阅读说明：本技术 一种抑制气单胞菌属的组合物及其制备方法与应用 (Composition for inhibiting aeromonas and preparation method and application thereof ) 是由 刘春� 李薇 苏友禄 江飚 马杰 秦真东 黄燕华 林蠡 于 2020-12-28 设计创作，主要内容包括：本发明提供了一种抑制气单胞菌属的组合物及其制备方法与应用,所述的方法为将鸡血藤提取物与千斤拔提取物混合均匀,然后向其中加入多西环素混合均匀后即成。本发明所述的一种抑制气单胞菌属的组合物中含有多西环素、鸡血藤提取物和千斤拔提取物,其三者的组合对气单胞菌属细菌有明显的抑制作用,该组合物可用于制备药剂与饲料添加剂。(The invention provides a composition for inhibiting aeromonas, a preparation method and an application thereof. The composition for inhibiting the aeromonas comprises doxycycline, the suberect spatholobus stem extract and the philippine flemingia root extract, the combination of the doxycycline, the suberect spatholobus stem extract and the flemingia root extract has an obvious inhibiting effect on the aeromonas bacteria, and the composition can be used for preparing medicaments and feed additives.)

1. A composition for inhibiting aeromonas, comprising: the composition is prepared from the following raw materials in parts by weight:

0.001 to 0.1 portion of doxycycline,

10-100 parts of a suberect spatholobus stem extract,

10-100 parts of philippine flemingia root extract.

2. The composition for inhibiting aeromonas according to claim 1, wherein: the composition is prepared from the following raw materials in parts by weight:

0.001 to 0.05 portion of doxycycline,

10-50 parts of a suberect spatholobus stem extract,

50-100 parts of philippine flemingia root extract.

3. The composition for inhibiting aeromonas according to claim 2, wherein: the composition is prepared from the following raw materials in parts by weight:

0.008 to 0.028 portion of doxycycline,

32-50 parts of a suberect spatholobus stem extract,

85-100 parts of philippine flemingia root extract.

4. The Aeromonas sp. The philippine flemingia root extract is prepared by the following steps: pulverizing and sieving philippine flemingia root to obtain philippine flemingia root powder, adding the philippine flemingia root powder into 10-30 times of 60-70% ethanol, leaching for 1-2 hours, extracting the obtained leaching liquor under reflux at 80-90 ℃, and concentrating to obtain the philippine flemingia root extract.

5. A process for the preparation of a composition for inhibiting Aeromonas according to any one of claims 1 to 4, wherein: the method comprises the following steps: mixing caulis Spatholobi extract and radix Flemingiae Philippinensis extract, adding doxycycline, and mixing.

6. A preparation method of a caulis spatholobi extract is characterized by comprising the following steps: the method comprises the following steps:

(1) pulverizing caulis Spatholobi, sieving to obtain caulis Spatholobi powder, and heating the powder at 60-90 deg.C for 5-30 min;

(2) then dividing the heated powder into three parts of caulis Spatholobi powder with the same mass, adding the first part of caulis Spatholobi powder into 10-30 times of 40-55% ethanol, leaching for 12-24 hours to obtain leaching liquor A, adding the second part of caulis Spatholobi powder into 10-30 times of 60-75% ethanol, leaching for 12-24 hours to obtain leaching liquor B, adding the third part of caulis Spatholobi powder into 10-30 times of 80-95% ethanol, leaching for 24-48 hours to obtain leaching liquor C;

(3) and (3) uniformly mixing the leaching liquor A, the leaching liquor B and the leaching liquor C, filtering, and rotatably steaming the obtained filtrate to obtain the leaching liquor.

7. The method for preparing spatholobus stem extract according to claim 6, wherein: the temperature of the heating step in the step (1) is 60-80 ℃, and the time is 15-30 minutes; the first part of the caulis spatholobi powder in the step (2) is added into 50-55% of ethanol, the second part of the caulis spatholobi powder is added into 60-65% of ethanol, and the third part of the caulis spatholobi powder is added into 80-90% of ethanol.

8. A caulis spatholobi extract is characterized in that: the extract is prepared by the method of claim 6 or 7.

9. An application of a composition for inhibiting aeromonas in preparing a medicament for inhibiting the aeromonas bacteria.

10. Use according to claim 9, characterized in that: the composition comprises doxycycline, caulis Spatholobi extract and radix Flemingiae Philippinensis extract.

Technical Field

The invention belongs to the field of aquatic products, and particularly relates to a composition for inhibiting aeromonas, a preparation method and an application thereof.

Background

China is a big aquaculture country, the aquaculture density and the yield of aquatic products are high, but the fish bacterial diseases frequently occur due to the laggard aquaculture technology and facilities. At present, the drug prevention and treatment mainly based on antibiotics is still the most effective and most direct method in the culture production, and plays an important role in preventing and treating fish bacterial diseases. However, the long-term use of antibiotics in production practice can lead to the emergence of various drug-resistant strains in a short time.

Disclosure of Invention

In view of the above, the present invention provides a composition for inhibiting aeromonas, a preparation method and an application thereof, aiming to overcome the defects in the prior art.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a composition for inhibiting aeromonas is prepared from the following raw materials in parts by weight:

0.001 to 0.1 portion of doxycycline,

10-100 parts of a suberect spatholobus stem extract,

10-100 parts of philippine flemingia root extract.

Preferably, the composition is prepared from the following raw materials in parts by weight:

0.001 to 0.05 portion of doxycycline,

10-50 parts of a suberect spatholobus stem extract,

50-100 parts of philippine flemingia root extract.

Preferably, the composition is prepared from the following raw materials in parts by weight:

0.008 to 0.028 portion of doxycycline,

32-50 parts of a suberect spatholobus stem extract,

85-100 parts of philippine flemingia root extract.

Further, the philippine flemingia root extract is prepared by the following steps: pulverizing and sieving philippine flemingia root to obtain philippine flemingia root powder, adding the philippine flemingia root powder into 10-30 times of 60-70% ethanol, leaching for 1-2 hours, extracting the obtained leaching liquor under reflux at 80-90 ℃, and concentrating to obtain the philippine flemingia root extract.

The preparation method of the composition for inhibiting the aeromonas comprises the following steps: mixing caulis Spatholobi extract and radix Flemingiae Philippinensis extract, adding doxycycline, and mixing.

A preparation method of a caulis spatholobi extract comprises the following steps:

(1) pulverizing caulis Spatholobi, sieving to obtain caulis Spatholobi powder, and heating the powder at 60-90 deg.C for 5-30 min;

(2) then dividing the heated powder into three parts of caulis Spatholobi powder with the same mass, adding the first part of caulis Spatholobi powder into 10-30 times of 40-55% ethanol, leaching for 12-24 hours to obtain leaching liquor A, adding the second part of caulis Spatholobi powder into 10-30 times of 60-75% ethanol, leaching for 12-24 hours to obtain leaching liquor B, adding the third part of caulis Spatholobi powder into 10-30 times of 80-95% ethanol, leaching for 24-48 hours to obtain leaching liquor C;

(3) and (3) uniformly mixing the leaching liquor A, the leaching liquor B and the leaching liquor C, filtering, and rotatably steaming the obtained filtrate to obtain the leaching liquor.

Further, the temperature of the heating step in the step (1) is 60-80 ℃, and the time is 15-30 minutes; the first part of the caulis spatholobi powder in the step (2) is added into 50-55% of ethanol, the second part of the caulis spatholobi powder is added into 60-65% of ethanol, and the third part of the caulis spatholobi powder is added into 80-90% of ethanol.

A caulis Spatholobi extract is prepared by the method.

An application of a composition for inhibiting aeromonas in preparing a medicament for inhibiting the aeromonas bacteria.

Further, the composition comprises doxycycline, caulis Spatholobi extract and Flemingia philippinensis extract.

Compared with the prior art, the invention has the following advantages:

the composition for inhibiting the aeromonas comprises doxycycline, the suberect spatholobus stem extract and the philippine flemingia root extract, the combination of the doxycycline, the suberect spatholobus stem extract and the flemingia root extract has an obvious inhibiting effect on the aeromonas bacteria, and the composition can be used for preparing medicaments and feed additives.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to examples.

The schubert aeromonas strain, the aeromonas hydrophila and the aeromonas spot described in the embodiment of the invention are presented by aquatic disease and immunity research rooms of the Zhujiang aquatic research institute of the Chinese aquatic science research institute.

EXAMPLE 1 preparation of extract of caulis Spatholobi

Pulverizing caulis Spatholobi, sieving to obtain caulis Spatholobi powder, accurately weighing 600g powder, and heating at 80 deg.C for 20 min; then dividing the heated powder into three parts of caulis Spatholobi powder with the same mass, adding the first part of caulis Spatholobi powder into 20 times of 50% ethanol, leaching for 12 hours to obtain leaching liquor A, adding the second part of caulis Spatholobi powder into 20 times of 60% ethanol, leaching for 12 hours to obtain leaching liquor B, adding the third part of caulis Spatholobi powder into 20 times of 90% ethanol, and leaching for 48 hours to obtain leaching liquor C; and uniformly mixing the leaching liquor A, the leaching liquor B and the leaching liquor C, filtering, and rotatably steaming the obtained filtrate to obtain the spatholobus stem extract A.

Pulverizing caulis Spatholobi, sieving to obtain caulis Spatholobi powder, accurately weighing 600g powder, and heating at 50 deg.C for 20 min; then dividing the heated powder into three parts of caulis Spatholobi powder with the same mass, adding the first part of caulis Spatholobi powder into 20 times of 50% ethanol, leaching for 12 hours to obtain leaching liquor A, adding the second part of caulis Spatholobi powder into 20 times of 60% ethanol, leaching for 12 hours to obtain leaching liquor B, adding the third part of caulis Spatholobi powder into 20 times of 90% ethanol, and leaching for 48 hours to obtain leaching liquor C; and uniformly mixing the leaching liquor A, the leaching liquor B and the leaching liquor C, filtering, and rotatably steaming the obtained filtrate to obtain the suberect spatholobus stem extract B.

Pulverizing caulis Spatholobi, sieving to obtain caulis Spatholobi powder, accurately weighing 600g powder, and heating at 50 deg.C for 20 min; then dividing the heated powder into three parts of caulis Spatholobi powder with the same mass, adding the first part of caulis Spatholobi powder into 20 times of 35% ethanol, leaching for 12 hours to obtain leaching liquor A, adding the second part of caulis Spatholobi powder into 20 times of 50% ethanol, leaching for 12 hours to obtain leaching liquor B, adding the third part of caulis Spatholobi powder into 20 times of 70% ethanol, and leaching for 48 hours to obtain leaching liquor C; and uniformly mixing the leaching liquor A, the leaching liquor B and the leaching liquor C, filtering, and rotatably steaming the obtained filtrate to obtain the spatholobus stem extract C.

Pulverizing caulis Spatholobi, sieving to obtain caulis Spatholobi powder, accurately weighing 1500g powder, and heating at 50 deg.C for 20 min; then dividing the heated powder into three parts of caulis Spatholobi powder with the same mass, adding the first part of caulis Spatholobi powder into 20 times of 60% ethanol, leaching for 12 hours to obtain leaching liquor A, adding the second part of caulis Spatholobi powder into 20 times of 80% ethanol, leaching for 12 hours to obtain leaching liquor B, adding the third part of caulis Spatholobi powder into 20 times of 95% ethanol, and leaching for 48 hours to obtain leaching liquor C; and uniformly mixing the leaching liquor A, the leaching liquor B and the leaching liquor C, filtering, and rotatably steaming the obtained filtrate to obtain a caulis spatholobi extract D.

Pulverizing and sieving philippine flemingia root to obtain philippine flemingia root powder, accurately weighing 1500g of philippine flemingia root powder, adding the philippine flemingia root powder into 20 times of 70% ethanol, leaching for 2 hours, performing reflux extraction on the obtained leaching solution at 80 ℃, and concentrating to obtain a philippine flemingia root extract.

And (3) uniformly mixing 48g of the suberect spatholobus stem extract A and 90g of the philippine flemingia root extract, adding 20mg of doxycycline into the mixture, and uniformly mixing to obtain a composition A.

And (3) uniformly mixing 48g of the suberect spatholobus stem extract B and 90g of the philippine flemingia root extract, adding 20mg of doxycycline into the mixture, and uniformly mixing to obtain a composition B.

And (3) uniformly mixing 48g of the suberect spatholobus stem extract C and 90g of the philippine flemingia root extract, adding 20mg of doxycycline into the mixture, and uniformly mixing to obtain a composition C.

And (3) uniformly mixing 48g of the suberect spatholobus stem extract D and 90g of the philippine flemingia root extract, adding 20mg of doxycycline into the mixture, and uniformly mixing to obtain a composition D.

Accurately weighing 90g of Flemingia philippinensis extract, adding 20mg of doxycycline, and mixing to obtain composition E.

Example 2 bacteriostatic Effect of caulis Spatholobi extract on Aeromonas hydrophila

1. Preparation of the culture Medium

BHI medium was purchased from BD, usa; 5% sheep blood agar plates, bacteria biochemical microassay tubes, etc. were purchased from Kyowa microorganism Co., Ltd.

2. Preparation of the bacterial suspension

And (3) test bacterium culture: activating aeromonas hydrophila strains, streaking on a solid culture medium by using an inoculating needle, culturing for 24 hours at 30 ℃, picking single bacterial colonies, inoculating the single bacterial colonies on a nutrient broth culture medium, and culturing for 24 hours by shaking. And diluting the bacterial liquid to a proper concentration by adopting a plate counting method, wherein the concentration of the bacterial liquid is 1 multiplied by 108 CFU/mL.

3. Drug susceptibility testing

The drug sensitivity test adopts an agar diffusion method, bacterial suspension cultured to a logarithmic phase is diluted to the concentration of about 1 × 103CFU/mL, a sterile cotton swab is used for dipping bacterial liquid and uniformly smearing the bacterial liquid on a culture medium, after the bacterial liquid is uniformly smeared, holes are uniformly punched on the plates, 4 holes are punched on each plate, and the marks are made. Adding 100 μ l (1g/mL) of the composition to each well, and culturing in an incubator at 30 ℃ for 24 h; and (4) after the flat plate is taken out, observing whether a bacteriostatic circle exists around the hole, and if so, measuring the diameter of the bacteriostatic circle by using a vernier caliper. Three replicates of each drug were run with a blank of sterile water. The result judgment standard is according to the standard introduced in the Chinese medicine pharmacology: the inhibition zone is more than or equal to 20mm, and the drug is extremely sensitive; the inhibition zone is 15-19mm, belonging to high sensitivity; the zone of inhibition is 10-14mm, belonging to the middle allergy; the inhibition zone is less than 10mm, and the low-sensitivity drug is low-sensitivity drug; has no bacteriostatic zone, and is insensitive (no bacteriostatic effect).

4. Secondary screening test

The extract was diluted in a two-fold dilution with nutrient broth medium, the bacterial suspension was added to each tube to achieve a bacterial concentration of 102CFU/mL, and composition A, composition B, composition C, and composition E were added to different tubes, each repeated 3 times without composition as a blank and without bacteria as a negative control. Shaking in a shaking table at 28 deg.C (180r/min) for 24 hr, taking out, comparing with a control tube, and observing, wherein the diameter of the inhibition zone and the inhibition grade are shown in Table 1.

TABLE 1 zone diameter and grade of inhibition

Item	Diameter of bacteriostatic circle (mm)	Grade of bacteriostasis
			Composition A	22.94±0.15	+++
Composition B	12.35±0.55	+
			Composition C	17.26±0.28	++
Composition D	16.63±0.41	++
			Composition E	9.26±0.42	+
Blank control	—	—
			Negative control	—	—

In the above tables, "+ + + + +" indicates extreme sensitivity, "+" indicates medium sensitivity or low sensitivity, and "-" indicates no sensitivity (no bacteriostatic effect).

As can be seen from table 1, composition a, composition B, composition C, composition D, and composition E all have bacteriostatic effects on aeromonas hydrophila, wherein the bacteriostatic level of composition a is extremely sensitive, the level of composition C and composition D is highly sensitive, and the level of composition B and composition E (not containing spatholobus stem extract) is medium sensitive or low sensitive, so that it can be seen that the composition containing spatholobus stem extract and philippine flemingia root extract has significant bacteriostatic effects on aeromonas hydrophila, wherein the bacteriostatic effect of composition a is particularly significant, while for composition B, when preparing the spatholobus stem extract, heating and activating at a lower temperature of 50 ℃ cannot sufficiently extract bacteriostatic components therein, so that the effect thereof has a larger difference compared with composition a, and for composition C and composition D, leaching is performed by ethanol with different concentration gradients, compared with the ethanol with concentration gradient in the preparation composition A, the effect has a certain difference, but the influence of the ethanol with different concentration gradient on the bacteriostasis effect of the suberect spatholobus stem extract is far lower than the influence of the activation temperature. Therefore, in the composition A, the combination of the caulis spatholobi extract, the philippine flemingia root extract and doxycycline has a remarkable bacteriostatic effect on the aeromonas hydrophila, and the change of the temperature of heating activation and the concentration gradient of ethanol also has a great influence on the bacteriostatic effect of the composition on the aeromonas hydrophila.

Example 3 Effect of the mixture ratio of caulis Spatholobi extract and radix Flemingiae Philippinensis extract on the antibacterial Effect

Pulverizing caulis Spatholobi, sieving to obtain caulis Spatholobi powder, accurately weighing 600g powder, and heating at 80 deg.C for 20 min; then dividing the heated powder into three parts of caulis Spatholobi powder with the same mass, adding the first part of caulis Spatholobi powder into 20 times of 50% ethanol, leaching for 12 hours to obtain leaching liquor A, adding the second part of caulis Spatholobi powder into 20 times of 60% ethanol, leaching for 12 hours to obtain leaching liquor B, adding the third part of caulis Spatholobi powder into 20 times of 90% ethanol, and leaching for 48 hours to obtain leaching liquor C; and uniformly mixing the leaching liquor A, the leaching liquor B and the leaching liquor C, filtering, and rotatably steaming the obtained filtrate to obtain the suberect spatholobus stem extract.

Pulverizing and sieving philippine flemingia root to obtain philippine flemingia root powder, accurately weighing 1500g of philippine flemingia root powder, adding the philippine flemingia root powder into 20 times of 70% ethanol, leaching for 2 hours, performing reflux extraction on the obtained leaching solution at 80 ℃, and concentrating to obtain a philippine flemingia root extract.

Mixing 48g caulis Spatholobi extract and 90g radix Flemingiae Philippinensis extract, adding 20mg doxycycline, and mixing to obtain composition A.

Mixing 5g caulis Spatholobi extract and 90g radix Flemingiae Philippinensis extract, adding 20mg doxycycline, and mixing to obtain composition B.

And (3) uniformly mixing 48g of the suberect spatholobus stem extract and 150g of the philippine flemingia root extract, adding 20mg of doxycycline into the mixture, and uniformly mixing to obtain a composition C.

Mixing 5g caulis Spatholobi extract and 150g radix Flemingiae Philippinensis extract, adding 20mg doxycycline, and mixing to obtain composition D.

Accurately weighing 90g of Flemingia philippinensis extract, adding 20mg of doxycycline, and mixing to obtain composition E.

48g of the spatholobus stem extract is accurately weighed, and then 20mg of doxycycline is added into the spatholobus stem extract and evenly mixed to obtain the composition F.

Accurately weighing 20mg of doxycycline, dissolving the doxycycline in 10 times of 60% ethanol, and uniformly mixing to obtain a composition G.

The bacterial suspension of Aeromonas hydrophila was added to each tube to achieve a bacterial concentration of 102CFU/mL, and composition A, composition B, composition C, composition E, composition F, and composition G were added to different tubes, each of which was repeated 3 times without adding composition as a blank and without adding bacteria as a negative control. Shaking in a shaker at 30 deg.C (180r/min) for 24 hr, taking out, comparing with control tube, and observing, wherein the diameter of inhibition zone and inhibition grade are shown in Table 2.

TABLE 2 zone of inhibition diameter and grade of inhibition

Item	Diameter of bacteriostatic circle (mm)	Grade of bacteriostasis
			Composition A	22.75±0.18	+++
Composition B	15.82±0.36	++
			Composition C	19.16±0.42	++
Composition D	12.35±0.22	+
			Composition E	8.36±0.15	+
Composition F	10.84±0.23	+
			Composition G	7.95±0.55	+
Blank control	—	—
			Negative control	—	—

In the above tables, "+ + + + +" indicates extreme sensitivity, "+" indicates medium sensitivity or low sensitivity, and "-" indicates no sensitivity (no bacteriostatic effect).

As can be seen from table 2, composition a, composition B, composition C, composition D, composition E, composition F and composition G all had bacteriostatic effects on aeromonas hydrophila, wherein the bacteriostatic rating of composition a was extremely sensitive, the rating of composition B and composition C was highly sensitive, and the rating of composition D, composition E (not containing spatholobus stem extract) and composition F (philippine flemingia root extract), composition G (doxycycline) was moderately sensitive or weakly sensitive. Therefore, the composition containing the caulis spatholobi extract and the philippine flemingia root extract has a remarkable bacteriostatic effect on aeromonas hydrophila, wherein the bacteriostatic effect of the composition A is particularly remarkable, the bacteriostatic effect of the composition B is obviously different from that of the composition A, and the composition B is different from the composition A in that the addition amount of the caulis spatholobi extract is 5g, so that the bacteriostatic effect of the composition can be reduced by the excessively low addition amount of the caulis spatholobi extract; compared with the composition A, the bacteriostatic effect of the composition C has obvious difference, but the effect is obviously superior to that of the composition B; the difference between the composition C and the composition A is that the addition amount of the philippine flemingia root extract is 150g, so that the antibacterial effect of the composition is reduced by excessively high addition amount of the philippine flemingia root extract, but the influence of the philippine flemingia root extract on the antibacterial effect of the composition is smaller than that of the suberect spatholobus stem extract. The composition D is different from the composition A in that the addition amount of the caulis spatholobi extract is 5g, the addition amount of the philippine flemingia root extract is 150g, the antibacterial effect of the composition D is different from that of the composition A, the composition B and the composition C, and the addition amounts of the caulis spatholobi extract and the philippine flemingia root extract need to be within a certain range to realize the optimal antibacterial effect; for the composition E and the composition F, the composition respectively added with the caulis spatholobi extract or the philippine flemingia root extract has a certain antibacterial effect on the aeromonas hydrophila, but the antibacterial effect is obviously different from the antibacterial effect of the composition simultaneously containing the caulis spatholobi extract and the philippine flemingia root extract, and for the composition G, the antibacterial effect of the single doxycycline on the aeromonas hydrophila is lower than that of the composition respectively added with the caulis spatholobi extract or the philippine flemingia root extract, which indicates that the composition simultaneously containing the doxycycline, the caulis spatholobi extract and the philippine flemingia root extract can realize the optimal antibacterial effect on the aeromonas hydrophil.

Example 4 bacteriostatic Effect of composition comprising caulis Spatholobi extract and Flemingia philippinensis extract on Aeromonas schubertii, Aeromonas hydrophila and Aeromonas spot

Pulverizing caulis Spatholobi, sieving to obtain caulis Spatholobi powder, accurately weighing 600g powder, and heating at 80 deg.C for 20 min; then dividing the heated powder into three parts of caulis Spatholobi powder with the same mass, adding the first part of caulis Spatholobi powder into 20 times of 50% ethanol, leaching for 12 hours to obtain leaching liquor A, adding the second part of caulis Spatholobi powder into 20 times of 60% ethanol, leaching for 12 hours to obtain leaching liquor B, adding the third part of caulis Spatholobi powder into 20 times of 90% ethanol, and leaching for 48 hours to obtain leaching liquor C; uniformly mixing the leaching liquor A, the leaching liquor B and the leaching liquor C, filtering, and rotatably steaming the obtained filtrate to obtain a caulis spatholobi extract;

pulverizing and sieving philippine flemingia root to obtain philippine flemingia root powder, accurately weighing 1500g of philippine flemingia root powder, adding the philippine flemingia root powder into 20 times of 70% ethanol, leaching for 2 hours, performing reflux extraction on the obtained leaching solution at 80 ℃, and concentrating to obtain a philippine flemingia root extract;

and (3) uniformly mixing 48g of the suberect spatholobus stem extract and 90g of the philippine flemingia root extract, adding 20mg of doxycycline into the mixture, and uniformly mixing to obtain the composition.

Respectively adding bacterial suspensions of the schubert aeromonas strain, the aeromonas hydrophila and the aeromonas spot into different test tubes to enable the bacterial concentration to reach 103CFU/mL, adding the composition into different test tubes, not adding the composition as a blank control, not adding the bacteria as a negative control, and repeating each test tube for 3 times. Shaking in a shaker at 28 deg.C (180r/min) for 24 hr, taking out, comparing with control tube, and observing, wherein the diameter of inhibition zone and inhibition grade are shown in Table 3.

TABLE 3 bacteriostatic effect of the compositions on different Aeromonas bacteria

In the above tables, "+ + + + +" indicates extreme sensitivity, "+" indicates medium sensitivity or low sensitivity, and "-" indicates no sensitivity (no bacteriostatic effect).

As can be seen from Table 3, the composition containing doxycycline, the extract of spatholobus stem and the extract of philippine flemingia root has significant bacteriostatic effects on Aeromonas schubertii, Aeromonas hydrophila and Aeromonas spot.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.