CN115778933A - Application of palmitic acid in inhibiting activity of vibrio alginolyticus alkaline serine protease - Google Patents
Application of palmitic acid in inhibiting activity of vibrio alginolyticus alkaline serine protease Download PDFInfo
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- CN115778933A CN115778933A CN202210839655.2A CN202210839655A CN115778933A CN 115778933 A CN115778933 A CN 115778933A CN 202210839655 A CN202210839655 A CN 202210839655A CN 115778933 A CN115778933 A CN 115778933A
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- Prior art keywords
- vibrio alginolyticus
- palmitic acid
- serine protease
- alkaline serine
- activity
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- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 title claims abstract description 101
- 241000607594 Vibrio alginolyticus Species 0.000 title claims abstract description 59
- 235000021314 Palmitic acid Nutrition 0.000 title claims abstract description 50
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 101710184263 Alkaline serine protease Proteins 0.000 title claims abstract description 33
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 19
- 230000000694 effects Effects 0.000 claims abstract description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000011550 stock solution Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 3
- 230000003313 weakening effect Effects 0.000 claims description 3
- 230000001018 virulence Effects 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 abstract description 10
- 238000002474 experimental method Methods 0.000 abstract description 9
- 238000002360 preparation method Methods 0.000 abstract description 7
- 229940079593 drug Drugs 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 5
- 206010059866 Drug resistance Diseases 0.000 abstract description 4
- 230000005764 inhibitory process Effects 0.000 abstract description 4
- 238000009364 mariculture Methods 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 229940122055 Serine protease inhibitor Drugs 0.000 abstract 1
- 239000003001 serine protease inhibitor Substances 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 4
- 238000009360 aquaculture Methods 0.000 description 3
- 244000144974 aquaculture Species 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005202 decontamination Methods 0.000 description 2
- 230000003588 decontaminative effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000007923 virulence factor Effects 0.000 description 2
- 239000000304 virulence factor Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 101710117021 Tyrosine-protein phosphatase YopH Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000012736 patent blue V Nutrition 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000010267 two-fold dilution method Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
The invention belongs to the field of microbial prevention and control preparations, and particularly discloses application of palmitic acid in inhibiting activity of vibrio alginolyticus alkaline serine protease. Experiments show that the palmitic acid has a good inhibition effect on the activity of the alkaline serine protease, so that the palmitic acid can be used as an inhibitor of the alkaline serine protease of vibrio alginolyticus. The invention opens up a new application of palmitic acid, and the palmitic acid is used as an alkaline serine protease inhibitor of vibrio alginolyticus, thereby providing a new candidate preparation for vibrio alginolyticus treatment; under the condition that the clinical Vibrio alginolyticus drug resistance is increasingly common and the drug resistance degree is increasingly serious, the anti-Vibrio alginolyticus inhibitor recovers the effect on the drug-resistant Vibrio alginolyticus, thereby saving the economic cost for the mariculture industry and reducing the toxic and side effects of other inhibitors.
Description
Technical Field
The invention belongs to the field of microbial prevention and control preparations, and particularly relates to application of palmitic acid in inhibiting activity of vibrio alginolyticus alkaline serine protease.
Background
The vibrio alginolyticus is common aquaculture pathogenic bacteria, can infect various marine organisms, and has the characteristics of high transmissibility, high pathogenicity, high drug resistance and the like. In recent years, the mariculture industry suffers from a significant economic loss caused by Vibrio alginolyticus. Therefore, it is important to study the pathogenic mechanism of Vibrio alginolyticus, in which alkaline serine protease is an important virulence factor in Vibrio alginolyticus. Nowadays, antibiotics are the main means for preventing and controlling vibrio alginolyticus, but abuse of antibiotics causes serious environmental pollution and the appearance of drug-resistant strains, so that a novel non-toxic and harmless natural inhibitor is urgently needed.
Disclosure of Invention
Alkaline serine protease is an important virulence factor of vibrio alginolyticus. The invention aims to provide application of palmitic acid in inhibiting activity of vibrio alginolyticus alkaline serine protease.
The invention is realized by the following technical scheme:
the invention discloses application of palmitic acid in inhibiting activity of vibrio alginolyticus alkaline serine protease.
Preferably, the vibrio alginolyticus is EPGS, and the preservation number of the strain is CCTCC No. AB209306.
Preferably, the inhibition of vibrio alginolyticus alkaline serine protease activity is performed to achieve the effect of weakening the virulence inducing ability of vibrio alginolyticus.
Preferably, a stock solution of 10 mg/mL is prepared from palmitic acid and absolute ethanol (with a concentration of more than 99.5%), and is stored at 4 ℃ for later use.
Preferably, the palmitic acid has a remarkable inhibitory effect on the activity of the vibrio alginolyticus alkaline serine protease within the concentration range of 1/8 MIC to 1/2 MIC (MIC = 0.6 mg/mL).
Compared with the existing inhibitor, the inhibitor has the following beneficial inhibiting effects:
the invention opens up a new application for palmitic acid, and the palmitic acid is used for inhibiting the activity of vibrio alginolyticus alkaline serine protease, including weakening the activity of vibrio alginolyticus alkaline serine protease, provides a new candidate inhibitor for inhibiting the vibrio alginolyticus alkaline serine protease, and enables the anti-vibrio alginolyticus inhibitor to recover the effect on the drug-resistant vibrio alginolyticus under the conditions that the clinical vibrio alginolyticus drug resistance is increasingly common and the drug-resistant degree is increasingly serious, thereby saving the economic cost for the marine aquaculture and reducing the toxic and side effects of other inhibitors.
The experiment for detecting the activity of the palmitic acid for inhibiting the vibrio alginolyticus alkaline serine protease shows that the palmitic acid has a strong inhibiting effect on the vibrio alginolyticus alkaline serine protease, and the activity of the vibrio alginolyticus alkaline serine protease can be obviously inhibited after the palmitic acid is added, so that the pathogenic capability of the vibrio alginolyticus is reduced. Palmitic acid can therefore be used as an anti-vibrio alginolyticus preparation.
Drawings
FIG. 1 graph of the effect of palmitic acid on the activity of Vibrio alginolyticus alkaline serine protease (". X.") indicates the significance of the three experimental groups to the control groupp＜0.01）。
Detailed Description
The present invention will now be described in further detail with reference to specific embodiments thereof, which are illustrated by way of example and not by way of limitation.
Examples
1. Raw materials:
palmitic acid (CAS No. 57-10-3, sigma-Aldrich): purchased from Shanghai Tantake technologies, inc.;
absolute ethanol: purchased from Tianjin, nature chemical Co., ltd;
the reagents were stored at 4 ℃. And taking out before the experiment, fully and uniformly mixing, and then inspecting.
2. Bacterial strains
The strain used in the research is vibrio alginolyticus EPGS (strain preservation number CCTCC No. AB 209306) provided by Arhua bioengineering research institute of university of east China Physiology, and the vibrio alginolyticus is stored at-80 ℃ for a long time.
3. Culture medium
LBS liquid culture medium: (3% (w/v) NaCl,1% (w/v) Tryptone,0.5% (w/v) Yeast Extract) were mixed in an aqueous solution, autoclaved (121 ℃,20 min), and stored at 4 ℃ for future use.
4. Instrumentation and equipment
LRH-250 biochemical incubator (Shanghai-Hengscientific instruments Co., ltd.);
ZD-85 gas bath constant temperature oscillator (Changzhou Jintanliang instruments, inc.);
SW-CJ-IFD single-person single-side decontamination workbench (Suzhou decontamination Equipment, inc.);
5. palmitic acid stock solutions
The palmitic acid was dissolved in absolute ethanol to prepare a stock solution with a concentration of 10 mg/mL, and stored at 4 ℃ for further use.
6. Preparation of bacterial liquid
During experiment, 1% of inoculum size of the strain is inoculated to LBS culture medium, 0.1% (v/v) ampicillin is added, and activation recovery culture is carried out for 12 h at 200r/min and 30 ℃ to ensure that the final concentration of the bacterial liquid is about 10 8 ~10 9 CFU/mL (4 ℃ storage) was used, and Vibrio alginolyticus was subjected to the above activation culture before all the tests of the present invention.
7. Preparation of drug sensitive plate
The invention adopts a two-fold dilution method to determine the MIC of the palmitic acid disturbance vibrio alginolyticus, and the specific operations are as follows: different milliliters of palmitic acid stock solution are added into LBS culture medium to make the final concentration of the palmitic acid CK (5 uL of absolute ethyl alcohol is added as a control group), 0.0375 mg/mL, 0.075 mg/mL, 0.15 mg/mL, 0.3 mg/mL, 0.6 mg/mL and 1.2 mg/mL, then the activated Vibrio alginolyticus is inoculated according to the inoculation amount of 1% (v/v) for shaking, LBS bacterial suspension containing the palmitic acid is transplanted into a 24-well plate, and meanwhile bacterial suspension without the palmitic acid is used as a control group of the experiment. The 24-well plate was incubated at a constant temperature in a 30 ℃ incubator.
And (3) culturing the vibrio alginolyticus for 24 hours in a constant-temperature incubator at 30 ℃, and observing the growth condition of the bacteria. The minimum concentration of the added palmitic acid when the culture solution is clear and the thalli can not be seen by naked eyes is the MIC of the palmitic acid to the vibrio alginolyticus. The experiments are all operated in parallel for 3 times, and the MIC value is accepted when the MIC value can be accurately repeated or only differs by one concentration, and the higher concentration is taken as the MIC value; when the MIC values differ by more than two concentrations, re-experiments are required until the requirements are met. The experimental results show that: the MIC of palmitic acid to Vibrio alginolyticus was 0.6 mg/mL.
8. Effect of sub-inhibitory concentration of palmitic acid on Activity of Vibrio alginolyticus alkaline serine protease
According to the MIC obtained by 7, the invention detects the activity of the alkaline serine protease of the vibrio alginolyticus under an experimental group with low-dose palmitic acid concentration of 1/8 MIC, 1/4 MIC and 1/2 MIC of palmitic acid and a control group without adding CK palmitic acid.
Alkaline serine protease (Asp) is a main extracellular virulence protein of vibrio alginolyticus, and the activity of the alkaline serine protease is quantitatively detected through the reaction of a sky blue powder (HPA) substrate.
Culturing at 30 deg.C for 9h to 1.5mL of secondary activated strain culture solution at late logarithmic growth stage in an EP tube, centrifuging at 12000rpm/min for 1min, transferring 1mL of supernatant into a culture flask containing 0.01g HPA and 1mL PBS, reacting at 37 deg.C and 200r/min for 2h, centrifuging at 12000rpm/min for 1min, taking a picture of the supernatant, and measuring the absorbance of the supernatant at 600 nm.
As a result, as shown in FIG. 1, the activity of Asp in the experimental group treated with palmitic acid was significantly decreased, and the decreased trend was inversely related to the concentration of palmitic acid. This result shows that: palmitic acid severely inhibits the activity of Vibrio alginolyticus Asp.
Experiments show that when the vibrio alginolyticus has the sub-inhibitory concentrations of 1/8 MIC, 1/4 MIC and 1/2 MIC of palmitic acid, the alkaline serine protein activity of the vibrio alginolyticus is respectively reduced by 59.38%, 84.37% and 90.63% relative to a control group, and the results show that the palmitic acid has a remarkable inhibition effect on the alkaline serine protease activity of the vibrio alginolyticus.
In conclusion, experiments show that the palmitic acid has a good inhibition effect on the activity of the vibrio alginolyticus alkaline serine protease, so that the palmitic acid can be used as a preparation for preventing and treating vibrio alginolyticus diseases. The invention opens up a new application for palmitic acid, can be used as an inhibitor for preventing and controlling vibrio alginolyticus diseases, and provides a new candidate antibiotic substitute for preventing and controlling the marine aquaculture industry.
Claims (5)
1. Use of palmitic acid for inhibiting vibrio alginolyticus alkaline serine protease activity.
2. The use of palmitic acid in inhibiting the activity of vibrio alginolyticus alkaline serine protease according to claim 1, wherein the vibrio alginolyticus is vibrio alginolyticus EPGS and the strain preservation number is CCTCC No. AB209306.
3. The use of palmitic acid according to claim 1 for inhibiting vibrio alginolyticus alkaline serine protease activity, wherein the palmitic acid is used for weakening the virulence of vibrio alginolyticus by inhibiting vibrio alginolyticus alkaline serine protease activity.
4. The use of palmitic acid according to claim 1 for inhibiting the activity of vibrio alginolyticus alkaline serine protease, wherein the palmitic acid is formulated with absolute ethanol into a stock solution of 10 mg/mL and stored at 4 ℃ for use.
5. The use of palmitic acid according to claim 3, for inhibiting vibrio alginolyticus alkaline serine protease activity, wherein the palmitic acid has a significant inhibitory effect on vibrio alginolyticus alkaline serine protease activity in a concentration range of 1/8 MIC to 1/2 MIC (MIC = 0.6 mg/mL).
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210839655.2A CN115778933A (en) | 2022-07-18 | 2022-07-18 | Application of palmitic acid in inhibiting activity of vibrio alginolyticus alkaline serine protease |
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| CN202210839655.2A CN115778933A (en) | 2022-07-18 | 2022-07-18 | Application of palmitic acid in inhibiting activity of vibrio alginolyticus alkaline serine protease |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100016430A1 (en) * | 2005-12-07 | 2010-01-21 | Malaysian Agriculteral Research And Development In | Modified coconut oils with broad antimicrobial spectrum |
| CN107188797A (en) * | 2017-05-23 | 2017-09-22 | 集美大学 | A kind of method that palmitic acid is extracted in the ballstone algae from ocean |
| CN111991385A (en) * | 2020-08-17 | 2020-11-27 | 暨南大学 | Application of palmitoleic acid in inhibiting aquatic pathogenic bacteria |
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- 2022-07-18 CN CN202210839655.2A patent/CN115778933A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100016430A1 (en) * | 2005-12-07 | 2010-01-21 | Malaysian Agriculteral Research And Development In | Modified coconut oils with broad antimicrobial spectrum |
| CN107188797A (en) * | 2017-05-23 | 2017-09-22 | 集美大学 | A kind of method that palmitic acid is extracted in the ballstone algae from ocean |
| CN111991385A (en) * | 2020-08-17 | 2020-11-27 | 暨南大学 | Application of palmitoleic acid in inhibiting aquatic pathogenic bacteria |
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