Fermentation Process for Producing Bioactive Pentapeptide by Penicillium oxalicum SG-4

Abstract

The present invention relates to the field of microbial fermentation technology, and particularly relates to a fermentation process for producing active pentapeptide by Penicillium oxalicum SG-4. The process comprises the following steps: preparing a seed culture medium; inoculating Penicillium oxalicum SG-4 into the seed culture medium and culturing to obtain a liquid fermentation strain; preparing a liquid fermentation medium; inoculating the liquid fermentation strain into the liquid fermentation medium, culturing and then ending the fermentation; filtering the fermentation broth and collecting the supernatant, which is the crude liquid. The present invention creatively adjusts the fermentation culture rotation speed and the liquid loading amount; through condition optimization, the pentapeptide yield in the metabolites of Penicillium oxalicum is greatly improved: the content of the Three Gorges peptide pentapeptide in the fermentation broth prepared by this production process is 1.77 g / L, which is 90% higher than that before process optimization, facilitating subsequent separation and purification; the production process of the present invention is simple, has high stability, and is convenient for large-scale production and promotion.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and particularly relates to an optimized fermentation process for producing a novel active pentapeptide by Penicillium oxalicum SG-4. Background Art

[0002] In our laboratory, an endophytic fungus SG-4 was screened from Myricaria laxiflora, a shrub specifically distributed in the Three Gorges area with a long history of citrus cultivation. Previous studies found that the metabolite active pentapeptide of the endophytic fungus Penicillium oxalicum SG-4 was named Three Gorges peptide. Three Gorges peptide has a good antagonistic effect on citrus spoilage-causing bacteria and can be used as a good biological preservative. Compared with other preservatives, it not only has good activity but also high safety, and has broad application prospects, providing a new way for the research of citrus anti-corrosion. However, the initial yield of the metabolite Three Gorges peptide produced by Penicillium oxalicum SG-4 in shake-flask fermentation is relatively low, only 0.14 g / L.

[0003] Based on this, it is of great significance to provide a strain that can ferment and produce a novel active pentapeptide and a fermentation process method that can significantly increase the yield. Summary of the Invention

[0004] Aiming at the problem of low yield of the strain that can ferment and produce a novel active pentapeptide in the prior art, the present invention provides an optimized fermentation process for producing a novel active pentapeptide by Penicillium oxalicum, which improves the yield by improving the medium composition and culture conditions.

[0005] A fermentation process for producing an active pentapeptide by Penicillium oxalicum SG-4, the process comprising the following steps:

[0006] S1: Prepare a seed medium, the seed medium comprising a carbon source, a nitrogen source, and inorganic salts;

[0007] S2: Inoculate Penicillium oxalicum SG-4 into the seed medium and culture to obtain a liquid fermentation strain;

[0008] S3: Prepare a liquid fermentation medium, the liquid fermentation medium comprising a carbon source, a nitrogen source, and inorganic salts;

[0009] S4: Inoculate the liquid fermentation strain into the liquid fermentation medium, culture and then end the fermentation;

[0010] S5: After filtering the fermentation broth, collect the supernatant, which is the crude liquid.

[0011] In step S1, the carbon source is dextrin, and its concentration is 30-50 g / L; the nitrogen source is a mixture of corn steep liquor and (NH3)2SO4, wherein the concentration of corn steep liquor is 20-25 g / L, the concentration of (NH3)2SO4 is 2-4 g / L, and the inorganic salt is magnesium sulfate, and the concentration of magnesium sulfate is 0.05-0.1 g / L.

[0012] In step S2, the strain culture conditions are pH 5 - 6.5, rotation speed 100 - 200 r / min, culture temperature 22 - 25°C, and culture time 6 - 8 days.

[0013] In the said S2, the strain culture conditions are pH 5.5, rotation speed 200 r / min, culture temperature 22°C, and culture time 6 days.

[0014] In step S3, the carbon source is dextrin with a mass concentration of 30 - 50 g / L dextrin, the nitrogen source is corn steep liquor with a mass concentration of 20 - 25 g / L, and the inorganic salt is ferric chloride with a mass concentration of 0.05 - 0.1 g / L.

[0015] In the said S3, the carbon source in the liquid fermentation medium is 40 g / L dextrin.

[0016] In the said S3, the nitrogen source in the liquid fermentation medium is 20 - 30 g / L corn steep liquor.

[0017] In the said S4, the strain culture conditions are pH 5 - 6.5, rotation speed 100 - 200 r / min, culture temperature 22 - 25°C, and liquid loading volume 30 - 40%.

[0018] In the said S4, the strain culture conditions are pH 5.0, rotation speed 200 r / min, culture temperature 22°C, and liquid loading volume 40%.

[0019] The beneficial effects of the present invention are as follows: The present invention provides an optimal liquid medium formulation, culture temperature, and rotation speed for the large - scale production of tricpeptide by Penicillium oxalicum SG - 4 strain, laying a foundation for the large - scale production and industrial application of Penicillium oxalicum SG - 4 strain, and having good application prospects.

[0020] The preservation information of Penicillium oxalicum SG - 4 refers to Patent 201510410093.X. Penicillium oxalicum SG4 was preserved at the China Center for Type Culture Collection on April 30, 2015, with the preservation number: CCTCC M2015270, and the preservation address is Wuhan University, Wuhan, Hubei. Description of the Drawings

[0021] Figure 1 Bar chart of the influence of 40 g / L different carbon sources on seed culture.

[0022] Figure 2 Bar chart of the influence of 2 g / L different nitrogen sources on seed culture.

[0023] Figure 3 Bar chart of the influence of 0.05 g / L different inorganic salts on seed culture.

[0024] Figure 4 Bar graph showing the effect of pH on seed culture.

[0025] Figure 5 Bar graph showing the effect of temperature on seed culture.

[0026] Figure 6 Line graph showing the effect of different seed ages on seed culture.

[0027] Figure 7 Bar graph showing the effect of different carbon sources at 40 g / L on fermentation.

[0028] Figure 8 Bar graph showing the effect of different nitrogen sources on fermentation.

[0029] Figure 9 Bar graph showing the effect of different inorganic salts on fermentation.

[0030] Figure 10 Bar graph showing the effect of different pH values on fermentation.

[0031] Figure 11 Bar graph showing the effect of different temperatures on fermentation.

[0032] Figure 12 Bar graph showing the effect of different rotation speeds on fermentation.

[0033] Figure 13 Bar graph showing the effect of different liquid volumes on fermentation. Detailed implementation method

[0034] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents, measurement methods, and equipment used in the present invention are all conventional reagents, methods, and equipment in the technical field.

[0035] Example 1

[0036] Determination of the components of the seed culture medium and the culture conditions of the strain

[0037] Based on the initial seed culture medium, with 24 °C, pH 6.0, 200 r / min for 6 days as the basic culture conditions, a single-factor optimization strategy was used to conduct experiments on optimizing the culture conditions of the strain. The optimal formula of the seed culture medium was determined by adding carbon sources, nitrogen sources, and inorganic salts, and the optimal culture conditions were determined by adjusting pH, culture temperature, rotation speed, and seed age.

[0038] Initial seed culture medium: PDA liquid medium

[0039] (1) Determination of carbon source

[0040] Based on the seed initial medium - PDA medium, using the single - factor optimization strategy, SG - 4 was respectively inoculated into 150 mL of liquid medium, cultured at 24 °C for 6 days, with a rotation speed of 200 r / min. After 6 days of culture, the bacteria in 150 mL of the liquid were dried and weighed for carbon source optimization, and different carbon sources were selected for optimization. Medium No. 1: Corn starch 40 g / L; Medium No. 2: Dextrin 40 g / L.

[0041] The effects of different carbon sources on the bacterial accumulation amount can be seen in Figure 1 . It can be seen from the figure that the most suitable carbon source for bacterial growth is dextrin

[0042] (2) Determination of nitrogen source

[0043] Based on Medium No. 1, using the single - factor optimization strategy, SG - 4 was respectively inoculated into 150 mL of liquid medium, cultured at 24 °C for 6 days, with a rotation speed of 200 r / min. After 6 days of culture, the bacteria in 150 mL of the liquid were dried and weighed for nitrogen source optimization, and different nitrogen sources were selected for optimization. Medium No. 1: Dextrin 40 g / L, corn steep liquor 25 g / L, sucrose 5 g / L; Medium No. 2: Dextrin 40 g / L, corn steep liquor 25 g / L, sucrose 5 g / L, (NH3)2SO4 2 g / L; Medium No. 3: Dextrin 40 g / L, corn steep liquor 25 g / L, sucrose 5 g / L, yeast powder 2 g / L.

[0044] The effects of different nitrogen sources on the bacterial accumulation amount can be seen in Figure 2 . It can be seen from the figure that the most suitable nitrogen source for bacterial growth is (NH3)2SO4

[0045] (3) Determination of inorganic salts

[0046] Based on Medium No. 0, using the single - factor optimization strategy, SG - 4 was respectively inoculated into 150 mL of liquid medium, cultured at 24 °C with a rotation speed of 200 r / min. After 6 days of culture, the bacteria in 150 mL of the liquid were dried and weighed for inorganic salt optimization, and different inorganic salts were selected for optimization.

[0047] Medium: Inorganic salts, corn starch 40 g / L, corn steep liquor 25 g / L, sucrose 5 g / L

[0048]

[0049]

[0050] The effects of different inorganic salts on the bacterial accumulation amount can be seen in Figure 3 . It can be seen from the figure that the most suitable inorganic salt for bacterial growth is Mg ion

[0051] (4) Determination of pH value

[0052] Through the optimization experiment of the seed culture medium, the optimal formula of the seed culture medium was determined as follows: corn starch 40 g / L, corn steep liquor 25 g / L, sucrose 5 g / L, MgSO4 0.05 g / L. On this basis, an optimization experiment of the seed culture conditions was carried out. The prepared seed culture medium was adjusted to pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, and pH 6.5 respectively, and cultured at 24 °C and 200 r / min for 6 days. The culture conditions are as Figure 4 shown, and pH 5.5 is the optimal culture condition.

[0053] (5) Determination of temperature

[0054] Through the optimization experiment of the seed culture medium, the optimal formula of the seed culture medium was determined as follows: dextrin 40 g / L, corn steep liquor 25 g / L, sucrose 5 g / L, MgSO4 0.05 g / L. On this basis, an optimization experiment of the seed culture conditions was carried out. The prepared seed culture medium was adjusted to pH 5.0 and cultured at 28 °C, 24 °C, and 22 °C for 6 days respectively. As Figure 5 shown, 22 °C is the optimal culture temperature.

[0055] (6) Determination of seed age

[0056] SG-4 was inoculated into 150 mL of PDA liquid medium. From the second day after inoculation, all the bacteria in 150 mL of the liquid were taken out and dried every day, and their weights were measured. As Figure 6 shown, the 6th day is the optimal seed age.

[0057] In summary, the optimal components of the seed culture medium and the bacterial strain culture conditions were screened as follows:

[0058] The seed culture medium includes dextrin 40 g / L, corn steep liquor 25 g / L, sucrose 5 g / L, and MgSO4 0.05 g / L

[0059] The bacterial strain culture conditions are pH 5.5, culture temperature 22 °C, and culture time 6 days.

[0060] Determination of the components of the fermentation culture medium and the bacterial strain culture conditions in Screening Example 2

[0061] On the basis of the initial fermentation medium, first, the bacterial strain liquid was prepared with the PDA liquid medium as the seed culture condition and inoculated into 150 mL of the initial fermentation medium. With 24 °C, pH 3.6, 120 rpm, and culturing for 6 days as the basic culture conditions, a single-factor optimization strategy was used to carry out an optimization experiment of the bacterial strain culture conditions. The optimal formula of the bacterial strain culture medium was determined by adding carbon sources, nitrogen sources, and inorganic salts, and the optimal culture conditions were determined by adjusting pH, culture temperature, rotation speed, and ventilation volume.

[0062] Initial fermentation medium: 40 g / L corn starch, 25 g / L corn steep liquor, 5 g / L sucrose

[0063] (1) Determination of carbon source

[0064] Based on the initial seed medium - PDA medium, using the single - factor optimization strategy, SG - 4 was inoculated into 150 mL of liquid medium respectively. At 24 °C and a rotation speed of 200 r / min, after culturing for 6 days, the fermentation broth was filtered. After centrifugation of the fermentation broth, the upper clarified liquid was diluted at a ratio of 1:5, and the content of Three Gorges peptide was detected by high - performance liquid chromatography. Different carbon sources were selected for optimization. Medium No. 1: 40 g / L corn starch; Medium No. 2: 40 g / L dextrin. The corresponding content of Three Gorges peptide is shown in Figure 7 . It can be seen from the figure that the most suitable carbon source for the production of pentapeptide by the bacteria is dextrin. Considering comprehensive factors, 40 g / L dextrin was selected as the carbon source of the fermentation medium.

[0065] (2) Determination of nitrogen source

[0066] Based on Medium No. 1, using the single - factor optimization strategy, SG - 4 was inoculated into 150 mL of liquid medium respectively. At 24 °C and a rotation speed of 200 r / min, after culturing for 6 days, the fermentation broth was filtered. After centrifugation of the fermentation broth, the upper clarified liquid was diluted at a ratio of 1:5, and the content of Three Gorges peptide was detected by high - performance liquid chromatography. Different nitrogen sources were selected for optimization. Medium No. 1: 40 g / L dextrin, 25 g / L corn steep liquor, 5 g / L sucrose; Medium No. 2: 40 g / L dextrin, 25 g / L corn steep liquor, 5 g / L sucrose, 2 g / L (NH3)2SO4; Medium No. 3: 40 g / L dextrin, 25 g / L corn steep liquor, 5 g / L sucrose, 2 g / L yeast powder.

[0067] The corresponding content of Three Gorges peptide is shown in Figure 8 . It can be seen from the figure that it is not suitable to add excessive nitrogen source for the production of Three Gorges peptide by the bacteria.

[0068] (3) Determination of inorganic salts

[0069] Based on Medium No. 0, using the single - factor optimization strategy, SG - 4 was inoculated into 150 mL of liquid medium respectively. At 24 °C and a rotation speed of 200 r / min, after culturing for 6 days, the fermentation broth was filtered. After centrifugation of the fermentation broth, the upper clarified liquid was diluted at a ratio of 1:5, and the content of Three Gorges peptide was detected by high - performance liquid chromatography. Different inorganic salt ions were selected for optimization.

[0070] Culture medium Inorganic salt Inorganic salt content g / L 0 Deionized water \ 1 Tap water \ 2 <![CDATA[Mg 2+ > 0.05 3 <![CDATA[Zn 2+ > 0.05 4 <![CDATA[Mn 2+ > 0.05 5 <![CDATA[Fe 2+ > 0.05 6 <![CDATA[Fe 3+ > 0.05 7 <![CDATA[Cu 2+ > 0.05

[0071] It was found through research that the most suitable inorganic salt for the fermentation production of Penicillium oxalicum SG - 4 is: FeCl3.

[0072] (4) Determination of pH value

[0073] Fermentation medium: 40 g / L corn starch, 25 g / L corn steep liquor, 5 g / L sucrose; Add 20% NaOH to the prepared fermentation medium respectively to adjust the pH to 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5 respectively; Then inoculate SG-4 into 150 mL of its liquid medium respectively, at 24 °C, with a rotation speed of 200 r / min. After culturing for 6 days, filter to obtain the fermentation broth; Centrifuge the fermentation broth and take the upper clarified liquid for dilution at a ratio of 1:5, and detect the content of Three Gorges peptide by high performance liquid chromatography. The yield of Three Gorges peptide is the highest at pH 5.5.

[0074] (5) Determination of temperature

[0075] Using the initial fermentation medium, the fermentation medium formula is: 40 g / L corn starch, 25 g / L corn steep liquor, 5 g / L sucrose. On this basis, optimize the fermentation culture conditions. Inoculate SG-4 into 150 mL of liquid medium respectively, at 28 °C, 24 °C, 22 °C, with a rotation speed of 200 r / min. After culturing for 6 days, filter to obtain the fermentation broth; Centrifuge the fermentation broth and take the upper clarified liquid for dilution at a ratio of 1:5, and detect the content of Three Gorges peptide by high performance liquid chromatography. The optimal temperature is 22 °C.

[0076] (6) Determination of rotation speed and liquid loading volume

[0077] ① Using PDA medium, the medium formula is: 200 g / L potatoes, 20 g / L sucrose. On this basis, optimize the fermentation culture conditions. Set the culture rotation speeds at 120 r / min, 160 r / min, and 200 r / min respectively, at 24 °C, with a liquid loading volume of 30%. After culturing for 6 days, filter to obtain the fermentation broth. After centrifuging the fermentation broth, take the upper clarified liquid for dilution at a ratio of 1:5, and then detect the content of Three Gorges peptide by high performance liquid chromatography. The culture conditions are as Figure 12 shown. 200 r / min is the optimal culture rotation speed.

[0078] ② Using PDA medium, the medium formula is: 200 g / L potatoes, 20 g / L sucrose. On this basis, optimize the fermentation culture conditions. Set the liquid loading volumes at 10%, 20%, 30%, and 40% respectively, at 24 °C, with a rotation speed of 200 r / min. After culturing for 6 days, filter to obtain the fermentation broth. After centrifuging the fermentation broth, take the upper clarified liquid for dilution at a ratio of 1:5, and then detect the content of Three Gorges peptide by high performance liquid chromatography. The culture conditions are as Figure 13 shown. The difference in the yield of Three Gorges peptide between 150 mL and 200 mL is not significant, so the more the liquid loading volume, the more the yield of Three Gorges peptide.

[0079] In summary, the optimal fermentation medium components and strain culture conditions screened are as follows:

[0080] The fermentation medium includes 40 g / L of dextrin, 25 g / L of corn steep liquor, 5 g / L of sucrose, and 0.05 g / L of FeCl3

[0081] The culture conditions for the strain are pH 5.5, culture temperature 22°C, liquid filling volume 40%, and rotation speed 200 r / min

[0082] Example 3

[0083] Based on the screening in Examples 1 and 2, in this case, an optimized fermentation process for a novel active pentapeptide produced by Penicillium oxalicum SG-4 includes the following steps:

[0084] S1: Prepare a seed medium, which includes a carbon source, a nitrogen source, and inorganic salts. The carbon source is 40 g / L of dextrin, the nitrogen source is 25 g / L of corn steep liquor plus (NH3)2SO4, and the inorganic salt is 0.05 g / L of magnesium sulfate;

[0085] S2: Inoculate Penicillium oxalicum SG-4 into the seed medium and culture to obtain a seed liquid;

[0086] S3: Prepare a liquid fermentation medium, which includes a carbon source, a nitrogen source, and inorganic salts. The carbon source is 40 g / L of dextrin, the nitrogen source is 25 g / L of corn steep liquor, and the inorganic salt is 0.05 g / L of ferric chloride; S4: Inoculate the seed liquid into the liquid fermentation medium and end the fermentation after culturing;

[0087] S5: After filtering the fermentation broth, collect the supernatant, which is the crude liquid.

[0088] In S1, use a 500 mL Erlenmeyer flask to hold 150 mL of the seed medium.

[0089] In S2, the optimal culture conditions for the seed liquid are pH 5.5, rotation speed 200 r / min, culture temperature 22°C, and culture time 6 days.

[0090] In S3, use a 500 mL Erlenmeyer flask to hold 150 mL of the liquid fermentation medium.

[0091] In S3, the carbon source in the liquid fermentation medium is 40 g / L of dextrin

[0092] In S3, the nitrogen source in the liquid fermentation medium is 25 g / L of corn steep liquor.

[0093] In S4, the optimal culture conditions for the strain fermentation are pH 5.0, culture temperature 22°C, for the rotation speed and liquid filling volume, rotation speed 200 r / min, liquid filling volume 40%.

[0094] Through the optimization of the culture medium components and culture conditions, the pentapeptide yield in the metabolites of Penicillium oxalicum has been greatly increased. The fermented liquid obtained by this production process contains 1.77 g / L of Three Gorges peptide, which is 90% higher than that before the process optimization.

[0095] Although the present invention has been described in detail by referring to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention. / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope described in the claims.

Claims

1. A fermentation process for producing active pentapeptide by Penicillium oxalicum SG-4, characterized in that, The process includes the following steps: S1: Prepare a seed culture medium, which includes a carbon source, a nitrogen source, and inorganic salts. The carbon source is dextrin with a concentration of 30 - 50 g / L; the nitrogen source is a mixture of corn steep liquor and (NH3)2SO4, where the concentration of corn steep liquor is 20 - 25 g / L and the concentration of (NH3)2SO4 is 2 - 4 g / L. The inorganic salt is magnesium sulfate with a concentration of 0.05 - 0.1 g / L; S2: Inoculate Penicillium oxalicum SG - 4 into the seed culture medium and cultivate to obtain a liquid fermentation strain. The preservation number of Penicillium oxalicum SG - 4 is: CCTCC NO: M 2015270; S3: Prepare a liquid fermentation medium, which includes a carbon source, a nitrogen source, and inorganic salts. The carbon source is dextrin with a mass concentration of 30 - 50 g / L, the nitrogen source is corn steep liquor with a mass concentration of 20 - 25 g / L, and the inorganic salt is ferric chloride with a mass concentration of 0.05 - 0.1 g / L; S4: Inoculate the liquid fermentation strain into the liquid fermentation medium, cultivate, and then end the fermentation; S5: Filter the fermentation broth and collect the supernatant, which is the crude liquid, In step S2, the strain cultivation conditions are pH 5 - 6.5, rotation speed 100 - 200 r / min, cultivation temperature 22 - 25 °C, and cultivation time 6 - 8 days, In step S4, the strain cultivation conditions are pH 5 - 6.5, rotation speed 100 - 200 r / min, cultivation temperature 22 - 25 °C, and liquid loading volume 30 - 40%.

2. The fermentation process for producing active pentapeptide by Penicillium oxalicum SG-4 according to claim 1, characterized in that, In S2, the strain cultivation conditions are pH 5.5, rotation speed 200 r / min, cultivation temperature 22 °C, and cultivation time 6 days.

3. The fermentation process for producing active pentapeptide by Penicillium oxalicum SG-4 as described in claim 1, wherein, In S3, the carbon source in the liquid fermentation medium is 40 g / L dextrin.

4. The fermentation process for producing active pentapeptide by Penicillium oxalicum SG-4 according to claim 1, characterized in that, In S3, the nitrogen source in the liquid fermentation medium is 20 - 30 g / L corn steep liquor.

5. The fermentation process for producing active pentapeptide by Penicillium oxalicum SG-4 according to claim 1, wherein, In S4, the strain cultivation conditions are pH 5.0, rotation speed 200 r / min, cultivation temperature 22 °C, and liquid loading volume 40%.

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