CN105238707B - A kind of strain and method of aurantiin of degrading - Google Patents
A kind of strain and method of aurantiin of degrading Download PDFInfo
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- CN105238707B CN105238707B CN201510520364.7A CN201510520364A CN105238707B CN 105238707 B CN105238707 B CN 105238707B CN 201510520364 A CN201510520364 A CN 201510520364A CN 105238707 B CN105238707 B CN 105238707B
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Abstract
The present invention relates to the strains and method of a kind of aurantiin of degrading, provide a kind of strain leaf bacillus Wb51b (Phyllobacterium sp.) of aurantiin that can degrade.Under certain condition, using the aurantiin of 2g/L as sole carbon source culture strain 72h, cell concentration reaches 0.45g/L, and naringin content is reduced to 0.0037g/L, is almost completely degraded.Directly with the aurantiin of free cell degradation 0.6g/L, reaction time 30min, the content of aurantiin is reduced to 0.1g/L, and degradation rate is up to 83.3%；With Immobilized Cells for Biodegradation 1.2g/L aurantiin, reaction time 60min, the content of aurantiin is reduced to 0.076g/L, and degradation rate is up to 93.7%.The strain can efficient degradation aurantiin, culture simple to nutritional requirement, easy；In addition, the strain no pathogenicity, may be directly applied to food industry.
Description
Technical field
The present invention relates to a kind of leaf bacillus, the methods for aurantiin of degrading more particularly, to it.
Background technique
Aurantiin (Naringenine-7-rhamnosidoglucoside) also known as naringin, naringin, aurantiin are
Most important bitter substance in honey pomelo pericarp.The content for reducing aurantiin, the industrialization technology for establishing grapefruit juice debitterizing will be to
Beverage industry brings great progress.In recent years, the method for microbial degradation aurantiin increasingly attracts people's attention, and is related to
The type of microorganism has fungi, and bacterium and yeast etc., wherein fungi production naringinase has been achieved with certain come aurantiin of degrading
Achievement；And the research of bacterial degradation aurantiin is also less.
As sweet shaddock cultivated area constantly expands, sweet shaddock yield is risen year by year, be singly every year the sweet shaddock output value in Fujian Province just
Reach 60,000,000 tons, but since deep process technology research lags, not yet realizes the deep processing of industrialization, production-supply-marketing contradiction is increasingly
It is prominent, cause a large amount of fruit to be dropped, the foul odour for abandoning fruit rot has seriously polluted the environment again.Sweet shaddock juicing is honey
The main means of shaddock deep processing, but the fruit drink obtained has apparent bitter taste, needs to remove aurantiin, carries out de- suffering reason
It can just drink afterwards.Therefore, aurantiin of how effectively degrading becomes a urgent project.
Honey shaddock juice, which takes off bitter main method, at present physisorphtion, the side such as food additives method and biological enzyme formulation
Method.Physisorphtion will also adsorb the nutritional ingredient in fruit juice while adsorbing bitter substance, and food additives method only will
Bitter substance masking, is not removed；And biological enzyme formulation method, 201010219099.6 (publication number of Chinese patent
CN101897452A, December 1 2010 time of disclosure) a kind of fermentation method for producing of grapefruit juice debitterizing enzyme is disclosed, it should
Method needs to isolate and purify enzyme, the higher cost of this process.
Summary of the invention
The purpose of the present invention is to provide a kind of strain leaf bacillus (Phyllobacterium sp.) of aurantiin of degrading
Wb51b。
The strain be leaf bacillus (Phyllobacterium sp.) Wb51b, screen and obtain from pedotheque, in
Be preserved within 2012 China General Microbiological bacterial strain preservation administration committee common micro-organisms center (abbreviation CGMCC, address are as follows:
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica), collection number of registering on the books is
CGMCC No.6472。
It is another object of the present invention to provide a kind of methods of aurantiin of degrading.
This method utilizes leaf bacillus (Phyllobacterium sp.) degradation aurantiin.Specific steps include: by leaf bacillus
(Phyllobacterium sp.) is inoculated with into the culture medium containing aurantiin, is cultivated, and use cultivated it is free thin
Born of the same parents, immobilize after cell or its mixed style, come aurantiin of degrading.
The present invention uses above-mentioned collection number for the leaf bacillus of CGMCC No.6472 degradation aurantiin.
The initial pH value cultivated is 3.5~8.5, preferably 3.5~6.5, more preferable 3.0~5.0.
The incubation time 12 hours~7 days, preferably for 24 hours~72h, more preferable 48h~72h.
Preferably 25~50 DEG C of the cultivation temperature, preferably 25~35 DEG C, more preferable 30~35 DEG C.
Cell is immobilized using sodium alginate.
Reusable Immobilized Cells for Biodegradation aurantiin.
Of the invention has the prominent advantages that:
1. strain used in the present invention be bacterium, can efficient degradation aurantiin, culture simple to nutritional requirement, easy.
2. strain no pathogenicity used in the present invention, the hidden danger in terms of inanimate object safety, may be directly applied to food work
Industry can reduce cost without isolating and purifying to relevant enzyme using free cell degradation aurantiin.
3. Immobilization in Sodium Alginate cell can be used come aurantiin of degrading in the present invention, can be used repeatedly.
Specific embodiment
The present invention provides a kind of leaf bacillus of degradable aurantiin.
The present invention discloses a kind of method using leaf bacillus degradation aurantiin.
The specific steps of the degradation aurantiin are as follows:
Leaf bacillus (Phyllobacterium sp.) CGMCC No.6472 is inoculated in the culture medium containing aurantiin
It is cultivated, aurantiin of degrading after using cultivated free cell or cell to immobilize measures its life in incubation
The degradation situation of long situation and aurantiin.
It include following components: aurantiin, ammonium sulfate, dipotassium hydrogen phosphate, sulfuric acid in the culture medium containing aurantiin
Magnesium, calcium chloride.The preferably following combination of the content of above-mentioned each component: 0.1~50g/L of naringin content, 0.05~2g/L of ammonium sulfate,
0.05~2g/L of dipotassium hydrogen phosphate, 0.05~2g/L of magnesium sulfate, 0.05~2g/L of calcium chloride.Wherein, naringin content preferably 0.1
~5g/L, more preferable 0.1~2g/L.
The initial pH value cultivated is 3.5~8.5, and preferably pH refers to be 3.5~6.5, more preferable 3.0~5.0.
The incubation time is not particularly limited, preferably for 24 hours~72h, more preferable 48h~72h.Incubation time is too short, then
It degrades not thorough enough；Incubation time is too long, uneconomical.
The cultivation temperature is not particularly limited, and preferably 25~35 DEG C, more preferable 30~35 DEG C.Cultivation temperature is high, thallus
Growth is fast, and too high thallus can be dead, and cultivation temperature is low, and thalli growth is slow, and too low thallus can be dead.
Free cell degradation aurantiin can be used in the method.
The cell degradation aurantiin after immobilization can be used in the method.Workable immobilization material does not limit especially
System, as long as the fixation to cell can be realized.
Reusable Immobilized Cells for Biodegradation aurantiin.
Also using the mixed form of free cell and immobilized cell degradation aurantiin.The mixed ratio and method
It is not particularly limited.
During fermented and cultured, the growing state of leaf bacillus and the degradation situation of aurantiin can be measured.
It is described further below with reference to some specific embodiments.Specific embodiment is that present invention be described in more detail,
Non-limiting protection scope of the present invention.
Embodiment 1
(1) separation and purifying of strain
It takes sweet shaddock orchard fresh soil samples 1g to be added in the enriched medium of 100mL and cultivates 48h, take 1mL enrichment training
Nutrient solution is transferred in new enriched medium, continuous 3 generation of enrichment culture.Enrichment culture liquid 1mL is taken, is carried out with 10 times of gradients continuous
Dilution selects appropriate dilution (10-1, 10-5, 10-6) bacteria suspension 0.2mL, be coated in isolation medium, be placed in 30 DEG C of trainings
It supports after cultivating 72h in case, the bacterial strain of all growths of picking carries out scribing line respectively and is separately cultured.Continuous scribing line was separately cultured for 3 generations, obtained
Bacterial strain must be purified.These pure bacterial strains are transferred to fluid nutrient medium, 30 DEG C, 150rpm, shaking table culture is pressed after cultivating 72h
State the situation of change of method measurement naringin content:
It is measured using high performance liquid chromatography, condition are as follows: mobile phase: acetonitrile: water=25: 75；Analytical column are as follows: C18
Reverse-phase chromatographic column；Temperature: 34 DEG C；Sample volume: 20 μ L；Flow velocity: 1.0mL/min；Instrument are as follows: Agilent 1200LC.
Used medium composition is as follows in the separation and purification process of bacterial strain:
Enriched medium, isolation medium, fluid nutrient medium (g/L): aurantiin 2；Ammonium sulfate 1；Dipotassium hydrogen phosphate 1；Nothing
Water calcium chloride 0.2；Magnesium sulfate 0.5；PH4.0~5.0；0.1MPa sterilizing 20min.
(2) bacterial strain is identified
The bacterial strain Wb51b that screening is obtained carries out morphological feature and the identification of 16S r DNA sequence dna, and detailed process is as follows:
Bacterial strain Wb51b is gram-Negative bacillus, and colony edge is white, irregular star；Middle part color is deeper, for soil
Yellow, it is round；Center is in orange-yellow.Bacterium colony surface is smooth and relatively wet, thallus be easy to choose from.
The 16S r DNA nucleotide sequence of bacterial strain Wb51b is as shown in sequence table.
The homology highest of itself and type strain Phyllobacterium sp., reaches 99.92%.
The bacterial strain Wb51b that step (1) screening obtains is accredited as leaf bacillus (Phyllobacterium based on features above
sp.)。
Embodiment 2
The measurement of growth curve and the degradation of aurantiin.
Strain: Phyllobacterium sp.Wb51b CGMCC No.6472.
Culture medium:
Seed culture medium (g/L): glucose 1；Yeast powder 2；Peptone 0.5；Aurantiin 0.5；PH 3.0~5.0,
0.1MPa, sterilize 20min.
Fermentation medium (g/L): aurantiin 2；Ammonium sulfate 1；Dipotassium hydrogen phosphate 1；Anhydrous calcium chloride 0.2；Magnesium sulfate 0.5；
PH 3.0~6.0,0.1MPa, sterilize 20min.
Seed culture: Phyllobacterium sp.Wb51b CGMCC No.6472 is inoculated into the shaddock containing 0.5g/L
In the culture medium of skin glycosides (250mL triangular flask, liquid amount 100mL), 30 DEG C of temperature, revolving speed 150rpm, shaking table culture is for 24 hours.
Fermented and cultured: by the Phyllobacterium sp.Wb51b CGMCC No.6472 after seed culture medium culture
Strain is seeded in fermentation medium, and condition of culture is identical as seed culture, primary every 4h sampling, measures biomass respectively
OD600nmAnd naringin content.
Experimental result:
At the end of experiment, when incubation time is 50h, biological magnitude reaches maximum value, and the concentration of biomass is 0.45g/
L。
At the end of experiment, respectively with the naringin content (g/L) of high performance liquid chromatography measurement each time point sample, difference
For 4h:0.22；8h:0.21；16h:0.19；For 24 hours: 0.15；28h；0.11；32h:0.065；36h:0.017；40h:0.0037.
Embodiment 3
Free cell degradation aurantiin
Strain: Phyllobacterium sp.Wb51b CGMCC No.6472.
Culture medium (g/L): aurantiin 2；Yeast extract 0.1；Ammonium sulfate 1: calcium chloride 0.2；Magnesium sulfate 0.5；Dipotassium hydrogen phosphate
1；PH 3.0~5.0,0.1MPa, sterilize 20min.
Phyllobacterium sp.Wb51b CGMCC No.6472 is connect in seed culture medium, 30 DEG C of temperature, revolving speed
150rpm, shaking table culture 72h.
The separation of free cell:
The Phyllobacterium sp.Wb51b CGMCC No.6472 microorganism collection of 72h will be cultivated, is 4 in temperature
DEG C, 10min is centrifuged in the centrifuge of revolving speed 8000rpm, 50mL capacity.It discards supernatant, washes three with the PBS buffer solution that pH is 6.0
Time, the aurantiin contained in thallus is removed, the final PBS buffer solution suspension thalline for using pH 6.0 is saved backup in 4 DEG C of refrigerators.
It is 0.6g/L in aurantiin concentration, cell concentration 10.6g/L measures shaddock under conditions of reaction temperature is 50 DEG C
The content that skin glycosides changes over time.
Experimental result:
When reaction time is 30min, naringin content 0.1g/L, the degradation rate of aurantiin is 83.3%.
Embodiment 4
Immobilized Cells for Biodegradation aurantiin
It is consistent in the preparation method and embodiment 3 of cell required for immobilization.
The preparation condition of immobilized cell are as follows: cell embedding amount is biomass/sodium alginate soln body of 15.9mg/10mL
Product, sodium alginate concentration 1.5%, calcium chloride concentration 0.04mol/L, curing time 6h.
The immobilized cell prepared according to the preparation condition of immobilized cell sterile water is washed three times, in 4 DEG C of ice
It is saved backup in case.
When temperature is 50 DEG C, the aurantiin solution and the isometric hybrid reaction of immobilized cell that are 1.2g/L by concentration,
Measure naringin content when different time points.
Experimental result:
When being 60min between when reacted, naringin content 0.076g/L, the degradation rate of aurantiin is 93.7%.
Embodiment 5
Reuse Immobilized Cells for Biodegradation aurantiin
The preparation method and embodiment 4 of immobilized cell are consistent.
The immobilized cell prepared is mixed at 50 DEG C with the aurantiin solution that concentration is 1.2g/L in equal volume, is measured
Naringin content changes with time situation.At the end of testing for the first time, reaction solution is outwelled, with aseptic water washing fixation cell
Born of the same parents, repeat more than experimental implementation, measurement naringin content change over time situation, be repeated 3 times.When occurring milkiness in reaction solution
When liquid phenomenon, with 0.004mol/L calcium chloride solution retightening bead, reuse 3 times.
Experimental result:
When immobilized cell is used three times, there is emulsion phenomenon in reaction solution, is solidified again with calcium chloride, immobilized cell
It can continue to reuse 3 times, the degradation rate of aurantiin is all larger than 90% in 6 batches that immobilized cell is reused.
The variation of many details is possible, therefore when without prejudice to the scope of the present invention, any technical field
The corresponding change that technical staff does it all should be regarded as the scope for not departing from the invention patent.
Claims (18)
1. it is a kind of degrade aurantiin strain, the strain be leaf bacillus (Phyllobacterium sp.) Wb51b, in
It is preserved in China General Microbiological culture presevation administrative center CGMCC, address on September 18th, 2012 are as follows: Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica, collection number of registering on the books be deposit number is CGMCC
No.6472。
2. a kind of method for aurantiin of degrading, which is characterized in that being inoculated with leaf bacillus (Phyllobacterium sp.) into containing
The culture medium of aurantiin, initial pH value be 3.5~8.5 under conditions of cultivated, and use cultivated free cell, into
Cell, free cell after row immobilization with immobilize after cell mixed style in it is any, come aurantiin of degrading；
The leaf bacillus is leaf bacillus (Phyllobacterium sp.) CGMCC No.6472.
3. the method for aurantiin of degrading according to claim 2, which is characterized in that the leaf bacillus is leaf bacillus
(Phyllobacterium sp.)CGMCC No.6472；Aurantiin concentration is 0.6g/L, cell concentration 10.6g/L, reaction
Temperature is 50 DEG C.
4. the method for aurantiin of degrading according to claim 2, which is characterized in that the initial pH value cultivated is
3.5~6.5.
5. the method for aurantiin of degrading according to claim 2, which is characterized in that the initial pH value cultivated is
3.5~5.0.
6. the method for aurantiin of degrading according to claim 2, which is characterized in that described in initial pH value is 3.5~8.5
Under the conditions of the incubation time cultivated be 12 hours~7 days.
7. the method for aurantiin of degrading according to claim 2, which is characterized in that described in initial pH value is 3.5~8.5
Under the conditions of the incubation time cultivated be for 24 hours~72h.
8. the method for aurantiin of degrading according to claim 2, which is characterized in that described in initial pH value is 3.5~8.5
Under the conditions of the incubation time cultivated be 48h~72h.
9. the method for aurantiin of degrading according to claim 2, which is characterized in that wrapped in the culture medium containing aurantiin
Containing following components: aurantiin, ammonium sulfate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride.
10. the method for aurantiin of degrading according to claim 9, it is characterised in that the content of each component are as follows: aurantiin
0.1~50g/L of content, 0.05~2g/L of ammonium sulfate, 0.05~2g/L of dipotassium hydrogen phosphate, 0.05~2g/L of magnesium sulfate, calcium chloride
0.05~2g/L.
11. the method for aurantiin of degrading according to claim 10, it is characterised in that the naringin content is 0.1~5g/
L。
12. the method for aurantiin of degrading according to claim 10, it is characterised in that the naringin content is 0.1~2g/
L。
13. the method for aurantiin of degrading according to claim 2, it is characterised in that the cultivation temperature described in it is 25~50
℃。
14. the method for aurantiin of degrading according to claim 2, it is characterised in that the cultivation temperature described in it is 25~35
℃。
15. the method for aurantiin of degrading according to claim 2, it is characterised in that the cultivation temperature described in it is 30~35
℃。
16. the method for aurantiin of degrading according to claim 2, which is characterized in that specific steps include: by leaf bacillus
(Phyllobacterium sp.) CGMCC No.6472 is inoculated in the culture medium containing aurantiin and is cultivated, initial pH value
Be 3.5~8.5, incubation time be 48~72h, cultivation temperature be 25~50 DEG C, use cultivated free cell or cell into
It degrades after row immobilization aurantiin.
17. the method for aurantiin of degrading according to claim 2, which is characterized in that using thin after Immobilization in Sodium Alginate
Born of the same parents' degradation aurantiin.
18. the method for aurantiin of degrading according to claim 2, which is characterized in that recycling Immobilized Cells for Biodegradation shaddock
Skin glycosides.
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Citations (1)
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6236660B2 (en) * | 2013-07-12 | 2017-11-29 | 国立研究開発法人農業・食品産業技術総合研究機構 | Plant growth promoter |
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Patent Citations (1)
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus;Maximo Sanchez 等;《International Journal of Systematic and Evolutionary Microbiology》;20141231;第64卷;第781-786页 * |
| 微生物来源柚苷酶的研究进展及应用;张林河 等;《化工进展》;20131231;第32卷(第5期);第1108-1115页 * |
| 新型产柚苷酶菌株phyllobacterium sp.Wb51b的筛选及其鉴定;新文秘网;《新文秘网》;20130829;第1-5页，尤其是第1页摘要部分、第2页1.4.1柚汁加工、第3页2材料与方法部分2.1.4培养基及溶液、第4页2.4实验方法 * |
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