CN105238707A - Microbial strain for degrading naringin and method - Google Patents
Microbial strain for degrading naringin and method Download PDFInfo
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- CN105238707A CN105238707A CN201510520364.7A CN201510520364A CN105238707A CN 105238707 A CN105238707 A CN 105238707A CN 201510520364 A CN201510520364 A CN 201510520364A CN 105238707 A CN105238707 A CN 105238707A
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Abstract
The invention relates to a microbial strain for degrading naringin and a method, and provides Wb51b (phyllobacterium sp.) capable of degrading the naringin. Under certain conditions, 2g/L of naringin serves as an unique carbon source and is used for culturing a microbial strain for 72 hours, concentration of thallus reaches 0.45 g/L, the content of the naringin is reduced to be 0.0037 g/L, and the naringin is nearly degraded completely. 0.6 g/L of naringin is directly degraded by free cells, reaction time is 30 minutes, the content of the naringin is reduced to be 0.1 g/L, and degradation rate reaches 83.3%; and 1.2 g/L of naringin is degraded by immobilized cells, reaction time is 60 minutes, the content of the naringin is reduced to be 0.076 g/L, and the degradation rate reaches 93.7%. The microbial strain can be used for degrading the naringin effectively, nutrition requirements are simple, and the microbial strain is easy to culture. In addition, the microbial strain does not have pathogenicity, and can be directly used in the food industry.
Description
Technical field
The present invention relates to a kind of leaf bacillus, especially relate to the method for its degraded naringin.
Background technology
Naringin (Naringenine-7-rhamnosidoglucoside), has another name called naringin, naringin, aurantiin, is topmost bitter substance in honey pomelo pericarp.Reduce the content of naringin, the industrialization technology setting up grapefruit juice debitterizing brings great progress by drink industry.In recent years, the method for microbiological deterioration naringin more and more causes the attention of people, and the kind relating to microorganism has fungi, bacterium and yeast etc., and wherein fungi produces naringinase naringin of degrading and obtained certain achievement; And the research of degradation by bacteria naringin is also less.
Along with sweet shaddock cultivated area constantly expands, honey shaddock output rises year by year, the sweet shaddock output value in annual Dan Shi Fujian Province just reaches 6,000 ten thousand tons, but because deep process technology research is delayed, not yet realize the deep processing of industrialization, production-supply-marketing contradiction, causes a large amount of fruits to be dropped, and the foul odour that abandons fruit rot is severe contamination environment again.It is the Main Means of sweet shaddock deep processing that honey shaddock is squeezed the juice, but the fruit drink obtained has obvious bitter taste, needs to remove naringin, just can drink after carrying out debitterize process.Therefore, naringin of how effectively degrading becomes a urgent problem.
The main method of current sweet shaddock juice debitterize has physisorphtion, the methods such as foodstuff additive method and biological enzyme formulation.Physisorphtion is while absorption bitter substance, and also by the nutritive ingredient in absorption fruit juice, bitter substance is just sheltered by foodstuff additive method, is not removed; And biological enzyme formulation method, Chinese patent 201010219099.6 (publication number CN101897452A, December 1 2010 time of disclosure) disclose a kind of fermentation method for producing of grapefruit juice debitterizing enzyme, the method needs to carry out separation and purification to enzyme, and the cost of this process is higher.
Summary of the invention
The object of the present invention is to provide a kind of bacterial classification leaf bacillus (Phyllobacteriumsp.) Wb51b of naringin of degrading.
Described bacterial classification is leaf bacillus (Phyllobacteriumsp.) Wb51b, screen from pedotheque and obtain, be preserved in China General Microbiological bacterial strain preservation management committee's common micro-organisms center in 2012 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica), register on the books and be numbered CGMCCNo.6472 in this preservation center.
Another object of the present invention is a kind of method providing naringin of degrading.
The method utilizes leaf bacillus (Phyllobacteriumsp.) to degrade naringin.Concrete steps comprise: inoculate leaf bacillus (Phyllobacteriumsp.) into the substratum containing naringin, cultivate, and use free cell, cell after being fixed or its mixed style cultivated, naringin of degrading.
The leaf bacillus degraded naringin that the present invention adopts above-mentioned collection number to be CGMCCNo.6472.
Described initial pH value of carrying out cultivating is 3.5 ~ 8.5, preferably 3.5 ~ 6.5, more preferably 3.0 ~ 5.0.
Described incubation time 12 hours ~ 7 days, preferred 24h ~ 72h, more preferably 48h ~ 72h.
Described culture temperature preferably 25 ~ 50 DEG C, preferably 25 ~ 35 DEG C, more preferably 30 ~ 35 DEG C.
Sodium alginate can be utilized being fixed of cell.
Immobilized Cells for Biodegradation naringin can be reused.
Outstanding advantages of the present invention is:
1. the present invention's bacterial classification used is bacterium, can efficient degradation naringin, simply, easily cultivates nutritional requirement.
2. the present invention's bacterial classification no pathogenicity used, the hidden danger of lifeless matter security aspect, can directly apply to foodstuffs industry, using free cell degraded naringin, without the need to carrying out separation and purification to involved enzyme, can reduce costs.
3. the present invention can use Immobilization in Sodium Alginate cell to naringin of degrading, and can repeatedly reuse.
Embodiment
The invention provides a kind of leaf bacillus of degradable naringin.
The present invention discloses a kind of method utilizing leaf bacillus degraded naringin.
The concrete steps of described degraded naringin are:
Leaf bacillus (Phyllobacteriumsp.) CGMCCNo.6472 is inoculated in the substratum containing naringin and cultivate, to degrade after using the free cell cultivated or being fixed of cell naringin, in culturing process, measure the degraded situation of its growing state and naringin.
Described containing in the substratum of naringin, comprise following component: naringin, ammonium sulfate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride.The content preferably following combination of above-mentioned each component: naringin content 0.1 ~ 50g/L, ammonium sulfate 0.05 ~ 2g/L, dipotassium hydrogen phosphate 0.05 ~ 2g/L, magnesium sulfate 0.05 ~ 2g/L, calcium chloride 0.05 ~ 2g/L.Wherein, naringin content is 0.1 ~ 5g/L preferably, more preferably 0.1 ~ 2g/L.
Described initial pH value of carrying out cultivating is 3.5 ~ 8.5, and preferred pH refers to be 3.5 ~ 6.5, more preferably 3.0 ~ 5.0.
Described incubation time is not particularly limited, preferred 24h ~ 72h, more preferably 48h ~ 72h.Incubation time is too short, then degrade thorough not; Incubation time is oversize, uneconomical.
Described culture temperature is not particularly limited, preferably 25 ~ 35 DEG C, more preferably 30 ~ 35 DEG C.Culture temperature is high, and thalli growth is fast, and too high thalline can be dead, and culture temperature is low, and thalli growth is slow, and too low thalline can be dead.
Described method can use free cell degraded naringin.
Described method can use the cell degradation naringin after immobilization.Spendable immobilization material is not particularly limited, as long as can realize fixing cell.
Immobilized Cells for Biodegradation naringin can be reused.
Also can utilize the mixed form degraded naringin of free cell and immobilized cell.Ratio and the method for described mixing are not particularly limited.
In fermentation culture process, the growing state of leaf bacillus and the degraded situation of naringin can be measured.
Be described further below in conjunction with some specific embodiments.Specific embodiment is for further describing the present invention, non-limiting protection scope of the present invention.
Embodiment 1
(1) abstraction and purification of bacterial classification
Get sweet shaddock orchard fresh soil samples 1g to join in the enrichment medium of 100mL and cultivate 48h, get 1mL enrichment culture liquid and proceed in new enrichment medium, continuous enrichment culture 3 generation.Get enrichment culture liquid 1mL, carry out continuous print dilution with 10 times of gradients, select appropriate dilution (10
-1, 10
-5, 10
-6) bacteria suspension 0.2mL, be coated with in isolation medium, be placed in after 30 DEG C of incubators cultivate 72h, the bacterial strain of all growths of picking carries out line separation and Culture respectively.In continuous line separation and Culture 3 generation, obtain purifying bacterial strain.These pure bacterial strains are transferred to liquid nutrient medium, 30 DEG C, 150rpm, shaking table is cultivated, and after cultivating 72h, measures the changing conditions of naringin content as follows:
Adopt high performance liquid chromatography to measure, condition is: moving phase: acetonitrile: water=25: 75; Analytical column is: C18 reverse-phase chromatographic column; Temperature: 34 DEG C; Sample size: 20 μ L; Flow velocity: 1.0mL/min; Instrument is: Agilent 1200LC.
In the abstraction and purification process of bacterial strain, used medium is composed as follows:
Enrichment medium, isolation medium, liquid nutrient medium (g/L): naringin 2; Ammonium sulfate 1; Dipotassium hydrogen phosphate 1; Calcium Chloride Powder Anhydrous 0.2; Magnesium sulfate 0.5; PH4.0 ~ 5.0; 0.1MPa sterilizing 20min.
(2) identification of strains
Carry out morphological specificity and 16SrDNA Sequence Identification by screening the bacterial strain Wb51b obtained, detailed process is as follows:
Bacterial strain Wb51b is gram negative bacillus, and colony edge is white, irregular star; Middle part color is comparatively dark, is khaki color, circular; Center is orange-yellow.Bacterium colony smooth surface and more moistening, thalline easily choose from.
The 16SrDNA nucleotide sequence of bacterial strain Wb51b is as shown in sequence table.
The homology of itself and type strain Phyllobacteriumsp. is the highest, reaches 99.92%.
Based on above feature, step (1) is screened the bacterial strain Wb51b obtained and be accredited as leaf bacillus (Phyllobacteriumsp.).
Embodiment 2
The mensuration of growth curve and the degraded of naringin.
Bacterial classification: Phyllobacteriumsp.Wb51bCGMCCNo.6472.
Substratum:
Seed culture medium (g/L): glucose 1; Yeast powder 2; Peptone 0.5; Naringin 0.5; PH3.0 ~ 5.0,0.1MPa, sterilizing 20min.
Fermention medium (g/L): naringin 2; Ammonium sulfate 1; Dipotassium hydrogen phosphate 1; Calcium Chloride Powder Anhydrous 0.2; Magnesium sulfate 0.5; PH3.0 ~ 6.0,0.1MPa, sterilizing 20min.
Seed culture: be inoculated into by Phyllobacteriumsp.Wb51bCGMCCNo.6472 (250mL triangular flask, liquid amount 100mL) in the substratum of the naringin containing 0.5g/L, temperature 30 DEG C, rotating speed 150rpm, 24h cultivated by shaking table.
Fermentation culture: the Phyllobacteriumsp.Wb51bCGMCCNo.6472 strain inoculation after being cultivated by seed culture medium is in fermention medium, and culture condition is identical with seed culture, every 4h sampling once, measures biomass OD respectively
600nmand naringin content.
Experimental result:
At the end of experiment, when incubation time is 50h, biological value reaches maximum value, and the concentration of biomass is 0.45g/L.
At the end of experiment, use the naringin content (g/L) of each time point sample of high-performance liquid chromatogram determination respectively, be respectively 4h:0.22; 8h:0.21; 16h:0.19; 24h:0.15; 28h; 0.11; 32h:0.065; 36h:0.017; 40h:0.0037.
Embodiment 3
Free cell degraded naringin
Bacterial classification: Phyllobacteriumsp.Wb51bCGMCCNo.6472.
Substratum (g/L): naringin 2; Yeast extract paste 0.1; Ammonium sulfate 1: calcium chloride 0.2; Magnesium sulfate 0.5; Dipotassium hydrogen phosphate 1; PH3.0 ~ 5.0,0.1MPa, sterilizing 20min.
Connect by Phyllobacteriumsp.Wb51bCGMCCNo.6472 in seed culture medium, temperature 30 DEG C, rotating speed 150rpm, 72h cultivated by shaking table.
The separation of free cell:
By cultivating the Phyllobacteriumsp.Wb51bCGMCCNo.6472 microorganism collection of 72h, be 4 DEG C in temperature, centrifugal 10min in the whizzer of rotating speed 8000rpm, 50mL capacity.Supernatant discarded, washes three times with the PBS damping fluid that pH is 6.0, the naringin contained in removing thalline, and the PBS damping fluid suspension thalline of final pH6.0 is for subsequent use in 4 DEG C of Refrigerator stores.
Be 0.6g/L in naringin concentration, cell concentration is 10.6g/L, and temperature of reaction is under the condition of 50 DEG C, measures the time dependent content of naringin.
Experimental result:
Reaction times, when being 30min, naringin content was 0.1g/L, and the degradation rate of naringin is 83.3%.
Embodiment 4
Immobilized Cells for Biodegradation naringin
The preparation method of cell required for immobilization is consistent with embodiment 3.
The preparation condition of immobilized cell is: cell embedding amount is the biomass/sodium alginate soln volume of 15.9mg/10mL, and sodium alginate concentration is 1.5%, and calcium chloride concentration is 0.04mol/L, and set time is 6h.
The immobilized cell sterilized water prepared by preparation condition according to immobilized cell washes three times, saves backup in 4 DEG C of refrigerators.
When temperature is 50 DEG C, be naringin solution and the immobilized cell equal-volume hybrid reaction of 1.2g/L by concentration, measure naringin content during different time points.
Experimental result:
When being when reacted 60min, naringin content is 0.076g/L, and the degradation rate of naringin is 93.7%.
Embodiment 5
Recycling Immobilized Cells for Biodegradation naringin
The preparation method of immobilized cell is consistent with embodiment 4.
Be that the naringin solution equal-volume of 1.2g/L mixes with concentration at 50 DEG C by the immobilized cell prepared, measure naringin content situation over time.At the end of first time experiment, reaction solution is outwelled, uses aseptic water washing immobilized cell, repeat above experimental implementation, measure naringin content changing conditions in time, repeat 3 times.When there is emulsion phenomenon in reaction solution, with retightening of 0.004mol/L calcium chloride solution bead, reuse 3 times.
Experimental result:
When immobilized cell uses three times, there is emulsion phenomenon in reaction solution, again solidifies with calcium chloride, and immobilized cell can continue to reuse 3 times, and in reusable 6 batches of immobilized cell, the degradation rate of naringin is all greater than 90%.
The change of a lot of details is possible, and therefore when without prejudice to scope of the present invention, the respective change that any person of ordinary skill in the field does it, all should be considered as the category not departing from patent of the present invention.
Claims (11)
1. the bacterial classification of a naringin of degrading, described bacterial classification is leaf bacillus (Phyllobacteriumsp.) Wb51b, be preserved in China General Microbiological culture presevation administrative center CGMCC on September 18th, 2012, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, registering on the books and be numbered preserving number in this preservation center is CGMCCNo.6472.
2. the method for a naringin of degrading, it is characterized in that, leaf bacillus (Phyllobacteriumsp.) is inoculated into the substratum containing naringin, be cultivate under the condition of 3.5 ~ 8.5 in initial pH value, and use free cell, cell after being fixed or its mixed style cultivated, naringin of degrading.
3. degrade according to claim 2 the method for naringin, it is characterized in that, described leaf bacillus is leaf bacillus (Phyllobacteriumsp.) CGMCCNo.6472.
4. to degrade according to claim 2 the method for naringin, it is characterized in that, described in the initial pH value of carrying out cultivating be 3.5 ~ 6.5, preferably 3.0 ~ 5.0.
5. to degrade according to claim 2 the method for naringin, it is characterized in that, described incubation time for being 12 hours ~ 7 days, preferred 24h ~ 72h, more preferably 48h ~ 72h.
6. to degrade according to claim 2 the method for naringin, it is characterized in that, describedly comprise following component containing in the substratum of naringin: naringin, ammonium sulfate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride.
7. to degrade according to claim 6 the method for naringin, it is characterized in that the content of described each component is: naringin content 0.1 ~ 50g/L, ammonium sulfate 0.05 ~ 2g/L, dipotassium hydrogen phosphate 0.05 ~ 2g/L, magnesium sulfate 0.05 ~ 2g/L, calcium chloride 0.05 ~ 2g/L; Wherein, naringin content preferably 0.1 ~ 5g/L.
8. to degrade according to claim 2 the method for naringin, it is characterized in that the culture temperature described in it is 25 ~ 50 DEG C, preferably 25 ~ 35 DEG C, more preferably 30 ~ 35 DEG C.
9. to degrade according to claim 2 the method for naringin, it is characterized in that, concrete steps comprise: inoculated by leaf bacillus (Phyllobacteriumsp.) CGMCCNo.6472 in the substratum containing naringin and cultivate, initial pH value is 3.5 ~ 8.5, incubation time is 48 ~ 72h, culture temperature is 25 ~ 50 DEG C, naringin of degrading after using the free cell or being fixed of cell cultivated.
10. to degrade according to claim 2 the method for naringin, it is characterized in that, use the cell degradation naringin after Immobilization in Sodium Alginate.
11. methods of degrading naringin according to claim 2, is characterized in that, recycling Immobilized Cells for Biodegradation naringin.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418268A (en) * | 2007-10-26 | 2009-04-29 | 中国海洋大学 | Production of novel bacterial exopolysaccharides by using Phyllobacterium sp.nov.921F bacterial strain |
| JP2015015933A (en) * | 2013-07-12 | 2015-01-29 | 独立行政法人農業・食品産業技術総合研究機構 | Plant growth promotion agent |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418268A (en) * | 2007-10-26 | 2009-04-29 | 中国海洋大学 | Production of novel bacterial exopolysaccharides by using Phyllobacterium sp.nov.921F bacterial strain |
| JP2015015933A (en) * | 2013-07-12 | 2015-01-29 | 独立行政法人農業・食品産業技術総合研究機構 | Plant growth promotion agent |
Non-Patent Citations (4)
| Title |
|---|
| MAXIMO SANCHEZ 等: "Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus", 《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》 * |
| 张林河 等: "微生物来源柚苷酶的研究进展及应用", 《化工进展》 * |
| 新文秘网: "新型产柚苷酶菌株phyllobacterium sp.Wb51b的筛选及其鉴定", 《新文秘网》 * |
| 陶天申 等主编: "《原核生物系统学》", 30 September 2007, 化学工业出版社 * |
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