CN110408607B - Fermentation optimization process for producing hyaluronidase by lactobacillus plantarum - Google Patents

Fermentation optimization process for producing hyaluronidase by lactobacillus plantarum Download PDF

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CN110408607B
CN110408607B CN201910822762.2A CN201910822762A CN110408607B CN 110408607 B CN110408607 B CN 110408607B CN 201910822762 A CN201910822762 A CN 201910822762A CN 110408607 B CN110408607 B CN 110408607B
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lactobacillus plantarum
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高瑞
韩鹏
韩秀云
齐宁
牛贵清
张彬
赵艳辉
郭弘彦
梁贵彬
王娜
刘文彬
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Abstract

The invention relates to the technical field of microbial fermentation, in particular to a fermentation optimization process for producing hyaluronidase by lactobacillus plantarum, which comprises the following steps: preparing a seed culture medium; inoculating lactobacillus plantarum into a seed culture medium, and culturing to obtain a liquid fermentation strain; preparing a liquid fermentation culture medium; inoculating the liquid fermentation strain into a liquid fermentation culture medium, and finishing fermentation after culture; centrifuging the fermentation liquor, and collecting supernatant to obtain crude enzyme solution. The invention creatively carries out the acclimation culture of the oxygen resistance of the bacteria by adjusting the fermentation culture rotating speed and the ventilation quantity, so that the lactobacillus plantarum can normally ferment and produce the hyaluronidase under the aerobic condition; through condition optimization, the yield of hyaluronidase in the lactobacillus plantarum metabolites is greatly improved: the hyaluronidase contained in the fermentation liquor prepared by the production process is 9604IU/mL, which is improved by 40 percent compared with that before the process optimization, thereby facilitating the subsequent separation and purification; the invention has simple and convenient production process and high stability, and is convenient for large-scale production and popularization.

Description

Fermentation optimization process for producing hyaluronidase by lactobacillus plantarum
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to an optimized fermentation process for producing hyaluronidase by lactobacillus plantarum.
Background
Hyaluronic acid is a major component constituting extracellular matrix of connective tissue of a host, and hyaluronidase (HAase) is a proteolytic enzyme capable of specifically decomposing Hyaluronic Acid (HA), which is an extracellular matrix component, to have a low molecular weight effect. The hyaluronidase acts on extracellular matrix to influence proliferation, differentiation and migration of cells, and plays a role in embryonic development and tumorigenesis development, so that the hyaluronidase has a very important role in effectively controlling pathogenic bacteria infection and preventing the pathogenic bacteria from spreading, and has an important significance in preventing and treating clinical bacterial infectious diseases.
Although the distribution of hyaluronidase is wide, the hyaluronidase is a neglected enzyme because the separation and purification are very difficult and the hyaluronidase is not well developed and utilized for a long time in the past. Meanwhile, the source of the hyaluronidase is very limited and the hyaluronidase is expensive, so that the application range of the hyaluronidase is limited to a great extent. Until the last decade, researchers have not begun to show a strong interest in HA and HAase, trying to explore their role in numerous biological processes. Research shows that hyaluronidase can promote the diffusion of injection by degrading HA in tissues, increasing the permeability of membranes, reducing viscosity and promoting the diffusion of the injection, and the phenomenon is called the diffusion effect of hyaluronidase. Based on the property, the hyaluronidase can be applied to the treatment process to accelerate the absorption speed, promote the absorption of injection, improve the local anesthesia effect and prevent the damage to tissues after subcutaneous and intramuscular injection. With the continuous and intensive understanding of the biological significance and importance of HA, low molecular weight HA and HA oligosaccharides, more and more evidence suggests that the biological activities of HA and HA oligosaccharides with low relative molecular mass are significantly different from HA.
Based on the above, the provided strain capable of producing hyaluronidase by fermentation and the fermentation process method capable of obviously improving the enzyme activity have very important significance.
Disclosure of Invention
Aiming at the problems of few strains and low enzyme activity of hyaluronidase fermentation production in the prior art, the invention provides a fermentation optimization process for producing hyaluronidase by lactobacillus plantarum, which improves the yield of hyaluronidase and improves the enzyme activity by 40% by improving the composition and culture conditions of a culture medium.
A fermentation optimization process for producing hyaluronidase by lactobacillus plantarum comprises the following steps:
s1: preparing a seed culture medium,
the seed culture medium comprises an inducer, a nitrogen source, inorganic salt and a surfactant,
the inducer can be simultaneously used as a carbon source and is hyaluronic acid with the molecular weight of 5g/L and 1000kDa-2000kDa,
the nitrogen source is a compound nitrogen source and comprises 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate,
the inorganic salt comprises 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride and 0.25g/L magnesium sulfate,
the surfactant is 1mL/L tween-80 or 0.05g/L disodium ethylene diamine tetraacetate;
s2: inoculating lactobacillus plantarum into a seed culture medium, and culturing to obtain a liquid fermentation strain;
s3: preparing a liquid fermentation culture medium,
the liquid fermentation culture medium comprises an inducer, a nitrogen source, inorganic salt and a surfactant,
the inducer can be used as a carbon source at the same time, is 1-10g/L hyaluronic acid with the molecular weight of 500KDa-1300kDa,
the nitrogen source is a compound nitrogen source and comprises 5g/L peptone, 10g/L yeast powder and 2g/L ammonium sulfate,
the inorganic salt comprises 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.02g/L manganese sulfate, 0.05g/L calcium chloride, 0.2g/L magnesium sulfate and 0.4g/L potassium dihydrogen phosphate,
the surfactant is 0.5mL/L-1.5mL/L tween-80 or 0.01g/L-0.08g/L disodium ethylene diamine tetraacetate;
s4: inoculating the liquid fermentation strain into a liquid fermentation culture medium, and finishing fermentation after culture;
s5: centrifuging the fermentation liquor, and collecting supernatant to obtain crude enzyme solution.
Furthermore, the Lactobacillus plantarum is Lactobacillus plantarum CnT012-56, the classified name of the strain is Lactobacillus plantarum, the preservation unit is China general microbiological culture Collection center, the preservation address is No. 3 of West Lu No.1 of the North Chen of the south-facing area in Beijing, the preservation time is 11 months and 28 days in 2018, and the preservation number is CGMCC NO. 16836.
Further, in S1, 200mL of seed culture medium was contained in a 500mL Erlenmeyer flask.
Furthermore, in the S2, the optimum culture conditions of the strain are pH6.0, the rotation speed is 50rpm, the culture temperature is 35 ℃, and the culture time is 18-24 h.
Further, in S3, a 5L fermenter was used to fill 4L of the liquid fermentation medium.
Further, in the S3, the inducer in the liquid fermentation medium is 5-8g/L hyaluronic acid with the molecular weight of 500KDa-1300 kDa.
Further, in the S3, the surfactant in the liquid fermentation medium is 1mL/L of Tween-80 or 0.05g/L of disodium ethylene diamine tetraacetate.
Further, in the S4, the optimum culture conditions of the strain are pH5.0-8.0, the culture temperature is 30-38 ℃, the culture is carried out for 48h, and the rotating speed and the ventilation volume are regulated in stages:
0-20h, the rotating speed is 50rpm, the ventilation volume is 2L/min,
20-30h, rotation speed of 100rpm and ventilation capacity of 4L/min
30-48h, the rotating speed is 200rpm, and the ventilation volume is 8L/min.
Further, in S4, the optimum culture conditions for the strain are preferably pH5.5 and the culture temperature is preferably 35 ℃.
Further, in the S4, the inoculation amount is 1% (v/v) to 10% (v/v), preferably 5% (v/v).
The invention provides a fermentation optimization process for producing hyaluronidase by lactobacillus plantarum, which has the advantages that,
(1) creatively adjusts the fermentation culture rotating speed and ventilation quantity to perform aerotolerant acclimation culture on the bacteria, so that the lactobacillus plantarum CnT012-56 can normally ferment and produce hyaluronidase under aerobic conditions;
(2) through condition optimization, the yield of hyaluronidase in the lactobacillus plantarum metabolites is greatly improved: the hyaluronidase contained in the fermentation liquor prepared by the production process is 9604IU/mL, which is improved by 40 percent compared with that before the process optimization, thereby facilitating the subsequent separation and purification;
(3) the invention has simple and convenient production process and high stability, and is convenient for large-scale production and popularization.
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In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a line graph showing the effect of 5g/L different carbon sources on seed culture in screening example 1;
FIG. 2 is a line graph showing the effect of different hyaluronic acid concentrations on seed culture in screening example 1;
FIG. 3 is a line graph showing the effect of different concentrations of Tween-80 on seed culture in screening example 1;
FIG. 4 is a line graph showing the effect of different concentrations of disodium EDTA on seed culture in screening example 1;
FIG. 5 is a line graph showing the effect of different ages on seed culture and HAase fermentation production in screening example 1;
FIG. 6 is a bar graph of the effect of different mutagens/carbon sources on HAase fermentation production in screening example 2.
OD in the figure600The absorption value of the bacterial liquid detected by an ultraviolet spectrophotometer at 600nm is used for reflecting the concentration of the thalli in the culture medium.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a hyaluronidase enzyme activity definition and enzyme activity detection method, which refers to the 'Chinese pharmacopoeia' (2015 edition appendix 1207) 'hyaluronidase determination method'.
Screening example 1 determination of seed Medium Components and Strain culture conditions
On the basis of a seed initial culture medium, performing a strain culture condition optimization experiment by using a single-factor optimization strategy and taking culture at 35 ℃, pH6.00 and 50rpm for 24 hours as a basic culture condition, determining an optimal strain culture medium formula by adding an inducer, a carbon source, a nitrogen source and inorganic salt, and determining an optimal culture condition by adjusting pH, culture temperature, rotation speed and seed age.
Seed initial culture medium: 10g/L peptone, 5g/L yeast powder, 5g/L hyaluronic acid (molecular weight 1000kDa-2000kDa), 3g/L glucose, 0.1g/L sodium chloride, 2g/L ammonium sulfate, 0.05g/L ferrous sulfate, 0.2g/L magnesium sulfate and 1mL/L Tween-80.
(1) Determination of inducer/carbon Source
On the basis of the initial culture medium of the seeds, a single-factor optimization strategy is utilized to optimize the inducer/carbon source, and different carbon sources and inducers HA with different concentrations are selected for optimization. The effects of the bacterial cell accumulation amounts corresponding to different carbon sources and different concentrations of the inducer HA on the hyaluronidase induction expression effect are shown in FIGS. 1 and 2. As can be seen from the figure, the most suitable carbon source for bacterial growth is glucose, the carbon source having a good induction effect on the hyaluronidase gene is hyaluronic acid, and the optimal concentration of hyaluronic acid is 5g/L, considering the combination of factors, as the inducer/carbon source of the seed culture medium.
(2) Determination of the Nitrogen Source
On the basis of the initial culture medium of the seeds, hyaluronic acid with an inducer/carbon source of 5g/L is determined, a nitrogen source is optimized by utilizing a single-factor optimization strategy, and different nitrogen sources are selected for optimization. The influence of different nitrogen sources on seed culture is shown in tables 1 and 2, wherein when the nitrogen source in table 1 is an organic nitrogen source with the total concentration of 15g/L, the influence of different proportions of peptone and yeast powder on seed culture is shown; table 2 shows the effect of 5g/L peptone, 10g/L yeast powder in combination with different concentrations of ammonium sulfate on seed culture. It can be seen that the most suitable nitrogen source for the growth of the cells is a complex nitrogen source: 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate.
TABLE 1 Effect of organic Nitrogen sources on seed culture
Figure BDA0002188050080000051
Figure BDA0002188050080000061
TABLE 2 Effect of Complex Nitrogen sources on seed culture
Figure BDA0002188050080000062
(3) Determination of inorganic salts
On the basis of the initial culture medium of the seeds, hyaluronic acid with an inducer/carbon source of 5g/L is determined, and a nitrogen source is a compound nitrogen source: 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate, and different inorganic salts are selected for optimization by utilizing an orthogonal test. The effect of different inorganic salts on seed culture is shown in Table 3. As can be seen from the table, the most suitable inorganic salts for bacterial growth are: 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride and 0.25g/L magnesium sulfate.
TABLE 3 Effect of inorganic salts on seed culture
Figure BDA0002188050080000063
Figure BDA0002188050080000071
(4) Determination of surfactants
On the basis of the initial culture medium of the seeds, an inducer/carbon source is determined to be 5g/L hyaluronic acid, and a nitrogen source is determined to be a compound nitrogen source: 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate, wherein the inorganic salts are as follows: 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride and 0.25g/L magnesium sulfate. And selecting different surfactants for optimization by utilizing a single-factor optimization strategy. The effect of different surfactants on seed culture is shown in fig. 3, 4. As can be seen from the figure, the surfactant most suitable for the growth of the thallus is 1mL/L Tween-80 or 0.05g/L disodium ethylene diamine tetraacetate.
(5) Determination of the pH value
Through a seed culture medium optimization experiment, the optimal seed culture medium formula is determined as follows: 5g/L of hyaluronic acid, and the nitrogen source is a composite nitrogen source: 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate, wherein the inorganic salts are as follows: 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride and 0.25g/L magnesium sulfate, and the surfactant is 1mL/L tween-8 or 0.05g/L disodium ethylenediamine tetraacetate. On the basis of this, optimization experiments of seed culture conditions were carried out by adjusting pH to 5.0, pH5.5, pH6.0, pH6.5, pH7.0, pH7.5, pH8.0 and pH8.5, respectively, in the prepared seed culture medium, and culturing at 35 ℃ and 50rpm for 24 hours, the culture conditions are shown in Table 4, and pH6.0 is the optimum culture condition.
TABLE 4 influence of pH on seed culture
pH value 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
OD600 0.6192 0.7954 0.9685 0.8767 0.6983 0.4551 0.1015 0.0989
(6) Determination of temperature
Through a seed culture medium optimization experiment, the optimal seed culture medium formula is determined as follows: 5g/L of hyaluronic acid, and the nitrogen source is a composite nitrogen source: 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate, wherein the inorganic salts are as follows: 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride and 0.25g/L magnesium sulfate, and the surfactant is 1mL/L tween-8 or 0.05g/L disodium ethylenediamine tetraacetate. On the basis of the optimization experiment of seed culture conditions, the prepared seed culture medium was adjusted to pH6.0, and cultured at 30 deg.C, 31 deg.C, 32 deg.C, 33 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 50rpm for 24 hours, the culture conditions are shown in Table 5, and 35 deg.C is the optimum culture temperature.
TABLE 5 Effect of temperature on seed culture
Figure BDA0002188050080000081
(7) Determination of rotational speed
Through a seed culture medium optimization experiment, the optimal seed culture medium formula is determined as follows: 5g/L of hyaluronic acid, and the nitrogen source is a composite nitrogen source: 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate, wherein the inorganic salts are as follows: 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride and 0.25g/L magnesium sulfate, and the surfactant is 1mL/L tween-8 or 0.05g/L disodium ethylenediamine tetraacetate. On the basis of this, optimization experiment of seed culture conditions was carried out by adjusting pH of the prepared seed culture medium to 6.0 and culturing at 35 ℃ for 24 hours at 0rpm, 50rpm, 100rpm, 150rpm, 200rpm, respectively, and the culturing conditions are shown in Table 6, where 50rpm is the optimum culturing speed.
TABLE 6 Effect of rotational speed on seed culture
Rotational speed (rpm) 0 50 100 150 200
OD600 0.7656 0.9476 0.7754 0.3211 0.1091
(8) Determination of seed age
Through a seed culture medium optimization experiment, the optimal seed culture medium formula is determined as follows: 5g/L of hyaluronic acid, and the nitrogen source is a composite nitrogen source: 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate, wherein the inorganic salts are as follows: 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride and 0.25g/L magnesium sulfate, and the surfactant is 1mL/L tween-8 or 0.05g/L disodium ethylenediamine tetraacetate. On the basis, an optimization experiment of seed culture conditions is carried out, and the optimal culture conditions of the seeds are determined to be pH6.0, 50rpm, 35 ℃. After 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, 48h, 50h, 52h, 54h, 56h, 58h and 60h of culture are respectively carried out under the conditions, the seeds are inoculated into a fermentation tank, and the result is shown in figure 5, when the growth state of the seeds cultured for 18h-24h is good and the seeds are transferred into the fermentation tank, the yield of hyaluronidase is highest.
In conclusion, the optimum seed culture medium components and strain culture conditions obtained by screening are as follows:
the seed culture medium comprises an inducer, a nitrogen source, inorganic salt and a surfactant, wherein the inducer can be simultaneously used as a carbon source and is hyaluronic acid with the molecular weight of 5g/L and 1000kDa-2000kDa, the nitrogen source is a composite nitrogen source and comprises 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate, the inorganic salt comprises 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride and 0.25g/L magnesium sulfate, and the surfactant is 1mL/L tween-80 or 0.05g/L disodium ethylene diamine tetraacetate;
the culture conditions of the strain are pH6.0, rotation speed of 50rpm, culture temperature of 35 deg.C, and culture time of 18-24 h.
Screening example 2 determination of fermentation Medium Components and Strain culture conditions
On the basis of a fermentation initial culture medium, firstly, strain liquid is prepared according to the optimum seed culture condition obtained by screening in screening example 1, the strain liquid is inoculated in a 5L fermentation tank, the strain liquid is cultured for 48 hours at 35 ℃, pH6.00, 50rpm and 2L/min as a basic culture condition, a strain culture condition optimization experiment is carried out by utilizing a single-factor optimization strategy, the optimum strain culture medium formula is determined by adding an inducer, a carbon source, a nitrogen source and inorganic salt, and the optimum culture condition is determined by adjusting pH, culture temperature, rotating speed and ventilation.
Fermentation initial medium: 10g/L peptone, 5g/L yeast powder, 5g/L hyaluronic acid (molecular weight 500kDa-1300kDa), 3g/L glucose, 0.1g/L sodium chloride, 2g/L ammonium sulfate, 0.05g/L ferrous sulfate, 0.2g/L magnesium sulfate and 1mL/L Tween-80.
(1) Determination of inducer/carbon Source
Glucose and hyaluronic acid are selected as an inducer/carbon source for optimization, and the corresponding thallus accumulation amount and the yield of hyaluronidase are shown in figure 6. As can be seen from the figure, the most suitable carbon source for the growth of the thallus is glucose, the carbon source for enabling the yield of the hyaluronidase to be the highest is hyaluronic acid, and the hyaluronic acid with the molecular weight of 500kDa-1300kDa is selected as a fermentation medium inducer/carbon source according to comprehensive factors, the most suitable concentration is 5-8g/L, and the enzyme activity can reach 7680 IU/mL.
(2) Determination of the Nitrogen Source
On the basis of a fermentation initial culture medium, an inducer/carbon source is determined to be 5-8g/L of hyaluronic acid with the relative molecular weight of 500-1300 kDa, and different nitrogen sources are selected for optimization by utilizing a single-factor optimization strategy. Researches show that the most suitable nitrogen source for fermentation production of hyaluronidase is a composite nitrogen source: 5g/L of peptone, 10g/L of yeast powder and 2g/L of ammonium sulfate, and the enzyme activity can reach 7705 IU/mL.
(3) Determination of inorganic salts
On the basis of an initial fermentation culture medium, an inducer/carbon source is 5-8g/L of hyaluronic acid with the relative molecular weight of 500KDa-1300kDa, and a nitrogen source is a compound nitrogen source: 5g/L peptone, 10g/L yeast powder and 2g/L ammonium sulfate, and different inorganic salts are selected for optimization by utilizing a single-factor optimization strategy. The research shows that the inorganic salt most suitable for the fermentation production of the hyaluronidase is as follows: 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.02g/L manganese sulfate, 0.05g/L calcium chloride, 0.2g/L magnesium sulfate and 0.4g/L potassium dihydrogen phosphate, and the enzyme activity can reach 7900IU/mL after optimization.
(4) Determination of surfactants
On the basis of an initial fermentation culture medium, an inducer/carbon source is 5-8g/L hyaluronic acid with a relative molecular weight of 500KDa-1300kDa, and a nitrogen source is a compound nitrogen source: 5g/L of peptone, 10g/L of yeast powder and 2g/L of ammonium sulfate, wherein the inorganic salt is: 0.1g/L of sodium chloride, 0.1g/L of ferrous sulfate, 0.02g/L of manganese sulfate, 0.05g/L of calcium chloride, 0.2g/L of magnesium sulfate and 0.4g/L of monopotassium phosphate. And selecting different surfactants for optimization by utilizing a single-factor optimization strategy. The research shows that the surfactant most suitable for the fermentation production of hyaluronidase is 1mL/L Tween-80 or 0.05g/L disodium ethylene diamine tetraacetate, and the enzyme activity is 7932 IU/mL.
(5) Determination of the pH value
Through fermentation medium optimization experiments, the optimal fermentation medium formula is determined as follows: 5-8g/L hyaluronic acid with a relative molecular weight of 500KDa-1300kDa, 5g/L peptone, 10g/L yeast powder, 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.02g/L manganese sulfate, 0.05g/L calcium chloride, 0.2g/L magnesium sulfate, 0.4g/L monopotassium phosphate, 1mL/L tween-80 or 0.05g/L disodium ethylenediamine tetraacetate. On the basis of this, optimization experiments of fermentation culture conditions were carried out by adjusting the pH of the fermentation medium to pH5.0, pH5.5, pH6.0, pH6.5, pH7.0, pH7.5, pH8.0, pH8.5, 35 ℃, 50rpm, and aeration rate 2L/min, and culturing for 48 hours, the culture conditions are shown in Table 7, and pH5.5 is the optimum culture condition.
TABLE 7 influence of pH on HAase fermentation production
Figure BDA0002188050080000111
(6) Determination of temperature
Through fermentation medium optimization experiments, the optimal fermentation medium formula is determined as follows: 5-8g/L hyaluronic acid with a relative molecular weight of 500KDa-1300kDa, 5g/L peptone, 10g/L yeast powder, 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.02g/L manganese sulfate, 0.05g/L calcium chloride, 0.2g/L magnesium sulfate, 0.4g/L monopotassium phosphate, 1mL/L tween-80 or 0.05g/L disodium ethylenediamine tetraacetate. On the basis, the optimization experiment of fermentation culture conditions is carried out, the pH of the prepared seed culture medium is adjusted to 5.5, and the culture temperatures are respectively set to be 30 ℃, 31 ℃, 32 ℃ and 33Culturing at 34 deg.C, 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 50rpm, and ventilation amount of 2L/min for 48 hr, and determining OD600And hyaluronidase activity, as shown in Table 8, 35 ℃ is the optimum culture temperature, so that the enzyme activity can reach 8179 IU/mL.
TABLE 8 Effect of temperature on HAase fermentation production
Figure BDA0002188050080000121
(7) Determination of rotational speed and ventilation
Firstly, through a fermentation culture medium optimization experiment, the optimal fermentation culture medium formula is determined as follows: 5-8g/L hyaluronic acid with a relative molecular weight of 500KDa-1300kDa, 5g/L peptone, 10g/L yeast powder, 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.02g/L manganese sulfate, 0.05g/L calcium chloride, 0.2g/L magnesium sulfate, 0.4g/L monopotassium phosphate, 1mL/L tween-80 or 0.05g/L disodium ethylenediamine tetraacetate. On the basis of this, optimization experiment of fermentation culture conditions was carried out by adjusting the pH of the prepared seed medium to 5.5, setting the culture rotation speed to 0rpm, 50rpm, 100rpm, 150rpm, 200rpm, 35 ℃, aeration rate to 2L/min, and culturing for 48 hours, wherein 50rpm was the optimum culture rotation speed, as shown in Table 6.
TABLE 9 Effect of rotation speed on HAase fermentation production
Rotational speed (rpm) 0 50 100 150 200
OD600 2.6509 2.7354 2.6988 2.6509 2.3722
HAase(IU/mL) 5433 8166 8031 6908 6344
Secondly, determining an optimal fermentation medium formula through a fermentation medium optimization experiment: 5-8g/L hyaluronic acid with a relative molecular weight of 500KDa-1300kDa, 5g/L peptone, 10g/L yeast powder, 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.02g/L manganese sulfate, 0.05g/L calcium chloride, 0.2g/L magnesium sulfate, 0.4g/L monopotassium phosphate, 1mL/L tween-80 or 0.05g/L disodium ethylenediamine tetraacetate. On the basis, the optimization experiment of fermentation culture conditions is carried out, the pH of the prepared seed culture medium is adjusted to 5.5, 200rpm is carried out, the temperature is 35 ℃, the ventilation volume is 1L/min, 2L/min, 3L/min, 4L/min, 5L/min, 6L/min, 7L/min and 8L/min, the culture is carried out for 48 hours, the culture condition is shown in the table 7, and 2L/min is the optimal ventilation volume.
TABLE 10 Effect of aeration on HAase fermentation production
Figure BDA0002188050080000122
Figure BDA0002188050080000131
③ from the table 2, the low dissolved oxygen in the fermentation broth is suitable for the growth and reproduction of lactobacillus plantarum CnT012-56, and the high dissolved oxygen is suitable for the production of hyaluronidase. The oxygen-resistant acclimation of the strain and the high yield of the hyaluronidase are realized by adjusting the fermentation rotating speed and the ventilation volume in stages and controlling the dissolved oxygen amount in the fermentation tank. The data in Table 11 show that the oxygen content in the fermentation broth is gradually increased to perform oxygen-resistant acclimation on the bacteria, so that the bacteria can produce hyaluronidase with high yield in the fermentation broth with higher dissolved oxygen. In the culture period of 0-20 hours, the dissolved oxygen is kept low, the proliferation of the thalli is realized, a large amount of proliferation is realized on the thalli, and the dissolved oxygen in the fermentation liquor is increased to obviously improve the yield of the hyaluronidase, for example, the enzyme activity can reach 9120IU/mL in the seventh group of experimental data in Table 11.
Based on this, experimental studies were conducted to adjust the rotational speed and the ventilation rate in three stages in detail based on the results of the seventh group of experiments in Table 11, and it was found that the adjustment of the rotational speed and the ventilation rate in three stages can provide a better culture effect (see Table 12). Table 12 the second set of experiments show that the hyaluronidase production can be higher, which can reach 9604IU/mL and 40% increase compared with that before optimization, by 50rpm and 2L/min ventilation during 0-20 hours of culture, 100rpm and 4L/min ventilation during 20-30 hours of culture, and 200rpm and 8L/min ventilation during 30-48 hours of culture.
TABLE 11 influence of two-stage regulation of the rotational speed and the aeration rate on the production of hyaluronidase by fermentation
Figure BDA0002188050080000132
Figure BDA0002188050080000141
TABLE 12 influence of three-stage regulation of the rotational speed and the aeration rate on the production of hyaluronidase by fermentation
Figure BDA0002188050080000142
In conclusion, the optimum fermentation medium components and strain culture conditions obtained by screening are as follows:
the fermentation medium comprises an inducer, a nitrogen source, inorganic salt and a surfactant, wherein the inducer can be simultaneously used as a carbon source and is hyaluronic acid with the molecular weight of 5-8g/L and 500-1300 kDa, the nitrogen source is a composite nitrogen source and comprises 5g/L peptone, 10g/L yeast powder and 2g/L ammonium sulfate, the inorganic salt comprises 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.02g/L manganese sulfate, 0.05g/L calcium chloride, 0.2g/L magnesium sulfate and 0.4g/L potassium dihydrogen phosphate, and the surfactant is 1mL/L tween-80 or 0.05g/L disodium ethylenediamine tetraacetate.
The culture conditions of the strains are pH5.5, the culture temperature is 35 ℃, the culture time is 48h, the rotating speed and the ventilation volume are regulated in stages,
0-20h, the rotating speed is 50rpm, the ventilation volume is 2L/min,
20-30h, rotation speed of 100rpm and ventilation capacity of 4L/min
30-48h, the rotating speed is 200rpm, and the ventilation volume is 8L/min.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (3)

1. The fermentation optimization process for producing hyaluronidase by lactobacillus plantarum is characterized in that the lactobacillus plantarum is lactobacillus plantarum CnT012-56, and the classification of the strain is named as lactobacillus plantarumLactobacillus plantarumThe preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang district, the preservation time is 11 months and 28 days in 2018,the preservation number is CGMCC NO. 16836;
the process comprises the following steps:
s1: preparing a seed culture medium, wherein the seed culture medium comprises an inducer, a nitrogen source, inorganic salt and a surfactant,
the inducer can be simultaneously used as a carbon source and is hyaluronic acid with the molecular weight of 5g/L and 1000kDa-2000kDa,
the nitrogen source is a compound nitrogen source and comprises 5g/L peptone, 10g/L yeast powder and 1.5g/L ammonium sulfate,
the inorganic salt comprises 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride and 0.25g/L magnesium sulfate,
the surfactant is 1mL/L tween-80 or 0.05g/L disodium ethylene diamine tetraacetate;
s2: inoculating lactobacillus plantarum into the seed culture medium, and culturing to obtain liquid fermentation strain under the conditions of pH6.0, rotation speed of 50rpm, culture temperature of 35 deg.C, and culture time of 18-24 hr;
s3: preparing a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises an inducer, a nitrogen source, inorganic salt and a surfactant,
the inducer is simultaneously used as a carbon source and is 5-8g/L hyaluronic acid with the molecular weight of 500KDa-1300kDa,
the nitrogen source is a compound nitrogen source and comprises 5g/L peptone, 10g/L yeast powder and 2g/L ammonium sulfate,
the inorganic salt comprises 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.02g/L manganese sulfate, 0.05g/L calcium chloride, 0.2g/L magnesium sulfate and 0.4g/L potassium dihydrogen phosphate,
the surfactant is 1mL/L tween-80 or 0.05g/L disodium ethylene diamine tetraacetate;
s4: inoculating liquid fermentation strain into liquid fermentation culture medium, inoculating 5% of the strain, culturing for 48 hr under the culture condition of pH5.5 and culture temperature of 35 deg.C, and adjusting rotation speed and ventilation by stages:
0-20h, the rotating speed is 50rpm, the ventilation volume is 2L/min,
20-30h, rotation speed of 100rpm and ventilation capacity of 4L/min
The rotation speed is 200rpm and the ventilation volume is 8L/min for 30-48 h;
s5: centrifuging the fermentation liquor, and collecting supernatant to obtain crude enzyme solution.
2. The optimized fermentation process for producing hyaluronidase by lactobacillus plantarum of claim 1, wherein 200mL of seed culture medium is contained in 500mL of triangular flask in S1.
3. The optimized fermentation process for producing hyaluronidase by lactobacillus plantarum of claim 1, wherein in S3, 4L of liquid fermentation medium is filled in 5L fermentation tank.
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