CN111109297A - Application of shell powder of mytilus coruscus in bacteriostasis of marine bacteria - Google Patents
Application of shell powder of mytilus coruscus in bacteriostasis of marine bacteria Download PDFInfo
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- CN111109297A CN111109297A CN201911411184.XA CN201911411184A CN111109297A CN 111109297 A CN111109297 A CN 111109297A CN 201911411184 A CN201911411184 A CN 201911411184A CN 111109297 A CN111109297 A CN 111109297A
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Abstract
The invention provides an application of shell powder of mytilus coruscus in the bacteriostasis aspect of marine bacteria, wherein waste mytilus coruscus shells are cleaned, dried, ground and calcined to obtain the shell powder; inoculating marine bacteria to be detected into a liquid culture medium for activation, then adding shell powder into the liquid culture medium for reaction, and sterilizing for later use; the shell powder has different bacteriostatic effects on Shewanella, vibrio splendidus and pseudoalteromonas at different concentrations, and the bacteriostatic rate in 24 hours reaches 93.6 percent, 92.1 percent and 95.7 percent respectively; the antibacterial effects of the calcined shell powder on Shewanella bacteria and vibrio splendidus tend to be stable within 3 hours and 6 hours respectively, and the antibacterial rate of the shell powder has no obvious difference along with the increase of time; therefore, the shell powder of the mytilus coruscus has bacteriostatic activity to certain marine bacteria, the bacteriostatic effect can tend to be stable in a short time, and a new direction is provided for resource utilization of the shells of the waste mytilus coruscus.
Description
Technical Field
The invention belongs to the technical field of antibacterial materials, and particularly relates to application of shell powder of mytilus coruscus in the aspect of bacteriostasis of marine bacteria.
Background
Bacteriostasis is the process of inhibiting the activity of microbes in the system to be treated, reducing their reproductive capacity or inhibiting their reproduction. In marine environments, certain marine bacteria are pathogenic bacteria of marine organisms; some bacteria can participate in the corrosion, denaturation, pollution and destruction processes of various marine substances; under certain conditions, the accumulation of marine bacterial metabolites can poison the aquaculture environment, such as the ammonia and hydrogen sulfide hazard to the aquaculture. With the increasing emphasis on environmental destruction of marine bacteria, many researchers have paid attention to bio-antibacterial materials that are non-toxic, efficient, and safe in performance.
The shell is a mollusk mantle, is a typical natural biomineralization material, has a very special internal microstructure, and has the characteristics of durability, chemical stability, heat resistance and the like. Wherein the stratum corneum has the characteristics of thinness, transparency, acid corrosion resistance and the like. The source of the shell is wide, the development potential is large, but the types of the shell related in the bacteriostatic research are few.
Mytilus coruscus belongs to the phylum Mollusca, the class Bivalvia, the order Mytilidae and the family Mytilidae, and is an important marine shellfish culture variety in China and is distributed along the coast of the yellow sea, the Bohai sea and the east sea. The mytilus coruscus has high nutritive value and has wide breeding prospect and market development potential. With the continuous expansion of the breeding yield of the mytilus coruscus in China, the environmental pressure is intensified by the waste of the shells.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the application of the shell powder of the mytilus coruscus in the aspect of bacteriostasis of marine bacteria.
In order to achieve the above purpose, the solution of the invention is as follows:
an application of shell powder of Mytilus coruscus in inhibiting marine bacteria is provided.
In a specific embodiment of the invention, the shells of the waste mytilus coruscus are cleaned, dried, ground and calcined to obtain the shell powder of the mytilus coruscus; inoculating marine bacteria to be detected into a liquid culture medium for activation, then adding shell powder of the mytilus coruscus into the liquid culture medium for reaction, and sterilizing at 121 +/-10 ℃ for later use.
In one embodiment of the invention, the shell powder has a diameter of less than or equal to 100 μm after grinding.
In one embodiment of the invention, the calcination temperature is 500 + -10 deg.C and the calcination time is 1.5 + -0.5 h.
In one embodiment of the invention, the marine bacteria to be tested are selected from the group consisting of Shewanella, Vibrio splendidus and Pseudomonas pseudoalteromonas.
In one embodiment of the present invention, the liquid medium is LB liquid medium.
In a specific embodiment of the present invention, the LB liquid medium comprises the following components: tryptone, yeast extract and agar.
In one embodiment of the invention, the activation temperature is 25 + -2 deg.C and the activation time is 12 + -1 h.
In one embodiment of the invention, the reaction temperature is 25 + -2 deg.C and the reaction time is 2 + -0.5 h.
Due to the adoption of the scheme, the invention has the beneficial effects that:
the shell powder of the calcined mytilus coruscus has different bacteriostatic effects on Shewanella, Vibrio splendidus and pseudoalteromonas at different concentrations, and the bacteriostatic rate in 24 hours reaches 93.6%, 92.1% and 95.7% respectively. In the growth of Shewanella bacteria and vibrio lautus, the antibacterial effects of calcined shell powder on Shewanella bacteria and vibrio lautus tend to be stable within 3h and 6h respectively, and the antibacterial rate of the shell powder has no obvious difference along with the increase of time. Therefore, the shell powder of the mytilus coruscus has bacteriostatic activity to certain marine bacteria, the bacteriostatic effect can tend to be stable in a short time, and a new direction is provided for resource utilization of the shells of the waste mytilus coruscus.
Drawings
FIG. 1 is a schematic diagram showing the effect of the shell powder of Mytilus coruscus with different concentrations on the inhibition rate of Shewanella.
FIG. 2 is a schematic diagram showing the effect of the shell powder of Mytilus coruscus with different concentrations on the bacteriostasis rate of Vibrio splendidus.
Fig. 3 is a schematic diagram showing the effect of the shell powder of the mytilus coruscus with different concentrations on the bacteriostasis rate of pseudoalteromonas.
FIG. 4 is a schematic diagram showing the effect of the shell powder of Mytilus coruscus on the inhibition rate of Shewanella and Vibrio lautus (left: Shewanella, right: Vibrio lautus, and a, b, and c are differential analyses).
Detailed Description
The invention provides application of shell powder of mytilus coruscus in bacteriostasis of marine bacteria.
The method comprises the steps of collecting waste mytilus coruscus shells, cleaning and drying the shells, grinding the shells into uniform sizes by using a ball mill to enable the diameters of the shell powder to be less than or equal to 100 micrometers, calcining the shells in a muffle furnace to obtain the shell powder of the mytilus coruscus, and taking out and storing the shells in a sealed mode for later use. Shewanella (Shewanella loihica), Vibrio splendidus (Vibrio spleendidus) and Pseudoalteromonas (Pseudomonas atlantic) were inoculated into LB liquid medium for activation, and shake-cultured at 25 ℃ for 12 hours for further use. Then adding the shell powder of the calcined mytilus coruscus into LB liquid culture medium for reaction, placing the liquid culture medium on a magnetic stirrer, operating at 1000rpm and 25 ℃ for 2h, measuring the pH value of the liquid culture medium to be 7.5, and sterilizing at 121 +/-10 ℃ for later use.
Wherein the calcining temperature is 500 +/-10 ℃, and the calcining time is 1.5 +/-0.5 h.
The LB liquid culture medium adopts a synthetic culture medium of Shanghai province company of biological engineering, and the prepared components are as follows: tryptone, yeast extract and agar.
The concentration of shell powder of Mytilus coruscus is 1mg/mL, 2mg/mL, 4mg/mL, 6mg/mL, 8mg/mL, and 10 mg/mL.
According to VBacterial liquid：VCulture medium1: 100 of the culture medium, wherein the culture medium is cultured Shewanella loihica, Vibrio spleendidus and Pseudomonas atlanticA single bacterial liquid; the preparation process comprises inoculating Shewanella loihica, Vibrio spleendidus and Pseudomonas atlantic to LB liquid medium respectively for activation, and performing shake culture at 25 deg.C for 12 h. The control group 1 is a pure LB liquid culture medium, the control group 2 is a LB liquid culture medium added with shell powder with different concentrations, the experimental group 1 is a pure LB liquid culture medium inoculated with experimental bacteria, and the experimental group 2 is a LB liquid culture medium added with shell powder with different concentrations and experimental bacteria. The control and experimental groups were incubated at 25 ℃ for 24h in a 200rpm incubator and the OD of the inoculum solution was measured at 600 nm. The bacteriostasis rate is calculated according to the following formula:
At＝1-(OD2-ODc2)/(OD1-ODc1)×100％
in the formula AtThe antibacterial rate, OD, is measured by turbidimetryc1Absorbance, OD, for control 11Absorbance, OD of Experimental group 1c2Absorbance, OD, of control 22The absorbance was that of experimental group 2.
The present invention will be further described with reference to the following examples.
Example 1:
in this example, Shewanella loihica, Vibrio spleendidus and Pseudoalteromonas lantica were inoculated into LB liquid medium for activation and shake-cultured at 25 ℃ for 12 hours for further use. Adding calcined shell powder of the mytilus coruscus into an LB liquid culture medium, wherein the adding concentrations of the shell powder are 1mg/mL, 2mg/mL, 4mg/mL, 6mg/mL, 8mg/mL and 10mg/mL respectively; placing the culture medium on a magnetic stirrer, and operating at 1000rpm and 25 deg.C for 2 h; the pH of the liquid medium was measured and sterilized at 121 ℃ for use.
According to VBacterial liquid：VCulture medium1: the ratio of 100 was added to the liquid medium. The control group 1 is a pure LB liquid culture medium, the control group 2 is an LB liquid culture medium added with shell powder with different concentrations, the experimental group 1 is a pure LB liquid culture medium inoculated with experimental bacteria, and the experimental group 2 is an LB liquid culture medium added with shell powder with different concentrations and experimental bacteria. After the control group and the experimental group were cultured at 25 ℃ for 24 hours in a shaker at 200rpm, 3mL of the bacterial solution was taken and the OD value of the bacterial solution was measured at 600 nm. And according to the above formula (i.e. by turbidity ratio)Comparing the bacteriostasis rates of the shell powder with different adding concentrations on different strains by a color method) to calculate the bacteriostasis rate of each concentration.
The inhibition rate of the shell powder for inhibiting 3 strains of marine bacteria is increased along with the increase of the concentration. Specifically, as can be seen from fig. 1, for shewanella, the maximum value of the inhibition rate reaches 93.6% after the addition concentration of shell powder reaches 10 mg/mL; as can be seen from FIGS. 2 and 3, for Vibrio splendidus and Pseudomonas pseudoalteromonas, the maximum bacteriostatic rate was reached at a concentration of 8mg/mL of shell powder added, which was 92.1% and 95.7%, respectively, and the bacteriostatic activity was not substantially enhanced with the increase in concentration when the concentration was increased to 10 mg/mL. In conclusion, the calcined shell powder of the mytilus coruscus has obvious bacteriostatic action on 3 marine bacteria adopted in the experiment, and has very obvious bacteriostatic effect when the concentration of the shell powder reaches 10 mg/mL.
Example 2:
the bacterial inhibition time variation experiment of the calcined shell powder of the mytilus coruscus of the embodiment is as follows. Shewanellaloihica and Vibrio spleendidus were inoculated into LB liquid medium for activation, and shake-cultured at 25 ℃ for 12 hours for further use. Adding calcined shell powder of the mytilus coruscus into an LB liquid culture medium, wherein the adding concentration of the shell powder is 10 mg/mL; placing the culture medium on a magnetic stirrer, and operating at 1000rpm and 25 deg.C for 2 h; the pH of the liquid medium was measured and sterilized at 121 ℃ for use.
According to VBacterial liquid：VCulture medium1: the ratio of 100 was added to the liquid medium. The control group 1 is a pure LB liquid culture medium, the control group 2 is an LB liquid culture medium with 10mg/mL of shell powder, the experimental group 1 is a pure LB liquid culture medium for inoculating the experimental bacteria, and the experimental group 2 is an LB liquid culture medium added with 10mg/mL of shell powder and the experimental bacteria. The control group and the experimental group are cultured in a shaker at 25 ℃ and 200rpm, and after 1h, 3h, 6h, 12h, 18h and 24h of culture, 3mL of bacterial liquid is taken to measure the OD value of the bacterial liquid at 600 nm. And calculating the bacteriostasis rate at each time according to the formula.
As shown in figure 4, in the culture medium with the fixed adding concentration of the shell powder, the shell powder can achieve the bacteriostatic effect in a short time. For Shewanella (a), the shell powder can have a stable bacteriostatic effect when acting on bacteria for 3 hours; for vibrio splendidus (b), the bacteriostasis effect is stable in 6 hours, and the bacteriostasis rate of the shell powder is not changed greatly after the concentration is increased. Therefore, the shell powder can ensure stable bacteriostatic effect within a certain time, and the bacteriostatic characteristic is very important for the practical application of the shell powder.
In conclusion, the three marine bacteria of Shewanella, Vibrio splendidus and pseudoalteromonas can achieve better bacteriostatic effect under the condition that the calcined shell powder concentration in the culture medium is 10mg/mL, 8mg/mL and 8mg/mL, and the bacteriostatic rate of 24h can respectively reach 93.6%, 92.1% and 95.7%. In the growth of Shewanella bacteria and vibrio lautus, the antibacterial effects of calcined shell powder on Shewanella bacteria and vibrio lautus tend to be stable within 3h and 6h respectively, and the antibacterial rate of the shell powder has no obvious difference along with the increase of time. Therefore, the shell powder of the mytilus coruscus has bacteriostasis to certain marine bacteria, and the bacteriostasis effect can be stable in a short time. In the culture medium added with the shell powder with fixed concentration, the shell powder can achieve the bacteriostatic effect in a short time, so that the shell powder can ensure the stable bacteriostatic effect in a certain time, and the bacteriostatic characteristic is very important for the practical application of the shell powder.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Claims (9)
1. An application of shell powder of Mytilus coruscus in inhibiting marine bacteria is provided.
2. Use according to claim 1, characterized in that: cleaning, drying, grinding and calcining the waste shells of the mytilus coruscus to obtain the shell powder of the mytilus coruscus;
inoculating marine bacteria to be detected into a liquid culture medium for activation, then adding shell powder of the mytilus coruscus into the liquid culture medium for reaction, and sterilizing at 121 +/-10 ℃ for later use.
3. Use according to claim 2, characterized in that: after grinding, the diameter of the shell powder is less than or equal to 100 mu m.
4. Use according to claim 2, characterized in that: the calcining temperature is 500 +/-10 ℃, and the calcining time is 1.5 +/-0.5 h.
5. Use according to claim 2, characterized in that: the marine bacteria to be detected are selected from Shewanella, Vibrio splendidus and pseudoalteromonas.
6. Use according to claim 2, characterized in that: the liquid culture medium is LB liquid culture medium.
7. Use according to claim 6, characterized in that: the LB liquid culture medium comprises the following components: tryptone, yeast extract and agar.
8. Use according to claim 2, characterized in that: the temperature of the activation is 25 +/-2 ℃, and the time of the activation is 12 +/-1 h.
9. Use according to claim 2, characterized in that: the reaction temperature is 25 +/-2 ℃, and the reaction time is 2 +/-0.5 h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115108779A (en) * | 2022-06-21 | 2022-09-27 | 青岛大学 | Waste active shellfish inactivated virus functional decorative wall material and preparation method thereof |
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