CN104988102B - The cultural method of one bacillus amyloliquefaciens and its application - Google Patents
The cultural method of one bacillus amyloliquefaciens and its application Download PDFInfo
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Abstract
The invention discloses the cultural method of a bacillus amyloliquefaciens and its application, belongs to microbial technology field, a bacillus amyloliquefaciens PC2, and deposit number is CCTCC NO:M2015435.Beneficial effects of the present invention：Bacillus amyloliquefaciens strain zymotic fluid provided by the invention has broad-spectrum antibacterial active, tomato can be effectively controlled to adopt rear reaping hook fruit rot bacterium, it is fresh-keeping for tomato postharvest storage, reduction is lost after adopting, and a kind of effective, nontoxic, noresidue, environment-friendly biological control new way are provided with safety to improve product quality.The strain fermentation culture medium requirement of the present invention is simple, and cost is cheap, and cultural method is simple, and the antistaling agent preparation technology being related to is simple, and application method is simple, suitably popularization and application in production.
Description
Technical field
The invention belongs to microbial technology field, and in particular to the cultural method of a bacillus amyloliquefaciens and its should
With.
Background technology
Fruits and vegetables are the important non-staple food of people's life, are the second largest agricultural products for being only second to grain, and food industry
Important source material.Tomato is important vegetable variety, and tomato postharvest storage disease species are more, and wherein tomato reaping hook fruit corruption bacterium is
In recent years the fruit postharvest pathogenic fungi found, mainly infects ripening fruits, causes the soft rotten corruption of fruit, is caused in circulation stores and transports
Larger economic loss.
China fruits and vegetable stock and preserving freshness more developed country falls behind relatively, and main use sprays the progress of chemical synthesis bactericide
Preserving fruit and vegetable utilizing.Long-term use can not only produce pathogen or the spoilage organisms resistance to the action of a drug, and fruit chemical residual is to health structure
Into potential threat.The material-flow methods such as other air conditioned storage, refrigeration are also the important technology of preserving fruit and vegetable utilizing, but its investment is big,
Operating cost is high, can not a wide range of popularization and application.Therefore, the fresh-keeping skill of postharvest fruit and vegetable safe efficient, conveniently, inexpensive is sought
Art method is significant.
Fresh-keeping microorganism is a kind of safe and nontoxic, effective biological way of keeping fresh, is had a extensive future.Using Antagonistic Fungi
Preparation carries out the fresh-keeping harmful effect that can reduce chemical synthesis bactericide to human health to fruits and vegetables, and effectively prevents phytopathy
The resistance to the action of a drug of opportunistic pathogen.The key core of Advance in Microbial Preservation Technology is to obtain efficient, safety, wide spectrum, stable antagonistic bacterial strains.Mesh
Preceding screened both at home and abroad obtains the larger antagonistic bacterial strains of many application potentials, including bacterium, yeast, mould etc..But
The very few of application is commercially produced.Wherein bacterial strain antimicrobial spectrum is narrower, and production cost is high, application environment condition is harsh,
The production application that the factors such as preventive effect is unstable limit a large amount of antagonistic strains is promoted.
Bacillus amyloliquefaciens are a kind of non-pathogenic bacterias being widely present in environment, and it can be produced in metabolic process
A variety of abundant Substances, there is extensive suppression bacterium and fungi activity, obtain Europe in agricultural production
The accreditation of the food security committee.The country studies it and synthesized using being concentrated mainly on to substitute in agricultural product production process at present
Agricultural chemicals is to all kinds of bacteriums and the disease-resistant growth-promoting function of fungal disease.Such as：Prevent and treat the bacillus amyloliquefaciens of wheat root disease
EA19 and its preparation (CN200910273498.8), a bacillus amyloliquefaciens and its application (CN201410389174.1).
Also have in recent years and post-harvest fresh-keeping is carried out to fruits and vegetables such as citrus, olive, fig, dendrobium candidums using bacillus amyloliquefaciens
Research.Current domestic patent of invention and document have no the phase that rear sickle-like bacteria fruit rot is adopted using bacillus amyloliquefaciens control tomato
Close research and application report.
Most of bacillus amyloliquefaciens production Substance culture medium nutritional requirement is of a relatively high, as NB culture mediums,
LB culture mediums etc..Therefore, production cost can be reduced by obtaining the more simple antagonistic strain of nutritional requirement, promote antagonistic strain related
The exploitation and popularization of product.
Prior art one related to the present invention
The technical scheme of prior art one
Agro-chemicals control is mainly used for tomato reaping hook fruit rot at present, conventional medicament has 25% pyrimethanil wettable
500~600 times of liquid of pulvis, 50% 500 times of liquid of Ghana's gold wettable powder, 70% thiophanate methyl wettable powder 1000~
1200 times of liquid etc..Field full leaf spraying, emphasis sprinkling fruit.
The shortcomings that prior art one
Though Agro-chemicals control can effectively control disease, chemical residual influences health, food security be present
Risk.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides the cultural method of a bacillus amyloliquefaciens and its application,
Can solve chemical agent fruit and vegetable preservation can produce chemical residual, the problem of so as to influence health.
To solve problem above, the technical solution adopted by the present invention is as follows, a bacillus amyloliquefaciens PC2, and preservation is compiled
Number it is CCTCC NO:M2015435.
Another technical solution used in the present invention is as follows, the method for bacterial strain PC2 Substance fermented and cultureds, including
Following steps：
S1 antagonistic strains isolate and purify
Respectively increasing bacterium is carried out with beef extract-peptone fluid nutrient medium, potato dextrose broth in 28~37 DEG C
24~48h is cultivated, microorganism separation is carried out using dilution-plate method, beef extract albumen is respectively adopted in wherein bacterium and fungi
On peptone fluid nutrient medium and potato dextrose broth flat board, 30~37 DEG C and 25~30 DEG C of incubator trainings are respectively placed in
2~3d is supported, according to features such as the forms, color, transparency of bacterium colony, the obvious single bacterium colony of picking morphological differences rule pure
Change, 4 DEG C of inclined-plane saves backup；
S2 antagonistic strains screen
It is prepared by S2.1 zymotic fluids
Bacterium bacterial strain to be sieved is inoculated in potato dextrose broth, 120 under the conditions of 30~37 DEG C~
180rpm shake flask fermentation 24~48h of culture, take nutrient solution zymotic fluid 10000~12000rpm refrigerated centrifuges 10 under the conditions of 4 DEG C
~20min, collect supernatant zymotic fluid and obtain sterile ferment filtrate through 0.22 μm of filtering with microporous membrane, 4 DEG C save backup；
S2.2 antagonisms spectrum measure
Antagonistic strain is screened using Oxford cup double-layer agar technique, indicator bacteria is made with staphylococcus aureus, with 10~15mL
NA culture mediums make lower floor's culture medium, uniformly place 4 Oxford cups that sterilized after solidification at the 2.5cm of flat board center, then every
The LB culture mediums that individual 10~15mL of flat-plate inverted is mixed with staphylococcus aureus make upper strata culture medium；100~150 are added in Oxford cup
μ L of supernatant ferment filtrates, put the presence or absence of 30~37 DEG C of 24~36h of culture, observation inhibition zone and measure its diameter；
S3 antagonism fermented liquids antagonism spectrum measure
Using Oxford cup double-layer agar technique determine antagonistic strain to staphylococcus aureus, Escherichia coli, micrococcus luteus,
The fungistatic effect of the bacterial strains such as bacillus subtilis, brewer's yeast, rhodotorula, tomato reaping hook fruit rot bacterium, tomato pythium rot bacterium, carefully
37 DEG C of bacterium is incubated, and 28 DEG C of fungi is incubated, and the presence or absence of inhibition zone is observed after 24h and measures its diameter, each place
Reason is in triplicate；
In the bacterial strain that separation obtains, filter out that bacteriostatic activity is most strong, one plant of most wide bacterial strain PC2 of antimicrobial spectrum.
Another technical solution used in the present invention is as follows, the preparation side of the bacteriostatic activity zymotic fluid antistaling agents of PC2 containing bacterial strain
Method, the sterile bacterial strain fermentation liquor distilled water of the PC2 prepared and following compositions are prepared by following concentration：Zymotic fluid 30~
50%, soybean protein isolate 3%~5%, phytic acid 0.1%~0.2%, sodium carboxymethylcellulose 0.5%~2%, calcium chloride 1%
~3%.
As preferred：Zymotic fluid 45%, soybean protein isolate 4%, phytic acid 0.15%, sodium carboxymethylcellulose 1.5%, chlorine
Change calcium 2.0%.
Another technical solution used in the present invention is as follows, the application method of the antistaling agent, choose outward appearance not damaged, into
Ripe degree and tamato fruit of the same size, 0.5~2min is handled with 1~2% liquor natrii hypochloritis's surface sterilization, then use sterilized water
Cleaning is multiple, dries；4 about 2cm gash are uniformly stabbed in the fruit equatorial plane with sterilizing toothpick, immerse the above-mentioned guarantor prepared
25~45s in fresh liquid, then immerse the tomato reaping hook fruit rot bacterium spore suspension (10 prepared6Individual/mL) 30s, or will directly protect
Fresh liquid sparges fruit surface.
Preservation information：A kind of bacillus amyloliquefaciens PC2, it is preserved in Wuhan University's Chinese Typical Representative on July 7th, 2015
Culture collection, deposit number are CCTCC NO:M2015435.
Beneficial effects of the present invention are as follows：
1. in order to overcome tomato to adopt the food safety risk that rear reaping hook fruit rot chemical prevention and control method is brought, there is provided one plant of battalion
Foster to require more simple, antagonism composes wide bacillus amyloliquefaciens, and bacterial strain fermentation liquor heat endurance, pH stability, ultraviolet
Stability is stronger；
2. providing the cultural method of bacillus amyloliquefaciens bacteriostatic activity zymotic fluid, this method is simple and convenient, and culture medium is matched somebody with somebody
Cheap and easy to get, the proper scale fermenting and producing in side, reduces production cost；
3. providing the bacterial strain control tomato method of adopting rear reaping hook fruit rot, this method is simple to operate, without special installation,
Safely, effectively, conveniently.
In summary, Bacillus amyloliquefaciens strain zymotic fluid provided by the invention has broad-spectrum antibacterial activity, can be effective
Control tomato adopt rear reaping hook fruit rot bacterium, be tomato postharvest storage it is fresh-keeping, reduction adopt after be lost, be improve product quality with peace
A kind of effective, nontoxic, noresidue, environment-friendly biological control new way are provided entirely.
The strain fermentation culture medium requirement of the present invention is simple, and cost is cheap, and cultural method is simple, prepared by the antistaling agent being related to
Technique is simple, and application method is simple, suitably application in production.
Brief description of the drawings
Fig. 1 is bacteriostasis design sketch of the bacterial strain PC2 without fermented liquids to tomato reaping hook fruit rot bacterium；
Fig. 2 is Antagonistic Fungi PC2 colonial morphology design sketch；
Fig. 3 is Antagonistic Fungi PC2 Gram's staining morphing effects figures；
Fig. 4 is the bacterial strain PC2 systematic evolution tree schematic diagrames based on 16S rDNA.
Embodiment
For the objects, technical solutions and advantages of the present invention are more clearly understood, develop simultaneously embodiment referring to the drawings, right
The present invention is described in further details.
Embodiment：
1 antagonistic strain isolates and purifies
Bee bread is carried out with beef extract-peptone fluid nutrient medium, potato dextrose broth in 28~37 DEG C respectively
24~48h of Zengjing Granule, microorganism separation is carried out using dilution-plate method, beef extract is respectively adopted in wherein bacterium and fungi
On peptone fluid nutrient medium (NA) and potato dextrose broth (PDA) flat board, 30~37 DEG C and 25 are respectively placed in
~30 DEG C of 2~3d of incubator culture.The features such as form, color, transparency according to bacterium colony, the obvious single bacterium of picking morphological differences
Drop into row line purifying, 4 DEG C of inclined-plane saves backup.56 plants of bacteriums are obtained altogether.
2 antagonistic strains screen
It is prepared by 2.1 zymotic fluids
Bacterium bacterial strain to be sieved is inoculated in potato dextrose broth, 120 under the conditions of 30~37 DEG C~
180rpm shake flask fermentation 24~48h of culture, take nutrient solution zymotic fluid 10000~12000rpm refrigerated centrifuges 10 under the conditions of 4 DEG C
~20min, collect supernatant zymotic fluid and obtain sterile ferment filtrate through 0.22 μm of filtering with microporous membrane, 4 DEG C save backup.
2.2 antagonisms spectrum measure
Antagonistic strain is screened using Oxford cup double-layer agar technique, indicator bacteria is made with staphylococcus aureus, with 10~15mL
NA culture mediums make lower floor's culture medium, uniformly place 4 Oxford cups that sterilized after solidification at the 2.5cm of flat board center, then every
The LB culture mediums that individual 10~15mL of flat-plate inverted is mixed with staphylococcus aureus make upper strata culture medium.100~150 are added in Oxford cup
μ L of supernatant ferment filtrates, put the presence or absence of 30~37 DEG C of 24~36h of culture, observation inhibition zone and measure its diameter.Obtain altogether
17 plants of antagonistic strains to staphylococcus aureus with stronger bacteriostatic activity.
3 antagonism fermented liquid antagonisms spectrum measure
Using Oxford cup double-layer agar technique determine antagonistic strain to staphylococcus aureus, Escherichia coli, micrococcus luteus,
The fungistatic effect of the bacterial strains such as bacillus subtilis, brewer's yeast, rhodotorula, tomato reaping hook fruit rot bacterium, tomato pythium rot bacterium.Carefully
37 DEG C of bacterium is incubated, and 28 DEG C of fungi is incubated, and the presence or absence of inhibition zone is observed after 24h and measures its diameter, each place
Reason is in triplicate.
In 17 plants of bacterial strains that separation obtains, there is that one plant of bacterial strain PC2 bacteriostatic activity is most strong, and antimicrobial spectrum is most wide.Table 1 is it
In PC2 bacterial strains antimicrobial spectrum, wherein the inhibition zone to tomato reaping hook fruit rot bacterium is 21.5mm (see Fig. 1).
The bacterial strain PC2 of table 1 bacteriostatic activity
4 antagonistic strain PC2 identification
By morphological feature, PC2 bacterial strains are accredited as solution starch bud by physio-biochemical characteristics and 16S rDNA sequence analyses
Spore bacillus (Bacillus amyloliquefaciens).
After PC2 cultivates 36h on PDA plate, single bacterium colony is formed, subcircular to ellipse, edge is irregular to be incised, table
Face fold, center straw hat shape protuberance, translucent, bacterium colony is creamy white (Fig. 2)；There is mycoderm to be formed when fluid nutrient medium is static.Mirror
Inspection cell is in rod-short or ellipticity, Gram-positive, produces gemma (Fig. 3).
The bacterial strain PC2 physio-biochemical characteristics of table 2
Note："+" represents reaction and is positive, and "-" represents reaction and is negative.
Fig. 4 is PC2 systematic evolution trees, it is seen that PC2 and 3 bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) gather in same branch.Bacterial strain PC2 is accredited as solution starch gemma by combining form, physiological and biochemical property
Bacillus.
5 bacterial strain fermentation liquor bacteriostatic activity stability
5.1 heat endurance
The sterile ferment filtrates of 10mL are taken in sterile tool plug test tube, the respectively water-bath at 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C
0.5h, 1h, 2h, 3h, high pressure steam sterilization 20min at 121 DEG C.Using tomato reaping hook fruit rot bacterium as instruction after room temperature to be restored
Bacterium, the bacteriostatic activity of the ferment filtrate of each processing is detected using Oxford cup double-layer agar technique, with the average diameter size of inhibition zone
Reflect the heat endurance of antibacterial substance.Each processing three is parallel, using the ferment filtrate of not thermally treated (25 DEG C of room temperatures) as
Blank control.
Table 3 is bacterial strain fermentation liquor thermal stability results.It can be seen that zymotic fluid heat endurance is good, 121 DEG C of processing 20min, it presses down
Bacterium activity still keeps original 71.1%.
Inhibition zone (mm) of the treatment of different temperature without fermented liquid of table 3 to tomato reaping hook fruit rot bacterium
5.2 ph stability
The sterile ferment filtrates of 10mL are taken in sterile tool plug test tube, respectively with 1.0mol/L HCl solutions or 1.0mol/L
NaOH solution is adjusted to different pH (pH1, pH3, pH5, pH8, pH10, pH12, pH14).Preliminary fermentation is recalled to after standing 2h at 25 DEG C
Filtrate pH7.0, is quantified with sterilized water, it is ensured that it is identical often to handle antibacterial substance concentration.Using tomato reaping hook fruit rot bacterium as indicator bacteria,
The bacteriostatic activity of the ferment filtrate of each processing is detected using Oxford cup double-layer agar technique, is reflected with the average diameter size of inhibition zone
The ph stability of antibacterial substance.Each processing three is parallel, is compared with original ferment filtrate (pH7.0).
Table 4 is bacterial strain fermentation liquor ph stability result.It can be seen that zymotic fluid has stronger absolute acid stability, particularly strong
Under the conditions of sour (pH1~4), bacteriostatic activity is held essentially constant.
The soda acid of table 4 handles inhibition zone (mm) of the without fermented liquid to tomato reaping hook fruit rot bacterium
5.3 UV stable
The sterile ferment filtrates of 10mL are taken in sterile tool plug test tube, irradiation 10 of being uncapped respectively under 15W uviol lamps at 30cm,
20、30、40、50、60min.Using tomato pythium rot bacterium as indicator bacteria, the fermentation of each processing is detected using Oxford cup double-layer agar technique
The bacteriostatic activity of filtrate, the ultraviolet stability of antibacterial substance is reflected with the average diameter size of inhibition zone.Each processing three is flat
OK, blank control is used as using the ferment filtrate without ultraviolet processing.
Table 5 is bacterial strain fermentation liquor UV stable result.It can be seen that zymotic fluid ultraviolet irradiation stability is stronger, ultraviolet photograph
10~60min is penetrated not make significant difference to zymotic fluid bacteriostatic activity.
Inhibition zone (mm) of the ultraviolet treatment with irradiation without fermented liquid of table 5 to tomato reaping hook fruit rot bacterium
6 antistaling agents of bacterial strain fermentation liquor containing PC2 determine to the prevention effect of tomato postharvest decay
The preparation of 6.1 antistaling agents：The sterile bacterial strain fermentation liquors of PC2 prepared by above-mentioned experiment are pressed with distilled water and following compositions
Following concentration are prepared：Zymotic fluid 30~50%, soybean protein isolate 3%~5%, phytic acid 0.1%~0.2%, carboxymethyl cellulose
Plain sodium 0.5%~2%, calcium chloride 1%~3%.Preferable formula：Zymotic fluid 45%, soybean protein isolate 4%, phytic acid
0.15%, sodium carboxymethylcellulose 1.5%, calcium chloride 2.0%.
6.2 fresh-keeping experiments：Outward appearance not damaged, maturity and tamato fruit of the same size are chosen, with 1~2% hypochlorous acid
Sodium solution surface sterilization handles 0.5~2min, then multiple with sterile water wash, dries.It is equal in the fruit equatorial plane with sterilizing toothpick
It is even to stab 4 about 2cm gash, 25~45s in the above-mentioned fresh-keeping liquid prepared is immersed, then immerse the tomato reaping hook fruit prepared
Maize ear rot bacterium spore suspension (106Individual/mL) 30s, or fresh-keeping liquid is directly sparged into fruit surface, control group only immerses fruit corruption bacterium spore
Sub- suspension, often handle 10 fruits.With carton is loaded after handling well, humidity 95% is put into, the ambient storage that 25 DEG C of temperature, one week
Fruit rot index and lesion diameter are counted afterwards.
Table 6
Table 6 is visible, and no matter composite preservative of the invention uses leaching fruit or spray pattern effectively tomato can be controlled to adopt
Fruit rot evil occurs afterwards, reduces fruit rot index and lesion diameter, extends storage period.
One of ordinary skill in the art will be appreciated that embodiment described here is to aid in reader and understands this hair
Bright implementation, it should be understood that protection scope of the present invention is not limited to such especially statement and embodiment.Ability
The those of ordinary skill in domain can be made according to these technical inspirations disclosed by the invention it is various do not depart from essence of the invention its
Its various specific deformations and combination, these deformations and combination are still within the scope of the present invention.
Claims (3)
- A 1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) PC2, it is characterised in that described one plant Bacillus amyloliquefaciens PC2 deposit number is CCTCC NO:M2015435.
- 2. a kind of preparation method of the antistaling agent of the bacteriostatic activities of PC2 containing bacterial strain zymotic fluid, it is characterised in that the bacterial strain PC2 is A bacillus amyloliquefaciens PC2 described in claim 1, by the sterile bacterial strain fermentation liquor distilled water of the PC2 prepared with Composition is stated to prepare by following concentration：Zymotic fluid 30~50%, soybean protein isolate 3%~5%, phytic acid 0.1%~0.2%, carboxylic Sodium carboxymethylcellulose pyce 0.5%~2%, calcium chloride 1%~3%.
- 3. the preparation method described in claim 2, it is characterised in that zymotic fluid 45%, soybean protein isolate 4%, phytic acid 0.15%, sodium carboxymethylcellulose 1.5%, calcium chloride 2.0%.
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| CN106434418A (en) * | 2016-08-05 | 2017-02-22 | 四川农业大学 | Culture medium for bacillus amyloliquefaciens culture medium for producing bacteriostatic active substances |
| CN106167777A (en) * | 2016-08-05 | 2016-11-30 | 四川农业大学 | A kind of cultural method of the bacillus amyloliquefaciens producing Substance |
| CN106479934B (en) * | 2016-11-23 | 2019-02-26 | 河北省科学院生物研究所 | A kind of ash arrhizus bacteria antagonistic strain and its screening technique and application |
| CN109984189B (en) * | 2019-04-29 | 2022-07-01 | 山西农业大学 | Fresh-cut fruit and vegetable fresh-keeping agent compounded with bacteriostats produced by bacillus licheniformis, bacillus atrophaeus and bacillus amyloliquefaciens |
| CN114015614B (en) * | 2021-12-02 | 2022-07-22 | 四川农业大学 | Actinomycete strain SCAUT013 and application thereof |
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