CN110408607A - A kind of lactobacillus plantarum production hyaluronidase fermentation optimization technique - Google Patents
A kind of lactobacillus plantarum production hyaluronidase fermentation optimization technique Download PDFInfo
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Abstract
The present invention relates to technical field of microbial fermentation, and in particular to a kind of lactobacillus plantarum production hyaluronidase fermentation optimization technique, technique is the following steps are included: prepare seed culture medium；Lactobacillus plantarum is accessed into seed culture medium, culture obtains liquid fermentation strain；Prepare liquid fermentation medium；Liquid fermentation strain is accessed in liquid fermentation medium, terminates fermentation after culture；After fermentation liquid is centrifuged, supernatant, as crude enzyme liquid are collected.The present invention carries out oxygen resistence domestication culture to thallus, lactobacillus plantarum normal fermentation under aerobic condition is enable to produce hyaluronidase creatively by adjusting fermented and cultured revolving speed, ventilatory capacity；By condition optimizing, hyaluronic acid production of enzyme in lactobacillus plantarum metabolite is substantially improved: hyaluronidase 9604IU/mL contained by fermentation liquid made from this production technology is isolated and purified than improving 40% before process optimization convenient for subsequent；Production technology of the present invention is easy, stability is high, convenient for expansion large-scale production and popularization.
Description
Technical field
The present invention relates to technical field of microbial fermentation, and in particular to a kind of lactobacillus plantarum production hyaluronic acid enzyme fermentation
Optimization technique.
Background technique
Hyaluronic acid is the main component for constituting host connective tissue cells epimatrix, and hyaluronidase
(hyaluronidase, HAase) is that energy specific cleavage extracellular matrix components --- hyaluronic acid (HA) makes its generate low point
A kind of proteolytic enzyme of sub-ization effect.Hyaluronidase influences the proliferation of cell, differentiation and acting on extracellular matrix
With migration, and play a role during embryonic development and tumor development, thus to effective control pathogenic bacterial infection, prevent
Its diffusion has a decisive role, and is also of great significance to the prevention and treatment of clinical bacteria infectious diseases.
Although hyaluronidase distribution is wide, extremely difficult due to isolating and purifying within a very long time in past, do not have
It is developed and used well, is a kind of ignored enzyme.Simultaneously because the source of hyaluronidase is very limited, price is high
It is expensive, largely also limit its application range.Until the nearly more than ten years, researcher just starts to show HA and HAase
Keen interest, it is intended to seek its effect in numerous biological processes.Research finds that hyaluronidase can be by degradation group
HA in knitting increases the permeability of film, reduces viscosity, promotes the diffusion of injection, these phenomenons are known as the expansion of hyaluronidase
Dissipate effect.Based on this property, hyaluronidase can be applied to therapeutic process, to accelerate infiltration rate, injection be promoted to absorb,
Local anaesthesia effect is improved, and is prevented after subcutaneous and intramuscular injection to disorganization.As people are to HA, low molecule HA and HA oligosaccharides
Biological significance and its understanding of importance deepen continuously, and more and more evidences show that low relative molecular mass HA and HA are few
The bioactivity and HA of sugar are dramatically different.
Based on this, provide it is a kind of can fermenting and producing hyaluronidase strain and fermentation that enzymatic activity can be made to significantly improve
Process has very important significance.
Summary of the invention
For the prior art can fermenting and producing hyaluronidase strain it is few and problem that enzymatic activity is lower, the present invention provide
A kind of lactobacillus plantarum production hyaluronidase fermentation optimization technique, which is made up of improvement culture medium and condition of culture,
Hyaluronic acid production of enzyme is improved, and enzyme activity is made to improve 40%.
A kind of lactobacillus plantarum produces hyaluronidase fermentation optimization technique, technique the following steps are included:
S1: preparing seed culture medium,
The seed culture medium includes inducer, nitrogen source, inorganic salts and surfactant,
The inducer is the hyaluronic acid of 5g/L molecular weight 1000kDa-2000kDa at the same time as utilization of carbon source,
The nitrogen source is compound nitrogen source, including 5g/L peptone, 10g/L yeast powder, 1.5g/L ammonium sulfate,
The inorganic salts include 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L chlorination
Calcium, 0.25g/L magnesium sulfate,
The surfactant is 1mL/L Tween-80 or 0.05g/L disodium ethylene diamine tetraacetate；
S2: accessing lactobacillus plantarum into seed culture medium, and culture obtains liquid fermentation strain；
S3: preparing liquid fermentation medium,
The liquid fermentation medium includes inducer, nitrogen source, inorganic salts and surfactant,
The inducer is the hyaluronic acid of 1-10g/L molecular weight 500KDa-1300kDa at the same time as utilization of carbon source,
The nitrogen source is compound nitrogen source, including 5g/L peptone, 10g/L yeast powder, 2g/L ammonium sulfate,
The inorganic salts include 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.02g/L manganese sulfate, 0.05g/L chlorination
Calcium, 0.2g/L magnesium sulfate, 0.4g/L potassium dihydrogen phosphate,
The surfactant is 0.5mL/L-1.5mL/L Tween-80 or 0.01g/L-0.08g/L ethylenediamine tetra-acetic acid two
Sodium；
S4: liquid fermentation strain is accessed in liquid fermentation medium, terminates fermentation after culture；
S5: after fermentation liquid is centrifuged, supernatant, as crude enzyme liquid are collected.
Further, the lactobacillus plantarum is lactobacillus plantarum CnT012-56, and the classification naming of the bacterial strain is plant cream
Bacillus Lactobacillus plantarum, depositary institution are China Committee for Culture Collection of Microorganisms's common micro-organisms
Center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the preservation time is on November 28th, 2018, deposit number
For CGMCC NO.16836.
Further, in the S1, using the bottled 200mL seed culture medium of 500mL triangle.
Further, in the S2, strain optimal culture conditions are pH6.0, revolving speed 50rpm, 35 DEG C of cultivation temperature, cultivate
Time 18-24h.
Further, in the S3, using the canned 4L liquid fermentation medium of 5L fermentation.
Further, in the S3, inducer is 5-8g/L molecular weight 500KDa-1300kDa in liquid fermentation medium
Hyaluronic acid.
Further, in the S3, surfactant is 1mL/L Tween-80 or 0.05g/L second in liquid fermentation medium
Edetate disodium.
Further, in the S4, strain optimal culture conditions are pH5.0-8.0,30 DEG C -38 DEG C of cultivation temperature, cultivate
48h adjusts revolving speed and ventilatory capacity stage by stage:
0-20h, revolving speed 50rpm, ventilatory capacity 2L/min,
20-30h, revolving speed 100rpm, ventilatory capacity 4L/min
30-48h, revolving speed 200rpm, ventilatory capacity 8L/min.
Further, in the S4, strain optimal culture conditions are preferably 35 DEG C of pH5.5, cultivation temperature.
Further, in the S4, inoculum concentration is 1% (v/v) -10% (v/v), preferably 5% (v/v).
The present invention provides a kind of lactobacillus plantarum production hyaluronidase fermentation optimization technique, the beneficial effect is that,
(1) creatively by adjusting fermented and cultured revolving speed, ventilatory capacity, oxygen resistence domestication culture is carried out to thallus, makes to plant
Object lactobacillus CnT012-56 can produce hyaluronidase in normal fermentation under aerobic condition；
(2) by condition optimizing, hyaluronic acid production of enzyme in lactobacillus plantarum metabolite is substantially improved: this production work
Hyaluronidase contained by fermentation liquid made from skill is 9604IU/mL, than improving 40% before process optimization, is convenient for subsequent separation
Purifying；
(3) production technology of the present invention is easy, stability is high, convenient for expansion large-scale production and popularization.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, for those of ordinary skill in the art
Speech, without creative efforts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is influence line chart of the 5g/L different carbon source to seed culture in screening example 1；
Fig. 2 is to screen influence line chart of the different hyaluronic acid concentrations to seed culture in example 1；
Fig. 3 is to screen influence line chart of the different Tween-80 concentration to seed culture in example 1；
Fig. 4 is to screen influence line chart of the different disodium ethylene diamine tetraacetate concentration to seed culture in example 1；
Fig. 5 is to screen in example 1 age not of the same race to the influence line chart of seed culture and HAase fermenting and producing；
Fig. 6 is to screen influence histogram of the different mutagens/carbon sources to HAase fermenting and producing in example 2.
OD in figure600For the absorption value that bacterium solution is detected in ultraviolet specrophotometer 600nm, for reflecting thallus in culture medium
Concentration.
Specific embodiment
Technical solution in order to enable those skilled in the art to better understand the present invention, below in conjunction with of the invention real
The attached drawing in example is applied, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work, all should belong to protection of the present invention
Range.
The definition of hyaluronidase enzyme activity and Enzyme activity assay method involved in the present invention arrived, with reference to " Chinese Pharmacopoeia " (2015
Version annex 1207) " hyaluronidase measuring method ".
Screen the determination of 1 seed culture medium component of example and Spawn incubation condition
On the basis of seed initial medium, with 35 DEG C, pH6.00,50rpm culture 24 hours are basic condition of culture,
Using single factor test optimisation strategy, the experiment of Spawn incubation condition optimizing is carried out, it is true by addition inducer, carbon source, nitrogen source, inorganic salts
Fixed best Spawn incubation based formulas determines optimal culture condition by adjusting pH, cultivation temperature, revolving speed, kind age.
Seed initial 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, 1mL/
L Tween-80.
(1) inducer/carbon source determination
On the basis of seed initial medium, using single factor test optimisation strategy, inducer/carbon source optimizing is carried out, is chosen
The inducer HA of different carbon source and various concentration is optimized.Thallus cumulant and various concentration corresponding to different carbon source
Fig. 1, Fig. 2 are shown in influence of the inducer HA to hyaluronidase inducing expression effect.It can be seen from the figure that being most suitable for thalli growth
Carbon source be glucose, and to hyaluronic acid enzyme gene play good inducing action carbon source be hyaluronic acid, composite factor
Consider, selects hyaluronic acid as seed culture medium inducer/carbon source, optimum concentration 5g/L.
(2) determination of nitrogen source
On the basis of seed initial medium, it is determined that inducer/carbon source is the hyaluronic acid of 5g/L, utilizes single factor test
Optimisation strategy carries out nitrogen source optimization, chooses different nitrogen sources and optimize.Influence of the different nitrogen sources to seed culture is shown in Table 1, table
2, wherein table 1 is nitrogen source when being the organic nitrogen source that total concentration is 15g/L, and peptone, yeast powder different ratio are to seed culture
It influences；Table 2 is 5g/L peptone, 10g/L yeast powder and influence of the various concentration ammonium sulfate compound tense to seed culture.It can see
Out, the nitrogen source for being most suitable for thalli growth is compound nitrogen source: 5g/L peptone, 10g/L yeast powder, 1.5g/L ammonium sulfate.
1 organic nitrogen source of table influences seed culture
2 compound nitrogen source of table influences seed culture
(3) determination of inorganic salts
On the basis of seed initial medium, it is determined that inducer/carbon source is the hyaluronic acid of 5g/L, and nitrogen source is compound
Nitrogen source: it is excellent to choose different inorganic salts progress using orthogonal test for 5g/L peptone, 10g/L yeast powder, 1.5g/L ammonium sulfate
Change.Different inorganic salts influence to seed culture be shown in Table 3.As can be seen from the table, it is most suitable for the inorganic salts of thalli growth are as follows:
0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride, 0.25g/L magnesium sulfate.
Influence of 3 inorganic salts of table to seed culture
(4) determination of surfactant
On the basis of seed initial medium, it is determined that inducer/carbon source is 5g/L hyaluronic acid, and nitrogen source is composite nitrogen
Source: 5g/L peptone, 10g/L yeast powder, 1.5g/L ammonium sulfate, 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, 0.25g/L magnesium sulfate.Using single factor test optimisation strategy, it is living to choose different surfaces
Property agent optimizes.Fig. 3, Fig. 4 are shown in influence of the different surfaces activating agent to seed culture.It can be seen from the figure that being most suitable for bacterium
The surfactant of body growth is 1mL/L Tween-80 or 0.05g/L disodium ethylene diamine tetraacetate.
(5) determination of pH value
Optimized by seed culture medium and tested, determines most suitable seed culture based formulas are as follows: 5g/L hyaluronic acid, nitrogen source are multiple
Close nitrogen source: 5g/L peptone, 10g/L yeast powder, 1.5g/L ammonium sulfate, inorganic salts are as follows: 0.1g/L sodium chloride, 0.1g/L sulfuric acid are sub-
Iron, 0.03g/L manganese sulfate, 0.04g/L calcium chloride, 0.25g/L magnesium sulfate, surfactant are 1mL/L tween -8 or 0.05g/L
Disodium ethylene diamine tetraacetate.The optimization experiment for carrying out seed culture condition on this basis, the seed culture medium prepared is distinguished
PH5.0, pH5.5, pH6.0, pH6.5, pH7.0, pH7.5, pH8.0, pH8.5 are adjusted, 35 DEG C, 50rpm culture 24 hours cultivate feelings
Condition is as shown in table 4, and pH6.0 is optimal culture conditions.
Influence of 4 pH value of table to 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
Optimized by seed culture medium and tested, determines most suitable seed culture based formulas are as follows: 5g/L hyaluronic acid, nitrogen source are multiple
Close nitrogen source: 5g/L peptone, 10g/L yeast powder, 1.5g/L ammonium sulfate, inorganic salts are as follows: 0.1g/L sodium chloride, 0.1g/L sulfuric acid are sub-
Iron, 0.03g/L manganese sulfate, 0.04g/L calcium chloride, 0.25g/L magnesium sulfate, surfactant are 1mL/L tween -8 or 0.05g/L
Disodium ethylene diamine tetraacetate.The optimization experiment of seed culture condition, the seed culture keynote that will be prepared are carried out on this basis
PH6.0 is respectively placed in 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, and 50rpm is cultivated 24 hours, training
Feeding situation is as shown in table 5, and 35 DEG C are most suitable cultivation temperatures.
Influence of 5 temperature of table to seed culture
(7) determination of revolving speed
Optimized by seed culture medium and tested, determines most suitable seed culture based formulas are as follows: 5g/L hyaluronic acid, nitrogen source are multiple
Close nitrogen source: 5g/L peptone, 10g/L yeast powder, 1.5g/L ammonium sulfate, inorganic salts are as follows: 0.1g/L sodium chloride, 0.1g/L sulfuric acid are sub-
Iron, 0.03g/L manganese sulfate, 0.04g/L calcium chloride, 0.25g/L magnesium sulfate, surfactant are 1mL/L tween -8 or 0.05g/L
Disodium ethylene diamine tetraacetate.The optimization experiment of seed culture condition, the seed culture keynote that will be prepared are carried out on this basis
PH6.0 is respectively placed in 0rpm, 50rpm, 100rpm, 150rpm, 200rpm, and 35 DEG C are cultivated 24 hours, cultivates situation such as 6 institute of table
Show, 50rpm is most suitable culture revolving speed.
Influence of 6 revolving speed of table to seed culture
| Revolving speed (rpm) | 0 | 50 | 100 | 150 | 200 |
| OD600 | 0.7656 | 0.9476 | 0.7754 | 0.3211 | 0.1091 |
(8) determination in age is planted
Optimized by seed culture medium and tested, determines most suitable seed culture based formulas are as follows: 5g/L hyaluronic acid, nitrogen source are multiple
Close nitrogen source: 5g/L peptone, 10g/L yeast powder, 1.5g/L ammonium sulfate, inorganic salts are as follows: 0.1g/L sodium chloride, 0.1g/L sulfuric acid are sub-
Iron, 0.03g/L manganese sulfate, 0.04g/L calcium chloride, 0.25g/L magnesium sulfate, surfactant are 1mL/L tween -8 or 0.05g/L
Disodium ethylene diamine tetraacetate.The optimization experiment for carrying out seed culture condition on this basis, determines that seed optimal culture conditions are
PH6.0,50rpm, 35 DEG C.With this condition respectively cultivate 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、60h
Afterwards, fermentor is accessed, as a result as shown in figure 5, when seed culture 18h-24h growth conditions are good, and after being transferred to fermentor, thoroughly
Bright matter acid production of enzyme highest.
To sum up, screening obtains most suitable seed culture medium component and Spawn incubation condition is as follows:
Seed culture medium includes inducer, nitrogen source, inorganic salts and surfactant, and inducer is at the same time as carbon source benefit
With for the hyaluronic acid of 5g/L molecular weight 1000kDa-2000kDa, nitrogen source is compound nitrogen source, including 5g/L peptone, 10g/L
Yeast powder, 1.5g/L ammonium sulfate, inorganic salts include 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate,
0.04g/L calcium chloride, 0.25g/L magnesium sulfate, surfactant are 1mL/L Tween-80 or 0.05g/L ethylenediamine tetra-acetic acid two
Sodium；
Spawn incubation condition is pH6.0, revolving speed 50rpm, 35 DEG C of cultivation temperature, incubation time 18-24h.
Screen the determination of 2 fermentation medium component of example and Spawn incubation condition
On the basis of fermenting initial medium, the preparation of obtained most suitable seed culture condition is screened to screen example 1 first
Strain liquid is inoculated in 5L fermentor, cultivates 48 hours with 35 DEG C, pH6.00,50rpm, 2L/min as basic condition of culture, benefit
With single factor test optimisation strategy, the experiment of Spawn incubation condition optimizing is carried out, is determined by addition inducer, carbon source, nitrogen source, inorganic salts
Best Spawn incubation based formulas determines optimal culture condition by adjusting pH, cultivation temperature, revolving speed, ventilatory capacity.
Ferment 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, 1mL/
L Tween-80.
(1) inducer/carbon source determination
Choose glucose, hyaluronic acid is optimized as inducer/carbon source, corresponding thallus cumulant and hyaluronic acid
Production of enzyme is shown in Fig. 6.It can be seen from the figure that the carbon source for being most suitable for thalli growth is glucose, and make hyaluronic acid production of enzyme most
High carbon source is hyaluronic acid, and composite factor considers, the hyaluronic acid of molecular weight 500kDa-1300kDa is selected to train as fermentation
Base inducer/carbon source is supported, optimum concentration 5-8g/L, enzyme activity is up to 7680IU/mL.
(2) determination of nitrogen source
On the basis of fermenting initial medium, it is determined that inducer/carbon source is relative molecular weight 500kDa-1300kDa
Hyaluronic acid 5-8g/L choose different nitrogen sources using single factor test optimisation strategy and optimize.It has been investigated that being most suitable for
The nitrogen source of hyaluronidase fermenting and producing is compound nitrogen source: 5g/L peptone, 10g/L yeast powder, 2g/L ammonium sulfate, enzyme activity are reachable
7705IU/mL。
(3) determination of inorganic salts
On the basis of fermenting initial medium, it is determined that inducer/carbon source is relative molecular weight 500KDa-1300kDa
Hyaluronic acid 5-8g/L, nitrogen source is compound nitrogen source: 5g/L peptone, 10g/L yeast powder, 2g/L ammonium sulfate utilize single factor test
Optimisation strategy is chosen different inorganic salts and is optimized.It has been investigated that being most suitable for the inorganic salts of hyaluronidase fermenting and producing
Are 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, 0.4g/
L potassium dihydrogen phosphate, optimized rear enzyme activity is up to 7900IU/mL.
(4) determination of surfactant
On the basis of fermenting initial medium, it is determined that inducer/carbon source is 5-8g/L relative molecular weight 500KDa-
The hyaluronic acid of 1300kDa, nitrogen source are compound nitrogen source: peptone 5g/L, yeast powder 10g/L, ammonium sulfate 2g/L, inorganic salts are as follows:
Sodium chloride 0.1g/L, ferrous sulfate 0.1g/L, manganese sulfate 0.02g/L, calcium chloride 0.05g/L, magnesium sulfate 0.2g/L, biphosphate
Potassium 0.4g/L.Using single factor test optimisation strategy, chooses different surfactants and optimize.It has been investigated that being most suitable for
The surfactant of bright matter acid enzyme fermentation production is 1mL/L Tween-80 or 0.05g/L disodium ethylene diamine tetraacetate, enzyme activity are
7932IU/mL。
(5) determination of pH value
Optimized by fermentation medium and tested, determines most suitable fermentative medium formula are as follows: 5-8g/L relative molecular weight
The hyaluronic acid 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 potassium dihydrogen phosphate, 1mL/L Tween-80 or
0.05g/L disodium ethylene diamine tetraacetate.The optimization experiment for carrying out fermentation culture conditions on this basis, fermentation medium is distinguished
PH5.0, pH5.5, pH6.0, pH6.5, pH7.0, pH7.5, pH8.0, pH8.5 are adjusted, 35 DEG C, 50rpm, ventilatory capacity 2L/min, is trained
It supports 48 hours, culture situation is as shown in table 7, and pH5.5 is optimal culture conditions.
Influence of 7 pH value of table to HAase fermenting and producing
(6) determination of temperature
Optimized by fermentation medium and tested, determines most suitable fermentative medium formula are as follows: 5-8g/L relative molecular weight
The hyaluronic acid 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 potassium dihydrogen phosphate, 1mL/L Tween-80 or
0.05g/L disodium ethylene diamine tetraacetate.The optimization experiment of fermentation culture conditions, the seed culture that will be prepared are carried out on this basis
Keynote pH5.5, is respectively set 30 DEG C of cultivation temperature, 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,
Ventilatory capacity 2L/min is cultivated 48 hours, measures OD600With hyaluronic acid enzyme activity, as shown in table 8,35 DEG C are most suitable cultivation temperatures,
Enzyme activity is set to can achieve 8179IU/mL.
Influence of 8 temperature of table to HAase fermenting and producing
(7) determination of revolving speed, ventilatory capacity
It is tested 1. being optimized by fermentation medium, determines most suitable fermentative medium formula are as follows: 5-8g/L relative molecular weight
The hyaluronic acid 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 potassium dihydrogen phosphate, 1mL/L Tween-80 or
0.05g/L disodium ethylene diamine tetraacetate.The optimization experiment of fermentation culture conditions, the seed culture that will be prepared are carried out on this basis
Keynote pH5.5 is respectively set and cultivates revolving speed 0rpm, 50rpm, 100rpm, 150rpm, 200rpm, and 35 DEG C, ventilatory capacity 2L/min,
Culture 48 hours, culture situation is as shown in table 6, and 50rpm is most suitable culture revolving speed.
Influence of 9 revolving speed of table to HAase fermenting and producing
| Revolving 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 |
It is tested 2. being optimized by fermentation medium, determines most suitable fermentative medium formula are as follows: 5-8g/L relative molecular weight
The hyaluronic acid 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 potassium dihydrogen phosphate, 1mL/L Tween-80 or
0.05g/L disodium ethylene diamine tetraacetate.The optimization experiment of fermentation culture conditions, the seed culture that will be prepared are carried out on this basis
Keynote pH5.5,200rpm, 35 DEG C, ventilatory capacity 1L/min, 2L/min, 3L/min, 4L/min, 5L/min, 6L/min, 7L/min,
8L/min is cultivated 48 hours, and culture situation is as shown in table 7, and 2L/min is most suitable ventilatory capacity.
Influence of 10 ventilatory capacity of table to HAase fermenting and producing
3. as shown in Table 2, low dissolved oxygen is suitable for lactobacillus plantarum CnT012-56 growth and breeding in fermentation liquid, high dissolved oxygen is suitable for
The generation of hyaluronidase.By adjusting fermentation revolving speed and ventilatory capacity stage by stage, dissolved oxygen amount in fermentor is controlled, realizes strain
The high yield of oxygen resistence domestication and hyaluronidase.Table 11 statistics indicate that, by gradually increasing oxygen content in fermentation liquid, to thallus
Oxygen resistence domestication has been carried out, thallus high yield hyaluronidase in the fermentation liquid with higher dissolved oxygen is enable.In the 0-20 of culture
During hour, low dissolved oxygen is kept, realizes the proliferation of thallus, realizes a large amount of proliferation in thallus, then increase dissolved oxygen amount in fermentation liquid
Hyaluronic acid production of enzyme can be significantly improved, such as the 7th group of experimental data of table 11, enzyme activity is up to 9120IU/mL.
Based on this, finer adjusting revolving speed and ventilatory capacity stage by stage are carried out on the basis of the 7th group of experimental result of table 11
Experimental study, discovery by revolving speed, ventilatory capacity divide three phases adjusting can obtain better culture effect (such as table 12).Table 12
Two groups of experiments are shown, during the 0-20 hour of culture, revolving speed 50rpm, ventilatory capacity 2L/min, in the 20-30 of culture small period
Between adjust revolving speed be 100rpm, ventilatory capacity 4L/min, during 30-48 hour of culture adjust revolving speed be 200rpm, ventilatory capacity
8L/min can obtain higher hyaluronic acid production of enzyme, up to 9604IU/mL, increase by 40% before relatively optimizing.
Table 11 adjusts the influence that revolving speed and ventilatory capacity produce hyaluronidase to fermentation in two stages
Table 12 adjusts the influence that revolving speed and ventilatory capacity produce hyaluronidase to fermentation in three stages
To sum up, screening obtains most suitable fermentation medium component and Spawn incubation condition is as follows:
Fermentation medium includes inducer, nitrogen source, inorganic salts and surfactant, and inducer is at the same time as carbon source benefit
With for the hyaluronic acid of 5-8g/L molecular weight 500kDa-1300kDa, nitrogen source is compound nitrogen source, including 5g/L peptone, 10g/L
Yeast powder, 2g/L ammonium sulfate, inorganic salts include 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 potassium dihydrogen phosphate, surfactant are 1mL/L Tween-80 or 0.05g/L ethylenediamine
Tetraacethyl disodium.
Spawn incubation condition is pH5.5,35 DEG C of cultivation temperature, incubation time 48h, adjusts revolving speed and ventilatory capacity stage by stage,
0-20h, revolving speed 50rpm, ventilatory capacity 2L/min,
20-30h, revolving speed 100rpm, ventilatory capacity 4L/min
30-48h, revolving speed 200rpm, ventilatory capacity 8L/min.
Although by reference to attached drawing and combining the mode of preferred embodiment to the present invention have been described in detail, the present invention
It is not limited to this.Without departing from the spirit and substance of the premise in the present invention, those of ordinary skill in the art can be to the present invention
Embodiment carry out various equivalent modifications or substitutions, and these modifications or substitutions all should in covering scope of the invention/appoint
What those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, answer
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection scope described in claim
It is quasi-.
Claims (10)
1. a kind of lactobacillus plantarum produces hyaluronidase fermentation optimization technique, which is characterized in that technique the following steps are included:
S1: preparing seed culture medium, and the seed culture medium includes inducer, nitrogen source, inorganic salts and surfactant,
The inducer is the hyaluronic acid of 5g/L molecular weight 1000kDa-2000kDa at the same time as utilization of carbon source,
The nitrogen source is compound nitrogen source, including 5g/L peptone, 10g/L yeast powder, 1.5g/L ammonium sulfate,
The inorganic salts include 0.1g/L sodium chloride, 0.1g/L ferrous sulfate, 0.03g/L manganese sulfate, 0.04g/L calcium chloride,
0.25g/L magnesium sulfate,
The surfactant is 1mL/L Tween-80 or 0.05g/L disodium ethylene diamine tetraacetate；
S2: accessing lactobacillus plantarum into seed culture medium, and culture obtains liquid fermentation strain；
S3: liquid fermentation medium is prepared, the liquid fermentation medium includes inducer, nitrogen source, inorganic salts and surface-active
Agent,
The inducer is used as utilization of carbon source simultaneously, is the hyaluronic acid of 1-10g/L molecular weight 500KDa-1300kDa,
The nitrogen source is compound nitrogen source, including 5g/L peptone, 10g/L yeast powder, 2g/L ammonium sulfate,
The inorganic salts include 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 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: liquid fermentation strain is accessed in liquid fermentation medium, terminates fermentation after culture；
S5: after fermentation liquid is centrifuged, supernatant, as crude enzyme liquid are collected.
2. a kind of lactobacillus plantarum as described in claim 1 produces hyaluronidase fermentation optimization technique, which is characterized in that institute
Stating lactobacillus plantarum is lactobacillus plantarum CnT012-56, and the classification naming of the bacterial strain is lactobacillus plantarum Lactobacillus
Plantarum, depositary institution are China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation address is Beijing
The institute 3 of city, North Star West Road, Chaoyang District 1, preservation time are on November 28th, 2018, and deposit number is CGMCC NO.16836.
3. a kind of lactobacillus plantarum as described in claim 1 produces hyaluronidase fermentation optimization technique, which is characterized in that institute
It states in S1, using the bottled 200mL seed culture medium of 500mL triangle.
4. a kind of lactobacillus plantarum as described in claim 1 produces hyaluronidase fermentation optimization technique, which is characterized in that institute
It states in S2, strain optimal culture conditions are pH6.0, revolving speed 50rpm, 35 DEG C of cultivation temperature, incubation time 18-24h.
5. a kind of lactobacillus plantarum as described in claim 1 produces hyaluronidase fermentation optimization technique, which is characterized in that institute
It states in S3, using the canned 4L liquid fermentation medium of 5L fermentation.
6. a kind of lactobacillus plantarum as described in claim 1 produces hyaluronidase fermentation optimization technique, which is characterized in that institute
It states in S3, inducer is the hyaluronic acid of 5-8g/L molecular weight 500KDa-1300kDa in liquid fermentation medium.
7. a kind of lactobacillus plantarum as described in claim 1 produces hyaluronidase fermentation optimization technique, which is characterized in that institute
It states in S3, surfactant is 1mL/L Tween-80 or 0.05g/L disodium ethylene diamine tetraacetate in liquid fermentation medium.
8. a kind of lactobacillus plantarum as described in claim 1 produces hyaluronidase fermentation optimization technique, which is characterized in that institute
State in S4, strain optimal culture conditions are pH5.0-8.0,30 DEG C -38 DEG C of cultivation temperature, culture 48h, to revolving speed and ventilatory capacity into
Row is adjusted stage by stage:
0-20h, revolving speed 50rpm, ventilatory capacity 2L/min,
20-30h, revolving speed 100rpm, ventilatory capacity 4L/min
30-48h, revolving speed 200rpm, ventilatory capacity 8L/min.
9. a kind of lactobacillus plantarum as claimed in claim 8 produces hyaluronidase fermentation optimization technique, which is characterized in that institute
It states in S4, strain optimal culture conditions are preferably 35 DEG C of pH5.5, cultivation temperature.
10. a kind of lactobacillus plantarum as described in claim 1 produces hyaluronidase fermentation optimization technique, which is characterized in that
In the S4, inoculum concentration 1%-10%, preferably 5%.
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| CN109880758A (en) * | 2019-03-05 | 2019-06-14 | 山东安华生物医药股份有限公司 | A kind of lactobacillus plantarum mutagenic strain and its method of mutagenesis and application |
| EP3730623A4 (en) * | 2019-03-05 | 2020-12-30 | Shandong Awa Biopharm Co., Ltd. | Small-molecule hyaluronic acid or salt thereof, and preparation method therefor |
| WO2021042634A1 (en) * | 2019-09-02 | 2021-03-11 | 山东安华生物医药股份有限公司 | Fermentation optimization process for producing hyaluronidase by lactobacillus plantarum |
| CN113789286A (en) * | 2021-07-30 | 2021-12-14 | 华熙生物科技股份有限公司 | Fermentation medium of animal bifidobacterium and application thereof |
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| CN110408607B (en) * | 2019-09-02 | 2021-05-18 | 山东安华生物医药股份有限公司 | Fermentation optimization process for producing hyaluronidase by lactobacillus plantarum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109880758A (en) * | 2019-03-05 | 2019-06-14 | 山东安华生物医药股份有限公司 | A kind of lactobacillus plantarum mutagenic strain and its method of mutagenesis and application |
| WO2020177456A1 (en) * | 2019-03-05 | 2020-09-10 | 山东安华生物医药股份有限公司 | Mutagenic strain of lactobacillus plantarum, mutagenesis method therefor and application thereof |
| EP3730623A4 (en) * | 2019-03-05 | 2020-12-30 | Shandong Awa Biopharm Co., Ltd. | Small-molecule hyaluronic acid or salt thereof, and preparation method therefor |
| WO2021042634A1 (en) * | 2019-09-02 | 2021-03-11 | 山东安华生物医药股份有限公司 | Fermentation optimization process for producing hyaluronidase by lactobacillus plantarum |
| CN113789286A (en) * | 2021-07-30 | 2021-12-14 | 华熙生物科技股份有限公司 | Fermentation medium of animal bifidobacterium and application thereof |
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