CN101897730B - Method for extracting streptomyces antibacterial product - Google Patents
Method for extracting streptomyces antibacterial product Download PDFInfo
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- CN101897730B CN101897730B CN2009102310529A CN200910231052A CN101897730B CN 101897730 B CN101897730 B CN 101897730B CN 2009102310529 A CN2009102310529 A CN 2009102310529A CN 200910231052 A CN200910231052 A CN 200910231052A CN 101897730 B CN101897730 B CN 101897730B
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Abstract
The invention relates to a method for extracting a streptomyces antibacterial product, and belongs to the technical field of biology. Streptomyces sp S24 is activated and fermented; the fermentation solution is sterilized, centrifuged, absorbed by macroporous absorption resin AB-8 and desorbed by 75 to 85 percent acetone; and a solvent is reclaimed from the desorption solution, and the desorption solution is evaporated and dried to obtain an antibacterial product extract. Experiments prove that the antibacterial product has broad-spectrum antibacterial activity for main mildewing fungi such as aspergillus flavus, aspergillus niger, aspergillus ochraceus and aspergillus fumigatus in food and feed and human pathogenic fungi such as aspergillus flavus and aspergillus fumigatus. Because the antibacterial product provided by the invention has obvious inhibiting effect on various food and geed mildewing fungi and human pathogenic fungi, the antibacterial product shows efficient broad-spectrum properties; and the method for extracting the antibacterial product is simple and convenient, has low cost, and has high practical application value and broad market prospect on the aspects of food preservative, feed additive and medicaments.
Description
(1) technical field
The present invention relates to extract from streptomycete S24 zymotic fluid with macroporous absorbent resin the method for the antibacterial product of its generation, belong to biological technical field.Be exactly the method for from streptomycete S24 zymotic fluid, extracting antibacterial product specifically.
(2) background technology
The object of the invention is to extract mainly go mouldy fungi and human body cause illness fungi aspergillus flavus that antibacterial product that streptomycete S24 fermentation produces is used for control grain and feed, aspergillus fumigatus etc.
Aspergillus fungi pollutes grain and feed can cause people and animals' generation aspergillus disease, and the toxin of generation has induced mutation, Immunosuppression and carcinogenesis, and since the sixties in last century, people conduct extensive research it, and are devoted to its effective control.The more improvement of detoxicated technology and the aspects such as optimization of detection method of concentrating on of domestic researcher, the method of in the past preventing and treating aspergillus fungi and endotoxin contamination grain and feed mainly is to remove aspergillus and the toxin that pollutes in the food with physics, chemistry and biology method, not only cost is high, and effect is also undesirable.
Streptomyces (Streptomyces) bacterial strain has abundant species diversity and metabolic type diversity, is the resource microorganism of extremely important generation natural activity product.Since Waksman found streptomycin, the separation of streptomycete and the research of metabolite thereof were subject to the extensive concern of microbiologist and pharmacy man.Have the microbial resources that Important Economic is worth as a class, more than 12000 of discovery plant in the microbe-derived physiological activator so far, produced by streptomyces bacterial strain more than 55%, comprising antibiotic, immunodepressant and enzyme inhibitor etc.The antibiotic about 2/3 of using clinically at present derives from streptomyces.The extraction active substance is study hotspot for many years always from streptomycete.We are take the aspergillus flavus reference culture as target, from Mount Taishan soil and grain storehouse dust, separate, screen acquisition Effective Anti aspergillus flavus streptomycete S24, research finds that the antibacterial product that this strain fermentation produces not only has the high-efficiency broad spectrum resistance to the fungi that mainly goes mouldy in grain and the feed, and human body cause illness fungi aspergillus flavus, aspergillus fumigatus are also had good inhibitory action.The present invention utilizes resin method to extract streptomycete S24 antibacterial product, has that equipment is simple, easy to operate, with short production cycle, low cost and other advantages, is beneficial to suitability for industrialized production.
(3) summary of the invention
The invention provides a kind of extracting method of streptomyces antibacterial product.
The present invention adopts following technical scheme to realize: a kind of streptomycete, this bacterial strain is expressed as streptomycete S24.This bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 13rd, 2009; The address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Bacterial strain deposit number: CGMCC No.3503; Strain classification name: streptomycete Streptomyces.Sp.
The research origin of streptomycete of the present invention (Streptomyces sp) S24 is in " aspergillus flavus biological and ecological methods to prevent plant disease, pests, and erosion screening ", sample is mainly taken from the Mount Taishan timbered soil, from wherein screening bacterial classification, altogether collect and obtain more than 200 of active bacterial strains, take the aspergillus flavus reference culture as target, screening aspergillus flavus Antagonistic Fungi, utilize its to the common fungi that goes mouldy in grain and the feed such as aspergillus flavus, Aspergillus ochraceus, aspergillus niger, aspergillus fumigatus, aspergillus oryzaes etc. and the common pathomycete of human body are such as aspergillus flavus, aspergillus fumigatus, the multiple encountered pathogenic fungi such as Fusarinm solani carries out the screening of plate face-off Antagonistic Fungi, strain fermentation, sieve again without fermented liquid, filter out at last a strain and have the streptomycete S24 that can produce the broad-spectrum high efficacy antibacterial product.
The characteristic of streptomycete S24:
1, strain morphology is learned feature
The S24 bacterial strain has thread mycelia, and the livings mycelia of base is more straight, and aerial hyphae slightly has bending and has than multi-branched, and the mycelium tabula is obvious, can the section of fragmenting into; Fibrillae of spores is straight, and secondary is verticillate, and the fibrillae of spores bunchiness is arranged, and spore is oval.
2, strain culturing feature
Streptomycete S24 growth differences on 8 kinds of different medium is larger, and its living mycelia of base and aerial hyphae color (seeing the following form) are different in the situation that different culture media produces pigment.The living mycelia of base is the multiple color take yellow as keynote; Aerial hyphae is the multiple color take white as keynote.
The cultural characteristic of streptomycete S24
Annotate: "-" represents poor growth; "+" representative is sparse; " ++ " representative is more; " +++" representative is more luxuriant; " ++ ++ " representative is luxuriant
3, physiological and biochemical property
Physiological and biochemical test shows: this bacterium can grow under 15～37 ℃ of environment, and growth was suppressed when environmental temperature reached 45 ℃; Its survival pH value is 4～12, and gelatin does not liquefy, milk solidifies, Starch Hydrolysis manifests the positive, can reduce nitrate, can grow at filter paper, but can not utilize cellulose, can produce H
2S and melanin, salt resistance is poor, and i.e. growth is very slow containing on the medium of NaCl3%; Streptomycete S24 can produce rennin, amylolytic enzyme, nitrate reductase in process of growth, can not produce protease, can not produce cellulase.
The physiological and biochemical property of streptomycete S24
Annotate: "-" represents feminine gender or mycelia does not grow; "+" represents the positive or mycelial growth amount
The objective of the invention is to provide the extracting method of streptomycete S24 antibacterial product, this antibacterial product not only has the high-efficiency broad spectrum resistance to the fungi that mainly goes mouldy in grain and the feed, and to human body cause illness fungi aspergillus flavus, aspergillus fumigatus, Fusarinm solani also has good inhibitory action, therefore, this antibacterial product has as food preservative, the potentiality of feed addictive and medicine, the present invention utilizes resin method to extract streptomycete S24 antibacterial product, it is simple to have equipment, easy to operate, with short production cycle, low cost and other advantages, be beneficial to suitability for industrialized production, it is used on a large scale.
A kind of extracting method of streptomyces antibacterial product, realize by following steps:
A, strain transfer that the inclined-plane is preserved be to dull and stereotyped PDA medium, 28 ℃ of constant temperature culture 48～72 hours; The PDA medium is: potato 200g, glucose 20g, agar 15g, (NH
4)
2SO
41g, MgSO
41g, KH
2PO
40.6g, CaCO
33g, water 1000ml;
B, with the dull and stereotyped strain transfer of PDA to seed culture medium, shaker fermentation 24 hours was forwarded to the fermentation medium shaker fermentation 84-96 hour by 2% inoculum concentration, collected zymotic fluid, 4200 rev/mins of low speed large capacity centrifuges centrifugal 30 minutes, are collected fermented supernatant fluid; Seed and fermentation culture conditions are: 28 ℃ of temperature, and 200 rev/mins of shaking speed, the shaking flask liquid amount is 50ml/250ml (or 100ml/500ml); Seed and fermentative medium formula are complex medium, are specially: glucose 16g, soluble starch 2.5g, peptone 10g, beancake powder 26g, NaCl 7.5g, (NH
4)
2SO
44g, water 1000ml;
C, macroporous absorbent resin is mixed by a certain percentage with fermented supernatant fluid, 150 rev/mins, vibration absorption 3-4 hour;
D, the resin after will adsorbing are collected the dress post, with removing partial impurities behind the finite concentration ethanol elution, then use finite concentration acetone elution antimicrobial product, flow velocity is 1.0ml/min, flow out from pigment zone and to begin to collect eluent, stop collection after collecting 1.2-1.5 times of column volume;
E, evaporating, concentrating and drying acetone eluent, resulting dry is antibacterial product;
Wherein:
Macroporous absorbent resin described in the step c is AB-8; Macroporous absorbent resin AB-8 and fermented supernatant fluid ratio are 1: 20 (m: V).
Eluent described in the steps d is acetone.
Concentration of alcohol described in the steps d is 15%-25%, and the eluent acetone concentration is 75%-85%.
The antibacterial product rate of recovery that streptomycete S24 produces is calculated:
Give birth to the preparation of master plate: after the fusing of PDA medium, when temperature is down to 50～55 ℃, add the aspergillus spore suspension, the concentration that makes spore is 2～4 * 10
4CFU/mL pours the medium spore suspension into culture dish, the 20mL/ culture dish, and the mixed bacterium of preparation is dull and stereotyped; The mensuration of tiring: adopt the Oxford agar diffusion method, with the Oxford cup put into give birth to survey mixed bacterium flat board after, 100 ℃ of sterilizations of antibacterial material solution 10 minutes, add in the cup of Oxford (250 μ L/ hole), cultivated 18 hours for 28 ℃, the right-angled intersection method is measured antibacterial circle diameter, calculates according to the diameter of inhibition zone and tires, antibacterial product accounting equation: the Y=10 that tires
(x+10.931)/10.797* n (Y: antibacterial product tire (U); X: antibacterial circle diameter (10.5mm＜x＜18.0mm); N: extension rate); Rate of recovery accounting equation: the antibacterial product rate of recovery=Y
1V
I/ YV (Y: tire by fermented supernatant fluid; V: fermented supernatant fluid volume; Y
1: eluent is tired; V
1: effluent volume).
The streptomycete S24 antibacterial product that extracts according to the present invention has the following advantages:
(1) broad-spectrum antibacterial property is good, and main fungi (such as aspergillus flavus, aspergillus niger, aspergillus fumigatus, Aspergillus ochraceus) and the human body cause illness fungi (aspergillus flavus, aspergillus fumigatus, Fusarinm solani) that causes grain and feed mold all had stronger inhibitory action.
(2) bacteriostatic activity is high, the antibacterial product that extracts with resin method presses down aspergillus flavus is minimum/and bacteriocidal concentration (MIC and MFC) is respectively 19.53 μ g/mL and 39.06 μ g/mL, aspergillus flavus is easy to infecting peanut when not having antibacterial product to exist and growth is very vigorous, and the growth of aspergillus flavus is subject to obvious inhibition in the situation that has antibacterial product to exist.
(3) the fermentation proterties is good, and fermentation period is short, and proterties is stable, and antibacterial product is to thermally-stabilised, storage endurance.
(4) macroporous absorbent resin selected of the present invention is good to the adsorptive selectivity of antibacterial product, and absorption is fast, and desorb is also fast, and adsorption capacity is larger, and solvent for use is recoverable all, is suitable for large-scale production.
(4) embodiment
Embodiment 1:
(1) streptomycete S24 cultivates and fermentation: after actication of culture is preserved on the inclined-plane, go to dull and stereotyped PDA medium growth 48 hours, punching is seeded to seed culture medium, seed culture condition: 28 ℃ of temperature, 200 rev/mins of shaking speed, incubation time 24 hours; Seed culture fluid is inoculated in fermentation medium, fermentation culture conditions by 2%: 28 ℃ of temperature, 200 rev/mins of shaking speed, shaking flask liquid amount 50ml/250ml, incubation time 96 hours; After the fermentation ends, collect zymotic fluid, 4200 rev/mins of low speed large capacity centrifuges, centrifugal 30 minutes, collect fermented supernatant fluid, volume is 2400ml, it is 4563 μ g/mL that living survey is tired;
(2) macroporous absorbent resin AB-8 adsorption antibacterial product: with polymeric adsorbent AB-8 and supernatant after zymotic fluid is centrifugal m/V=1 in proportion: 20 mix, 150 rev/mins of vibration absorption 4 hours; Resin after the absorption is collected the chromatographic column of packing into;
(3) eluant, eluent desorb antibacterial product: 2-3 times of column volume of deionized water wash-out, with removing partial impurities behind 15% ethanol elution, then be that 80% acetone carries out the wash-out activated product with concentration, flow velocity 1.0ml/min, pigment zone flows out and begins to collect the acetone eluent, obtain altogether eluent 180ml, it is 54135.43 μ g/mL that living survey is tired;
(4) mensuration of the acquisition of antibacterial product and the rate of recovery: the dry that will collect the acetone eluent concentrate drying acquisition that obtains is the antibacterial product that streptomycete S24 produces; Measure respectively former fermented supernatant fluid, AB-8 resin adsorption after fermentation raffinate and acetone eluent are tired, and recording the AB-8 resin is 98.62% to the adsorption rate of antibacterial product, and strippant is 93.78% to the desorption efficiency of antibacterial product, and the antibacterial product rate of recovery is 88.98%.
Embodiment 2
(1) streptomycete S24 cultivates and fermentation: after actication of culture is preserved on the inclined-plane, go to dull and stereotyped PDA medium growth 56 hours, punching is seeded to seed culture medium, seed culture condition: 28 ℃ of temperature, 200 rev/mins of shaking speed, incubation time 24 hours; Seed culture fluid is inoculated in fermentation medium, fermentation culture conditions by 2%: 28 ℃ of temperature, 200 rev/mins of shaking speed, shaking flask liquid amount 100ml/500ml, incubation time 96 hours; After the fermentation ends, collect zymotic fluid, 4200 rev/mins of low speed large capacity centrifuges, centrifugal 30 minutes, collect fermented supernatant fluid, volume is 4600ml, it is 4325.5 μ g/mL that living survey is tired;
(2) macroporous absorbent resin AB-8 adsorption antibacterial product: with polymeric adsorbent AB-8 and supernatant after zymotic fluid is centrifugal m/V=1 in proportion: 20 mix, 150 rev/mins of vibration absorption 4 hours; Resin after the absorption is collected the chromatographic column of packing into;
(3) eluant, eluent desorb antibacterial product: 2-3 times of column volume of deionized water wash-out, with removing partial impurities behind 20% ethanol elution, then be that 85% acetone carries out the wash-out activated product with concentration, flow velocity 1.0ml/min, the pigment zone outflow begins to collect the acetone eluent and obtains altogether eluent 320ml, and it is 54058.48 μ g/mL that living survey is tired;
(4) mensuration of the acquisition of antibacterial product and the rate of recovery: the dry that will collect the acetone eluent concentrate drying acquisition that obtains is the antibacterial product that streptomycete S24 produces.Measure respectively former fermented supernatant fluid, AB-8 resin adsorption after fermentation raffinate and acetone eluent are tired, and recording the AB-8 resin is 96.89% to the adsorption rate of antibacterial product, and strippant is 91.82% to the desorption efficiency of antibacterial product, and the antibacterial product rate of recovery is 86.94%.
Claims (1)
1. the extracting method of a streptomyces antibacterial product is characterized in that may further comprise the steps:
A, strain transfer that the inclined-plane is preserved be to dull and stereotyped PDA medium, 28 ℃ of constant temperature culture 48～72 hours; The PDA medium is: potato 200g, glucose 20g, agar 15g, (NH
4)
2SO
41g, MgSO
41g, KH
2PO
40.6g, CaCO
33g, water 1000ml;
B, the dull and stereotyped activated spawn of PDA is forwarded to seed culture medium, shaker fermentation 24 hours was forwarded to the fermentation medium shaker fermentation 84-96 hour by 2% inoculum concentration, collect zymotic fluid, 4200 rev/mins of low speed large capacity centrifuges centrifugal 30 minutes, are collected fermented supernatant fluid; Seed and fermentation culture conditions are: 28 ℃ of temperature, and 200 rev/mins of shaking speed, the shaking flask liquid amount is 50ml/250ml (or 100ml/500ml); Seed and fermentative medium formula are complex medium, are specially: glucose 16g, soluble starch 2.5g, peptone 10g, beancake powder 26g, NaCl 7.5g, (NH
4)
2SO
44g, water 1000ml;
C, with macroporous absorbent resin AB-8 and fermented supernatant fluid in proportion 1: 20M: V mixes, 150 rev/mins, vibration absorption 3-4 hour;
D, the resin after will adsorbing are collected the dress post, be to remove partial impurities behind the ethanol elution of 15%-25% with concentration, then be the acetone elution antimicrobial product of 75%-85% with concentration, flow velocity is 1.0ml/min, flow out from pigment zone and to begin to collect eluent, stop collection after collecting 1.2-1.5 times of column volume;
E, evaporating, concentrating and drying acetone eluent, resulting dry is antibacterial product.
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| CN106489342A (en) * | 2016-10-19 | 2017-03-15 | 山东旺盛园林股份有限公司 | A kind of local improving method of salt-soda soil planting soil |
| CN113957015B (en) * | 2021-11-23 | 2023-01-24 | 山东省花生研究所 | Streptomyces katsuradai YY-2S and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1872854A (en) * | 2005-05-31 | 2006-12-06 | 西北农林科技大学农药研究所 | Antibiotic in lactam class, and prepartion method |
| CN101024688A (en) * | 2007-02-13 | 2007-08-29 | 吴光耀 | Natural antibiotic antiseptic polylysine and its preparing method |
| CN101100686A (en) * | 2007-06-29 | 2008-01-09 | 华中科技大学 | Water-soluble streptomycete polysaccharide and application thereof |
| CN101153287A (en) * | 2007-09-18 | 2008-04-02 | 南京农业大学 | Streptomycete antimicrobial production and method of preparing the same |
| CN101182485A (en) * | 2007-09-29 | 2008-05-21 | 北京市农林科学院 | Streptomyces lydicus producing natamycin and uses thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1872854A (en) * | 2005-05-31 | 2006-12-06 | 西北农林科技大学农药研究所 | Antibiotic in lactam class, and prepartion method |
| CN101024688A (en) * | 2007-02-13 | 2007-08-29 | 吴光耀 | Natural antibiotic antiseptic polylysine and its preparing method |
| CN101100686A (en) * | 2007-06-29 | 2008-01-09 | 华中科技大学 | Water-soluble streptomycete polysaccharide and application thereof |
| CN101153287A (en) * | 2007-09-18 | 2008-04-02 | 南京农业大学 | Streptomycete antimicrobial production and method of preparing the same |
| CN101182485A (en) * | 2007-09-29 | 2008-05-21 | 北京市农林科学院 | Streptomyces lydicus producing natamycin and uses thereof |
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