CN1793331A - Lichen spore bacillus strain and application of bacillocin produced by it - Google Patents
Lichen spore bacillus strain and application of bacillocin produced by it Download PDFInfo
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- CN1793331A CN1793331A CN 200510061554 CN200510061554A CN1793331A CN 1793331 A CN1793331 A CN 1793331A CN 200510061554 CN200510061554 CN 200510061554 CN 200510061554 A CN200510061554 A CN 200510061554A CN 1793331 A CN1793331 A CN 1793331A
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Abstract
The invention discloses bacillus licheniformis strain. Its preservation name is Bacillus licheniformis ZJU12. The preservation unit is Chinese typical culture preservation center. The data is July fourth in 2005 yare. The number is CCTCC M 205072. The invention also discloses the produced bacteriocin which has antagonism for gram-positive bacteria, plant pathogenic fungi, plant pathogeny gram-negative bacteria.
Description
Technical field
The present invention relates to the purposes of the bacteriocin of a kind of lichem bacillus strain and generation thereof at aspects such as biological food anti-corrosive fresh-keeping, animals and plants disease control and medicine.
Background technology
Bacillus licheniformis (Bacillus licheniformis) is the amphimicrobian saprophytic microorganism that a class nature extensively exists.This bacterium is used as the industrial production bacterium of proteolytic ferment, amylase, biologically active agent, microbiotic and other particularization materials etc. always for a long time, and HUMAN HEALTH and environment are had very low toxicity.A kind of ring type polypeptide class antagonistic substance that the peptide class antagonistic substance that Bacillus licheniformis produces is separated to from Bacillus subtilis var Tracy (Meleney was renamed as Bacilluslicheniformis Var Tracy in 1994) bacterial strain in 1945 for people such as Johnson the earliest, called after bacitracin (Bacitracin) has been widely used in medicine and field of veterinary.This bacterium has good antagonistic action to multiple gram positive bacterium, part gram negative bacterium.So far, people have separated some peptide antibioticses from different Bacillus licheniformis.These peptide antibioticses are secondary metabolic products, are not genes encodings, and the multiple protein enzyme is had resistance.
Bacteriocin be a class by the bacterium excretory, rrna synthetic, the protein that close bacterium is had antagonistic action.Recently, from different lichem bacillus strains 26 L-10/3RA, 490/5 and P40 be separated to three kinds of bacteriocin Lichenin, Bacillocin 490and P40 respectively.These bacteriocins are inequality at aspects such as molecular weight, thermostability, culture condition, action spectrums.But they all can not have antagonistic action to golden staphylococci and fungi, and insensitive to trypsinase.
Summary of the invention
At the deficiencies in the prior art part, the invention provides a kind of lichem bacillus strain, with and the bacteriocin that can have antagonistic action to numerous food spoilage organism, phytopathogen and human body pathogenic bacteria that produces.
The present invention is for reaching above purpose, be to realize: a kind of lichem bacillus strain is provided by such technical scheme, this Bacillus licheniformis (Bacillus licheniformis) bacterial strain, the preservation name is called: Bacillus licheniformis ZJU12, depositary institution: Chinese typical culture collection center, preservation date: on July 4th, 2005, preserving number: CCTCC M 205072.
The bacterial classification of this lichens genus bacillus ZJU12 grows fine on normal temperature and conventional substratum; Bacterium colony is canescence on the LB substratum, smooth surface, toughness.
The present invention also provides the bacteriocin that above-mentioned lichem bacillus strain produced, and this bacteriocin has anti-microbial activity.
This bacteriocin has antagonistic action to gram positive bacterium, plant pathogenic fungi, pathogenic gram negative bacterium.Gram positive bacterium is subtilis, streptococcus aureus, staphylococcus epidermidis, enterococcus faecalis or micrococcus flavus etc.Plant pathogenic fungi is Chinese cabbage alternaria, cotton-wilt fusarium, stem of noble dendrobium blackspot pathogenic bacteria, the former bacterium of rice blast or rice banded sclerotial blight cause of disease bacterium etc.The pathogenic gram negative bacterium is a rice leaf spot bacteria etc.
Antagonistic bacterium provided by the present invention is Bacillus licheniformis (Bacillus licheniformis) ZJU12 CCTCC M 205072, and this bacterium separates from Zhejiang University's Hua Jiachi school district soil.
The experiment proved that the bacteriocin that Bacillus licheniformis ZJU12 is produced detects in exponential growth mid-term, and its activity reaches maximum value in the early stage of steadily growing.
The cultivation of Bacillus licheniformis ZJU12 goes bacterium liquid to subtilis (Bacilliussubtilis), streptococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis), enterococcus faecalis (Enterococcus faecium), micrococcus flavus gram positive bacteriums such as (Micrococcus flavus), to Chinese cabbage alternaria (Alternaria brassicae), cotton-wilt fusarium (Fusarium oxysporum), stem of noble dendrobium blackspot pathogenic bacteria (Guignardia sp), former bacterium of rice blast (Pyricularia grisea Sacc.) and rice banded sclerotial blight cause of disease bacterium plant pathogenic fungis such as (Rhizoctonia solani) all have antagonistic action, but to intestinal bacteria (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), catarrh Blanc Chinese bacterium (Branhamella catarrhalis), Acinetobacter bauamnnii (Acinetobacterbaumanii), enterobacter cloacae (Enterobacter cloacae) and Bacillus proteus gram negative bacteriums such as (Proteus sp) do not have antagonistic action, but this pathogenic gram negative bacterium of rice leaf spot bacteria (Xanthomonas oryzaepv.oryzae) is had strong antagonistic action.
With the micrococcus flavus is indicator, adopts the stepwise dilution method to measure this cultivation and goes the antagonism vigor of bacterium liquid to be about 1000AU/ml.
The cultivation of Bacillus licheniformis ZJU12 goes bacterium liquid activity after Proteinase K and trypsin treatment to completely lose.100 ℃ handle 30 minutes after the antagonism vigor do not have considerable change, almost do not have vigor at 121 ℃ after handling 15 minutes.Going the bacteria solution active scope is pH3-11, and optimum pH is about 7.
The antagonism crude extract of Bacillus licheniformis ZJU12 is tested mouse when the dosage of 0.8g/20g do not have acute toxicity.Therefore can be used for aspects such as biological food anti-corrosive fresh-keeping, animals and plants disease control and medicine.
Description of drawings
Fig. 1 is that Bacillus licheniformis ZJU12 growth curve and bacteriocin produce curve.
Embodiment
1, for the examination material:
1.1 antagonism bacterium
Bacillus licheniformis ZJU12 is separated from Zhejiang University's Hua Jiachi school district soil by this laboratory, and culture condition is LB liquid or solid substratum (peptone 10g/L, NaC110g/L, yeast extract 5g/L, solid add agar 15-20g/L again, pH7.0), 30 ℃, aerobic.
1.2 pathogenic bacteria
Acinetobacter bauamnnii (Acinetobacter baumanii), subtilis (Bacilliussubtilis), catarrh Blanc Chinese bacterium (Branhamella catarrhalis), enterobacter cloacae (Enterobacter cloacae), enterococcus faecalis (Enterococcus faecium), intestinal bacteria (Escherichia coli), micrococcus flavus (Micrococcus flavus), Bacillus proteus (Proteussp), Pseudomonas aeruginosa (Pseudomonas aeruginosa), streptococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis).Culture condition is LB liquid or solid substratum, and is 30 ℃, aerobic.
Chinese cabbage alternaria (Alternaria brassicae), rice leaf spot bacteria (Xanthomonasoryzae pv.oryzae), rice blast pathogenic bacteria (Pyricularia grisea Sacc.), cotton-wilt fusarium (Fusarium oxysporum), rice banded sclerotial blight cause of disease bacterium (Rhizoctonia solani), stem of noble dendrobium blackspot pathogenic bacteria (Guignardia sp), culture condition are PSA solid medium (potato 200g/L, sucrose 10g/L, agar 15-20g/L, pH7.0), 30 ℃, aerobic.
With above-mentioned pathogenic bacteria as indicator.
2, remove bacterium liquid extracting method:
Bacillus licheniformis ZJU12 is inoculated in the LB liquid nutrient medium, cultivates 12 hours as seed culture fluid for 30 ℃.Inoculation 5% was cultivated 34 hours in another LB liquid nutrient medium.5000 * g obtained supernatant liquor in centrifugal 10 minutes for removing bacterium liquid, as antagonism liquid.
3, the measuring method of bacteriocin antagonism vigor:
Get above-mentioned indicator respectively and be inoculated in the LB liquid nutrient medium, overnight incubation is got 0.1ml bacterium liquid and is evenly distributed on the LB flat board, and air-dry back is the punch tool punching of 5mm with the aperture, and every hole adds 20 μ l antagonism liquid, and overnight incubation is observed antagonistic action, and the pH of this moment is 7.0.Concrete outcome sees Table 1.
The antagonism spectrum (pH 7.0) of removing bacterium liquid that table 1 Bacillus licheniformis ZJU12 cultivates
| Indicator | Antibacterial circle diameter (mm) | Antagonistic ability | ||
| Bacterium | Gram-positive bacteria | Subtilis (Bacillius subtilis) | 18 | +++ |
| Enterococcus faecalis (Enterococcus faecium) | 10 | ++ | ||
| Micrococcus flavus (Micrococcus flavus) | 24 | ++++ | ||
| Streptococcus aureus ATCC25923 (Staphylococcus aureus) | 22 | ++++ | ||
| Streptococcus aureus (Staphylococcus aureus) | 22 | ++++ | ||
| Staphylococcus epidermidis (Staphylococcus epidermidis) | 20 | +++ | ||
| Gram-negative bacteria | Acinetobacter bauamnnii (Acinetobacter baumanii) | - | - | |
| Catarrh Blanc Chinese bacterium (Branhamella catarrhalis) | - | - | ||
| Enterobacter cloacae (Enterobacter cloacae) | - | - | ||
| Intestinal bacteria (Escherichia coli) | - | - | ||
| Bacillus proteus (Proteus sp) | - | - | ||
| Pseudomonas aeruginosa (Pseudomonas aeruginosa) | - | - | ||
| Rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) | 26 | +++++ | ||
| Fungi | Chinese cabbage alternaria (Alternaria brassicae) | 20 | +++ | |
| Cotton-wilt fusarium (Fusarium oxysporum) | 23 | ++++ | ||
| Stem of noble dendrobium blackspot pathogenic bacteria (Guignardia sp) | 26 | +++++ | ||
| The former bacterium of rice blast (Pyricularia grisea Sacc.) | 25 | ++++ | ||
| Rice banded sclerotial blight cause of disease bacterium (Rhizoctonia solani) | 25 | ++++ | ||
Annotate: when antibacterial circle diameter was 6-9mm, antagonistic ability was expressed as "+"; When antibacterial circle diameter was 10-15mm, antagonistic ability was expressed as " ++ "; When antibacterial circle diameter was 16-20mm, antagonistic ability was expressed as " +++"; When antibacterial circle diameter was 21-25mm, antagonistic ability was expressed as " ++ ++ "; When antibacterial circle diameter during greater than 26mm, antagonistic ability is expressed as " ++ +++"; The no antagonistic action of "-" expression.
4, the research of Bacillus licheniformis ZJU12 growth curve and bacteriocin formation curve:
Bacillus licheniformis ZJU12 is inoculated in the LB liquid nutrient medium, cultivates 12 hours as fermentation culture for 30 ℃.Get 3 aseptic 100ml triangular flasks, pipette 50ml fermentation culture in bottle 30 ℃ respectively, 150r/min cultivated 6 hours, then every two hours, with nonvaccinated nutrient solution as blank determination OD
600Value.Simultaneously, 5000 * g got supernatant in centrifugal 3 minutes, measured its antibacterial circle diameter to micrococcus flavus (mm).Concrete outcome as described in Figure 1.(◆) among Fig. 1 represents OD
600(■) represent antibacterial circle diameter.
Above-mentioned experiment shows: the bacteriocin that Bacillus licheniformis ZJU12 is produced detects in exponential growth mid-term, and its activity reaches maximum value in the early stage of steadily growing.
5, the quantitative assay of bacteriocin antagonism vigor:
With the micrococcus flavus is indicator, adopts the stepwise dilution method to measure Bacillus licheniformis and cultivates the antagonism vigor that removes bacterium liquid.With AU/ml is unit, and the antagonism vigor is represented with the inverse of maximum bacteriostatic extension rate.Record its antagonism vigor and be about 1000AU/ml.
6, go bacterium liquid stability to measure:
6.1 the proteolytic enzyme effect is measured:
With Bacillus licheniformis cultivate go bacterium liquid respectively through Proteinase K (37 ℃, pH7), 30 ℃ of trypsin pH7) were handled 6 hours, final enzyme concn is 1mg/ml.To be untreated is contrast, measures its antagonistic activity to micrococcus flavus.Concrete outcome sees Table 2.
The effect of table 2 proteolytic enzyme is measured
| Processing mode | Be untreated | Proteinase K | Trypsinase |
| Antibacterial circle diameter (mm) | 22 | 0 | 0 |
Experiment shows: Bacillus licheniformis ZJU12 cultivates and goes bacterium liquid activity after Proteinase K and trypsin treatment almost completely to lose.
6.2 the mensuration of thermostability:
Get 1ml respectively and go bacterium liquid in test tube, handle 30min and 1h down at 37,60,80,100 ℃, 121 ℃ of processing are cooled to room temperature, and (CK) compares with undressed sample.
Table 3 thermal stability determination
| Temperature (℃) | CK | 37 | 60 | 80 | 100 | 121 | ||||
| Time (min) | - | 30 | 60 | 30 | 60 | 30 | 60 | 30 | 60 | 15 |
| Antibacterial circle diameter (mm) | 22 | 22 | 22 | 22 | 22 | 22 | 21.5 | 21 | 20.5 | 0 |
Experiment shows: 100 ℃ handle 30 minutes after the antagonism vigor do not have considerable change, almost do not have vigor at 121 ℃ after handling 15 minutes.
6.3 ph stability is measured:
Add the 1ml sample in test tube, with 1mol/L sodium hydroxide or 1mol/L hydrochloric acid sample solution being transferred to pH value respectively is 3,5,7,9,11, again the pH of each pipe is recalled to about pH7.0 the detection bacteriostatic activity behind the room temperature maintenance 24h.Concrete outcome is as shown in table 4.
Table 4 ph stability is measured
| pH | 3 | 5 | 7 | 9 | 11 |
| Antibacterial circle diameter (mm) | 18 | 20 | 22 | 18 | 10 |
Experiment shows: going the bacteria solution active scope is pH3-11, and optimum pH is about 7.0.
7, the mensuration of antagonism crude extract acute toxicology:
Select 30 of SPF level ICR mouse for use, be divided into 6 groups at random, every group female 3, male 2.Temperature: 22 ± 1 ℃, humidity: 50-70%, illumination: 150-200Lx, light and shade replaced in 12 hours, noise＜50dB.Irritated before the stomach fasting 8 hours, irritating the body of stomach integration is not 0.80ml/20g, 0.64ml/20g, 0.51ml/20g, 0.41ml/20g, 0.33ml/20g, 0.26ml/20g, irritates after stomach finishes, and observes the death state of mouse in 7 days, carries out record.
Concrete outcome sees Table 5.
The measurement result of table 5, antagonism crude extract acute toxicology
| Concentration (mg/ml) | Dosage (ml/20g) | Number of animals | Death toll in 7 days | ||
| Male | Female | Male | Female | ||
| 1.0 | 0.80 | 2 | 3 | 0 | 0 |
| 1.0 | 0.64 | 2 | 3 | 0 | 0 |
| 1.0 | 0.51 | 2 | 3 | 0 | 0 |
| 1.0 | 0.41 | 2 | 3 | 0 | 0 |
| 1.0 | 0.33 | 2 | 3 | 0 | 0 |
| 1.0 | 0.26 | 2 | 3 | 0 | 0 |
More than experimental results show that: the antagonism crude extract of Bacillus licheniformis ZJU12 is the highest when the dosage of 0.8g/20g, and the experiment mouse does not have acute toxicity; Show that it can be used for aspects such as biological food anti-corrosive fresh-keeping, animals and plants disease control and medicine safely.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (7)
1, a kind of lichem bacillus strain, it is characterized in that: this Bacillus licheniformis (Bacilluslicheniformis) bacterial strain, the preservation name is called: Bacillus licheniformis ZJU12, depositary institution: Chinese typical culture collection center, preservation date: on July 4th, 2005, preserving number: CCTCC M205072.
2, a kind of lichem bacillus strain according to claim 1 is characterized in that: the bacterial classification of described Bacillus licheniformis ZJU12, grow fine on normal temperature and conventional substratum; Bacterium colony is canescence on the LB substratum, smooth surface, toughness.
3, a kind of bacteriocin that is produced as the described lichem bacillus strain of claim 1 to 2, it is characterized in that: described bacteriocin has anti-microbial activity.
4, a kind of purposes of bacteriocin as claimed in claim 3 is characterized in that: described bacteriocin has antagonistic action to gram positive bacterium, plant pathogenic fungi, pathogenic gram negative bacterium.
5, the purposes of bacteriocin according to claim 4 is characterized in that: described gram positive bacterium is subtilis, streptococcus aureus, staphylococcus epidermidis, enterococcus faecalis or micrococcus flavus.
6, the purposes of bacteriocin according to claim 4 is characterized in that: described plant pathogenic fungi is Chinese cabbage alternaria, cotton-wilt fusarium, stem of noble dendrobium blackspot pathogenic bacteria, the former bacterium of rice blast or rice banded sclerotial blight cause of disease bacterium.
7, the purposes of bacteriocin according to claim 4 is characterized in that: described pathogenic gram negative bacterium is a rice leaf spot bacteria.
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| CNB2005100615543A CN100352916C (en) | 2005-11-14 | 2005-11-14 | Lichen spore bacillus strain and application of bacillocin produced by it |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603015B (en) * | 2008-06-13 | 2010-11-10 | 河北维尔康制药有限公司 | Bacillus licheniformis strain, application thereof and method for producing poly-gamma-glutamic acid thereby |
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Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1032570C (en) * | 1993-01-20 | 1996-08-21 | 河南华能科技发展公司 | Method for producing biologic pesticide by lichen gemma |
| US6569425B2 (en) * | 2001-06-22 | 2003-05-27 | David Joseph Drahos | Bacillus licheniformis biofungicide |
| CN1219052C (en) * | 2003-08-14 | 2005-09-14 | 南京农业大学 | Rice wine lactobacillus strain for producing bacteriocin and its use method |
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| CN101603015B (en) * | 2008-06-13 | 2010-11-10 | 河北维尔康制药有限公司 | Bacillus licheniformis strain, application thereof and method for producing poly-gamma-glutamic acid thereby |
| CN101775360B (en) * | 2009-09-04 | 2012-02-01 | 武汉科诺生物科技股份有限公司 | Preparation method of Bacillus licheniformis and raw materials |
| CN101935628A (en) * | 2010-06-13 | 2011-01-05 | 浙江大学 | Bacillus subtilis and application thereof |
| CN103966283A (en) * | 2013-01-30 | 2014-08-06 | 大连海洋大学 | Kelp endophytic bacillus licheniformis protein, application thereof to preservation and application method thereof |
| CN105907655A (en) * | 2016-03-02 | 2016-08-31 | 西北师范大学 | Typhonium giganteum Engl endogenous Bacillus licheniformis with antagonism effects on a plurality of plant pathogenic fungi and separation method thereof |
| CN105820987A (en) * | 2016-05-31 | 2016-08-03 | 四川大工汇能纵横环境技术有限责任公司 | Bacillus licheniformis strain and microecologics |
| CN107494639A (en) * | 2017-09-27 | 2017-12-22 | 印江彬彬百香果种植发展有限公司 | A kind of composition for treating passion fruit black spot and preparation method thereof |
| CN108192851A (en) * | 2018-03-13 | 2018-06-22 | 长沙艾格里生物肥料技术开发有限公司 | Dongfanghong bacteria strain, Dongfanghong bacteria agent and its application |
| CN108192851B (en) * | 2018-03-13 | 2021-03-19 | 长沙艾格里生物肥料技术开发有限公司 | Oriental rhodobacter strain, oriental rhodobacter agent and application thereof |
| CN109182215A (en) * | 2018-10-17 | 2019-01-11 | 湖南省林业科学院 | One bacillus licheniformis bacterial strain and its application in inhibition bacterial pathogen |
| CN109182215B (en) * | 2018-10-17 | 2021-09-24 | 湖南省林业科学院 | Bacillus licheniformis strain and application thereof in inhibition of bacterial pathogenic bacteria |
| CN110184224A (en) * | 2019-06-18 | 2019-08-30 | 上海交通大学 | One staphylococcus epidermidis and its application |
| CN110577915A (en) * | 2019-09-29 | 2019-12-17 | 中南民族大学 | Bacillus licheniformis CZ186 strain and application thereof |
| CN114395511A (en) * | 2022-02-18 | 2022-04-26 | 安徽科技学院 | Bacillus licheniformis FY1 and application thereof |
| CN114395511B (en) * | 2022-02-18 | 2023-04-25 | 安徽科技学院 | Bacillus licheniformis FY1 and application thereof |
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