CN101665769A - Bacterial strain with high protopectinase yield and method for extracting pectin by fermentation - Google Patents
Bacterial strain with high protopectinase yield and method for extracting pectin by fermentation Download PDFInfo
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- CN101665769A CN101665769A CN200910043038A CN200910043038A CN101665769A CN 101665769 A CN101665769 A CN 101665769A CN 200910043038 A CN200910043038 A CN 200910043038A CN 200910043038 A CN200910043038 A CN 200910043038A CN 101665769 A CN101665769 A CN 101665769A
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Abstract
The invention discloses Aspergillus niger strain with high protopectinase yield and a method for extracting pectin by fermenting the same. The Aspergillus niger CD-01 is screened out from soil filledwith rotten oranges in Changde of Hunan province, and the preservation number is CCTCC NO: M209036. The method for extracting pectin by fermentation comprises the steps of slant culture, fermentation,pectin extraction and the like; the obtained products have large high molecular weight, high jelly grade and stable quality; and all the indexes accord with the pectin standard. The method is superior to other pectin extraction methods in production cost, and is higher in pectin yield in comparison with previous extraction methods by microbial fermentation.
Description
Technical field
The invention belongs to fermentation technical field, be specifically related to a plant height and produce the bacterial strain of protopectinase and adopt this strain fermentation to extract the method for pectin.
Background technology
Pectin is distributed widely in fruit, rhizome and the leaf of plant with forms such as protopectin-, soluble pectin, pectic acid.As a kind of important foodstuff additive, pectin lacks on market very much, and international market price is about 17,000 dollars/ton at present.Tangerine peel is the by product of orange deep processing etc., is one of main raw material of industrial production pectin, wherein contains the pectin of have an appointment 30% (accounting for the tangerine peel dry weight).At present, the method from extraction of pectin mainly contains several classes such as acid hydrolyzation, microwave method, ion-exchange-resin process, microbial method.
Acid hydrolyzation is that the most traditional method of pectin is extracted in industry.Its principle is to adopt diluted acid that the water-insoluble protopectin-in the pericarp membrane is hydrolyzed to water soluble pectin.This method has fast, simple, advantage such as cost is lower, and the pectin productive rate can reach more than 26%, but local hydrolysis can take place pectin molecule in the leaching process, has reduced the pectin relative molecular mass, thereby influences pectin yield and quality; The pectin extracting soln viscosity causes filtration difficulty greatly; The waste residue that produces in the production process is difficult to handle; Simultaneously because the extensive application of acid is also bigger to the corrosion of equipment.
Microwave method is to utilize high-power hertzian wave to penetrate to be extracted inner vascular bundle of medium and cell system, owing to absorb micro-wave energy, the cell interior temperature rises rapidly, the internal pressure that cell is subjected to surpasses the cell walls ability to bear and causes cell rupture, thereby effective constituent freely flows out in the cell, by spe medium, dissolving under lower temperature condition, further filter also, separate, can obtain extract.The microwave method extraction method not only has advantages such as simple, quick, and output is very high, almost can extract the pectin in the pericarp fully.But energy consumption is bigger, also because reaction conditions is violent, causes the difficult separation of later stage solid-liquid.
The technical process of ion-exchange-resin process is: pulverize the peel of Citrus reticulata Blanco that will handle dehydration back, and the underflow liquid made from ion exchange resin and water is heating and filtering under agitation, isolates insoluble ion-exchanger and waste residue, obtains containing the filtrate of pectin.With the pectin in this method extraction tangerine peel, productive rate can reach 22%～23%, and the quality of pectin is also better, but has adopted the acid of higher concentration equally, and is also bigger to the corrosion of equipment.
The microorganism ratio juris is to utilize that the excretory protopectinase is decomposed into soluble pectin with protopectin-in the plant tissue in the microorganism growth process, so can be with filtering fermentation liquor, concentrate and the precipitation of the pectin in the fermented liquid extracted with precipitation agent.Microbial method is at first by propositions such as Japanese sakai, and it produces protopectinase bacterial strain Trichosporon penicillatum with a strain and is applied in the extraction of tangerine peel pectin, and the pectin productive rate can reach 13.3% (with the ratio of tangerine peel dry weight).After this, people such as JC Contreras Esquivel utilize from Aspergillus kawachii IFO 4308 substratum protopectinase solution and extract tangerine peel pectin, and productive rate reaches 17.7% (with the ratio of tangerine peel dry weight).Adopt the pectin molecule amount of microbe fermentation method extraction big, pectin grade is high, steady quality, extracting solution mesocarp not broken (being easy to solid-liquid separation), need not to heat-treat or acid treatment, have series of advantages such as low consumption, low pollution, constant product quality.But with regard to present technology, also there is the not high deficiency of productive rate in this method.
Summary of the invention
The object of the present invention is to provide a plant height to produce the Aspergillus niger strain Aspergillusniger CD-01 of protopectinase.
The Aspergillus niger strain that the present invention relates to has been submitted preservation on March 4th, 2009.Depositary institution's title: Chinese typical culture collection center; Preservation address: China, Wuhan, Wuhan University; Deposit number is CCTCC NO:M 209036, classification called after: aspergillus niger CD-01, Aspergillusniger CD-01.
Another object of the present invention is to the deficiency that exists at existing pectin extractive technique, the method for provide that a kind of production cost is low, good product quality, output height and the high microbial fermentation of byproduct economic worth extracting pectin.
The method that microbial fermentation of the present invention extracts pectin may further comprise the steps:
1) slant culture: aspergillus niger spore is seeded on the slant medium, cultivated 3-5 days down for 34-36 ℃, wash aspergillus niger spore, make spore suspension and be diluted to 1.0-2.5 * 10 with sterilized water
6Individual/mL, refrigerate in 4 ℃ standby;
2) fermentation: getting this season, not have the dried tangerine peel that goes mouldy be 10-20 by water material mass ratio: 1 puts in the distilled water, leave standstill 25-35min, the pH value is transferred to 4.5-5.5, autoclaving 15-25min, the urea that mass ratio adds 0.1-0.2% is pressed in the cooling back, presses the aspergillus niger spore suspension of mass ratio inoculation 5-15%, cultivates 24-48h in the constant temperature shaking table, temperature is 34-36 ℃, and rotating speed is 150-210r/min;
3) pectin extracts: nutrient solution is filtered or centrifugal, the fermented soln that separation is obtained adopts 95% ethanol of 2.4 times of volumes to precipitate extraction, and with 70% washing with alcohol twice, lyophilize promptly gets product after the precipitate and separate.
Described slant culture top condition is: cultivated 4 days down for 34-36 ℃, inoculation with the spore suspension preferred concentration is: 1.0-2.5 * 10
6Individual/mL.
Optimal ph is 4.5-5.5 in the described fermentation step, and the preferred addition of urea is 0.1-0.2%, and aspergillus niger spore is seeded under the aseptic condition and carries out, and optimum inoculation amount is 5-15%.
Optimal culture condition is in the described fermentation step: cultivate 24-28h in the constant temperature shaking table, temperature is 34-36 ℃, and rotating speed is 150-210r/min.
The a large amount of high reactivity polygalacturonases that exist in the described filtrate are put the pectin molecule that can decompose wherein for a long time, influence the pectin quality, therefore after centrifugal or filtration obtain pectin solution, should add ethanol immediately and precipitate.
Compared with prior art, the present invention has following advantage:
1) cost is lower: whole fermented extracted process condition gentleness is not high and consume energy low to equipment requirements; Therefore the tangerine peel raw material does not need fragmentation in addition, and the fermented soln solid-liquid separation is easier, and to compare cost much lower with additive method;
2) productive rate is higher: pectin of the present invention extracts productive rate up to 24.5% (with the ratio of tangerine peel dry weight), extracts the pectin method with existing microorganism and compares, and productive rate improves greatly, and is suitable with traditional pectin extraction method productive rate;
3) good product quality: the pectin product molecular weight is big, the degree of gelation height, and steady quality, every index all meets pectin standard QB 2484-2000;
4) byproduct economic worth height: also have a large amount of protopectinases and polygalacturonase in the fermented liquid except pectin, very high economic worth is all arranged, this is that other pectin extraction methods are not available.
Embodiment
Following examples are intended to illustrate the present invention rather than limitation of the invention further, and the present invention can implement by the described arbitrary mode of summary of the invention.
Embodiment 1:
Aspergillus niger spore is inoculated on the slant medium in 35 ℃ of growths after 4 days, washes aspergillus niger spore, make spore suspension and be diluted to 1.75 * 10 with sterilized water
6Individual/mL, refrigerate in 4 ℃ standby.
Getting this season does not have the dried tangerine peel 6.67g that goes mouldy and puts in the 250mL triangular flask that 100mL distilled water is housed, and leaves standstill 30min, the pH value is accurately transferred to 5.0, autoclaving 25min; The cooling back adds urea 0.14g under aseptic condition, add aspergillus niger spore suspension 10mL again, and spore concentration is 1.75 * 10
6Individual/mL; The postvaccinal bottle that shakes is cultivated 36h in the constant temperature shaking table, it is 35 ℃ that temperature is set, and rotating speed is 180r/min; Nutrient solution is filtered or centrifugal (get 1mL and be used for determination of yield), get filtrate and pectin is precipitated, filters, with pectin washing of precipitate twice, will last the pectin that obtains be precipitated lyophilize with 20mL, 70% ethanol with 95% ethanol of 2.4 times of volumes.Adopt sulfuric acid carbazole method to measure pectin content, recording the pectin productive rate is 24.5% (with the ratio of tangerine peel dry weight).
Embodiment 2:
Aspergillus niger spore is inoculated on the slant medium in 35 ℃ of growths after 4 days, washes aspergillus niger spore, make spore suspension and be diluted to 1.75 * 10 with sterilized water
6Individual/mL, refrigerate in 4 ℃ standby.
Getting this season does not have the dried tangerine peel 6.67g that goes mouldy and puts in the 250mL triangular flask that 100mL distilled water is housed, and leaves standstill 35min, the pH value is accurately transferred to 5.5, autoclaving 20min; The cooling back adds urea 0.15g under aseptic condition, add aspergillus niger spore suspension 10mL again, and spore concentration is 1.75 * 10
6Individual/mL; The postvaccinal bottle that shakes is cultivated 32h in the constant temperature shaking table, it is 36 ℃ that temperature is set, and rotating speed is 150r/min; Nutrient solution is filtered or centrifugal (get 1mL and be used for determination of yield), get filtrate and pectin is precipitated, filters, with pectin washing of precipitate twice, will last the pectin that obtains be precipitated lyophilize with 20mL, 70% ethanol with 95% ethanol of 2.4 times of volumes.Adopt sulfuric acid carbazole method to measure pectin content, recording the pectin productive rate is 22.8% (with the ratio of tangerine peel dry weight).
Embodiment 3:
Aspergillus niger spore is inoculated on the slant medium in 35 ℃ of growths after 4 days, washes aspergillus niger spore, make spore suspension and be diluted to 1.75 * 10 with sterilized water
6Individual/mL, refrigerate in 4 ℃ standby.
Getting this season does not have the dried tangerine peel 6.67g that goes mouldy and puts in the 250mL triangular flask that 100mL distilled water is housed, and leaves standstill 25min, the pH value is accurately transferred to 4.5, autoclaving 15min; The cooling back adds urea 0.14g under aseptic condition, add aspergillus niger spore suspension 10mL again, and spore concentration is 1.7 * 10
6Individual/mL; The postvaccinal bottle that shakes is cultivated 40h in the constant temperature shaking table, it is 34 ℃ that temperature is set, and rotating speed is 200r/min; Nutrient solution is filtered or centrifugal (get 1mL and be used for determination of yield), get filtrate and pectin is precipitated, filters, with pectin washing of precipitate twice, will last the pectin that obtains be precipitated lyophilize with 20mL, 70% ethanol with 95% ethanol of 2.4 times of volumes.Adopt sulfuric acid carbazole method to measure pectin content, recording the pectin productive rate is 23.1% (with the ratio of tangerine peel dry weight).
Claims (10)
1, a plant height produces the Aspergillus niger strain Aspergillus niger CD-01 of protopectinase, and its deposit number is CCTCC NO:M 209036.
2, a kind of method that adopts the described Aspergillus niger strain fermented extracted of claim 1 pectin is characterized in that may further comprise the steps:
1) slant culture: Aspergillus niger strain Aspergillus niger CD-01 spore inoculating to slant medium, was cultivated 3-5 days down for 34-36 ℃, washed aspergillus niger spore, make spore suspension and be diluted to 1.0-2.5 * 10 with sterilized water
6Individual/mL, refrigerate in 4 ℃ standby;
2) fermentation: getting dried tangerine peel is 10-20 by water material mass ratio: 1 puts in the distilled water, leave standstill 25-35min, the pH value is transferred to 4.5-5.5, autoclaving 15-25min, the urea that mass ratio adds 0.1-0.2% is pressed in the cooling back, presses the aspergillus niger spore suspension of mass ratio inoculation 5-15%, cultivates 24-48h in the constant temperature shaking table, temperature is 34-36 ℃, and rotating speed is 150-210r/min;
3) pectin extracts: nutrient solution is filtered or centrifugal, the fermented soln that separation is obtained adopts 95% ethanol of 2.4 times of volumes to precipitate extraction, and with 70% washing with alcohol twice, lyophilize promptly gets product after the precipitate and separate.
3, a kind of microbial fermentation according to claim 2 extracts the method for pectin, and it is characterized in that: the slant culture condition is: cultivated 3-5 days for 34-36 ℃.
4, a kind of microbial fermentation according to claim 2 extracts the method for pectin, it is characterized in that: inoculation is 1.0-2.5 * 10 with spore suspension concentration
6Individual/mL.
5, a kind of microbial fermentation according to claim 2 extracts the method for pectin, it is characterized in that: dried tangerine peel need not be broken, directly adds.
6, a kind of microbial fermentation according to claim 2 extracts the method for pectin, it is characterized in that: in the fermentation step pH value is transferred to 4.5-5.5.
7, a kind of microbial fermentation according to claim 2 extracts the method for pectin, and it is characterized in that: the addition of urea is 0.1-0.2%.
8, a kind of microbial fermentation according to claim 2 extracts the method for pectin, and it is characterized in that: aspergillus niger spore is seeded under the aseptic condition and carries out in the fermentation step, and inoculum size is 5-15%.
9, a kind of microbial fermentation according to claim 2 extracts the method for pectin, and it is characterized in that: culture condition is in the fermentation step: cultivate 24-48h in the constant temperature shaking table, temperature is 34-36 ℃, and rotating speed is 150-210r/min.
10, a kind of microbial fermentation according to claim 2 extracts the method for pectin, it is characterized in that: centrifugal or filter the pectin solution that obtains after add ethanol immediately.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660526A (en) * | 2012-05-30 | 2012-09-12 | 山东大学 | Method for producing pectate lyase by two-section pH (potential of hydrogen) control |
| CN103468582A (en) * | 2013-08-13 | 2013-12-25 | 湖南省农产品加工研究所 | Pectinase preparation producing Aspergillus japonicus PJ01 and enzyme production method |
| CN103833467A (en) * | 2014-03-04 | 2014-06-04 | 重庆圣沛农业科技有限公司 | Organic compound fertilizer with citrus pomace and preparation method of organic compound fertilizer |
| CN104388361A (en) * | 2014-12-03 | 2015-03-04 | 厦门大学 | Marine bacterium for producing pectinase and application of marine bacterium |
| CN106265846A (en) * | 2016-09-18 | 2017-01-04 | 天津北洋百川生物技术有限公司 | A kind of fermentation process improving Radix Rhodiolae oxidation resistance |
| CN108148825A (en) * | 2016-12-05 | 2018-06-12 | 漳州市皓康生物科技有限公司 | A kind of protopectinase preparation method for being used to extract pectin |
| CN110699402A (en) * | 2019-11-25 | 2020-01-17 | 巢湖学院 | Method for preparing low-methoxyl pectin by using pomace |
-
2009
- 2009-04-03 CN CN200910043038A patent/CN101665769A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660526A (en) * | 2012-05-30 | 2012-09-12 | 山东大学 | Method for producing pectate lyase by two-section pH (potential of hydrogen) control |
| CN102660526B (en) * | 2012-05-30 | 2013-05-29 | 山东大学 | Method for producing pectate lyase by two-section pH (potential of hydrogen) control |
| CN103468582A (en) * | 2013-08-13 | 2013-12-25 | 湖南省农产品加工研究所 | Pectinase preparation producing Aspergillus japonicus PJ01 and enzyme production method |
| CN103468582B (en) * | 2013-08-13 | 2015-04-08 | 湖南省农产品加工研究所 | Pectinase preparation producing Aspergillus japonicus PJ01 and enzyme production method |
| CN103833467A (en) * | 2014-03-04 | 2014-06-04 | 重庆圣沛农业科技有限公司 | Organic compound fertilizer with citrus pomace and preparation method of organic compound fertilizer |
| CN103833467B (en) * | 2014-03-04 | 2015-10-14 | 重庆圣沛农业科技有限公司 | A kind of citrus peel residue compoiste fertilizer and preparation method thereof |
| CN104388361A (en) * | 2014-12-03 | 2015-03-04 | 厦门大学 | Marine bacterium for producing pectinase and application of marine bacterium |
| CN104388361B (en) * | 2014-12-03 | 2017-03-08 | 厦门大学 | The marine bacteria of one plant of product pectase and its application |
| CN106265846A (en) * | 2016-09-18 | 2017-01-04 | 天津北洋百川生物技术有限公司 | A kind of fermentation process improving Radix Rhodiolae oxidation resistance |
| CN108148825A (en) * | 2016-12-05 | 2018-06-12 | 漳州市皓康生物科技有限公司 | A kind of protopectinase preparation method for being used to extract pectin |
| CN110699402A (en) * | 2019-11-25 | 2020-01-17 | 巢湖学院 | Method for preparing low-methoxyl pectin by using pomace |
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Open date: 20100310 |