CN103589655A - Preparation method for Bacillus amyloliquefaciens HRH 317 and antibacterial substances thereof - Google Patents
Preparation method for Bacillus amyloliquefaciens HRH 317 and antibacterial substances thereof Download PDFInfo
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Abstract
从土壤中分离筛选的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HRH.317菌株于2013年3月15日保藏，保藏号CGMCCNo.7314。该菌株在牛肉膏蛋白胨培养基上培养，菌体形成大量芽孢，形成芽孢前菌体呈杆状，排列为单个或成链，革兰氏阳性；培养3天，菌落形态乳白色，直径约6.75mm，表面有褶皱，光学特征不透明、有凸起、圆形、边缘呈波状或花瓣状；液体静置培养时有菌膜生成；与已知菌株的16SrDNA基因序列进行同源性比对，与解淀粉芽孢杆菌的同源性达到了99%-100%，从16SrDNA序列的系统进化树显示，与解淀粉芽孢杆菌（Bacillus amyloliquefaciens strain BGP20）的相似性最高，经生理生化鉴定，鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。
The HRH.317 strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) isolated and screened from the soil was preserved on March 15, 2013, with the preservation number CGMCCNo.7314. The strain is cultured on beef extract peptone medium, the bacteria form a large number of spores, and the bacteria before the formation of the spores are rod-shaped, arranged in single or in chains, Gram-positive; after 3 days of cultivation, the colony is milky white, with a diameter of about 6.75mm , with wrinkles on the surface, optical features are opaque, raised, round, and the edges are wavy or petal-shaped; when the liquid is cultured statically, a bacterial film is formed; the homology comparison with the 16SrDNA gene sequence of the known strain is compared with the solution The homology of Bacillus amyloliquefaciens reaches 99%-100%. According to the phylogenetic tree of 16SrDNA sequence, it has the highest similarity with Bacillus amyloliquefaciens strain BGP20. After physiological and biochemical identification, it is identified as Bacillus amyloliquefaciens Bacillus ( Bacillus amyloliquefaciens ).
Description
Technical field
The present invention relates to microorganism field, specifically relate to the new bacterial strain of bacillus amyloliquefaciens of a strain broad-spectrum antibacterial and the preparation method of the antibacterial substance that produces thereof.
Background technology
In recent years, food sanitation safe problem is more outstanding, and the cry that the people looks forward to being able to eat quality-assured food, safety food is more and more higher, but food-safety problem is never well solved.In food-safety problem, one of topmost problem is exactly food spoilage ，Ju the World Health Organization (WHO) statistics, and the annual whole world is only because the financial loss that food spoilage causes just reaches hundred million dollars of hundreds ofs.Food spoilage normally causes due to microorganism, going rotten of fruit and vegetable food caused by fungi especially, and at present fungi control mainly be take chemosynthesis sanitas and chemical pesticide as main, the environmental pollution that chemical pesticide causes causes potential threat to human health, urgently have new, microbiological antiseptic and pesticide new variety or novel form are put on market efficiently, to meet the demand in this field.
Summary of the invention
The invention provides the preparation method of a bacillus amyloliquefaciens HRH.317 and antibacterial substance thereof, this antibacterial substance has that antimicrobial spectrum is wide, bacteriostatic activity is high, can suppress multiple fungus and bacterium.
Technical scheme of the present invention is as follows:
One bacillus amyloliquefaciens (
bacillus amyloliquefaciens) HRH.317 bacterial strain, on March 15th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.7314, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Described bacillus amyloliquefaciens HRH.317 separation screening from field soil obtains.The beef-protein medium forming the mass/volume part by following ingredients (0.5 part of extractum carnis, 1 part of peptone, 0.5 part of NaCl, 1.5 parts, agar, 100 parts of distilled water, the pH value of nutrient solution is 7.2-7.4) upper 37 ℃ of constant temperature culture 24 hours, thalline forms a large amount of gemma, form the front thalline shaft-like (0.9 ~ 1.4 * 2.7 ~ 3.9 μ m) of gemma, be arranged as single or chaining (see figure 1), Gram-positive; Cultivate 3 days, colonial morphology is: oyster white, and diameter is about 6.75mm, roughly has a fold, and optical signature is opaque, have projection, circle, edge to be wavy or petal-shaped (see figure 2); During the standing cultivation of liquid, there is mycoderm to generate; Percutaneous puncture-inoculation is cultivated thalline cloud diffusion growth in the substratum of test tube, and showing has mobility; Through Physiology and biochemistry, identify, its feature is in Table 1;
The physiological and biochemical property of table 1 bacillus amyloliquefaciens HRH.317
| Detect index | Result | Detect index | Result |
| Nitrate reduction | + | Citrate trianion utilizes | + |
| Catalase | + | Phenylalanine deaminase | — |
| V-P measures | + | Produce indoles | — |
| Starch Hydrolysis | + | Indolepyruvic acid (IPA) is measured | — |
| Salt tolerance and need salt | ? | Lecithinase | + |
| 2%NaCl | + | Glucose oxidase fermentation | Fermented type |
| 5%NaCl | + | Gelatine liquefication | — |
| 7%NaCl | + | Casein hydrolysis | + |
| 10%NaCl | — | Hydrolysis urobenzoic acid | + |
| ? | ? | Methyl red (M.R) | — |
Note: "+" represents that this index is positive; "-" represents that this index is negative.
The 16S rDNA sequence of extracting gained bacillus amyloliquefaciens HRH.317 is inputted in the GeneBank database of NCBI, carry out sequence analysis with the 16S rDNA gene order of known bacterial strain, the 16S rDNA sequence of known bacillus amyloliquefaciens HRH.317 and bacillus amyloliquefaciens (
bacillus amyloliquefaciens) homology the highest, reached 99%-100% (in Table 2).
Table 2 bacillus amyloliquefaciens HRH.317 sequence homology comparison result
From the systematic evolution tree of 16S rDNA sequence, show (seeing Fig. 3), the 16S rDNA sequence of bacillus amyloliquefaciens and bacillus amyloliquefaciens (
bacillus amyloliquefaciensstrain BGP20) similarity is the highest.Therefore, be accredited as bacillus amyloliquefaciens (
bacillus amyloliquefaciens), and called after bacillus amyloliquefaciens (
bacillus amyloliquefaciens) HRH.317.
The preparation method of bacillus amyloliquefaciens HRH.317 antibacterial substance that bacterial strain produces, comprises the steps:
(1) prepare fermented liquid by bacillus amyloliquefaciens HRH.317 inoculation to the fermentation after sterilizing with in nutrient solution, under 36-38 ℃, 160 r/min conditions, cultivate 20-24 hour, obtain fermented liquid;
(2) prepare the fermented liquid that antibacterial substance liquid prepares (1) and get supernatant liquor after centrifugal 20 min of 5000r/min, obtain liquid antibacterial substance.
Fermented liquid of the present invention is comprised of the raw material of following mass/volume part: 0.5 part of extractum carnis, and 1 part of peptone, 0.5 part of NaCl, 100 parts of distilled water, pH 7.2.
The characteristic that shows the antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces by following test
1, Antibacterial Activity and scope of restraining fungi
The antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces is tested by Oxford agar diffusion method, is about to 15mL beef-protein medium and is laid in diameter 90mm, in the culture dish of dark 20mm, is placed in standing solidifying on level table, then gets and be diluted to 10
6on the beef-protein medium flat board of indicator bacteria suspension 0.2 mL of cfu/mL after above-mentioned solidifying, then with aseptic spreading rod, indicator bacteria suspension is smoothened, culture dish lid is opened and is made the sterile air about 40min that circulates, after bacterium bacteria suspension to be instructed is slightly dry by the dull and stereotyped fully absorption of beef-protein medium, by internal diameter 6mm, external diameter 8mm, the aseptic Oxford cup of high 10mm is positioned on flat board with tweezers gently, 200 μ L bacillus amyloliquefaciens HRH.317 fermented liquid supernatant liquid are added after the cup of Oxford, under 4-5 ℃ of condition, spread 12 hours, after fully spreading, cultivates 3-5 days (when indicator is bacterium 3 days in 30 ℃ fermented liquid, when indicator is yeast or mould 5 days) observe inhibition zone situation.Test-results shows, the antibacterial substance of this bacterium has higher Antibacterial Activity, its tire for 128AU/mL(indicator be genus bacillus (
bacillus.sp) H108), and antimicrobial spectrum is wide, Gram-positive and the gram negative bacterium that can suppress multiple food-borne pathogens and cause food spoilage, and quenchable gram positive bacterial strain comprises: subtilis (
bacillus subtilis), Bacillus coagulans (
bacillus coagulans), streptococcus aureus (
staphyloccocus aureus); Quenchable gram negative strain comprises: intestinal bacteria (
escherichia coli), Salmonella enteritidis (
salmonella enteritidis); Also can suppress the multiple fungies such as mould in yeast saccharomyces cerevisiae, rhodotorula, wine yeast, Pear black spot bacterium Kikuchi chain lattice spore, grape downy mildew, aspergillus niger, mould.Test-results refers to table 3, Fig. 4-Figure 16.
The antimicrobial spectrum of table 3 bacillus amyloliquefaciens antibacterial substance that HRH.317 produces
| Sequence number | Indicator | Source | Antibacterial circle diameter (mean value: mm) |
| 1 | Genus bacillus ( Bacillus.sp）H108 | The bioengineering dept preservation of Food Science and Engineering institute of Agricultural University Of Shanxi | 31 |
| 2 | Bacillus coagulans ( Bcillus coagulans)CICC 10144 | Chinese industrial microbial strains preservation administrative center | 19 |
| 3 | Subtilis ( Bcillus coagulans)CMCC 63501-2aq | Chinese medicine microbial strains preservation administrative center | 15 |
| 4 | Streptococcus aureus ( Staphlococcus aureus)CMCC 26003-5a1 | Chinese medicine microbial strains preservation administrative center | 17 |
| 5 | Colon bacillus ( Escherichia coli)CMCC 44102-3a11 | Chinese medicine microbial strains preservation administrative center | 15 |
| 6 | Salmonella enteritidis ( Salmonella enteritidis） | The bioengineering dept preservation of Food Science and Engineering institute of Agricultural University Of Shanxi | 15 |
| 7 | Yeast saccharomyces cerevisiae ( Saccharomyces cerevisiae)H226 | The bioengineering dept preservation of Food Science and Engineering institute of Agricultural University Of Shanxi | 16 |
| 8 | Rhodotorula ( Rhodotorula）H215 | The bioengineering dept preservation of Food Science and Engineering institute of Agricultural University Of Shanxi | 17 |
| 9 | Wine yeast ( Saccharomyces ellipsoideus)H222 | The bioengineering dept preservation of Food Science and Engineering institute of Agricultural University Of Shanxi | 16 |
| 10 | Pear black spot bacterium Kikuchi chain lattice spore ( Alternaria kikuchi-ana) | The bioengineering dept preservation of Food Science and Engineering institute of Agricultural University Of Shanxi | 24.5 |
| 11 | Grape downy mildew ( Plasmopara uiticola） | The bioengineering dept preservation of Food Science and Engineering institute of Agricultural University Of Shanxi | 20.5 |
| 12 | Aspergillus niger ( Aspergillus niger) | The bioengineering dept preservation of Food Science and Engineering institute of Agricultural University Of Shanxi | 23 |
| 13 | Mould ( Penicillium.sp) | The bioengineering dept preservation of Food Science and Engineering institute of Agricultural University Of Shanxi | 20 |
2, acid suppresses to get rid of test, gets rid of hydrogen peroxide test and protease digestion test
The liquid antibacterial substance producing with bacillus amyloliquefaciens HRH.317 of the present invention, with mould, it is indicator, the acid of carrying out the antibacterial substance of bacillus amyloliquefaciens HRH.317 generation suppresses to get rid of test, gets rid of hydrogen peroxide test and protease digestion test, and test method and result are as follows:
(1) test method suppress is got rid of in acid: it is 7 that the pH of the liquid antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention is produced is adjusted to 1mol/L HCl and 1mol/L NaOH, and the mould of take is made bacteriostatic test with Oxford agar diffusion method as indicator.Test-results is shown in Figure 17, and in figure, a is before acid suppresses to get rid of, antibacterial circle diameter 14 ㎜; B is that after acid suppresses to get rid of, antibacterial circle diameter 13.7 ㎜, between the two without significant difference.
(2) get rid of hydrogen peroxide test method: get the liquid antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces, add catalase, water-bath after 4 hours at 37 ℃, the mould of take is done bacteriostatic test with Oxford agar diffusion method as indicator.Test-results is shown in Figure 18, and in figure, a gets rid of before hydrogen peroxide, antibacterial circle diameter 14.5 ㎜; B gets rid of after hydrogen peroxide, antibacterial circle diameter 2.2 ㎜, significant difference between the two (P < 0.05).
(3) protease digestion test method: get the liquid antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces, first with 1mol/L NaOH and 1mol/L HCL, adjust the suitableeest action pH 5-9 that pH is Proteinase K, by 0.5mol/L amount, add Proteinase K, at 37 ℃, constant temperature water bath is 2 hours, again pH value is recalled to original numerical value, the mould of take is done bacteriostatic test with Oxford agar diffusion method as indicator.Test-results 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 between the two (P < 0.05).
Test-results shows: fermented supernatant fluid suppresses to get rid of test through acid, and the bacteriostatic activity that shows this bacterial strain fermentation liquor supernatant liquor is not to come from acid; After catalase is processed, antibacterial circle diameter significantly dwindles; Proteinase K is processed rear inhibition zone and is reduced to some extent.Show bacillus amyloliquefaciens (
bacillus amyloliquefaciens) antibacterial substance that produces of HRH.317 may be hydrogen peroxide and a kind of protein or polypeptides matter.
3, antimicrobial stability
(1) antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces has good thermostability
The antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention is produced thermal treatment 30min at 60-90 ℃, its fungistatic effect does not have noticeable change; Thermal treatment 15min at 100 ℃, its bacteriostatic activity be original (CK) 67%; Thermal treatment 30min at 100 ℃, bacteriostatic activity be original (CK) 57%; Thermal treatment 15min at 121 ℃, bacteriostatic activity still can keep the 57%(of original (CK) in Table 4).
The thermostability of table 4 bacillus amyloliquefaciens antibacterial substance that HRH.317 produces
| Process | ? | 60℃ | 70℃ | 80℃ | 90℃ | 100℃ | 121℃ |
| Thermal treatment 15min antibacterial circle diameter (mm) | ? | 20 | 19 | 18 | 18 | 14 | 12 |
| Thermal treatment 30min antibacterial circle diameter (mm) | ? | 19 | 18 | 16.5 | 15 | 12 | ? |
| CK antibacterial circle diameter (mm) | 21 | ? | ? | ? | ? | ? | ? |
Therefore, the antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces has good thermostability, and this feature determines that it has very large using value in foodstuffs industry is produced.
(2) antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces has good ph stability
The antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention is produced is processed 30min in the scope of pH 5-9, and the difference of fungistatic effect is not remarkable; Under pH 2 conditions, process 30min, bacteriostatic activity still can retain 77% of original activity (CK); Under pH 11 conditions, process 30min, bacteriostatic activity still can retain 82% of original activity (CK), in Table 5.
The ph stability of table 5 bacillus amyloliquefaciens antibacterial substance that HRH.317 produces
| 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 | ? | ? | ? | ? | ? | ? | ? | ? | ? | ? |
The bacteriostatic activity that shows this antibacterial substance has the subject range of wider pH.Therefore can in the condition of slant acidity, neutrality, meta-alkalescence, use.
(3) the antibacterial substance Surfactant that bacillus amyloliquefaciens HRH.317 of the present invention produces has satisfactory stability
Adopt polyoxyethylene glycol, tween 80, three kinds of tensio-active agents of polysorbas20 as test materials, with the antibacterial substance bacterium liquid that three kinds of tensio-active agents produce with bacillus amyloliquefaciens HRH.317 of the present invention respectively, mix, the mixed solution that the concentration that is formulated as tensio-active agent is 1%, distilled water substitution list surface-active agent for contrast (CK), the mould of take is respectively done bacteriostatic experiment as indicator, at 37 ℃, cultivate 4 hours, survey its antibacterial circle diameter.Test-results shows, through polyoxyethylene glycol, tween 80 is processed and contrasted there is no significant difference; After polysorbas20 is processed, the activity of bacillus amyloliquefaciens antibacterial substance that HRH.317 produces obviously reduces, for 76.6% of contrast, in Table 6.
The stability that table 6 bacillus amyloliquefaciens HRH.317 produces antibacterial substance Surfactant
| Tensio-active agent | CK | Polysorbas20 | Tween 80 | Polyoxyethylene glycol |
| Antibacterial circle diameter (mm) | 23.5 | 18 | 23 | 22 |
Beneficial effect of the present invention
The antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces has higher Antibacterial Activity, its tire for 128AU/mL(indicator be genus bacillus H108), antimicrobial spectrum is wide, can suppress the multiple gram-positive microorganism that causes food spoilage, as genus bacillus, subtilis, Bacillus coagulans, streptococcus aureus etc., also can suppress to cause the Gram-negative bacteria of food spoilage, as intestinal bacteria and Salmonella enteritidis; Also can suppress the multiple fungies such as mould in yeast saccharomyces cerevisiae, rhodotorula, wine yeast, Pear black spot bacterium Kikuchi chain lattice spore, grape downy mildew, aspergillus niger, mould.This antibacterial substance has good thermostability and ph stability, and polyoxyethylene glycol, tween 80, three kinds of tensio-active agents of polysorbas20 are also had to good stability.The antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces has a good application prospect as the natural antiseptic agent of fruits and vegetables and food.
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 that the 16S rDNA sequential system relevant to bacillus amyloliquefaciens HRH.317 of the present invention grown tree.
Fig. 4 be bacillus amyloliquefaciens antibacterial substance that HRH.317 produces of the present invention suppress genus bacillus (
bacillus.sp) H108 result.
Fig. 5 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress Bacillus coagulans (
bcillus coagulans) CICC 10144 results.
Fig. 6 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress subtilis (
bcillus coagulans) CMCC 63501-2aq result.
Fig. 7 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress streptococcus aureus (
staphlococcus aureus) CMCC 26003-5a1 result.
Fig. 8 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress colon bacillus (
escherichia coli) CMCC 44102-3a11 result.
Fig. 9 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress Salmonella enteritidis (
salmonella enteritidis) result.
Figure 10 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) H226 result.
Figure 11 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress rhodotorula (
rhodotorula) H215 result.
Figure 12 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress wine yeast (
saccharomyces ellipsoideus) H222 result.
Figure 13 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress Pear black spot bacterium Kikuchi chain lattice spore (
alternaria kikuchi-ana) result.
Figure 14 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress grape downy mildew (
plasmopara uiticola) result.
Figure 15 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress aspergillus niger (
aspergillus niger) result.
Figure 16 be the liquid antibacterial substance that produces of bacillus amyloliquefaciens HRH.317 of the present invention suppress mould (
penicillium) result.
Figure 17 is that the liquid antibacterial substance acid that bacillus amyloliquefaciens HRH.317 of the present invention produces suppresses to get rid of test-results.
Figure 18 is that the liquid antibacterial substance that bacillus amyloliquefaciens HRH.317 of the present invention produces is got rid of hydrogen peroxide test result.
Figure 19 is the liquid antibacterial substance protease K digesting test-results that bacillus amyloliquefaciens HRH.317 of the present invention produces.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
embodiment 1
One bacillus amyloliquefaciens (
bacillus amyloliquefaciens) HRH.317, on March 15th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature be bacillus amyloliquefaciens (
bacillus amyloliquefaciens), preserving number is: CGMCC No.7314.
Described bacillus amyloliquefaciens (
bacillus amyloliquefaciens) HRH.317 is not intended to separation screening and obtains from field soil.Beef-protein medium (extractum carnis 0.5g, peptone 1.0g, NaCl 0.5g, agar 1.5g, water 100mL,
p7.2,121 ℃ of H, sterilizing 20 minutes) upper 37 ℃ of constant temperature culture are 24 hours, and thalline forms a large amount of gemma, form thalline shaft-like (0.9 ~ 1.4 * 2.7 ~ 3.9 μ m) before gemma, are arranged as single or chaining, Gram-positive; Cultivate 3 days, colonial morphology is: oyster white, and diameter is about 6.75mm, roughly has a fold, and optical signature is opaque, have projection, circle, edge to be wavy or petal-shaped; During the standing cultivation of liquid, there is mycoderm to generate; Percutaneous puncture-inoculation is cultivated thalline cloud diffusion growth in the substratum of test tube, and showing has mobility.
Through Physiology and biochemistry, identify and to comprise: nitrate reduction, catalase, V-P mensuration, Starch Hydrolysis, Citrate trianion utilization, phenylalanine deaminase, salt tolerance and need salt, produce indoles, test and the 16S rDNA Phylogenetic Analysis such as indolepyruvic acid (IPA) mensuration, lecithinase, glucose oxidase fermentation, gelatine liquefication, casein hydrolysis, hydrolysis urobenzoic acid, methyl red (M.R), determine this bacterial strain be bacillus amyloliquefaciens (
bacillus amyloliquefaciens).
embodiment 2
Bacillus amyloliquefaciens (
bacillus amyloliquefaciens) preparation method of the antibacterial substance that produces of HRH.317, first bacillus amyloliquefaciens HRH.317 is inoculated into nutrient solution (extractum carnis 0.5g, peptone 1.0g, NaCl 0.5g for fermentation, water 100ml, 7.2,121 ℃ of pH, sterilizing 20min) in, at 37 ℃, under 160 r/min conditions, cultivate 24 hours, obtain fermented liquid; Then the fermented liquid of preparation is got to supernatant liquor after centrifugal 20 min of 5000r/min, obtain crude liquid antibacterial substance.
Claims (5)
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