CN103951736B - A kind of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product and preparation method thereof - Google Patents
A kind of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product and preparation method thereof.It is by bacillus amyloliquefaciens NCPSJ7 fermented liquid collected by centrifugation supernatant liquor, and fermented supernatant fluid, through ammonium sulfate precipitation and desalination and concentration by ultrafiltration, then through anion exchange chromatography and desalination and concentration by ultrafiltration, and carries out spraying dry and obtains antibacterial protein product.Antibacterial protein product prepared by aforesaid method, its main component is: 24kDa and 36kDa two kinds of antibacterial proteins, the total content of these two kinds of antibacterial proteins accounts for more than 60% of spray powder quality.The rehydration of this antibacterial protein product is functional, by force water-soluble, and bacteriostasis property is good simultaneously, has industrial applications prospect, can be applied to the fields such as antiseptic preservation of fruits and vegetables, foodstuff additive, fish medicine, veterinary drug.
Description
Technical field
The invention belongs to microbial fermentation solution separation and purification preparing technical field, particularly a kind of bacillus amyloliquefaciens antibacterial protein product and preparation method thereof.
Background technology
Plant diseases is a global problem, causes huge financial loss.The statistic data of Food and Argriculture OrganizationFAO (FAO) shows, the underproduction loss that Plant diseases causes every year on average is about the 10%-15% of ultimate production, wherein accounts for 80% by fungus-caused Plant diseases.The main chemical bactericide that uses controls Plant diseases at present, although chemical bactericide can reduce or remit Plant diseases effectively, chemical bactericide also causes negative impact.The residual health that threaten people and animals of chemical agent, endangers environment.Therefore the novel method of alternative chemical bactericide is researched and developed to control the importance that Plant diseases is research at present.
Along with continuous enhancing and the application of new and high technology in agriculture field of people's environmental consciousness, beneficial organism is utilized more and more to earn widespread respect to control Plant diseases.The research of China to biological control starts from the initial stage fifties, there is investigator that excellent antagonistic microbe thalline is applied to the biological control of Plant diseases at present, and obtain remarkable effect, but antagonistic microbe produces antagonist Quality Research and mostly rests on laboratory stage, main research be that Antagonistic Fungi produces the kind of antimicrobial substance, the separation purification method of antimicrobial substance and antifungal mechanism etc., only have minority such as nisin, tennecetin etc. to obtain Commercialization application.
Bacillus amyloliquefaciens BacillusamyloliquefaciensNCPSJ7 system present invention applicant is separated and obtains in Shandong Province's Laiwu City ginger field soil, be deposited in China typical culture collection center at present, deposit number: CCTCCNO:M2013098 (number of patent application: 201310112580.9, denomination of invention: broad-spectrum antimicrobial Bacillus amyloliquefaciens strain and application thereof).The thalline of this bacterial strain and fermented liquid have the effect of the microbial Plant diseasess of pathogenic such as antagonism peach head mold canker, cucumber fusarium axysporum, graw mold of tomato, jujube anthrax, pears penicilliosis, apple brown rot, alternaria leaf spot of apple, watermelon blight and the food-borne pathogens such as fruit vegetable post-harvest diseases and antagonism streptococcus aureus, Salmonella paratyphi A, Vibrio parahemolyticus, yeast and spoilage organism.The document does not study its various preparation further.
Summary of the invention
The object of the invention is to study further bacillus amyloliquefaciens BacillusamyloliquefaciensNCPSJ7, a kind of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product and preparation method thereof is provided.The separating and purifying technologies such as ammonium sulfate precipitation, ultrafiltration, column chromatography and spray drying technology organically combine by the present invention, provide a kind of purifying of simple, efficient, low cost and prepare the method for bacillus amyloliquefaciens NCPSJ7 antibacterial protein.Prepared antibacterial protein product purity is higher, water-soluble good with bacteriostasis property, has industrial applications prospect.
Technical scheme of the present invention is: a kind of preparation method of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product, is characterized in that, comprise the following steps:
(1) ammonium sulfate precipitation
By bacillus amyloliquefaciens NCPSJ7 fermented liquid in 4 DEG C, the centrifugal 10-20 minute of 12000-13000rpm, collect fermented supernatant fluid, adding solid ammonium sulfate to ammonium sulfate concentrations is while stirring 50-60% saturation ratio, 4 DEG C of hold over night; The centrifugal 10-20 minute of 12000-13000rpm, collecting precipitation, the phosphate buffered saline buffer adding 20-50mMpH6.5-8.0 redissolves, and in the centrifugal 10-20 minute of 12000-13000rpm, collects supernatant liquor, be antibacterial protein crude extract;
(2) desalination and concentration by ultrafiltration
The antibacterial protein crude extract of step (1) is placed in ultra-filtration equipment, in 4 DEG C, adopt the ultrafiltration membrance filter of 5000Da, retain the phosphate buffered saline buffer that part adds 20-50mMpH6.5-8.0, repeat ultrafiltration 2-4 time, collection retains part, is the antibacterial protein crude extract after desalination and concentration, concentrates 10-60 doubly relative to fermented supernatant fluid;
(3) being further purified of antibacterial protein
Adopt DEAESepharoseFastFlow anion exchange chromatography, by the antibacterial protein crude extract loading after step (2) desalination and concentration, then phosphate buffered saline buffer wash-out 1 column volume of 50mMpH6.5-7.0 is first adopted, adopt the NaCl solution of 0.25M (adopting the phosphate buffered saline of a 50mMpH6.5-7.0) wash-out 1-2 column volume again, wash-out is except foreigh protein removing; Finally adopt the NaCl solution of 0.25-0.6M (adopting the phosphate buffered saline of a 50mMpH6.5-7.0) gradient elution 4-5 column volume, collect 2 elution peaks, be antibacterial protein sample;
(4) desalination and concentration by ultrafiltration again
The antibacterial protein sample of step (3) is placed in ultra-filtration equipment, in 4 DEG C, adopts the ultrafiltration membrance filter of 5000Da, retain the phosphate buffered saline buffer that part adds appropriate 20-50mMpH6.5-8.0, repeat 2-4 time, collect and retain part, be the antibacterial protein sample after desalination and concentration;
(5) spraying dry
By the antibacterial protein sample after step (4) desalination and concentration inlet temperature 130 DEG C, under material flow 3.3mL/min condition, carry out spraying dry, collect spray powder, be antibacterial protein product.
Antibacterial protein product prepared by aforesaid method, molecular weight is 20-40kDa, and its main component is: 24kDa and 36kDa two kinds of antibacterial proteins, the total content of these two kinds of antibacterial proteins accounts for more than 60% of spray powder quality.Its morphological appearance is brownish-yellow powder.The experiment proved that, this antibacterial protein has broad spectrum antibacterial, especially obvious to the bacteriostatic action of pears penicilliosis pathogenic bacteria ACCC37275 and ring rot of apple pathogenic bacteria LW182.
Present invention also offers and a kind ofly adopt above-mentioned antibacterial protein product to be the oranges and tangerines antisepsis antistaling agent of main component, it is characterized in that, be made up of the raw material of following weight parts: bacillus amyloliquefaciens NCPSJ7 antibacterial protein 50-90 part, amino-oligosaccharide 10-20 part, APG 3-5 part, Fructus Zanthoxyli oil 1-2 part, glycerine 2-3 part.
Preparation and using method: fully mixed by above-mentioned raw materials, dry with after the 400-4000 times of aqueous solution spraying during use.
Amino-oligosaccharide, also referred to as the special oligochitosan of agricultural, oligochitosan has direct repression to a certain degree to various plants pathogenic bacteria.There is trace, efficient, low cost, the feature such as nuisanceless, significant to China's Agricul tural Sustain able Development.
Alkyl glycoside, APG for short, synthesized by renewable resources natural fat alcohol and glucose, it is the more comprehensive new non-ionic surfactants of a kind of performance, have the characteristic of conventional nonionic and anion surfactant concurrently, having high surface, good ecological security and intermiscibility, is internationally recognized first-selection " green " functional surfactant.There is stronger broad spectrum antibiotic activity, product thickening effectiveness significantly, be easy to dilution, gel-free phenomenon, easy to use.
Fructus Zanthoxyli oil: the oil of directly being squeezed out by Semen Pericarpium Zanthoxyli, has certain expelling parasite and moisture retention.
Above-mentioned antisepsis antistaling agent has good anticorrosion, fresh-keeping effect to the oranges and tangerines that low temperature and normal temperature are placed, obviously can reduce the generation of the diseases such as Penicillium italicum, green mould, anthrax, blossom-end rot, preserve 30 days at normal temperatures, healthy fruit >=95%, there is good fresh-keeping effect simultaneously, compared with not adding the oranges and tangerines of any antisepsis antistaling agent, fruit percentage of water loss at least have dropped 20%.
Simultaneously, antibacterial protein provided by the invention and amino-oligosaccharide, alkyl glycoside etc. composite after, there is obvious antibacterial synergism, compared with not adding the formula of antibacterial protein, the healthy fruit that oranges and tangerines are preserved 30 days at normal temperatures at least can improve 25%, and the security of this formula is high, does not develop immunity to drugs, so have a good application prospect.
NCPSJ7 antibacterial protein provided by the invention is the Extracellular metabolism of bacillus amyloliquefaciens NCPSJ7, DNA extration is simple, only centrifugal for fermented liquid removing thalline need be the study containing antibacterial protein, then by the protein in ammonium sulfate precipitation high efficiency extraction fermented supernatant fluid, play the effect of extracting concentrated antibacterial protein, the ultra-filtration membrane in suitable aperture is selected to carry out desalination and concentration by ultrafiltration subsequently, serve desalination simultaneously, concentrate and remove the effect of lower molecular weight foreign protein, then the anion exchange properties of such antibacterial protein is utilized, adopt anion exchange chromatography pointedly, by continuing to optimize parameter, obtain the elution requirement be suitable for, antibacterial protein is purified to greatest extent, substantially increase the purity of product.Production efficiency spray drying technology that is higher, relative inexpensiveness is finally selected to prepare antibacterial protein pulvis, the anti-microbial activity of antibacterial protein is easily subject to the impact of extraneous extreme environment, especially under the hot conditions of drying process with atomizing, easily make anti-microbial activity reduce even inactivation, thus the drying process with atomizing parameter of antibacterial protein to the anti-microbial activity of product and quality most important.
The beneficial effect of this formula is: bacillus amyloliquefaciens antibacterial protein product preparation method technique provided by the invention is simple, cost is lower, prepared antibacterial protein product is mainly containing 24kDa and 36kDa two kinds of antibacterial proteins, the total content of these two kinds of antibacterial proteins accounts for more than 60% of spray powder quality, the rehydration of spray powder is functional, by force water-soluble, the solubleness under normal temperature in every 100mL water is greater than 50g.Bacteriostasis property is good simultaneously, has industrial applications prospect, can be applied to the fields such as antiseptic preservation of fruits and vegetables, foodstuff additive, fish medicine, veterinary drug.
Accompanying drawing explanation
Fig. 1 is DEAE anion-exchange chromatography figure; Peak 1:0.05M phosphate buffered saline buffer elution peak; Peak 2:0.25MNaCl elution peak; Peak 3:0.25-0.40MNaCl elution peak; Peak 4:0.40-0.60MNaCl elution peak;
Fig. 2 is that the bacteriostatic activity of each elution peak of DEAE anionresin detects; 1: antibacterial protein crude extract; 2:DEAE anion exchange layer elution peak 1; 3:DEAE anion exchange layer elution peak 2; 4:DEAE anion exchange layer elution peak 3; 5:DEAE anion exchange layer elution peak 4;
Fig. 3 is that antibacterial protein product is to pears penicilliosis pathogenic bacteria ACCC37275 bacteriostatic action;
Fig. 4 is the bacteriostatic action of antibacterial protein product to ring rot of apple pathogenic bacteria LW182.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further described, so that those skilled in the art understand the present invention.
Embodiment 1: the preparation of antibacterial protein crude extract
The Culture and fermentation conditions of bacillus amyloliquefaciens BacillusamyloliquefaciensNCPSJ7 is shown in the embodiment 2 of Chinese patent application 201310112580.9 (denomination of invention: broad-spectrum antimicrobial Bacillus amyloliquefaciens strain and application thereof), finally by shake-flask culture 6 days, obtain fermented liquid.
By 10L fermented liquid in 4 DEG C, centrifugal 20 minutes of 13000rpm, collect fermented supernatant fluid, slowly add solid ammonium sulfate to 60% saturation ratio while stirring, 4 DEG C of hold over night, centrifugal 20 minutes of 13000rpm, collecting precipitation, the phosphate buffer 1 L adding 50mMpH7.0 redissolves, and in 13000rpm centrifugal 20 minutes, collect supernatant liquor, be antibacterial protein crude extract;
1L antibacterial protein crude extract is placed in ultra-filtration equipment, in 4 DEG C, adopt the ultrafiltration membrance filter of 5000Da, when retaining some residual volume and being about 0.1L, add the phosphate buffered saline buffer of 50mMpH7.0 to original volume, repeat 3 times, retain some residual liquid 0.2L for the last time, collect and retain part, be the antibacterial protein crude extract after desalination and concentration, relative to the volume of fermented supernatant fluid, cycles of concentration is 50 times.
Embodiment 2: the DEAESepharoseFastFlow anion exchange chromatography of antibacterial protein
Antibacterial protein crude extract nature flow velocity loading, adopts phosphate buffered saline buffer wash-out 1 column volume of 50mMpH7.0, then adopts 0.25MNaCl solution (adopting the phosphate buffered saline of a 50mMpH7.0) wash-out 1-2 column volume to remove foreigh protein removing; Then adopt the 0.25-0.6MNaCl of 4-5 column volume (adopting the phosphate buffered saline of 50mMpH7.0) solution gradient wash-out, obtain two elution peaks (0.25-0.4MNaCl elution peak, 0.4-0.6MNaCl elution peak).Elution speed 1.0ml/min, determined wavelength 280nm; Collect two elution peaks of the phosphate buffered saline buffer elution peak of 50mMpH7.0,0.25MNaCl elution peak and 0.25-0.6MNaCl, its tomographic map is shown in Fig. 1, adopts Odontothrips loti to detect the bacteriostatic activity of each elution peak to watermelon blight pathogenic bacteria, sees Fig. 2.
As can be seen from Figure 2: in Fig. 2, the antibacterial circle diameter of antibacterial protein crude extract is about 20mm, the antibacterial circle diameter of elution peak 3,4 is about 16 and 20mm respectively, and elution peak 1,2 does not have obvious inhibition zone.As can be seen from Figure 2: finally adopt the fungistatic effect of two elution peaks of the 0.25-0.6MNaCl solution gradient wash-out of 4-5 column volume good, collected, as antibacterial protein sample.The sample at peak 3 and peak 4 is through SDS-PAGE detected through gel electrophoresis simultaneously, and its molecular weight is respectively 36kDa and 24kDa.
Embodiment 3: the preparation of antibacterial protein spray powder
Above-mentioned antibacterial protein sample is placed in ultra-filtration equipment, in 4 DEG C, adopts the ultrafiltration membrance filter of 5000Da, retain part and add the phosphate buffered saline buffer of 50mMpH6.5-8.0 to original volume, repeat 3 times, collect and retain part, be the antibacterial protein sample after desalination and concentration;
Antibacterial protein crude extract sample is inlet temperature 130 DEG C, and temperature out 65 DEG C, carries out spraying dry under material flow 3.3mL/min condition, collects spray powder, is antibacterial protein product.After testing, its main component is: 24kDa and 36kDa two kinds of antibacterial proteins, the total content of these two kinds of antibacterial proteins accounts for spray powder quality more than 65%.
Embodiment 4: the bacteriostatic activity of antibacterial protein product detects
Adopt Odontothrips loti to detect bacteriostatic activity, take antibacterial protein spray powder 100mg in 10ml centrifuge tube by precision electronic balance, add 5ml ultrapure water and redissolve, degerming with 0.22 μm of water system membrane filtration.
Be inoculated in PDA flat board after being activated by pears penicilliosis pathogenic bacteria ACCC37275 and ring rot of apple pathogenic bacteria LW182, cultivate 7 days for 28 DEG C; Put into the 1.5mLEP pipe of sterilizing under aseptic condition with the punching of diameter 7mm punch tool, shred by sterile razor blade and add the mixing of 1mL sterilized water, obtained pathogenic bacteria pityrosporion ovale suspension is for subsequent use.
Get 100 μ L pathogenic bacteria pityrosporion ovale suspensions in sterilized blank plate, pour the mixing of PDA substratum into, after to be solidified, 1 Oxford cup is put in each flat board, add above-mentioned prepared antibacterial protein redissolution liquid 200 μ L respectively, in 28 DEG C of mold incubators, cultivate the antibacterial circle diameter observed afterwards for 3-7 days around the cup of Oxford.Fig. 3 and Fig. 4 is shown in the bacteriostatic action of pears penicilliosis pathogenic bacteria ACCC37275 and ring rot of apple pathogenic bacteria LW182.The antibacterial circle diameter of Fig. 3 is about 25mm, and the antibacterial circle diameter in Fig. 4 is about 19mm.Compare the fermented liquid after except thalline fungistatic effect (to the antibacterial circle diameter of pears penicilliosis pathogenic bacteria ACCC37275 and ring rot of apple pathogenic bacteria LW182 be about respectively 13.5 and 11mm), its fungistatic effect obviously strengthens.
Embodiment 5 oranges and tangerines antisepsis antistaling agent
Formula (weight part): bacillus amyloliquefaciens NCPSJ7 antibacterial protein 75 parts, amino-oligosaccharide 16 parts, APG 4.5 parts, Fructus Zanthoxyli oil 1.5 parts, glycerine 3 parts.
Preparation and using method: fully mixed by above-mentioned raw materials, dry with after the aqueous solution spraying of 2000 times during use.
Adopt this citrus preservative to preserve 30 days at normal temperatures, healthy fruit >=95%, have good fresh-keeping effect simultaneously, compared with not adding the oranges and tangerines of any antisepsis antistaling agent, fruit percentage of water loss declines >=20%.
Claims (5)
1. a preparation method for bacillus amyloliquefaciens NCPSJ7 antibacterial protein product, is characterized in that, comprise the following steps:
(1) ammonium sulfate precipitation
By centrifugal for bacillus amyloliquefaciens NCPSJ7 fermented liquid, collect fermented supernatant fluid, adding solid ammonium sulfate to ammonium sulfate concentrations is while stirring 50-60% saturation ratio, 4 DEG C of hold over night; Centrifugal, collecting precipitation, the phosphate buffered saline buffer adding 20-50mMpH6.5-8.0 redissolves, centrifugal, collects supernatant liquor, is antibacterial protein crude extract;
(2) desalination and concentration by ultrafiltration
The antibacterial protein crude extract of step (1) is placed in ultra-filtration equipment, in 4 DEG C, adopts the ultrafiltration membrance filter of 5000Da, retain the phosphate buffered saline buffer that part adds 20-50mMpH6.5-8.0, repeat ultrafiltration 2-4 time, collect and retain part, be the antibacterial protein crude extract after desalination and concentration;
(3) being further purified of antibacterial protein
Adopt DEAESepharoseFastFlow anion exchange chromatography, by the antibacterial protein crude extract loading after step (2) desalination and concentration, then phosphate buffered saline buffer wash-out 1 column volume of 50mMpH6.5-7.0 is first adopted, adopt NaCl solution wash-out 1-2 column volume of 0.25M again, wash-out is except foreigh protein removing; Finally adopt NaCl solution gradient elution 4-5 column volume of 0.25-0.6M, collect 2 elution peaks, be antibacterial protein sample;
(4) desalination and concentration by ultrafiltration again
The antibacterial protein sample of step (3) is placed in ultra-filtration equipment, in 4 DEG C, adopts the ultrafiltration membrance filter of 5000Da, retain the phosphate buffered saline buffer that part adds 20-50mMpH6.5-8.0, repeat 2-4 time, collect and retain part, be the antibacterial protein sample after desalination and concentration;
(5) spraying dry
By the antibacterial protein sample after step (4) desalination and concentration inlet temperature 130 DEG C, under material flow 3.3mL/min condition, carry out spraying dry, collect spray powder, be the antibacterial protein product that molecular weight is 20-40kDa.
2. the preparation method of a kind of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product as claimed in claim 1, it is characterized in that, the NaCl solution of described step (3) all adopts the phosphate buffered saline of 50mMpH6.5-7.0.
3. the preparation method of a kind of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product as claimed in claim 1, it is characterized in that, the centrifugal of described step (1) is: the centrifugal 10-20 minute of 12000-13000rpm.
4. the bacillus amyloliquefaciens NCPSJ7 antibacterial protein product in claim 1-3 prepared by any one, it is characterized in that, main component is: two kinds of antibacterial proteins of 24kDa and 36kDa, and the total content of these two kinds of antibacterial proteins accounts for more than 60% of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product population.
5. the oranges and tangerines antisepsis antistaling agent that is main component with bacillus amyloliquefaciens NCPSJ7 antibacterial protein product according to claim 4, it is characterized in that, be made up of the raw material of following weight parts: bacillus amyloliquefaciens NCPSJ7 antibacterial protein product 50-90 part, amino-oligosaccharide 10-20 part, APG 3-5 part, Fructus Zanthoxyli oil 1-2 part, glycerine 2-3 part.
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| CN106544309B (en) * | 2017-01-17 | 2019-05-31 | 华南师范大学 | One bacillus amyloliquefaciens and its antibacterial protein and the application in prawn is fresh-keeping |
| CN110669812B (en) * | 2019-11-18 | 2023-01-20 | 河北省微生物研究所有限公司 | Application of bacillus amyloliquefaciens in preventing and treating cucumber anthracnose |
| CN113462614B (en) * | 2021-08-04 | 2023-04-07 | 青岛百奥安泰生物科技有限公司 | Deep-sea-derived Bacillus belgii RS6-14 and extraction method and application of antibacterial extract produced by same |
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