A fermentation medium and a fermentation method for expressing bovine lactoferrin

Abstract

The application discloses a fermentation medium and a fermentation method for expressing bovine lactoferrin, and belongs to the technical field of fermentation. The fermentation medium comprises the following components: yeast extract 10-20 g / L, proteose peptone 20-30 g / L, glycerol or glucose 10-20 g / L, yeast nitrogen base without amino acid 12-15 g / L, dipotassium hydrogen phosphate 4-6 g / L, potassium dihydrogen phosphate 2-3 g / L, biotin 0.0002-0.0005 g / L, amino acid 4-46 g / L, ferrous salt 2-5 g / L, sugar material 5-10 g / L and sugar material derivative 5-10 g / L. The fermentation medium can meet the growth and metabolism requirements of the bacteria and improve the expression amount of bovine lactoferrin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation, in particular to a fermentation medium and a fermentation method for expressing bovine lactoferrin. BACKGROUND

[0002] Lactoferrin is a natural antibacterial glycoprotein mainly existing in human milk. Due to its specific iron ion binding capacity, it has strong bactericidal and bacteriostatic capacity for gram-positive and gram-negative bacteria. In addition, lactoferrin also has multiple effects such as antiviral, antioxidant and immunomodulation. Therefore, lactoferrin is widely used in various fields such as milk powder, food and medicine. The United States FDA, Japan, South Korea and other countries currently allow lactoferrin to be used as a food additive in sports and functional foods. In China, the National Standard "Hygienic Standard for the Use of Food Additives GB2760-19966" added lactoferrin to infant formula milk powder in 2004.

[0003] Studies have found that bovine lactoferrin has extremely similar biological activity to human lactoferrin, but the separation and preparation of bovine lactoferrin from bovine milk is difficult and costly. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a fermentation medium and a fermentation method for expressing bovine lactoferrin, which can overcome the problems of high cost and difficulty in preparing bovine lactoferrin in the prior art.

[0005] In a first aspect, the present application provides a fermentation medium for expressing bovine lactoferrin, comprising the following components: yeast extract 10-20 g / L, peptone 20-30 g / L, glycerol or glucose 10-20 g / L, yeast nitrogen base (YNB) without amino acids 12-15 g / L, dipotassium hydrogen phosphate 4-6 g / L, potassium dihydrogen phosphate 2-3 g / L, biotin 0.0002-0.0005 g / L, amino acids 4-46 g / L, ferrous salt 2-5 g / L, sugar substance 5-10 g / L and sugar substance derivative 5-10 g / L.

[0006] Compared with the prior art, the fermentation medium for expressing bovine lactoferrin contains 4-46 g / L of amino acids, 2-5 g / L of ferrous salt, 5-10 g / L of saccharide substances and 5-10 g / L of saccharide substance derivatives, which jointly improve the expression amount of bovine lactoferrin. In addition, the initial concentration of glycerol or glucose as the carbon source is low, which reduces the concentration inhibition of the substrate, improves the growth rate of the bacteria, shortens the lag phase of the bacteria, and thus shortens the growth cycle. Furthermore, the yeast extract and the proteose peptone in the fermentation medium can improve the stability of the secreted protein, prevent or reduce the degradation of the secreted protein, and the biotin in the fermentation medium plays a role as a coenzyme of various enzymes in the metabolic process of the bacteria, which ensures the activity of the enzyme system in the bacteria.

[0007] Further, the amino acids include L-histidine, and the content of the L-histidine in the fermentation medium is 4-6 g / L.

[0008] Compared with the prior art, the content of the amino acid in the fermentation medium is limited to 4-6 g / L of L-histidine in the technical scheme, and since the bovine lactoferrin in the application uses histidine as a selection marker expression system, the addition of L-histidine in the medium can promote the induced expression of bovine lactoferrin in the late fermentation.

[0009] Further, the amino acids further include 1-2 kinds of glutamic acid, aspartic acid, leucine or alanine.

[0010] The content of the glutamic acid in the fermentation medium is 5-10 g / L.

[0011] The content of the aspartic acid in the fermentation medium is 15-20 g / L.

[0012] The content of the leucine in the fermentation medium is 5-15 g / L.

[0013] The content of the alanine in the fermentation medium is 10-20 g / L.

[0014] Compared with the prior art, the amino acid further includes 1-2 kinds of glutamic acid, aspartic acid, leucine or alanine in the technical scheme, and the amino acid added in the medium has a synergistic effect with L-histidine, which can further promote the synthesis of bovine lactoferrin by the bacteria.

[0015] Further, the saccharide substances include monosaccharides; the monosaccharides include mannose and / or galactose; and / or the ferrous salt is ferrous sulfate.

[0016] Compared with the prior art, the above technical scheme adds mannose and / or galactose and ferrous sulfate in the culture medium. The above specific types of sugar substances can improve the synthesis efficiency of bovine lactoferritin and increase the expression amount of bovine lactoferritin. In addition, bovine lactoferritin is a glycoprotein containing iron ions, and the ferrous ions in ferrous sulfate can play a role in the synthesis of bovine lactoferritin and also improve the enzyme activity of the fermentation bacteria and participate in the metabolic reaction of the bacteria.

[0017] The derivative of the sugar substance is N-acetylglucosamine, and the content of N-acetylglucosamine in the fermentation medium is 5-10 g / L.

[0018] Compared with the prior art, the above technical scheme limits the type and content of the derivative of the sugar substance. The appropriate amount of N-acetylglucosamine in the culture medium cooperates with other substances in the culture medium to promote the synthesis of bovine lactoferritin, thereby increasing the expression amount of bovine lactoferritin.

[0019] In a second aspect, the present application provides a fermentation method for producing bovine lactoferritin, comprising the following steps:

[0020] Inoculating the seed liquid of the recombinant Pichia pastoris containing the bovine lactoferritin coding gene into the above fermentation medium to start fermentation;

[0021] After 30-35 h of culture, feeding medium A is added, and the OD value of the fermentation broth is detected by sampling; 600 When the OD 600 value reaches 150-200, the feeding medium A is stopped; the feeding medium A for the culture stage comprises 500-600 g / L of glycerol and 5-8 g / L of yeast extract;

[0022] The temperature is reduced to 22-24℃, the feeding medium B for the induction stage is added, and the expression of bovine lactoferritin by the recombinant Pichia pastoris is started; the feeding medium B for the induction stage comprises 0.5-1 L / L of methanol and 5-10 g / L of proteose peptone; and the induction culture is performed for 35-40 h.

[0023] Compared with the prior art, the present application effectively improves the growth rate of the bacteria and the expression level of the target protein by controlling the ratio of the carbon source and the nitrogen source in the feeding medium. In addition, the carbon source and the nitrogen source are combined in the present application, which brings great convenience to the fermentation process personnel and enables them to accurately control the addition amount of the feeding medium.

[0024] Further, ammonia water is used to control the pH of the fermentation broth to be 5-6 during the fermentation culture process; the pH is maintained at 5.5-6 during the bacteria culture stage, and the pH is maintained at 5-5.5 during the induction stage.

[0025] Compared with the prior art, the above technical scheme maintains different pH values in the bacterial culture stage and the induction stage respectively, which is beneficial to the growth and reproduction in the bacterial culture stage and the protein expression in the induction stage.

[0026] Further, the feeding rate of the culture medium A in the culture stage is 20-40 g / L / h in terms of the volume of the fermentation liquid.

[0027] The feeding rate of the culture medium B in the induction stage is 1-3 ml / L / h in the first 0-5 h and 4-6 ml / L / h after 5 h.

[0028] Compared with the prior art, the above technical scheme limits the feeding rate of the culture medium A in the culture stage and the culture medium B in the induction stage, and the fermentation bacteria can be reasonably provided with nutrients at the above specific feeding rate, so as to promote the growth and reproduction of the bacteria and the expression of the target protein.

[0029] Further, the recombinant Pichia pastoris is obtained by connecting the bovine lactoferrin gene, the PAOX1 promoter and the vector pPIC9K and then transforming them into the GS115 type Pichia pastoris.

[0030] The bovine lactoferrin is selected by using histidine as a marker.

[0031] Compared with the prior art, the above technical scheme limits the selection marker of the bovine lactoferrin, and the bovine lactoferrin selected by using histidine as a marker is more suitable for the above fermentation medium and fermentation method, has a higher expression amount, and is beneficial to subsequent protein purification. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical schemes and beneficial effects of the present application clearer, the present application will be further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0033] In a first aspect, the embodiments of the present application provide a fermentation medium for expressing bovine lactoferrin, which comprises the following components: yeast extract 10-20 g / L, proteose peptone 20-30 g / L, glycerol or glucose 10-20 g / L, yeast nitrogen base (YNB) without amino acid 12-15 g / L, dipotassium hydrogen phosphate 4-6 g / L, potassium dihydrogen phosphate 2-3 g / L, biotin 0.0002-0.0005 g / L, amino acid 4-46 g / L, ferrous salt 2-5 g / L, sugar substance 5-10 g / L and sugar substance derivative 5-10 g / L.

[0034] For example, the concentration of the yeast extract in the fermentation medium for expressing bovine lactoferritin can be 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L or 20 g / L, preferably 12-18 g / L.

[0035] For example, the concentration of the protein peptone in the fermentation medium for expressing bovine lactoferritin can be 20 g / L, 22 g / L, 24 g / L, 26 g / L, 28 g / L or 30 g / L, preferably 24-28 g / L.

[0036] For example, the concentration of the glycerol or glucose in the fermentation medium for expressing bovine lactoferritin can be 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L or 20 g / L, preferably 12-16 g / L.

[0037] The amino acid-free yeast nitrogen source can be a commercially available product, for example, the amino acid-free yeast nitrogen source (YNB) produced by Solarbio Company (Cat#Y8040) can be selected.

[0038] For example, the concentration of the amino acid-free yeast nitrogen source in the fermentation medium for expressing bovine lactoferritin can be 12 g / L, 13 g / L, 14 g / L or 15 g / L, preferably 13-14 g / L.

[0039] For example, the concentration of the potassium phosphate dibasic in the fermentation medium for expressing bovine lactoferritin can be 4 g / L, 5 g / L or 6 g / L, preferably 5 g / L.

[0040] For example, the concentration of the potassium phosphate dibasic in the fermentation medium for expressing bovine lactoferritin can be 2 g / L or 3 g / L.

[0041] For example, the concentration of the biotin in the fermentation medium for expressing bovine lactoferritin can be 0.0002 g / L, 0.0003 g / L, 0.0004 g / L or 0.0005 g / L, preferably 0.0003-0.0004 g / L.

[0042] The fermentation medium for expressing bovine lactoferritin provided in the technical solution can meet the growth and metabolism requirements of the bacteria and promote the expression of bovine lactoferritin by the bacteria, in addition, the growth cycle of the bacteria can be shortened by 3-8 h, and the expression amount of bovine lactoferritin can be increased to 4-5 g / L.

[0043] Further, on the basis of the above embodiment, the amino acid in the embodiment of the present application can be L-histidine, and the content of L-histidine in the fermentation medium can be 4-6 g / L.

[0044] For example, the content of L-histidine in the fermentation medium can be 4 g / L, 5 g / L or 6 g / L, preferably 5 g / L.

[0045] The L-histidine in the technical solution can maintain the intracellular environment of the bacterial cells stable, promote the healthy growth of the cells, and reduce the stress response, and can also promote the induced expression of bovine lactoferrin in the late fermentation period, especially for bovine lactoferrin with a histidine selection marker.

[0046] Further, on the basis of the above embodiment, the amino acid in the embodiment of the present application can further include 1-2 kinds of glutamic acid, aspartic acid, leucine or alanine.

[0047] The content of glutamic acid in the fermentation medium is 5-10 g / L, for example, which can be specifically selected as 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, preferably 6-8 g / L.

[0048] The content of aspartic acid in the fermentation medium is 15-20 g / L, for example, which can be specifically selected as 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L, preferably 16-18 g / L.

[0049] The content of leucine in the fermentation medium is 5-15 g / L, for example, which can be specifically selected as 5 g / L, 8 g / L, 10 g / L, 13 g / L or 15 g / L, preferably 8-13 g / L.

[0050] The content of alanine in the fermentation medium is 10-20 g / L, for example, which can be specifically selected as 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L or 20 g / L, preferably 12-18 g / L.

[0051] The glutamic acid, aspartic acid, leucine or alanine in the technical solution has a synergistic effect with the L-histidine, and can further promote the growth and reproduction of the bacterial cells and the expression of the target protein.

[0052] Further, on the basis of the above embodiment, the sugar substance in the embodiment of the present application includes monosaccharide, and the monosaccharide includes mannose and / or galactose; and / or, the ferrous salt is ferrous sulfate.

[0053] The content of the above-mentioned sugar substance in the fermentation medium is 5-10 g / L, for example, which can be specifically selected as 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, preferably 6-8 g / L.

[0054] The content of the above-mentioned ferrous salt in the fermentation medium is 2-5 g / L, for example, 2 g / L, 3 g / L, 4 g / L or 5 g / L, preferably 3-4 g / L.

[0055] The mannose, galactose and sugar substance derivative N-acetylglucosamine in the above technical solution cooperate with each other to improve the expression amount of bovine lactoferritin.

[0056] Further, on the basis of the above embodiment, the sugar substance derivative in the embodiment of the present application is N-acetylglucosamine; the content of N-acetylglucosamine in the fermentation medium is 5-10 g / L, for example, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, preferably 6-8 g / L.

[0057] The above technical solution adds an appropriate amount of N-acetylglucosamine in the fermentation medium, which specifically cooperates with mannose and / or galactose to further improve the expression amount of bovine lactoferritin.

[0058] In a second aspect, the embodiment of the present application provides a fermentation method for generating bovine lactoferritin, comprising the following steps:

[0059] S1, inoculate the seed liquid of the recombinant Pichia pastoris strain containing the bovine lactoferritin coding gene into the above-mentioned fermentation medium to start fermentation.

[0060] The preparation method of the above-mentioned recombinant Pichia pastoris can be referred to as follows: after the bovine lactoferritin gene with a histidine tag, the PAOX1 promoter and the vector pPIC9K are connected, they are transformed into GS115 type Pichia pastoris to obtain the recombinant Pichia pastoris strain GS115 / pPIC9K-blf. The GenbanK ID of the bovine lactoferritin gene is JX294418.1.

[0061] The preparation method of the above-mentioned seed liquid of the recombinant Pichia pastoris can be referred to as follows:

[0062] The Pichia pastoris recombinant bacteria are coated on the YPD solid culture medium plate, placed in an incubator at 30±1℃ and cultured for 2-4 days, and the smooth, regular shape and larger colonies are picked and inoculated into 200±20 mL YPD liquid medium in a shaking flask, placed in a shaking bed, the temperature is controlled at 30±1℃, the rotation speed is 200-220 rpm, and the culture is carried out for 18-24 h to obtain the seed liquid with OD 600 8-12.

[0063] The preparation of the above-mentioned YPD solid culture medium can be referred to as follows:

[0064] 1-3% yeast extract, 2-4% peptone, 2-3% glucose, 1.5-2% agar, 1500-2000 μg / mL geneticin (G418), sterilized by 121±1℃, 20-30 min high temperature, cooled to 50±1℃, sterile operation in super-clean bench, pour into flat plate, each containing 20±1 mL of medium; % is the mass percentage.

[0065] The preparation of the shake flask containing the above-mentioned YPD liquid medium can be referred to as follows:

[0066] 1-3% yeast extract, 2-4% peptone, 2-3% glucose, 1500-2000 μg / mL geneticin (G418), 400 ml is prepared, 200 ml of medium is contained in each 1000 ml shake flask, and the shake flask is placed in a high-pressure steam sterilization pot, which is set at 121±1℃ for high-temperature sterilization for 20-30 min; % is the mass percentage.

[0067] The specific operation process of this step can be referred to as follows: the prepared shake flask seed liquid is sterilely inoculated into the fermentation tank with sterilized bacteria and adjusted pH, the medium in the fermentation tank is the above-mentioned fermentation medium for expressing bovine lactoferritin, the initial aeration ratio is controlled to be 1-2 VVM, the initial stirring speed is controlled to be 100-200 rpm, the tank pressure is controlled to be 0.03-0.06 MPa, the DO is corrected to be 100%, the DO is controlled to be 10-60% by increasing the stirring speed and the aeration ratio, and the pH is controlled to be 5-6 by using ammonia water.

[0068] S2, after 30-35 h of culture, feeding medium A is added, and the OD 600 value of the fermentation broth is detected every 2 hours, when the OD 600 value reaches 150-200, the feeding of the medium A is stopped; the feeding medium A in the culture stage comprises 500-600 g / L of glycerol and 5-8 g / L of yeast extract. In the above-mentioned culture stage, ammonia water is used to maintain the pH at 5.5-6.

[0069] The preparation of the feeding medium A in the above-mentioned culture stage can be referred to as follows: 500-600 g / L of glycerol and 5-8 g / L of yeast extract, and the sterilization parameters are set to be 121±1℃ and 20-30 min.

[0070] For example, the concentration of glycerol in the feeding medium A in the culture stage can be specifically selected to be 500 g / L, 520 g / L, 540 g / L, 560 g / L, 580 g / L or 600 g / L, and preferably 520-560 g / L.

[0071] For example, the concentration of yeast extract in the feeding medium A in the culture stage can be specifically selected to be 5 g / L, 6 g / L, 7 g / L or 8 g / L, and preferably 6-7 g / L.

[0072] The flow rate of the inducible stage feed medium B is 1-3 ml / L / h in the first 0-5 h and 4-6 ml / L / h after 5 h, based on the volume of the fermentation broth.

[0073] S3, the temperature is reduced to 22-24℃, and the inducible stage feed medium B is added to induce the expression of recombinant Pichia pastoris to express bovine lactoferrin, and the induction culture is performed for 35-40 h. The pH is maintained at 5-5.5 by using ammonia water in the above induction stage.

[0074] The inducible stage feed medium B includes 0.5-1 L / L of methanol and 5-10 g / L of proteose peptone. For example, the concentration of methanol can be specifically selected to be 0.5 L / L, 0.6 L / L, 0.7 L / L, 0.8 L / L, 0.9 L / L or 1 L / L, and preferably 0.6-0.8 L / L; and the concentration of proteose peptone can be specifically selected to be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, and preferably 6-9 g / L.

[0075] The flow rate of the inducible stage feed medium B is 1-3 ml / L / h in the first 0-5 h and 4-6 ml / L / h after 5 h, based on the volume of the fermentation broth.

[0076] For example, the flow rate in the first 0-5 h can be selected to be 1 ml / L / h, 1.5 ml / L / h, 2 ml / L / h, 2.5 ml / L / h or 3 ml / L / h.

[0077] For example, the flow rate after 5 h can be selected to be 4 ml / L / h, 4.5 ml / L / h, 5 ml / L / h, 5.5 ml / L / h or 6 ml / L / h, and preferably 4.5-5.5 ml / L / h.

[0078] The above technical solution combines the carbon source and nitrogen source in the feed medium, which is more convenient than separately feeding the carbon source and nitrogen source, and can more accurately control the ratio of the carbon source and nitrogen source during feeding. At the same time, the above technical solution also optimizes the ratio of the carbon source and nitrogen source in the feed medium, and the above specific ratio and flow rate of the feed medium can effectively improve the growth rate of the bacteria and the expression level of the target protein.

[0079] In order to better illustrate the technical solutions of the present application, the present application also provides the following specific examples. It should be understood that the raw materials used in the following examples are commercially available unless otherwise specified.

[0080] Example 1

[0081] The present example provides a fermentation medium for expressing bovine lactoferrin, comprising the following components: yeast extract 10 g / L, proteose peptone 20 g / L, glucose 10 g / L, YNB 12 g / L, potassium phosphate dibasic 4 g / L, potassium phosphate monobasic 2 g / L, biotin 0.0002 g / L, L-histidine 4 g / L, ferrous sulfate 2 g / L, galactose 5 g / L, and N-acetylglucosamine 5 g / L.

[0082] The present example also provides a fermentation method for producing bovine lactoferrin using the fermentation medium, comprising the following steps:

[0083] S1, the bovine lactoferrin gene with a histidine tag, PAOX1 promoter and vector pPIC9K are connected and then transformed into GS115 Pichia pastoris to obtain recombinant Pichia pastoris bacteria GS115 / pPIC9K-blf. The GenBank ID of the bovine lactoferrin gene is JX294418.1.

[0084] S2, the Pichia pastoris recombinant bacteria are inoculated on YPD solid medium plates and placed in a 30℃ incubator for 3 days. Larger colonies are picked and inoculated into 200 mL YPD liquid medium in a shake flask. The temperature is controlled at 30℃, the rotation speed is 220 rpm, and the culture is performed for 20 h to obtain an OD 600 of 8-12.

[0085] The YPD solid medium comprises the following components: 1% yeast extract, 2% proteose peptone, 2% glucose, 2% agar, and 2000 μg / mL geneticin (G418). The percentage is the mass percentage.

[0086] The YPD liquid medium comprises the following components: 1% yeast extract, 2% proteose peptone, 2% glucose, and 2000 μg / mL geneticin (G418). The percentage is the mass percentage.

[0087] S3, the prepared seed liquid is aseptically inoculated into a sterilized fermentation tank with adjusted pH. The medium in the fermentation tank is the fermentation medium for expressing bovine lactoferrin described above. The initial aeration ratio is controlled at 2 VVM, the initial stirring speed is 200 rpm, the tank pressure is 0.03 MPa, the DO is corrected to 100%, the DO is controlled at 10-60% by increasing the stirring speed and aeration ratio, and the pH is controlled at 5-6 using ammonia water.

[0088] After 30 hours of cultivation, fed-batch culture medium A was introduced for the cell culture phase. This culture medium A consisted of 500 g / L glycerol and 5 g / L yeast extract, fed at a rate of 40 g / L / h. Oddi concentration (OD) of the fermentation broth was measured every 2 hours. 600 Value, OD of fermentation broth 600 When the temperature reaches 150-200, stop feeding medium A during the fed-batch culture phase and start preparing medium B for the fed-batch induction phase.

[0089] The temperature was lowered to 23°C, and feed medium B for the induction phase was added to begin inducing the expression of bovine lactoferrin in recombinant Pichia pastoris. Feed medium B for the induction phase consisted of 0.5 L / L methanol and 5 g / L peptone. The feed medium was added at a rate of 1.5 ml / L / h from 0 to 5 h, and the feed rate after 5 h was 5 ml / L / h.

[0090] During fermentation, the pH was maintained at 5.5–6 before induction by adding ammonia during the cell culture stage, and at 5–5.5 during the induction stage.

[0091] Fermentation was completed after 35 hours of induction, and bovine lactoferrin was then tested.

[0092] Example 2

[0093] This embodiment provides a fermentation medium for expressing bovine lactoferrin, comprising the following components: yeast extract 20 g / L, peptone 30 g / L, glycerol 20 g / L, YNB 15 g / L, dipotassium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 3 g / L, biotin 0.0005 g / L, L-histidine 6 g / L, aspartic acid 20 g / L, alanine 20 g / L, ferrous sulfate 5 g / L, mannose 5 g / L, and N-acetylglucosamine 10 g / L.

[0094] The fermentation method for producing bovine lactoferrin using this fermentation medium differs from that in Example 1 in the following ways:

[0095] The culture medium A for the incubation phase consisted of 600 g / L glycerol and 8 g / L yeast extract, fed at a rate of 20 g / L / h. The temperature was lowered to 22°C, and the induction phase feed medium B, consisting of 1 L / L methanol and 10 g / L peptone, was added at a rate of 1 ml / L / h for the first 0-5 hours, increasing to 4 ml / L / h after 5 hours.

[0096] Example 3

[0097] The present example provides a fermentation medium for expressing bovine lactoferrin, comprising the following components: yeast extract 15 g / L, peptone 25 g / L, glucose 15 g / L, YNB 13 g / L, potassium phosphate dibasic 5 g / L, potassium phosphate monobasic 2.5 g / L, biotin 0.0003 g / L, L-histidine 5 g / L, glutamic acid 7 g / L, leucine 10 g / L, ferrous sulfate 3 g / L, galactose 5 g / L, mannose 5 g / L, and N-acetylglucosamine 7 g / L.

[0098] The fermentation method for producing bovine lactoferrin using the fermentation medium is compared with the fermentation method of Example 1, the difference being that:

[0099] The feeding medium A for the culture stage comprises glycerol 550 g / L, yeast extract 6 g / L, and the feeding rate is 30 g / L / h. The temperature is lowered to 24°C, and the induction stage feeding medium B is added, which comprises methanol 1 L / L, peptone 7 g / L, the feeding rate is 3 ml / L / h for 0-5 h, and the feeding rate is 6 ml / L / h after 5 h.

[0100] Comparative Example 1

[0101] The fermentation method for producing bovine lactoferrin is carried out according to the fermentation method of Example 3, the difference being that the fermentation medium for expressing bovine lactoferrin is different, and in the present comparative example, L-histidine in the fermentation medium for expressing bovine lactoferrin is replaced by an equivalent amount of leucine.

[0102] Comparative Example 2

[0103] The fermentation method for producing bovine lactoferrin is carried out according to the fermentation method of Example 1, the difference being that the fermentation medium for expressing bovine lactoferrin is different, and in the present comparative example, N-acetylglucosamine in the fermentation medium for expressing bovine lactoferrin is replaced by an equivalent amount of mannose.

[0104] Comparative Example 3

[0105] The fermentation method for producing bovine lactoferrin is carried out according to the fermentation method of Example 2, the difference being that the fermentation medium for expressing bovine lactoferrin is different, and in the present comparative example, the content of mannose in the fermentation medium for expressing bovine lactoferrin is 0.

[0106] Comparative Example 4

[0107] The fermentation method for producing bovine lactoferrin is carried out according to the fermentation method of Example 3, the difference being that the fermentation medium for expressing bovine lactoferrin is different, and in the present comparative example, the content of ferrous sulfate in the fermentation medium for expressing bovine lactoferrin is 0.

[0108] Test Example 1

[0109] The expression level of bovine lactoferrin in the fermentation broth after fermentation was completed was detected according to GB 1903.17-2016, and the results are shown in Table 1.

[0110] Table 1. Expression levels of bovine lactoferrin in fermentation broth

[0111]

[0112]

[0113] The concentration of Pichia pastoris cells in the fermentation broth after fermentation was completed was measured, and the results are shown in Table 2.

[0114] Table 2. Pichia pastoris cell concentration in fermentation broth

[0115] Test Example OD 600 ]]> Example 1 652 Example 2 661 Example 3 658 Comparative Example 1 596 Comparative Example 2 586 Comparative Example 3 582 Comparative Example 4 576

[0116] The test results above show that the culture medium and fermentation method used in Examples 1-3 can effectively increase the expression level of bovine lactoferrin, reaching a maximum of 4.5 g / L. The OD of the fermentation broth after fermentation was completed... 600 The values ​​can reach between 500 and 600. In Comparative Examples 1-4, after adjusting the composition of the fermentation medium, the expression level of bovine lactoferrin could only reach 2.7-2.9 g / L. However, in Comparative Example 1, replacing L-histidine with an equal amount of leucine resulted in a decrease in bovine lactoferrin expression, indicating that L-histidine has a synergistic effect with other amino acids in increasing bovine lactoferrin expression. In Comparative Example 2, replacing N-acetylglucosamine with an equal amount of mannose resulted in a decrease in bovine lactoferrin expression, indicating that N-acetylglucosamine and galactose have a synergistic effect in increasing bovine lactoferrin expression. The above experiments show that Comparative Example 3, which did not contain mannose, and Comparative Example 4, which did not contain ferrous sulfate, also showed a significant decrease in bovine lactoferrin expression in Comparative Examples 3-4, indicating that the addition of mannose and ferrous sulfate can increase bovine lactoferrin expression. In summary, amino acids (L-histidine, aspartic acid, alanine, etc.), ferrous salts, sugars (mannose and / or galactose) and N-acetylglucosamine in the fermentation medium work together to enhance the expression of bovine lactoferrin.

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fermentation medium for recombinant Pichia pastoris expressing bovine lactoferricin containing a bovine lactoferricin-encoding gene, characterized in that, The fermentation medium comprises the following components: yeast extract 10-20 g / L, peptone 20-30 g / L, glycerol or glucose 10-20 g / L, yeast nitrogen base without amino acid 12-15 g / L, dipotassium hydrogen phosphate 4-6 g / L, potassium dihydrogen phosphate 2-3 g / L, biotin 0.0002-0.0005 g / L, amino acid 4-46 g / L, ferrous salt 2-5 g / L, saccharide 5-10 g / L, and saccharide derivative 5-10 g / L; The amino acid comprises L-histidine, and the content of the L-histidine in the fermentation medium is 4-6 g / L; The saccharide comprises monosaccharide, and the monosaccharide comprises mannose and / or galactose; The saccharide derivative is N-acetylglucosamine.

2. The fermentation medium according to claim 1, characterized in that, The amino acid further comprises 1-2 kinds of glutamic acid, aspartic acid, leucine or alanine; The content of the glutamic acid in the fermentation medium is 5-10 g / L; The content of the aspartic acid in the fermentation medium is 15-20 g / L; The content of the leucine in the fermentation medium is 5-15 g / L; The content of the alanine in the fermentation medium is 10-20 g / L.

3. The fermentation medium of claim 1, wherein, The ferrous salt is ferrous sulfate.

4. A fermentation process for the production of bovine lactoferrin, characterized in that, The method comprises the following steps: Seeding the seed liquid of the recombinant Pichia pastoris containing the bovine lactoferricin coding gene into the fermentation medium according to any one of claims 1-3 to start fermentation; After 30~35h hours of cultivation, the feed medium A is added to the bacterial culture stage, and the OD value of the fermentation broth is detected by sampling 600 When the OD 600 value reaches 150~200, stop adding the feed medium A to the culture stage; the feed medium A for the culture stage includes glycerol 500~600g / L, yeast extract 5~8g / L; Reducing the temperature to 22-24 ℃, adding the induction stage feed medium B to start inducing the recombinant Pichia pastoris to express bovine lactoferricin; the induction stage feed medium B comprises 0.5-1 L / L of methanol and 5-10 g / L of peptone; and the induction culture is performed for 35-40 h.

5. The fermentation process of claim 4, wherein, The pH of the fermentation liquid is controlled to be 5-6 by using ammonia water during the fermentation culture process; the pH is maintained to be 5.5-6 during the cell culture stage, and the pH is maintained to be 5-5.5 during the induction stage.

6. The fermentation process of claim 4, wherein, The feeding rate of the culture stage feed medium A is 20-40 g / L / h in terms of the volume of the fermentation liquid; The feeding rate of the induction stage feed medium B is 1-3 ml / L / h during 0-5 h, and the feeding rate is 4-6 ml / L / h after 5 h.

7. The fermentation process of claim 4, wherein, The recombinant Pichia pastoris is obtained by linking the bovine lactoferricin gene, the PAOX1 promoter and the vector pPIC9K, and then transforming the combination into GS115 type Pichia pastoris; The bovine lactoferricin is selected by using histidine as a marker.

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