 ± 0.1, temperature 30 ± 0.5℃, dissolved oxygen DO 50 ± 5% (agitation rate 400 ± 50 rpm), the bacteria are cultured to an OD600 of 15-20 (0-18 hours); Stage 2: The pH is lowered to 3.8 ± 0.1 (controlled by automatic addition of 2M H2SO4), the temperature is lowered to 28 ± 0.5°C, and the dissolved oxygen (DO) is lowered to 20 ± 5% for small molecule peptide synthesis (stirring rate 200 ± 50 rpm), and the reaction time is 18-60 hours; at the beginning of stage 2, 0.1-0.2 mM inducer IPTG (isopropyl-β-D-thiogalactopyranoside) is added to continuously induce exogenous gene expression, and the residual sugar concentration is maintained at 2-5 g / L during the synthesis process; 3) The fermentation broth obtained in step 2) is centrifuged, ultrafiltered, and freeze-dried to obtain a white powdery small molecule peptide product.

[0008] Specifically, in step 1), the seed culture medium (YPD) is composed of 20-30 g / L glucose, 10-15 g / L yeast extract powder, and 20-30 g / L peptone. Culture conditions: 30±1°C, 200 rpm, and culture until the logarithmic growth phase (OD600≈8-10, approximately 12-16 hours).

[0009] Specifically, in step 2), the fermentation basal medium is composed of: Carbon source: glucose 50-60 g / L, Nitrogen source: ammonium sulfate 5-8 g / L, yeast extract 10-15 g / L, Trace elements: MgSO4 1-3g / L, KH2PO4 3-5g / L.

[0010] Furthermore, in step 2), when the residual sugar concentration drops to 2 g / L, the feed pump is started to add feed solution to maintain the residual sugar concentration at 2-5 g / L. The feed solution composition is: 200-220 g / L glucose, 5-8 g / L L-cysteine, and 10-15 g / L glycine (pH 5.0, filtered and sterilized). The dynamic feeding strategy is as follows: Feeding trigger condition: When the residual sugar concentration drops to 5 g / L, the feeding pump is started. Feed rate: 0.1-0.2 mL / min (adjusted by online glucose sensor feedback), Feeding goal: maintain the residual sugar concentration at 2-5 g / L to avoid excess carbon source inhibiting peptide synthesis.

[0011] Specifically, in step 3), the fermentation broth is pretreated by centrifuging at 8000×g for 10-20 minutes at 4±1° C. to remove the bacterial cells.

[0012] Furthermore, in step 3), ultrafiltration is performed in two steps: the first step uses an 8-10 kDa ultrafiltration membrane to remove large molecular proteins; the second step uses a 3-5 kDa ultrafiltration membrane to retain the target small molecular peptide. The freeze-drying in step 3) specifically involves prefreezing at -80±5°C for 8-15 hours, followed by freeze-drying at -40°C to -30°C for 12-36 hours, with a moisture content of ≤5%. This yields the product of the present invention (a white powdered small molecular peptide).

[0013] In the method of the present invention, the reaction process is monitored, specifically as follows: (1) Online detection: pH, DO and temperature are monitored in real time through the built-in probe in the fermenter. The residual sugar concentration was analyzed by HPLC (high performance liquid chromatography) sampling every hour; (2) Offline detection: Samples were taken every 6 hours to measure bacterial density (OD600), small molecule peptide concentration (BCA method) and metabolic byproducts (such as ethanol, GC detection).

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1) The present invention maximizes bacterial growth through two-stage pH control, inhibits impurity proteins and activates peptide synthase, greatly increasing the proportion of target product; 2) The present invention uses a dynamic feeding optimization strategy to stabilize the carbon-nitrogen ratio, avoid substrate inhibition and improve yield; 3) The small molecule peptide product of the present invention has a molecular weight of 800-1200 Da, a purity of ≥95%, a free radical scavenging rate of 92%, and strong antibacterial activity; 4) Through precise fermentation control and efficient purification technology, the method of the present invention achieves efficient, targeted, and large-scale production of small molecule peptides, with stable product quality and potential for industrial production. DETAILED DESCRIPTION

[0015] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0016] In the following examples, the raw materials used are all commercially available products that can be directly purchased, or can be prepared using conventional techniques in the art, such as yeast extract powder purchased from Angel Yeast Co., Ltd. (Model FM902).

[0017] Example 1 A method for preparing a small molecule peptide mediated by yeast metabolism The preparation method of the present invention is as follows: 1. Fermentation 1. Culture medium configuration (1) Seed culture medium (YPD): glucose 20 g / L, yeast extract powder 15 g / L, peptone 20 g / L; Culture conditions: 30°C, 200 rpm, culture until the logarithmic growth phase (OD600≈8-10, about 12-16 hours).

[0018] (2) Fermentation basal medium: Carbon source: glucose 60 g / L; Nitrogen source: ammonium sulfate 5 g / L, yeast extract powder 15 g / L; Trace elements: MgSO4 3g / L, KH2PO4 3g / L.

[0019] (3) Feed solution: glucose 200 g / L, L-cysteine 8 g / L, glycine 10 g / L (pH 5.0, filter sterilized).

[0020] Cultivation process: Prepare seed culture medium in a dedicated seed tank and incubate at 30°C until the logarithmic growth phase. Pump the seed solution through sterile piping into a pre-sterilized fermentation tank filled with basal medium for a two-stage pH-controlled fermentation. The inoculum volume should be 8% of the tank volume.

[0021] 2. Two-stage pH controlled fermentation Stage 1: Bacterial proliferation (12 hours) Parameters: pH 5.5 ± 0.1, temperature 30 ± 0.5°C, dissolved oxygen (DO) 50 ± 5%, stirring rate 400 rpm; Objective: Rapid accumulation of biomass (OD600 reaches 20).

[0022] Phase 2: Small molecule peptide synthesis (40 hours) Parameter switching: The pH dropped to 3.8 ± 0.1 (controlled by automatic feeding of 2 M H2SO4); The temperature dropped to 28 ± 0.5°C; DO was reduced to 20 ± 5%, stirring rate was 200 rpm; Inducer addition: At the beginning of stage 2, 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to continuously induce the expression of the exogenous gene; Dynamic feeding strategy Feeding trigger condition: When the residual sugar concentration drops to 5g / L, the feeding pump is started to add feeding liquid; Feed rate: 0.1 mL / min (regulated by online glucose sensor feedback); Feeding goal: maintain the residual sugar concentration at 2-5 g / L to avoid excess carbon source inhibiting peptide synthesis.

[0023] 3. Process monitoring (1) Online detection: pH, DO and temperature are monitored in real time through built-in probes in the fermenter.

[0024] The residual sugar concentration was analyzed by HPLC (high performance liquid chromatography) by sampling every hour.

[0025] (2) Offline detection: Samples were taken every 6 hours to measure bacterial density (OD600), small molecule peptide concentration (BCA method) and metabolic byproducts (such as ethanol, GC detection).

[0026] 2. Product separation and purification 1. Fermentation broth pretreatment Centrifugation: 4°C, 8000 × g for 15 minutes to remove bacteria; 2. Ultrafiltration: Step 1: 10 kDa ultrafiltration membrane to remove large molecular proteins; Step 2: 3 kDa ultrafiltration membrane to retain the target small molecule peptide; 3. Freeze-drying: pre-freeze at -80°C for 12 hours, freeze-dry at -30°C for 24 hours, with moisture ≤5%, to obtain the product of the present invention (white powdery small molecule peptide).

[0027] Example 2 A method for preparing a small molecule peptide mediated by yeast metabolism The preparation method of the present invention is as follows: 1. Fermentation 1. Culture medium configuration (1) Seed culture medium (YPD): glucose 30 g / L, yeast extract powder 10 g / L, peptone 30 g / L; Culture conditions: 30°C, 200 rpm, culture to the logarithmic growth phase (OD600≈8-10, about 12-16 hours).

[0028] (2) Fermentation basal medium: Carbon source: glucose 50 g / L; Nitrogen source: ammonium sulfate 8 g / L, yeast extract powder 10 g / L; Trace elements: MgSO4 1g / L, KH2PO4 5g / L.

[0029] (3) Feed solution: glucose 220 g / L, L-cysteine 5 g / L, glycine 15 g / L (pH 5.0, filter sterilized).

[0030] Cultivation process: Prepare seed culture medium in a dedicated seed tank and incubate at 30°C until the logarithmic growth phase. Pump the seed solution through sterile piping into a pre-sterilized fermentation tank filled with basal medium for a two-stage pH-controlled fermentation. The inoculum volume should be 8% of the tank volume.

[0031] 2. Two-stage pH controlled fermentation Phase 1: Bacterial proliferation (15 hours) Parameters: pH 5.5 ± 0.1, temperature 30 ± 0.5°C, dissolved oxygen (DO) 50 ± 5%, stirring rate 400 rpm; Objective: Rapid accumulation of biomass (OD600 reaches 18); Phase 2: Small molecule peptide synthesis (45 hours) Parameter switching: The pH dropped to 3.8 ± 0.1 (controlled by automatic feeding of 2 M H2SO4); The temperature dropped to 28 ± 0.5°C; DO was reduced to 20 ± 5%, stirring rate was 200 rpm; Inducer addition: At the beginning of stage 2, 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to continuously induce the expression of the exogenous gene; Dynamic feeding strategy Feeding trigger condition: When the residual sugar concentration drops to 5 g / L, the feeding pump is started to add feeding liquid; Feed rate: 0.1 mL / min (regulated by online glucose sensor feedback); Feeding goal: maintain the residual sugar concentration at 2-5 g / L to avoid excess carbon source inhibiting peptide synthesis.

[0032] 3. Process monitoring (1) Online detection: pH, DO and temperature are monitored in real time through built-in probes in the fermenter.

[0033] The residual sugar concentration was analyzed by HPLC (high performance liquid chromatography) by sampling every hour.

[0034] (2) Offline detection: Samples were taken every 6 hours to measure bacterial density (OD600), small molecule peptide concentration (BCA method) and metabolic byproducts (such as ethanol, GC detection).

[0035] 2. Product separation and purification 1. Fermentation broth pretreatment Centrifugation: 4°C, 8000 × g for 15 minutes to remove bacteria; 2. Ultrafiltration: Step 1: 10 kDa ultrafiltration membrane to remove large molecular proteins; Step 2: 3 kDa ultrafiltration membrane to retain the target small molecule peptide; 3. Freeze-drying: Pre-freeze at -80°C for 12 hours, freeze-dry at -35°C for 24 hours, and the moisture content is ≤5% to obtain the product of the present invention (white powdery small molecule peptide).

[0036] Example 3 A method for preparing a small molecule peptide mediated by yeast metabolism The preparation method of the present invention is as follows: 1. Fermentation 1. Culture medium configuration (1) Seed culture medium (YPD): glucose 25 g / L, yeast extract powder 13 g / L, peptone 25 g / L; Culture conditions: 30°C, 200 rpm, culture to the logarithmic growth phase (OD600≈8-10, about 12-16 hours).

[0037] (2) Fermentation basal medium: Carbon source: glucose 55 g / L; Nitrogen source: ammonium sulfate 7g / L, yeast extract powder 13g / L; Trace elements: MgSO4 2g / L, KH2PO4 4g / L.

[0038] (3) Feed solution: glucose 210 g / L, L-cysteine 7 g / L, glycine 12 g / L (pH 5.0, filter sterilized).

[0039] Cultivation process: Prepare seed culture medium in a dedicated seed tank and incubate at 30°C until the logarithmic growth phase. Pump the seed solution through sterile piping into a pre-sterilized fermentation tank filled with basal medium for a two-stage pH-controlled fermentation. The inoculum volume should be 8% of the tank volume.

[0040] 2. Two-stage pH controlled fermentation Phase 1: Bacterial proliferation (18 hours) Parameters: pH 5.5 ± 0.1, temperature 30 ± 0.5°C, dissolved oxygen (DO) 50 ± 5%, stirring rate 400 rpm; Objective: Rapid accumulation of biomass (OD600 reaches 20); Phase 2: Small molecule peptide synthesis (60 hours) Parameter switching: The pH dropped to 3.8 ± 0.1 (controlled by automatic feeding of 2 M H2SO4); The temperature dropped to 28 ± 0.5°C; DO was reduced to 20 ± 5%, stirring rate was 200 rpm; Inducer addition: At the beginning of stage 2, 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to continuously induce exogenous gene expression.

[0041] Dynamic feeding strategy Feeding trigger condition: When the residual sugar concentration drops to 5 g / L, the feeding pump is started to add feeding liquid; Feed rate: 0.1 mL / min (regulated by online glucose sensor feedback); Feeding goal: maintain the residual sugar concentration at 2-5 g / L to avoid excess carbon source inhibiting peptide synthesis.

[0042] 3. Process monitoring (1) Online detection: pH, DO and temperature are monitored in real time through built-in probes in the fermenter.

[0043] The residual sugar concentration was analyzed by HPLC (high performance liquid chromatography) by sampling every hour.

[0044] (2) Offline detection: Samples were taken every 6 hours to measure bacterial density (OD600), small molecule peptide concentration (BCA method) and metabolic byproducts (such as ethanol, GC detection).

[0045] 2. Product separation and purification 1. Fermentation broth pretreatment Centrifugation: 4°C, 8000 × g for 15 minutes to remove bacteria; 2. Ultrafiltration: Step 1: 10 kDa ultrafiltration membrane to remove large molecular proteins; Step 2: 3 kDa ultrafiltration membrane to retain the target small molecule peptide; 3. Freeze-drying: Pre-freeze at -80°C for 12 hours, freeze-dry at -40°C for 24 hours, and the moisture content is ≤5% to obtain the product of the present invention (white powdery small molecule peptide).

[0046] Comparative Example 1: A method for preparing a small molecule peptide mediated by yeast The preparation method of the present invention is as follows: 1. Fermentation 1. Culture medium configuration (1) Seed culture medium (YPD): glucose 25 g / L, yeast extract powder 13 g / L, peptone 25 g / L; Culture conditions: 30°C, 200 rpm, culture to the logarithmic growth phase (OD600≈8-10, about 12-16 hours).

[0047] (2) Fermentation basal medium: Carbon source: glucose 55 g / L; Nitrogen source: ammonium sulfate 7g / L, yeast extract powder 13g / L; Trace elements: MgSO4 2g / L, KH2PO4 4g / L.

[0048] (3) Feed solution: glucose 210 g / L, L-cysteine 7 g / L, glycine 12 g / L (pH 5.0, filter sterilized).

[0049] Cultivation process: Prepare seed culture medium in a dedicated seed tank and incubate at 30°C until the logarithmic growth phase. Pump the seed solution through sterile piping into a pre-sterilized fermentation tank filled with basal culture medium for fermentation. The inoculum volume should be 8% of the tank volume.

[0050] 2. Fermentation process pH 5.5 ± 0.1, temperature 30 ± 0.5°C, dissolved oxygen (DO) 50 ± 5%, agitation 400 rpm. Fermentation for 48 hours.

[0051] 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added at the beginning of the fermentation; Dynamic feeding strategy Feeding trigger condition: When the residual sugar concentration drops to 5g / L, the feeding pump is started to add feeding liquid.

[0052] Feed rate: 0.1 mL / min (regulated by online glucose sensor feedback); Feeding goal: maintain the residual sugar concentration at 2-5 g / L to avoid excess carbon source inhibiting peptide synthesis.

[0053] 3. Process monitoring (1) Online detection: pH, DO and temperature are monitored in real time through built-in probes in the fermenter.

[0054] The residual sugar concentration was analyzed by HPLC (high performance liquid chromatography) by sampling every hour.

[0055] (2) Offline detection: Samples were taken every 6 hours to measure bacterial density (OD600), small molecule peptide concentration (BCA method) and metabolic byproducts (such as ethanol, GC detection).

[0056] 2. Product separation and purification 1. Fermentation broth pretreatment Centrifugation: 4°C, 8000 × g for 15 minutes to remove bacteria; 2. Ultrafiltration: Step 1: 10 kDa ultrafiltration membrane to remove large molecular proteins; Step 2: 3 kDa ultrafiltration membrane to retain the target small molecule peptide; 3. Freeze-drying: Pre-freeze at -80℃ for 12 hours, freeze-dry at -40℃ for 24 hours, and obtain this product when the moisture content is ≤5%.

[0057] Comparative Example 2: A method for preparing a small molecule peptide derived from yeast-directed metabolism The preparation method of the present invention is as follows: 1. Fermentation 1. Culture medium configuration (1) Seed culture medium (YPD): glucose 25 g / L, yeast extract powder 13 g / L, peptone 25 g / L; Culture conditions: 30°C, 200 rpm, culture to the logarithmic growth phase (OD600≈8-10, about 12-16 hours).

[0058] (2) Fermentation basal medium: Carbon source: glucose 55 g / L; Nitrogen source: ammonium sulfate 7g / L, yeast extract powder 13g / L; Trace elements: MgSO4 2g / L, KH2PO4 4g / L.

[0059] (3) Feed solution: glucose 210 g / L, L-cysteine 7 g / L, glycine 12 g / L (pH 5.0, filter sterilized).

[0060] Cultivation process: Prepare seed culture medium in a dedicated seed tank and incubate at 30°C until the logarithmic growth phase. Pump the seed solution through sterile piping into a pre-sterilized fermentation tank filled with basal culture medium for fermentation. The inoculum volume should be 8% of the tank volume.

[0061] 2. Fermentation process pH 3.8 ± 0.1, temperature 28 ± 0.5°C, dissolved oxygen (DO) 20 ± 5%, agitation 200 rpm. Fermentation for 48 hours.

[0062] 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added at the beginning of the fermentation; Dynamic feeding strategy Feeding trigger condition: When the residual sugar concentration drops to 5g / L, the feeding pump is started to add feeding liquid; Feed rate: 0.1 mL / min (regulated by online glucose sensor feedback); Feeding goal: maintain the residual sugar concentration at 2-5 g / L to avoid excess carbon source inhibiting peptide synthesis.

[0063] 3. Process monitoring (1) Online detection: pH, DO and temperature are monitored in real time through built-in probes in the fermenter.

[0064] The residual sugar concentration was analyzed by HPLC (high performance liquid chromatography) by sampling every hour.

[0065] (2) Offline detection: Samples were taken every 6 hours to measure bacterial density (OD600), small molecule peptide concentration (BCA method) and metabolic byproducts (such as ethanol, GC detection).

[0066] 2. Product separation and purification 1. Fermentation broth pretreatment Centrifugation: 4°C, 8000 × g for 15 minutes to remove bacteria; 2. Ultrafiltration: Step 1: 10 kDa ultrafiltration membrane to remove large molecular proteins; Step 2: 3 kDa ultrafiltration membrane to retain the target small molecule peptide; 3. Freeze-drying: Pre-freeze at -80℃ for 12 hours, freeze-dry at -40℃ for 24 hours, and obtain this product when the moisture content is ≤5%.

[0067] Experimental determination The products prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 were tested for physicochemical properties, biological activity, and antibacterial activity. The testing methods are as follows (testing steps not described in detail may be performed using conventional techniques in the art). The test results are shown in the table below.

[0068] 1. Physical and chemical properties testing: Molecular weight distribution: MALDI-TOF mass spectrometry was used to confirm the molecular weight of the main peak.

[0069] Purity: HPLC analytical purity (C18 column, acetonitrile-water gradient elution).

[0070] 2. Biological activity test Antioxidant activity: A 1 mg / mL sample was prepared and the DPPH free radical scavenging rate and ORAC value (oxygen radical absorbance capacity) were tested.

[0071] 3. Antimicrobial activity: A 1 mg / mL sample was prepared and the diameter of the inhibition zone against Escherichia coli (ATCC 25922) was detected by disk diffusion method.

[0072] From the results in the above table, it can be seen that the molecular weight distribution, purity, free radical scavenging rate, oxygen free radical absorption capacity and inhibition zone diameter test results for Escherichia coli of the products prepared in the examples and comparative examples can be seen: the products prepared by the method of the present invention have a high content of small molecule peptides, and both biological activity and antibacterial activity are relatively strong, indicating that through two-stage pH control, the growth of the bacteria can be maximized, and the impurity protein can be effectively inhibited and the peptide synthetase can be activated, thereby greatly increasing the proportion of the target product.

Claims

1. A method for preparing a small molecule peptide through yeast-directed metabolism, characterized in that: The steps include: 1) Prepare seed culture medium, then culture with shaking at 30±1°C until the logarithmic growth phase to obtain seed solution; 2) adding the seed solution to a fermentation tank filled with fermentation basal medium for two-stage pH-controlled fermentation; Stage 1: Under the conditions of pH 5.5 ± 0.1, temperature 30 ± 0.5℃, and dissolved oxygen DO 50 ± 5%, the bacteria were cultured to an OD600 of 15-20; Stage 2: The pH is lowered to 3.8 ± 0.1, the temperature to 28 ± 0.5°C, and the dissolved oxygen (DO) to 20 ± 5% for small molecule peptide synthesis. The reaction time is 18-60 hours. At the beginning of stage 2, 0.1-0.2 mM isopropyl-β-D-thiogalactoside is added to continuously induce exogenous gene expression, and the residual sugar concentration is maintained at 2-5 g / L during synthesis. 3) The fermentation broth obtained in step 2) is centrifuged, ultrafiltered, and freeze-dried to obtain the product.

2. The method for preparing a small molecule peptide through yeast-directed metabolism according to claim 1, wherein: In step 1), the seed culture medium comprises 20-30 g / L glucose, 10-15 g / L yeast extract powder, and 20-30 g / L peptone.

3. The method for preparing a small molecule peptide through yeast-directed metabolism according to claim 1, wherein: In step 2), the fermentation basal medium is composed of: 50-60 g / L glucose, 5-8 g / L ammonium sulfate, 10-15 g / L yeast extract, 1-3 g / L MgSO4, and 3-5 g / L KH2PO4.

4. The method for preparing a small molecule peptide through yeast-directed metabolism according to claim 1, wherein: In step 2), when the residual sugar concentration drops to 2 g / L, the feed pump is started to add feed solution to maintain the residual sugar concentration at 2-5 g / L. The feed solution comprises 200-220 g / L of glucose, 5-8 g / L of L-cysteine, and 10-15 g / L of glycine.

5. The method for preparing a small molecule peptide through yeast-directed metabolism according to claim 1, wherein: In step 3), the fermentation broth is centrifuged at 4±1°C for 10-20 minutes to remove the bacteria.

6. The method for preparing a small molecule peptide through yeast-directed metabolism according to claim 1, wherein: In step 3), ultrafiltration is performed in two steps. The first step uses an 8-10 kDa ultrafiltration membrane to remove large molecular proteins; the second step uses a 3-5 kDa ultrafiltration membrane to retain the target small molecular peptides.

7. The method for preparing a small molecule peptide through yeast-directed metabolism according to claim 1, wherein: The freeze-drying in step 3) is specifically as follows: pre-freezing at -80±5°C for 8-15 hours and freeze-drying for 12-36 hours.

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