Corn steep liquor enzymatic hydrolysate for promoting cordycepin production by cordyceps militaris and preparation method and application thereof

Abstract

The application provides corn steep liquor enzymolysis matter for promoting Cordyceps militaris to produce cordycepin and a preparation method and application thereof, and belongs to the technical field of cordycepin production. The preparation method of the corn steep liquor enzymolysis matter provided by the application comprises the following steps: corn steep liquor is mixed with protease for enzymolysis, and then the enzyme is inactivated; the supernatant is taken out and vacuum concentrated to obtain corn steep liquor enzymolysis liquid concentrate; and the corn steep liquor enzymolysis liquid concentrate is dried to obtain the corn steep liquor enzymolysis matter; the protease comprises pepsin, neutral protease or alkaline protease. The preparation method can obtain active peptide segments in corn protein which are beneficial to the metabolism of Cordyceps militaris, and thus the yield and biological activity of metabolites of Cordyceps militaris are improved. The corn steep liquor and the microbial source food industry enzyme with low price are selected, and the corn steep liquor enzymolysis liquid prepared can partially replace expensive nitrogen sources such as proteose peptone in microbial fermentation, so that the production cost of cordycepin is significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of cordycepin production technology, and particularly relates to a corn steep liquor enzymatic hydrolysate that promotes cordycepin production by Cordyceps militaris, its preparation method and application. Background Technology

[0002] Cordyceps militaris, also known as North Cordyceps, is a fungus belonging to the genus Cordyceps in the family Clavicipitaceae of the subphylum Ascomycotina. It shares the same family and genus as the rare and precious traditional Chinese medicine, Cordyceps sinensis. Because Cordyceps militaris contains similar active ingredients to Cordyceps sinensis, including various amino acids, superoxide dismutase, cordycepin, adenosine, cordyceps polysaccharides, and cordycepic acid, it has been considered a substitute for Cordyceps sinensis. Cordycepin, also known as 3-deoxyadenosine, is a nucleoside analogue and a major bioactive compound found in Cordyceps militaris. It possesses multiple effects, including antioxidant, anti-inflammatory, immune-boosting, and anti-tumor properties. Recent studies have shown that cordycepin has anti-COVID-19 effects. In addition, cordycepin has been found to have anti-photoaging and anti-pigmentation properties in cosmeceutical products. Therefore, in recent years, cordycepin has been in high demand in the food, pharmaceutical, and cosmetic industries. However, the low production efficiency and high price of cordycepin greatly limit its widespread application in the food and pharmaceutical fields.

[0003] Currently, wild Cordyceps militaris is relatively scarce, and cordycepin mainly comes from artificially cultivated Cordyceps militaris. There are two methods for cultivating artificial Cordyceps militaris: solid-state fermentation and liquid culture, which can be used for the production and enrichment of cordycepin. Although some technological progress has been made in the liquid fermentation of Cordyceps militaris for cordycepin production in recent years, existing methods are mainly affected by factors such as the Cordyceps militaris strain, culture medium composition, and fermentation process. In particular, low-cost, high-efficiency nitrogen source culture medium growth factors play a key role in improving cordycepin yield and reducing production costs. Meanwhile, significantly increasing cordycepin yield remains a pressing technical challenge for the cordycepin industry. Therefore, exploring and developing new Cordyceps militaris growth promoters is an effective way to achieve efficient cordycepin production.

[0004] Corn steep liquor, a byproduct of corn starch processing, is rich in nutrients. Currently, it is mainly used in feed production or discarded as industrial waste, causing waste and environmental pollution. Studies have shown that corn steep liquor can be used as a culture medium in fermentation processes for biopharmaceuticals, antibiotics, and enzyme preparations. However, due to the large molecular weight of corn steep liquor proteins and their low microbial absorption and utilization rate, it cannot currently be used as an effective nutrient to replace high-quality nitrogen sources such as peptone. There are also few reports on the application of corn steep liquor in improving the cordycepin production efficiency of Cordyceps militaris. Summary of the Invention

[0005] In view of this, the purpose of this invention is to solve the key problems of low yield and high cost of existing cordycepin fermentation, and to provide a method for preparing corn steep liquor hydrolysate that promotes high cordycepin production by Cordyceps militaris. The corn steep liquor hydrolysate prepared by the method of this invention can induce Cordyceps militaris to produce cordycepin efficiently and reduce the production cost of cordycepin.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing corn steep liquor hydrolysate that promotes cordycepin production by Cordyceps militaris, comprising the following steps: mixing corn steep liquor with a protease for enzymatic hydrolysis, inactivating the enzyme, taking the supernatant for vacuum concentration to obtain corn steep liquor hydrolysate concentrate, and drying the corn steep liquor hydrolysate concentrate to obtain corn steep liquor hydrolysate; wherein the protease includes pepsin, neutral protease or alkaline protease.

[0008] Preferably, when the protease is pepsin, the enzymatic hydrolysis temperature is 35-45°C, the hydrolysis time is 2.0-3.0 h, and the hydrolysis pH is 2.1.

[0009] Preferably, when the protease is a neutral protease, the enzymatic hydrolysis temperature is 45–55°C, the enzymatic hydrolysis time is 2.0–3.0 h, and the enzymatic hydrolysis pH is 7.5.

[0010] Preferably, when the protease is an alkaline protease, the enzymatic hydrolysis temperature is 55–60°C, the enzymatic hydrolysis time is 2.0–3.0 h, and the enzymatic hydrolysis pH is 9.0.

[0011] Preferably, the corn steep liquor is pretreated corn steep liquor, and the pretreatment method includes the following steps: after removing precipitated impurities from the corn steep liquor, the supernatant is diluted to a corn steep liquor protein concentration of 0.08-0.2 g / mL to obtain pretreated corn steep liquor.

[0012] Preferably, the enzyme inactivation temperature is 90–100°C, and the enzyme inactivation time is 10–30 min.

[0013] Preferably, after vacuum concentration, a corn steep liquor enzymatic hydrolysate concentrate with a solid content of 20-30 wt% is obtained.

[0014] The present invention also provides a corn steep liquor hydrolysate that can promote the production of cordycepin by Cordyceps militaris, the corn steep liquor hydrolysate being prepared by the above-described preparation method.

[0015] The present invention also provides the application of the above preparation method or the above corn steep liquor enzymatic hydrolysate in cordycepin production by Cordyceps militaris.

[0016] The present invention also provides a method for producing cordycepin, comprising the following steps: adding the above-mentioned corn steep liquor hydrolysate to a Cordyceps militaris fermentation culture medium at an amount of 0.5 to 3.5 g / L, inoculating Cordyceps militaris for fermentation, and obtaining cordycepin.

[0017] The beneficial effects of this invention are:

[0018] The preparation method of this invention can enzymatically hydrolyze corn protein to obtain active peptides beneficial to the metabolism of Cordyceps militaris, thereby improving the yield and bioactivity of Cordyceps militaris metabolites. Furthermore, the corn steep liquor hydrolysate prepared by this method contains abundant amino acids and active corn protein peptides, which are easily absorbed and utilized by microorganisms, significantly promoting the growth of Cordyceps militaris and the production of cordycepin. The preparation method provided by this invention uses inexpensive corn steep liquor and microbial-derived food industry enzymes. The resulting corn steep liquor hydrolysate can partially replace expensive nitrogen sources such as peptone in microbial fermentation, significantly reducing the production cost of cordycepin. The corn steep liquor hydrolysate preparation process provided by this invention is simple, the product has no toxic side effects, is suitable for large-scale production, and is easy to promote and apply. Attached Figure Description

[0019] Figure 1 The effect of different proteases on the enzymatic hydrolysis of corn steep liquor;

[0020] Figure 2 Cordycepin standard curve;

[0021] Figure 3 Cordycepin yield in different groups;

[0022] Figure 4 The effect of different amounts of corn steep liquor hydrolysate powder added on cordycepin formation;

[0023] Figure 5 The effect of different amounts of corn steep liquor hydrolysate powder added on the biomass of Cordyceps militaris. Detailed Implementation

[0024] This invention provides a method for preparing corn steep liquor hydrolysate that promotes cordycepin production by Cordyceps militaris, comprising the following steps: mixing corn steep liquor with a protease for enzymatic hydrolysis, inactivating the enzyme, taking the supernatant for vacuum concentration to obtain corn steep liquor hydrolysate concentrate, and drying the corn steep liquor hydrolysate concentrate to obtain corn steep liquor hydrolysate; wherein the protease includes pepsin, neutral protease or alkaline protease.

[0025] This invention does not specifically limit the source of the corn steep liquor. Preferably, the corn steep liquor is pretreated before being mixed with the protease for enzymatic hydrolysis. The pretreatment preferably includes the following steps: removing precipitated impurities from the corn steep liquor, and then diluting the supernatant to a corn steep liquor protein concentration of 0.08–0.2 g / mL to obtain pretreated corn steep liquor. This pretreatment step ensures that the corn steep liquor to be hydrolyzed has a suitable protein concentration, thereby preventing protein precipitation and facilitating successful enzymatic hydrolysis. During pretreatment, centrifugation at 4000 rpm for 10 min at room temperature is preferred to remove precipitated impurities. The dilution is preferably performed with distilled water to a corn steep liquor protein concentration preferably of 0.1–0.18 g / mL.

[0026] In this invention, the enzymatic hydrolysis is preferably carried out under constant temperature and stirring. When the protease is pepsin, the hydrolysis temperature is preferably 35–45°C, more preferably 38–42°C, the hydrolysis time is preferably 2.0–3.0 h, more preferably 2.5 h, and the hydrolysis pH is preferably 2.1. When the protease is a neutral protease, the hydrolysis temperature is preferably 45–55°C, more preferably 48–52°C, the hydrolysis time is preferably 2.0–3.0 h, more preferably 2.5 h, and the hydrolysis pH is preferably 7.5. When the protease is an alkaline protease, the hydrolysis temperature is preferably 55–60°C, more preferably 56–58°C, the hydrolysis time is preferably 2.0–3.0 h, more preferably 2.5 h, and the hydrolysis pH is preferably 9.0. This invention does not specifically limit the source of pepsin, neutral protease, and alkaline protease. The enzyme activity of the alkaline protease is preferably 20,000 U / g, the enzyme activity of the pepsin is preferably 250,000 U / g, and the enzyme activity of the neutral protease is preferably 200,000 U / g. In this invention, the added amounts of pepsin, neutral protease, and alkaline protease are preferably 2.0% to 3.0% of the protein content, where protein content refers to the weight of protein in the corn steep liquor solution.

[0027] After enzymatic hydrolysis, enzyme inactivation is preferably performed. The inactivation temperature is preferably 90–100°C, more preferably 95–98°C, and the inactivation time is preferably 10–30 min, more preferably 15–25 min. After enzyme inactivation, the hydrolysate is allowed to cool, and the pH is preferably adjusted to 7.0–8.0. The supernatant is preferably collected by centrifugation at 2000–4000 r / min for 10 min. After collecting the supernatant, it is concentrated under vacuum, preferably to a solid content of 20–30 wt%. The present invention does not have a specific limitation on the specific method of vacuum concentration; conventional vacuum concentration methods in the art can be used. After obtaining the corn steep liquor enzymatic hydrolysate concentrate, it is dried, preferably by vacuum freeze-drying. The present invention does not have a specific limitation on the specific conditions of vacuum freeze-drying; conventional vacuum freeze-drying methods in the art can be used.

[0028] This invention also provides a corn steep liquor hydrolysate that can promote cordycepin production by Cordyceps militaris, the hydrolysate being prepared by the above-described method. This invention utilizes the inexpensive byproduct of the corn starch industry, corn steep liquor, through enzymatic hydrolysis to obtain a hydrolysate rich in amino acids and small-molecule proteins. Microbial fermentation experiments have demonstrated that the obtained hydrolysate can significantly promote the growth of Cordyceps militaris and increase the cordycepin content. Specifically, it can increase the dry weight of Cordyceps militaris mycelial cells from 7.09 g / L to 10.71 g / L and the cordycepin content from 41.24 mg / L to 131.26 mg / L.

[0029] The present invention also provides the application of the above preparation method or the above corn steep liquor enzymatic hydrolysate in cordycepin production by Cordyceps militaris.

[0030] The present invention also provides a method for producing cordycepin, comprising the following steps: adding the above-mentioned corn steep liquor hydrolysate to a Cordyceps militaris fermentation culture medium at an amount of 0.5 to 3.5 g / L, inoculating Cordyceps militaris for fermentation, and obtaining cordycepin.

[0031] In this invention, the preferred amount of corn steep liquor hydrolysate added is 1–2.5 g / L, more preferably 1.5–2 g / L. In this invention, the preferred fermentation medium for *Cordyceps militaris* includes the following raw materials: 10 g / L glucose, 5 g / L peptone, 1 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate. In this invention, the inoculation amount of *Cordyceps militaris* is 5–10 mL of seed solution / 100 mL (each mL of seed solution contains 1 × 10⁻⁶... 5 ~1×10 6The fermentation temperature is preferably 25°C, the fermentation time is preferably 8 days, and the fermentation is preferably carried out at a rotation speed of 120 r / min. This invention does not specifically limit the source of Cordyceps militaris, and the method for producing cordycepin described in this invention is generally applicable to all Cordyceps militaris in the art.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the following embodiments are all conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Example 1

[0036] (1) The corn starch processing by-product corn syrup stock solution (Qiqihar Longjiang Fufeng Biotechnology Co., Ltd.) was centrifuged at 4000 r / min for 10 min at room temperature to remove corn syrup precipitate impurities. The supernatant was then diluted with distilled water to a corn syrup protein concentration of 0.1 g / mL.

[0037] (2) Add pepsin (enzyme activity 250000U / g, Sigma) to corn steep liquor solution. The amount of pepsin added is 2.0% of the weight of protein in corn steep liquor solution. Adjust the pH value to 2.1 with 6.0mol / L HCl. Enzymatic hydrolyze for 2 hours under constant temperature and stirring at 45℃ to obtain corn steep liquor hydrolysate.

[0038] (3) The corn steep liquor hydrolysate was heated to 90°C for 10 min to inactivate it. After cooling, the pH was adjusted to 7.0, centrifuged at 2000 r / min for 10 min, and the supernatant was collected and concentrated under vacuum to obtain a corn steep liquor hydrolysate concentrate with a solid content of 20 wt%. Then, it was freeze-dried under vacuum to obtain corn steep liquor hydrolysate powder, which was stored at 4°C for later use.

[0039] Example 2

[0040] The difference from Example 1 is that the protease added in step (2) is a neutral protease (enzyme activity 200000U / g, Shanghai Lanji Biotechnology Co., Ltd.), the pH value is adjusted to 7.5 with 1.0mol / L HCl, and the enzyme is hydrolyzed for 2h under constant temperature stirring at 55℃. The rest is the same as in Example 1.

[0041] Example 3

[0042] The difference from Example 1 is that the protease added in step (2) is an alkaline protease (enzyme activity 200000U / g, Novozymes), the pH value is adjusted to 9.0 with 0.4mol / L NaOH, and the enzyme is hydrolyzed for 2 hours under constant temperature and stirring at 55℃. The rest is the same as in Example 1.

[0043] Example 4

[0044] The degree of hydrolysis of the corn steep liquor hydrolysate obtained in step (2) of Examples 1-3 was determined respectively. The specific determination methods are as follows:

[0045] The degree of hydrolysis (DH) was determined using the pH-stat method.

[0046]

[0047] In the formula: V NAOH —Volume of alkali consumed in the titration (mL); N NAOH —The molar concentration of the alkali consumed (mol / L); M p —Total amount of protein involved in hydrolysis (g); H tot —The number of gram equivalents of peptide bonds per gram of protein; the average degree of dissociation of α-α-amino acids.

[0048]

[0049] Where: pH: initial pH value;

[0050]

[0051] T = 273.15 + t

[0052] Where: t: reaction environment temperature.

[0053] The results are as follows Figure 1 As shown. The degree of hydrolysis of water-soluble proteins in the enzymatic hydrolysis products of pepsin, neutral protease and alkaline protease ranged from 20.16% to 27.7%, with the order of degree of hydrolysis being alkaline protease (27.7%), neutral protease (23.45%), and pepsin (20.16%).

[0054] Example 5

[0055] Cordyceps militaris fermentation experiment

[0056] The corn steep liquor hydrolysate powders obtained in Examples 1-3 were added to Cordyceps militaris fermentation medium (10 g / L glucose, 5 g / L peptone, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate) at a rate of 2.5 g / L, respectively, at a ratio of 5 mL seed culture / 100 mL (1 × 10⁻⁶). 5Cordyceps militaris (CGMCC No. 2577) was inoculated at an inoculum size of 1 spore cells / mL. The fermentation was carried out at 25℃ and 120 r / min for 10 days to obtain the fermentation broth. The cordycepin content was then determined using cordycepin as an indicator.

[0057] The group without corn steep liquor hydrolysate powder was designated as the control group, and the group with corn steep liquor concentrate was designated as the corn steep liquor group. The amount of corn steep liquor concentrate added was the same as the amount of protease added.

[0058] The method for determining cordycepin content is as follows:

[0059] HPLC chromatographic conditions and preparation of standard curves

[0060] Column: C 18 The column is 250 × 4.6 nm and 5 μm in diameter.

[0061] Chromatographic conditions: mobile phase: water:methanol = 85:15, flow rate: 1 mL / min, detection wavelength: 260 nm, column temperature: 30 ℃, injection volume: 20 μL. Weigh 25 mg of cordycepin standard, dissolve in double-distilled water, and dilute to 25 mL to prepare a 100 μg / mL mixed standard solution. Under the above chromatographic conditions, construct a standard curve with the standard concentration as the x-axis and peak area as the y-axis. The regression equation is y = 33625x - 11319, R0 2 =0.9999 (e.g.) Figure 2 ).

[0062] Determination of cordycepin in the sample: The fermentation broth was centrifuged at 4000 r / min for 10 min at room temperature, and 0.5 mL of the supernatant was taken. The sample was diluted by a certain factor, added to the mobile phase and mixed. After centrifugation at 10000 r / min for 5 min at room temperature, the supernatant was filtered through a 0.22 μm microporous membrane to prepare the sample solution. The sample solution was then tested under the above chromatographic conditions, and the cordycepin content in the fermentation broth was calculated based on the standard curve.

[0063] The results are as follows Figure 3 As shown, after adding the enzymes from Example 1 (pepsyte), Example 2 (neutral protease hydrolysate), and Example 3 (alkaline protease hydrolysate) to the Cordyceps militaris fermentation medium, the cordycepin yields in the fermentation broth were 120.13 mg / L, 130.8 mg / L, and 154.9 mg / L, respectively. Alkaline protease hydrolysis showed the strongest effect and significantly increased the cordycepin yield.

[0064] Example 6

[0065] The amino acid content of corn steep liquor before and after enzymatic hydrolysis (corn steep liquor hydrolysate) in Example 3 was determined. The amino acid content was determined using an amino acid analyzer according to the method in GB5009.124—2016, "National Food Safety Standard - Determination of Amino Acids in Food". The corn steep liquor and its enzymatic hydrolysate were freeze-dried and stored for later use. 0.05 g of sample was accurately weighed into each ampoule, 9 mL of 6.67 M hydrochloric acid was added, nitrogen gas was introduced, and the ampoule was sealed. The sealed ampoule was placed at 110℃ for hydrolysis for 20 h. After removal, the solution in the ampoule was adjusted to pH 2.2, and sodium citrate buffer solution was added to bring the volume to 100 mL. The solution was filtered twice through a 0.22 μm filter membrane, and the amino acid content in the sample was determined using a fully automated amino acid analyzer. The results are shown in Table 1.

[0066] Table 1. Amino acid content of corn steep liquor before and after hydrolysis

[0067]

[0068] Table 1 shows that the amino acid content in corn steep liquor was 21.86 g / 100g (P<0.5), and after enzymatic hydrolysis, the amino acid content was 26.52 g / 100g (P<0.5), an increase of 4.67 g / 100g (P<0.5). Seventeen amino acids were detected in both the corn steep liquor and the enzymatic hydrolysate, indicating a rich variety and high nutritional value. The alkaline protease cleavage site is a peptide bond with an aromatic amino acid or a hydrophobic amino acid (tryptophan, tyrosine, phenylalanine, etc.) on the carboxyl side, producing small peptides or amino acids. The content of hydrophobic amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine) in the enzymatic hydrolysate all increased. The most abundant amino acid in the corn steep liquor was alanine, at 3.71 g / 100g, which increased to 4.42 g / 100g after hydrolysis, an increase of 0.71 g / 100g. Glutamic acid, glycine, and aspartic acid, as precursors for the synthesis of purine nucleotides, showed increased content after hydrolysis, increasing by 0.51 g / 100 g, 0.32 g / 100 g, and 0.24 g / 100 g, respectively. Since the production of cordycepin follows the purine nucleotide metabolic pathway, the increase in glutamic acid, glycine, and aspartic acid in the enzymatic hydrolysate provides abundant substrate raw materials for the later synthesis of cordycepin in Cordyceps militaris.

[0069] Example 7

[0070] The corn steep liquor hydrolysate powder (CSLH) obtained in Example 3 was added to the Cordyceps militaris fermentation medium (10 g / L glucose, 5 g / L peptone, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate) at concentrations of 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, and 3.5 g / L, respectively, at a ratio of 8 mL seed culture / 100 mL (1 × 10⁻⁶). 6Cordyceps militaris was inoculated at an inoculum size of 1 spore cells / mL, fermented at 25℃ and 120 rpm for 10 days to obtain the fermentation broth. The cordycepin content and mycelial dry weight were then determined. The cordycepin content was determined using the same method as in Example 5. The mycelial dry weight was determined as follows: the fermentation broth was centrifuged at 4000 rpm for 10 min at room temperature to obtain mycelium. The mycelium was washed 2-3 times with distilled water and filtered to obtain the mycelial sample. The sample was then placed in a petri dish and dried at 60-90℃ until constant weight. The measured weight was the mycelial dry weight (mg), and the mycelial content in the fermentation broth was the biomass (mg / L).

[0071] The results are as follows Figure 4 and Figure 5 As shown, adding 0.5–3.5 g / L of CSLH to the Cordyceps militaris fermentation medium can effectively improve the growth of Cordyceps militaris and promote the production of cordycepin. The cordycepin content is more than 3 times that of the untreated medium, and in some cases more than 10 times that of the untreated medium. The biomass can reach up to 1.5 times that of the original. This indicates that CSLH can induce Cordyceps militaris to produce more cordycepin, thereby improving the production efficiency of cordycepin and effectively reducing production costs.

[0072] Example 8

[0073] The difference from Example 3 is that the corn steep liquor protein concentration in step (1) is 0.15 g / mL. All other steps are the same as in Example 3.

[0074] Example 9

[0075] The difference from Example 3 is that the fermentation temperature in step (2) is 60°C, while the rest is the same as Example 3.

[0076] Example 10

[0077] The difference from Example 3 is that the fermentation time in step (2) is 2.5 hours, while the rest is the same as Example 3.

[0078] Example 11

[0079] The difference from Example 3 is that the enzyme inactivation temperature in step (3) is 98°C and the enzyme inactivation time is 15 min, while the rest are the same as in Example 3.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing corn steep liquor hydrolysate that promotes cordycepin production by Cordyceps militaris, characterized in that, The process includes the following steps: mixing corn steep liquor with protease for enzymatic hydrolysis, inactivating the enzyme, taking the supernatant and concentrating it under vacuum to obtain corn steep liquor hydrolysate concentrate, and drying the corn steep liquor hydrolysate concentrate to obtain corn steep liquor hydrolysate; the protease includes pepsin, neutral protease or alkaline protease; When the protease is pepsin, the enzymatic hydrolysis temperature is 35~45℃, the enzymatic hydrolysis time is 2.0~3.0h, and the enzymatic hydrolysis pH is 2.1; When the protease is a neutral protease, the enzymatic hydrolysis temperature is 45~55℃, the enzymatic hydrolysis time is 2.0~3.0h, and the enzymatic hydrolysis pH is 7.5; When the protease is an alkaline protease, the enzymatic hydrolysis temperature is 55~60℃, the enzymatic hydrolysis time is 2.0~3.0h, and the enzymatic hydrolysis pH is 9.0; The amount of pepsin, neutral protease, or alkaline protease added is 2.0% to 3.0% of the protein content.

2. The preparation method according to claim 1, characterized in that, The corn steep liquor is pretreated corn steep liquor. The pretreatment method includes the following steps: after removing precipitated impurities from the corn steep liquor, the supernatant is diluted to a corn steep liquor protein concentration of 0.08~0.2g / mL to obtain pretreated corn steep liquor.

3. The preparation method according to claim 1, characterized in that, The enzyme inactivation temperature is 90~100℃, and the enzyme inactivation time is 10~30min.

4. The preparation method according to claim 1, characterized in that, After vacuum concentration, a corn steep liquor hydrolysate concentrate with a solid content of 20-30 wt% is obtained.

5. A corn steep liquor hydrolysate capable of promoting cordycepin production by Cordyceps militaris, characterized in that, The corn steep liquor hydrolysate was prepared by the preparation method described in any one of claims 1 to 4.

6. The preparation method according to any one of claims 1 to 4 or the corn steep liquor hydrolysate according to claim 5 in the production of cordycepin by Cordyceps militaris.

7. A method for producing cordycepin, characterized in that, The method includes the following steps: adding the corn steep liquor hydrolysate of claim 5 to the Cordyceps militaris fermentation medium at a concentration of 0.5~3.5 g / L, inoculating with Cordyceps militaris for fermentation, and obtaining cordycepin.

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