CN103589655B - Bacillus amyloliquefaciens HRH317 and the preparation method of antibacterial substance thereof - Google Patents
Bacillus amyloliquefaciens HRH317 and the preparation method of antibacterial substance thereof Download PDFInfo
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Abstract
From soil separation screening bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HRH.317 bacterial strain is in preservation on March 15 in 2013, preserving number CGMCC No.7314.This bacterial strain is cultivated on beef-protein medium, and thalline forms a large amount of spore, and before formation spore, thalline is shaft-like, is arranged as single or chaining, Gram-positive；Cultivating 3 days, colonial morphology milky, diameter about 6.75mm, there is fold on surface, and optical signature is opaque, have projection, circle, edge is wavy or petal-shaped；Mycoderma is had to generate during Liquid static culture；Carry out sequence analysis with the 16S rDNA gene order of known bacterial strain, reached 99% 100% with the homology of bacillus amyloliquefaciens, shown from the systematic evolution tree of 16S rDNA sequence, with bacillus amyloliquefaciens (Bacillus amyloliquefaciens Strain BGP20) similarity the highest, through Physiology and biochemistry identify, be accredited as bacillus amyloliquefaciens (Bacillus amyloliquefaciens)。
Description
Technical field
The present invention relates to microorganism field, concretely relate to the bacillus amyloliquefaciens new strains of a strain broad-spectrum antibacterial
And the preparation method of produced antibacterial substance.
Background technology
In recent years, food sanitation safe problem ratio is more prominent, and the people looks forward to being able to eat quality-assured food, the exhaling of safety food
Sound is more and more higher, but food-safety problem is never well solved.In food-safety problem topmost problem it
One is exactly food apoilage, adds up according to the World Health Organization (WHO), the warp that the annual whole world is only caused because of food apoilage
Ji loss the most hundreds of hundred million dollars.Food apoilage is often as what microorganism caused, going rotten especially of fruit and vegetable food
Caused by fungus, and fungus control is mainly based on chemosynthesis preservative and chemical pesticide at present, the ring that chemical pesticide causes
Human health has been caused potential threat by environment pollution, it would be highly desirable to have new, efficient microbiological antiseptic and pesticide new variety
Or novel form puts on market, to meet the demand in this field.
Summary of the invention
The present invention provides a bacillus amyloliquefaciens HRH.317 and the preparation method of antibacterial substance thereof, this antibacterial substance
Have that antimicrobial spectrum is wide, bacteriostatic activity is high, multiple fungus and antibacterial can be suppressed.
Technical scheme is as follows:
One bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) HRH.317 bacterial strain, in 2013
March 15 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC
No.7314, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.
Described bacillus amyloliquefaciens HRH.317 separation screening from field soil obtains.By following ingredients
Mass/volume part composition beef-protein medium (Carnis Bovis seu Bubali cream 0.5 part, peptone 1 part, NaCl 0.5 part, agar
1.5 parts, distilled water 100 parts, the pH value of culture fluid is 7.2-7.4) upper 37 DEG C of constant temperature culture 24 hours, thalline forms a large amount of bud
Spore, forms thalline shaft-like (0.9 ~ 1.4 × 2.7 ~ 3.9 μm) before spore, is arranged as single or chaining (see figure 1), Gram-positive；
Cultivating 3 days, colonial morphology is: milky, diameter about 6.75mm, rough has fold, and optical signature is opaque, there have to be convex
Rise, circle, edge are wavy or petal-shaped (see figure 2)；During Liquid static culture, Mycoderma is had to generate；Percutaneous puncture-inoculation cultivates thalline
In the culture medium of test tube, cloud diffusion growth, shows there is mobility；Identifying through Physiology and biochemistry, its feature is shown in Table 1；
The physiological and biochemical property of table 1 bacillus amyloliquefaciens HRH.317
| Testing index | Result | Testing index | Result |
| Nitrate reduction | + | Citrate utilizes | + |
| Catalase | + | Phenylalanine deaminase | — |
| V-P measures | + | Produce indole | — |
| Starch Hydrolysis | + | Indolepyruvic acid (IPA) measures | — |
| Salt tolerance and need salt | Lecithinase | + | |
| 2%NaCl | + | Glucose oxidative fermentation | Fermented type |
| 5%NaCl | + | Gelatin liquefaction | — |
| 7%NaCl | + | Casein hydrolyzes | + |
| 10%NaCl | — | Hydrolysis hippuric acid | + |
| C.I. 13020. (M.R) | — |
Note: "+" represent that this index is for the positive；" " represents that this index is for feminine gender.
The GeneBank data base of the 16S rDNA sequence inputting NCBI of gained bacillus amyloliquefaciens HRH.317 will be extracted
In, carry out sequence analysis with the 16S rDNA gene order of known bacterial strain, it is known that the 16S of bacillus amyloliquefaciens HRH.317
RDNA sequence and bacillus amyloliquefaciens (Bacillus amyloliquefaciens ) homology the highest, reached 99%-
100% (being shown in Table 2).
Table 2 bacillus amyloliquefaciens HRH.317 sequence homology comparison result
Show (see Fig. 3) from the systematic evolution tree of 16S rDNA sequence, the 16S rDNA sequence of bacillus amyloliquefaciens with
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens Strain BGP20) similarity the highest.Therefore, by it
Be accredited as bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and named bacillus amyloliquefaciens
(Bacillus amyloliquefaciens ) HRH.317。
The preparation method of the produced antibacterial substance of bacillus amyloliquefaciens HRH.317 bacterial strain, comprises the steps:
(1) fermentation liquid is prepared by the fermentation culture fluid after bacillus amyloliquefaciens HRH.317 inoculation to sterilizing
In, 36-38 DEG C, under the conditions of 160 r/min, cultivate 20-24 hour, obtain fermentation liquid；
(2) prepare after fermentation liquor 5000r/min prepared by (1) is centrifuged 20 min by antibacterial substance liquid and take supernatant,
Obtain liquid antibacterial substance.
Fermentation liquid of the present invention is made up of the raw material of following masses/parts by volume: Carnis Bovis seu Bubali cream 0.5 part, peptone 1
Part, NaCl 0.5 part, distilled water 100 parts, pH 7.2.
By the characteristic of the antibacterial substance that following test display bacillus amyloliquefaciens HRH.317 of the present invention is produced
1, Antibacterial Activity and scope of restraining fungi
The antibacterial substance that the bacillus amyloliquefaciens HRH.317 of the present invention is produced is tested by Odontothrips loti, will 15mL
Beef-protein medium is laid in diameter 90mm, in the culture dish of deep 20mm, is placed on level table standing solidification, then takes
It is diluted to 106On indicator bacteria bacteria suspension 0.2 mL of cfu/mL beef-protein medium flat board after above-mentioned solidification, so
Being smoothened by indicator bacteria bacteria suspension with aseptic spreading rod afterwards, culture dish lid is opened and is made filtrated air circulate about 40min, bacterium bacterium to be instructed
Suspension by beef-protein medium flat board fully absorb the most dry after, by internal diameter 6mm, external diameter 8mm, the aseptic Oxford of high 10mm
Cup tweezers are positioned on flat board gently, and 200 μ L bacillus amyloliquefaciens HRH.317 fermented liquid supernatant liquid are added Oxford cup
After, spread 12 hours under the conditions of 4-5 DEG C, after fermentation liquid fully spreads, cultivate 3-5 days (when indicator bacteria is antibacterial 3 in 30 DEG C
My god；When indicator bacteria is yeast or mycete 5 days) observe inhibition zone situation.Result of the test shows, the antibacterial substance of this antibacterial has relatively
High Antibacterial Activity, its titer be 128AU/mL(indicator bacteria be bacillus cereus (Bacillus.sp) H108), and antimicrobial spectrum is wide,
Multiple food-borne pathogens can be suppressed and cause Gram-positive and the gram negative bacteria of food apoilage, can suppress
Gram positive bacterial strain include: bacillus subtilis (Bacillus subtilis), Bacillus coagulans (Bacillus coagulans), staphylococcus aureus (Staphyloccocus aureus)；Quenchable gram negative strain includes:
Escherichia coli (Escherichia coli), Salmonella enteritidis (Salmonella enteritidis)；May also suppress as made
Brewer yeast, Rhodothece glutinis, wine yeast, Pear black spot bacterium Kikuchi rod method is mould, Fructus Vitis viniferae downy mildew, aspergillus niger, penicillium sp etc. are multiple very
Bacterium.Result of the test refers to table 3, Fig. 4-Figure 16.
The antimicrobial spectrum of the produced antibacterial substance of table 3 bacillus amyloliquefaciens HRH.317
| Sequence number | Indicator bacteria | Source | Antibacterial circle diameter (meansigma methods: Mm) |
| 1 | Bacillus cereus (Bacillus.sp) H108 | Food Science and Engineering institute of Agricultural University Of Shanxi bioengineering dept Preservation | 31 |
| 2 | Bacillus coagulans (Bcillus coagulans)CICC 10144 | Chinese industrial Microbiological Culture Collection administrative center | 19 |
| 3 | Bacillus subtilis (Bcillus coagulans)CMCC 63501-2aq | Chinese medicine Microbiological Culture Collection administrative center | 15 |
| 4 | Staphylococcus aureus (Staphlococcus aureus)CMCC 26003-5a1 | Chinese medicine Microbiological Culture Collection administrative center | 17 |
| 5 | Colon bacillus (Escherichia coli)CMCC 44102-3a11 | Chinese medicine Microbiological Culture Collection administrative center | 15 |
| 6 | Salmonella enteritidis (Salmonella enteritidis) | Food Science and Engineering institute of Agricultural University Of Shanxi bioengineering dept Preservation | 15 |
| 7 | Saccharomyces cerevisiae (Saccharomyces cerevisiae)H226 | Food Science and Engineering institute of Agricultural University Of Shanxi bioengineering dept Preservation | 16 |
| 8 | Rhodothece glutinis (Rhodotorula) H215 | Food Science and Engineering institute of Agricultural University Of Shanxi bioengineering dept Preservation | 17 |
| 9 | Wine yeast (Saccharomyces ellipsoideus)H222 | Food Science and Engineering institute of Agricultural University Of Shanxi bioengineering dept Preservation | 16 |
| 10 | Pear black spot bacterium Kikuchi rod method (Alternaria kikuchi-ana) | Food Science and Engineering institute of Agricultural University Of Shanxi bioengineering dept Preservation | 24.5 |
| 11 | Fructus Vitis viniferae downy mildew (Plasmopara uiticola) | Food Science and Engineering institute of Agricultural University Of Shanxi bioengineering dept Preservation | 20.5 |
| 12 | Aspergillus niger (Aspergillus niger) | Food Science and Engineering institute of Agricultural University Of Shanxi bioengineering dept Preservation | 23 |
| 13 | Penicillium sp (Penicillium.sp) | Food Science and Engineering institute of Agricultural University Of Shanxi bioengineering dept Preservation | 20 |
2, acid suppression is got rid of test, is got rid of hydrogen peroxide test and protease digestion test
The liquid antibacterial substance produced with the bacillus amyloliquefaciens HRH.317 of the present invention, is indicator bacteria with penicillium sp, carries out
The acid suppression of the antibacterial substance that bacillus amyloliquefaciens HRH.317 produces is got rid of test, is got rid of hydrogen peroxide test and protease
Digestion trial, test method and result are as follows:
(1) acid suppression eliminating test method: the liquid bateriostatics that bacillus amyloliquefaciens HRH.317 of the present invention is produced
The pH 1mol/L HCl of matter and 1mol/L NaOH is adjusted to be 7, makees bacteriostatic test with penicillium sp for indicator bacteria Odontothrips loti.Examination
Testing result and see Figure 17, in figure, a is before acid suppression is got rid of, antibacterial circle diameter 14；B is after acid suppression is got rid of, antibacterial circle diameter
13.7, between the two without significant difference.
(2) hydrogen peroxide test method is got rid of: take the liquid that bacillus amyloliquefaciens HRH.317 of the present invention produced antibacterial
Material, adds catalase, at 37 DEG C after water-bath 4 hours, does bacteriostatic test with penicillium sp for indicator bacteria Odontothrips loti.Test
Result is shown in Figure 18, before in figure, a is for getting rid of hydrogen peroxide, and antibacterial circle diameter 14.5；After b is for getting rid of hydrogen peroxide, inhibition zone is straight
Footpath 2.2, significant difference (P ＜ 0.05) between the two.
(3) protease digestion test method: take the liquid bateriostatics that bacillus amyloliquefaciens HRH.317 of the present invention is produced
Matter, the suitableeest action pH 5-9 first adjusting pH to be E.C. 3.4.21.64 with 1mol/L NaOH and 1mol/L HCL, add egg by 0.5mol/L amount
White enzyme K, constant temperature water bath 2 hours at 37 DEG C, then pH value is recalled to original numerical value, do with penicillium sp for indicator bacteria Odontothrips loti
Bacteriostatic test.Result of the test is shown in Figure 19, before in figure, a is protease K digesting, and antibacterial circle diameter 16；After b is protease K digesting,
Antibacterial circle diameter 14, significant difference (P ＜ 0.05) between the two.
Result of the test shows: fermented supernatant fluid gets rid of test through acid suppression, shows the antibacterial of this bacterial strain fermentation liquor supernatant
Activity is not from acid；After hydrogen peroxide ferment treatment, antibacterial circle diameter is reduced significantly；After E.C. 3.4.21.64 process, inhibition zone is
Reduce.Show bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) antibacterial substance produced by HRH.317
It is probably hydrogen peroxide and a kind of protide or polypeptides matter.
3, antimicrobial stability
(1) antibacterial substance that the bacillus amyloliquefaciens HRH.317 of the present invention is produced has good heat stability
Antibacterial substance heat treatment 30min at 60-90 DEG C that the bacillus amyloliquefaciens HRH.317 of the present invention is produced,
Its fungistatic effect does not has significant change；Heat treatment 15min at 100 DEG C, its bacteriostatic activity is the 67% of original (CK)；At 100 DEG C
Lower heat treatment 30min, bacteriostatic activity is the 57% of original (CK)；Heat treatment 15min at 121 DEG C, bacteriostatic activity still can keep original
(CK) 57%(is shown in Table 4).
The heat stability of the produced antibacterial substance of table 4 bacillus amyloliquefaciens HRH.317
| Process | 60℃ | 70℃ | 80℃ | 90℃ | 100℃ | 121℃ | |
| Heat treatment 15min antibacterial circle diameter (mm) | 20 | 19 | 18 | 18 | 14 | 12 | |
| Heat treatment 30min antibacterial circle diameter (mm) | 19 | 18 | 16.5 | 15 | 12 | ||
| CK antibacterial circle diameter (mm) | 21 |
Therefore, the antibacterial substance that the bacillus amyloliquefaciens HRH.317 of the present invention is produced has good heat stability, should
Feature determines that it has the biggest using value in food industry produces.
(2) antibacterial substance that the bacillus amyloliquefaciens HRH.317 of the present invention is produced has good ph stability
The antibacterial substance produced by the bacillus amyloliquefaciens HRH.317 of the present invention processes in the range of pH 5-9
30min, the difference of fungistatic effect is the most notable；Processing 30min under the conditions of pH 2, bacteriostatic activity still can retain original activity (CK)
77%；Processing 30min under the conditions of pH 11, bacteriostatic activity still can retain the 82% of original activity (CK), be shown in Table 5.
The ph stability of the produced antibacterial substance of table 5 bacillus amyloliquefaciens HRH.317
| Process | pH2 | pH3 | pH4 | pH5 | pH6 | pH7 | pH8 | PH9 | pH10 | pH11 | |
| Antibacterial circle diameter (mm) | 17 | 18 | 19 | 22 | 21 | 22 | 21.5 | 23 | 19 | 18 | |
| CK | 22 |
Show that the bacteriostatic activity of this antibacterial substance has the subject range of wider pH.Therefore can be in slant acidity, neutrality, meta-alkali
Property condition in use.
(3) the antibacterial substance Surfactant that the bacillus amyloliquefaciens HRH.317 of the present invention is produced has good
Stability
Employing Polyethylene Glycol, Tween 80, three kinds of surfactants of polysorbas20 are as test material, with three kinds of surfactants
The antibacterial substance bacterium solution produced with the bacillus amyloliquefaciens HRH.317 of the present invention respectively is mixed homogeneously, and is formulated as surface activity
The concentration of agent is the mixed liquor of 1%, and comparison (CK) replaces surfactant with distilled water, does antibacterial for indicator bacteria with penicillium sp respectively
Experiment, cultivates 4 hours at 37 DEG C, surveys its antibacterial circle diameter.Result of the test shows, processes through Polyethylene Glycol, Tween 80 and compares
It is not significantly different from；After polysorbas20 processes, the activity of the produced antibacterial substance of bacillus amyloliquefaciens HRH.317 substantially reduces,
For the 76.6% of comparison, it is shown in Table 6.
The stability of table 6 bacillus amyloliquefaciens HRH.317 produced antibacterial substance Surfactant
| Surfactant | CK | Polysorbas20 | Tween 80 | Polyethylene Glycol |
| Antibacterial circle diameter (mm) | 23.5 | 18 | 23 | 22 |
Beneficial effects of the present invention
The antibacterial substance that the bacillus amyloliquefaciens HRH.317 of the present invention produces has higher Antibacterial Activity, its titer
It is bacillus cereus H108 for 128AU/mL(indicator bacteria), antimicrobial spectrum is wide, can suppress the multiple gram causing food apoilage
Positive bacteria, such as bacillus cereus, bacillus subtilis, Bacillus coagulans, staphylococcus aureus etc., it is possible to suppression causes food
Putrid and deteriorated gram negative bacteria, such as escherichia coli and Salmonella enteritidis；May also suppress such as saccharomyces cerevisiae, Rhodothece glutinis, Portugal
The multiple funguses such as grape brewer yeast, Pear black spot bacterium Kikuchi rod method mould, Fructus Vitis viniferae downy mildew, aspergillus niger, penicillium sp.This antibacterial substance has
Polyethylene Glycol, Tween 80, three kinds of surfactants of polysorbas20 are also had the most steady by good heat stability and ph stability
Qualitative.The antibacterial substance that the bacillus amyloliquefaciens HRH.317 of the present invention produces has as the natural antiseptic agent of fruit and vegerable and food
There is good application prospect.
Accompanying drawing explanation
Fig. 1 is the thalli morphology of bacillus amyloliquefaciens HRH.317 of the present invention.
Fig. 2 is the colonial morphology of bacillus amyloliquefaciens HRH.317 of the present invention.
Fig. 3 is to grow tree to 16S rDNA sequential system relevant for bacillus amyloliquefaciens HRH.317 of the present invention.
Fig. 4 be the produced antibacterial substance of bacillus amyloliquefaciens HRH.317 of the present invention suppression bacillus cereus (Bacillus.sp)
H108 result.
Fig. 5 is the liquid antibacterial substance suppression Bacillus coagulans that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Bcillus coagulans) CICC 10144 result.
Fig. 6 is the liquid antibacterial substance suppression bacillus subtilis that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Bcillus coagulans) CMCC 63501-2aq result.
Fig. 7 is the liquid antibacterial substance suppression staphylococcus aureus that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Staphlococcus aureus) CMCC 26003-5a1 result.
Fig. 8 is the liquid antibacterial substance suppression colon bacillus that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Escherichia coli) CMCC 44102-3a11 result.
Fig. 9 is the liquid antibacterial substance suppression Salmonella enteritidis that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Salmonella enteritidis) result.
Figure 10 is the liquid antibacterial substance suppression saccharomyces cerevisiae that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Saccharomyces cerevisiae) H226 result.
Figure 11 is the liquid antibacterial substance suppression Rhodothece glutinis that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Rhodotorula) H215 result.
Figure 12 is the liquid antibacterial substance suppression wine yeast that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Saccharomyces ellipsoideus) H222 result.
Figure 13 is the liquid antibacterial substance suppression Pear black spot bacterium Kikuchi that bacillus amyloliquefaciens HRH.317 of the present invention is produced
Rod method (Alternaria kikuchi-ana) result.
Figure 14 is the liquid antibacterial substance suppression Fructus Vitis viniferae downy mildew that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Plasmopara uiticola) result.
Figure 15 is the liquid antibacterial substance suppression aspergillus niger that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Aspergillus niger) result.
Figure 16 is the liquid antibacterial substance suppression penicillium sp that bacillus amyloliquefaciens HRH.317 of the present invention is produced
(Penicillium) result.
Figure 17 is that test knot is got rid of in the liquid antibacterial substance acid suppression that bacillus amyloliquefaciens HRH.317 of the present invention is produced
Really.
Figure 18 is that the liquid antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention is produced gets rid of hydrogen peroxide test knot
Really.
Figure 19 is the liquid antibacterial substance protease K digesting test knot that bacillus amyloliquefaciens HRH.317 of the present invention is produced
Really.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1
One bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) HRH.317, in 2013 3
Within 15th, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center by the moon and (is called for short CGMCC, address: Beijing
North Star West Road, Chaoyang District, city 1 institute 3, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is for solving starch
Bacillus cereus ( Bacillus amyloliquefaciens), preserving number is: CGMCC No.7314.
Described bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) HRH.317 is from field soil
In be not intended to separation screening and obtain.At beef-protein medium (Carnis Bovis seu Bubali cream 0.5g, peptone 1.0g, NaCl 0.5g, fine jade
Fat 1.5g, water 100mL,PH 7.2,121 DEG C, sterilizing 20 minutes) upper 37 DEG C of constant temperature culture 24 hours, thalline forms a large amount of spore,
Form thalline shaft-like (0.9 ~ 1.4 × 2.7 ~ 3.9 μm) before spore, be arranged as single or chaining, Gram-positive；Cultivate 3 days, bacterium
The form that falls is: milky, diameter about 6.75mm, rough has fold, and optical signature is opaque, have projection, circle, limit
Edge is wavy or petal-shaped；During Liquid static culture, Mycoderma is had to generate；Percutaneous puncture-inoculation cultivates thalline cloud in the culture medium of test tube
Vaporific diffusion growth, shows there is mobility.
Identify through Physiology and biochemistry and include: the utilization of nitrate reduction, catalase, V-P mensuration, Starch Hydrolysis, citrate,
Phenylalanine deaminase, salt tolerance and need salt, produce indole, indolepyruvic acid (IPA) mensurations, lecithinase, glucose aoxidize
Fermentation, gelatin liquefaction, casein hydrolysis, the hydrolysis test such as hippuric acid, C.I. 13020. (M.R) and 16S rDNA Phylogenetic Analysis, really
This bacterial strain fixed be bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) 。
Embodiment 2
Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) system of antibacterial substance produced of HRH.317
Preparation Method, first bacillus amyloliquefaciens HRH.317 is inoculated into fermentation culture fluid (Carnis Bovis seu Bubali cream 0.5g, peptone 1.0g,
NaCl 0.5g, water 100ml, pH 7.2,121 DEG C, sterilizing 20min) in, at 37 DEG C, under the conditions of 160 r/min, cultivate 24 little
Time, obtain fermentation liquid；Then take supernatant after fermentation liquor 5000r/min of preparation being centrifuged 20 min, obtain crude liquid
Antibacterial substance.
Claims (2)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HRH.317 bacterial strain, is characterized in that,
Being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 15th, 2013, preserving number is
CGMCC No.7314。
Bacillus amyloliquefaciens the most according to claim 1 (Bacillus amyloliquefaciens) HRH.317
Bacterial strain, the system of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the produced antibacterial substance of HRH.317 bacterial strain
Preparation Method, comprises the steps:
(1) prepare fermentation liquid to be connect by bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HRH.317 bacterial strain
Kind in the fermentation culture fluid after sterilizing, 36-38 DEG C, under the conditions of 160 r/min, cultivate 20-24 hour, fermented
Liquid；
(2) prepare after fermentation liquor 5000r/min prepared by (1) is centrifuged 20 min by antibacterial substance liquid and take supernatant, to obtain final product
Liquid antibacterial substance.
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