A fermentation process suitable for type Ⅲ collagen expression

Abstract

The application discloses a fermentation process suitable for type III collagen expression, and relates to the technical field of recombinant genetic engineering bacteria fermentation. The application uses an alginine and methanol double carbon source mixed flow adding strategy, solves the problem that cells grow and protein expression jointly compete for carbon source and energy when Pichia pastoris expresses an exogenous target protein by taking methanol as the only carbon source and energy, increases the metabolic burden of the cells, and causes low expression efficiency; the three-step methanol flow adding strategy improves the expression efficiency and reduces the toxic effect of methanol on the cells, so that the yield of type III collagen is further improved. The fermentation process provided by the application solves the problem of improving high-efficiency and high-quality expression of a target product while reducing production cost under the condition of high-density bacterial body fermentation.

Description

Technical Field

[0001] This invention relates to the field of recombinant genetically engineered bacterial fermentation technology, and in particular to a fermentation process suitable for the expression of type III collagen. Background Technology

[0002] Collagen is the most abundant protein in animals, distributed in tissues and organs such as the cardiovascular system, bones, and skin, and plays an important physiological role. Currently, the mainstream method for obtaining collagen involves breaking down animal hides and connective tissues, such as those of cattle and pigs, through acid, alkali, and enzymatic hydrolysis to obtain a mixture of collagen peptides. This process mainly poses risks of immunogenicity and animal viral infections, thus limiting its application in many fields.

[0003] At least 27 types of collagen have been discovered by researchers. Based on their main functions, they are mainly divided into two categories: fibroblastic collagen and non-fibroblastic collagen. Fibroblastic collagen is widely distributed in tissues, accounting for over 90%, and includes type I, type II, type III, type IV, type V, and type VI collagen. Type I and type III collagen are most widely distributed in the dermis. Type I collagen consists of two α1 chains and one α2 chain, and has the function of protecting mucous membranes and repairing ulcers. Type III collagen is found in new blood vessels, embryonic dermis, and scar tissue, and consists of three identical α1 chains. Based on the tissue characteristics of collagen fiber formation, it can be classified into fibrous, glassy, ​​cartilaginous, elastic, and fish roe collagen, etc. According to its solubility, it is divided into soluble and insoluble collagen. Soluble collagen can be further divided into acidic, alkaline, and neutral categories based on its pH value.

[0004] Genetic engineering technology for producing recombinant collagen offers advantages such as a single component, high safety, and a controllable production process. Given the broad application prospects of collagen, particularly the high demand for high-quality collagen in advanced medical materials and health products, the recombinant expression of collagen using genetic engineering technology has become a significant research direction. Genetic engineering involves various expression systems, including E. coli, yeast, insect cells, mammalian cells, and transgenic crops. However, mammalian and insect cell expression systems are costly and time-consuming, making them unsuitable for industrialization. In contrast, microbial fermentation for producing recombinant collagen is low-cost, quick, and easier to cultivate, making it more suitable for commercial production.

[0005] In recent years, large-scale cultivation of Pichia pastoris has become a crucial technology in the biopharmaceutical field, continuously driving the rapid development of the biotechnology industry. The Pichia pastoris expression system possesses significant advantages in expressing exogenous proteins, such as relatively simple structure and clear genetic background, the presence of the AOX1 promoter with strong initiation ability and strict regulation, absence of endotoxins and other harmful substances, post-translational modification capabilities characteristic of eukaryotic cells, the ability to achieve both intracellular and extracellular secretory expression, and high genetic stability due to the integration of exogenous genes into chromosomes. However, some problems still exist in the current cultivation process, such as long growth cycles, low production efficiency, and high fermentation energy consumption. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] Therefore, one of the main objectives of this invention is to provide a fermentation process suitable for the expression of type III collagen. The fermentation process provided by this invention solves the problem of improving the efficient and high-quality expression of the target product while reducing production costs under high-density cell fermentation conditions.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the present invention provides a fermentation process suitable for the expression of type III collagen, the fermentation process comprising: adding methanol, alanine and / or vitamin C during the fermentation induction phase.

[0010] In one embodiment, the fermentation process specifically includes adding methanol and alanine during the fermentation induction phase.

[0011] In one embodiment, the methanol addition rate is 1-3 g / L in the early stage of fermentation induction, 3-5 g / L in the middle stage of fermentation induction, and 3-5 g / L in the late stage of fermentation induction.

[0012] In one embodiment, the methanol addition rate is 3 g / L in the early stage of fermentation induction, 5 g / L in the middle stage of fermentation induction, and 3 g / L in the late stage of fermentation induction.

[0013] In one embodiment, the start time of the middle stage of fermentation induction is 25-35 hours into the fermentation induction stage and / or the start time of the late stage of fermentation induction is 43-53 hours into the fermentation induction stage.

[0014] In one embodiment, the start time of the middle stage of fermentation induction is the 30th hour of the fermentation induction stage and / or the start time of the late stage of fermentation induction is the 48th hour of the fermentation induction stage.

[0015] In one embodiment, the alanine is added at a rate of 0.1~1 mM / h.

[0016] In one embodiment, the alanine is added at a rate of 0.5 mM / h.

[0017] In one embodiment, the fermentation process specifically includes: adding methanol and alanine during the fermentation induction stage; the methanol addition rate is 1~3 g / L in the early stage of the fermentation induction stage, 3~5 g / L in the middle stage of the fermentation induction stage, and 3~5 g / L in the late stage of the fermentation induction stage; the alanine addition rate is 0.1~1 mM / h.

[0018] In one embodiment, the fermentation process specifically includes: adding methanol and alanine during the fermentation induction stage; the methanol addition rate is 1~3 g / L in the early stage of the fermentation induction stage, adjusted to 3~5 g / L during the 25~35 h of the fermentation induction stage, and adjusted to 3~5 g / L during the 43~53 h of the fermentation induction stage; the alanine addition rate is 0.1~1 mM / h.

[0019] In one embodiment, the fermentation process specifically includes: adding methanol and alanine during the fermentation induction stage; the methanol addition rate is 3 g / L in the early stage of the fermentation induction stage, adjusted to 5 g / L at the 30th hour of the fermentation induction stage, and adjusted to 3 g / L at the 48th hour of the fermentation induction stage; the alanine addition rate is 0.5 mM / h.

[0020] In one embodiment, the fermentation process further includes:

[0021] The fermentation strains are inoculated into the fermenter to enter the growth stage;

[0022] Once the glycerin is depleted, add more glycerin to initiate the feeding phase.

[0023] After stopping the addition of glycerol and starving the plant, methanol, alanine, and / or vitamin C are added to initiate the fermentation induction phase.

[0024] In one embodiment, the fermentation strain is Pichia pastoris.

[0025] In another aspect, the present invention provides the application of the above-mentioned fermentation process in the preparation of type III collagen.

[0026] In another aspect, the present invention also provides a type III collagen, which is prepared by the fermentation process described above.

[0027] (III) Beneficial Effects

[0028] This invention provides a fermentation process suitable for type III collagen expression. Compared with existing technologies, it has the following advantages:

[0029] 1. An optimized fermentation process was adopted to solve the problem that when Pichia pastoris uses methanol as the sole carbon and energy source to express exogenous target proteins, cell growth and protein expression compete for carbon and energy, increasing the metabolic burden on cells and leading to low expression efficiency.

[0030] 2. Based on the dual carbon source feeding strategy of alanine and methanol, a three-step methanol feeding strategy was adopted to improve the yield of type III collagen, enhance expression efficiency, and reduce the toxic effects of methanol on cells, thereby further increasing the yield of type III collagen.

[0031] 3. Under high-density cell fermentation conditions, improve the efficient and high-quality expression of the target product while reducing production costs. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Terms and Definitions

[0034] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0036] Example 1 Fermentation for type III collagen expression #1:

[0037] Strain: JY3-8.

[0038] Culture medium: Yeast peptone: 2 g / L; Yeast extract: 2 g / L; Phosphate: 10 ml / L; Calcium sulfate: 0.46 g / L; Potassium sulfate: 9.1 g / L; Magnesium sulfate heptahydrate: 7.5 g / L; Potassium hydroxide: 2 g / L; Glycerol: 40 g / L; Defoamer: 0.5 g / L; Biotin: 5 mg / L; PTM1: 4.35 ml / L.

[0039] Inoculate with primary seed culture medium: Take the glycerol tube out of the -80℃ freezer and inoculate 2 ml into a liquid shake flask (containing 50 ml of culture medium) in a clean bench. Incubate at 30℃ on a shaker for 18 h.

[0040] Inoculate with secondary seed culture medium: Take 10 ml of the above-cultured bacterial solution and inoculate it into a shake flask (containing 100 ml of culture medium), and incubate at 30°C on a shaker for 8 hours.

[0041] Inoculate the fermenter: Inoculate all the cultured bacterial solution into the fermenter (containing 2L of culture medium).

[0042] Fermentation control:

[0043] Growth stage: 5% inoculum, pH 5.2, 30℃, flow rate 1-4 vvm, tank pressure 0 MPa, DO 25%.

[0044] Feeding stage: When the base glycerol is exhausted, start adding glycerol. When DO rebounds, start adding glycerol. DO is automatically controlled at 25%, and pH and temperature remain unchanged.

[0045] Induction phase: glycerol is fed and wet weight is measured. When the wet weight reaches 180-220 g / L, induction is ready. Glycerol feeding is stopped and DO rebound is waited for. After starvation for 0.5 h, 3 g / L of methanol is added. DO is automatically controlled at 25%, pH 5.8. After induction for 12 h, the temperature is lowered to 25℃.

[0046] Example 2 Fermentation for Type III Collagen Expression #2:

[0047] Strain: JY3-8.

[0048] Culture medium: Yeast peptone: 2 g / L; Yeast extract: 2 g / L; Phosphate: 10 ml / L; Calcium sulfate: 0.46 g / L; Potassium sulfate: 9.1 g / L; Magnesium sulfate heptahydrate: 7.5 g / L; Potassium hydroxide: 2 g / L; Glycerol: 40 g / L; Defoamer: 0.5 g / L; Biotin: 5 mg / L; PTM1: 4.35 ml / L.

[0049] Inoculate with primary seed culture medium: Take the glycerol tube out of the -80℃ freezer and inoculate it into a liquid shake flask (containing 50ml of culture medium) in a clean bench, and incubate on a shaker at 30℃.

[0050] Inoculate with secondary seed culture medium: Take 10 ml of the above-cultured bacterial solution and inoculate it into a shake flask (containing 100 ml of culture medium), and incubate in a shaker at 30°C.

[0051] Inoculate the fermenter: Inoculate all the cultured bacterial solution into the fermenter (containing 2L of culture medium).

[0052] Fermentation control:

[0053] Growth stage: 5% inoculum, pH 5.2, 30℃, flow rate 1-4 vvm, tank pressure 0 MPa, DO 25%.

[0054] Feeding stage: When the base glycerol is exhausted, start adding glycerol. When DO rebounds, start adding glycerol. DO is automatically controlled at 25%, and pH and temperature remain unchanged.

[0055] Induction phase: glycerol is added and the wet weight is measured. When the wet weight reaches 180-220 g / L, induction is ready. Glycerol addition is stopped and DO rebound is waited for. After starvation for 0.5 h, 3 g / L methanol and 0.5 mM / h alanine are added. DO is controlled at 25% and pH is 5.8. After induction for 12 h, the temperature is lowered to 25℃.

[0056] Example 3 Fermentation for Type III Collagen Expression #3:

[0057] Strain: JY3-8.

[0058] Culture medium: Yeast peptone: 2 g / L; Yeast extract: 2 g / L; Phosphate: 10 ml / L; Calcium sulfate: 0.46 g / L; Potassium sulfate: 9.1 g / L; Magnesium sulfate heptahydrate: 7.5 g / L; Potassium hydroxide: 2 g / L; Glycerol: 40 g / L; Defoamer: 0.5 g / L; Biotin: 5 mg / L; PTM1: 4.35 ml / L.

[0059] Inoculate with primary seed culture medium: Take the glycerol tube out of the -80℃ freezer and inoculate it into a liquid shake flask (containing 50ml of culture medium) in a clean bench, and incubate on a shaker at 30℃.

[0060] Inoculate with secondary seed culture medium: Take 10 ml of the above-cultured bacterial solution and inoculate it into a shake flask (containing 100 ml of culture medium), and incubate in a shaker at 30°C.

[0061] Inoculate the fermenter: Inoculate all the cultured bacterial solution into the fermenter (containing 2L of culture medium).

[0062] Fermentation control:

[0063] Growth stage: 5% inoculum, pH 5.2, 30℃, flow rate 1-4 vvm, tank pressure 0 MPa, DO 25%.

[0064] Feeding stage: When the base glycerol is exhausted, start adding glycerol. When DO rebounds, start adding glycerol. DO is automatically controlled at 25%, and pH and temperature remain unchanged.

[0065] Induction phase: glycerol is added and wet weight is measured. When the wet weight reaches 180-220 g / L, induction is prepared. Glycerol addition is stopped and DO rebound is waited for. After starvation for 0.5 h, 3 g / L methanol and 0.016 mM / h vitamin C are added. DO is controlled at 25% and pH is 5.8. After induction for 12 h, the temperature is lowered to 25℃.

[0066] The fermentation yields of Examples 1-3 were tested, and the results are shown in Table 1.

[0067] Table 1

[0068]

[0069] As shown in Table 1, under the same culture conditions, after the seeds were inoculated into the fermenter and fermented for 92 hours, the mixed feeding of methanol and alanine significantly increased the collagen yield.

[0070] Example 4 Fermentation for Type III Collagen Expression #4:

[0071] Strain: JY3-8.

[0072] Culture medium: Yeast peptone: 2 g / L; Yeast extract: 2 g / L; Phosphate: 10 ml / L; Calcium sulfate: 0.46 g / L; Potassium sulfate: 9.1 g / L; Magnesium sulfate heptahydrate: 7.5 g / L; Potassium hydroxide: 2 g / L; Glycerol: 40 g / L; Defoamer: 0.5 g / L; Biotin: 5 mg / L; PTM1: 4.35 ml / L.

[0073] Inoculate with primary seed culture medium: Take the glycerol tube out of the -80℃ freezer and inoculate it into a liquid shake flask (containing 50ml of culture medium) in a clean bench, and incubate on a shaker at 30℃.

[0074] Inoculate with secondary seed culture medium: Take 10 ml of the above-cultured bacterial solution and inoculate it into a shake flask (containing 100 ml of culture medium), and incubate in a shaker at 30°C.

[0075] Inoculate the fermenter: Inoculate all the cultured bacterial solution into the fermenter (containing 2L of culture medium).

[0076] Fermentation control:

[0077] Growth stage: 5% inoculum, pH 5.2, 30℃, flow rate 1-4 vvm, tank pressure 0 MPa, DO 25%.

[0078] Feeding stage: When the base glycerol is exhausted, start adding glycerol. When DO rebounds, start adding glycerol. DO is automatically controlled at 25%, and pH and temperature remain unchanged.

[0079] Induction phase: glycerol is fed and wet weight is measured. When the wet weight reaches 180-220 g / L, induction is prepared. Glycerol feeding is stopped and DO rebound is waited for. After starvation for 0.5 h, 3 g / L methanol and 0.5 mM / h alanine are added. DO is controlled at 25%, pH 5.8. After induction for 12 h, the temperature is lowered to 25℃. After 30 h, the methanol concentration is adjusted to 5 g / L.

[0080] Example 5 Fermentation for Type III Collagen Expression #5:

[0081] Strain: JY3-8.

[0082] Culture medium: Yeast peptone: 2 g / L; Yeast extract: 2 g / L; Phosphate: 10 ml / L; Calcium sulfate: 0.46 g / L; Potassium sulfate: 9.1 g / L; Magnesium sulfate heptahydrate: 7.5 g / L; Potassium hydroxide: 2 g / L; Glycerol: 40 g / L; Defoamer: 0.5 g / L; Biotin: 5 mg / L; PTM1: 4.35 ml / L.

[0083] Inoculate with primary seed culture medium: Take the glycerol tube out of the -80℃ freezer and inoculate it into a liquid shake flask (containing 50ml of culture medium) in a clean bench, and incubate on a shaker at 30℃.

[0084] Inoculate with secondary seed culture medium: Take 10 ml of the above-cultured bacterial solution and inoculate it into a shake flask (containing 100 ml of culture medium), and incubate in a shaker at 30°C.

[0085] Inoculate the fermenter: Inoculate all the cultured bacterial solution into the fermenter (containing 2L of culture medium).

[0086] Fermentation control:

[0087] Growth stage: 5% inoculum, pH 5.2, 30℃, flow rate 1-4 vvm, tank pressure 0 MPa, DO 25%.

[0088] Feeding stage: When the base glycerol is exhausted, start adding glycerol. When DO rebounds, start adding glycerol. DO is automatically controlled at 25%, and pH and temperature remain unchanged.

[0089] Induction phase: glycerol is fed and wet weight is measured. When the wet weight reaches 180-220 g / L, induction is ready. Glycerol feeding is stopped and DO rebound is waited for. After starvation for 0.5 h, 3 g / L methanol and 0.5 mM / h alanine are added. DO is controlled at 25%, pH 5.8. After induction for 12 h, the temperature is lowered to 25℃. The methanol concentration is adjusted to 5 g / L after 30-48 h, and then adjusted to 3 g / L after 48 h.

[0090] Example 6 Fermentation for Type III Collagen Expression #6:

[0091] Strain: JY3-8.

[0092] Culture medium: Yeast peptone: 2 g / L; Yeast extract: 2 g / L; Phosphate: 10 ml / L; Calcium sulfate: 0.46 g / L; Potassium sulfate: 9.1 g / L; Magnesium sulfate heptahydrate: 7.5 g / L; Potassium hydroxide: 2 g / L; Glycerol: 40 g / L; Defoamer: 0.5 g / L; Biotin: 5 mg / L; PTM1: 4.35 ml / L.

[0093] Inoculate with primary seed culture medium: Take the glycerol tube out of the -80℃ freezer and inoculate it into a liquid shake flask (containing 50ml of culture medium) in a clean bench, and incubate on a shaker at 30℃.

[0094] Inoculate with secondary seed culture medium: Take 10 ml of the above-cultured bacterial solution and inoculate it into a shake flask (containing 100 ml of culture medium), and incubate in a shaker at 30°C.

[0095] Inoculate the fermenter: Inoculate all the cultured bacterial solution into the fermenter (containing 2L of culture medium).

[0096] Fermentation control:

[0097] Growth stage: 5% inoculum, pH 5.2, 30℃, flow rate 1-4 vvm, tank pressure 0 MPa, DO 25%.

[0098] Feeding stage: When the base glycerol is exhausted, start adding glycerol. When DO rebounds, start adding glycerol. DO is automatically controlled at 25%, and pH and temperature remain unchanged.

[0099] Induction phase: glycerol is fed and wet weight is measured. When the wet weight reaches 180-220 g / L, induction is ready. Glycerol feeding is stopped and DO rebound is waited for. After starvation for 0.5 h, 3 g / L methanol and 0.5 mM / h alanine are added. DO is controlled at 25%, pH 5.8. After induction for 12 h, the temperature is lowered to 25℃. The methanol concentration is adjusted to 4 g / L after 30-48 h, and then adjusted to 3 g / L after 48 h.

[0100] Example 7 Fermentation for Type III Collagen Expression #7:

[0101] Strain: JY3-8.

[0102] Culture medium: Yeast peptone: 2 g / L; Yeast extract: 2 g / L; Phosphate: 10 ml / L; Calcium sulfate: 0.46 g / L; Potassium sulfate: 9.1 g / L; Magnesium sulfate heptahydrate: 7.5 g / L; Potassium hydroxide: 2 g / L; Glycerol: 40 g / L; Defoamer: 0.5 g / L; Biotin: 5 mg / L; PTM1: 4.35 ml / L.

[0103] Inoculate with primary seed culture medium: Take the glycerol tube out of the -80℃ freezer and inoculate it into a liquid shake flask (containing 50ml of culture medium) in a clean bench, and incubate on a shaker at 30℃.

[0104] Inoculate with secondary seed culture medium: Take 10 ml of the above-cultured bacterial solution and inoculate it into a shake flask (containing 100 ml of culture medium), and incubate in a shaker at 30°C.

[0105] Inoculate the fermenter: Inoculate all the cultured bacterial solution into the fermenter (containing 2L of culture medium).

[0106] Fermentation control:

[0107] Growth stage: 5% inoculum, pH 5.2, 30℃, flow rate 1-4 vvm, tank pressure 0 MPa, DO 25%.

[0108] Feeding stage: When the base glycerol is exhausted, start adding glycerol. When DO rebounds, start adding glycerol. DO is automatically controlled at 25%, and pH and temperature remain unchanged.

[0109] Induction phase: glycerol is fed and wet weight is measured. When the wet weight reaches 180-220 g / L, induction is ready. Glycerol feeding is stopped and DO rebound is waited for. After starvation for 0.5 h, 3 g / L methanol and 0.5 mM / h alanine are added. DO is controlled at 25%, pH 5.8. After induction for 12 h, the temperature is lowered to 25℃. The methanol concentration is adjusted to 7 g / L after 30-48 h, and then adjusted to 3 g / L after 48 h.

[0110] The fermentation yields of Examples 4-6 were tested, and the results are shown in Table 2.

[0111] Table 2

[0112]

[0113] As shown in Table 2, under the same culture conditions, after the seeds were inoculated into the fermenter and fermented for 92 hours, the three-step method (maintaining a methanol concentration of 3 g / L for the first 30 hours of induction, 5 g / L for 30-48 hours, and 3 g / L for 48 hours) significantly increased the collagen yield.

[0114] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0115] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermentation process suitable for type III collagen expression, characterized in that, The fermentation process includes: adding methanol and alanine during the fermentation induction stage; The methanol addition rate is 3 g / L in the early stage of fermentation induction, 3-5 g / L in the middle stage of fermentation induction, and 3-5 g / L in the late stage of fermentation induction. The mid-term start time of the fermentation induction phase is the 30th hour of the fermentation induction phase. The start time of the later stage of fermentation induction is 48 hours after the fermentation induction stage. The fermentation strain is Pichia pastoris.

2. The fermentation process according to claim 1, characterized in that, The methanol addition rate is 3 g / L in the early stage of fermentation induction, 5 g / L in the middle stage of fermentation induction, and / or 3 g / L in the late stage of fermentation induction.

3. The fermentation process according to claim 2, characterized in that, The methanol addition rate was 3 g / L in the early stage of fermentation induction, 5 g / L in the middle stage of fermentation induction, and 3 g / L in the late stage of fermentation induction.

4. The fermentation process according to claim 1, characterized in that, The alanine is added at a rate of 0.1~1 mM / h.

5. The fermentation process according to claim 4, characterized in that, The alanine was added at a rate of 0.5 mM / h.

6. The fermentation process according to claim 1, characterized in that, The fermentation process also includes: The fermentation strains are inoculated into the fermenter to enter the growth stage; Once the glycerin is depleted, add more glycerin to initiate the feeding phase. After stopping the addition of glycerol and starving the body, add methanol and alanine.

7. The application of the fermentation process according to any one of claims 1-6 in the preparation of type III collagen.

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