LU500238B1 - Efficient inhibitor of microcystis aeruginosa and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of biological technology, and specifically discloses an inhibitor of Microcystis aeruginosa, which is composed of a berberin mother liquor and an azone mother liquor. The present invention increases the permeability of Microcystis aeruginosa cells to berberine through the addition of azone, the rate of algae inhibition, and at the same time in the water quality control link of aquaculture, reduce the usage of berberine and cultivation costs.

Description

DESCRIPTION EFFICIENT INHIBITOR OF MICROCYSTIS AERUGINOSA AND

PREPARATION METHOD AND APPLICATION THEREOF Technical Field The present invention relates to the technical field of microalgae, in particular to the inhibition of Microcystis aeruginosa, especially to an inhibitor of Microcystis aeruginosa and a preparation method thereof.

Background The outbreak of Microcystis aeruginosa bloom reduces the dissolved oxygen content in the water body, deteriorates water quality, reduces biodiversity, and destroys the biological chain. During the digestion process, it will emit a fishy and unpleasant odor and will diffuse with the air (Schindler D W. Recent advances in the understanding and management of eutrophication. Limnol & Oceanogr. 2006,51(1):356-363.), and release a cyclic hepeptide exotoxin—microcystin, which not only damages aquatic animal liver seriously, but also accumulates in aquatic animals, and transfers toxins into the human body through the action of the food chain, thereby threatening human health.

The prevention and control of Microcystis bloom mainly includes physical, chemical and biological control methods. Biological control is widely used because of its advantages of green, environmental protection and no secondary pollution. Biological control mainly uses higher aquatic organisms, Chinese herbal medicine and other aquatic plants to inhibit algae.

The alkaloids in Chinese herbal medicine have significant antibacterial effects. The berberine in the isoquinoline alkaloids has a very significant inhibitory effect on Microcystis aeruginosa (Study on the inhibitory effect of berberine on Microcystis; Yang Pan, 2012), and has the advantages of quick effect, long-lasting and stable effect.

Berberine has abundant reserves in nature, and the extraction process thereof is relatively simple. It can even be obtained by chemical synthesis, and because bacteria hardly develop resistance to it, as a natural antibiotic, the berberine is widely used in medicine, hygiene and agriculture.

Summary of the Invention For aquaculture, berberine can be used to inhibit the outbreak of Microcystis aeruginosa, but it has the disadvantages of large dosage, high cost and long action time when used alone.

In order to overcome the above shortcomings, the present invention provides an inhibitor of Microcystis aeruginosa, which is composed of a berberine and an azone.

By adding the azone to increase the berberine absorption efficiency of Microcystis aeruginosa, thereby increasing the inhibition rate of berberine to Microcystis, there is no relevant report at present. Applying it to the prevention and control of Microcystis bloom in the aquaculture industry will help reduce the usage of berberine and cultivation costs, shorten the duration of action, improve the quality of aquatic products and human health.

The present invention further provides a method for the use of the above inhibitor (application), the inhibitor is mainly used to inhibit the outbreak of Microcystis aeruginosa in aquaculture.

The method specifically includes: preparing a berberine mother liquor; preparing an azone mother liquor; adding the berberine mother liquor into a Microcystis aeruginosa liquid, and then adding the azone mother liquor; wherein a concentration of the berberine is 0.1-5.0 mg/L, and a concentration of the azone is 1.0- 50.0 mg/L.

The berberine is a berberine hydrochloride.

The inhibitor of Microcystis aeruginosa and its application provided by the present invention increase the permeability of Microcystis aeruginosa cells to berberine through the addition of azone, increase the rate of algae inhibition, and at the same time in the water quality control link of aquaculture, reduce the usage of berberine and cultivation costs.

Brief Description of Figures The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

Fig.1 is a structural formula of berberine; Fig.2 is a graph showing the cell density of Microcystis aeruginosa under the action of berberine in Comparative Example 1; Fig.3 is a graph showing the inhibition rate of berberine on Microcystis aeruginosa in Comparative Example 1; Fig.4 is a graph showing the cell density of Microcystis aeruginosa under the action of azone in Comparative Example 2; Fig. 5 is a graph showing the cell density of Microcystis aeruginosa in Embodiment 1; Fig.6 is a graph showing the synergistic inhibition rate of berberine and azone on Microcystis aeruginosa in Embodiment 1 .

Detailed Description The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the protection scope of the present invention.

In at least one embodiment, the present invention provides an inhibitor of Microcystis aeruginosa, the inhibitor of Microcystis aeruginosa is composed of a berberine and an azone.

Wherein the concentration of the berberine is 0.1-5.0 mg/L, and the concentration of the azone is 1.0- 50.0 mg/L.

In at least one embodiment, the present invention further provides a preparation method for the inhibitor of Microcystis aeruginosa, the method specifically comprises: preparing a berberine mother liquor, the berberine mother liquor is prepared by adding berberine into distilled water; then adding azone into the berberine mother liquor. In some embodiments, before adding azone into the berberine mother liquor, azone is added into distilled water to form an azone mother liquor.

In at least one embodiment, the present invention further provides a method for using the inhibitor of Microcystis aeruginosa, the method specifically comprises: preparing a berberine mother liquor; preparing an azone mother liquor; adding the berberine mother liquor into a Microcystis aeruginosa liquid, and then adding the azone mother liquor; wherein the concentration of the berberine is 0.1-5.0 mg/L, and the concentration of the azone is 1.0- 50.0 mg/L.

The inhibitor of the embodiments of the present invention and application thereof will be further described in detail below in conjunction with specific embodiments.

Comparative Example 1 In this comparative example, different volumes of berberine mother liquor were added into Microcystis aeruginosa liquid to form different berberine gradients, Microcystis aeruginosa was cultured under the same conditions, and samples were taken at different times to determine cell density, so as to determine the inhibition rate of different concentrations of berberine on Microcystis aeruginosa, specifically comprises: (1) preparation of berberine mother liquor 100 mL of distilled water was added into a clean 250 mL conical flask, 1 g of berberine was added into it, then magnetic stirring was performed for 20 minutes to obtain the berberine mother liquor.

(2) determination of the inhibitory effect of berberine on Microcystis aeruginosa different volumes of berberine mother liquor prepared in step (1) were added into Microcystis aeruginosa liquid with a initial concentration of 2.0 X 10° cell * mL”, so that the berberine concentration in each treatment group is 0 mg/L (blank group), 0.1 mg/L, 0.2 mg/L, 1.0 mg/L, 2.0 mg/L and 5.0 mg/L respectively. BG11 medium was used for culture, and the Microcystis aeruginosa were cultured at a temperature of 27°C, a light-dark ratio of 12 h : 12 h, and a light intensity of 38 u mol * (m° * s)"! for 7 days. The cell density of Microcystis aeruginosa was determined by taking samples at Oh, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, the inhibition rate of berberine on Microcystis aeruginosa was calculated.

Refer to Fig. 2 and Fig. 3, it can be seen that there is no significant difference in cell density between the blank group and the treatment groups with berberine concentrations of 0.1 mg/L, 0.2 mg/L, and 1.0 mg/L (P>0.05), the inhibitory effect of the treatment group with the berberine concentration of 2 mg/L was significantly different than that of the blank group and several low-concentration treatment groups (P = 0.00 <0.01). At the end of the 7-day test, the cell density was about 7.34 x 10° cell * mL”; It can also be observed that the growth of Microcystis aeruginosa is significantly inhibited when the berberine concentration is 5 mg/L (P = 0.00 <0.01), and the cell density of Microcystis aeruginosa is about 4.65 X 10° cell * mL” at the end of the 7-day test. Therefore, berberine has a certain inhibitory effect on the growth of Microcystis aeruginosa cells, but there is no significant difference in the low-concentration groups, such as 0.1 mg/L, 0.2 mg/L, and 1.0 mg/L, which has low inhibition rate, with no more than 16%; while the inhibition rate of berberine on Microcystis aeruginosa after 7-day in the 2 mg/L and 5 mg/L treatment groups were

30.96% and 56.27%, respectively.

Comparative Example 2 Same as Comparative Example 1, the azone mother liquor was added into Microcystis aeruginosa liquid with the initial concentration of 2.0X 10° cell * mL to make the concentration of the azone is 20.0 mg/L without adding berberine (neither berberine nor azone was added in blank group). BG11 medium was used for culture, and the Microcystis aeruginosa were cultured at a temperature of 27°C, a light-dark ratio of 12 h : 12 h, and a light intensity of 38 Lmol * (m? * s) for 7 days. The cell density of Microcystis aeruginosa was determined by taking samples at O h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, to determine whether azone affect the growth of Microcystis aeruginosa.

Referring to Fig. 4, when Microcystis aeruginosa was cultured without addition of azone and with addition of 20mg/L of azone, there was no significant difference in the cell density of Microcystis aeruginosa between the two groups (P>0.05), indicating that azone will not affect the growth of Microcystis aeruginosa.

Embodiment 1 In this embodiment, different volumes of azone mother liquor and same volumes of berberine mother liquor were added into Microcystis aeruginosa liquid to form same berberine gradients and different azone gradients, the Microcystis aeruginosa were cultured under the same conditions, and samples were taken at different times to determine cell density, so as to determine the inhibition rate of different concentrations of azone on Microcystis aeruginosa, details are as follows: the berberine mother liquor was added into Microcystis aeruginosa liquid with the initial concentration of 2.0 X 10° cell * mL"! to make the berberine concentration is

2.0 mg/L, then different volumes of azone mother liquor were added to make the final azone concentrations are 0 mg/L (blank group), 1.0 mg/L, 2.0 mg/L, 5.0 mg/L, 10.0 mg/L and 20.0 mg/L. BG11 medium was used for culture, and the Microcystis aeruginosa were cultured at a temperature of 27°C, a light-dark ratio of 12 h : 12 h, and a light intensity of 38 1 mol (m? es)! for 7 days. The cell density of Microcystis aeruginosa was determined by taking samples at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, so as to determine the inhibition rate of berberine on Microcystis aeruginosa under different azone concentrations.

Refer to Fig. 5 and Fig. 6, it can be seen that under the combined action of berberine (concentration of 2.0 mg/L) and azone, the growth trend of Microcystis aeruginosa in the 6 groups was basically similar, and under the condition of same initial density (2 X 10° cell * mL”), the cell density of Microcystis aeruginosa cultured in the treatment groups was significantly lower than that in the blank group, and this phenomenon continued until the end of the experiment. One-way analysis of variance showed that when the concentration of azone was 20.0 mg/L, the growth of Microcystis aeruginosa was significantly inhibited by berberine and azone (P=0.001 <0.05); when the concentration of azone was lower, the growth of Microcystis aeruginosa was not significantly inhibited (P> 0.05). Under the combined action of berberine (concentration of 2.0 mg/L) and azone, the inhibition rate of the two on Microcystis aeruginosa gradually increases with the increase of the concentration of azone, when the concentration of azone is 20mg/L, on the 3rd day of culture, the inhibition rate of the two on Microcystis aeruginosa is as high as 74.30%, which was significantly higher than that of other treatment groups (P<0.01).

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, it should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims (7)

Claims

1. An inhibitor of Microcystis aeruginosa, characterized in that the inhibitor of Microcystis aeruginosa 1s composed of a berberine and an azone.

2. The inhibitor of Microcystis aeruginosa as claimed in claim 1, characterized in that a concentration of the berberine is 0.1-5.0 mg/L, and a concentration of the azone is

1.0- 50.0 mg/L.

3. The inhibitor of Microcystis aeruginosa as claimed in claim 1, characterized in that a concentration of the berberine is 2.0-5.0 mg/L, and a concentration of the azone is

10.0- 20.0 mg/L.

4. The inhibitor of Microcystis aeruginosa as claimed in claim 1, characterized in that the berberine is a berberine hydrochloride.

5. A method for preparing the inhibitor of Microcystis aeruginosa as claimed in any one of claims 1 to 4, characterized in that comprising: preparing a berberine mother liquor, the berberine mother liquor is prepared by adding the berberine into distilled water; adding the azone into the berberine mother liquor.

6. A use of the inhibitor of Microcystis aeruginosa as claimed in any one of claims 1 to 4 in preventing Microcystis aeruginosa blooms.

7. The use as claimed in claim 6, characterized in that comprising: preparing the berberine mother liquor; preparing the azone mother liquor; adding the berberine mother liquor into a Microcystis aeruginosa liquid, and then adding the azone mother liquor; wherein the concentration of the berberine is 0.1-5.0 mg/L, and the concentration of the azone is 1.0- 50.0 mg/L.