CN115975882B - Bacillus subtilis and application thereof - Google Patents
Bacillus subtilis and application thereof Download PDFInfo
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- CN115975882B CN115975882B CN202211640528.6A CN202211640528A CN115975882B CN 115975882 B CN115975882 B CN 115975882B CN 202211640528 A CN202211640528 A CN 202211640528A CN 115975882 B CN115975882 B CN 115975882B
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Abstract
The application discloses bacillus subtilis and application thereof. The bacillus subtilis (Bacillus subtilis) No. 1 CGMCC No.26006 can obviously inhibit the rot or deterioration of tomato fruits, has simple culture conditions, is easy for industrial production and has good application prospect.
Description
Technical Field
The application relates to bacillus subtilis and application thereof, and belongs to the technical field of biology.
Background
Bacillus subtilis (Bacillus subtilis) is a bacterium of the genus bacillus, a gram-positive aerobic bacterium, the surface of which has flagella, and which forms in vivo endospores that survive against a harsh external environment. Bacillus subtilis is widely used in foods, composite bactericides and feed additives. The bacillus subtilis is used as a microbial preservative, can effectively inhibit the propagation of microorganisms, and can be used as a preservative for fruits and vegetables, but the main microorganisms polluted in different fruits and vegetables are usually different, and microorganisms in different tissues (such as roots, leaves and fruits) of the fruits and vegetables are also different, so that the effect is also greatly different when the preservative containing the bacillus subtilis is used for preserving different fruits and vegetables or different tissues of the fruits and vegetables.
Tomato (Lycopersicom esculentum Mill.) is originally produced in south America. The tomato fruit has attractive color and luster, delicious juice and extremely high nutritive value, and contains basic nutrients such as sugar, protein, organic acid, cellulose, mineral substances and the like, and also contains rich carotene, VC, vitamin B group, manganese, copper, iodine, zinc and the like. However, since the appearance and eating quality of tomato fruit are continuously reduced in the whole process of postharvest packaging, transportation, storage and consumption, the economic benefit is seriously affected, and how to maintain the postharvest quality is also a concern.
In the research and development process of the tomato fruit preservative, some preservatives have good preservation effect in short-term storage, and some preservatives have advantages in long-term storage; some preservatives are effective in inhibiting loss of internal nutrient molecules, and some are effective in preventing decay or loss of water, and some are effective in preventing external quality. That is, different tomato preservatives may be suitable for different preservation purposes. On the one hand, people pay more attention to the preservation effect of a long period (more than 10 days, especially more than 2 weeks) compared with the short-period storage preservation; on the other hand, in the postharvest process, the change of the internal quality (such as respiration intensity and the like) of the tomato fruit needs to be measured by an instrument, and the external quality (such as decay, weightlessness and the like) can be intuitively perceived by consumers, so that the preservative with an intuitive preservation effect on the external quality of the tomato fruit is favored by the market.
At present, a method for keeping tomato fruits fresh for a long time, especially for external quality is needed in the market.
Disclosure of Invention
In order to solve the technical problems, the application firstly provides the bacillus subtilis.
The registration number of the bacillus subtilis (Bacillus subtilis) No. 1 in the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No.26006.
The application also provides application of the bacillus subtilis in tomato preservation.
The application also provides application of the bacillus subtilis in inhibiting mould.
The application also provides a composite microbial inoculant, which comprises the bacillus subtilis, and/or bacterial suspension of the bacillus subtilis, and/or metabolite of the bacillus subtilis, and/or fermentation product of the bacillus subtilis, and/or filtrate of cell culture of the bacillus subtilis, and/or one or more mixtures thereof.
The application also provides a preservative which comprises the microbial inoculum and other preservatives; or comprises the active ingredients of the microbial inoculum and other antistaling agents.
The application also provides a method for preparing the preservative or the composite microbial inoculum, which comprises the step of culturing the bacillus subtilis.
The application also provides application of the preservative or the composite microbial inoculum in preservation of tomato fruits.
The application also provides application of the preservative or the composite microbial inoculum to inhibiting mould.
The application also provides a method for preserving tomato fruits, which enables the tomato fruits to be contacted with the preservative or the composite microbial inoculum.
In some embodiments, the tomato fruit is kept fresh to inhibit respiration intensity, and/or to inhibit decay, and/or to inhibit weight loss.
The application has the outstanding beneficial effects that: the bacillus subtilis has remarkable long-term fresh-keeping effect on the quality of tomato fruits: can make the weight loss rate of tomato fruits lower, the decay rate lower and the respiration intensity lower after more than 10 days, especially after 2 weeks after harvest.
Preservation description
Strain name: bacillus subtilis
Latin name: bacillus subtilis
Strain number: no. 1
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Preservation date: 2022, 11/2
Accession numbers of the preservation center: CGMCC No.26006.
Drawings
The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate the application and together with the embodiments of the application, serve to explain the application.
Fig. 1: strain No. 1 detection and identification report.
Fig. 2: phylogenetic tree of strain No. 1.
Fig. 3: comparing the weight loss rate of the picked tomato fruits.
Fig. 4: and comparing the rot rate of the picked tomato fruits.
Fig. 5: and comparing the respiration intensity of the picked tomato fruits.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The application provides a bacillus subtilis (Bacillus subtilis) No. 1, which has a registration number of CGMCC No.26006 in the common microorganism center of China Committee for culture Collection of microorganisms.
The application also provides a microbial inoculum comprising the bacillus subtilis, and/or a bacterial suspension of the bacillus subtilis, and/or a metabolite of the bacillus subtilis, and/or a fermentation product of the bacillus subtilis, and/or a filtrate of a cell culture of the bacillus subtilis, and/or one or more mixtures thereof.
The application also provides a preservative, which comprises the microbial inoculum and other preservatives; or comprises the active ingredients of the microbial inoculum and other antistaling agents.
The application also provides a method for preparing the preservative or the microbial inoculum, which comprises the step of culturing the bacillus subtilis.
The application also provides a tomato fresh-keeping method, which enables tomato fruits to be contacted with the bacillus subtilis, the preservative or the microbial inoculum.
The application also provides the bacillus subtilis, or the preservative, or the application of the microbial inoculum in tomato preservation.
In some embodiments, the tomato fruit is kept fresh to inhibit respiration intensity, and/or to inhibit decay, and/or to inhibit weight loss. Preferably, tomatoes are of the Tianfeng series.
The application also provides the application of the bacillus subtilis, the preservative and the microbial inoculum in inhibiting mould.
In some embodiments, the mold is aspergillus flavus or aspergillus niger.
In some embodiments, the bacillus subtilis is inoculated at a concentration of 3% -4%.
Definitions and terms
"bulk quality" includes both internal quality and external quality. For example, the quality of respiration intensity, weight loss rate, decay rate, and the like are included.
"tomato" includes any tomato variety, such as the Tianfeng series (e.g., tianfeng No.2, tianfeng No. 601, etc.), the Weidi series (e.g., weidi No. 8), etc. In some embodiments, the tomato refers to tomato fruit, tomato plant, or tomato seedling.
"fermentation product": the method is characterized in that strains are inoculated into a culture medium, after a period of culture, the strains are fermented by utilizing nutrient components in the culture medium, and fermented products, such as high-density fermentation liquor and the like, are formed, and the bacteria play the same or similar antibacterial or fresh-keeping role as that of the No. 1 bacteria.
"metabolite": also referred to as metabolites, refers to metabolites produced by the strain, including primary and secondary metabolites, such as mixtures including secretions, cyclic lipopeptides, and the like, which exert the same or similar bacteriostatic or preservative effects as the bacteria of the species 1.
"filtrate of cell culture": refers to a cell group or a growth of bacteria in a certain space for a certain time. Such as slant cultures, shake flask cultures, etc., of bacteria, which function as or near to the bacteriostatic or preservative effect of bacteria No. 1.
"bacterial suspension": the supernatant after passing through the filter membrane after fermentation plays the same or similar role of bacteriostasis or fresh-keeping as the bacteria No. 1.
"Medium": any suitable medium may be used, for example:
LB solid medium: prepared according to the proportion of 1000mL deionized water, 15g agar powder, 10g sodium chloride, 5g yeast extract powder and 10g peptone, and sterilized by high-pressure steam at 121 ℃ for 30min.
LB liquid medium: prepared in a ratio of 1000mL deionized water, 10g sodium chloride, 5g yeast extract, 10g peptone, and autoclaved at 121℃for 30min.
NA medium: prepared in a proportion of 1000mL deionized water, 15g glucose, 5g sodium chloride, 5g yeast extract, 10g peptone, and autoclaved at 121℃for 30min.
PDA (potato dextrose agar) medium: prepared according to the proportion of 1000mL deionized water and 35g PDA agar powder, dissolved in boiling water bath and sterilized by high pressure steam at 121 ℃ for 30min.
High-density fermentation medium: 1000mL deionized water, 10.0g glucose, 5.0g yeast powder, 10.75g corn powder, 9.0g soybean powder, 7.0g,KH2PO4 0.66g,NaCl 4g,MgSO4. 7H2O 0.6g,CaCl2 3g peptone, 0.6g MnSO4.H2O and 200mL liquid loading (500 mL conical flask), adjusting pH to 6.5-6.7, and sterilizing under high pressure15min)。
By "contacting" is meant any method by which a preservative may be administered to tomato fruit, for example spraying or coating the preservative on the surface of the tomato fruit, or immersing the tomato fruit in the preservative.
"other preservative" refers to existing preservatives other than the preservative or microbial agent of the present application.
The "effective component" refers to a component having a bacteriostatic or antistaling function.
Example 1 isolation and identification of Bacillus subtilis (Bacillus subtilis) No. 1 CGMCC No.26006
The inventor accidentally obtains a bacillus subtilis from experiments, and compared with other microorganisms, the bacillus subtilis has quite remarkable preservative and fresh-keeping effects on tomato fruits, so that the inventor separates and stores the bacillus subtilis and then carries out molecular biological identification to obtain bacillus subtilis (Bacillus subtilis) No. 1 CGMCCNo.26006. Then, the inventors also determined the bacterial morphology and physicochemical properties of the strain, gyrB gene sequence, 16SrRNA gene sequence, and drawn phylogenetic tree by the results of the gene sequence determination. Wherein, the identification result is shown in fig. 1, and the analysis result of the evolutionary tree is shown in fig. 2.
EXAMPLE 2 antibacterial property study of Bacillus subtilis (Bacillus subtilis) No. 1 CGMCC No.26006
1. The experimental method comprises the following steps:
(1) single colony of 1-2 loop bacillus subtilis (Bacillus subtilis) No. 1 CGMCC No.26006 is inoculated in a sterilized LB culture medium and cultured for 24 hours at 37 ℃. Culturing single colony bacterial liquid in an NA culture medium with the inoculum size of 4% -5% (namely, if the NA culture medium is 100mL, 4mL-5mL of single colony bacterial liquid is taken), and culturing at the constant temperature of 37 ℃ for 24h; taking out, centrifuging at 7000rpm/min at 4deg.C for 15min, collecting supernatant, filtering with 0.22 μm microporous membrane to obtain Bacillus subtilis fermentation broth, collecting supernatant after 5-10mL centrifugation with syringe, injecting supernatant onto 0.22 μm microporous membrane, filtering, and collecting Bacillus subtilis suspension after filtering. Can be stored in a refrigerator at 4 ℃ for standby.
(2) 100-150. Mu.L of sterile water was applied to an Aspergillus niger/Aspergillus flavus plate and spores were gently rolled with a glass rod into the sterile water at the location where the mold colonies were more. Transferring the strain to PDA solid culture medium, and uniformly coating the strain liquid by using a glass rod.
(3) The oxford cup method is used for measuring the antibacterial activity: punching holes on the oxford cup at the right central position of the PDA solid culture medium by using forceps, adding 100 mu L of bacterial suspension into the oxford cup by using a liquid-transferring gun, and culturing in a constant-temperature incubator at 37 ℃ for 48-72h, and 96-120h. After culturing, observing experimental phenomena, recording the diameter of the inhibition zone, and judging the antibacterial effect of the bacillus subtilis on the mould by the size of the inhibition zone.
2. Experimental results: the bacillus subtilis has an inhibition effect on aspergillus niger and aspergillus flavus, and has a better inhibition effect on aspergillus flavus.
Example 3 preservative and fresh-keeping effects of Bacillus subtilis (Bacillus subtilis) No. 1 CGMCC No.26006 on tomato fruits
1. Main experimental materials:
(1) "Tianfeng 601" tomato is picked from Beijing cis, picked with stalks and transported to laboratory for experiment.
(2) Fermentation broth of bacillus subtilis (Bacillus subtilis) No. 1 CGMCC No. 26006: and (3) performing high-density fermentation culture on the bacillus subtilis, setting two experimental groups, respectively performing shake culture for 24 hours at 37 ℃ and 200rpm/min with seed liquid inoculum size of 3% and 4%, performing stationary culture for 24 hours to obtain a bacillus subtilis culture, centrifuging the culture, and filtering with a 0.22 mu m filter membrane to obtain the bacillus subtilis.
2. The experimental method comprises the following steps:
fruits which are fresh, uniform in size and have no mechanical damage and diseases and similar maturity are selected and divided into three groups: one group is CK, and no treatment is carried out; the other two groups are treatment groups, respectively filling sterilized watering cans into fermentation liquor of bacillus subtilis 1 CGMCC No.26006, uniformly spraying the fermentation liquor on the surfaces of tomatoes, standing for 5 hours until the surfaces are completely dried, filling the tomatoes into PE packaging bags, sealing, punching 10 fruits, and storing the tomatoes at normal temperature for 16 days. The initial value was sampled and measured as index 0d, and the index was measured every 0d, 4d, 8d, 12d, and 16d. Repeated three times.
3. Evaluation index
(1) Weight loss ratio: weight loss (%) = (mass of tomato before storage-mass of tomato after storage)/mass of tomato before storage x 100.
(2) Decay rate: % rotten = (real number of tomato without commercial value/real number of total tomato fruit) ×100.
(3) Respiration intensity: 1kg of tomatoes are placed in a chamber of a breath strength direct reader, measured for 15min and the result is recorded.
4. Experimental results
(1) Weight loss ratio: after 1 week, for example from day 8 to day 16, the inhibition effect of the fermentation broth of bacillus subtilis No. 1 cgmccno.26006 on the tomato weight loss rate was significantly better than the control see table 1 (showing only the results with significant differences) and fig. 3.
Table 1 weight loss rate significance analysis
(2) Decay rate: whether short-term (e.g., within 1 week) or long-term (e.g., over 2 weeks), the fermentation broth of bacillus subtilis No. 1 CGMCC No.26006 showed significantly better inhibition effect on tomato decay rate than the control, see table 2 (showing only the significantly different results) and fig. 4.
TABLE 2 analysis of the significance of decay rate
(3) Respiration intensity: after storage for more than 2 weeks, for example 16 days, the fermentation broth of bacillus subtilis No. 1 cgmccno.26006 showed significantly better inhibition effect on tomato breath intensity than the control, see table 3 (showing only the significantly different results) and fig. 5.
Table 3 breath intensity significance analysis
In conclusion, the bacillus subtilis has remarkable long-term fresh-keeping effect on the quality of tomato fruits: can make the weight loss rate of tomato fruits lower, the decay rate lower and the respiration intensity lower after more than 10 days, especially after 2 weeks after harvest.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (8)
1. A bacillus subtilis, characterized in that: the bacillus subtilis is [ ]Bacillus subtilis) Is No. 1, and the registration number of the microbial agent in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No.26006.
2. A microbial inoculum, characterized in that: comprising the bacillus subtilis of claim 1, a bacterial suspension of the bacillus subtilis, a fermentation broth of the bacillus subtilis, and/or one or more mixtures thereof, wherein the fermentation broth of the bacillus subtilis does not contain the bacillus subtilis of claim 1.
3. A preservative, which is characterized in that: comprising the microbial agent of claim 2 and other preservative agents; or comprises the microbial inoculum of claim 2 and other active ingredients of fresh-keeping agents.
4. A method for preparing the microbial agent of claim 2 or the preservative of claim 3, which is characterized in that: comprising the step of culturing the Bacillus subtilis of claim 1.
5. A tomato fruit fresh-keeping method is characterized in that: contacting tomato fruits with a fermentation broth of the bacillus subtilis according to claim 1, wherein the fermentation broth is obtained by centrifugally filtering a culture of the bacillus subtilis and does not contain the bacillus subtilis according to claim 1.
6. Use of the bacillus subtilis according to claim 1, the microbial inoculum according to claim 2, or the preservative according to claim 3 for preserving tomato fruits.
7. The method according to claim 5 or the use according to claim 6, characterized in that the tomato fruit is kept fresh for inhibition of respiration intensity, and/or inhibition of decay, and/or inhibition of weight loss.
8. Use of the bacillus subtilis of claim 1, the microbial inoculum of claim 2, or the preservative of claim 3 for inhibiting aspergillus niger and aspergillus flavus.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005343823A (en) * | 2004-06-03 | 2005-12-15 | Ogawa & Co Ltd | Antibacterial composition |
| CN102816725A (en) * | 2012-09-07 | 2012-12-12 | 江苏省农业科学院 | Bacillus subtilis and application thereof |
| CN105441350A (en) * | 2015-06-15 | 2016-03-30 | 北京农学院 | Bacillus subtilis capable of producing substance with multiple enzyme activities and application of Bacillus subtilis |
| CN113528377A (en) * | 2021-06-21 | 2021-10-22 | 山东碧蓝生物科技有限公司 | Bacillus subtilis with growth promoting and corrosion preventing effects and application thereof |
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- 2022-12-19 CN CN202211640528.6A patent/CN115975882B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005343823A (en) * | 2004-06-03 | 2005-12-15 | Ogawa & Co Ltd | Antibacterial composition |
| CN102816725A (en) * | 2012-09-07 | 2012-12-12 | 江苏省农业科学院 | Bacillus subtilis and application thereof |
| CN105441350A (en) * | 2015-06-15 | 2016-03-30 | 北京农学院 | Bacillus subtilis capable of producing substance with multiple enzyme activities and application of Bacillus subtilis |
| CN113528377A (en) * | 2021-06-21 | 2021-10-22 | 山东碧蓝生物科技有限公司 | Bacillus subtilis with growth promoting and corrosion preventing effects and application thereof |
Non-Patent Citations (1)
| Title |
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| 枯草芽孢杆菌BS-1菌株对猕猴桃采后软腐病的抑制和保鲜效果评价;刘奎等;《保鲜与加工》;参见全文 * |
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