CN101307299B - Method for breeding high yield strain of natamycin and high-efficiency fermenting, abstracting and purifying method of natamycin - Google Patents
Method for breeding high yield strain of natamycin and high-efficiency fermenting, abstracting and purifying method of natamycin Download PDFInfo
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Abstract
The invention proposes a seed selection of a high-yield natamycin production strain and a fermentation culture production process and an extraction purification process method for the same. After screening, the invention obtains the natamycin high-yield strain Streptomyces gilvosporeus HBJ591 which is sent to China Center of Type Culture Collection for preservation with a collection number of CCTCC No.M208028. The fermentation process comprises the following steps: 1) producing seed culture solution through seed culture; 2) carrying out natamycin fermentation in the seed culture solution, andproducing natamycin with a yield as high as 15.5g/L and an average yield of about 14g/L through the fermentation of natamycin high-yield strain Streptomyces gilvosporeus HBJ591. The natamycin extraction and purification method comprises the following steps: (1) carrying out subacidity separation and precipitation; (2) dilution; (3) dissolution under an acidity condition; (4) taking supernatant after filtration; (5) neutralization for crystallization; (6) separation and drying to obtain a natamycin product. The method can realize the production of natamycin with low cost, high yield and high quality.
Description
Technical field
The present invention relates to a plant height and produce screening, the hereditary mutagenesis of tennecetin bacterial strain and utilize this superior strain to carry out the processing method of tennecetin fermentation and extraction purifying, belong to the industrial microbial technology field.
Background technology
Tennecetin (Natamycin) also claims trip Streptomycin sulphate (Pimaricin), it is a kind of important polyenes, this antibiotic is a kind of very strong antifungal agents, because it can narrow spectrum inhibition yeast and mould, stops the formation of aflatoxin in the filamentous fungus.People such as nineteen fifty-five Struyk separation near the soil the Pietermaritzburg town, Natal state, South Africa obtains Natal streptomycete Streptomyces natalensis, and therefrom separation has first obtained tennecetin.Now can by
Streptomycete fermentations such as Streptomyces natalensis and StrePtomyces chatanoogensis are through microbial fermentation production.
Tennecetin appearance white (or cream-colored) is colourless, tasteless crystalline powder, and molecular weight is 665.73, structural formula such as figure below.Tennecetin is a kind of amphiprotic substance, and a basic group and an acidic-group are arranged in the molecule, and iso-electric point is 6.5, and fusing point is 280 ℃.Be slightly soluble in water, methyl alcohol, be dissolved in diluted acid, Glacial acetic acid and dimethylamino benzophenone acid amides, be insoluble in most of organic solvent.Solubleness under the room temperature in water is 30～100mg/L.Be higher than 9 or be lower than at 3 o'clock in the pH value, its solubleness can increase, and is highly stable in the pH of most of food scope.Tennecetin has certain heat resistanceheat resistant processing power, and is relatively stable under drying regime, can tolerate of short duration high temperature (100 ℃).
The tennecetin structural formula
Natamycin is widely used in milk-product production, garden spgarden stuff production, meat product processing, bakes many fields such as based food and feed processing as food preservatives, additive, also be widely used in field of medicaments because of it is antimycotic, have a good application prospect.The subject matter of restriction Natamycin widespread use is its higher production cost and expensive price.
Relevant patent (the publication number: CN1070688A that the tennecetin fermentative production has been arranged at present; CN1071460A; CN1072959A; 1891834A) with extraction purifying patent (publication number: CN101062934A; CN1515678A; CN1891709A).In the disclosed patent, the fermentation level of tennecetin and needs to carry out the control of pH value all below 10g/L in the whole fermentation process, make the technological process trouble like this, and yield and purifying rate are not high.
Summary of the invention
The objective of the invention is to propose a kind of high yield tennecetin and produce seed selection and the fermentation culture production technique and the extraction and purification process method of bacterial strain, thereby realize natamycin with low cost, high yield, high-quality production.
One, the seed selection of tennecetin superior strain
Streptomycete HBJ016 with this laboratory screening and preservation is a starting strain, carries out genetic breeding with UV mutagenesis in conjunction with the genome rearrangement technology that protoplastis merges.In the UV mutagenic processes, streptomycete spore suspension concentration is 10
6～10
8Individual/ml, 20W ultraviolet lamp 20～30cm is apart from irradiation, (the UV irradiation is after 1～2 minute to adopt repeatedly irradiation at interval, incubated at room is 2～3 minutes under dark condition, UV irradiation is 1～2 minute once more, 3～6 times so repeatedly) method improve its mutation frequency, under the situation of high lethality rate (lethality rate is controlled between 80%～90%), the strain of screening high density streptomycin resistant mutation, this method can effectively improve the probability that screening obtains gain mutant.The different mutant strains that UV mutagenesis is obtained adopt conventional protoplastis to merge and parents' deactivation Protoplast Fusion Technique, carry out genome rearrangement, obtain a strain tennecetin superior strain Streptomyces gilvosporeus HBJ591 through multi-turns screen and (be preserved in Chinese typical culture collection center, preserving number: CCTCCNo.M208028).Through repeated experiments repeatedly, the output of this mutant strain shake flask fermentation all remains on the level about 10.6g/L, shows genetic stability preferably, amplifies fermentation in 10L～100L fermentor tank, and output reaches the high yield level of 15.5g/L.
The bacteria characteristic of HBJ591 bacterial strain:
A, morphological specificity: on nutrient agar, grow aerial hyphae prosperity, little brown or light brown, edge white.
B, physiological and biochemical property: the energy liquefy gelatin, hydrolyzed starch does not produce class melanochrome, 28 ℃～30 ℃ of optimum growth temperatures.
Two, tennecetin zymotechnique
Be to be the zymotechnique of bacterial classification below with HBJ591 tennecetin superior strain Streptomyces gilvosporeus, can in 10L～100L fermentor tank, amplify or dwindle fermentation volume, fermentation period is between 96～160 hours, fermentation yield is in the level of 13～15.5g/L, compares with shake flask fermentation and improved about 4g/L.This technology is compared outstanding feature with other disclosed tennecetin zymotechniques, and the whole process from seed culture to fermentation need not to carry out the control of pH value, has avoided the use of soda acid, has simplified zymotechnique greatly, has reduced environmental protection pressure.This technology is applicable to 100L～100, and the technology of 000L fermentor tank is amplified.Concrete technology is as follows:
One) seed culture
A. seed culture based formulas
Peptone 10g/L, sodium-chlor 10g/L, glucose 15g/L, all the other are water.
After peptone and sodium-chlor are prepared together, regulate pH to 7.0 with KOH, sterilized 30 minutes for 121 ℃, glucose is prepared separately, adds under aseptic condition after the overanxious degerming; After perhaps three kinds of components are prepared together, regulate pH to 7.0, sterilized 30～40 minutes for 115 ℃, to avoid the high temperature cabonization of glucose with KOH.
B. seed liquor is cultivated
The mode that the seed liquor cultivation adopts amplification step by step to spread cultivation is carried out, and culture temperature is 29 ℃, and whole process is not carried out the control of pH value.Fs: with the 1.5ml HBJ591 tennecetin superior strain spore suspension (spore concentration about 10 of above-mentioned seed selection
8Individual/as ml) to insert the 100ml seed culture medium, shaking table 200～250rpm cultivated 12～16 hours.Subordinate phase: the fs seed culture fluid by 0.5% inoculum size, is inserted the fresh seeds substratum, shook bottle or 1L～10L fermentor cultivation 16～18 hours.As use shake-flask culture, loading amount is no more than 10%, when using fermentor cultivation, looks the defoamer that particular case adds 0.2g/L in good time.Phase III: the seed culture fluid of subordinate phase by 1% inoculum size, was inserted 5L～30L fermentor cultivation 24～28 hours.The natural pH value of cultivating the back seed liquor is between 4.0～5.0.
Two) tennecetin fermentation
A. fermentative medium formula
Soybean protein powder 35～40g/L, yeast powder 6～10g/L, glucose 40～50g/L, defoamer 0.2g/L.
After soybean protein powder and yeast powder are prepared together, regulate pH to 7.5 with KOH, sterilized 40 minutes for 121 ℃, glucose is prepared separately, adds after 115 ℃ of sterilizations; After perhaps three kinds of components are prepared together, regulate pH to 7.5, sterilized 40 minutes for 115 ℃ with KOH.The initial concentration that guarantees glucose in the fermention medium is not less than 40g/L.In addition, separately compound concentration is 60% glucose sterilization, is used for fermenting process stream and adds glucose in the afterfermentation liquid.
B. fermenting process control
The HBJ591 tennecetin superior strain spore seed liquor that will spread cultivation through three steps, the inoculum size by 2% inserts fermention medium.Leavening temperature is 29 ℃, and whole fermentation process is not carried out the control of pH value, is 20%～30% by dissolved oxygen in rotating speed or the air flow control fermentor tank.Detect the consumption of glucose in the fermenting process, stream adds glucose mother liquid in good time, makes that the content of glucose is not less than 20g/L in the fermenting process, and the residual sugar in the fermented liquid can be used back one batch fermentation by quilt cover, to reduce production costs.The 6th day of fermentation or (analyze tennecetin content in the fermented liquid) when tennecetin content no longer raises in fermented liquid, put and jar collect a fermented liquid by HPLC.This fermentation technology process need not to carry out the control of pH value, simplified zymotechnique, through multiple batches of 10L～100L fermentor tank experiment, utilize HBJ591 tennecetin superior strain to ferment, tennecetin output is up to the level of 15.5g/L, average level at about 14g/L is far above the fermentation level of tennecetin in the present publication.
Three, tennecetin extracts and purifying process
Tennecetin is water insoluble under usual conditions, and organic solvents more commonly used extract, as United States Patent (USP) 3,892, and 850, all take the method for organic solvent extraction among WO92/07998, the WO92/1058.These methods need be used inflammable, volatile organic solvents such as methyl alcohol, Virahol, certainly will cause that higher solvent loss, recovery cost and higher safety features require, high expenses of environmental protection.And the use of methyl alcohol, cause this production of by-products of tennecetin methyl esters easily, thereby influence the purity of product.In China national patent CN101062934A, a kind of method of with an organic solvent not extracting tennecetin from fermented liquid is disclosed.This patent is to utilize the character of tennecetin different solubility in the aqueous solution under the condition of different pH, realizes the extraction of tennecetin.In its alkalization process, the pH value of fermented liquid all requires more than 11, certainly will use a large amount of alkali lye to regulate like this and show the tart fermented liquid originally.Though this method has been avoided the use of organic solvent, the use of a large amount of soda acids can bring consumption, the recovery problem of unpredictable environmental issue and soda acid equally.In addition, because the content of tennecetin is lower in the fermented liquid, these methods all need to carry out the concentration of fermented liquid before extracting, and extract yield and purity to improve, and certainly will increase the energy consumption in the production like this.
The step of conventional employing methanol extraction tennecetin is: concentrated broth → adding methyl alcohol, rising pH value (more than 10) → removal mycelium → reduction pH value (about 7.0), evaporation methyl alcohol → recovery precipitation, the dry product that gets.This technology be the pH value of rising fermented liquid to alkalescence, use the dissolve with methanol tennecetin again, remove to reduce behind the thalline pH and obtain the tennecetin precipitation.Though fermented liquid is through concentrating, volume is still bigger, the large usage quantity of soda acid and methyl alcohol.The present invention is utilizing the superior strain fermentation, obtain on the basis of high-content natamycin fermentation liquor, comprehensive utilization organic solvent, pH value are regulated and temperature controlled method, and the method that adopts raffinate to apply mechanically second extraction, secondary purifying, realize the high efficiency extraction and the purifying of tennecetin, extract yield and reach 90%, purity reaches 95%.The present invention is adjusted to slightly acidic with script acid fermentation liquid earlier, tennecetin is adhered to and thalline, under sour environment, use the dissolve with methanol tennecetin after collecting thalline, avoided a large amount of uses of methyl alcohol and soda acid like this, and improved the yield of extraction and the purity of purifying.Concrete technology is as follows:
1. slightly acidic precipitation separation.Fermented liquid is regulated pH value to 6.0 (slightly acidic) with NaOH, and 50 ℃ of insulations 10～12 hours.Centrifugal removal supernatant keeps precipitation, or Plate Filtration is removed part moisture content.
2. dilution.Measure moisture content in precipitation or the filter residue, suspend with methanol/water solution and precipitate, making the ratio of methyl alcohol and water (comprising remaining water in precipitation or the filter residue) is 65/35 (v/v), is cooled to 5～10 ℃.
3. acidic conditions dissolving down.Above-mentioned suspension is regulated pH value to 3.0 with HCL, under 5～10 ℃ of conditions, stirred 30～50 minutes, tennecetin is dissolved fully.
4. cross the leaching supernatant liquor.With the centrifugal or Plate Filtration of suspension in 3, keep supernatant.To precipitate or filter residue with methanol/water solution (70/30, v/v) washing keeps supernatant, and with the first time supernatant merge, to improve the tennecetin yield.
5. adjust back to neutral crystallization.With the supernatant liquor pH regulator to 7.0 in 4, and incubated at room 16～20 hours makes the complete crystallization of tennecetin with NaOH, and the remaining tennecetin of solution should be lower than 1.5g/L.
6. separate dry.Centrifugal or Plate Filtration is collected the tennecetin crystal, washes dry or other modes of twice final vacuum and removes moisture, makes moisture content be lower than 9%, obtains the tennecetin product.Tennecetin once extracts yield and arrives 85%, and purity reaches 90%.Raffinate in 5 is collected the back second extraction, and total yield reaches 90%; The secondary purifying can make purity reach 95%.
The concrete embodiment of tennecetin fermented extracted:
Example 1:10L ferment tank, and tennecetin extract purifying
Shake the amplification culture step by step that bottle, 1L fermentor tank, 5L fermentor tank carry out seed liquor at 1L respectively by above-mentioned technology, incubation times at different levels were respectively 12 hours, 16 hours and 26 hours, obtained seed liquor 1L, and seed liquor final pH value is 4.7.Fermention medium 5L (soybean protein powder, 38g/L pack in the 10L fermentor tank; Yeast powder, 8g/L; Glucose 40/L), inserts seed liquor 1L, and fermenting process stream altogether adds 60% glucose 2.6L.Control dissolved oxygen between 20%～30% by rotating speed and air flow, fermentation period 144 hours (6 days), final fermentating liquid volume 7.8L, biomass 45.3g/L, tennecetin content 13.8g/L.
The 7.8L fermented liquid is extracted by said extracted, purifying process, and disposable extraction obtains tennecetin product 92.3g, and extracting yield is 85.7%.Purity is 93.8% after the secondary purifying is carried out in dissolving, crystallization again under sour environment.
Example 2:30L ferment tank, and tennecetin extract purifying
Shake the amplification culture step by step that bottle, 5L fermentor tank, 10L fermentor tank carry out seed liquor at 1L respectively by above-mentioned technology, incubation times at different levels were respectively 12 hours, 18 hours and 24 hours, obtained seed liquor 4L, and seed liquor final pH value is 4.6.Fermention medium 20L (soybean protein powder, 40g/L pack in the 30L fermentor tank; Yeast powder, 8g/L; Glucose 45/L), inserts seed liquor 4L, and fermenting process stream altogether adds 60% glucose 8.2L.Control dissolved oxygen between 20%～30% by rotating speed and air flow, fermentation period 150 hours, final fermentating liquid volume 25.3L, biomass 46.1g/L, tennecetin content 15.1g/L.
The 25.3L fermented liquid is extracted by said extracted, purifying process, and disposable extraction obtains tennecetin product 330.3g, and extracting yield is 87.5%.Purity is 94.5% after the secondary purifying is carried out in dissolving, crystallization again under sour environment.
Example 3:100L ferment tank, and tennecetin extract purifying
Shake the amplification culture step by step that bottle, 10L fermentor tank, 30L fermentor tank carry out seed liquor at 1L respectively by above-mentioned technology, incubation times at different levels were respectively 12 hours, 18 hours and 26 hours, obtained seed liquor 14L, and seed liquor final pH value is 4.6.Fermention medium 70L (soybean protein powder, 38g/L pack in the 100L fermentor tank; Yeast powder, 8g/L; Glucose 45/L), inserts seed liquor 14L, and fermenting process stream altogether adds 60% glucose 26.7L.Control dissolved oxygen between 20%～30% by rotating speed and air flow, fermentation period 158 hours, final fermentating liquid volume 87.4L, biomass 45.8g/L, tennecetin content 14.8g/L.
The 85L fermented liquid is extracted by said extracted, purifying process, and disposable extraction obtains tennecetin product 1093.2g, and extracting yield is 86.9%.Purity is 93.6% after the secondary purifying is carried out in dissolving, crystallization again under sour environment.
Claims (2)
1. tennecetin superior strain Streptomyces gilvosporeus HBJ591, preserving number is CCTCC NO:M208028.
2. the zymotechnique of tennecetin superior strain HBJ591 according to claim 1 is characterized in that step is:
One) seed culture
A. seed culture based formulas:
Peptone 10g/L, sodium-chlor 10g/L, glucose 15g/L, all the other are water;
After peptone and sodium-chlor are prepared together, regulate pH to 7.0 with KOH, sterilized 30 minutes for 121 ℃, glucose is prepared separately, adds under aseptic condition after the filtration sterilization; After perhaps three kinds of components are prepared together, regulate pH to 7.0, sterilized 30～40 minutes for 115 ℃, to avoid the high temperature cabonization of glucose with KOH;
B. seed liquor is cultivated: the mode that the seed liquor cultivation adopts amplification step by step to spread cultivation is carried out, and culture temperature is 29 ℃, and whole process is not carried out the control of pH value;
Fs: with spore concentration about 10
8The HBJ591 tennecetin superior strain spore suspension 1.5ml of individual/ml inserts the 100ml seed culture medium, and shaking table 200～250rpm cultivated 12～16 hours;
Subordinate phase: the fs seed culture fluid by 0.5% inoculum size, is inserted the fresh seeds substratum, shook bottle or 1L～10L fermentor cultivation 16～18 hours; When using shake-flask culture, loading amount is no more than 10%; When using fermentor cultivation, look the defoamer that particular case adds 0.2g/L in good time;
Phase III: the seed culture fluid of subordinate phase by 1% inoculum size, was inserted 5L～30L fermentor cultivation 24～28 hours; The natural pH value of cultivating the back seed liquor is between 4.0～5.0;
Two) tennecetin fermentation
A. fermentative medium formula
Soybean protein powder 35～40g/L, yeast powder 6～10g/L, glucose 40～50g/L, defoamer 0.2g/L after soybean protein powder and yeast powder are prepared together, regulates pH to 7.5 with KOH, sterilizes 40 minutes for 121 ℃, and glucose is prepared separately, adds after 115 ℃ of sterilizations; After perhaps three kinds of components are prepared together, regulate pH to 7.5, sterilized 40 minutes for 115 ℃ with KOH, the initial concentration that guarantees glucose in the fermention medium is not less than 40g/L, in addition, separately compound concentration is 60% glucose sterilization, is used for fermenting process stream and adds glucose in the afterfermentation liquid;
B. fermenting process control
The HBJ591 tennecetin superior strain spore seed liquor that will spread cultivation through three steps, inoculum size by 2% inserts fermention medium, leavening temperature is 29 ℃, whole fermentation process is not carried out the control of pH value, by dissolved oxygen in rotating speed or the air flow control fermentor tank is 20%～30%, detect the consumption of glucose in the fermenting process, stream adds glucose mother liquid in good time, make that the content of glucose is not less than 20g/L in the fermenting process, residual sugar quilt cover in the fermented liquid is used back one batch fermentation, reducing production costs, and the 6th day of fermentation or when tennecetin content no longer raises in fermented liquid, put a jar collection fermented liquid, tennecetin output reaches the level of 14g/L----15.5g/L.
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| JP2011181793A (en) * | 2010-03-03 | 2011-09-15 | Koito Mfg Co Ltd | Light emitting device |
| CN102746988A (en) * | 2010-08-13 | 2012-10-24 | 安泰生物工程股份有限公司 | Method for hydrolyzing streptococcus lactis and method for producing natamycin with streptococcus lactis hydrolysate |
| CN104611283B (en) * | 2012-12-27 | 2018-09-21 | 北京市农林科学院 | A kind of recombination streptomyces lydicus and its application |
| CN103665074B (en) * | 2014-01-07 | 2016-05-18 | 厦门大学 | The method for extraction and purification of natamycin in a kind of zymotic fluid |
| CN107828837A (en) * | 2017-12-12 | 2018-03-23 | 山东福瑞达生物科技有限公司 | A kind of process for producing of the liquid natamycin of stabilization |
| CN109943610B (en) * | 2019-05-06 | 2021-05-25 | 淮北师范大学 | Natamycin fermentation process based on exogenous saturated fatty acid addition |
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| CN1515678A (en) * | 2003-08-25 | 2004-07-28 | 天津科技大学 | Preparation method of natamycin |
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