Composite moss inhibitor and preparation and application thereof

Abstract

The invention discloses a composite moss inhibitor as well as preparation and application thereof. The composite moss inhibitor comprises L-proline, L-tyrosine and catechol in a mass ratio of (10-30): (0.1-0.3): (10-30), and can be applied to inhibiting or killing spirogyra in a water body. After the L-proline, the L-tyrosine and the catechol are compounded, the growth of spirogyra can be effectively inhibited, the spirogyra can be obviously inhibited under the condition of lower dosage, and the inhibition rate on the spirogyra is up to 90% or above. Meanwhile, the method is safe to crustacean aquaculture animals, water pollution and phytotoxicity phenomena can be effectively avoided, and the method has a good application prospect in aquaculture.

Description

TECHNICAL FIELD

[0001] The present application relates to an algal inhibitor, in particular to a compound spirogyra inhibitor and its preparation and application. BACKGROUND

[0002] Spirogyra, also known as water mite (Spirogyra sp.) (Wang Tao, Du Guozhi. Water mite prevention and control of rice yield-increasing factor test summary [J]. Reclamation and rice, 2001 (3): 35.) is a common harmful filamentous algae in aquaculture water. It not only consumes a large amount of nutrient salts in water, causing water to be lean, inhibiting the growth of water bait organisms, and causing water quality deterioration (Zhang Man, Gai Xiaolong, Wang Yifan, He Zijie, Wang Kang, Chang Huachu, Jia Qi, Wang Yuming, Zeng Daqing, Zhou Chuanjiang. Biological manipulation method for regulation effect of spirogyra [J]. Acta Scientiae Circumstantiae, 2019, 39 (3): 722-729.) but also can adhere to the cheek, gill and other parts of crustacean aquaculture animals such as shrimps and crabs, affecting the activity and feeding of crustacean aquaculture animals (Tang Yuhua, Cai Jianzhong, Zheng Guang, Li Hongjin. Crab pond spirogyra hazard and prevention and control [J]. Aquaculture, 2013, 34 (12): 48-49.) (Ding Changyun. Crab pond spirogyra prevention and control [J]. Aquaculture, 2012, 33 (8): 52.) (Zhong Shiqun. Spirogyra on the hazard of freshwater crayfish and its prevention and control technology [J]. Scientific fish culture, 2020 (3): 51-52.)

[0003] At present, although chemical algicides with less side effects such as chlorine dioxide, copper sulfate, diuron, chlorhexidine, glutaraldehyde and sodium dichloroisocyanurate have good algae inhibition effect on Microcystis aeruginosa and other cyanobacteria, the algae inhibition rate is as high as 100%, but the water sponge is different from the blue-green algae as a unicellular prokaryote, which belongs to perennial filamentous body connected by many vegetative cells, does not branch, occasionally has false root-like branches, and often gathers into a group (Wang Tao, Du Guozhi. Summary of water sponge control experiment by rice yield enhancer [J]. Reclamation and rice production, 2001 (3): 35.), leading to poor algae inhibition effect of these chemical algicides with less side effects on water sponge, and even no effect, and the algae inhibition rate is only about 30% or even less than 22% when the dosage is 10 mg / L (Gao Wei. Effects of nine algicides and three compound algicides on the growth of Microcystis aeruginosa, Chlorella vulgaris and water sponge [D]. Henan: Henan Normal University, 2016.). Therefore, aquaculture industry often uses pesticides such as prometryne and green algae net to control water sponge (Liu Jiamin, Zhou Zhijie, Huang Minsheng, Yuan Yuxin, He Wenhui. Comparative analysis of the control effect of three pesticides on water sponge [J]. Chemical World, 2024, 65 (3): 163-170. Gao Junjie, Quan Xinfang. Experience of water sponge control in Jinhui Lake Scenic Area in Baoji [J]. Water Resources Development and Management, 2018, 4 (4): 77-79.), but the use of these pesticides is easy to cause secondary pollution and destroy the ecological balance (Liao Zhengjun, An Zhenhua, Gu Rongrong, Dou Jingyi, Qin Shu lei, Lu Weny an, Wu Yuheng. Acute toxicity of rice shrimp special green algae net to crayfish and its effect on green algae growth [J]. Aquaculture, 2022, 43 (6): 33-36.), and even cause pesticide poisoning accidents. For example, in 2019, aquaculture farmers in Jiangsu, Hunan and Hubei used prometryne to kill water sponge, which caused the death of river crab, green shrimp and crayfish (Ge Jiachun, Shen Weijian, Wang Zhihong, Yu Keyao, Fu Longlong, Ma Xingkong, Tianjian. Investigation on green algae pesticide poisoning in ponds in Gaochun District of Nanjing [J]. Aquaculture, 2021, 42 (11): 66-67, 69.). Therefore, how to obtain a preparation that can effectively inhibit the growth of water sponge in aquaculture water and has no pesticide poisoning risk and water pollution risk to crustacean aquaculture animals such as shrimps and crabs has become a technical problem to be solved. SUMMARY

[0004] The purpose of the present application is to provide a composite green algae inhibitor, solve the problems of poor algae inhibition effect of existing chemical algicides or pesticides on water sponge and easy pollution of water body and production of aquaculture pesticide poisoning. The second purpose of the present application is to provide a preparation method of the composite green algae inhibitor, and solve the problem of how to prepare the composite green algae inhibitor. The third purpose of the present application is to provide the application of the composite green algae inhibitor in inhibiting or killing water sponge, and solve the problem of how to efficiently inhibit or kill water sponge in water.

[0005] Technical solution: The composite moss inhibitor provided by the application comprises proline, tyrosine and catechol in a mass ratio of 10-30:0.1-0.3:10-30.

[0006] Preferably, the proline and tyrosine are both L-type amino acids.

[0007] Preferably, the mass ratio of the proline, tyrosine and catechol is 15-25:0.1-0.2:15-25.

[0008] The second aspect of the application provides a preparation method of the composite moss inhibitor, comprising the following steps:

[0009] The proline powder, tyrosine powder and catechol powder are mixed in proportion to obtain the composite moss inhibitor.

[0010] In some embodiments, the method further comprises the step of dissolving the composite moss inhibitor in water to prepare an aqueous agent. The composite moss inhibitor can be an aqueous agent or a dry powder agent, or other common pesticide formulations.

[0011] The third aspect of the application provides the use of the above-mentioned composite moss inhibitor in inhibiting or killing water moss.

[0012] Preferably, the method for using the above-mentioned composite moss inhibitor to inhibit or kill water moss comprises the following steps:

[0013] (1) dissolving the composite moss inhibitor in water to obtain a mother liquor;

[0014] (2) adding the mother liquor into a water body containing water moss to inhibit or kill the water moss.

[0015] Preferably, in step (1), the ratio of the composite moss inhibitor to water is 500-50000 mg:1 L.

[0016] Preferably, in step (2), the volume ratio of the mother liquor to the water body containing water moss is 1:9-999.

[0017] Preferably, in step (2), the final concentration of the composite moss inhibitor in the water body containing water moss is 0.5-100 mg / L.

[0018] Preferably, the final concentration of chlorophyll a contained in the water moss in the water body containing water moss is not more than 50 mg / L.

[0019] Advantages: Compared with the prior art, the application has the following advantages:

[0020] The L-proline, L-tyrosine and catechol are compounded to effectively inhibit the growth of Spirogyra, and the Spirogyra can be inhibited by a lower amount, and the inhibition rate of Spirogyra can reach more than 90%. Meanwhile, the Spirogyra is safe for crustacean aquaculture animals, can effectively avoid water pollution and drug damage, and has a good application prospect in aquaculture. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Algal inhibition effect of different final concentrations of the compound green moss inhibitor on Spirogyra. DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be further described below in combination with the drawings.

[0023] Example 1: The determination method of the compound green moss inhibitor and the ratio is as follows:

[0024] (1) Experimental materials

[0025] Spirogyra FACHB-737 was purchased from the Freshwater Algae Culture Collection Center of the Chinese Academy of Sciences; L-proline, L-tyrosine and catechol were purchased from Hebei Rencan Biotechnology Co., Ltd.; BG11 medium was prepared according to the method of Li Xinzhou et al. (Li Xinzhou, Zhuang Sina. Effect of culture medium on the growth and oil production of Chlorella sp. KM-201305 [J]. Chemical and Biological Engineering, 2019, 36(9): 35-38.); NaNO3, K2HPO4, acetone, CaCl2·2H2O, citric acid, ferric ammonium citrate, CuSO4·5H2O, EDTA-2Na, Na2CO3, NaCl, H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, Co(NO3)2·6H2O and MgSO4·7H2O were purchased from the National Pharmaceutical Group (Shanghai) Chemical Reagent Co., Ltd.

[0026] (2) Optimization of compound components and ratio

[0027] Spirogyra FACHB-737 was used as a representative Spirogyra species. Before the experiment, Spirogyra FACHB-737 was inoculated into BG11 medium under sterile conditions, and then cultured at 25℃, light intensity of 2 000lx, light-dark cycle ratio of 14h:10h for 7d to prepare Spirogyra FACHB-737 algal liquid with chlorophyll a concentration of 0.88mg / L.

[0028] The orthogonal test design was used to optimize the compounding ratio of L-proline, L-tyrosine and catechol. The orthogonal test factor level table is shown in Table 1. According to the compounding ratio in the orthogonal test design table (Table 2), L-proline, L-tyrosine and catechol were weighed to a total mass of 50 g, and a composite preparation was prepared in a small mixing machine (15 r / min mixing for 30 min). Then, sterile distilled water was added to make up to 1 L, and a magnetic stirring heater was used to stir at 50 ℃ for 30 min to make the mixture uniform, and a 50000 mg / L mother liquor was prepared. After cooling at 121 ℃ for 20 min, the mother liquor was diluted 10 times with sterile distilled water to prepare a dilution solution with a concentration of 500 mg / L. Then, 2 mL of the dilution solution of each compounding ratio of the composite moss inhibitor was added to 18 mL of Batrachospermum FACHB-737 algal liquid with a chlorophyll a concentration of 0.88 mg / L. Under the same conditions, 2 mL of sterile distilled water was added as a blank control, and the Batrachospermum FACHB-737 algal liquid containing only a final concentration of 24.937 mg / L L-proline was used as a comparative example 1, the Batrachospermum FACHB-737 algal liquid containing only a final concentration of 0.125 mg / L L-tyrosine was used as a comparative example 2, the Batrachospermum FACHB-737 algal liquid containing only a final concentration of 24.937 mg / L catechol was used as a comparative example 3, the Batrachospermum FACHB-737 algal liquid containing a final concentration of 24.937 mg / L L-proline and a final concentration of 0.125 mg / L L-tyrosine was used as a comparative example 4, the Batrachospermum FACHB-737 algal liquid containing a final concentration of 24.937 mg / L catechol and a final concentration of 0.125 mg / L L-tyrosine was used as a comparative example 5, and the Batrachospermum FACHB-737 algal liquid containing a final concentration of 24.937 mg / L L-proline and a final concentration of 24.937 mg / L catechol was used as a comparative example 6.

[0029] All treatments were cultured under the conditions of a temperature of 25 ℃, a light intensity of 2000 lx, and a 14 h:10 h (light:dark) cycle, and the algal liquid was shaken 3 times a day during the culture period. After 7 days of continuous culture, the chlorophyll a content (mg / L) of the algal liquid of each treatment was determined by spectrophotometry (Ministry of Environmental Protection. Determination of chlorophyll a in water by spectrophotometry: [S]. 2017.). The alga inhibition rate was calculated according to the following formula:

[0030]

[0031] Each treatment was repeated three times. Statistical analysis was performed using IBM SPSS Statistics 27 statistical software, and P<0.05 indicated a significant difference. The experimental data are expressed as mean ± standard deviation (Mean ± SD).

[0032] Table 1 Orthogonal test factor level table

[0033]

[0034] Table 2 Orthogonal test design table

[0035]

[0036] The algae-inhibiting effect of the compound green algae inhibitor on Spirogyra was as follows:

[0037] Table 3 Algae-inhibiting effect of the compound green algae inhibitor on Spirogyra

[0038]

[0039]

[0040] Note: The same letter means no significant difference (P>0.05), and different letters mean significant difference (P<0.05).

[0041] As can be seen from the results in Table 3, the compound green algae inhibitor prepared by compounding L-proline, L-tyrosine and catechol at a ratio of 20:0.1:20 had an algae-inhibiting rate of 90.76% on Spirogyra at a dosage of 50 mg / L, which was increased by 67.49% (P<0.05), 79.40% (P<0.05) and 62.99% (P<0.05) compared with the algae-inhibiting rate of L-proline, L-tyrosine and catechol alone, and was increased by 68.36% (P<0.05), 71.93% (P<0.05) and 66.03% (P<0.05) compared with the algae-inhibiting rate of L-proline+L-tyrosine, catechol+L-tyrosine and L-proline+catechol in combination, and was increased by 5.76% (P<0.05) to 29.81% (P<0.05) compared with the algae-inhibiting rate of the compound green algae inhibitor prepared at other compounding ratios. This indicates that the compounding of L-proline, L-tyrosine and catechol at a ratio of 20:0.1:20 in the compound green algae inhibitor has a significant synergistic effect on inhibiting Spirogyra. As can be seen from Comparative Examples 4-6, the algae-inhibiting effect of the compound green algae inhibitor on Spirogyra depends on the simultaneous presence of L-proline, L-tyrosine and catechol, and the absence of any of them will greatly weaken the algae-inhibiting effect of the compounding preparation, further confirming the mutual synergistic effect between the three components.

[0042] Example 2: Safety investigation experiment of the compound green algae inhibitor

[0043] The safety of the compound green algae inhibitor was analyzed by static in vitro exposure method with Eriocheir sinensis as a representative animal of crustacean aquaculture, referring to the method of Wu Song (Wu Song, Huang Xiaoli, Tao Yue, et al. Acute toxicity of chlorantraniliprole to Eriocheir sinensis [J]. Journal of Aquaculture, 2023, 36(3): 46-51.). The experimental design was 1 control group and 6 experimental groups, with 3 replicates in each group. The experimental aquarium was 70 cm x 50 cm x 50 cm in size, and 20 healthy Eriocheir sinensis were placed in each tank, with 10 L of aerated tap water. The compound green algae inhibitor prepared in Example 1 with the best compound ratio (No. 4) was added to the tank in the experimental group, so that the final concentration of the compound green algae inhibitor in each tank was 0.05, 0.5, 5, 50, 500, 5000 mg / L. The control group did not add any substance. During the test, the Eriocheir sinensis was not fed, and the oxygen was not stopped. The aquaculture water was replaced once a day to ensure the stability of the final concentration of the compound green algae inhibitor in the aquaculture water every day. The health status of Eriocheir sinensis in each group was observed for 14 consecutive days, and the number of deaths of Eriocheir sinensis in each group was recorded. 50 The half lethal concentration (LD 50 ) of the compound green algae inhibitor on Eriocheir sinensis was calculated by Bliss method (Dong Xuehong, Tian Min, Ji Ce, et al. Comparison of two LD 50 calculation methods for Vibrio parahaemolyticus virulence [J]. Journal of Shanghai Ocean University, 2016, 25(1): 86-96.).

[0044] Table 4 Safety of compound green algae inhibitor on Eriocheir sinensis

[0045]

[0046] Note: The same letter means no significant difference (P>0.05).

[0047] The experimental results (Table 4) showed that Eriocheir sinensis in the control group and the experimental group did not die after 14 days, and grew well and fed normally, without any abnormal symptoms. This indicates that the LD 50 of the compound green algae inhibitor on Eriocheir sinensis treated for 14 days is greater than 5000 mg / L. The compound green algae inhibitor is safe for crustacean aquaculture animals.

[0048] Example 3: Evaluation of the algal inhibition effect of the compound green algae inhibitor on Spirogyra under different dosages

[0049] The water moss FACHB-737 was used as a representative algae of water moss. Before the experiment, the water moss FACHB-737 strain was inoculated into BG11 medium through sterile operation, and then cultured at 25°C for 7 days under light intensity of 2,000 lx and 14h:10h (light:dark). The concentration of chlorophyll a of the water moss FACHB-737 algae liquid was 0.51 mg / L. The mother liquor of the composite green moss inhibitor prepared in Example 1 under the optimal compound ratio (No. 4) was diluted with sterile distilled water to prepare diluents with concentrations of 5, 10, 15, 20, and 25 mg / L. Then, 2 mL of each diluent of the composite green moss inhibitor was added to 18 mL of the water moss FACHB-737 algae liquid with a chlorophyll a concentration of 0.51 mg / L, respectively. The water moss FACHB-737 algae liquid to which 2 mL of sterile distilled water was added under the same conditions was used as a control. All treatments were cultured under the same conditions of temperature of 25°C, light intensity of 2,000 lx, and 14h:10h (light:dark). The algae liquid was shaken 3 times a day during the culture period. After continuous culture for 7 days, the content of chlorophyll a (mg / L) of the water moss FACHB-737 algae liquid in each treatment was determined by reference to the spectrophotometric method (Ministry of Environmental Protection. Determination of chlorophyll a in water by spectrophotometry: [S]. 2017.). Each treatment was performed in triplicate.

[0050] The experimental results are shown in Table 1. Figure 1 Figure 1 In Table 1, the same letter indicates that the difference is not significant (P>0.05), and different letters indicate that the difference is significant (P<0.05). Figure 1 The results show that the composite green moss inhibitor has a significant inhibitory effect on the growth of water moss at a final concentration of 0.5-2.5 mg / L. Compared with the control group, the concentration of chlorophyll a is reduced by 0.26 mg / L (P<0.05), 0.35 mg / L (P<0.05), 0.44 mg / L (P<0.05), 0.45 mg / L (P<0.05), and 0.53 mg / L (P<0.05), respectively. This indicates that the algae inhibition rate of the composite green moss inhibitor on water moss is 27.97%, 36.98%, 46.97%, 47.75%, and 56.70% at concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 mg / L, respectively. According to the effect evaluation standard for aquaculture environmental improver published by the Ministry of Agriculture and Rural Affairs, a significant effect is achieved when the main indicators are improved by more than 50%, and an effective effect is achieved when the main indicators are improved by 10%-40%. The effective inhibitory dose of the composite green moss inhibitor on water moss is 0.5-2.0 mg / L, and the significant inhibitory dose is 2.5 mg / L.​

Claims

1. A composite moss inhibitor, characterized by comprising: The proline, tyrosine and catechol are in a mass ratio of 10-30:0.1-0.3:10-30.

2. The compound of claim 1, wherein The proline and tyrosine are both L-type amino acids.

3. The compound of claim 1, wherein The mass ratio of the proline, tyrosine and catechol is 15-25:0.1-0.2:15-25.

4. The preparation method of the compound algae inhibitor according to claim 1, characterized in that, The method comprises the following steps: mixing proline powder, tyrosine powder and catechol powder in proportion to obtain a composite moss inhibitor.

5. Use of the composite moss inhibitor according to any one of claims 1-4 in inhibiting or killing water moss.

6. Use according to claim 5, characterized in that, The method comprises the following steps: (1) dissolving the composite moss inhibitor in water to obtain a mother liquor; (2) adding the mother liquor into a water body containing water moss to inhibit or kill the water moss.

7. Use according to claim 6, characterized in that, In step (1), the ratio of the composite moss inhibitor to water is 500-50000 mg:1 L.

8. Use according to claim 6, characterized in that, In step (2), the volume ratio of the mother liquor to the water body containing water moss is 1:9-999.

9. Use according to claim 6, characterized in that, In step (2), the final concentration of the composite moss inhibitor in the water body containing water moss is 0.5-100 mg / L.

10. Use according to claim 6, characterized in that, The final concentration of chlorophyll a contained in the water moss in the water body containing water moss is not more than 50 mg / L.

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