CN103146794B - Method for improving sisal hemp saponin productivity with microbial fermentation - Google Patents
Method for improving sisal hemp saponin productivity with microbial fermentation Download PDFInfo
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- CN103146794B CN103146794B CN201210561720.6A CN201210561720A CN103146794B CN 103146794 B CN103146794 B CN 103146794B CN 201210561720 A CN201210561720 A CN 201210561720A CN 103146794 B CN103146794 B CN 103146794B
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Abstract
The invention discloses a method for improving sisal hemp saponin productivity with microbial fermentation. Objective bacterial strain used is preserved in China Center for Type Culture Collection. A preserving date of the bacterial strain is February 25th, 2011; a preserving number of the bacterial strain is CCTCC NO: M2011050; and a classification name is bacillus GLHZB21, namely, Corynebacterium sp. GLHZB21. After being fermented and cultivated, the objective bacterial strain is added into sisal hemp waste for fermentation and centrifugation so that supernate is obtained and then is dried in a vacuum drying oven so that sisal hemp saponin is obtained. According to the microbial fermentation, the saponin productivity is obviously improved. Compared with an acid-base chemical extraction method which is popular currently, the productivity is increased by about 30%. The microorganism has the advantages of being safe, environment-friendly, low in cost, rapid in degradation velocity, high in degradation rate and the like. The method is capable of being used in traditional saponin industrial production to replace the current acid-base chemical extraction technique method.
Description
Technical field:
The invention belongs to the field such as extraction and purifying of life science, biotechnology, biological chemistry, natural product chemistry, Effective Component of Chinese Medicine saponin(e, be particularly related to a kind of method of utilizing microorganism fermentation to improve sisal hemp saponin(e productive rate, specifically from soil, filter out a kind of bacterial strain and can improve sisal hemp saponin(e productive rate for extracting sisal hemp saponin(e.
Background technology:
Sisal hemp contains saponin(e, sisal hemp has another name called Yucca gloriosa L., is a kind of common agave, perennial leaf fibres crop, sisal hemp saponin(e derives from numb juice and the dregs of linseeds or sesame seeds after sisal fibers traditional mode of production product, discarded, is medicine intermediate and the important source material of synthesizing steroid hormone medicine; From the juice of fresh leaves squeezing, can obtain protein, pectin polysaccharide, saponin(e etc.; 5%-9% crude protein; The pectin polysaccharides such as arabogalactan, xylan, xyloglucan; The saponin(es such as A, B, C, D, E, F, G, wherein sisal hemp saponin(e B is made up of hecogenin and glucose, semi-lactosi, wood sugar, and sisal hemp saponin D is made up of hecogenin and glucose, semi-lactosi, wood sugar, rhamnosyl; Sapogenin has Chlorogenin, Rockogenin, Ba Erbo sapogenin.
Along with improving constantly of standard of living, the use of natural drug is more and more subject to people's favor.Saponin(e has multiple biological activity and is widely used in food, healthcare products, medicine with it; As antitumor, anti-inflammatory, antibacterial, hemostasis, anti-ageing, hypoglycemic, protect the liver, and feed fortifier etc., has very large researching value and DEVELOPMENT PROSPECT in a lot of fields.
The method that at present traditional saponin separation extracts mainly contains: the solvent method of acid and alkali hydrolysis and the precipitator method, its limitation is that the extraction amount of separating effective component and the degree of purifying are all relatively low, alkali extraction and acid precipitation, because acid and alkali hydrolysis law part is more violent, the normal side reaction such as dehydration, configuration conversion with saponin(e, and to note selecting suitable reagent to avoid the destruction to aglycon; Ultrasonic extraction method can be avoided the destruction of High Temperature High Pressure to effective constituent, but its thickness to wall of container and the putting position of container have very high requirement, otherwise will have influence on leaching effect; Supercritical extraction, material is had to good perviousness and stronger dissolving power, to material component, some extraction has higher efficiency, but because saponins compound hydroxyl is many, polarity is large, need intensified pressure and add suitable catalyzer to improve extraction yield, so want the saponin(e of scale operation high density, still cost is higher.Above-mentioned showing, urgently develop the extraction and separation method of new-type environmental protection, economy, Sustainable development.
The method of biotechnology fermentation is maked rapid progress, more and more extensive in the application of biological field; By adding object bacterial strain, accelerate the effective constituent of abolishing and discharge as much as possible Mierocrystalline cellulose the inside of plant cell wall, thereby reduce the extraction difficulty of sisal hemp saponin(e, by adopting the most adaptive ratio of the factors such as feed liquid bacterium, temperature, fermentation time, can reach economize in raw materials, the object of reagent, power, labour and minimizing environmental pollution, fermentation costs is dropped to minimum, for scale operation provides more economical method.
Summary of the invention:
The object of the invention is, for reducing sisal hemp saponin extraction difficulty, provides a kind of method of utilizing microorganism fermentation to improve sisal hemp saponin(e productive rate.
Concrete steps are:
(1) prepare substratum:
A. take peeled potatoes 100-250 gram, and by potato dice, add 0.5-2.0 premium on currency post-heating and boil 20-40 minute, be cooled to afterwards room temperature, by four layers of filtered through gauze, gained filtrate is settled to 0.5-2.0 liter, for subsequent use.
B. get the prepared filtrate 0.05-1.5 liter for subsequent use of step (1) a step and be placed in triangular flask, again to the agar powder that adds 1.0-2.0 gram in triangular flask, with kraft paper wrapping triangle bottleneck, sterilizing 20 minutes under 1.103MPa, 121 DEG C of conditions, then product in triangular flask is promptly poured into diameter and be in the culture dish of 9 centimetres, add a cover, after agar solidifies, make solid medium, for subsequent use.
C. remaining filtrate 0.5-1.95 liter in the prepared filtrate for subsequent use of step (1) a step being moved to 1.0-2.5 rises in container, wrap up bottleneck with kraft paper, under 1.103MPa, the condition of 121 DEG C, sterilizing 20 minutes, makes liquid fermentation medium, for subsequent use.
(2) plane of object bacterial strain is cultivated and fermentation culture:
Under aseptic condition, the object bacterial strain that inclined-plane is preserved is coated on step (1) b and walks on the solid medium making, at 20-35 DEG C, cultivate 36 hours, then take out bacterial strain, being inoculated into 50-200 milliliter step (1) c walks in the liquid fermentation medium making, at 28-36 DEG C, with the speed shaking table oscillation and fermentation cultivation of 100-300 rev/min 72 hours, make fermented liquid; Described object strain microorganism is now preserved in Chinese Typical Representative culture collection center, preservation date: on February 25th, 2011, deposit number: CCTCC NO:M2011050, Classification And Nomenclature: excellent bacillus GLHZB21(Corynebacterium sp.GLHZB21).
(3) extraction of sisal hemp saponin(e in sisal hemp waste material:
Get 30 milliliters of fermented liquids that step (2) makes and be placed in centrifuge tube, centrifugal 20 minutes of the rotating speed of 4000-6000 rev/min, abandon supernatant liquor, remainder joins in 100 ml sterile waters and dilutes, in bacterium liquid after dilution, add 5-20 gram of sisal hemp waste material, after fermenting 24 hours with the hunting speed of 200 revs/min on shaking table, get 5 milliliters of fermented liquids, with the rotating speed of 4000-6000 rev/min centrifugal 15 minutes, get supernatant liquor, the oven dry of anhydrating in vacuum drying oven, extracts sisal hemp saponin(e.
Test by extinction, add the solid-liquid ratio of thalline not add the feed liquid of thalline probably can increase by 30% left and right at the light absorption value of 450nm, illustrate that sisal hemp saponin(e productive rate improves.
The present invention utilizes microorganism fermentation can obviously improve saponin(e productive rate, compared with the existing methods, this microorganism has safety and environmental protection, cost is low, degradation speed is fast, degradation rate high, and therefore this content can be for the chemical extraction processing method in traditional saponin(e industrial production.
Brief description of the drawings
Fig. 1 is the form photo that is respectively the microorganism of the present invention observing under 10 times and 100 times at eyepiece and object lens.
Fig. 2 is microbe colony photo of the present invention.
Embodiment
Embodiment:
(1) prepare substratum:
A. take 200 grams of peeled potatoes, and by potato dice, add 1 premium on currency post-heating and boil 30 minutes, be cooled to afterwards room temperature, by four layers of filtered through gauze, gained filtrate is settled to 1 liter, for subsequent use.
B. get 0.1 liter of the prepared filtrate for subsequent use of step (1) a step and be placed in the triangular flask of 250 milliliters, again to the agar powder that adds 1.5 grams in triangular flask, with kraft paper wrapping triangle bottleneck, sterilizing 20 minutes under 1.103MPa, 121 DEG C of conditions, then product in triangular flask is promptly poured into diameter and be in the culture dish of 9 centimetres, add a cover, after agar solidifies, make solid medium, for subsequent use.
C. 0.9 liter of remaining filtrate in the prepared filtrate for subsequent use of step (1) a step is moved in 1.5 liters of containers, wrap up bottleneck with kraft paper, under 1.103MPa, the condition of 121 DEG C, sterilizing 20 minutes, makes liquid fermentation medium, for subsequent use.
(2) plane of object bacterial strain is cultivated and fermentation culture:
Under aseptic condition, the object bacterial strain that inclined-plane is preserved is coated on step (1) b and walks on the solid medium making, at 30 DEG C, cultivate 36 hours, then take out bacterial strain, being inoculated into 200 milliliters of step (1) c walks in the liquid fermentation medium making, at 30 DEG C, with the speed shaking table oscillation and fermentation cultivation of 200 revs/min 72 hours, make fermented liquid; Described object strain microorganism is now preserved in Chinese Typical Representative culture collection center, preservation date: on February 25th, 2011, deposit number: CCTCC NO:M2011050, Classification And Nomenclature: excellent bacillus GLHZB21 (Corynebacterium sp.GLHZB21).
(3) extraction of sisal hemp saponin(e in sisal hemp waste material:
Get 30 milliliters of fermented liquids that step (2) makes and be placed in centrifuge tube, centrifugal 20 minutes of the rotating speed of 4500 revs/min, abandon supernatant liquor, remainder joins in 100 ml sterile waters and dilutes, and adds 10 grams of sisal hemp waste materials in the bacterium liquid after dilution, after fermenting 24 hours with the hunting speed of 200 revs/min on shaking table, get 5 milliliters of fermented liquids, with the rotating speeds of 4500 revs/min centrifugal 15 minutes, get supernatant liquor, the oven dry of anhydrating in vacuum drying oven, extracts sisal hemp saponin(e.
This microbial fermentation processes is processed sisal hemp residue compared with classical acid alkali chemical process, and productive rate improves 30.1%.
Claims (1)
1. utilize microorganism fermentation to improve a method for sisal hemp saponin(e productive rate, it is characterized in that concrete steps are:
(1) prepare substratum:
A. take peeled potatoes 100-250 gram, and by potato dice, add 0.5-2.0 premium on currency post-heating and boil 20-40 minute, be cooled to afterwards room temperature, by four layers of filtered through gauze, gained filtrate is settled to 0.5-2.0 liter, for subsequent use;
B. get the prepared filtrate 0.05-1.5 liter for subsequent use of step (1) a step and be placed in triangular flask, again to the agar powder that adds 1.0-2.0 gram in triangular flask, with kraft paper wrapping triangle bottleneck, sterilizing 20 minutes under 1.103MPa, 121 DEG C of conditions, then product in triangular flask is promptly poured into diameter and be in the culture dish of 9 centimetres, add a cover, after agar solidifies, make solid medium, for subsequent use;
C. remaining filtrate 0.5-1.95 liter in the prepared filtrate for subsequent use of step (1) a step being moved to 1.0-2.5 rises in container, wrap up bottleneck with kraft paper, under 1.103MPa, the condition of 121 DEG C, sterilizing 20 minutes, makes liquid fermentation medium, for subsequent use;
(2) plane of object bacterial strain is cultivated and fermentation culture:
Under aseptic condition, the object bacterial strain that inclined-plane is preserved is coated on step (1) b and walks on the solid medium making, at 20-35 DEG C, cultivate 36 hours, then take out bacterial strain, being inoculated into 50-200 milliliter step (1) c walks in the liquid fermentation medium making, at 28-36 DEG C, with the speed shaking table oscillation and fermentation cultivation of 100-300 rev/min 72 hours, make fermented liquid; Described object strain microorganism is now preserved in Chinese Typical Representative culture collection center, preservation date: on February 25th, 2011, deposit number: CCTCC NO:M2011050, Classification And Nomenclature: excellent bacillus GLHZB21 is Corynebacterium sp.GLHZB21;
(3) extraction of sisal hemp saponin(e in sisal hemp waste material:
Get 30 milliliters of fermented liquids that step (2) makes and be placed in centrifuge tube, centrifugal 20 minutes of the rotating speed of 4000-6000 rev/min, abandon supernatant liquor, remainder joins in 100 ml sterile waters and dilutes, in bacterium liquid after dilution, add 5-20 gram of sisal hemp waste material, after fermenting 24 hours with the hunting speed of 200 revs/min on shaking table, get 5 milliliters of fermented liquids, with the rotating speed of 4000-6000 rev/min centrifugal 15 minutes, get supernatant liquor, the oven dry of anhydrating in vacuum drying oven, extracts sisal hemp saponin(e.
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| CN103966300B (en) * | 2014-05-25 | 2016-09-28 | 桂林理工大学 | A kind of method utilizing fermentable that Folium Agaves Sisalanae saponin is changed into sapogenin |
| CN106367371B (en) * | 2016-09-18 | 2019-04-02 | 中南民族大学 | The enterobacter cloacae clx-14 bacterial strain and application thereof of one plant of production chonglou saponin I |
| CN106421203A (en) * | 2016-12-07 | 2017-02-22 | 中国农业科学院麻类研究所 | Sisal hemp-plant leaf cream production method |
| CN109006294B (en) * | 2018-08-14 | 2022-02-25 | 云南唯七诺健康科技有限公司 | Panax notoginseng cultivation method without pesticide residue |
| CN109864177A (en) * | 2019-03-26 | 2019-06-11 | 河南科技大学 | A kind of fermentation processing method increasing saponin extraction amount in clover |
| CN114853821B (en) * | 2022-06-08 | 2024-07-05 | 中华全国供销合作总社济南果品研究所 | Method for separating saponin from canned asparagus processing wastewater |
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| CN1570133A (en) * | 2004-04-27 | 2005-01-26 | 复旦大学 | Ginsenoside Compound-K preparing method |
| CN101139562A (en) * | 2007-07-02 | 2008-03-12 | 昆明诺唯金参生物工程有限责任公司 | Process for preparing rare ginsenoside Compound K by fermenting panax notoginseng saponins with streptomycete |
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| CN1570133A (en) * | 2004-04-27 | 2005-01-26 | 复旦大学 | Ginsenoside Compound-K preparing method |
| CN101139562A (en) * | 2007-07-02 | 2008-03-12 | 昆明诺唯金参生物工程有限责任公司 | Process for preparing rare ginsenoside Compound K by fermenting panax notoginseng saponins with streptomycete |
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Owner name: GUANGXI SANTO PHARMACEUTICAL CO., LTD. Free format text: FORMER OWNER: GUILIN UNIVERSITY OF TECHNOLOGY Effective date: 20150703 |
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Effective date of registration: 20150703 Address after: 545200 No. 72 Yang Yang middle road, Dapu Town, Liucheng County, the Guangxi Zhuang Autonomous Region, Liuzhou Patentee after: Guangxi Shengte Pharmaceutical Co.,Ltd. Address before: 541004 the Guangxi Zhuang Autonomous Region Guilin Construction Road No. 12 Patentee before: Guilin University of Technology |
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