A method for high-density fermentation industrial production of succinamidyl-alanyl-tyrosyl-prolyl-glycyl-arginyl-arginyl-arginyl-arginyl-arginyl

Abstract

The application provides a high-density fermentation industrial production method of the scytalone 37 peptide, which comprises the following steps: rejuvenating and activating the fermentation strain of the scytalone 37 peptide, Bacillus subtilis, and then carrying out primary seed, secondary seed, 30 L seed tank, 3 m3 seed tank, 30 m3 fermentation tank fermentation, adding a supplement in the 30 m3 fermentation process and discharging the tank. The segmented supplement before the scytalone 37 peptide production can avoid the excessive growth of the Bacillus subtilis and reduce the secretion of other metabolites; the segmented supplement in the middle and late stages of the scytalone 37 peptide production can meet the nutritional needs of the Bacillus subtilis for producing the scytalone 37 peptide. The method can improve the content of the scytalone 37 peptide in the fermentation liquid, shorten the fermentation time and improve the production efficiency.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to a method for the industrial production of kiwifruit peptides through high-density fermentation. Background Technology

[0002] Antimicrobial peptides (AMPs) are among the oldest antimicrobial peptides in biological evolution. They are important components of innate immune regulation in various organisms, from prokaryotes to humans, and are innate immune effector molecules with direct antibacterial and microbiome-regulating effects. Historically, the antimicrobial function of antimicrobial peptides was the first to be recognized, hence the initial naming. With a deeper understanding of their structure and function, recent years have revealed their crucial role in regulating the body's immune response; many antimicrobial peptides act as a bridge between innate and acquired immunity. Antimicrobial peptides exert their anti-infective effects through direct bactericidal / bacteriostatic actions and by enhancing the body's immune response, which is a key advantage over antibiotics. Therefore, antimicrobial peptides are now also referred to as immune defense peptides.

[0003] Current research indicates that antimicrobial peptides possess broad-spectrum antibacterial, antiviral, antitumor, antiprotozoal, and immunomodulatory activities, and are less prone to inducing drug resistance. Antimicrobial peptides exhibit various immunomodulatory effects, including regulating inflammatory responses, chemotaxis of immune cells, promotion of cell differentiation, and activation of both innate and adaptive immune systems. These diverse immunomodulatory properties make antimicrobial peptides a promising candidate for treating infectious and immune diseases.

[0004] Sublancin, also known as thiamphetapeptide, is composed of 37 amino acid residues with a molecular weight of 3879.8 Da (based on the 2018 international relative atomic mass). Its structure is stable, consisting of five cysteine ​​residues forming two disulfide bonds, and exhibits good resistance to high temperatures, acids, alkalis, and pepsin. Experiments have shown that sublancin can enhance the phagocytic function of RAW264.7 macrophages. Clinical trials have demonstrated that sublancin can enhance the immune function of livestock, poultry, and aquatic animals, improve the efficacy of vaccines, and has high safety with no drug residues.

[0005] There have been some studies on heptacapeptide both domestically and internationally. In 1998, Paik SH et al. studied the chemical and biological structural characteristics and identification of the transporter protein gene of heptacapeptide produced by Bacillus subtilis 168. They concluded that heptacapeptide is a polypeptide composed of 37 amino acid residues containing two disulfide bonds. Its aqueous solution showed no degradation or inactivation after two years of storage at room temperature, indicating its relative stability. Although heptacapeptide has great potential to replace traditional antibiotics, the yield of heptacapeptide produced by Bacillus subtilis is very low, which cannot meet the needs of large-scale use. Therefore, scholars at home and abroad have been committed to improving the yield of heptacapeptide. Shengyue Ji et al. improved the yield of heptacapeptide by introducing three specific promoters and recombinant Bacillus subtilis operons, ultimately increasing the yield of heptacapeptide produced by recombinant Bacillus subtilis to 642 mg / L. Shengyue Ji further increased the yield of heptacapeptide to 968 mg / L by culturing the recombinant strain in a 5 L fermenter using the selected optimal culture conditions. There is also patent CN 112029697 which increases the yield of subtilisin to 3100 mg / L through fermentation with recombinant Bacillus subtilis JY011802. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the yield of subtilis 37-peptide produced by Bacillus subtilis in the prior art is low and cannot meet the needs of large-scale use.

[0007] To solve this technical problem, the present invention provides a method for high-density fermentation industrial production of 37-peptide of kiwifruit, the method comprising the following steps: revitalizing and activating the fermentation strain Bacillus subtilis for 37-peptide of kiwifruit, using primary seed, secondary seed, fermentation in a 30 L seed tank, a 3 m³ seed tank, and a 30 m³ fermentation tank, adding feed during the 30 m³ fermentation process, and then releasing the fermenter. (1) Activation of Bacillus subtilis: Bacillus subtilis strain was taken out from -80 ℃, warmed, and then streaked onto nutrient broth agar plates and incubated at 35 ℃ for 12 hours; (2) Primary seed: Pick a single colony from the plate and inoculate it into the primary seed culture medium at a volume of 30 mL / 150 mL. Incubate at 37°C for 6-8 h in a shaker at 180 r / min. Measure the OD value of the fermentation broth. Once it reaches 1.6-1.8, the primary seed liquid is obtained. (3) Secondary seed: Take 1 mL from the primary seed liquid and inoculate it into two secondary seed culture media, with a volume of 100 mL / 500 mL. Incubate in a shaker at 220 r / min at 37℃ for 5-7 h. Measure the OD value of the fermentation broth. After it reaches 1.7-2.0, the secondary seed liquid is obtained. (4) 30 L seed tank: The secondary seed liquid was inoculated into a 30 L seed tank containing 15 L of seed culture medium at an inoculation rate of 1%. The fermentation was carried out at 160 r / min, tank pressure of 0.05 MPa, and a ventilation rate of 1:1 for 5-7 h. After the OD value of the fermentation liquid reached 3.2-3.6, the 30 L seed tank liquid was obtained. (5) 3 m³ seed tank: 30 L of seed tank liquid was inoculated into a 3 m³ seed tank containing 1.5 m³ of seed culture medium at an inoculation rate of 1%. The fermentation was carried out at 160 r / min, tank pressure of 0.05 MPa, and ventilation rate of 1:1 for 5-7 h. After the OD value of the fermentation liquid reached 3.5-4.0, the Bacillus subtilis seed liquid producing subtilis 37-peptide was obtained. (6) Feeding during 30 m³ fermentation: Feeding is added during 30 m³ fermentation based on the changes in the OD value of the fermentation broth and the content of thiamphetamine. (7) Removal from the tank: The time for removal from the tank is determined by monitoring the content of 37-peptide every 0.5 hours after fermentation for 32 hours. When the increase in the content of 37-peptide is less than 50 mg / L in two separate tests, the tank is removed immediately and fermentation is stopped to obtain 37-peptide fermentation liquid.

[0008] The culture medium for activating the microbial strain was formulated as follows: 2 g beef extract, 2 g yeast extract, 10 g peptone, 5 g NaCl, 15 g agar, diluted with water to a final volume of 1000 mL and adjusted to pH 7.4. The primary and secondary seed culture media were also formulated as follows: 2 g beef extract, 2 g yeast extract, 10 g peptone, 5 g NaCl, 15 g agar, diluted with water to a final volume of 1000 mL and adjusted to pH 7.4. The 30 L seed tank and 3 m³ culture medium were formulated as follows: 13 g / L yeast extract, 10 g / L peptone, 4 g / L water-soluble corn starch, 8 g / L corn steep liquor, 5 g / L glucose, 1.5 g / L dipotassium hydrogen phosphate, 2 g / L sodium chloride, 0.5 g / L magnesium sulfate, with the remainder being drinking water.

[0009] The 30 m³ fermentation medium contained yeast extract at a concentration of 5–10 g / L, glucose at a concentration of 0.2–2 g / L, water-soluble corn starch at a concentration of 2–10 g / L, corn steep liquor at a concentration of 8–20 g / L, ammonium chloride at a concentration of 1–2 g / L, sodium chloride at a concentration of 1–3 g / L, and defoamer at a concentration of 2–5 g / L, with the remainder being drinking water. The feed contains 5-15 g / L glucose, 4-15 g / L corn steep liquor, and the remainder is drinking water.

[0010] The sterilization time for adding the culture medium into the fermenter is 8–12 minutes.

[0011] The fed-batch method in step (6) is as follows: During the early stage of basidipotassium production, the fed-batch amount is adjusted based on the OD value of the fermentation broth; during the middle and late stages of basidipotassium production, the fed-batch amount is adjusted based on the basidipotassium content in the fermentation broth. The specific steps are as follows: In the early stage of fermentation of 37-peptide-producing chlorophyll: continuous fed-batch feeding was implemented. Specifically, from 4 to 18 hours after the start of fermentation, the feed was fed every 2 hours based on the OD value of the fermentation broth. When the OD value was between 2 and 20, the feed rate was 7 to 8 mL / L·h; when the OD value was between 20 and 40, the feed rate was 8 to 10 mL / L·h; when the OD value was between 40 and 60, the feed rate was 10 to 12 mL / L·h; when the OD value was between 60 and 80, the feed rate was 13 to 15 mL / L·h; and when the OD value was between 80 and 110, the feed rate was 18 to 20 mL / L·h. During the mid-stage of thiamphetamine production: continuous fed-batch operation was implemented. Specifically, between 18 and 28 hours of fermentation, the fed-batch rate was adjusted based on the thiamphetamine content in the fermentation broth, monitored every 2 hours. The feeding rate was 20–25 mL / L·h when the thiamphetamine content was 200–2000 mg / L; 25–30 mL / L·h when the content was 2000–3000 mg / L; 30–40 mL / L·h when the content was 3000–5000 mg / L; and 40–50 mL / L·h when the content was above 5000 mg / L. c. Late stage of thiamphetapeptide production: Continuous fed-batch feeding. Specifically, from 28 h of fermentation to discharge, fed-batch feeding is carried out based on the thiamphetapeptide content in the fermentation broth monitored every 1 h. When the thiamphetapeptide content is below 6000 mg / L, the feeding rate is 50-55 mL / L·h; when the thiamphetapeptide content is 6000-8000 mg / L, the feeding rate is 60-65 mL / L·h.

[0012] The specific steps of the feeding process in step (6) are as follows: In the early stage of α-fermentation of 37-peptide: intermittent fed-batch feeding was used. Specifically, from 4 to 18 hours of fermentation, fed-batch feeding was performed every 2 hours based on the OD value of the fermentation broth. When the OD value was between 2 and 20, 300 mL of feed was fed; when the OD value was between 20 and 40, 380 mL of feed was fed; when the OD value was between 40 and 60, 440 mL of feed was fed; when the OD value was between 60 and 80, 560 mL of feed was fed; and when the OD value was between 80 and 110, 760 mL of feed was fed. b. Mid-stage production of thiamphetamine: Intermittent fed-batch feeding, specifically, between 18 and 28 hours of fermentation, fed-batch feeding is performed according to the thiamphetamine content in the fermentation broth monitored every 2 hours. When the thiamphetamine content is 200–2000 mg / L, 900 mL of feed is added; when the thiamphetamine content is 2000–3000 mg / L, 1100 mL of feed is added; when the thiamphetamine content is 3000–5000 mg / L, 1400 mL of feed is added; and when the thiamphetamine content is above 5000 mg / L, 1800 mL of feed is added. c. Late stage of thiamphetapeptide production: Intermittent feed addition. Specifically, from 28 hours of fermentation to discharge, feed is added according to the thiamphetapeptide content in the fermentation broth monitored every hour. When the thiamphetapeptide content is below 6000 mg / L, 1050 mL of feed is added; when the thiamphetapeptide content is between 6000 and 8000 mg / L, 1200 mL of feed is added.

[0013] The feeding step in step (6) is specifically as follows: Continuous feeding was employed, specifically starting 4 hours after fermentation, at a rate of 20–30 mL / L·h.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a method suitable for industrial high-density fermentation of sublancin. The fermentation strain of sublancin, Bacillus subtilis (CGMCC No. 13941), is rejuvenated and activated, and then subjected to primary seeding, secondary seeding, fermentation in a 30 L seed tank, a 3 m³ seed tank, and a 30 m³ fermentation tank. During the 30 m³ fermentation process, feed is added and the tank is discharged. Using the method of the present invention to produce sublancin can increase the content of sublancin in the fermentation broth, shorten the fermentation time, and improve production efficiency. Attached Figure Description

[0015] Figure 1 This is a flowchart of the industrial production process of high-density fermentation of 37-peptide from calcined kiwifruit.

[0016] Figure 2 This is a schematic diagram of the primary structure of 37-peptide of calcinolone.

[0017] Figure 3 This is a high-performance liquid chromatogram of thiamphetamine in the fermentation broth.

[0018] Figure 4 This is a high-performance liquid chromatogram of the standard solution of calcinyl thiamphetamine.

[0019] Figure 5The high-performance liquid chromatogram (125 μg / mL) of the standard solution of 37-peptide of calcinolone. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified in this instruction manual, all fermentation conditions and methods are conventional methods, including all culture media that have undergone conventional sterilization.

[0022] Preparation Example This preparation example illustrates the method for preparing Bacillus subtilis fermentation broth that produces subtilisin.

[0023] like Figures 1-5 As shown, this invention provides a method for the high-density industrial production of tahinitrofurantoin via fermentation. The method includes the following steps: Bacillus subtilis (CGMCC No. 13941), the fermentation strain for tahinitrofurantoin, undergoes strain rejuvenation and activation, primary seed culture, secondary seed culture, fermentation in a 30 L seed tank, a 3 m³ seed tank, and a 30 m³ fermentation tank; during the 30 m³ fermentation process, feed is added; and the mixture is discharged from the tank. (1) Activation of Bacillus subtilis: Bacillus subtilis strain was taken out from -80 ℃, warmed, and then streaked onto nutrient broth agar plates and incubated at 35 ℃ for 12 h. (2) Primary seed: Pick a single colony from the plate and inoculate it into the primary seed culture medium at a volume of 30 mL / 150 mL. Incubate at 37°C for 6-8 h in a shaker at 180 r / min. Measure the OD value of the fermentation broth. Once it reaches 1.6-1.8, the primary seed liquid is obtained. (3) Secondary seed: Take 1 mL from the primary seed liquid and inoculate it into two secondary seed culture media, with a volume of 100 mL / 500 mL. Incubate in a shaker at 220 r / min at 37 °C for 5 to 7 h. Measure the OD value of the fermentation broth. After it reaches 1.7 to 2.0, the secondary seed liquid is obtained. (4) 30 L seed tank: The secondary seed liquid was inoculated into a 30 L seed tank containing 15 L of seed culture medium at an inoculation rate of 1%. The fermentation was carried out at 160 r / min, tank pressure of 0.05 MPa, and a ventilation rate of 1:1 for 5-7 h. After the OD value of the fermentation liquid reached 3.2-3.6, the 30 L seed tank liquid was obtained. (5) 3 m³ seed tank: 30 L of seed tank liquid was inoculated into a 3 m³ seed tank containing 1.5 m³ of seed culture medium at an inoculation rate of 1%. The fermentation was carried out at 160 r / min, tank pressure of 0.05 Mpa, and ventilation rate of 1:1 for 5-7 h. After the OD value of the fermentation liquid reached 3.5-4.0, the Bacillus subtilis seed liquid producing subtilis 37-peptide was obtained. (6) Feeding during 30 m³ fermentation: Feeding is added during 30 m³ fermentation based on the changes in the OD value of the fermentation broth and the content of thiamphetamine. (7) Removal from the tank: The time for removal from the tank is determined by monitoring the content of 37-peptide every 0.5 hours after fermentation for 32 hours. When the increase in the content of 37-peptide is less than 50 mg / L in two separate tests, the tank is removed immediately and fermentation is stopped to obtain 37-peptide fermentation liquid.

[0024] The culture medium for activating the microbial strain consisted of 2 g beef extract, 2 g yeast extract, 10 g peptone, 5 g NaCl, and 15 g agar, diluted to 1000 mL with water and adjusted to pH 7.4. The primary and secondary seed culture media consisted of the same formula: 2 g beef extract, 2 g yeast extract, 10 g peptone, 5 g NaCl, and 15 g agar, diluted to 1000 mL with water and adjusted to pH 7.4. The 30 L seed tank and 3 m³ culture medium consisted of 13 g / L yeast extract, 10 g / L peptone, 4 g / L water-soluble corn starch, 8 g / L corn steep liquor, 5 g / L glucose, 1.5 g / L dipotassium hydrogen phosphate, 2 g / L sodium chloride, 0.5 g / L magnesium sulfate, with the remainder being drinking water. All cultures were sterilized at 121 °C for 30 min.

[0025] The 30 m³ fermentation medium contained yeast extract at a concentration of 5–10 g / L, glucose at a concentration of 0.2–2 g / L, water-soluble corn starch at a concentration of 2–10 g / L, corn steep liquor at a concentration of 8–20 g / L, ammonium chloride at a concentration of 1–2 g / L, sodium chloride at a concentration of 1–3 g / L, and defoamer at a concentration of 2–5 g / L, with the remainder being drinking water. The feed contains 5-15 g / L glucose, 4-15 g / L corn steep liquor, and the remainder is drinking water.

[0026] The sterilization time for adding the culture medium into the fermenter is 8 to 12 minutes, such as 8 minutes, 10 minutes, or 12 minutes.

[0027] Example 1 This embodiment illustrates a method for the industrial production of kiwifruit peptides using high-density fermentation.

[0028] The 30 m³ fermentation medium consisted of 8 g / L yeast extract, 1 g / L glucose, 5 g / L water-soluble corn starch, 13 g / L corn steep liquor, 1.5 g / L ammonium chloride, 2 g / L sodium chloride, 3 g / L defoamer, and the remainder being drinking water.

[0029] The feed formulation consisted of 10 g / L glucose, 8 g / L corn steep liquor, and the remainder was drinking water, with the pH adjusted to between 7.2 and 7.6. The feed was sterilized at 115 ℃ for 20 min.

[0030] Disinfection of fermentation tank before fermentation: Add 20 m³ of fermentation medium to a 30 m³ fermentation tank, adjust the pH to between 7.2 and 7.6, and sterilize by steaming at 115 ℃ for 10 min.

[0031] The prepared Bacillus subtilis seed culture producing subtilis 37-peptide was inoculated at an inoculation rate of 1%. The fermentation conditions were: temperature 37 ℃, aeration ratio of 1:0.5, rotation speed of 180 r / min, and tank pressure of 0.05 MPa.

[0032] Feeding method The feeding step (6) of the preparation example is as follows: In the early stage of fermentation of 37-peptide-producing chlorophyll: continuous fed-batch feeding was implemented. Specifically, from 4 to 18 hours after the start of fermentation, the feed was fed every 2 hours based on the OD value of the fermentation broth. When the OD value was between 2 and 20, the feed rate was 7 to 8 mL / L·h; when the OD value was between 20 and 40, the feed rate was 8 to 10 mL / L·h; when the OD value was between 40 and 60, the feed rate was 10 to 12 mL / L·h; when the OD value was between 60 and 80, the feed rate was 13 to 15 mL / L·h; and when the OD value was between 80 and 110, the feed rate was 18 to 20 mL / L·h. During the mid-stage of thiamphetamine production: continuous fed-batch operation was implemented. Specifically, between 18 and 28 hours of fermentation, the fed-batch rate was adjusted based on the thiamphetamine content in the fermentation broth, monitored every 2 hours. The feeding rate was 20–25 mL / L·h when the thiamphetamine content was 200–2000 mg / L; 25–30 mL / L·h when the content was 2000–3000 mg / L; 30–40 mL / L·h when the content was 3000–5000 mg / L; and 40–50 mL / L·h when the content was above 5000 mg / L. c. Late stage of thiamphetapeptide production: Continuous fed-batch feeding. Specifically, from 28 h of fermentation to discharge, fed-batch feeding is carried out based on the thiamphetapeptide content in the fermentation broth monitored every 1 h. When the thiamphetapeptide content is below 6000 mg / L, the feeding rate is 50-55 mL / L·h; when the thiamphetapeptide content is 6000-8000 mg / L, the feeding rate is 60-65 mL / L·h.

[0033] Determining the time for discharge: After 32 hours of fermentation, monitor the content of 37-butyrate every 0.5 hours. When the increase in the content of 37-butyrate between two tests is less than 50 mg / L, immediately discharge the product from the tank and stop fermentation to obtain the 37-butyrate fermentation broth.

[0034] The OD value and tahinitrophenylephrine content of the fermentation broth were recorded at 18 h and when the broth was removed from the tank, and the fermentation time was also recorded. The results are shown in Table 1.

[0035] Example 2 This embodiment illustrates a method for the industrial production of kiwifruit peptides using high-density fermentation.

[0036] The 30 m³ fermentation medium consisted of 10 g / L yeast extract, 0.2 g / L glucose, 2 g / L water-soluble corn starch, 16 g / L corn steep liquor, 1 g / L ammonium chloride, 1 g / L sodium chloride, 2 g / L defoamer, and the remainder being drinking water. The feed formulation consisted of 5 g / L glucose, 15 g / L corn steep liquor, and the remainder was drinking water, with the pH adjusted to between 7.2 and 7.6. The feed was sterilized at 115 ℃ for 20 min.

[0037] Before fermentation, the fermenter was sterilized by adding 20 m³ of fermentation medium to a 30 m³ fermenter, adjusting the pH to between 7.2 and 7.6, and then passing steam at 121 ℃ for 30 min.

[0038] The prepared Bacillus subtilis seed culture producing subtilis 37-peptide was inoculated at an inoculation rate of 1.0%. The fermentation conditions were: temperature 37 ℃, aeration ratio of 1:0.5, rotation speed of 180 r / min, and tank pressure of 0.05 MPa.

[0039] Feeding method The feeding step (6) of the preparation example is as follows: In the early stage of fermentation of 37-peptide-producing chlorophyll: continuous fed-batch feeding was implemented. Specifically, from 4 to 18 hours after the start of fermentation, the feed was fed every 2 hours based on the OD value of the fermentation broth. When the OD value was between 2 and 20, the feed rate was 7 to 8 mL / L·h; when the OD value was between 20 and 40, the feed rate was 8 to 10 mL / L·h; when the OD value was between 40 and 60, the feed rate was 10 to 12 mL / L·h; when the OD value was between 60 and 80, the feed rate was 13 to 15 mL / L·h; and when the OD value was between 80 and 110, the feed rate was 18 to 20 mL / L·h. b. Mid-stage of thiamethoxam production: Continuous fed-batch feeding, specifically, between 18 and 28 hours of fermentation, fed-batch feeding is conducted based on monitoring the thiamethoxam content in the fermentation broth every 2 hours. The feeding rate is 20–25 mL / L·h when the thiamethoxam content is 200–2000 mg / L; 25–30 mL / L·h when the content is 2000–3000 mg / L; 30–40 mL / L·h when the content is 3000–5000 mg / L; and 40–50 mL / L·h when the content is above 5000 mg / L. c. Late stage of thiamphetapeptide production: Continuous fed-batch feeding. Specifically, from 28 h of fermentation to discharge, fed-batch feeding is carried out based on the thiamphetapeptide content in the fermentation broth monitored every 1 h. When the thiamphetapeptide content is below 6000 mg / L, the feeding rate is 50-55 mL / L·h; when the thiamphetapeptide content is 6000-8000 mg / L, the feeding rate is 60-65 mL / L·h.

[0040] Determining the time for discharge: After 32 hours of fermentation, monitor the content of 37-butyrate every 0.5 hours. When the increase in the content of 37-butyrate between two tests is less than 50 mg / L, immediately discharge the product from the tank and stop fermentation to obtain the 37-butyrate fermentation broth.

[0041] The OD value and tahinitrophenylephrine content of the fermentation broth were recorded at 18 h and when the broth was removed from the tank, and the fermentation time was also recorded. The results are shown in Table 1.

[0042] Example 3 This embodiment illustrates a method for the industrial production of kiwifruit peptides using high-density fermentation.

[0043] The 30 m³ fermentation medium consisted of 5 g / L yeast extract, 2 g / L glucose, 10 g / L water-soluble corn starch, 12 g / L corn steep liquor, 2 g / L ammonium chloride, 3 g / L sodium chloride, 5 g / L defoamer, and the remainder being drinking water.

[0044] The feed formulation consisted of 15 g / L glucose, 4 g / L corn steep liquor, and the remainder was drinking water. The pH was adjusted to between 7.2 and 7.6. The feed was sterilized at 115 ℃ for 20 min.

[0045] Before fermentation, the fermenter was sterilized by adding 20 m³ of fermentation medium to a 30 m³ fermenter, adjusting the pH to between 7.2 and 7.6, and then passing steam at 121 ℃ for 30 min.

[0046] The prepared Bacillus subtilis seed culture producing subtilis 37-peptide was inoculated at an inoculation rate of 1.0%. The fermentation conditions were: temperature 37 ℃, aeration ratio of 1:0.5, rotation speed of 180 r / min, and tank pressure of 0.05 MPa.

[0047] Feeding method The feeding step (6) of the preparation example is as follows: In the early stage of fermentation of 37-peptide-producing α-carboxylic acid: continuous fed-batch feeding was implemented. Specifically, from 4 to 18 hours after the start of fermentation, the feed rate was increased every 2 hours based on the OD value of the fermentation broth. When the OD value was between 2 and 20, the feed rate was 7 to 8 mL / L·h; when the OD value was between 20 and 40, the feed rate was 8 to 10 mL / L·h; when the OD value was between 40 and 60, the feed rate was 10 to 12 mL / L·h; when the OD value was between 60 and 80, the feed rate was 13 to 15 mL / L·h; and when the OD value was between 80 and 110, the feed rate was 18 to 20 mL / L·h. During the mid-stage of thiamphetamine production: continuous fed-batch operation was implemented. Specifically, between 18 and 28 hours of fermentation, the fed-batch rate was adjusted based on the thiamphetamine content in the fermentation broth, monitored every 2 hours. The feeding rate was 20–25 mL / L·h when the thiamphetamine content was 200–2000 mg / L; 25–30 mL / L·h when the content was 2000–3000 mg / L; 30–40 mL / L·h when the content was 3000–5000 mg / L; and 40–50 mL / L·h when the content was above 5000 mg / L. c. Late stage of thiamphetapeptide production: Continuous fed-batch feeding. Specifically, from 28 h of fermentation to discharge, fed-batch feeding is carried out based on the thiamphetapeptide content in the fermentation broth monitored every 1 h. When the thiamphetapeptide content is below 6000 mg / L, the feeding rate is 50-55 mL / L·h; when the thiamphetapeptide content is 6000-8000 mg / L, the feeding rate is 60-65 mL / L·h.

[0048] Determining the time for discharge: After 32 hours of fermentation, monitor the content of 37-butyrate every 0.5 hours. When the increase in the content of 37-butyrate between two tests is less than 50 mg / L, immediately discharge the product from the tank and stop fermentation to obtain the 37-butyrate fermentation broth.

[0049] The OD value and tahinitrophenylephrine content of the fermentation broth were recorded at 18 h and when the broth was removed from the tank, and the fermentation time was also recorded. The results are shown in Table 1.

[0050] Example 4 This embodiment illustrates a method for the industrial production of kiwifruit peptides using high-density fermentation.

[0051] The high-density fermentation industrial production of 37-peptide of kiwifruit was carried out according to the method of Example 1, except that the feed-in step (6) of the preparation example was added directly in batches, as detailed below: In the early stage of α-fermentation of 37-peptide: intermittent fed-batch feeding was used. Specifically, from 4 to 18 hours of fermentation, fed-batch feeding was performed every 2 hours based on the OD value of the fermentation broth. When the OD value was between 2 and 20, 300 mL of feed was fed; when the OD value was between 20 and 40, 380 mL of feed was fed; when the OD value was between 40 and 60, 440 mL of feed was fed; when the OD value was between 60 and 80, 560 mL of feed was fed; and when the OD value was between 80 and 110, 760 mL of feed was fed. b. Mid-stage production of thiamphetamine: Intermittent fed-batch feeding, specifically, between 18 and 28 hours of fermentation, fed-batch feeding is performed according to the thiamphetamine content in the fermentation broth monitored every 2 hours. When the thiamphetamine content is 200–2000 mg / L, 900 mL of feed is added; when the thiamphetamine content is 2000–3000 mg / L, 1100 mL of feed is added; when the thiamphetamine content is 3000–5000 mg / L, 1400 mL of feed is added; and when the thiamphetamine content is above 5000 mg / L, 1800 mL of feed is added. c. Late stage of thiamphetapeptide production: Intermittent feed addition. Specifically, from 28 hours of fermentation to discharge, feed is added according to the thiamphetapeptide content in the fermentation broth monitored every hour. When the thiamphetapeptide content is below 6000 mg / L, 1050 mL of feed is added; when the thiamphetapeptide content is between 6000 and 8000 mg / L, 1200 mL of feed is added.

[0052] Determining the time for discharge: After 32 hours of fermentation, monitor the content of 37-butyrate every 0.5 hours. When the increase in the content of 37-butyrate between two tests is less than 50 mg / L, immediately discharge the product from the tank and stop fermentation to obtain the 37-butyrate fermentation broth.

[0053] The OD value and tahinitrophenylephrine content of the fermentation broth were recorded at 18 h and when the broth was removed from the tank, and the fermentation time was also recorded. The results are shown in Table 1.

[0054] Example 5 This embodiment illustrates a method for the industrial production of kiwifruit peptides using high-density fermentation.

[0055] The high-density fermentation of 37-peptide of kiwifruit was carried out in an industrial production according to the method of Example 1. The difference is that the feeding step (6) of the preparation example is to start feeding 4 h after fermentation, and the feeding rate is 20-30 mL / L·h.

[0056] The OD value and tahinitrophenylephrine content of the fermentation broth were recorded at 18 h and when the broth was removed from the tank, and the fermentation time was also recorded. The results are shown in Table 1.

[0057] Example 6 This embodiment illustrates a method for the industrial production of kiwifruit peptides using high-density fermentation.

[0058] The high-density fermentation of 37-peptide was carried out industrially according to the method of Example 1, except that fed-batch feeding was not performed.

[0059] The OD value and tahinitrophenylephrine content of the fermentation broth were recorded at 18 h and when the broth was removed from the tank, and the fermentation time was also recorded. The results are shown in Table 1.

[0060] Example 7 This embodiment illustrates a method for the industrial production of kiwifruit peptides using high-density fermentation.

[0061] The high-density fermentation industrial production of 37-peptide was carried out according to the method of Example 1, except that the fermentation tank culture medium and feed formulation were different. The culture medium formulation of the 30 m³ fermentation tank in this example was 30.2 g / L water-soluble corn starch, 23.6 g / L peptone, 18.2 g / L corn steep liquor, 5 g / L dipotassium hydrogen phosphate, 10 g / L magnesium sulfate, and the remainder was drinking water.

[0062] The feed formulation consisted of 10 g / L glucose, 8 g / L yeast extract, and the remainder was drinking water.

[0063] The OD value and tahinitrophenylephrine content of the fermentation broth were recorded at 18 h and when the broth was removed from the tank, and the fermentation time was also recorded. The results are shown in Table 1.

[0064] Example 8 This embodiment illustrates a method for the industrial production of kiwifruit peptides using high-density fermentation.

[0065] The high-density fermentation industrial production of 37-peptide was carried out according to the method of Example 4, except that the fermentation tank culture medium and feed formulation were different. The culture medium formulation of the 30 m³ fermentation tank in this example was 30.2 g / L water-soluble corn starch, 23.6 g / L peptone, 18.2 g / L corn steep liquor, 5 g / L dipotassium hydrogen phosphate, 10 g / L magnesium sulfate, and the remainder was drinking water.

[0066] The feed formulation consisted of 10 g / L glucose, 8 g / L yeast extract, and the remainder was drinking water.

[0067] The OD value and tahinitrophenylephrine content of the fermentation broth were recorded at 18 h and when the broth was removed from the tank, and the fermentation time was also recorded. The results are shown in Table 1.

[0068] Table 1. OD values ​​and trihexyphenidyl content of fermentation broth after 18 h of fermentation and at the time of discharge.

[0069] As can be seen from the results in Table 1, in the high-density industrial fermentation process of Bacillus subtilis producing 37-peptide, the preferred fermentation medium and fed-batch medium of the present invention, as well as the preferred fed-batch method, can increase the content of 37-peptide in the fermentation broth and shorten the fermentation time.

[0070] Example 9 This embodiment illustrates a method for detecting the OD value of fermentation broth.

[0071] The OD value of the fermentation broth was measured using a UV spectrophotometer with a wavelength of 630 nm. Distilled water was used as a blank control. The freshly obtained fermentation broth was diluted several times with distilled water so that the absorbance of the diluted liquid was between 0.1 and 1.0. The OD value of the fermentation broth was the measured absorbance multiplied by the corresponding dilution ratio.

[0072] Example 10 This embodiment illustrates a method for detecting the content of thiamphetamine in fermentation broth.

[0073] The content of thiamphetamine in the fermentation broth was determined by high performance liquid chromatography.

[0074] A.1 Principle After centrifugation, the thiamphetamine in the fermentation broth was separated by a high-performance liquid chromatography (HPLC) reversed-phase chromatography system, and determined by an ultraviolet detector or a diode array detector. The external standard method was used for quantification.

[0075] A.2 Reagents or materials Unless otherwise specified, use only analytical grade reagents.

[0076] A.2.1 Water: GB / T 6682, Grade I.

[0077] A.2.2 Trifluoroacetic acid: chromatographic grade.

[0078] A.2.3 Acetonitrile: chromatographic grade.

[0079] A.2.4 Mobile phase A: Transfer 1 mL of trifluoroacetic acid (A.2.2) to 1000 mL of water, filter through a 0.22 μm aqueous phase filter membrane, degas for 10 min, and seal for later use.

[0080] A.2.5 Mobile phase B: Measure 800 mL of acetonitrile (A.2.3), add 200 mL of water, mix well, then add 0.85 mL of trifluoroacetic acid (A.2.2) and mix well. Filter through a 0.22 μm organic phase filter membrane, degas for 10 min, and seal for later use.

[0081] A.2.6 37-peptide reference standard: content 99.30% ± 0.22%.

[0082] A.2.7 Substantiopeptide Standard Stock Solution: Accurately weigh 350 mg (accurate to 0.01 mg) of substantiopeptide reference standard (A.2.6), place it in a 10 mL centrifuge tube, add 5 mL of water, and vortex for 3-5 min until completely dissolved. Transfer the solution to a 50 mL volumetric flask, rinse the centrifuge tube three times with water, and transfer the rinsing solution to the volumetric flask. Continue sonicating for 15-30 min, then dilute to volume with water and mix well. The concentration of substantiopeptide in this solution is 7000 μg / mL. Store in a sealed container at 2 ℃-8 ℃, with a shelf life of 7 days.

[0083] A.2.8 Standard series solutions of tahinitrofurantoin: Accurately measure an appropriate amount of tahinitrofurantoin standard stock solution (A.2.7), dilute with water, and prepare a series of tahinitrofurantoin standard solutions with concentrations of 50 μg / mL, 300 μg / mL, 875 μg / mL, 1750 μg / mL, 3500 μg / mL, and 7000 μg / mL. Prepare fresh before use.

[0084] A.2.9 Filter membrane: 0.22 μm, aqueous phase and organic phase.

[0085] A.3 Instruments and Equipment A.3.1 High-performance liquid chromatograph: equipped with an ultraviolet detector or a diode array detector.

[0086] A.3.2 Ultrasonic cleaner.

[0087] A.3.3 Vortex mixer.

[0088] A.3.4 Centrifuge: Rotation speed not less than 12000 r / min.

[0089] A.4 Test Procedure.

[0090] A.4.1 Sample Solution Preparation Perform two parallel experiments; take an appropriate amount of fresh fermentation broth into a centrifuge tube, centrifuge at 12000 r / min for 2 min, use a syringe to draw about 2.5 mL of the supernatant, filter through a 0.22 μm filter membrane, discard the first 1 mL of filtrate, and collect the subsequent filtrate in a sample bottle for high performance liquid chromatography (HPLC) determination; if the content of trichogramma-7-peptide in the fermentation broth is >1000 μg / mL, dilute the supernatant after centrifugation with water 10 times, use a syringe to draw about 2.5 mL of the supernatant, filter through a 0.22 μm filter membrane, discard the first 1 mL of filtrate, and collect the subsequent filtrate in a sample bottle for HPLC determination.

[0091] A.4.2 Measurement.

[0092] A.4.2.1 Reference conditions for high performance liquid chromatography.

[0093] The reference conditions for high performance liquid chromatography are as follows: a) Chromatographic column: C8 column, particle size 5 μm, inner diameter 4.6 mm, column length 150 mm, or equivalent performance; b) Mobile phase: Mobile phase A: (A.2.4); Mobile phase B: (A.2.5); Gradient elution, the elution gradient is shown in Table A.1; c) Flow rate: 1.0 mL / min; d) Column temperature: 22 ℃; e) Injection volume: 20 μL; f) Detection wavelength: 280 nm.

[0094] Table A.1 Elution gradient of mobile phase

[0095] A.4.2.2 Determination of standard series solutions and sample solutions.

[0096] Under optimal instrument conditions, the standard series solutions and sample solutions were analyzed separately. A standard curve was plotted with the peak area of ​​basidiophore peptide on the x-axis and the concentration of the standard series solutions on the y-axis. The correlation coefficient of the standard curve was not less than 0.995. The liquid chromatogram of basidiophore peptide is attached. Figure 5 .

[0097] A.5 Experimental Data Processing.

[0098] The content of thiamphetamine in the fermentation broth is calculated by multiplying the concentration of thiamphetamine in the sample solution by the dilution factor, as calculated by the standard curve, in micrograms per milliliter (μg / mL) or milligrams per liter (mg / L).

[0099] The measurement results are expressed as the arithmetic mean of parallel measurements.

[0100] A.6 Precision.

[0101] Under repeatability conditions, the absolute difference between two independent measurements should not exceed 10.0% of the arithmetic mean.

[0102] The advantages of this invention's segmented fed-batch method are as follows: Segmented feeding during the early stage of basidiopeptide production avoids excessive growth of Bacillus subtilis and reduces the secretion of other metabolites; segmented feeding during the middle and late stages of basidiopeptide production meets the nutritional needs of Bacillus subtilis for basidiopeptide production. The synthesis of basidiopeptide is directly related to the carbon source and various metal ions in the fermentation broth, such as Mg... 2+ Mn 2+ Metal ions are important cofactors for the synthesis of subtilisin synthase. Appropriate concentrations of metal ions can promote the synthesis of subtilisin, but high concentrations will inhibit the growth of the host.

[0103] The method of this invention for producing sublancin can increase the content of sublancin in the fermentation broth, shorten the fermentation time, and improve production efficiency.

[0104] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention. This includes any combination of specific technical features. To avoid unnecessary repetition, the present invention will not further describe the various possible combinations. However, these simple modifications and combinations should also be considered as part of the content disclosed in this invention and are all within the protection scope of this invention.

Claims

1. A method for the industrial-scale production of kiwifruit peptides through high-density fermentation, characterized in that, The method includes the following steps: Bacillus subtilis, the fermentation strain for subtilis 37-peptide, is revitalized and activated; primary seed, secondary seed, fermented in a 30 L seed tank, a 3 m³ seed tank, and a 30 m³ fermentation tank; supplementary feed is added during the 30 m³ fermentation process; and the tank is then discharged. (1) Activation of Bacillus subtilis: Bacillus subtilis strain was taken out from -80 ℃, warmed, and then streaked onto nutrient broth agar plates and incubated at 35 ℃ for 12 hours; (2) Primary seed: Pick a single colony from the plate and inoculate it into the primary seed culture medium at a volume of 30 mL / 150 mL. Incubate at 37 °C for 6–8 h in a shaker at 180 r / min. Measure the OD value of the fermentation broth. Once it reaches 1.6–1.8, the primary seed liquid is obtained. (3) Secondary seed: Take 1 mL from the primary seed liquid and inoculate it into two secondary seed culture media, with a volume of 100 mL / 500 mL. Incubate in a shaker at 220 r / min at 37 ℃ for 5-7 h. Measure the OD value of the fermentation broth. After it reaches 1.7-2.0, the secondary seed liquid is obtained. (4) 30 L seed tank: The secondary seed liquid was inoculated into a 30 L seed tank containing 15 L of seed culture medium at an inoculation rate of 1%. The fermentation was carried out at 160 r / min, tank pressure of 0.05 MPa, and a ventilation rate of 1:1 for 5-7 h. After the OD value of the fermentation liquid reached 3.2-3.6, the 30 L seed tank liquid was obtained. (5) 3 m³ seed tank: 30 L of seed tank liquid was inoculated into a 3 m³ seed tank containing 1.5 m³ of seed culture medium at an inoculation rate of 1%. The fermentation was carried out at 160 r / min, tank pressure of 0.05 MPa, and ventilation rate of 1:1 for 5-7 h. After the OD value of the fermentation liquid reached 3.5-4.0, the Bacillus subtilis seed liquid producing subtilis 37-peptide was obtained. (6) Feeding during 30 m³ fermentation: Feeding was added during 30 m³ fermentation based on the changes in the OD value of the fermentation broth and the content of thiamphetamine. (7) Release from the tank: The release time is determined by monitoring the content of 37-peptide every 0.5 h after fermentation for 32 h. When the increase in the content of 37-peptide is less than 50 mg / L, the tank is immediately released and fermentation is stopped to obtain 37-peptide fermentation liquid. The feeding method in step (6) is one of the following two: The first method: the fed-batch feeding method in step (6) is as follows: the amount of fed-batch feed is adjusted according to the OD value of the fermentation broth in the early stage of basidiopeptide production, and the amount of fed-batch feed is adjusted according to the content of basidiopeptide in the fermentation broth in the middle and late stages of basidiopeptide production; the specific steps are as follows: In the early stage of fermentation of 37-peptide-producing chlorophyll: continuous fed-batch feeding was implemented. Specifically, from 4 to 18 hours after the start of fermentation, the feed was fed every 2 hours based on the OD value of the fermentation broth. When the OD value was between 2 and 20, the feed rate was 7 to 8 mL / L·h; when the OD value was between 20 and 40, the feed rate was 8 to 10 mL / L·h; when the OD value was between 40 and 60, the feed rate was 10 to 12 mL / L·h; when the OD value was between 60 and 80, the feed rate was 13 to 15 mL / L·h; and when the OD value was between 80 and 110, the feed rate was 18 to 20 mL / L·h. During the mid-stage of thiamphetamine production: continuous fed-batch operation was implemented. Specifically, between 18 and 28 hours of fermentation, the fed-batch rate was adjusted based on the thiamphetamine content in the fermentation broth, monitored every 2 hours. The feeding rate was 20–25 mL / L·h when the thiamphetamine content was 200–2000 mg / L; 25–30 mL / L·h when the content was 2000–3000 mg / L; 30–40 mL / L·h when the content was 3000–5000 mg / L; and 40–50 mL / L·h when the content was above 5000 mg / L. c. Late stage of thiamphetamine production: Continuous fed-batch operation. Specifically, from 28 h of fermentation to discharge, fed-batch operation is performed based on the thiamphetamine content in the fermentation broth monitored every 1 h. When the thiamphetamine content is below 6000 mg / L, the fed-batch rate is 50–55 mL / L·h; when the thiamphetamine content is 6000–8000 mg / L, the fed-batch rate is 60–65 mL / L·h. The second method: The feeding step in step (6) is as follows: In the early stage of α-fermentation of 37-peptide: intermittent fed-batch feeding was used. Specifically, from 4 to 18 hours of fermentation, fed-batch feeding was performed every 2 hours based on the OD value of the fermentation broth. When the OD value was between 2 and 20, 300 mL of feed was fed; when the OD value was between 20 and 40, 380 mL of feed was fed; when the OD value was between 40 and 60, 440 mL of feed was fed; when the OD value was between 60 and 80, 560 mL of feed was fed; and when the OD value was between 80 and 110, 760 mL of feed was fed. b. Mid-stage production of thiamphetamine: Intermittent fed-batch feeding, specifically, between 18 and 28 hours of fermentation, fed-batch feeding is performed according to the thiamphetamine content in the fermentation broth monitored every 2 hours. When the thiamphetamine content is 200–2000 mg / L, 900 mL of feed is added; when the thiamphetamine content is 2000–3000 mg / L, 1100 mL of feed is added; when the thiamphetamine content is 3000–5000 mg / L, 1400 mL of feed is added; and when the thiamphetamine content is above 5000 mg / L, 1800 mL of feed is added. c. Late stage of thiamphetamine production: Intermittent fed-batch feeding. Specifically, from 28 hours of fermentation until discharge, fed-batch feeding is performed according to the thiamphetamine content in the fermentation broth monitored every hour. When the thiamphetamine content is below 6000 mg / L, 1050 mL of fed-batch feeding is added; when the thiamphetamine content is between 6000 and 8000 mg / L, 1200 mL of fed-batch feeding is added. The feed contains 10 g / L glucose, 8 g / L corn steep liquor, and the remainder is drinking water; The 30 m³ fermentation medium contained yeast extract at a concentration of 8 g / L, glucose at a concentration of 1 g / L, water-soluble corn starch at a concentration of 5 g / L, corn steep liquor at a concentration of 13 g / L, ammonium chloride at a concentration of 1.5 g / L, sodium chloride at a concentration of 2 g / L, and defoamer at a concentration of 3 g / L, with the remainder being drinking water.

2. The method for high-density fermentation industrial production of kiwifruit peptide as described in claim 1, characterized in that, The culture medium for activating the microbial strain was formulated as follows: 2 g beef extract, 2 g yeast extract, 10 g peptone, 5 g NaCl, 15 g agar, and water was added to a final volume of 1000 mL to adjust the pH to 7.

4. The culture medium for both the 30 L and 3 m³ seed tanks was formulated as follows: 13 g / L yeast extract, 10 g / L peptone, 4 g / L water-soluble corn starch, 8 g / L corn steep liquor, 5 g / L glucose, 1.5 g / L dipotassium hydrogen phosphate, 2 g / L sodium chloride, 0.5 g / L magnesium sulfate, with the remainder being drinking water.

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