CN101298623B - Fermentation production method of validacin - Google Patents
Fermentation production method of validacin Download PDFInfo
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Abstract
The invention relates to a ferment production method of validamycin in the field of biotechnology and comprises following steps: (1) the activation and the plate cultivation of microorganism strains are implemented; (2) the seed cultivation is implemented; (3) the ferment cultivation is implemented. By adopting a method of ferment at a higher temperature of 42 DEG C, about half of the ferment period can be shortened, and higher ferment titer is still ensured, thus greatly improving the ferment productivity of validamycin and achieving the mycoprotein content that is higher than conventional ferment by about 70 percent when the ferment is finished. The ferment production method of validamycin of the invention shortens the ferment period of the validamycin production, improves the utilization rate of devices, lowers unit energy consumption, and simultaneously increases the mycoprotein content, thus having good industrial application prospect.
Description
Technical field
The present invention relates to a kind of fermentation method for producing of biological technical field, particularly, relate to a kind of fermentation method for producing of validamycin.
Background technology
Streptomyces hygroscopicus well ridge mutation (Streptomyces hygroscopicus var.jingganggensis Yen) is that a kind of that area, Jinggang Mountain, pesticide research place, Shanghai in 1973 screens can produce the validamycin bacterial strain of (having another name called jingganmycin).The validamycin of its fermentative production since have efficient, nontoxic, be easy to characteristics such as popularizations, obtained successful exploitation and promoted rapidly in China, become in paddy rice producing region, Asia and used the most a kind of anti-rice fungus diseases to do harm to agricultural antibiotic.
According to bibliographical information, validamycin comprises A, B, C, D, E, F, eight components of G, H, the A ingredients constitute about 70% that wherein anti-microbial activity is the strongest.Its chemical name is: N-((1S)-(1,4,6/5)-3-methylol-4,5,6-trihydroxy--2-is vinyl also)-(O--D-glucopyranosyl-(1 → 3)-(1S)-(1,2,4/3,5)-2,3,4-trihydroxy--5-methylol hexahydroaniline; English by name: validamycin A or jinggangmycin A; Its chemical molecular formula is as follows:
Its relative molecular weight is 515.5, and no definite melting point is 135 ℃ of decomposition.
Iwasa in 1970 have explored the fermentation condition of validamycin, can obtain the biological value of 35000u/ml in 5 days 37 ℃ of fermentations.The Shanghai agricultural chemicals was explored 40 ℃ of thermophilic fermentation conditions that are suitable for well ridge Streptomycin sulphate fermentative production validamycin in 1977, obtained the biological value of 3000g/ml in 40 hours at this temperature bottom fermentation.Wang Shishan etc. utilize single factor and experiment of many factor to study the industrial optimum carbon nitrogen ratio of streptomyces hygroscopicus well ridge mutation 7-11 bacterial strain, have optimized substratum, and comparatively detailed research the influence of leavening temperature and rotating speed to the validamycin fermentation.Bacterium slag after the fermentation is the excellent protein source.Fermented liquid reclaims the bacterium slag through press filtration can dry the back as fodder protein additive, or directly as fish meal.
Find by prior art documents, the leavening temperature that adopts in the industrial production of validamycin is 39 ℃～40 ℃ at present, adopt 72～96 hours longer cycles progressively to accumulate during the fermentation and finally reach high yield, also have and ferment through 40～50 hours periods under adopting 40 ℃, but output can only reach about 60% (Wang Shishan of the whole output of above-mentioned fermentation, explain sub-ox, Chen Shouwen etc., nitrogenous source and carbon-nitrogen ratio is preferred in the fermentation of streptomyces hygroscopicus jingganmycin, Hua Zhong Agriculture University's journal, 2001,20 (6): 554-556).If adopt the long period fermentation, some secondary metabolites are often in thalline nutritive deficiency or a large amount of synthetic through just beginning after a series of atomizations, these secondary metabolites that present well ridge streptomycete is produced are also not exclusively clear and definite, so a large amount of secondary metabolite of the long meeting accumulation of fermentation period.Simultaneously, at present industrial production often utilizes ion exchange method directly to extract validamycin from fermented liquid, and makes aqua or use as pulvis by spraying drying.Use in the field of the direct concentrate formulation of fermented liquid, and the waste liquid that perhaps utilizes ion exchange method to extract the back generation all can be discharged into these metabolites in the environment, brings the potential environmental pollution.It is longer to produce the fermentation period that then needs under 40 ℃, declines to a great extent at fermentation intracellular protein content in latter stage, and the protein content that can be utilized in the fermented liquid also declines to a great extent.Therefore how can obtain higher effective mycin output in than the bob ferment cycle, it is that suitability for industrialized production validamycin institute problem demanding prompt solution is carried out in this area that simultaneously concrete protein content still can keep higher level.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of fermentation method for producing of validamycin is provided.Because there is threshold phenomenon in validamycin output to temperature response, so the present invention adopts the method for the higher leavening temperature that is higher than threshold temperature effectively to improve fermentative production speed and intracellular protein content.Method of the present invention is when obtaining higher effective mycin output and zymophyte body protein content, and fermentation period has shortened half, has improved plant factor, significantly reduces energy consumption; And because the shortening of fermentation period, contained other meta-bolitess of fermented liquid are few, use the pollution that can reduce environment if directly be condensed into preparation.
The present invention is achieved by the following technical solutions:
The present invention includes following steps:
(1) actication of culture is cultivated with dull and stereotyped
Spore suspension thawing with the validamycin of freezing preservation produces bacterium is coated on spore suspension on the solid medium flat board then; Coating finishes and is placed on 30 ℃ of spores of cultivating the plate surface coverage of making even after 5 to 6 days, makes spore suspension;
(2) seed culture
With the activatory spore suspension, be inoculated in the seed culture medium according to the ratio that inserts the 200l spore suspension in per 1 liter of seed culture medium, fermentation culture was carried out 20 hours in the inoculation back under 42 ℃;
(3) fermentation culture
With the cultured seed nutrient solution, be inoculated in the fermention medium according to the ratio that inserts the 100ml seed culture fluid in per 1 liter of fermention medium, cultivate down in 42 ℃ the inoculation back; Short period under 42 ℃ is fermented, and full 36 hours finish to cultivate.
Solid medium in the described step (1) is meant the YMS solid medium, and the mass percent of its integral part is: yeast extract 0.39%, solid solubility starch 0.39%, maltose 0.96%, agar 1.93%, CoCl6H
2O 0.0005%, deionized water 96.33%.
The mass percent of the integral part of the seed culture medium in the described step (2) is: Semen Maydis powder 2.81%, soybean cake powder 2.07%, yeast extract 0.94%, sodium-chlor 0.19%, potassium primary phosphate 0.08%, deionized water 93.91%.
The mass percent of the integral part of the fermention medium in the described step (3) is: Semen Maydis powder 7.92%, soybean cake powder 3.52%, yeast extract 0.44%, sodium-chlor 0.09%, potassium primary phosphate 0.09%, deionized water 87.94%.
The utilization of the present invention little rule of whole volume variance of fermenting when being higher than threshold temperature uses the method for the higher leavening temperature that is higher than threshold temperature effectively to improve the fermentative production speed and the intracellular protein content of validamycin.The present invention utilizes 42 ℃ as leavening temperature, the validamycin of the about 10g/L of accumulation absolute content after 36 hours fermentation culture, and 40 ℃ of bottom fermentations reporting at present in the time of 36 hours the accumulation absolute content of validamycin be about 8g/L.Use among the present invention fermentation process can shorten fermentation time, obtain higher relative potency.Significantly cut down the consumption of energy.Simultaneously,, reduced the consumption of tropina in the lengthy fermentation process, therefore can when guaranteeing validamycin output, increase comprehensive benefit because fermentation period shortens.
Embodiment
Below embodiments of the invention are elaborated: present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1:
1. experiment material:
The bacterial classification that adopts: streptomyces hygroscopicus 5008 bacterial strains (Streptomyces hygroscopicusvar.jingganggensis 5008)
2. experimental procedure:
(1) actication of culture is cultivated with dull and stereotyped
Get intensive evenly being coated with of frozen glycerine spore suspension at the YMS flat board.Inoculation finish be placed on 30 ℃ cultivate 5～6 days can cyan or gray spore.Toward producing the sterilized water that the plentiful flat board of spore adds 3～4ml, scrape the spore that planar surface covers gently with cotton swab simultaneously, it is suspended in the sterilized water.
(2) seed culture
Stainless steel spring is curled into ring-type, places the 250ml triangular flask, the 50ml seed culture medium of packing into then, sterilization.After treating the substratum cooling, draw spore suspension 10l adding and shake in the bottle, inoculate.After the inoculation, will shake bottle respectively as for 28 ℃, 30 ℃, 220 change cultivation, 20 hours under 35 ℃ of temperature.Each temperature is provided with 3 parallel laboratory tests.
(3) fermentation culture
Fermentation shake flask is 250ml, puts into the fermention medium 50ml that packs into behind the spring, sterilization.During switching, at first three parallel seeds are shaken bottle and mix, then transfer to fermentation shake flask with the seed of 5ml pipette, extract 5ml, cultivate from 28 ℃, 30 ℃, 35 ℃ seed is respectively inoculated 9 and is shaken bottle.Place under the corresponding temperature condition, 220 change cultivation.Got 3 shook bottle and carries out various detections at full 36,60,96 hours.
(4) validamycin output detects
Fermented liquid is centrifugal remove thalline after, add isopyknic chloroform and shake strongly, 12000g removed albumen precipitation in centrifugal 10 minutes subsequently.Supernatant liquor is analyzed with HPLC after with the 0.2m membrane filtration.Chromatographic condition 5mM/L, pH=7.0, phosphate buffered saline buffer: chromatographically pure methyl alcohol=98: 2 (volume ratio), flow velocity 1ml/min detects wavelength 210nm, Eclipse XDB-184.6mm * 150mm analytical column, 25 ℃ of control column temperatures, retention time is about 5min.Do more quantitative by the validamycin standard substance than typical curve.(seeing Table 1)
Under the culture temperature that the detection of table 1HPLC method is 28 ℃-35 ℃, the output of the validamycin of different incubation times
In the table: NA represents that the HPLC method does not record the output validamycin.
3. interpretation:
In the present embodiment, 28 ℃ to 30 ℃ temperature increase by 2 ℃, and the whole output of fermenting has improved 0.12g/L.When from 30 ℃ to 35 ℃, temperature increases by 5 ℃, and output improves about 0.9g/L.Along with temperature since 28 ℃ of raisings, observe fermentation titer and improve thereupon.
Embodiment 2:
Select 35 ℃, 37 ℃ and 40 ℃ are carried out fermentation test.Implementation step and result are as follows:
1. the bacterial classification of Cai Yonging: with embodiment 1
2. actication of culture and dull and stereotyped cultural method: with embodiment 1
3. seed culture:
To shake bottle after the inoculation respectively as for 35 ℃, 37 ℃, cultivate 20 hours under 40 ℃ of temperature.All the other are with embodiment 1.
4. fermentation culture
35 ℃, 37 ℃, cultivate the seed that obtains for 40 ℃, insert fermention medium, place corresponding 35 ℃ respectively, 37 ℃, under 40 ℃ of temperature condition, 220 commentaries on classics are cultivated.Got 3 shook bottle and carries out various detections at 84 hours.All the other method stepss are with embodiment 1.
4. fermenting process detects
(1) detect validamycin output, method is with embodiment 1 (the results are shown in Table 2).
Under the culture temperature that the detection of table 2HPLC method is 35 ℃-40 ℃, the validamycin output of different incubation times
| Sample time (h) | 35 ℃ of output (g/L) | 37 ℃ of output (g/L) | 40 ℃ of output (g/L) |
| ?84 | 0.92±0.05 | 10.05±0.98 | 12.20±0.73 |
Interpretation:
In the present embodiment, exist a huge output to rise between 35 ℃ and 37 ℃, temperature has only improved 2 ℃, and the whole output of fermenting in 84 hours is from the 0.92g/L 10.05g/L that ascends to heaven.There is a temperature threshold in this explanation between 35 ℃ and 37 ℃, surpass this threshold value when controlling culture temperature, and envrionment temperature acts on the validamycin route of synthesis by specific mechanism, has caused its output to rise to.
(2) detection of tropina amount: investigate protein content in the bacterium slag, assessment bacterium slag is as the utility value of protein raw materials
Get 1ml fermented liquid 5000g, remove supernatant liquor after 5min is centrifugal and collect thalline in the little centrifuge tube of 2ml, suspend after the washing.Handle sample and water with N,O-Diacetylmuramidase and replace N,O-Diacetylmuramidase to put into 37 ℃ of water-bath kinds insulations together hatching 1 hour, add 10% sodium laurylsulfonate (SDS) solution progressive lysing cell in the sample as blank.Behind the high speed centrifugation, draw supernatant liquor and in measurement range, utilize standard Xylene Brilliant Cyanine G method to measure protein concentration (seeing Table 3) for 100～200 times according to the dilution of concentration situation.
Table 335 ℃-40 ℃ of culture temperature hypothallus protein contents
| Sample time (h) | 35 ℃ of protein contents (g/L) | 37 ℃ of protein contents (g/L) | 40 ℃ of protein contents (g/L) |
| 84 | 7.33±0.46 | 6.40±0.22 | 3.29±0.41 |
Interpretation:
In the present embodiment, raising along with leavening temperature, tropina content raise along with leavening temperature and reduces when fermentation stopped, particularly be elevated to 40 ℃ from 37 ℃ when temperature, protein content when fermentation stops has almost descended one times, and this shows that the bacterium slag for comprehensive utility value after adopting 40 ℃ to ferment 84 hours is lower.
Embodiment 3:
Selection is higher than 37 ℃ of the leavening temperatures of temperature response threshold value, and 40 ℃ and 42 ℃ are carried out fermentation test.Implementation step and result are as follows:
1. the bacterial classification of Cai Yonging: with embodiment 1
2. actication of culture and dull and stereotyped cultural method: with embodiment 1
3. seed culture
To shake bottle after the inoculation respectively as for 37 ℃, 40 ℃, cultivate 20 hours under 42 ℃ of temperature.All the other are with embodiment 1.
4. fermentation culture
37 ℃, 40 ℃, cultivate the seed that obtains for 42 ℃, insert fermention medium, place corresponding 37 ℃ respectively, 40 ℃, under 42 ℃ of temperature condition, 220 rev/mins of cultivations.12,24, to get 3 in 36,60,96 hours and shake bottle and carry out various detections, 42 ℃ of temperature condition promptly finished fermentation in full 36 hours.All the other method stepss are with embodiment 1.
4. fermenting process detects
The detection index comprises: residual sugar and pH, validamycin output, intracellular protein content.Residual sugar adopts the standard sulfuric acid-phynol method to measure, and pH measures with pH meter.All the other methods are with embodiment 2.
5. experimental result
(1) residual sugar and pH detected result (see Table 4 and table 5):
The residual sugar amount of table 437 ℃-42 ℃ of following different incubation times
| Sample time (h) | 37 ℃ of residual sugars (g/L) | 40 ℃ of residual sugars (g/L) | 42 ℃ of residual sugars (g/L) |
| 36 | 18.32±4.06 | 14.4±3.72 | 15.07±4.61 |
| 96 | 4.60±1.06 | 5.55±2.53 | - |
The pH value of table 537 ℃-42 ℃ of following different incubation times
| Sample time (h) | 37℃pH | 40℃pH | 42℃pH |
| 36 | 8.13±0.06 | 8.16±0.06 | 8.28±0.10 |
| 96 | 8.39±0.02 | 8.63±0.11 | - |
Interpretation:
Residual sugar concentration is higher at 36 hours, can further optimize substratum, and the ratio that increases quick-acting carbon sources increases the utilization ratio of sugar.And the pH value difference is not little, can not impact downstream processing.
(2) validamycin output detected result (seeing Table 6):
Utilize HPLC to measure validamycin content, method is with example 1.
The validamycin output of table 637 ℃-42 ℃ of following different incubation times
| Sample time (h) | 37 ℃ of output (g/L) | 40 ℃ of output (g/L) | 42 ℃ of output (g/L) |
| ?12 | 0.899±0.06 | 3.21±0.19 | 4.21±0.09 |
| ?24 | 2.543±0.08 | 6.18±0.27 | 7.10±0.39 |
| ?36 | 4.469±0.44 | 8.74±0.39 | 10.14±0.48 |
| ?60 | 8.573±0.74 | 10.63±0.39 | - |
| ?96 | 10.66±0.97 | 12.56±0.30 | - |
Interpretation:
In the present embodiment, the validamycin resultant velocity of 42 ℃ of earlier fermentations is fast, at the validamycin of 36 hours accumulation 10.14g/L, reaches 80.1% of 40 ℃ of fermentation 96 hourly output 12.56g/L; And 40 ℃ of fermentations have only 69.5% of its 96 hourly output 12.56 at 36 hours accumulation 8.74g/L validamycins.Adopt 42 ℃ of fermentations of comparatively high temps, fermentation period shortens dramatically.If consider the industrial fermentation energy consumption problem, per hour power consumption is 1 unit, then ferments to consume 96 unit electric power in 96 hours, obtains 100% relative potency.If adopt 42 ℃, 36 hours, then 72 unit electric power can supply 2 batch fermentations, obtained the relative potency more than 160%, can significantly reduce energy consumption.
(3) detection of tropina amount
Get 1ml fermented liquid 5000g, remove supernatant liquor after 5min is centrifugal and collect thalline in 2ml EP pipe, suspend after the washing.Handle sample and water with N,O-Diacetylmuramidase and replace N,O-Diacetylmuramidase to put into 37 ℃ of water-bath kinds insulations together hatching 1 hour, add 10% a SDS progressive lysing cell in the sample as blank.Behind the high speed centrifugation, draw supernatant liquor and in measurement range, utilize the Xylene Brilliant Cyanine G method to measure protein concentration for 100～200 times according to the dilution of concentration situation.(seeing Table 7)
The tropina amount of table 737 ℃-42 ℃ of following different incubation times
| Sample time (h) | 37 ℃ of protein contents (g/L) | 40 ℃ of protein contents (g/L) | 42 ℃ of protein contents (g/L) |
| ?12 | 4.11±0.55 | 4.93±0.60 | 4.85±0.48 |
| ?24 | 6.06±0.87 | 5.85±0.83 | 5.50±0.68 |
| ?36 | 6.74±0.67 | 5.50±0.50 | 5.43±0.29 |
| ?60 | 6.78±0.36 | 5.32±0.40 | - |
| ?96 | 5.43±0.63 | 2.70±0.64 | - |
Interpretation:
In the present embodiment, tropina is the highest in 37 ℃ of fermenting processs, but the validamycin output of 37 ℃ of fermentations is starkly lower than 40 ℃ and 42 ℃, so and is not suitable for producing.When fermenting for 40 ℃ and 42 ℃, 36 hours protein content is about 5.5g/L.Ferment by 96 hours at 40 ℃, then tropina content only is left 2.7g/L, and a large amount of valuable albumen are consumed, and have reduced the comprehensive utilization value of bacterium slag as protein source.Therefore, if use 42 ℃ high temperature short period fermentation in 36 hours, then can when guaranteeing validamycin output and productivity, increase comprehensive utilization benefit.
Claims (4)
1. the fermentation method for producing of a validamycin is characterized in that, comprises the steps:
(1) spore suspension that the validamycin of freezing preservation is produced bacterium melts, and then spore suspension is coated on the solid medium flat board, and coating finishes and is placed on 30 ℃ of spores of cultivating the plate surface coverage of making even after 5 to 6 days, makes spore suspension;
It is streptomyces hygroscopicus 5008 bacterial strains that described validamycin produces bacterium;
(2) with the activatory spore suspension, be inoculated in the seed culture medium according to the ratio that inserts 200 microlitre spore suspensions in per 1 liter of seed culture medium, fermentation culture was carried out 20 hours in the inoculation back under 42 ℃;
(3) with the cultured seed nutrient solution, be inoculated in the fermention medium according to the ratio that inserts the 100ml seed culture fluid in per 1 liter of fermention medium, cultivate down in 42 ℃ the inoculation back, 42 ℃ of following 36 hours short period fermentations, full 36 hours finish to cultivate.
2. the fermentation method for producing of validamycin as claimed in claim 1 is characterized in that, the mass percent of the moiety of the solid medium described in the step (1) is: yeast extract 0.39%, solid solubility starch 0.39%, maltose 0.96%, agar 1.93%, CoCl6H
2O 0.0005%, deionized water 96.3295%.
3. the fermentation method for producing of validamycin as claimed in claim 1, it is characterized in that, the mass percent of the moiety of the seed culture medium described in the step (2) is: Semen Maydis powder 2.81%, soybean cake powder 2.07%, yeast extract 0.94%, sodium-chlor 0.19%, potassium primary phosphate 0.08%, deionized water 93.91%.
4. the fermentation method for producing of validamycin as claimed in claim 1, it is characterized in that, the mass percent of the moiety of the fermention medium described in the step (3) is: Semen Maydis powder 7.92%, soybean cake powder 3.52%, yeast extract 0.44%, sodium-chlor 0.09%, potassium primary phosphate 0.09%, deionized water 87.94%.
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| CN101851653B (en) * | 2010-05-17 | 2013-05-29 | 上海交通大学 | Fermentation production method of validamycin A |
| CN101914600A (en) * | 2010-08-27 | 2010-12-15 | 上海交通大学 | Validamycin optimal fermentation method based on hydrogen peroxide |
| CN103805543B (en) * | 2014-01-26 | 2016-06-01 | 福州大学 | A kind of bacterial strain and application thereof producing herbimycin |
| CN109777851B (en) * | 2017-11-15 | 2022-08-23 | 华东理工大学 | Method for improving penicillin biological fermentation yield |
| CN109321616B (en) * | 2018-12-11 | 2022-04-08 | 浙江省桐庐汇丰生物科技有限公司 | Validamycin fermentation medium and method for fermenting Validamycin by using same |
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| CN1772914A (en) * | 2005-11-01 | 2006-05-17 | 浙江工业大学 | Microbial process of producing validamycin anamine and validamycin amine |
| CN101134976A (en) * | 2006-08-29 | 2008-03-05 | 武汉天惠生物工程有限公司 | Method for producing validamycin by circulating fermentation |
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| CN1772914A (en) * | 2005-11-01 | 2006-05-17 | 浙江工业大学 | Microbial process of producing validamycin anamine and validamycin amine |
| CN101134976A (en) * | 2006-08-29 | 2008-03-05 | 武汉天惠生物工程有限公司 | Method for producing validamycin by circulating fermentation |
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