CN104531555A - Bunge pricklyash leaf endophytic bacillus safensis, method for screening and purifying bacillus safensis as well as application thereof - Google Patents
Bunge pricklyash leaf endophytic bacillus safensis, method for screening and purifying bacillus safensis as well as application thereof Download PDFInfo
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Abstract
The invention discloses a method for screening and purifying bunge pricklyash leaf endophytic bacillus safensis GHF4 and an application thereof, the bacillus safensis is preserved in China Center for Type Culture Collection with a preservation number CCTCC NO: M2014535, and is named as GHF4. The method is characterized by comprising the following steps: taking fresh bunge pricklyash leaf, disinfecting and cleaning, performing plate enrichment culture, performing gradient dilution plate coating, primary screening, secondary screening and the like to realize screening and purifying. its fermentation metabolites has strong antioxidant activity, when the concentration of a broth is 3.75mg/mL, clearance to ABTS free radical by endophytic bacillus safensis can reach 99.48%, and IC50 value is 0.80mg/mL. The endophytic bacillus safensis has good application prospect on production aspect of a natural anti-oxidant.
Description
Technical field
The present invention relates to a kind of Folium Zanthoxyli Bungeani Nei Shengshafenxi genus bacillus and screen purification process and purposes.
The bacterial strain that the present invention obtains is in China typical culture collection center preservation on October 31 in 2014, depositary institution address: Wuhan, China Wuhan University, deposit number: CCTCC M 2014535, specific name: Sha Fenxi genus bacillus GHF4 (Bacillus safensis GHF4).
Background technology
Along with the development of society, people have had new demand to health, and natural antioxidants is because of its applied range, and toxic side effect is little, more and more concerned, and extract the Natural Antioxidants focus always studied of numerous scholar especially.In recent years, endophyte of plant receives much attention, much domestic and international research confirms, endophyte of plant is a kind of new type resource developed, it, with host's interaction process, not only has Promoting plant growth, improves the ability of disease resistance of plant, but also the active substance that energy metabolism is identical or similar with host, endophyte of plant meta-bolites is a kind of potential resource, mostly has biologic activity, as the effect (Schulz such as antibacterial, antiviral, B., Boyle, C., Draeger, S.
a.K., & Krohn, K.Endophytic fungi:A source of novel biologically active secondary metabolites [J] .Mycological Research, 2002,106:996 – 1004.).Because it has high yield, environmental friendliness, the features such as safety, so make its application in the industry such as food, medicine have bright prospects.
At present, there are no about being separated the patent and non-patent literature report that obtain Nei Shengshafenxi genus bacillus from Folium Zanthoxyli Bungeani, the research of oxidation-resistance is not had to report about Folium Zanthoxyli Bungeani endophyte meta-bolites yet.
Summary of the invention
The object of this invention is to provide a strain Folium Zanthoxyli Bungeani Nei Shengshafenxi genus bacillus, and in China typical culture collection center preservation, deposit number is CCTCC NO.:M2014535; Another object of the present invention is the application of the anti-oxidant activity of this Folium Zanthoxyli Bungeani endophyte.
Chinese prickly ash endophyte of the present invention is that contriver is separated the strain Sha Fenxi genus bacillus obtained from Folium Zanthoxyli Bungeani, shows that it belongs to Nei Shengshafenxi genus bacillus through traditional method qualification and Molecular Identification, and by its called after GHF4.
Folium Zanthoxyli Bungeani endophyte of the present invention---GHF4 is deposited in China typical culture collection center (CCTCC) on October 31st, 2014, and deposit number is CCTCC M 2014535.
Screening and the purifying of Folium Zanthoxyli Bungeani Nei Shengshafenxi genus bacillus comprise the following steps:
(1) cleaning and sterilizing
In aseptic operating platform, with the fresh Folium Zanthoxyli Bungeani of aseptic water washing 3 times, then being that 0.5 ~ 1.5% chlorine bleach liquor soaks fresh pericarpium zanthoxyli bungeani leaf 3 ~ 8min by concentration, is the ethanol disinfection 5 ~ 10min of 75% afterwards by concentration, aseptic water washing 3 times, and retain last bath water in contrast;
(2) enrichment culture
By the fresh pericarpium zanthoxyli bungeani leaf of above-mentioned cleaning and sterilizing process, grind in mortar and smash to pieces, be inoculated in the nutrient agar medium solid medium containing 1% potassium bichromate, cultivate 5 ~ 7 days under temperature 28 ~ 30 DEG C of constant incubators, observe and have bacterium colony to grow;
(3) dull and stereotyped coating
Picking characteristic of bacteria bacterium colony in bacterium colony after above-mentioned nutrient agar medium solid medium is cultivated, gradient dilution rear plate coating in stroke-physiological saline solution, is placed in temperature 28 ~ 30 DEG C of constant incubators cultivation constant incubators that after 5 ~ 7 days, picking bacterial colony is placed on temperature 28 ~ 30 DEG C in the line of nutrient agar medium solid medium upper flat plate and cultivates 5 days;
(4) primary dcreening operation
Picking characteristic of bacteria bacterium colony in bacterium colony after above-mentioned nutrient agar medium solid medium is cultivated, gradient dilution rear plate coating in stroke-physiological saline solution, being placed in temperature 28 ~ 30 DEG C of constant incubators cultivates after 5 ~ 7 days, picking bacterial colony is in the line of nutrient agar medium solid medium upper flat plate, and the constant incubator being placed in 28 ~ 30 DEG C is cultivated 5 ~ 7 days; Select single bacterium colony that above step obtains, streak inoculation is in new nutrient agar medium solid medium;
(5) multiple sieve
Step (3) obtained strains is inoculated in nutrient agar medium liquid nutrient medium, fermentation culture in temperature 28 ~ 30 DEG C of shaking tables, supernatant liquor is got after fermented liquid is centrifugal, by 2, after 2-joins nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) free radical scavenging experimental evaluation fermented liquid oxidation-resistance, by the strongest strain number of fermented liquid anti-oxidant activity and preservation;
(6) physiological and biochemical property and 16S rDNA analyze
Physiological and biochemical property and 16S rDNA analyze, and determine the kind of bacterium.
Described enrichment medium: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, agar 1.5%, pH is 7.0 ~ 7.6, adds 1% potassium bichromate after sterilizing; Nutrient agar medium solid medium: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, agar 1.5%, pH is 7.0 ~ 7.6; Nutrient agar medium liquid nutrient medium: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, pH is 7.0 ~ 7.6.
Performance test and structural characterization
Physiological and biochemical property: as shown in table 1,16S rDNA analyzes: refer to SEQUENCE LISTING.
Through above-mentioned 6 screening step obtained strains respectively on opticmicroscope (after gramstaining) and observed under electron microscope bacterium, its morphological specificity: bacterial classification of the present invention is gram-positive microorganism, the single rod-short of thalline, smooth surface, long 1.0 ~ 1.1 μm, wide 0.5 μm, well-grown on nutrient agar, colonial morphology is creamy white, smooth surface, colony edge unfairness; Growth temperature is 28 ~ 30 DEG C, the growth of bacterial classification can utilize glucose, melibiose, cellobiose as sole carbon source, through molecular cloning order-checking obtain the 16S rDNA sequence of this bacterial strain CCTCC M 2014535 and Sha Fenxi bacillus safensis (GENEBANK accession number is NR_113945.1) homology the highest, reach more than 99%.
The fermented liquid of the present invention to Sha Fenxi genus bacillus CCTCC M 2014535 carries out HPLC analysis, and efficient liquid phase chromatographic analysis condition is as follows: ODS-3 chromatographic column (
4.6 × 250mm, 5 μm); Flow velocity, 1mL/min; Column temperature, 25 DEG C; Determined wavelength, 254nm; Gradient eluent: 0min, 0% methyl alcohol (0.1% formic acid); 10min, 20% methyl alcohol (0.1% formic acid); 20min, 25% methyl alcohol (0.1% formic acid); 30min ~ 40min, 100% methyl alcohol (0.1% formic acid).
The antioxygenation of Sha Fenxi genus bacillus CCTCC M 2014535 fermented liquid of the present invention comprises the following steps:
(1) nutrient agar medium liquid nutrient medium is sub-packed in triangular flask, with the bacterial classification after a little purifying of transfering loop picking in culturing bottle, 48h is cultivated at temperature 28 ~ 30 DEG C of shaking tables, the centrifugal 10min of fermentation liquor 4000r/min, get supernatant liquor, after concentrating under reduced pressure drying, obtain Folium Zanthoxyli Bungeani endophyte fermentation broth extract;
(2) by Folium Zanthoxyli Bungeani endophyte fermentation broth extract respectively with 18.75mg/mL, 12.5mg/mL, 6.25mg/mL, 3.125mg/mL, the concentration of 1.25mg/mL adds 96 orifice plates, and add-on is 50 μ L/ holes, then in 96 orifice plates, add 200 μ LABTS working fluids.Under lucifuge condition, carry out free radical scavenging experiment reaction 30min, measure the absorbancy of each reaction solution after reaction terminates immediately, and calculate clearance rate and 503nhibiting concentration value (IC
50).
Tool of the present invention has the following advantages:
1, screening method is simple, easy to operate.
2, screening the Folium Zanthoxyli Bungeani endophyte GHF4 obtained is Sha Fenxi genus bacillus.
3, screen the Folium Zanthoxyli Bungeani endophyte bacterial strain fermentation liquor obtained and there is good anti-oxidant activity.
Accompanying drawing explanation
Fig. 1 a is the scanning electron microscope (SEM) photograph of bacterial strain CCTCC M 2014535
Fig. 1 b is the scanning electron microscope enlarged view of bacterial strain CCTCC M 2014535
Fig. 2 is the ABTS free radical scavenging activity figure of bacterial strain CCTCC M 2014535
Fig. 3 is that the HPLC of the fermented liquid of bacterial strain CCTCC M 2014535 analyzes collection of illustrative plates
Embodiment
Below by embodiment, the present invention is specifically described; what be necessary to herein means out is that the present embodiment is only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.
Embodiment 1:
(1) cleaning and sterilizing
In aseptic operating platform, aseptic water washing three times, after producing fresh safflower Folium Zanthoxyli Bungeani 8min with 0.5% clorox immersion Hanyuan, with the ethanol disinfection 10min of 75%, aseptic water washing three times, and retain last bath water in contrast.
(2) enrichment culture
Be inoculated in nutrient agar flat board by after the Folium Zanthoxyli Bungeani grinding after surface sterilization, cultivate 5 days in temperature 28 DEG C of constant incubators.
(3) dull and stereotyped coating
Picking characteristic of bacteria bacterium colony in bacterium colony after above-mentioned nutrient agar medium solid medium is cultivated, gradient dilution rear plate coating in stroke-physiological saline solution, is placed in temperature 28 DEG C of constant incubators cultivation constant incubators that after 7 days, picking bacterial colony is placed on temperature 28 DEG C in the line of nutrient agar medium solid medium upper flat plate and cultivates 5 days;
(4) primary dcreening operation
Picking characteristic of bacteria bacterium colony from the bacterium colony after the cultivation of nutrient agar medium solid medium, gradient dilution rear plate coating in stroke-physiological saline solution, being placed in temperature 28 DEG C of constant incubators cultivates after 7 days, picking bacterial colony is in the line of nutrient agar medium solid medium upper flat plate, and the constant incubator being placed in temperature 28 DEG C is cultivated 7 days; Select single bacterium colony that above step obtains, streak inoculation is in new nutrient agar medium solid medium.
(5) multiple sieve
Step (3) obtained strains is inoculated in nutrient agar medium liquid nutrient medium, fermentation culture in temperature 28 DEG C of shaking tables.Get supernatant liquor after fermented liquid is centrifugal, detected after its fermented liquid oxidation-resistance by ABTS free radical scavenging experiment, by the strongest strain number of fermented liquid anti-oxidant activity and preservation.
(6) physiological and biochemical property and 16S rDNA analyze:
Physiological and biochemical property and 16S rDNA analyze, and determine the kind of bacterium.
Embodiment 2
(1) cleaning and sterilizing:
In aseptic operating platform, aseptic water washing three times, after producing fresh pericarpium zanthoxyli bungeani leaf 5min with 1% clorox immersion Hanyuan, with the ethanol disinfection 8min of 75%, aseptic water washing three times, and retain last bath water in contrast.
(2) enrichment culture: be inoculated in nutrient agar flat board by after the Folium Zanthoxyli Bungeani grinding after surface sterilization, cultivate 6 days in temperature 29 DEG C of constant incubators.
(3) dull and stereotyped coating
Picking characteristic of bacteria bacterium colony in bacterium colony after above-mentioned nutrient agar medium solid medium is cultivated, gradient dilution rear plate coating in stroke-physiological saline solution, is placed in temperature 29 DEG C of constant incubators cultivation constant incubators that after 6 days, picking bacterial colony is placed on temperature 29 DEG C in the line of nutrient agar medium solid medium upper flat plate and cultivates 5 days;
(4) primary dcreening operation:
Picking characteristic of bacteria bacterium colony from the bacterium colony after the cultivation of nutrient agar medium solid medium, gradient dilution rear plate coating in stroke-physiological saline solution, being placed in temperature 29 DEG C of constant incubators cultivates after 6 days, picking bacterial colony is in the line of nutrient agar medium solid medium upper flat plate, and the constant incubator being placed in 29 DEG C is cultivated 6 days; Select single bacterium colony that above step obtains, streak inoculation is in new nutrient agar medium solid medium.
(5) multiple sieve:
Step (3) obtained strains is inoculated in nutrient agar medium liquid nutrient medium, fermentation culture in temperature 29 DEG C of shaking tables.Get supernatant liquor after fermented liquid is centrifugal, detected after its fermented liquid oxidation-resistance by ABTS free radical scavenging experiment, by the strongest strain number of fermented liquid anti-oxidant activity and preservation.
(6) physiological and biochemical property and 16S rDNA analyze: physiological and biochemical property and 16S rDNA analyze, and determine the kind of bacterium.
Embodiment 3
(1) cleaning and sterilizing:
In aseptic operating platform, aseptic water washing three times, after producing fresh pericarpium zanthoxyli bungeani leaf 3min with 1.5% clorox immersion Hanyuan, with the ethanol disinfection 5min of 75%, aseptic water washing three times, and retain last bath water in contrast.
(2) enrichment culture:
Be inoculated in nutrient agar flat board by after the Folium Zanthoxyli Bungeani grinding after surface sterilization, cultivate 7 days in temperature 30 DEG C of constant incubators.
(3) dull and stereotyped coating
Picking characteristic of bacteria bacterium colony in bacterium colony after above-mentioned nutrient agar medium solid medium is cultivated, gradient dilution rear plate coating in stroke-physiological saline solution, is placed in temperature 30 DEG C of constant incubators cultivation constant incubators that after 5 days, picking bacterial colony is placed on temperature 30 DEG C in the line of nutrient agar medium solid medium upper flat plate and cultivates 5 days;
(4) primary dcreening operation:
Picking characteristic of bacteria bacterium colony from the bacterium colony after the cultivation of nutrient agar medium solid medium, gradient dilution rear plate coating in stroke-physiological saline solution, being placed in temperature 30 DEG C of constant incubators cultivates after 5 days, picking bacterial colony is in the line of nutrient agar medium solid medium upper flat plate, and the constant incubator being placed in temperature 30 DEG C is cultivated 5 days; Select single bacterium colony that above step obtains, streak inoculation is in new nutrient agar medium solid medium.
(5) multiple sieve:
Step (4) obtained strains is inoculated in nutrient agar medium liquid nutrient medium, fermentation culture in temperature 30 DEG C of shaking tables.Get supernatant liquor after fermented liquid is centrifugal, detected after its fermented liquid oxidation-resistance by ABTS free radical scavenging experiment, by the strongest strain number of fermented liquid anti-oxidant activity and preservation.
(6) physiological and biochemical property and 16S rDNA analyze: physiological and biochemical property and 16S rDNA analyze, and determine the kind of bacterium.
Application example 1
One of anti-oxidant activity detection method of Folium Zanthoxyli Bungeani endophyte fermented liquid:
(1) by the separation and purification scheme of example 1, endophyte bacterial strain separation and purification from Folium Zanthoxyli Bungeani obtained carries out liquid fermentation and culture: Folium Zanthoxyli Bungeani endogenetic bacteria substratum is nutrient agar medium liquid nutrient medium, nutrient agar medium liquid nutrient medium is sub-packed in triangular flask, with the bacterial classification after a little purifying of transfering loop picking in culturing bottle, 48h is cultivated at temperature 28 ~ 30 DEG C of shaking tables, the centrifugal 10min of fermentation liquor 4000r/min, get supernatant liquor, after concentrating under reduced pressure drying, obtain Folium Zanthoxyli Bungeani endophyte fermentation broth extract.
(2) Antioxidative Activity Determination: add 200 μ L ABTS working fluids in 96 orifice plates.Folium Zanthoxyli Bungeani endophyte fermentation broth extract is added 96 orifice plates with 18.75mg/mL, 12.5mg/mL, 6.25mg/mL, 3.125mg/mL, 1.25mg/mL concentration respectively, and add-on is 50 μ L/ holes.Under lucifuge condition, carry out free radical scavenging experiment reaction 30min, measure the absorbancy of each reaction solution after reaction terminates immediately, and calculate clearance rate and IC
50.
(3) Antioxidative Activity Determination experimental result as shown in Figure 2.Result shows, when Folium Zanthoxyli Bungeani endophyte fermented liquid concentration reaches 3.75mg/mL, can reach 99.48% to the clearance rate of ABTS free radical.Folium Zanthoxyli Bungeani endophyte fermented liquid is to the IC of ABTS free radical as calculated
50=0.802mg/mL.
The physiological and biochemical property of table 1 GHF4
Claims (5)
1. a strain Folium Zanthoxyli Bungeani endophyte, is characterized in that: this bacterium is Sha Fenxi genus bacillus (Bacillus safensis), depositary institution: China typical culture collection center, preserving number: CCTCC M 2014535.
2., according to Folium Zanthoxyli Bungeani endophyte according to claim 1, it is characterized in that being:
(1) morphological specificity: bacterial strain of the present invention is gram-positive microorganism, the single rod-short of thalline, smooth surface, long 1.0 ~ 1.1 μm, wide 0.5 μm, well-grown on nutrient agar, colonial morphology is creamy white, smooth surface, colony edge unfairness;
(2) physio-biochemical characteristics
(3) homology analysis: be 16S rDNA and analyze, the highest with the Sha Fenxi bacillus safensis recorded in international data center Genbank (GENEBANK accession number is NR_113945.1) homology, reach more than 99%.
3. the screening purification process of Folium Zanthoxyli Bungeani endophyte according to claim 2, is characterized in that the screening of Folium Zanthoxyli Bungeani Nei Shengshafenxi genus bacillus and purification process comprise the following steps:
(1) cleaning and sterilizing
In aseptic operating platform, with the fresh Folium Zanthoxyli Bungeani of aseptic water washing 3 times, then being that 0.5 ~ 1.5% chlorine bleach liquor soaks fresh pericarpium zanthoxyli bungeani leaf 3 ~ 8min by concentration, is the ethanol disinfection 5 ~ 10min of 75% afterwards by concentration, aseptic water washing 3 times, and retain last bath water in contrast;
(2) enrichment culture
By the fresh pericarpium zanthoxyli bungeani leaf of above-mentioned cleaning and sterilizing process, grind in mortar and smash to pieces, be inoculated in the nutrient agar medium solid medium containing 1% potassium bichromate, cultivate 5 ~ 7 days under temperature 28 ~ 30 DEG C of constant incubators, observe and have bacterium colony to grow;
(3) dull and stereotyped coating
Picking characteristic of bacteria bacterium colony in bacterium colony after above-mentioned nutrient agar medium solid medium is cultivated, gradient dilution rear plate coating in stroke-physiological saline solution, is placed in temperature 28 ~ 30 DEG C of constant incubators cultivation constant incubators that after 5 ~ 7 days, picking bacterial colony is placed on temperature 28 ~ 30 DEG C in the line of nutrient agar medium solid medium upper flat plate and cultivates 5 days;
(4) primary dcreening operation
Picking characteristic of bacteria bacterium colony in bacterium colony after above-mentioned nutrient agar medium solid medium is cultivated, gradient dilution rear plate coating in stroke-physiological saline solution, being placed in temperature 28 ~ 30 DEG C of constant incubators cultivates after 5 ~ 7 days, picking bacterial colony is in the line of nutrient agar medium solid medium upper flat plate, and the constant incubator being placed in 28 ~ 30 DEG C is cultivated 5 ~ 7 days; Select single bacterium colony that above step obtains, streak inoculation is in new nutrient agar medium solid medium;
(5) multiple sieve
Step (3) obtained strains is inoculated in nutrient agar medium liquid nutrient medium, fermentation culture in temperature 28 ~ 30 DEG C of shaking tables, supernatant liquor is got after fermented liquid is centrifugal, by 2, after 2-joins nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts free radical scavenging experimental evaluation fermented liquid oxidation-resistance, by the strongest strain number of fermented liquid anti-oxidant activity and preservation;
(6) physiological and biochemical property and 16S rDNA analyze
Physiological and biochemical property and 16S rDNA analyze, and determine the kind of bacterium.
4. the screening purification process of Folium Zanthoxyli Bungeani endophyte according to claim 2, it is characterized in that enrichment medium is: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, agar 1.5%, pH is 7.0 ~ 7.6, adds 1% potassium bichromate after sterilizing; Nutrient agar medium solid medium is: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, and agar 1.5%, pH is 7.0 ~ 7.6; Nutrient agar medium liquid nutrient medium is: peptone 1%, yeast extract 0.5%, and sodium-chlor 1%, pH is 7.0 ~ 7.6.
5. the purposes of Folium Zanthoxyli Bungeani endophyte according to claim 1, is characterized in that the antioxygenation of this endophyte fermented liquid comprises the following steps:
(1) nutrient agar medium liquid nutrient medium is sub-packed in triangular flask, with the bacterial classification after a small amount of purifying of transfering loop picking in culturing bottle, 48h is cultivated in temperature 28 ~ 30 DEG C of shaking tables, the centrifugal 10min of fermentation liquor 4000r/min, get supernatant liquor, concentrating under reduced pressure postlyophilization obtains Folium Zanthoxyli Bungeani endophyte fermentation broth extract;
(2) by Folium Zanthoxyli Bungeani endophyte fermentation broth extract respectively with 18.75mg/mL, 12.5mg/mL, 6.25mg/mL, 3.125mg/mL, the concentration of 1.25mg/mL adds 96 orifice plates, add-on is 50 μ L/ holes, then in 96 orifice plates, adds 200 μ L ABTS working fluids, under lucifuge condition, carry out free radical scavenging experiment, reaction 30min, measures the absorbancy of each orifice plate reaction solution immediately, and calculates clearance rate and IC after reaction terminates
50.
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| CN112961746A (en) * | 2021-03-09 | 2021-06-15 | 四川大学 | Brewing method for improving flavor characteristics of cider |
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| CN111647531A (en) * | 2020-06-03 | 2020-09-11 | 青岛北方茶仓茶文化有限公司 | Siamese bacillus LBP for increasing antioxidant activity, and fermentation product and application thereof |
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| CN111718868A (en) * | 2020-06-03 | 2020-09-29 | 青岛北方茶仓茶文化有限公司 | Edinglake terribacillus LBX capable of improving free radical scavenging capacity and fermentation product and application thereof |
| CN111718868B (en) * | 2020-06-03 | 2022-03-11 | 青岛北方茶仓茶文化有限公司 | Edinglake terribacillus LBX capable of improving free radical scavenging capacity and fermentation product and application thereof |
| CN111647531B (en) * | 2020-06-03 | 2022-03-11 | 青岛北方茶仓茶文化有限公司 | Siamese bacillus LBP for increasing antioxidant activity, and fermentation product and application thereof |
| CN112961746A (en) * | 2021-03-09 | 2021-06-15 | 四川大学 | Brewing method for improving flavor characteristics of cider |
| CN113387861A (en) * | 2021-06-16 | 2021-09-14 | 四川大学 | Method for preparing pyrrole-2-carboxylic acid by using pepper endogenous bacillus cereus and application |
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| CN104531555B (en) | 2017-02-01 |
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