CN112691184A - Bacteriostatic composition for inhibiting saprolegnia and preparation method thereof - Google Patents
Bacteriostatic composition for inhibiting saprolegnia and preparation method thereof Download PDFInfo
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a bacteriostatic composition for inhibiting saprolegnia and a preparation method thereof, wherein the composition comprises the following components in percentage by mass: fructus Cnidii extract, Lonicera Japonica extract and pupa Bombycis protein peptide at ratio of 0.1-10: 1-100. The bacteriostatic composition for inhibiting the saprolegnia dicranostii provided by the invention combines the fructus cnidii extract, the honeysuckle extract and the silkworm pupa protein peptide, has a remarkable effect on inhibiting the saprolegnia dicranostii, and reduces the death rate of the saprolegnia dicranostii.
Description
Technical Field
The invention belongs to the field of aquatic products, and particularly relates to a bacteriostatic composition for inhibiting saprolegnia and a preparation method thereof.
Background
Schizothorax (Schizothorax o' connori Lloyd) belongs to the subfamily of Schizothorax (Cypriniae) Schizothorax (Schizothorax-cinae) Schizothorax (Schizothorax ), also known as Pseudosciaena clavus. The main and branch streams and the auxiliary water bodies are distributed in the upstream and midstream of the Yalu Tibetan Bujiang. 2006-2007 investigation shows that schizothorax bigarvifolia accounts for 35% -42% of the Yalu Tibet Bujiang fishery harvest and is one of the main economic fishes in the producing area.
At the end of the 70 s in the 20 th century, the schizothorax bigarviensis body mass of Yaluzang Bujiang was 1000 g/tail, and the fishery body mass of 2010 was 200 g/tail, which indicates that the resource quantity of the schizothorax bigarviensis of Yaluzang Bugjiang is being miniaturized. The fish egg hatchability is extremely low due to severe water mold of the schizothorax prenanti, and a large amount of the schizothorax prenanti dies.
Disclosure of Invention
In view of the above, the present invention provides a bacteriostatic composition for inhibiting saprolegnia and a preparation method thereof, which aims 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 bacteriostatic composition for inhibiting saprolegnia, which comprises the following components in a mass ratio of 1: fructus Cnidii extract, Lonicera Japonica extract and pupa Bombycis protein peptide at ratio of 0.1-10: 1-100.
Preferably, the composition comprises the following components in a mass ratio of 1: 0.5-8 parts of fructus cnidii extract, honeysuckle extract and silkworm pupa protein peptide, wherein the weight parts of the fructus cnidii extract and the honeysuckle extract are 20-80 parts.
Preferably, the composition comprises the following components in a mass ratio of 1: 0.5-5 parts of fructus cnidii extract, honeysuckle extract and silkworm pupa protein peptide, and the ratio is 30-62.
Further, the fructus cnidii extract is prepared by the method comprising the following steps: drying fructus Cnidii, pulverizing into powder, adding into 10-20 times of 60-90% ethanol, ultrasonic extracting for 1-5 hr, and filtering to obtain the final product; the temperature of the ultrasonic step is 30-50 ℃.
Further, the honeysuckle extract is prepared by a method comprising the following steps: drying honeysuckle flower buds, pulverizing into powder, adding the powder into 1-10 times of 60-90% ethanol, performing ultrasonic treatment for 0.5-1 hr, adjusting pH to 3-5, and performing Soxhlet extraction at 50-80 deg.C.
Further, the silkworm pupa protein peptide is prepared by the method comprising the following steps: adding silkworm pupa protein powder into 10-20 times of water, adjusting the pH value of the solution to 1-5, heating in water bath, adding 5000-.
Further, the temperature of the water bath heating step is 40-60 ℃; the time of the enzymolysis step is 3-8 hours.
The preparation method of the bacteriostatic composition for inhibiting the saprolegnia comprises the following steps: mixing the honeysuckle extract, the cnidium fruit extract and the silkworm pupa protein peptide uniformly.
Furthermore, the composition is applied to preparing a medicine for inhibiting the Saprolegnia parasitica.
Furthermore, the composition is applied to preparing feed with the function of inhibiting the Saprolegnia parasitica of the schizothorax prenanti.
Compared with the prior art, the invention has the following advantages:
the bacteriostatic composition for inhibiting the saprolegnia dicranostii provided by the invention combines the fructus cnidii extract, the honeysuckle extract and the silkworm pupa protein peptide, has a remarkable effect on inhibiting the saprolegnia dicranostii, and reduces the death rate of the saprolegnia dicranostii.
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.
Example 1 preparation of Torilis fructus extract, Lonicera japonica extract and silkworm pupa protein peptide
Weighing 100g of fructus cnidii, drying, pulverizing into powder, adding the powder into 1000mL of 90% ethanol, performing ultrasonic extraction at 35 ℃ for 2 hours, and filtering to obtain the fructus cnidii extract.
Weighing 100g of honeysuckle flower buds, drying, crushing into powder, adding the powder into 100mL of 90% ethanol, performing ultrasonic treatment for 0.5 hour, adjusting the pH value of the solution to 3.5, and performing Soxhlet extraction at 60 ℃ to obtain the honeysuckle extract.
Weighing 100g of silkworm chrysalis protein powder, adding the silkworm chrysalis protein powder into 1000mL of water, adjusting the pH value of the solution to 2, heating in a water bath at 50 ℃, adding 8000U/g of pepsin, carrying out enzymolysis for 5 hours, and carrying out boiling water bath enzyme deactivation after the enzymolysis is finished to obtain the silkworm chrysalis protein peptide.
EXAMPLE 2 preparation of bacteriostatic compositions
Weighing 10g of fructus cnidii extract, 10g of honeysuckle extract and 400g of silkworm pupa protein peptide, and mixing to obtain the antibacterial composition A.
Weighing 10g of fructus cnidii extract, 60g of honeysuckle extract and 200g of silkworm pupa protein peptide, and mixing to obtain the antibacterial composition B.
Weighing 10g of fructus cnidii extract, 2g of honeysuckle extract and 900g of silkworm pupa protein peptide, and mixing to obtain the antibacterial composition C.
Weighing 10g of fructus cnidii extract, 120g of honeysuckle extract and 1200g of silkworm pupa protein peptide, and mixing to obtain the antibacterial composition D.
Weighing 10g of fructus cnidii extract and 400g of silkworm pupa protein peptide, and mixing to obtain the antibacterial composition E.
Weighing 10g of honeysuckle extract and 400g of silkworm pupa protein peptide, and mixing to obtain the antibacterial composition F.
Weighing 10G of fructus cnidii extract and 10G of honeysuckle extract, and mixing to obtain the antibacterial composition G.
EXAMPLE 3 bacteriostatic Effect of the bacteriostatic composition
And (3) test bacterium culture: activating the strain of saprolegnia, inoculating to solid culture medium, culturing at 15 deg.C for 24 hr, picking out single colony, inoculating to nutrient broth culture medium, and shake culturing for 24 hr. And (3) 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 103 CFU/mL.
Adding the bacteriostatic composition A, the bacteriostatic composition B, the bacteriostatic composition C, the bacteriostatic composition D, the bacteriostatic composition E, the bacteriostatic composition F, the bacteriostatic composition G and a conventional bacteriostatic agent into different test tubes, wherein the compositions are not added as blank controls, and bacteria are not added as negative controls, and each test tube is repeated for 3 times. Shaking (180r/min) in a shaking table at 15 ℃ for 24h, wherein the diameter of the inhibition zone and the inhibition grade are shown in Table 1. 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).
TABLE 1 zone diameter and grade of inhibition
| Item | Diameter of bacteriostatic circle (mm) | Grade of bacteriostasis |
| Bacteriostatic composition A | 24.36±0.35 | +++ |
| Bacteriostatic composition B | 23.64±0.10 | +++ |
| Bacteriostatic composition C | 20.46±0.85 | +++ |
| Bacteriostatic composition D | 16.36±0.32 | ++ |
| Bacteriostatic composition E | 14.96±0.15 | + |
| Bacteriostatic composition F | 15.67±0.49 | ++ |
| Bacteriostatic composition G | 11.95±0.10 | + |
| Conventional bacteriostatic agent | 10.39±0.35 | + |
| 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, the bacteriostatic compositions a-G all have bacteriostatic effects on saprolegnia, wherein the bacteriostatic grades of the bacteriostatic composition a and the bacteriostatic composition B on saprolegnia are extremely sensitive, which indicates that the bacteriostatic composition comprises the following components in a mass ratio of 1: the bacteriostatic composition of the fructus cnidii extract, the honeysuckle extract and the silkworm pupa protein peptide has a remarkable inhibition effect on saprolegnia, and the mass ratio of the fructus cnidii extract to the honeysuckle extract to the silkworm pupa protein peptide is 1: the fructus cnidii extract, the honeysuckle extract and the silkworm pupa protein peptide have more excellent antibacterial effect only by 0.1-10:1-100 of the antibacterial composition; the bacteriostatic results of the bacteriostatic composition E, the bacteriostatic composition F and the bacteriostatic composition G show that the bacteriostatic effect of the bacteriostatic composition containing the silkworm pupa protein peptide is obvious; chlorogenic acid in the fructus cnidii extract, organic acid and flavonoid compounds in the honeysuckle extract have certain antibacterial effect, and the antibacterial composition containing only the fructus cnidii extract and the honeysuckle extract cannot achieve the antibacterial effect of the antibacterial composition A.
Example 4 Effect of the bacteriostatic composition on the wild Schizothorax heterodentata Saprolegnia
Weighing 100g of fructus cnidii, drying, pulverizing into powder, adding the powder into 1000mL of 90% ethanol, performing ultrasonic extraction at 35 ℃ for 2 hours, and filtering to obtain the fructus cnidii extract.
Weighing 100g of honeysuckle flower buds, drying, crushing into powder, adding the powder into 100mL of 90% ethanol, performing ultrasonic treatment for 0.5 hour, adjusting the pH value of the solution to 3.5, and performing Soxhlet extraction at 60 ℃ to obtain the honeysuckle extract.
Weighing 100g of silkworm chrysalis protein powder, adding the silkworm chrysalis protein powder into 1000mL of water, adjusting the pH value of the solution to 2, heating in a water bath at 50 ℃, adding 8000U/g of pepsin, carrying out enzymolysis for 5 hours, and carrying out boiling water bath enzyme deactivation after the enzymolysis is finished to obtain the silkworm chrysalis protein peptide.
Weighing 10g of fructus cnidii extract, 10g of honeysuckle extract and 400g of silkworm pupa protein peptide, and mixing to obtain the antibacterial composition A.
Weighing 10g of fructus cnidii extract to obtain the antibacterial composition B.
Weighing 10g of honeysuckle extract to obtain the bacteriostatic composition C.
Weighing 10g of silkworm pupa protein peptide to obtain the antibacterial composition D.
Weighing 10g of fructus cnidii extract and 10g of honeysuckle extract, and mixing to obtain the antibacterial composition E.
10g of conventional bacteriostatic agent was weighed as a control group.
The experimental heteroodontic schizothorax prenanti parent fish is captured from the karst section of Bujiang river in Yalu Tibetan in 2020-1-8 months, fishing is carried out for 1 time (fishing is carried out for 3 times in the first ten days, the middle ten days and the last ten days in 4 and 5 months) per month, and the sexual maturity gynoecial heteroodontic schizothorax prenanti parent fish is more than 30 tails each. Temporarily culturing the captured test fish in a breeding base of an aquatic science research institute of agrestics academy of sciences in autonomous region of Tibet, temporarily culturing the test fish in a cement pond with the area of 4m multiplied by 3m multiplied by 1m, and starting the test after the fish is stable (after temporary culture for 7d, mechanical damage is caused by fish transportation and selection, and more than 95% of fish bodies have water mold). Selecting 100 fish as test fish, dividing into 6 groups, adding antibacterial composition A, antibacterial composition B, antibacterial composition C, antibacterial composition D, antibacterial composition E and control group, closing water inlet and outlet during administration, draining liquid medicine, and injecting fresh water. The mortality, the cure rate and the recurrence rate of Saprolegnia after 7 days of administration of the drugs in each experimental group were counted, and the results are shown in Table 2.
TABLE 2 bacteriostatic results of the bacteriostatic composition against Saprolegnia denticulata
| Mortality (%) | Saprolegnia cure rate (%) | 7d Saprolegnia recurrence rate (%) | |
| Bacteriostatic composition A | 3.41±0.08 | 93.24±0.52 | 2.07±0.21 |
| Bacteriostatic composition B | 18.39±0.26 | 66.41±0.26 | 12.96±0.08 |
| Bacteriostatic composition C | 23.95±0.15 | 52.74±0.80 | 7.63±0.71 |
| Bacteriostatic composition D | 43.64±0.28 | 31.93±0.74 | 18.36±0.24 |
| Bacteriostatic composition E | 16.18±0.36 | 73.69±0.48 | 10.69±0.78 |
| Control group | 37.21±0.40 | 51.36±0.41 | 26.39±0.49 |
As can be seen from table 2, the bacteriostatic composition a has a significant inhibitory effect on wild schizothorax prenanti saprolegnia, and has significant mortality, cure rate and recurrence rate, which indicates that the mass ratio of the components is 1: the fructus cnidii extract, the honeysuckle extract and the silkworm pupa protein peptide antibacterial composition has an obvious antibacterial effect on the saprolegnia at a ratio of 0.5-5: 30-62; the effect of singly adopting the fructus cnidii extract, the honeysuckle extract or the silkworm pupa protein peptide as the antibacterial component on wild schizothorax prenanti-aquatica is not ideal all the time, the effect of adopting the combination of the fructus cnidii extract and the honeysuckle extract to inhibit the aquatica is obviously different from that of the antibacterial composition A, the results show that the fructus cnidii extract and the honeysuckle extract can achieve good antibacterial effect only by being combined with the silkworm pupa protein peptide, and the silkworm pupa protein peptide as the small molecular peptide can carry a large amount of effective antibacterial components in the fructus cnidii extract and the honeysuckle extract to enter the body of a test fish, thereby improving the antibacterial effect.
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.
Claims (10)
1. A bacteriostatic composition for inhibiting saprolegnia, characterized in that: the composition comprises the following components in a mass ratio of 1: fructus Cnidii extract, Lonicera Japonica extract and pupa Bombycis protein peptide at ratio of 0.1-10: 1-100.
2. The bacteriostatic composition for inhibiting saprolegnia according to claim 1, characterized in that: the composition comprises the following components in a mass ratio of 1: 0.5-8 parts of fructus cnidii extract, honeysuckle extract and silkworm pupa protein peptide, wherein the weight parts of the fructus cnidii extract and the honeysuckle extract are 20-80 parts.
3. The bacteriostatic composition for inhibiting saprolegnia according to claim 2, characterized in that: the composition comprises the following components in a mass ratio of 1: 0.5-5 parts of fructus cnidii extract, honeysuckle extract and silkworm pupa protein peptide, and the ratio is 30-62.
4. The bacteriostatic composition for inhibiting saprolegnia according to claim 3, characterized in that: the fructus cnidii extract is prepared by the following steps: drying fructus Cnidii, pulverizing into powder, adding into 10-20 times of 60-90% ethanol, ultrasonic extracting for 1-5 hr, and filtering to obtain the final product; the temperature of the ultrasonic step is 30-50 ℃.
5. The bacteriostatic composition for inhibiting saprolegnia according to claim 3, characterized in that: the honeysuckle extract is prepared by the method comprising the following steps: drying honeysuckle flower buds, pulverizing into powder, adding the powder into 1-10 times of 60-90% ethanol, performing ultrasonic treatment for 0.5-1 hr, adjusting pH to 3-5, and performing Soxhlet extraction at 50-80 deg.C.
6. The bacteriostatic composition for inhibiting saprolegnia according to claim 3, characterized in that: the silkworm pupa protein peptide is prepared by the method comprising the following steps: adding silkworm pupa protein powder into 10-20 times of water, adjusting the pH value of the solution to 1-5, heating in water bath, adding 5000-.
7. The bacteriostatic composition for inhibiting saprolegnia according to claim 6, wherein: the temperature of the water bath heating step is 40-60 ℃; the time of the enzymolysis step is 3-8 hours.
8. The method for preparing a bacteriostatic composition for inhibiting saprolegnia according to any one of claims 1 to 7, characterized in that: the method comprises the following steps: mixing the honeysuckle extract, the cnidium fruit extract and the silkworm pupa protein peptide uniformly.
9. The application of a bacteriostatic composition for inhibiting saprolegnia is characterized in that: the composition is applied to the preparation of the medicine for inhibiting the Saprolegnia parasitica of the schizothorax prenanti.
10. The application of a bacteriostatic composition for inhibiting saprolegnia is characterized in that: the composition is applied to preparing feed with the function of inhibiting the Saprolegnia parasitica of the schizothorax prenanti.
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