Fermentation medium for expressing bovine lactoferrin and fermentation method

Abstract

The invention discloses a fermentation medium for expressing bovine lactoferrin and a fermentation method, and belongs to the technical field of fermentation. The fermentation medium comprises the following components: 10 to 20 g / L of yeast extract, 20 to 30 g / L of peptone, 10 to 20 g / L of glycerol or glucose, 12 to 15 g / L of yeast nitrogen group without amino acid, 4 to 6 g / L of dipotassium phosphate, 2 to 3 g / L of monopotassium phosphate, 0.0002 to 0.0005 g / L of biotin, 4 to 46 g / L of amino acid, 2 to 5 g / L of ferrite, 5 to 10 g / L of carbohydrate and 5 to 10 g / L of carbohydrate derivative. By using the fermentation medium disclosed by the invention, the expression quantity of bovine lactoferrin can be increased while the growth and metabolism requirements of thalli are met.

Description

Technical Field

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

[0002] Lactoferrin is a natural antibacterial glycoprotein mainly found in human milk. Due to its specific iron ion binding ability, it has strong bactericidal and antibacterial abilities against both Gram-positive and Gram-negative bacteria. Lactoferrin also has multiple effects such as antiviral, antioxidant and immunomodulatory. Therefore, lactoferrin is widely used in many fields such as milk powder, food and medicine. The US FDA, Japan, South Korea and other countries currently allow lactoferrin to be used as a food additive in sports and functional foods. my country allows the addition of lactoferrin to infant formula milk powder in the 2004 supplement to the national standard "Food Additives Hygiene Standard GB2760-19966".

[0003] Studies have found that bovine lactoferrin and human lactoferrin have extremely similar biological activities, but the separation and preparation of bovine lactoferrin from cow's milk is difficult and costly. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a fermentation medium and a fermentation method for expressing bovine lactoferrin, so as to overcome the problems of difficulty and high cost in the preparation of bovine lactoferrin in the prior art.

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

[0006] Compared with the prior art, the fermentation medium for expressing bovine lactoferrin of the present invention contains 4-46 g / L of amino acids, 2-5 g / L of ferrous salts, 5-10 g / L of carbohydrates and 5-10 g / L of carbohydrate derivatives, which together play the role of increasing the expression of bovine lactoferrin. In addition, the initial concentration of glycerol or glucose used as a carbon source in the present invention is low, which reduces the concentration inhibition of the substrate, increases the growth rate of the bacteria, shortens the lag phase of the growth of the bacteria, and thus shortens the growth cycle. In addition, the yeast extract and 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 acts as a cofactor of multiple enzymes in the process of bacterial metabolism, ensuring the activity of the enzyme system in the bacteria.

[0007] Furthermore, the amino acid includes L-histidine, and the content of L-histidine in the fermentation medium is 4-6 g / L.

[0008] Compared with the prior art, the above technical solution defines the amino acid in the culture medium as L-histidine with a content of 4 to 6 g / L. Since the bovine lactoferrin in the present invention uses histidine as an expression system for screening markers, adding L-histidine to the culture medium can promote the induced expression of bovine lactoferrin in the late fermentation stage.

[0009] Furthermore, the amino acids include 1 to 2 of glutamic acid, aspartic acid, leucine or alanine;

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

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

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

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

[0014] Compared with the prior art, the above technical solution specifies that the amino acids also include 1-2 of glutamic acid, aspartic acid, leucine or alanine. After adding the amino acids to the culture medium, they have a synergistic effect with L-histidine, which can further promote the synthesis of bovine lactoferrin by bacteria.

[0015] Furthermore, the carbohydrate substance includes 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 solution adds mannose and / or galactose, as well as ferrous sulfate, to the culture medium. The above specific types of carbohydrate substances can improve the synthesis efficiency of bovine lactoferrin and increase the expression of bovine lactoferrin. In addition, bovine lactoferrin is a glycoprotein containing iron ions. The ferrous ions in ferrous sulfate can play a role in the synthesis of bovine lactoferrin, and can also increase the enzyme activity of the fermented bacteria and participate in the metabolic reaction of the bacteria.

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

[0018] Compared with the prior art, the above technical solution limits the type and content of sugar derivatives, and an appropriate amount of N-acetylglucosamine in the culture medium cooperates with other substances in the culture medium to promote the synthesis of bovine lactoferrin, thereby increasing the expression level of bovine lactoferrin.

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

[0020] Inoculating the recombinant Pichia pastoris seed solution containing the bovine lactoferrin encoding gene into the fermentation medium to start fermentation;

[0021] After 30-35h of cultivation, feed medium A was added during the bacterial culture stage, and the OD of the fermentation liquid was tested by sampling. 600 Value, when OD 600 When the concentration reaches 150-200, the feed medium A in the culture stage is stopped; the feed medium A in the culture stage includes 500-600 g / L of glycerol and 5-8 g / L of yeast extract;

[0022] The temperature is lowered to 22-24° C., and the induction stage feed medium B is fed to start inducing the recombinant Pichia pastoris to express bovine lactoferrin; the induction stage feed medium B includes 0.5-1 L / L methanol and 5-10 g / L peptone; the induction culture is carried out for 35-40 hours.

[0023] Compared with the prior art, the present invention effectively improves the growth rate of bacteria and the expression level of the target protein by controlling the ratio of carbon source and nitrogen source in the feed medium. In addition, the carbon source and nitrogen source are combined in the feed, which brings greater convenience to the operation of the fermentation process personnel, so that they can accurately control the addition amount of the feed medium.

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

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

[0026] Furthermore, the flow rate of feed medium A during the culture stage is 20 to 40 g / L / h based on the volume of the fermentation broth;

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

[0028] Compared with the prior art, the above technical scheme limits the flow acceleration rate of feed medium A in the cultivation stage and feed medium B in the induction stage. Adding feed medium at the above specific flow acceleration rate can reasonably provide nutrition to the fermentation bacteria, promote the growth and reproduction of the bacteria and the expression of the target protein.

[0029] Furthermore, 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] Bovine lactoferrin uses histidine as a selection marker.

[0031] Compared with the prior art, the above technical solution defines the screening marker of bovine lactoferrin. Bovine lactoferrin with histidine as the screening marker is more suitable for the above fermentation medium and fermentation method, has a higher expression level, and is conducive to subsequent protein purification. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] In a first aspect, an embodiment of the present invention provides a fermentation medium for expressing bovine lactoferrin, comprising the following components: 10-20 g / L yeast extract, 20-30 g / L peptone, 10-20 g / L glycerol or glucose, 12-15 g / L yeast nitrogen base (YNB) without amino acids, 4-6 g / L dipotassium hydrogen phosphate, 2-3 g / L potassium dihydrogen phosphate, 0.0002-0.0005 g / L biotin, 4-46 g / L amino acids, 2-5 g / L ferrous salts, 5-10 g / L carbohydrates, and 5-10 g / L carbohydrate derivatives.

[0034] For example, the concentration of the yeast extract in the fermentation medium for expressing bovine lactoferrin 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 peptone in the fermentation medium for expressing bovine lactoferrin 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 lactoferrin 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 yeast nitrogen base without amino acids can be a directly purchased commodity. For example, the amino acid-free yeast nitrogen base (YNB) (Cat#Y8040) produced by Solarbio can be selected.

[0038] For example, the concentration of the yeast nitrogen base without amino acids in the fermentation medium for expressing bovine lactoferrin 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 above-mentioned dipotassium hydrogen phosphate in the fermentation medium for expressing bovine lactoferrin can be 4 g / L, 5 g / L or 6 g / L, preferably 5 g / L.

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

[0041] For example, the concentration of the biotin in the fermentation medium for expressing bovine lactoferrin may 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 lactoferrin provided in the above technical solution can promote the expression of bovine lactoferrin by bacteria while meeting the growth and metabolic requirements of bacteria. In addition, the above fermentation medium can shorten the growth period of bacteria by 3 to 8 hours, and increase the expression level of bovine lactoferrin to 4 to 5 g / L.

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

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

[0045] L-histidine in the above technical solution can maintain the stability of the intracellular environment of the bacteria, promote healthy cell growth, and reduce stress response. On the other hand, it can also promote the induced expression of bovine lactoferrin in the late fermentation period, especially bovine lactoferrin with a histidine selection marker.

[0046] Furthermore, based on the above embodiments, the amino acids in the embodiments of the present invention may also include 1 to 2 of glutamic acid, aspartic acid, leucine or alanine.

[0047] The content of glutamic acid in the fermentation medium is 5 to 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 to 8 g / L.

[0048] The content of aspartic acid in the fermentation medium is 15-20 g / L. For example, it can be 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 to 15 g / L, for example, it can be 5 g / L, 8 g / L, 10 g / L, 13 g / L or 15 g / L, preferably 8 to 13 g / L.

[0050] The content of alanine in the fermentation medium is 10-20 g / L, for example, it 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 above technical solution has a synergistic effect with L-histidine, which can further promote the growth and reproduction of bacteria and the expression of target proteins.

[0052] Further, based on the above embodiments, the carbohydrate substance in the embodiments of the present invention includes monosaccharides, and the monosaccharides include mannose and / or galactose; and / or the ferrous salt is ferrous sulfate.

[0053] The content of the above-mentioned sugar substances in the fermentation medium is 5-10 g / L. For example, it can be 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 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 the carbohydrate derivative N-acetylglucosamine in the above technical solution cooperate with each other to increase the expression level of bovine lactoferrin.

[0056] Further, based on the above embodiments, the carbohydrate derivative in the embodiments of the present invention is N-acetylglucosamine; the content of N-acetylglucosamine in the fermentation medium is 5 to 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 to 8 g / L.

[0057] The above technical solution adds an appropriate amount of N-acetylglucosamine to the fermentation medium, which works synergistically with mannose and / or galactose to further increase the expression level of bovine lactoferrin.

[0058] In a second aspect, an embodiment of the present invention provides a fermentation method for producing bovine lactoferrin, comprising the following steps:

[0059] S1. Inoculate the recombinant Pichia pastoris seed solution containing the bovine lactoferrin encoding gene into the above fermentation medium to start fermentation.

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

[0061] The method for preparing the above-mentioned recombinant Pichia yeast seed solution can be referred to as follows:

[0062] The recombinant Pichia pastoris was spread on a YPD solid medium plate and placed in an incubator at 30±1°C for 2-4 days. The smooth, regular-shaped, and larger colonies were picked and inoculated into a 200±20 mL shaking bottle containing YPD liquid medium. The shaker was placed at a controlled temperature of 30±1°C and a rotation speed of 200-220 rpm for 18-24 h to obtain the OD 600 It is 8 to 12 shake flask seed liquid.

[0063] The preparation of the above YPD solid 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), sterilize at 121±1℃ for 20-30min, cool to 50±1℃, perform aseptic operation in a clean bench, pour into plates, each containing 20±1mL of culture medium; % is the mass percentage.

[0065] The preparation of the shake flask containing the 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), prepare 400 ml, each 1000 ml shake flask contains 200 ml culture medium, put it into a high pressure steam sterilizer, set it at 121±1℃ and sterilize it at high temperature for 20-30 minutes; % 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 aseptically introduced into a sterilized and pH-adjusted fermentation tank, the culture medium in the fermentation tank is the above-mentioned fermentation medium for expressing bovine lactoferrin, the initial ventilation ratio is controlled to be 1-2VVM, the initial stirring speed is 100-200rpm, the tank pressure is 0.03-0.06MPa, the DO is corrected to 100%, and the DO is controlled to be 10-60% by increasing the stirring speed and the ventilation ratio, and the pH is controlled to be 5-6 using ammonia water.

[0068] S2. After 30-35 hours of cultivation, feed medium A was added during the bacterial culture stage, and samples were taken every 2 hours to detect the OD of the fermentation liquid. 600 Value, when OD 600 When the pH reaches 150-200, the feed medium A in the feeding culture stage is stopped; the feed medium A in the culture stage includes 500-600 g / L glycerol and 5-8 g / L yeast extract. In the above bacterial cell culture stage, ammonia water is used to maintain the pH at 5.5-6.

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

[0070] For example, the glycerol concentration in the feed medium A during 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, preferably 520-560 g / L.

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

[0072] In the above technical scheme, when the feed medium A is added during the flow-added bacterial culture stage, the flow acceleration rate of the feed medium A during the culture stage is 20 to 40 g / L / h, based on the volume of the fermentation liquid. For example, it can be 20 g / L / h, 25 g / L / h, 30 g / L / h, 35 g / L / h or 40 g / L / h, preferably 25 to 35 g / L / h.

[0073] S3, lower the temperature to 22-24°C, feed the induction stage feed medium B, start inducing the recombinant Pichia pastoris to express bovine lactoferrin, and carry out induction culture for 35-40 hours. Ammonia water is used to maintain the pH at 5-5.5 during the induction stage.

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

[0075] In the above technical solution, when feeding medium B during the feeding induction stage, the flow acceleration rate is 1-3 ml / L / h in the 0th to 5th hour and 4-6 ml / L / h after 5 hours, based on the volume of the fermentation liquid.

[0076] For example, the flow rate in the first 0 to 5 hours can be selected as 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 hours can be selected as 4 ml / L / h, 4.5 ml / L / h, 5 ml / L / h, 5.5 ml / L / h or 6 ml / L / h, preferably 4.5 to 5.5 ml / L / h.

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

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

[0080] Example 1

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

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

[0083] S1. The bovine lactoferrin gene with histidine tag and PAOX1 promoter were connected to the vector pPIC9K and transformed into GS115 type Pichia pastoris to obtain recombinant Pichia pastoris GS115 / pPIC9K-blf. The GenbanK ID of the bovine lactoferrin gene is JX294418.1.

[0084] S2. Spread the recombinant Pichia pastoris on a YPD solid medium plate, place it in an incubator at 30°C for 3 days, pick the smooth, regular-shaped, and larger colonies and inoculate them into a 200 mL shake flask containing YPD liquid medium, place it in a shaker, control the temperature at 301°C, and the rotation speed at 220 rpm for 20 h to obtain the OD 600 It is 8 to 12 shake flask seed liquid.

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

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

[0087] S3. Aseptically transfer the prepared shake flask seed liquid into a sterilized and pH-adjusted fermentation tank, wherein the culture medium in the fermentation tank is the fermentation medium for expressing bovine lactoferrin, and the initial aeration ratio is controlled to be 2VVM, the initial stirring speed is 200 rpm, the tank pressure is 0.03 MPa, the DO is corrected to 100%, and 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 using aqueous ammonia.

[0088] After 30 hours of cultivation, feed medium A for the bacterial culture phase was added. The feed medium A for the culture phase included 500 g / L glycerol and 5 g / L yeast extract, and the feeding rate was 40 g / L / h. The OD of the fermentation broth was tested every 2 hours. 600 Value, fermentation liquid OD 600 When the concentration reaches 150-200, the feed medium A in the fed-batch culture stage is stopped, and the feed medium B in the fed-batch induction stage is started.

[0089] The temperature was lowered to 23°C, and the induction stage feed medium B was added to begin inducing the recombinant Pichia pastoris to express bovine lactoferrin. The induction stage feed medium B included 0.5 L / L methanol and 5 g / L peptone, which were added at a rate of 1.5 ml / L / h from 0 to 5 h, and the flow rate after 5 h was 5 ml / L / h.

[0090] During the fermentation process, the pH value was maintained at 5.5-6 before induction by adding ammonia water during the bacterial culture stage, and at 5-5.5 during the induction stage.

[0091] The fermentation was completed after 35 hours of induction, and bovine lactoferrin was detected.

[0092] Example 2

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

[0094] The fermentation method for producing bovine lactoferrin using the fermentation medium is different from that in Example 1 in that:

[0095] The culture stage feed medium A includes 600 g / L glycerol and 8 g / L yeast extract, and the feed rate is 20 g / L / h. The temperature is lowered to 22°C, and the induction stage feed medium B is fed. The induction stage feed medium B includes 1 L / L methanol and 10 g / L peptone, and is fed at a rate of 1 ml / L / h from 0 to 5 hours, and the flow rate after 5 hours is 4 ml / L / h.

[0096] Example 3

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

[0098] The fermentation method for producing bovine lactoferrin using the fermentation medium is different from that in Example 1 in that:

[0099] The culture stage feed medium A includes 550 g / L glycerol and 6 g / L yeast extract, and the feed rate is 30 g / L / h. The temperature is lowered to 24°C, and the induction stage feed medium B is fed, which includes 1 L / L methanol and 7 g / L peptone, and the flow rate is 3 ml / L / h from 0 to 5 hours, and the flow rate after 5 hours is 6 ml / L / h.

[0100] Comparative Example 1

[0101] Fermentation was carried out according to the fermentation method for producing bovine lactoferrin in Example 3, except that the fermentation medium used for expressing bovine lactoferrin was different. In this comparative example, an equal amount of L-histidine in the fermentation medium used for expressing bovine lactoferrin was replaced by leucine.

[0102] Comparative Example 2

[0103] Fermentation was carried out according to the fermentation method for producing bovine lactoferrin in Example 1, except that the fermentation medium used for expressing bovine lactoferrin was different. In this comparative example, an equal amount of N-acetylglucosamine in the fermentation medium used for expressing bovine lactoferrin was replaced with mannose.

[0104] Comparative Example 3

[0105] Fermentation was carried out according to the fermentation method for producing bovine lactoferrin in Example 2, with the only difference being that the fermentation medium used for expressing bovine lactoferrin was different. In this comparative example, the content of mannose in the fermentation medium used for expressing bovine lactoferrin was 0.

[0106] Comparative Example 4

[0107] Fermentation was carried out according to the fermentation method for producing bovine lactoferrin in Example 3, with the only difference being that the fermentation medium used for expressing bovine lactoferrin was different. The content of ferrous sulfate in the fermentation medium used for expressing bovine lactoferrin in this comparative example was 0.

[0108] Test Example 1

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

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

[0111]

[0112]

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

[0114] Table 2 Pichia yeast cell concentration in fermentation broth

[0115] Test Case <![CDATA[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 above test results show that the culture medium and fermentation method used in Examples 1 to 3 can effectively increase the expression level of bovine lactoferrin, up to 4.5 g / L, and the OD of the fermentation liquid after the fermentation is completed. 600 The value can reach between 500 and 600. After adjusting the components of the fermentation medium in Comparative Examples 1 to 4, the expression level of bovine lactoferrin can only reach 2.7 to 2.9 g / L. In Comparative Example 1, after replacing an equal amount of L-histidine with leucine, the expression level of bovine lactoferrin decreased, indicating that L-histidine and other amino acids have a synergistic effect in increasing the expression level of bovine lactoferrin. In Comparative Example 2, after replacing an equal amount of N-acetylglucosamine with mannose, the expression level of bovine lactoferrin decreased, indicating that N-acetylglucosamine and galactose have a synergistic effect in increasing the expression level of bovine lactoferrin. The above experiments show that Comparative Example 3 does not contain mannose, and Comparative Example 4 does not contain ferrous sulfate. The expression level of bovine lactoferrin in Comparative Examples 3 to 4 also decreases significantly, indicating that the addition of mannose and ferrous sulfate can increase the expression level of bovine lactoferrin. In short, amino acids (L-histidine, aspartic acid, alanine, etc.), ferrous salts, carbohydrates (mannose and / or galactose) and N-acetylglucosamine in the fermentation medium work together to increase the expression of bovine lactoferrin.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fermentation medium for expressing bovine lactoferrin, characterized in that: The invention comprises the following components: 10-20 g / L yeast extract, 20-30 g / L peptone, 10-20 g / L glycerol or glucose, 12-15 g / L yeast nitrogen base without amino acid, 4-6 g / L dipotassium hydrogen phosphate, 2-3 g / L potassium dihydrogen phosphate, 0.0002-0.0005 g / L biotin, 4-46 g / L amino acid, 2-5 g / L ferrous salt, 5-10 g / L sugar substance and 5-10 g / L sugar substance derivative.

2. The fermentation medium according to claim 1, characterized in that The amino acids include L-histidine, and the content of the L-histidine in the fermentation medium is 4-6 g / L.

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

4. The fermentation medium according to claim 1, characterized in that The carbohydrate substance includes monosaccharide; the monosaccharide includes mannose and / or galactose; and / or the ferrous salt is ferrous sulfate.

5. The fermentation medium according to claim 1, characterized in that The sugar substance derivative is N-acetylglucosamine; the content of the N-acetylglucosamine in the fermentation medium is 5-10 g / L.

6. A fermentation method for producing bovine lactoferrin, characterized in that: The following steps are involved: Inoculating a recombinant Pichia pastoris seed solution containing a bovine lactoferrin encoding gene into the fermentation medium of any one of claims 1 to 5 to start fermentation; After 30-35h of cultivation, feed medium A was added during the bacterial culture stage, and the OD of the fermentation liquid was tested by sampling. 600 Value, when OD 600 When the concentration reaches 150-200, the feed medium A in the culture stage is stopped; the feed medium A in the culture stage includes 500-600 g / L of glycerol and 5-8 g / L of yeast extract; The temperature is lowered to 22-24° C., and the induction stage feed medium B is fed to start inducing the recombinant Pichia pastoris to express bovine lactoferrin; the induction stage feed medium B includes 0.5-1 L / L methanol and 5-10 g / L peptone; the induction culture is carried out for 35-40 hours.

7. The fermentation method according to claim 6, characterized in that Ammonia water is used to control the pH of the fermentation liquid to 5-6 during the fermentation culture process; the pH is maintained at 5.5-6 during the bacterial culture stage, and the pH is maintained at 5-5.5 during the induction stage.

8. The fermentation method according to claim 6, characterized in that The flow rate of feed medium A during the culture stage is 20 to 40 g / L / h based on the volume of the fermentation broth; The flow rate of the feed medium B in the induction stage is 1-3 ml / L / h in the first 0-5 hours, and the flow rate after 5 hours is 4-6 ml / L / h.

9. The fermentation method according to claim 6, characterized in that: The recombinant Pichia pastoris is obtained by connecting the bovine lactoferrin gene, the PAOX1 promoter and the vector pPIC9K and then transforming them into GS115 type Pichia pastoris; The bovine lactoferrin uses histidine as a screening marker.

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