CN111991385A - Application of palmitoleic acid in inhibiting aquatic pathogenic bacteria - Google Patents
Application of palmitoleic acid in inhibiting aquatic pathogenic bacteria Download PDFInfo
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- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The invention discloses an application of palmitoleic acid in inhibiting aquatic pathogenic bacteria, and relates to a new application of palmitoleic acid in microalgae. The palmitoleic acid extracted from the microalgae has obvious antibacterial effects on vibrio parahaemolyticus, vibrio alginolyticus and streptococcus agalactiae, and the microalgae is originally sourced from a water body and is an important participant in a food chain of an ecological culture environment. Therefore, the development of a novel fishing medicine product based on the antibacterial palmitoleic acid from microalgae can reduce the use of antibiotics, promote the green development of fishery and lay a foundation for the development of green fishing medicines.
Description
Technical Field
The invention relates to a new application of palmitoleic acid in microalgae, in particular to an application of palmitoleic acid in inhibiting aquatic pathogenic bacteria.
Background
Palmitoleic acid, a functional monounsaturated fatty acid of omega 7, of formula C16H30O2It is widely used in plant, animal fat, yeast and microalgae. Wherein the microalgae is used as natural source of palmitoleic acid, and has the advantages of rapid growth, high photosynthetic efficiency and carbon fixationEmission reduction and the like; in recent years, research shows that microalgae such as euglena, chrysophyceae, phaeodactylum tricornutum and the like can accumulate a large amount of palmitoleic acid, the highest content of palmitoleic acid can reach 30% of the dry weight of cells, and the content of palmitoleic acid is obviously higher than that of the microalgae from traditional animals and plants (fish oil and sea buckthorn). In addition, the separation and concentration (ZL201810970871.4) of palmitoleic acid in the xanthomonas has been realized in earlier researches by the inventor, and a foundation is laid for the application of palmitoleic acid from microalgae.
Vibrio and streptococcus agalactiae are high-risk pathogenic bacteria causing aquaculture diseases, particularly vibrio parahaemolyticus, are food-borne pathogenic bacteria, and can cause diseases of aquatic animals and human diseases. At present, antibiotic medicines are mostly adopted for prevention and treatment in aquaculture, which is not beneficial to the development of green ecological culture. Palmitoleic acid, as an antibacterial fatty acid, has been reported in inhibiting human pathogenic bacteria such as drug-resistant staphylococcus aureus, streptococcus salivarius, candida albicans and the like, but has very few reports on bactericidal activity against aquatic pathogenic bacteria.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the application of palmitoleic acid in inhibiting aquatic pathogenic bacteria.
In order to reduce the use of antibiotics in aquaculture, the invention discovers the antibacterial activity of palmitoleic acid in microalgae on vibrio parahaemolyticus, vibrio alginolyticus and streptococcus agalactiae, and lays a foundation for the development of green fishery drugs.
The purpose of the invention is realized by the following technical scheme:
the invention provides an application of palmitoleic acid in inhibiting aquatic pathogenic bacteria.
Preferably, palmitoleic acid is used in the preparation of products for inhibiting aquatic pathogenic bacteria.
The palmitoleic acid is concentrated palmitic acid from microalgae; further, the concentrated palmitic acid is derived from the yellow silk algae, and the purity is about 81 percent.
The aquatic pathogenic bacteria is at least one of aquatic pathogenic vibrio and aquatic pathogenic streptococcus;
preferably, the aquatic pathogenic bacteria is at least one of vibrio parahaemolyticus, vibrio alginolyticus and streptococcus agalactiae.
The effective concentration of the palmitoleic acid is more than or equal to 31.25 mug/mL; further 31.25 to 500. mu.g/mL.
The dosage form of the product is liquid medicine, ointment or powder;
the product contains one or more pharmaceutically acceptable carriers or excipients.
The antibacterial activity of palmitoleic acid on aquatic pathogenic bacteria is obtained by the following steps:
1) activation of pathogenic bacteria
Respectively inoculating aquatic pathogenic bacteria frozen at-80 deg.C onto brain heart infusion Broth (BHI) solid plate by plate streaking method (adding 37g BHI into 1L deionized water, sterilizing 15g agar powder at 121 deg.C under high pressure for 20min, pouring plate), culturing at 28 deg.C for 24 hr, selecting monoclonal by inoculating loop, inoculating into BHI liquid culture medium (without adding agar powder), culturing at 28 deg.C and 180rpm under constant temperature shaking until OD of bacterial liquid is reached750＝1.0。
2) Preparation of samples
Dissolving a certain mass of concentrated palmitoleic acid (separated from chrysosporium) and a palmitoleic acid standard substance (more than or equal to 98%) in 5% DMSO to prepare 20mg/mL mother liquor; then diluted to the corresponding final concentration by an equal dilution method.
3) Antibacterial zone determination by filter paper sheet method
Firstly, sterile water is used for adjusting the concentration of fresh seed liquid of aquatic pathogenic bacteria to 1 multiplied by 106cfu/mL, then sucking 100 mu L of bacterial liquid, dripping the bacterial liquid on a BHI solid flat plate, and uniformly coating the bacterial liquid on the BHI solid flat plate by using a glass coating rod; secondly, clamping a sterile circular filter paper sheet (the diameter is 6cm) by using tweezers, gently placing the sterile circular filter paper sheet on the surface of a solid flat plate (the sterile circular filter paper sheet is triangular and symmetrical), marking, and slightly compressing by using the tweezers; then, concentrated palmitoleic acid, palmitoleic acid standards, and 5% DMSO (blank) at known concentrations were dropped onto corresponding pieces of filter paper, respectively; standing for 30min, transferring to a constant temperature incubator at 28 deg.C after the medicinal liquid is fully diffused, and culturing for 24 h; after the culture was completed, the diameter of the zone of inhibition was measured using a ruler.
4) Determination of Minimum Inhibitory Concentration (MIC) by Millipore dilution method
Weighing a certain mass of concentrated palmitoleic acid separated from chrysophyceae, dissolving in DMSO to prepare a mother liquor with a final concentration of 10mg/mL, diluting by equal times to concentrations of 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125 and 0.0390625mg/mL respectively, and sucking 20 mu L of the solution respectively and adding into a 96-well plate; secondly, diluting fresh seed liquid of aquatic pathogenic bacteria into a liquid with a thallus concentration of 1 × 10 by adopting BHI liquid culture medium6cfu/mL, 180. mu.L of the bacterial suspension was added to one well, so that the final concentrations of palmitoleic acid were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91. mu.g/mL, respectively. The experiment was additionally set up with two sets of controls: control a-20 μ L DMSO +180 μ L bacterial suspension; control B20 μ L sterile water +180 μ L of broth. Finally, the 96-well plate is placed in a constant temperature shaking table at 28 ℃ and the rotating speed is 45rpm, and the culture is carried out for 24h, and the MIC is the concentration corresponding to the complete bacteria-free growth in the 96-well plate.
Compared with the prior art, the invention has the following advantages and effects:
the palmitoleic acid extracted from the microalgae has obvious antibacterial effects on vibrio parahaemolyticus, vibrio alginolyticus and streptococcus agalactiae, and the microalgae is originally sourced from a water body and is an important participant in a food chain of an ecological culture environment. Therefore, the development of novel fishery medicine products based on the antibacterial palmitoleic acid from microalgae can reduce the use of antibiotics and promote the green development of fishery.
Drawings
FIG. 1 is the bacteriostatic activity of palmitoleic acid on Vibrio parahaemolyticus; wherein Ctrl: 5% DMSO; centralized PA: concentrating the palmitoleic acid; PA standard: palmitoleic acid standard.
FIG. 2 is the bacteriostatic activity of palmitoleic acid on Vibrio alginolyticus; wherein Ctrl: 5% DMSO; centralized PA: concentrating the palmitoleic acid; PA standard: palmitoleic acid standard.
FIG. 3 is the bacteriostatic activity of palmitoleic acid against Streptococcus agalactiae; wherein Ctrl: 5% DMSO; centralized PA: concentrating the palmitoleic acid; PA standard: palmitoleic acid standard.
FIG. 4 is palmitoleic acid vs. no milk chainMinimal inhibitory concentration of cocci; wherein, CPA: the concentration range of the concentrated palmitoleic acid is 3.91-500 mu g/mL (equal-time dilution); DMSO and H2O served as control groups, respectively.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus agalactiae used in examples;
vibrio parahaemolyticus is disclosed in "Minxia Yang, Jun Zhang, Qiting Liang, et al, 2019. Antagonitic activity of marine Streptomyces sp.073 on pathogenic Vibrio parahaemolyticus. Fisheies Science,85(3): 533-543";
streptococcus agalactiae in the literature "south China, Chenghui, Zhang, 2015.100 Chinese herbal medicine aqueous extracts inhibit and kill Streptococcus agalactiae in vitro pharmacodynamic studies [ J ]. ecology science, 34 (3): 26-30. ";
in the literature, "chenhui", wherein 2012.100 kinds of Chinese herbal medicines are used for in vitro bacteriostasis experiment of vibrio alginolyticus [ J ]. Chinese veterinary medical science, 34 (6): 452-455.
Palmitoleic acid in the examples (purity ≈ 81%) was derived from xanthothrice sp (ZL 201810970871.4).
Example 1: inhibitory Effect of palmitoleic acid on Vibrio parahaemolyticus
Diluting freshly prepared Vibrio parahaemolyticus seed solution to 1 × 10 with sterile water6cfu/mL, pipette 100. mu.L and spread evenly on BHI solid plates; clamping three sterile circular filter paper sheets with the diameter of 6mm, placing the sterile circular filter paper sheets on the surface of a culture medium coated with a bacterial solution in a triangular symmetry manner, respectively dropwise adding 7 mu L of a palmitoleic acid standard substance (PA standard) and Concentrated palmitoleic acid (Concentrated PA) which are dissolved in 5% DMSO and have the concentration of 10mg/mL, and blankControl (Ctrl) was 5% DMSO; standing for 30min, transferring to a constant temperature incubator at 28 deg.C after the medicinal liquid is fully diffused, and culturing for 24 h; after the culture was completed, the diameter of the zone of inhibition was measured using a ruler.
As shown in FIG. 1, both the palmitoleic acid standard and the concentrated palmitoleic acid isolated from xanthothrips significantly inhibited the growth of Vibrio parahaemolyticus and formed transparent zones of 10.43. + -. 0.33mm and 11.43. + -. 0.49mm in diameter, whereas the blank control did not find a zone of inhibition, thus demonstrating that palmitoleic acid has biological activity of inhibiting the growth of Vibrio parahaemolyticus; wherein the diameter of the inhibition zone of the concentrated palmitoleic acid is slightly smaller than that of the palmitoleic acid standard product, which is caused by low purity.
Example 2: inhibitory Effect of palmitoleic acid on Vibrio alginolyticus
Diluting freshly prepared seed solution of Vibrio alginolyticus to 1 × 10 with sterile water6cfu/mL, pipette 100. mu.L and spread evenly on BHI solid plates; clamping 3 sterile circular filter paper sheets with the diameter of 6mm, placing the sterile circular filter paper sheets on the surface of a culture medium coated with bacterial liquid in a triangular symmetry manner, respectively dropwise adding 7 mu L of palmitoleic acid standard substance (more than or equal to 98%) and concentrated palmitoleic acid which are dissolved in 5% DMSO and have the concentration of 10mg/mL, wherein the blank control group is 5% DMSO; standing for 30min, transferring to a constant temperature incubator at 28 deg.C after the medicinal liquid is fully diffused, and culturing for 24 h; after the culture was completed, the diameter of the zone of inhibition was measured using a ruler.
The results are shown in fig. 2, that the palmitoleic acid standard and the concentrated palmitoleic acid isolated from xanthothrips significantly inhibited the growth of vibrio alginolyticus and formed transparent zones of 10.29 ± 0.40mm and 11.33 ± 0.29mm in diameter, while the blank control did not find a zone of inhibition, thus demonstrating that palmitoleic acid has biological activity of inhibiting the growth of vibrio alginolyticus; wherein the diameter of the inhibition zone of the concentrated palmitoleic acid is slightly smaller than that of the palmitoleic acid standard product, which is caused by low purity.
Example 3: inhibitory Effect of palmitoleic acid on Streptococcus agalactiae
Diluting the freshly prepared Streptococcus agalactiae seed solution to 1 × 10 with sterile water6cfu/mL, pipetted 100. mu.L, was evenly coated onto BHI solid platesThe above step (1); clamping 3 sterile circular filter paper sheets with the diameter of 6mm, placing the sterile circular filter paper sheets on the surface of a culture medium coated with bacterial liquid in a triangular symmetry manner, respectively dropwise adding 7 mu L of palmitoleic acid standard substance (more than or equal to 98%) and concentrated palmitoleic acid which are dissolved in 5% DMSO and have the concentration of 10mg/mL, wherein the blank control group is 5% DMSO; standing for 30min, transferring to a constant temperature incubator at 28 deg.C after the medicinal liquid is fully diffused, and culturing for 24 h; after the culture was completed, the diameter of the zone of inhibition was measured using a ruler.
The results are shown in fig. 3, where the same concentration of palmitoleic acid standard and the concentrated palmitoleic acid isolated from xanthothrips significantly inhibited the growth of streptococcus agalactiae and formed clear zones of 12.10 ± 0.39mm and 13.12 ± 0.16mm in diameter, whereas the blank control did not find a zone of inhibition, thus demonstrating that palmitoleic acid has biological activity of inhibiting the growth of streptococcus agalactiae; wherein the diameter of the inhibition zone of the concentrated palmitoleic acid is slightly smaller than that of the palmitoleic acid standard product, which is caused by low purity.
Example 4: determination of minimum inhibitory concentration of palmitoleic acid on Streptococcus agalactiae
Weighing a certain mass of concentrated palmitoleic acid separated from xanthomonas campestris, dissolving in DMSO to prepare a mother solution with a final concentration of 10mg/mL, diluting the mother solution with equal times to concentrations of 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125 and 0.0390625mg/mL respectively, and sucking 20 mu L of the solution to add into a 96-well plate; secondly, the fresh seed liquid of the streptococcus agalactiae is diluted into the cell concentration of 1 multiplied by 10 by adopting a BHI liquid culture medium6cfu/mL, 180. mu.L of the bacterial suspension was added to one well, so that the final concentrations of palmitoleic acid were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91. mu.g/mL, respectively. The experiment was additionally set up with 2 controls: control a-20 μ L DMSO +180 μ L bacterial suspension; control B20 μ L sterile water +180 μ L of broth. Finally, the 96-well plate is placed in a constant temperature shaking table at 28 ℃ and the rotating speed is 45rpm, and the culture is carried out for 24h, and the MIC is the concentration corresponding to the complete bacteria-free growth in the 96-well plate.
As shown in FIG. 4, the growth of Streptococcus agalactiae was significantly inhibited, i.e., the MIC was 31.25. mu.g/mL, when the concentration of the concentrated palmitoleic acid was 31.25. mu.g/mL or more, depending on the turbidity of the culture solution.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. Application of palmitoleic acid in inhibiting aquatic pathogenic bacteria is provided.
2. Use according to claim 1, characterized in that:
application of palmitoleic acid in preparation of product for inhibiting aquatic pathogenic bacteria is provided.
3. Use according to claim 1 or 2, characterized in that:
the palmitoleic acid is concentrated palmitic acid from microalgae.
4. Use according to claim 1 or 2, characterized in that:
the aquatic pathogenic bacteria is at least one of aquatic pathogenic vibrio and aquatic pathogenic streptococcus.
5. Use according to claim 1 or 2, characterized in that:
the aquatic pathogenic bacteria is at least one of vibrio parahaemolyticus, vibrio alginolyticus and streptococcus agalactiae.
6. Use according to claim 1 or 2, characterized in that:
the effective concentration of the palmitoleic acid is more than or equal to 31.25 mu g/mL.
7. Use according to claim 6, characterized in that:
the effective concentration of the palmitoleic acid is 31.25-500 mu g/mL.
8. Use according to claim 2, characterized in that:
the dosage form of the product is liquid medicine, ointment or powder.
9. Use according to claim 2, characterized in that:
the product contains one or more pharmaceutically acceptable carriers or excipients.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115778933A (en) * | 2022-07-18 | 2023-03-14 | 陕西科技大学 | Application of palmitic acid in inhibiting activity of vibrio alginolyticus alkaline serine protease |
| EP4126809A4 (en) * | 2020-04-01 | 2024-08-28 | Univ Cornell | Compositions and methods for inhibiting vibrio infection |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989008981A1 (en) * | 1988-03-21 | 1989-10-05 | Daratech Pty Ltd | Teat dip |
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