CN108823123A - It is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method - Google Patents
It is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method Download PDFInfo
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- CN108823123A CN108823123A CN201810603938.0A CN201810603938A CN108823123A CN 108823123 A CN108823123 A CN 108823123A CN 201810603938 A CN201810603938 A CN 201810603938A CN 108823123 A CN108823123 A CN 108823123A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
- C12P19/626—Natamycin; Pimaricin; Tennecetin
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Abstract
It is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method, be related to technical field of bioengineering.Actication of culture, the strain are brown yellow spore streptomycete, Streptomyces gilvosporeus, ATCC 13326；First order seed culture；Shake flask fermentation.Influence of 3 kinds of difference plant hormone IAA, GA and 6-BA to brown yellow spore streptomyces cell growth and natamycin synthesis, and compound addition precursor substance sodium propionate during the fermentation are investigated.To understand the regulating and controlling effect of plant hormone and precursor substance in brown yellow spore streptomyces cell growth and natamycin synthesis, experimental basis is provided to improve the efficiency that brown yellow spore streptomycete fermentation produces natamycin using plant hormone and precursor substance.
Description
Technical field
The present invention relates to technical field of bioengineering, receive him more particularly, to a kind of brown yellow spore streptomycete fermentation production of regulation
The preparation method of mycin.
Background technique
In food industry, microbial contamination can bring significant damage to production and preservation, if food is by fungal contamination
Generated fungimycin afterwards has and causes bleeding, teratogenesis, carcinogenic etc. pathogenic, damages to human health.In order to solve this
One problem, people start to introduce food preservative.The production method of food preservative mainly has chemical synthesis and biology at present
Technology law, wherein biotechnology method low toxicity, wide spectrum and economical and practical natural sandy gravel produced is gradually by people
Favor (Iraj Rasooli.et al.2007；Zhang JL.et al.2010).
Natamycin (Natamycin) is a kind of polyene macrolides compound, molecular formula C13H47NO13, white
It is a kind of odorless, tasteless, low dosage and highly-safe food preservative to creamy crystalline powder.Can effectively it inhibit
The growth of saccharomycete and mould simultaneously prevents aflatoxin in filamentous fungi from being formed, and reduces infringement of the pathogenic fungus to the mankind.
Compared with other antimicrobial products, natamycin is extremely low to mammalian toxicity, can widely be applied by safety；And he is mould due to receiving
Plain solubility is extremely low, will not influence the taste and flavor of food when handling food surface, therefore natamycin is extensive
Applied to the fields such as food, control of plant disease, feed and medical treatment (PrajnaNV.et al.2010；Cong F.et
al.2017；Bhatta RS.et al.2012).Natamycin is mainly by Streptomyces natalensis (Streptomyces
Natalensus), brown yellow spore streptomycete (Streptomyces gilvosporeus) and streptomyces chatanoogensis
(the Farid MA.et al.2015 such as (Streptomyces chattanoogensis) producing strains fermenting and producing；Liang J.et
al.2008；Liu SP.et al.2015).Brown Huang spore streptomycete is easy to cultivate because having, and fermentation period is short, fast growing, nothing
Light requirement shines, and using several kinds of carbon source, and can realize many advantages such as High Density Cultivation in the fermenter, therefore is a kind of great
Microorganism (the Liang J.et al.2008 for thering is business development to be worth；WangY.et al.2016).But due to brown yellow spore strepto-
Bacterium fermentation yield is low to cause natamycin price higher, so that its application prospect be made to be very limited.So improving fermentation liquid
The yield of middle natamycin, to realization, its industrialized production is of great significance.
In order to improve the yield of natamycin, researcher has carried out mutagenic and breeding, gene work to brown yellow spore streptomyces species
Journey is transformed and the culture medium and condition of culture of thallus is optimized.Chen etc. (Chen GQ.et al.2008) passes through optimization
Culture medium composition and improvement zymotechnique make natamycin yield reach 2.45g/L, improve 90% compared with control group.Wang etc.
(Wang M.et al.2016) is transformed by genetic engineering, and yield of the natamycin in 1L fermentor is made to reach 7.0g/L.
Wang's etc. (Wang S.et al.2015) studies have shown that addition hexadecane etc. is remarkably improved brown yellow spore as the carrier of oxygen
Dissolved oxygen level when streptomycete fermentation promotes the synthesis of natamycin.Hao Xiaobing's etc. (Hao Xiaobing waits .2009) studies have shown that
The addition of precursor substance sodium propionate also has significant impact to brown yellow spore streptomycete, can make the output increased of natamycin
84.7%.
Plant hormone be plant own metabolism generate organic substance, can by adjust plant cell division with stretch
Long, tissue differentiation, the mature regulating plant growths such as with aging, development and metabolin accumulation.Plant hormone is in low concentration
There is apparent facilitation (Park WK.et al.2013) for cell in range.Studies have shown that being properly added plant hormone
There is growth-promoting functions (LuYD.et al.2015) to marine microalgae and photosynthetic bacteria.In addition, plant hormone is to fungi
Growth and metabolism aspect also have a certain impact.Yu (Yu XJ.et al.2015) etc. has studied 6-BA to thraustochytriale cell
The influence of growth and docosahexaenoic acid (DHA) accumulation, the results showed that, 6-BA can promote oil and fat accumulation and the synthesis of DHA, when
When adding 3mg/L 6-BA, the yield of its oil and fat accumulation and DHA can be made to improve 25.9% and 52.8% compared with control group respectively.Mesh
Before, the research of brown yellow spore streptomycete effect is reported it is not yet found that closing plant hormone.Heteroauxin (IAA), gibberellic acid (GA)
It is 3 kinds of typical plant hormones with 6-benzyladenine (6-BA), is belonging respectively to auxin in plant hormone, gibberellin and thin
Born of the same parents' mitogen three categories.
Summary of the invention
The purpose of the present invention is to provide it is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method.
The present invention includes the following steps：
1) actication of culture, the strain are brown yellow spore streptomycete, Streptomycesgilvosporeus, ATCC
13326；
In step 1), the specific method of the actication of culture can be：The strain transfer for being preserved in -70 DEG C is trained in plate
It supports in base, 28 DEG C of 5~7d of culture, observes colonial morphology, stored in 4 DEG C of refrigerators spare；The plating medium forms
(g/L)：Magnesium sulfate 0.5, ferrous sulfate 0.01, dipotassium hydrogen phosphate 0.5, sodium chloride 0.5, potassium nitrate 1.0, soluble starch
20.0, agar 20.0, pH value 7.0.
2) first order seed culture；
In step 2), the specific method of the first order seed culture can be：By the flat-plate bacterial colony in step 1), with inoculation
The access of the strain spore of activation is equipped with the 250mL conical flask of 50mL seed culture medium by ring picking, 28 DEG C, 200rpm culture 24~
48h；The seed culture medium composition can be (g/L)：Calcium carbonate 2.0, peptone 5.0, yeast powder 8.0, glucose 8.0, soya bean
Cake powder 10.0, soluble starch 20.0, pH value 7.0.
3) shake flask fermentation；
In step 3), the specific method of the shake flask fermentation can be：By the seed culture medium in step 2), with 6%
Inoculum concentration access is equipped in the 250mL conical flask of fermentation medium, 28 DEG C, 200rpm culture 120h；The fermentation medium group
Cheng Kewei (g/L)：Sodium chloride 2.0, yeast powder 5.0, peptone 5.0, glucose 10.0, soybean cake powder 15.0, calcium carbonate 15.0,
Soluble starch 50.0, pH value 7.2.
The present invention has investigated 3 kinds of difference plant hormone IAA, GA and 6-BA to brown yellow spore streptomyces cell growth and has received that he is mould
The influence of element synthesis, and compound addition precursor substance sodium propionate during the fermentation.To understand plant hormone and precursor substance
Regulating and controlling effect in brown yellow spore streptomyces cell growth and natamycin synthesis, to be improved using plant hormone and precursor substance
The efficiency of brown Huang spore streptomycete fermentation production natamycin provides experimental basis.
The present invention activates brown yellow spore streptomycete by plate and first order seed, prepares seed liquor, is inoculated in fermented and cultured
Base, experiment by adding exogenous plant hormones in the initial stage, optimize the addition type of plant hormone, are optimizing first
Experiment basis on, and then ferment to for 24 hours when compound addition precursor substance sodium propionate again.Pass through compound addition plant hormone 6-BA
With precursor substance sodium propionate, influence of the substrate to fermentation process is studied, optimal supplying technics condition is obtained.Using the fermentation work
Skill produces natamycin, effectively improves the yield of natamycin, to reduce production cost, is conducive to industrial applications.
Detailed description of the invention
Fig. 1 is that 0h adds influence of the different plant hormones to brown yellow spore strepto- bacteria biomass.
Fig. 2 is that 0h adds influence of the different plant hormones to brown yellow spore streptomycete natamycin yield.
Fig. 3 is that 0h adds influence of the different plant hormones to brown yellow spore streptomycete natamycin yield.
Fig. 4 is that 0h adds influence of the compound addition sodium propionate for 24 hours of 6-BA to brown yellow spore strepto- bacteria biomass.In Fig. 4, A
It is 0h+1mg/L 6-BA for Control, B；C is 0h+1mg/L 6-BA+0.6%sodiumpropionate.
Fig. 5 is that 0h adds influence of the compound addition sodium propionate for 24 hours of 6-BA to brown yellow spore streptomycete natamycin yield.Scheming
In 5, A Control, B are 0h+1mg/L 6-BA；C is 0h+1mg/L 6-BA+0.6%sodiumpropionate.
Fig. 6 is that 0h adds influence of the compound addition sodium propionate for 24 hours of 6-BA to brown yellow spore streptomycete natamycin yield.Scheming
In 6, A Control, B are 0h+1mg/L 6-BA；C is 0h+1mg/L 6-BA+0.6%sodiumpropionate.
Specific embodiment
It elaborates below by embodiment to the present invention.
Embodiment 1
0h adds different plant hormones to the growth of brown yellow spore streptomycete and the influence of natamycin yield
Brown Huang spore streptomycete is accessed with the strain spore that oese picking activates and is trained equipped with 50mL seed after plate activates
Support the 250mL conical flask of base, 28 DEG C, 24~48h of 200rpm culture.Bacterial strain after activation is accessed into the culture of 250mL shaking flask, carrier fluid
Amount is 50mL, adds 1mg/LIAA, GA and 6-BA respectively in the fermentation medium, 28 DEG C, 200rpm is cultivated.It is every in incubation
A sample is taken for 24 hours, measures the biomass and natamycin yield of each experimental group, as a result as shown in Figures 1 to 3.
As shown in Figure 1, it is not significant to the growth-promoting effect of somatic cells to add 3 Plant Hormones by 0h, but wherein adds
The experimental group of 6-BA is in the advantageous growth in somatic cells of 24~72h.72h adds the experimental group biomass highest of 6-BA, reaches
6% is improved compared to the 35.2g/L of control group to 37.3g/L, adds the experimental group of GA and IAA to brown yellow spore streptomycete
Growth nothing is obviously promoted effect.
By Fig. 2 and 3 it is found that being synthetically produced different influences to natamycin after 0h adds 3 Plant Hormones：Addition
1mg/L 6-BA all has apparent improvement effect to the productivity and yield of natamycin, and maximum value is reached in 96h, is received at this time
His mycin yield is 1.39g/L, and the 1.26g/L compared with control group improves 10.3%, and natamycin yield is also improved compared with control group
24.3%；And the experimental group for adding 1mg/L GA and IAA also has certain facilitation to the synthesis of natamycin, the result
Show to add the accumulation that 3 Plant Hormones are conducive to natamycin in 0h, wherein 0h add 1mg/L 6-BA most beneficial for
Increase the yield of natamycin.
Embodiment 2
The compound influence for adding 0.6% sodium propionate for 24 hours to brown yellow spore streptomycete fermentation of 0h addition 1mg/L 6-BA
On the basis of embodiment 1, experiment investigates 0h and adds 1mg/L further in 0.6% sodium propionate of compound addition for 24 hours
6-BA is compound to add influence of 0.6% sodium propionate to the brown yellow growth of spore streptomyces cell and natamycin accumulation for 24 hours, into one
Step improves the yield of natamycin, and when shake flask fermentation culture to 72,96 and 120h is separately sampled, measures the biomass of each experimental group
And natamycin yield, as a result as shown in figures 4-6.
From fig. 4, it can be seen that compound addition precursor substance sodium propionate has the growth of somatic cells on the basis of adding 6-BA
There is facilitation, 96h biomass is 33.0g/L, and the 29.0g/L compared with control group improves 13.8%.It can be seen from Figures 5 and 6
Compound addition precursor substance sodium propionate has apparent rush to the productivity and yield of natamycin on the basis of 0h adds 6-BA
Into effect, in 96h, natamycin yield is 1.75g/L, and the 1.30g/L compared with control group improves 34.6%, and natamycin produces
Rate also improves 19.9% compared with control group
Influence by research plant hormone to brown yellow spore streptomycete, discovery 6-BA are affected to it, about its metabolism
Mechanism is still unintelligible, thinks that the addition of 6-BA can accelerate brown yellow spore streptomyces cell multiple fission, thus to brown yellow spore chain
The growth of fungal cell and natamycin synthesis all have certain facilitation.Further, since the addition of 6-BA, may promote
Into the variation of related enzyme activity in natamycin synthesis process, to affect the metabolic process of natamycin synthesis.
The present invention, which tests, investigates compound addition external source plant during brown yellow spore streptomycete fermentation production natamycin
The influence that hormone and precursor substance sodium propionate accumulate thalli growth and natamycin.The result shows that 0h addition 1mg/L plant swashs
Under the conditions of plain 6-benzyladenine (6-BA), the maximum production and yield of natamycin are respectively 1.39g/L and 53.5mg/g, compared with
10.3% and 27.3% has been respectively increased in control group.At this time in fermentation to the sodium propionate of compound addition 0.6% for 24 hours, make to receive that he is mould
The maximum production and yield of element respectively reach 1.75g/L and 51.5mg/g, and 34.6% and 19.9% has been respectively increased compared with control group,
This explanation adds plant hormone in cell growth process and precursor substance is conducive to the accumulation of natamycin, this is further to explain
The metabolic regulation network of bright brown yellow spore streptomycete synthesis natamycin is laid a good foundation, while also being provided for its industrialized production
New way.
Claims (7)
1. it is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method, it is characterised in that include the following steps：
1) actication of culture, the strain are brown yellow spore streptomycete, Streptomycesgilvosporeus, ATCC 13326；
2) first order seed culture；
3) shake flask fermentation.
2. as described in claim 1 it is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method, feature exists
In in step 1), the specific method of the actication of culture is：- 70 DEG C of strain transfer will be preserved in plating medium,
28 DEG C of 5~7d of culture observe colonial morphology, store in 4 DEG C of refrigerators spare.
3. as claimed in claim 2 it is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method, feature exists
Become in the plating medium group：Magnesium sulfate 0.5, ferrous sulfate 0.01, dipotassium hydrogen phosphate 0.5, sodium chloride 0.5, potassium nitrate
1.0, soluble starch 20.0, agar 20.0, unit g/L, pH value 7.0.
4. as described in claim 1 it is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method, feature exists
In in step 2), the specific method of the first order seed culture is：It, will with oese picking by the flat-plate bacterial colony in step 1)
The strain spore access of activation is equipped with the 250mL conical flask of 50mL seed culture medium, 28 DEG C, 24~48h of 200rpm culture.
5. as claimed in claim 4 it is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method, feature exists
Become in the seed culture medium group：Calcium carbonate 2.0, peptone 5.0, yeast powder 8.0, glucose 8.0, soybean cake powder 10.0,
Soluble starch 20.0, unit g/L, pH value 7.0.
6. as described in claim 1 it is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method, feature exists
In in step 3), the specific method of the shake flask fermentation is：By the seed culture medium in step 2), connect with 6% inoculum concentration
Enter in the 250mL conical flask equipped with fermentation medium, 28 DEG C, 200rpm culture 120h.
7. as claimed in claim 6 it is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method, feature exists
Become in the fermentation medium group：Sodium chloride 2.0, yeast powder 5.0, peptone 5.0, glucose 10.0, soybean cake powder 15.0,
Calcium carbonate 15.0, soluble starch 50.0, unit g/L, pH value 7.2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957590A (en) * | 2019-05-06 | 2019-07-02 | 淮北师范大学 | A kind of natamycin synthesis technology based on mycelium pellet structure regulating |
| CN110269175A (en) * | 2019-06-05 | 2019-09-24 | 嘉兴浙华质检技术服务有限公司 | A kind of spraying micro-ecology preparation and preparation method thereof for feed anticorrosion |
| CN114875100A (en) * | 2022-06-27 | 2022-08-09 | 山东第一医科大学(山东省医学科学院) | Method for improving natamycin fermentation yield by activating natamycin synthesis in advance |
-
2018
- 2018-06-12 CN CN201810603938.0A patent/CN108823123A/en active Pending
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| 杨东靖: "纳他霉素生产菌株的选育", 《天津科技大学硕士论文》 * |
| 郝晓兵等: "褐黄孢链霉菌生产纳他霉素工艺条件研究", 《厦门大学学报(自然科学版)》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957590A (en) * | 2019-05-06 | 2019-07-02 | 淮北师范大学 | A kind of natamycin synthesis technology based on mycelium pellet structure regulating |
| CN110269175A (en) * | 2019-06-05 | 2019-09-24 | 嘉兴浙华质检技术服务有限公司 | A kind of spraying micro-ecology preparation and preparation method thereof for feed anticorrosion |
| CN114875100A (en) * | 2022-06-27 | 2022-08-09 | 山东第一医科大学(山东省医学科学院) | Method for improving natamycin fermentation yield by activating natamycin synthesis in advance |
| CN114875100B (en) * | 2022-06-27 | 2023-06-16 | 山东第一医科大学(山东省医学科学院) | Method for improving fermentation yield of natamycin by activating natamycin synthesis in advance |
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