Fermentation production method for enhancing epsilon-polylysine by supplementing epigallocatechin

Abstract

The invention discloses a fermentation production method for enhancing epsilon-polylysine by supplementing epigallocatechin, which comprises the following steps of: carrying out fed-batch fermentation to produce epsilon-polylysine when the concentration of the supplemented epigallocatechin is 0.4-2.0 L / d; and when epigallocatechin is added, the yield of epsilon-polylysine is respectively improved by 15.9% and 35.227% at most compared with the control group. In 5L tank fed-batch fermentation, the yield of epsilon-polylysine reaches up to 42.8 g / L when fermentation is performed for 184 hours, and the yield is 5.59 g / d; meanwhile, through transcriptome, metabolome and some key enzyme activity tests, the gallocatechin can enhance the tricarboxylic acid cycle pathway activity and antioxidant activity, and provides richer raw materials and energy substances for epsilon-polylysine synthesis.

Description

Technical Field

[0001] The invention relates to a method for enhancing the fermentation production of epsilon-polylysine by adding epigallocatechin, and belongs to the technical field of fermentation engineering. Background Art

[0002] ε-poly-L-lysine (ε-PL) is a new type of biopolymer formed by the condensation of ε-NH2 and α-COOH. It generally contains 25-35 L-lysine residues and is generally in the form of light yellow powder with a slightly bitter taste. It has strong hygroscopicity, is easily soluble in water, slightly soluble in ethanol, has good thermal stability, and has a wide pH range (pH <9). ε-PL has a unique molecular structure and many excellent properties, such as good antibacterial properties, thermal stability, edibility, water solubility, biodegradability, and non-toxicity to humans and the environment. ε-PL also has a wide antibacterial spectrum and has varying degrees of inhibition against Gram-negative bacteria, Gram-positive bacteria, yeast, mold, certain viruses and pathogens; at the same time, the minimum inhibitory concentration (MIC) of ε-PL against most Gram-negative and Gram-positive bacteria is less than 100μg / mL, which has a good antibacterial effect.

[0003] Based on the above advantages of ε-PL, ε-PL has a wide range of potential applications. The excellent antibacterial properties made ε-PL the first to be used in food preservation. At present, ε-PL has been used as a food preservative additive in many countries and regions. In addition, ε-PL can also be used in industries such as antibacterial materials, cosmetics, medicine and agriculture.

[0004] Streptomyces albulus is a highly aerobic microorganism. The production of ε-PL is mainly achieved through aerobic fermentation of Streptomyces albulus. Since the wild-type strain has a low ability to produce ε-PL, traditional methods such as physical and chemical mutagenesis, ribosome engineering, genome rearrangement, atmospheric pressure room temperature plasma and genetic engineering are usually used to select ε-PL high-yielding bacteria. For example, Japanese scholar Hiraki et al. first used resistance screening combined with mutagenesis in 1998 to select a mutant strain, which increased the yield of ε-PL by 10 times compared with the wild strain. Liu and Wang et al. used multiple streptomycin resistance breeding to obtain mutant strains with yields increased by 1.79 times and 2.75 times, respectively.

[0005] With the in-depth analysis of the mechanism of ε-PL synthesis pathway, more and more studies have emerged on the use of genetic engineering methods to modify white Streptomyces to improve its ability to produce ε-PL. For example, Hamano et al. (2007) cloned the Ask gene from S. albulus and mutated it, and then homologously expressed it in S. albulus, so that the modified engineered bacteria could increase the production of ε-PL to a certain extent. Subsequently, Wang Ze et al. (2020) homologously overexpressed the pls gene in S. albulus M-Z18, which increased its pls transcription level by 16.8 times. Wang et al. (2021) used kasOp * Promoter binding phage The ribosome binding site of the capsid protein was used to overexpress the ε-PL synthase gene (pls), which increased the ε-PL production by 88.2%. Fumihito et al. (2023) homologously overexpressed two genes, dapB and dapE, in the diaminopimelate (DAP) pathway, increasing the ε-PL production of the resulting engineered strain by 14%.

[0006] However, it is far from enough to use genetic engineering methods to transform engineered bacteria to increase the yield of ε-PL. Therefore, Kahar et al. (2001) established a two-stage pH control method for ε-PL fermentation production, and more and more studies have been conducted on the establishment and improvement of ε-PL fermentation processes. For example, Wang et al. (2018) used glucose and glycerol as mixed carbon sources, and adopted an improved "pH shock" and optimized oxygen supply method to obtain an ε-PL yield of 70.3 g / L. Liu et al. (2021) used cell immobilization combined with in situ separation technology (ISPR) to increase the ε-PL yield to 3.51 g / L in batch fermentation. Zeng et al. (2022) used an extract of Botrytis cinerea to induce Streptomyces albus, which ultimately increased the ε-PL yield by 1.34 times. Recently, Zhu et al. (2024) constructed an engineered strain overexpressing the L-lysine transporter gene lysP and the ε-PL polymerase gene pls, and obtained the current highest yield of 81.4±5.2 g / L under the condition of adding exogenous L-lysine in fed-batch fermentation.

[0007] Patent CN116751822A provides a method for promoting polylysine fermentation production by adding tea polyphenols. The method can effectively produce ε-PL by batch fermentation according to the concentration of added tea polyphenols in the range of 0.5-2.5 g / L. The ε-PL yield and bacterial concentration reach 43.4 g / L and 42.0 g / L, respectively, and the product yield reaches 5.21 g / (L·d); however, the ε-PL fermentation production cost is still relatively high. Therefore, the art is in urgent need of a fermentation method for improving the production efficiency of ε-PL and reducing its production cost. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a method for enhancing the fermentation production of ε-polylysine by adding epigallocatechin (EGC). Epigallocatechin (EGC) is an organic polyphenol compound with multiple physiological activities such as antioxidant, anti-inflammatory, antibacterial, anti-allergic and anti-diabetic. Studies have shown that this type of polyphenol compound has a strong antioxidant capacity and can effectively remove free radicals in cells, thereby playing a protective role, which is of great significance for maintaining human health and normal physiological metabolism of cells. However, there is no relevant report on the effect of epigallocatechin on the physiological metabolism and ε-PL biosynthesis of Streptomyces albus.

[0009] Therefore, the present invention adds a certain concentration of epigallocatechin components to the ε-polylysine fermentation medium and during the fermentation process, and uses white Streptomyces to perform batch or fed-batch fermentation, thereby effectively increasing the ε-polylysine yield and reducing its production cost, shortening the fermentation time of ε-polylysine to a certain extent, and at the same time, the present invention explores the effect of epigallocatechin on the physiological and biochemical mechanisms of the production bacteria.

[0010] In order to solve the above technical problems, the present invention provides a method for enhancing the fermentation production of ε-polylysine by adding epigallocatechin, and the specific scheme is as follows:

[0011] The fermentation medium used in the fermentation method of the present invention is added with 0.4-2.0 g / L of epigallocatechin.

[0012] Furthermore, the fermentation medium also includes 30-50 g / L of glucose, 2-6 g / L of yeast powder, 6-10 g / L of ammonium sulfate, 0.2-0.6 g / L of magnesium sulfate, 0.5-0.9 g / L of dipotassium hydrogen phosphate, 1.0-1.50 g / L of potassium dihydrogen phosphate, 0.015-0.035 g / L of ferrous sulfate and 0.02-0.05 g / L of zinc sulfate.

[0013] Furthermore, the strain producing ε-polylysine is Streptomyces albus, Streptomyces hygroscopicus, Streptomyces graminearum, Streptomyces chrysanthemi or Streptomyces griseus.

[0014] Furthermore, the fermentation process is to inoculate the production strain into the fermentation medium at an inoculation rate of 4-12%, and ferment at a temperature of 26-33°C, a rotation speed of 130-490rpm, and a dissolved oxygen of 10-30%.

[0015] Furthermore, the fermentation is batch fermentation or fed-batch fermentation.

[0016] Furthermore, during batch fermentation, the following steps are included: when the pH naturally drops from the initial 6.5-7.6 to 3.6-4.0, the pH is maintained at 3.6-4.0 by adding a pH regulator until the batch fermentation is completed.

[0017] Furthermore, during the fed-batch fermentation, the pH is controlled by the following steps: when the pH naturally drops from the initial 6.5-7.6 to 4.5-5.6, a pH regulator is added to maintain the pH at 4.5-5.6 for 6-10 hours; then, when the pH naturally drops to 3.4-3.9, a pH regulator is added to maintain the pH at 3.4-3.9 until the fed-batch fermentation is completed.

[0018] Furthermore, the fed-batch fermentation comprises the following steps: when the residual glucose concentration in the fermentation broth drops below 8.0 g / L, adding a glucose solution to control the glucose concentration in the fermentation broth at 7-11 g / L, and adding a nitrogen source solution to maintain the amino nitrogen concentration in the fermentation broth at 0.5-1.0 g / L; and between 72 and 160 hours of fermentation, adding an epigallocatechin solution to maintain the epigallocatechin concentration in the fermentation broth at 0.4-2.0 g / L.

[0019] Furthermore, the concentration of the glucose solution is 300-700 g / L; the nitrogen source solution is a mixed nitrogen source solution of 50-90 g / L ammonium sulfate and 70-100 g / L yeast powder, and the concentration of the epigallocatechin solution is 30-70 g / L.

[0020] Beneficial Effects

[0021] (1) The present invention produces ε-polylysine by adding epigallocatechin to the fermentation medium and during the fermentation process. The added epigallocatechin has a low price, thus saving costs.

[0022] (2) The fermentation method of the present invention increases the yield of ε-polylysine. Under the same conditions, the maximum yield of ε-polylysine in shake flask and tank batch fermentation with epigallocatechin added by the present invention is 15.9% higher than that of comparative example 1 as a control, and 35.227% higher than that of comparative example 4. In fed-batch fermentation, the maximum yield of ε-polylysine of the present invention reaches 42.8 g / L, and the yield reaches 5.59 g L / d, which has good economic benefits.

[0023] (3) The fermentation method of the present invention is simple and feasible. In the fed-batch fermentation process, the fermentation time for obtaining the highest ε-polylysine production is 184 h, at which the maximum ε-polylysine production reaches 42.8 g / L, and the productivity of ε-polylysine reaches 5.59 g L / d, which shortens the fermentation time to a certain extent. At the same time, because the maximum production is achieved earlier in the fed-batch fermentation process of the present invention, in actual production, the energy consumption required for the operation of the fermentation process equipment (such as fermentation tank stirring, gas supply, cooling and heating, etc.) can be saved, further saving costs.

[0024] (4) The culture medium used in the fermentation method of the present invention has a low nutrient content. The concentrations of glucose and yeast extract of the present invention are 40 g / L and 4 g / L, respectively. That is, the present invention has a greater cost advantage in terms of raw materials.

[0025] (6) The fermentation method of the present invention has a demand for oxygen (dissolved oxygen) of 10-30%, which means that the demand for stirring rate and air flow rate in the fermentation process of the present invention is relatively low, and the energy consumption is also reduced accordingly. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a morphological diagram of a single colony of Streptomyces albus on a plate at different culture times of the present invention;

[0027] Figure 2 is a scanning electron microscope image of the hyphae of Streptomyces albus when fermented for 24 hours by the method of the present invention;

[0028] Figure 3 It is a bar graph of important metabolites and key enzyme activities in the fermentation process of the present invention;

[0029] Figure 4 is a volcano plot of differentially expressed genes in the transcriptome of the present invention;

[0030] Figure 5 These are the top 10 significantly differentially expressed genes in the three main GO terms of the transcriptome of the present invention;

[0031] Figure 6 The top 30 significantly differentially expressed genes in the KEGG analysis of the transcriptome of the present invention;

[0032] Figure 7 is a chemical classification diagram of metabolites in the cells of the present invention;

[0033] Figure 8 It is the differential metabolite profile of the present invention under the KEGG classification. DETAILED DESCRIPTION

[0034] The test methods involved in the following embodiments are as follows:

[0035] 1. ε-polylysine content determination method:

[0036] The present invention adopts methyl orange colorimetry to determine the content of ε-polylysine;

[0037] (1) Preparation of standard curve: Accurately weigh a certain amount of ε-polylysine standard to prepare standard solutions with ε-polylysine concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, 0.10 mg / mL, 0.12 mg / mL, 0.14 mg / mL and 0.16 mg / mL, respectively. Then use a UV spectrophotometer to detect the OD values ​​of the solutions. 465 Absorbance value, make standard curve;

[0038] (2) Measure the OD of the sample using the same method 465 The absorbance value is used to calculate the content of ε-polylysine using the standard curve prepared in step (1).

[0039] The culture medium and solution configuration methods involved in the following embodiments are as follows:

[0040] 1. Preparation of fermentation medium (g / L):

[0041] Glucose 40, yeast powder 4, ammonium sulfate 8, magnesium sulfate 0.4, dipotassium hydrogen phosphate 0.7, potassium dihydrogen phosphate 1.2, ferrous sulfate 0.02, zinc sulfate 0.03; adjust pH to 6.8 and sterilize at 121℃ for 20 minutes.

[0042] 2. Preparation of fermentation medium with epigallocatechin-3-phosphate (g / L):

[0043] Glucose 40, yeast powder 4, ammonium sulfate 8, magnesium sulfate 0.4, dipotassium hydrogen phosphate 0.7, potassium dihydrogen phosphate 1.2, ferrous sulfate 0.02, zinc sulfate 0.03, epigallocatechin 1.5; adjust the pH to 6.8 and sterilize at 121℃ for 20 minutes.

[0044] 3. Preparation of 0.75mmol / L methyl orange solution:

[0045] Accurately weigh 0.491 g of the recrystallized and freeze-dried methyl orange, dissolve it in distilled water, make the volume to 2 L, transfer it to a brown narrow-necked bottle, and store it at 4°C away from light for later use.

[0046] 4. Preparation of Epigallocatechin (EGC) Stock Solution:

[0047] Weigh 0.1 g of epigallocatechin powder, dissolve it in sterile water and dilute to 1 ml to prepare a 100 g / L epigallocatechin mother solution, then filter and sterilize it with a filter membrane and transfer it to a sterile brown narrow-necked bottle, and store it at 4°C away from light for future use.

[0048] 5. Preparation of Epigallocatechin Gallate (EGCG) Stock Solution:

[0049] Weigh 0.1 g of epigallocatechin gallate, dissolve it in an appropriate amount of sterile water, and dilute to 10 ml. Shake well to prepare a 10 g / L epigallocatechin gallate mother solution and filter aseptically to remove bacteria in the solution to ensure sterility. Store properly at 4°C for future use.

[0050] 6. Preparation of epicatechin gallate (ECG) stock solution:

[0051] Weigh 0.1 g of epicatechin gallate, dissolve it in an appropriate amount of sterile water, and dilute to 10 ml. Shake well to prepare a 10 g / L epicatechin gallate mother solution and filter aseptically to remove bacteria in the solution to ensure sterility. Store properly at 4°C for future use.

[0052] 7. White Streptomyces seed liquid culture method:

[0053] After 40 ml of fermentation medium is placed in a 250 ml Erlenmeyer flask and sterilized, Streptomyces albus is inoculated into the fermentation medium under sterile conditions using an inoculation loop and stirred until all the bacteria are suspended in the medium; then, it is placed in a constant temperature shaker and cultured at 30°C and 200 r / min for 24 hours to obtain a seed solution.

[0054] Raw materials and equipment involved in the embodiments:

[0055] Epigallocatechin (EGC) involved in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with the product number S31356;

[0056] Epigallocatechin gallate (EGCG) involved in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with the product number S25528;

[0057] The epicatechin gallate (ECG) involved in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with the product number S22728;

[0058] The strain used in the following examples is Streptomyces albus;

[0059] The fermentation tank involved in the following examples was purchased from Shanghai Baoxing Biological Equipment Engineering Co., Ltd., model BIOTECH-5JG-7000A5L.

[0060] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0061] Embodiment 1:

[0062] Fermentation culture of ε-PL by adding epigallocatechin to fermentation medium:

[0063] (1) Add 40 mL of fermentation medium into a 250 mL shake flask, add 0.75 mL of epigallocatechin mother solution and 0.25 mL of sterile water, so that the concentration of epigallocatechin in the fermentation medium is 1.5 g / L;

[0064] (2) 4 mL of the seed solution was inoculated into the fermentation medium supplemented with epigallocatechin obtained in step (1), and fermented for 72 h at a fermentation temperature of 30° C. and a shaking speed of 180 rpm.

[0065] The results showed that the ε-PL yield and bacterial cell volume were 1.31 g / L and 8.79 g / L, respectively.

[0066] Comparative Example 1:

[0067] Add sterile water to fermentation medium to ferment ε-PL:

[0068] (1) Add 40 mL of fermentation medium to a 250 mL shake flask and add 1 mL of sterile water;

[0069] (2) 4 mL of the seed solution was inoculated into the culture medium obtained in step (1), and fermented for 72 h at a fermentation temperature of 30° C. and a shaking speed of 180 rpm.

[0070] The results showed that the ε-PL yield and bacterial cell volume were 1.13 g / L and 5.79 g / L, respectively.

[0071] Comparative Example 2:

[0072] Add EGCG to fermentation medium to ferment ε-PL:

[0073] (1) Add 40 mL of fermentation medium to a 250 mL shake flask, add 0.75 mL of epigallocatechin gallate (EGCG) stock solution and 0.25 mL of sterile water to make the EGCG concentration in the fermentation medium 1.5 g / L;

[0074] (2) 4 mL of the seed solution was inoculated into the culture medium obtained in step (1), and fermented for 72 h at a fermentation temperature of 30° C. and a shaking speed of 180 rpm.

[0075] The results showed that the ε-PL yield and bacterial cell volume were 1.09 g / L and 6.37 g / L, respectively.

[0076] Comparative Example 3:

[0077] Add ECG fermentation medium to ferment ε-PL:

[0078] (1) Add 40 mL of fermentation medium to a 250 mL shake flask, add 0.75 mL of epicatechin gallate (ECG) mother solution and 0.25 mL of sterile water to make the ECG concentration in the fermentation medium 1.5 g / L;

[0079] (2) 4 mL of the seed solution was inoculated into the culture medium obtained in step (1), and fermented for 72 h at a fermentation temperature of 30° C. and a shaking speed of 180 rpm.

[0080] The results showed that the ε-PL yield and bacterial cell volume were 1.11 g / L and 7.12 g / L, respectively.

[0081] Embodiment 2:

[0082] Production of ε-PL by fermentation using fermentation medium supplemented with epigallocatechin:

[0083] (1) Add 3 L of epigallocatechin-containing fermentation medium to a 5 L fermentation tank, then inoculate 0.3 L of seed solution, adjust the pH value to 7.0, adjust the fermentation temperature to 30° C., adjust the stirring speed to 180 rpm, the ventilation volume to 0.3 vvm, control the dissolved oxygen content to above 10%, and the tank pressure to 0.03 MPa (gauge pressure);

[0084] (2) In the early stage of fermentation, when the pH spontaneously dropped to 3.8, the pH was maintained at 3.8 by automatically adding 12.5% ​​(w / w) ammonia solution until the product concentration began to decrease and the fermentation ended [Note: fermentation end point sign: generally the product concentration began to decrease]. The yield reached the highest level after 72 hours of fermentation; the total fermentation time was 96 hours.

[0085] The results showed that the ε-PL yield and bacterial cell volume were 2.38g / L and 9.87g / L, respectively.

[0086] Comparative Example 4:

[0087] Batch fermentation production of ε-PL using fermentation medium:

[0088] (1) Add 3 L of fermentation medium to a 5 L fermentation tank, then inoculate 0.3 L of seed solution, adjust the pH value to 7.0, adjust the fermentation temperature to 30° C., adjust the stirring speed to 180 rpm, the ventilation volume to 0.3 vvm, control the dissolved oxygen content to more than 10%, and the pressure in the tank to 0.03 MPa (gauge pressure);

[0089] (2) In the early stage of fermentation, when the pH spontaneously dropped to 3.8, the pH was maintained at 3.8 by automatically adding 12.5% ​​(w / w) ammonia solution until the product concentration began to decrease and the fermentation ended [Note: fermentation end point sign: generally the product concentration began to decrease]; the yield reached the highest level after 72 hours of fermentation; the total fermentation time was 96 hours.

[0090] The results showed that the ε-PL yield and bacterial cell volume were 1.76 g / L and 8.67 g / L, respectively.

[0091] Embodiment 3:

[0092] Production of ε-PL by fed-batch fermentation with epigallocatechin-3-phosphate

[0093] (1) Add 3 L of epigallocatechin-containing fermentation medium to a 5 L fermentation tank, then inoculate 0.3 L of seed solution, adjust the pH value to 7.0, adjust the fermentation temperature to 30° C., adjust the stirring speed to 180 rpm, the ventilation volume to 0.3 vvm, control the dissolved oxygen content to above 10%, and the tank pressure to 0.03 MPa (gauge pressure);

[0094] (2) The pH was adjusted by a three-stage pH control method: when the pH dropped naturally from the initial 6.8 to 5.0, the pH was maintained at 5.0 for about 8 hours; then, when the pH dropped naturally from 5.0 to 3.8, 12.5% ​​(w / w) ammonia water was automatically added to maintain the pH at 3.8 until the fermentation was completed;

[0095] Feeding method: when the glucose concentration in the fermentation liquid drops below 10 g / L, a sterilized concentrated 600 g / L glucose solution is automatically added to control the glucose concentration in the fermentation liquid at about 10 g / L; at the same time, a sterilized mixed nitrogen source solution containing 80 g / L ammonium sulfate and 100 g / L fish meal is automatically added to maintain the amino nitrogen concentration in the fermentation liquid at about 1 g / L; from the 3rd to the 7th day of fermentation, 6.5 mL of a filtered and sterilized 60 g / L epigallocatechin solution is added every 24 hours to maintain the epigallocatechin concentration in the fermentation liquid at about 1.48 g / L; the total fermentation time is about 200 h.

[0096] The results showed that the fermentation time for obtaining the highest ε-polylysine production was 184 h, at which time the ε-PL production was 42.8 g / L and the ε-PL yield was 5.59 g / L / d; the fermentation time for obtaining the highest bacterial cell volume was 176 h, at which time the bacterial cell volume was 42.8 g / L.

[0097] Patent CN116751822A provides a method for adding tea polyphenols to promote polylysine fermentation production. The specific embodiments of the present invention are different from the patent CN116751822A in that: (1) the nutrients required by the method of the present invention are lower than those in CN116751822A. The glucose concentration in the fermentation medium used in the present invention is 40 g / L, while that in the reference document is 50 g / L. The contents of yeast powder and ammonium sulfate are also reduced to a certain extent. Therefore, the present invention has a greater cost advantage in terms of raw material requirements;

[0098] (2) During the fermentation process, the oxygen demand (dissolved oxygen rate) of the present invention is above 10%, which is lower than the dissolved oxygen rate of CN116751822A of above 20%, indicating that the fermentation process of the present invention has lower requirements for stirring rate and air flow, and correspondingly, energy consumption is also reduced;

[0099] (3) In the fed-batch fermentation process, although the maximum ε-polylysine yield (42.8 g / L) of the present invention is slightly lower than the maximum ε-polylysine yield (43.4 g / L) of CN116751822A, the time to obtain the maximum ε-polylysine yield (184 h) is 16 h earlier than that of CN116751822A (200 h), and the productivity of ε-polylysine (5.59 g / L / d) is higher than that of CN116751822A (5.2 g / L / d); at the same time, because the present invention reaches the maximum yield earlier, in actual production, the method of the present invention can be used to release the tank 16 h earlier than CN116751822A, saving the energy consumption required for the operation of the fermentation process equipment (such as fermentation tank stirring, gas supply, cooling and heating, etc.).

[0100] Embodiment 4:

[0101] Observation of colony and mycelial ball morphology:

[0102] The fermentation broths in Example 1 and Comparative Example 1 were taken respectively, and single colonies of Streptomyces albus were isolated on the culture medium by streaking culture on a plate, and the colonies were cultured for 4 days, 6 days and 10 days respectively, and the morphology of single colonies of Streptomyces albus on the plate was observed;

[0103] The bacterial liquid of Example 1 and Comparative Example 1 was fermented in a shake flask for 48 hours, and the morphology of the mycelial balls in the bacterial liquid of Example 1 and Comparative Example 1 was observed under a scanning electron microscope with magnifications of 1000× (50.0 μm), 10000× (5.00 μm) and 20000× (2.00 μm), respectively.

[0104] The results are as follows:

[0105] The results of single colony morphology observation on the plate are as follows Figure 1As shown, CK is the blank control group comparative example 1, and T is the experimental group example 1; on the 4th day of culture, there were significant differences between the two groups of single colonies, the edges of the colonies in the experimental group were light yellow, and the protrusion in the middle was obvious, while the colonies in the control group were white, and the surfaces of the colonies in the two groups were smooth; on the 6th day of culture, wrinkles appeared on the surfaces of the colonies in the two groups, the colonies in the blank group were still white, the colonies in the experimental group were light yellow as a whole, and some colonies gradually turned green; on the 10th day of culture, the edges of the colonies in the blank group were light yellow, and the colonies in the experimental group were slightly green; overall, the colonies in the experimental group were darker in color, larger in diameter, more obvious in the middle, faster in colony growth, and better in growth than the blank group, which indicates that gallic acid tea has a significant effect on the morphology of white Streptomyces, which is beneficial to the growth of the bacteria.

[0106] The results of observation on the morphology of mycelial balls in liquid fermentation culture are as follows Figure 2 As shown, CK is the blank control group comparative example 1, and T is the experimental group example 1; in the culture medium with added epigallocatechin (T), the mycelium density is larger, the mycelium breaks are less, and the mycelium surface is smooth; while in the control culture medium, the mycelium density is smaller, the mycelium breaks are more, and the mycelium surface is obviously damaged; the results show that epigallocatechin has a good protective effect on the mycelium morphology of Streptomyces albus in liquid fermentation culture, and can provide a good environment for the normal physiological metabolism of Streptomyces albus cells.

[0107] Embodiment 5:

[0108] Determination of important metabolites and key enzyme activities during the growth and product synthesis of Streptomyces albus:

[0109] 5 mL of the bacterial solution from Example 1 and Comparative Example 1 after 24 h, 48 h and 72 h of fermentation culture were taken respectively, and the activities of important metabolites and key enzymes in the bacterial growth and product synthesis processes were determined respectively; the important metabolites and key enzymes determined included malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and ε-polylysine synthase (PLS).

[0110] The results are as follows:

[0111] The results are as follows Figure 3 As shown, CK is the blank control group comparative example 1, and T is the experimental group example 1; the results show that the enzyme activity of the experimental group with the addition of epigallocatechin is improved to varying degrees compared with the control group, indicating that epigallocatechin can resist the intracellular oxidative stress reaction, so that the physiological metabolism of the strain is significantly improved, that is, epigallocatechin helps to increase the supply of intermediate metabolites such as precursor L-lysine, improve the activity of ε-polylysine synthase used for product polymerization, and finally increase the yield of ε-PL.

[0112] In the initial stage of fermentation, the intracellular lipid peroxidation level MDA content of the experimental group showed a higher level than that of the control group; however, as the fermentation entered the late logarithmic growth phase, although the number of bacteria in the T group increased rapidly and proliferated significantly, its MDA activity decreased and was lower than that of the control group, which shows that the addition of epigallocatechin can effectively reduce MDA and may play a role in resisting oxidative damage caused by acid stress; at the end of fermentation, the MDA content of the two was basically at the same level, indicating that epigallocatechin can indeed reduce the MDA content.

[0113] The SOD and CAT enzyme activities in the experimental group were always higher than those in the control group, which indicated that epigallocatechin had a certain degree of antioxidant effect on S. albus.

[0114] Embodiment 6:

[0115] Transcriptome differentially expressed gene determination:

[0116] 10 mL of bacterial solution from Example 1 and Comparative Example 1 after fermentation for 24 h was taken respectively, and transcriptome differentially expressed genes were determined to reveal the molecular mechanism of epigallocatechin on ε-PL synthesis at the transcriptional level.

[0117] The results are as follows:

[0118] 1. The results are as follows Figure 4 As shown, Figure 4 It is a volcano plot of differentially expressed genes, where CK is the blank control group comparative example 1, and T is the experimental group example 1. It was found that there were 3315 differentially expressed genes, of which 1436 genes were upregulated in the experimental group relative to the control group, while 1879 genes were downregulated.

[0119] 2. GO analysis of significantly differentially expressed genes:

[0120] GO analysis (Gene Ontology) is divided into three parts: biological process BP, molecular function MF and cell composition CC. In each GO classification, this example selects the top 10 GO term entries with the most obvious enrichment for analysis. The results are as follows: Figure 5 As shown. It can be seen that epigallocatechin-3-glucopyranoside has a significant effect on the cell metabolism, structural components and transcription of genes related to material transport of Streptomyces albus.

[0121] Through the DEGs analysis of up-regulated genes, it was found that the significantly enriched item in cellular components was "membrane" (GO: 0016020), and the significantly concentrated molecular function areas mainly included "transmembrane transporter activity" (GO: 0022857), "transporter activity" (GO: 0005215) and "FAD binding" (GO: 0071949); the significantly enriched items in biological processes were "establishing positioning" (GO: 0051234), "transmembrane transport" (GO: 0055085), "localization" (GO: 0051179) and "transport" (GO: 0006810); this indicates that epigallocatechin may further affect the metabolic process of Streptomyces albus by affecting the transmembrane transport and signal transduction processes of Streptomyces albus.

[0122] Through the DEGs analysis of down-regulated genes, it was found that in terms of cellular components, the significantly enriched item was "intracellular anatomical knot" (GO: 0005622); in terms of molecular function, the prominent enriched items were "structural molecular activity" (GO: 0005198) and "structural component of ribosome" (GO: 0003735); the significantly enriched items in biological processes were "protein metabolic process" (GO: 0044267), "translation" (GO: 0006412), "peptide metabolic process" (GO: 0006518), "peptide biosynthesis process" (GO: 0043043) and "cellular metabolic process" (GO: 0044237); the functional annotation results of these down-regulated genes showed that epigallocatechin-3 had a great effect on the ribosome and material metabolism of Streptomyces albus.

[0123] 3. KEGG analysis of significantly differentially expressed genes:

[0124] Based on the KEGG enrichment analysis results of differentially expressed genes, this example selects the top 30 metabolic pathways with the lowest p-value, that is, the most obvious enrichment, for presentation. The results are as follows: Figure 6 As shown; KEGG enrichment analysis results showed that DEGs were mainly concentrated in "bacterial secretion system", "ribosome" and "protein export";

[0125] KEGG enrichment analysis of up-regulated genes revealed that DEGs were enriched in key pathways such as “quorum sensing,” “ABC transporters,” and “biosynthesis of vancomycin group antibiotics”;

[0126] Among them, the up-regulation of quorum sensing-related genes was mainly concentrated in secD and yidC genes. secD is a protein translocase subunit, and yidC is a membrane protein insertase. The Sec apparatus is a multiprotein complex that promotes the insertion of certain membrane proteins. It is a means for many extracytoplasmic proteins to translocate across the cytoplasmic membrane. secD helps protein export.

[0127] The up-regulated expression of ABC transporter-related genes was mainly concentrated in pstA, pstC, pstS and cbiQ genes. ABC transporters are major active transporters that can consume ATP to transport small and large molecules. ABC transporters help improve the utilization efficiency of nutrients and also reduce the content of harmful substances in cells.

[0128] The up-regulated expression of genes related to the biosynthesis of vancomycin antibiotics was mainly concentrated in the hppD and rfbB genes, hppD is 4-hydroxyphenylpyruvate dioxygenase, and rfbB is dTDP-glucose 4,6-dehydratase; 4-hydroxyphenylpyruvate dioxygenase (hppD) is an Fe(II)-dependent non-heme oxygenase that catalyzes the second step of the tyrosine catabolic pathway, which is basically the common denominator of all aerobic life forms; rfbB uses NAD coenzyme to produce dTDP-4-keto-6-deoxyglucose from dTDP-α-d-glucose through a series of oxidation, dehydration and reduction steps; From the above analysis, it can be seen that epigallocatechin mainly affects the transporter genes of Streptomyces albus. It up-regulated some transporter genes and improved the material transport and enzyme decomposition capabilities of Streptomyces albus to a certain extent;

[0129] The results of KEGG enrichment analysis of down-regulated genes showed that the differentially expressed genes were mainly concentrated in the field of "ribosome", which was consistent with the results of GO enrichment analysis; the down-regulation of ribosome-related genes mainly involved protein genes composed of 50S large subunit and 30S small subunit, which indicated that the biosynthesis of ribosomes was inhibited to a certain extent; this inhibition may reduce protein production in cells, thereby limiting the excessive growth of bacteria, reducing the consumption of ATP in cells, and reserving energy for subsequent growth metabolism, which may trigger the specific stress response mechanism of Streptomyces albus;

[0130] Streptomyces albus uses a two-component system (TCS) signal transduction system to sense and respond to environmental stress, which is an important adaptive mechanism. In this system, TCS uses membrane proteins as pressure sensors and regulates gene expression through transcription factors, allowing bacteria to respond quickly and effectively to changes in the external environment. Epigallocatechin, as a plant secondary metabolite, has been shown to cause changes in many signal transduction-related genes in Streptomyces albus, indicating that epigallocatechin may indirectly regulate the bacterial signal transduction network by affecting the activity of the TCS system. Since TCS plays a major role in signal transduction, bacteria can adapt to different stress environments and maintain the stability of survival and growth.

[0131] Embodiment 7:

[0132] Intracellular metabolomics assays:

[0133] 10 mL of bacterial solution from Example 1 and Comparative Example 1 after fermentation for 48 h was taken respectively for intracellular metabolomics determination.

[0134] The results are as follows:

[0135] 1. Chemical classification of metabolites in Streptomyces albus cells Figure 7 As shown, the results showed that a total of 412 metabolites were identified in Streptomyces albus cells; lipids and lipid molecules occupied a significant proportion, followed by organic oxygen compounds.

[0136] The OPLS-DA method was used to screen for significantly differential metabolites, and the VIP value was set to be greater than 1 and the P value was set to be less than 0.05 as the screening criteria. A total of 149 differential metabolites were detected between the CK and T groups, including 60 significantly up-regulated metabolites and 89 significantly down-regulated metabolites. The significantly up-regulated metabolites mainly included citric acid, malic acid, catechins, 5'-pyridoxine phosphate and uridine-5'-monophosphate, and the significantly down-regulated metabolites included deoxyinosine, agaric acid, succinic acid and glucoside. Citric acid and malic acid are core intermediate metabolites in the tricarboxylic acid cycle, and their significant upregulation is often regarded as an enhancement of the activity of the tricarboxylic acid cycle pathway. Citric acid is the first substance synthesized in the tricarboxylic acid cycle. It is generated by the condensation of acetyl CoA and oxaloacetic acid under the catalysis of citrate synthase. This step is a key step in the tricarboxylic acid cycle and is a highly exergonic reaction.

[0137] Malic acid is the penultimate substance synthesized in the tricarboxylic acid cycle. The metabolite after malic acid is oxaloacetic acid, which is the last substance synthesized in the tricarboxylic acid cycle. The significant increase in malic acid content provides a possibility for the increase in the synthesis of oxaloacetic acid. Oxaloacetic acid can condense with acetyl CoA to continue to generate citric acid. The supply of oxaloacetic acid is conducive to the smooth progress of the tricarboxylic acid cycle. Oxaloacetic acid can also provide raw materials for the synthesis of other metabolites. For example, oxaloacetic acid can form aspartic acid under the action of aspartate aminotransferase. Aspartic acid then enters the diaminopimelic acid (DAP) pathway through a series of reactions to synthesize lysine. Lysine is then synthesized into ε-polylysine under the action of ε-polylysine synthase.

[0138] Therefore, the significant increase in citric acid and malic acid provides more intermediate raw materials for the synthesis of ε-polylysine, providing raw material guarantee for the increase in ε-polylysine production; at the same time, one citric acid molecule participates in the tricarboxylic acid cycle, and can generate a total of 10 ATP molecules until the end of the cycle. The activity of the tricarboxylic acid cycle pathway can be enhanced by the increase in citric acid content, producing more ATP, and providing more energy for the synthesis of ε-polylysine that requires ATP consumption;

[0139] Bankar et al. found that adding L-aspartic acid and citric acid can significantly increase the production of ε-polylysine from 97.08 mg / L to 409.94 mg / L. This is because the increase in the content of citric acid inside the cell effectively promotes the synthesis of ATP in the cell and enhances the stability of the pH value in the cell. This stability has a significant effect on improving the cell's resistance to the acidic environment; therefore, the upregulation of citric acid is also one of the important mechanisms for cells to adapt to the acidic environment and maintain the stability of their physiological functions.

[0140] Pyridoxamine phosphate, as an active ingredient of vitamin B6, plays a key coenzyme role in the metabolism of amino acids, especially in transamination and decarboxylation reactions. Some amino acids (such as aspartic acid) and oxaloacetate can be converted through transamination reactions. Oxaloacetate is a precursor for the synthesis of L-lysine, which is positively correlated with 5'-pyridoxamine phosphate in metabolism and has an increasing content. This trend promotes the accumulation of aspartic acid and ultimately promotes the increase in the production of ε-polylysine.

[0141] If succinate is upregulated in the tricarboxylic acid cycle, it will inhibit the reaction of fumarate to malate and oxaloacetate to aspartate. Therefore, downregulation of succinate is beneficial to increase the production of ε-polylysine.

[0142] 2. KEGG enrichment pathway analysis of differential metabolites:

[0143] The KEGG database was used to perform functional annotation and enrichment analysis on the differential metabolites. The results of differential metabolite profiles of the CK and T groups under the KEGG classification were as follows: Figure 8 As shown in the figure, the differential metabolites between the CK group and the T group are mainly concentrated in multiple key metabolic pathways, including but not limited to the tricarboxylic acid cycle, purine nucleotide metabolism, carbon source metabolism, oxidative phosphorylation process, multiple core metabolic pathways and pyrimidine nucleotide metabolism; among them, the most significant difference is the tricarboxylic acid cycle pathway, with a P value of 0.00042. Combined with the above results, compared with the CK group, the contents of citric acid and malic acid in the T group showed an upward trend, and succinic acid showed a downward trend, which provided more intermediate metabolites and energy substances for promoting the synthesis of ε-polylysine.

[0144] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A fermentation method for producing ε-polylysine, characterized in that: The method comprises the following steps: when the epsilon-polylysine producing strain is fermented to produce epsilon-polylysine, 0.4-2.0 g / L of epigallocatechin is added into the fermentation medium.

2. The fermentation method according to claim 1, characterized in that The fermentation medium comprises 30-50 g / L of glucose, 2-6 g / L of yeast powder, 6-10 g / L of ammonium sulfate, 0.2-0.6 g / L of magnesium sulfate, 0.5-0.9 g / L of dipotassium hydrogen phosphate, 1.0-1.50 g / L of potassium dihydrogen phosphate, 0.015-0.035 g / L of ferrous sulfate and 0.02-0.05 g / L of zinc sulfate.

3. The fermentation method according to claim 1, characterized in that The ε-polylysine producing strain may be any one of Streptomyces albus, Streptomyces hygroscopicus, Streptomyces graminearum, Streptomyces chrysanthemi or Streptomyces griseus.

4. The fermentation method according to claim 1, characterized in that The steps of producing ε-polylysine by fermentation are as follows: inoculating the ε-polylysine producing strain into a fermentation medium added with 0.4-2.0 g / L epigallocatechin at an inoculum amount of 4-12%, and fermenting at a temperature of 26-33° C., a rotation speed of 130-490 rpm, and a dissolved oxygen content of 10-30%.

5. The fermentation method according to claim 4, characterized in that The fermentation is batch fermentation or fed-batch fermentation.

6. The fermentation method according to claim 4 or 5, characterized in that: The batch fermentation comprises the following steps: when the pH naturally drops from the initial 6.5 to 7.6 to 3.6-4.0, the pH is maintained at 3.6-4.0 by adding a pH regulator until the batch fermentation is completed.

7. The fermentation method according to claim 4 or 5, characterized in that: During fed-batch fermentation, the pH is regulated by the following steps: when the pH naturally drops from the initial 6.5-7.6 to 4.5-5.6, a pH regulator is added to maintain the pH at 4.5-5.6 for 6-10 hours; then, when the pH naturally drops to 3.4-3.9, a pH regulator is added to maintain the pH at 3.4-3.9 until the fed-batch fermentation is completed.

8. The fermentation method according to claim 7, characterized in that During the fed-batch fermentation, the method further comprises the following steps: when the residual glucose concentration in the fermentation broth drops below 8.0 g / L, a glucose solution is added to control the glucose concentration in the fermentation broth at 7-11 g / L, and a nitrogen source solution is added to maintain the amino nitrogen concentration in the fermentation broth at 0.5-1.0 g / L; and at 72-160 hours of fermentation, an epigallocatechin solution is added to maintain the epigallocatechin concentration in the fermentation broth at 0.4-2.0 g / L.

9. The fermentation method according to claim 8, characterized in that The concentration of the glucose solution is 300-700 g / L; the nitrogen source solution is a mixed nitrogen source solution of 50-90 g / L ammonium sulfate and 70-100 g / L yeast powder.

10. The fermentation method according to claim 8, characterized in that The concentration of the epigallocatechin solution is 30-70 g / L.

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