CN114873739A

CN114873739A - Nitrite reductase promoter and application thereof

Info

Publication number: CN114873739A
Application number: CN202210536195.6A
Authority: CN
Inventors: 谷劲松; 张艳楠; 陈立; 刘�文
Original assignee: Shandong Sheng'ao Biotechnology Co ltd
Current assignee: Shandong Sheng'ao Biotechnology Co ltd
Priority date: 2022-05-17
Filing date: 2022-05-17
Publication date: 2022-08-09
Anticipated expiration: 2042-05-17
Also published as: CN114873739B

Abstract

The invention relates to the technical field of nitrite reductase, in particular to a nitrite reductase promoter and application thereof. Comprises 60 to 80 parts by weight of 0.1mmol/L FeCl ₂ Solution and 40-80 parts of an enzyme-based stabilizing mixture, wherein the enzyme-based stabilizing mixture is prepared by a method comprising the following steps: 25: 25: 2: 1 the mass ratio of glucose, laminarin, sodium hydrosulfite and MnSO ₄ ·H ₂ O and CaCl ₂ ·2H ₂ O and then dissolving the mixture with an equal mass of water. The nitrite reductase promoter provided by the invention can efficiently improve the probiotic activity of nitrite reductase produced in the compound fermentation liquor used in cooperation, and improve the yield and the yield of the nitrite reductaseThe activity of the enzyme can effectively improve the decomposition rate of nitrite reductase on nitrite.

Description

Nitrite reductase promoter and application thereof

Technical Field

The invention relates to the technical field of nitrite reductase, in particular to a nitrite reductase promoter and application thereof.

Background

Aquaculture has gradually evolved into a high-density intensive model in pursuit of the enormous profits brought by the huge consumer markets. Meanwhile, the concept of consumers is changed, consumers pursuing quality of life need a large amount of aquatic products, and the requirements on the quality of the aquatic products are higher and higher, the quality problems of the aquatic product cultivation that the consumers and farmers worry about are caused by the over-standard ammonia nitrogen, nitrite, pathogenic bacteria, toxic elements and the like caused by intensive cultivation, the problem of nitrite content rise is common, the water environment is seriously polluted due to the over-high nitrite content in the cultivation process, and even aquatic animals are induced to be ill and killed. In order to solve the problem, various chemical aquatic preparations such as disinfectants, water quality modifiers and algicides are layered, the water quality modifiers can quickly reduce the content of excessively high nitrite to a certain extent, but for the ecological environment of cultivation and aquatic animals, long-term use of the water quality modifiers causes adverse effects such as environmental pollution, ecological damage, drug resistance, drug residue and the like, and the adverse effects not only limit the cultivation capacity of the water body, but also greatly influence the quality of the cultivated animals. Therefore, the application of the biological control method gradually becomes a hotspot, and the application of the microecological preparation before the nitrite problem is controllable is found through visiting, so that the water body is obviously improved, but once the nitrite problem is outbreak, the oxidation-reduction potential of the water body is increased, the microecological preparation is difficult to exert efficient degradation, the growth of cultured animals is influenced, and the growth and the planting of part of probiotics in the water body are threatened. Therefore, biological control methods in water body treatment have great limitations.

In order to meet the great safety challenge on the breeding of animals and the probiotics in water, a nitrite reductase (NiR) with high specificity and efficiency is the focus of research. It was found that nitrite reductase degrades nitrite to N ₂ Or NH ₄ ⁺ And the accumulation of nitrite nitrogen in the aquatic environment is reduced. Currently, nirs are classified into two categories according to their molecular structure and the metal ions contained:copper-type nitrite reductase (CuNiR) encoded by the nirK gene and cytochrome cd 1-nitrite reductase (cd1-NiR) encoded by the nirS gene. The CuNiR is widely distributed and exists in Bacillus megaterium (Bacillus megaterium), Bacillus cereus (Bacillus cereus), Lactobacillus plantarum (Lactobacillus plantarum) and the like. CuNiR is a trimeric protein composed of 3 identical subunits, each monomer containing two types of copper atom active centers, i.e., electron donating T ₁ Cu center and catalytic T ₂ A Cu center. At T ₁ A Cu position, Cu coordinated by 2 His, 1 Cys and axial Met to form a tetrahedral geometry; t is ₂ Cu is in a resting state (His) ₃ -H ₂ An O tetrahedral geometry.

However, nitrite reductase also has a problem of low activity in poor water bodies with high burst of nitrite. Based on this, there is a need for an accelerator that can increase the yield and activity of nitrite reductase and accelerate the rate of nitrite reductase decomposing.

Disclosure of Invention

Aiming at the technical problem that the activity of nitrite reductase is not high in poor water with high nitrite outbreak, the invention provides a nitrite reductase promoter and application thereof, the nitrite reductase promoter provided by the invention can efficiently improve the yield and activity of nitrite reductase, provide a proper action environment for nitrite reductase and effectively improve the decomposition rate of nitrite reductase on nitrite; the components contained in the accelerant are environment-friendly, so that the breeding environment can be stabilized and improved; effectively reduces the content of nitrite in the aquaculture water body and promotes the green and healthy growth of the aquaculture animals.

In a first aspect, the invention provides a nitrite reductase promoter, which comprises 60-80 parts by weight of 0.1mmol/L FeCl ₂ Solution and 40-80 parts of enzyme-based stable mixture, wherein the enzyme-based stable mixture is prepared by mixing glucose, laminarin, sodium dithionite and MnSO according to the mass ratio of 50: 25: 2: 1 ₄ ·H ₂ O and CaCl ₂ ·2H ₂ O is mixed and thenThe mixture was dissolved with an equal mass of water.

Further, FeCl ₂ 75 parts of solution;

the weight portion of the enzyme-based stable mixture is 50 portions.

In a second aspect, the invention provides an application of the nitrite reductase promoter as a water quality regulator.

Further, adding the compound fermentation broth and a nitrite reductase promoter into the culture water body, incubating at normal temperature to obtain a ferrous fermentation broth chelate, and splashing the ferrous fermentation broth chelate into the culture environment;

the composite fermentation broth is a mixed fermentation broth of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), Bacillus megaterium (Bacillus megaterium) and Enterococcus faecium (Enterococcus faecium).

Furthermore, the volume ratio of the fermentation broth ferrous chelate to the aquaculture water body is 5%.

Further, the preparation method of the composite fermentation liquid comprises the steps of inoculating a colony mixture of lactobacillus plantarum, lactobacillus casei, bacillus megaterium and enterococcus faecium in a fermentation culture medium with the pH value of 6.0-6.8 according to the volume ratio of 3-4%, and carrying out anaerobic culture on the colony mixture for 3 days at the temperature of 28 ℃, wherein the moisture content reaches 80-90%, so as to obtain the composite fermentation liquid.

Further, the fermentation medium contains 7.50g meat and bone meal, 24.38g wheat bran, MgSO ₄ ·7H ₂ O 0.313g、(NH ₄ ) ₂ SO ₄ 1.0g、KH ₂ PO ₄ 2.0g and 1000mL of deionized water, and the preparation method comprises sterilizing the components of the fermentation medium at 121 ℃ under high temperature and high pressure, keeping for 15min, standing, cooling to 28 ℃ to form the medium, wherein the pH value of the fermentation medium is 6.0-6.8.

Further, the volume ratio of the nitrite reductase promoter to the composite fermentation broth is 1: 400.

the invention has the beneficial effects that:

ferrous ions in the nitrite reductase promoter have the effect of promoting the yield and activity of nitrite reductase generated by thalli. And the other effective component of the nitrite reductase promoter, namely the stable mixture, contains reductive substances, so that the reduction state of ferrous iron can be ensured, and the utilization rate of the ferrous iron for nitrite reductase is improved. All components of the nitrite reductase promoter are environment-friendly, and the nitrite content is efficiently degraded on the basis of ensuring the steady state of a water body.

The composite fermentation liquor contains a plurality of biological components with extremely strong reducibility, including organic acids such as formic acid, acetic acid, lactic acid and citric acid, and amino acids such as tyrosine, cysteine and glutamic acid, after the composite fermentation liquor is mixed with the nitrite reductase promoter, ferrous iron can be chelated with the reducibility substances in the composite fermentation liquor, can be absorbed and utilized by probiotics more easily, and participates in the synthesis of the nitrite reductase, and meanwhile, the components can also obviously improve the oxidation-reduction potential of a water body in the incubation process, activate the activity of the probiotics to the optimal state and adapt to the environment of a culture water body so as to improve the field planting rate of the probiotics in the culture water body, and provide proper catalytic conditions for the nitrite reductase.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 shows NaNO in example 1 and blank groups for different time periods ₂ The change curve of the concentration.

FIG. 2 shows NaNO in example 2 and blank in Experimental example 1 during different periods of time ₂ The change curve of the concentration.

FIG. 3 shows NaNO in example 3 and blank groups of Experimental example 1 during different time periods ₂ The change curve of the concentration.

FIG. 4 shows NaNO in each group for different periods of time in Experimental example 1 ₂ The change curve of the concentration.

FIG. 5 is a graph showing the change of enzyme activities in the example 1 group and the blank group in Experimental example 3 at different time periods.

FIG. 6 is a graph showing the change in enzyme activity in example 2 and blank in Experimental example 3 at different time periods.

FIG. 7 is a graph showing the change in enzyme activity in example 3 and blank groups at different time periods in Experimental example 3.

FIG. 8 is a graph showing the change in enzyme activity in each group of the experimental example 3 at different time periods.

In fig. 1-4, the solid line in the middle of the curve represents the mean, and the shading above and below the mean represents the data error.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The Lactobacillus plantarum (Lactobacillus plantarum) used in the specific embodiment of the invention is purchased from China general microbiological culture Collection center (CGMCC), and the strain number is 1.16089;

lactobacillus casei (Lactobacillus casei) is purchased from China general microbiological culture Collection center (CGMCC), and the strain number is 1.3206;

bacillus megaterium (Bacillus megaterium) is purchased from China general microbiological culture Collection center (CGMCC), and the strain number is 1.8802;

enterococcus faecium (Enterococcus faecium) is purchased from China general microbiological culture Collection center (CGMCC), and the strain number is 1.2025.

The fermentation medium used in the embodiment of the invention contains 7.50g of meat and bone meal, 24.38g of wheat bran and MgSO ₄ ·7H ₂ O 0.313g、(NH ₄ ) ₂ SO ₄ 1.0g、KH ₂ PO ₄ 2.0g and 1000mL of deionized water, and the preparation method comprises mixing the components of the fermentation medium in 1Sterilizing at 21 deg.C under high temperature and high pressure, maintaining for 15min, standing, cooling to 28 deg.C to form culture medium with pH of 6.0-6.8;

the simple culture medium contains 50g of anhydrous glucose, 30g of yeast extract powder and 1000mL of deionized water, and the preparation method comprises high-temperature high-pressure sterilization at 121 ℃ for 20 min.

The preparation method of the sulfanilic acid solution used in the specific embodiment of the invention comprises the following steps: weighing 0.5g of sulfanilic acid, dissolving in 150mL of 10% acetic acid, and storing in a brown bottle at normal temperature, wherein the solution can be stabilized for a plurality of weeks;

the preparation method of the alpha-naphthylamine solution comprises the following steps: 0.1g of alpha-naphthylamine is weighed out and dissolved in 150mL of 10% acetic acid, then 20mL of distilled water are added and the solution is stored at 4 ℃ in a brown bottle, can be stable for several weeks and is reconstituted if it becomes dark brown.

Example 1

Nitrite reductase promoter comprises 60 parts of 0.1mmol/L FeCl ₂ The solution and 80 parts of an enzyme-based stabilizing mixture, wherein the enzyme-based stabilizing mixture is prepared by

According to the following weight ratio of 50: 25: 25: 2: 1, respectively weighing glucose, laminarin, sodium hydrosulfite and MnSO ₄ ·H ₂ O and CaCl ₂ ·2H ₂ Mixing O, dissolving the mixture with water of equal mass, stirring for 15min, transferring to container, and sealing to obtain enzyme-based stable mixture containing glucose 0.5g, laminarin 0.25g, sodium dithionite 0.25g, and MnSO 0.02g ₄ ·H ₂ O、0.01g CaCl ₂ ·2H ₂ O。

Example 2

Example 2 differs from example 1 in that FeCl in example 2 ₂ The parts by weight of the solution and the enzyme-based stabilizing mixture are 75 parts and 50 parts, respectively.

Example 3

Example 3 differs from example 1 in that FeCl in example 3 ₂ The weight parts of the solution and the enzyme-based stabilizing mixture are respectively 80 parts and 40 parts.

Experimental example 1

The effect of the nitrite reductase promoter prepared in examples 1 to 3 on nitrite reductase degradation of lactobacillus plantarum was determined by the α -naphthylamine method. The experimental method is as follows:

s1 plots a standard curve: 0.01g NaNO was weighed ₂ Dissolving in 500mL of water, quantitatively transferring into a 1000mL volumetric flask, diluting with distilled water to a marked line to prepare a standard stock solution, wherein the mass concentration of the standard stock solution is 10 mg/L. Accurately transferring 0.00mL, 0.40mL, 0.80mL, 1.20mL, 1.60mL, 2.00mL of NaNO ₂ Putting the standard stock solution into a 6-piece 50mL colorimetric tube, then respectively adding 30mL of distilled water and 2mL of sulfanilic acid solution, shaking up, standing for 3min, respectively adding 2mL of alpha-naphthylamine solution, adding water to a constant volume, shaking up, and standing for 20 min. By using ultraviolet-visible spectrophotometer, quartz cuvette with reagent blank as reference at wavelength lambda _max The absorbance of each solution was measured at 540nm (3 replicates were averaged) and a standard curve of absorbance-nitrite mass concentration was plotted, y being 0.5504x-0.0046, R ² ＝0.9972。

S2 preparation of composite fermentation liquor: stirring and mixing lactobacillus plantarum, lactobacillus casei, bacillus megaterium and enterococcus faecium according to the weight ratio of 3:3:2:2 to obtain a colony mixture, inoculating the colony mixture into a fermentation culture medium according to the inoculation amount of 4% of the volume ratio, and carrying out anaerobic culture on the colony mixture at the temperature of 28 ℃ for 3 days until the water content reaches 80-90%;

s3 nitrite degradation: with 20mg/L sterile NaNO ₂ NaNO in simple solution-adjusted culture medium ₂ Taking the mixture with the concentration of 10mg/L as a simulated water body, inoculating the composite fermentation liquor according to the inoculation amount of 4% by volume, then respectively adding the nitrite reductase accelerant of each embodiment with the volume ratio of 0.01% to culture at 25 ℃, correspondingly taking the mixture as the group of the embodiment 1, the group of the embodiment 2 and the group of the embodiment 3, and sampling and measuring NaNO at 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 26h, 30h, 32h, 34h and 36h ₂ Concentration;

meanwhile, a simulated water body inoculated with 4% of composite fermentation liquor in volume ratio and without nitrite reductase promoter is used as a blank group.

And (S4) sample detection:

accurately sucking 1.0mL of sample into a triangular flask, adding 12.5mL of saturated borax solution, shaking up, adding 50mL of distilled water, heating in a boiling water bath for 15min, taking out, rapidly cooling to room temperature with tap water, transferring into a 100mL volumetric flask, adding 5mL of zinc acetate solution, mixing uniformly, adding 5mL of potassium ferrocyanide solution, fully mixing uniformly, fixing the volume, standing until protein precipitates in the volumetric flask, taking 4mL of supernatant into a centrifugal tube, centrifuging at 3000r/min for 10min, and obtaining the supernatant as the sample to be detected.

Accurately transferring 2mL of a sample to be detected into a 50mL colorimetric tube, adding 30mL of distilled water, adding 2mL of sulfanilic acid solution, shaking up, standing for 3min, adding 2mL of alpha-naphthylamine solution respectively, adding water to a constant volume, shaking up, and standing for 20 min. Zeroing (adding reagent) with a quartz cuvette and distilled water as a blank at wavelength lambda by using an ultraviolet-visible spectrophotometer _max The absorbance of the solution was measured at 540nm (3 replicates were averaged). After the absorbance of the sample at 540nm is measured, the nitrite content of the sample is obtained according to a standard curve, and the degradation rate formula is as follows:

η＝(N ₀ －N _t )/N ₀ ×100％；

in the formula: eta is the nitrite degradation rate; n is a radical of ₀ The mass concentration of the nitrite before treatment is mg/L; n is a radical of _t The nitrite mass concentration was determined as mg/L.

The results are shown in Table 1 below and FIGS. 1 to 3.

TABLE 1 NaNO in samples at different time periods ₂ Concentration detection data statistics (unit: mg/L)

The detection data in Table 1 are processed to obtain the residual NaNO at different times for the blank group and the parallel experimental samples in the groups of examples 1-3 ₂ The AVG. + -. SD values of the concentrations are shown in Table 2 and FIG. 4.

TABLE 2 NaNO in samples at different time periods ₂ Statistical analysis of AVG + -SD concentration (unit: mg)/L)

Measuring time	Blank group	EXAMPLE 1 group	EXAMPLE 2 group	EXAMPLE 3 group
					1h	0.387±0.005	0.374±0.007	0.361±0.005	0.368±0.005
2h	0.343±0.006	0.334±0.008	0.316±0.007	0.340±0.007
					4h	0.327±0.008	0.300±0.015	0.283±0.007	0.302±0.012
6h	0.254±0.006	0.269±0.017	0.236±0.012	0.267±0.013
					8h	0.239±0.008	0.231±0.011	0.198±0.015	0.222±0.016
10h	0.227±0.01	0.192±0.017	0.151±0.016	0.186±0.007
					12h	0.194±0.009	0.160±0.012	0.135±0.012	0.162±0.009
24h	0.124±0.007	0.086±0.010	0.053±0.007	0.088±0.007
					26h	0.118±0.006	0.080±0.006	0.047±0.007	0.077±0.005
30h	0.109±0.004	0.072±0.007	0.041±0.005	0.063±0.006
					32h	0.105±0.004	0.066±0.006	0.034±0.004	0.057±0.003
34h	0.102±0.003	0.058±0.004	0.029±0.003	0.048±0.004
					36h	0.100±0.003	0.052±0.005	0.030±0.003	0.045±0.003

Calculated, after 36 hours, blank group is matched with NaNO ₂ The degradation rate was 74.16%.

As shown in FIG. 1, in the lag phase 1-4h, the blank group and the compound strain in example 1 have substantially the same metabolic absorption effect, and can complete the growth and proliferation of the bacterial cells by assimilating simple nutrient components for NaNO ₂ The degradation effect of the microbial cells has no obvious difference and shows a trend of fluctuation and decline until the logarithmic growth period is increased by 5-12 hours, the metabolic absorption capacity of the microbial cells is enhanced, the utilization rate of nutritional ingredients such as a ferrous chelator and the like is increased, and NaNO is contained in the group of example 1 in the period ₂ The content of the NaNO is obviously lower than that of a blank group, and the NaNO is treated after 36 hours in the experiment ₂ The degradation rate was 86.09%.

As shown in FIG. 2, NaNO was present in the group of example 2 during the lag phase of 1-4h ₂ The concentration of (A) is significantly lower than that of the blank group, because in example 2, the ratio of the ferrous chelate compound is relatively increased, and the bacterial cells grow to take in enough ferrous chelate compound for NaNO ₂ Synthesis of reductase, therefore, the later group of example 2 sets of NaNO ₂ The degradation effect is better than that of the blank group, and example 2 group has NaNO after 36h ₂ The degradation rate reaches 91.68 percent.

As shown in FIG. 3, example 3 group of prophase NaNO ₂ The degradation of the iron chelate complex is crossed with the blank group, because the proportion of the iron chelate complex is larger in the embodiment 3, the proportion of the enzyme-based stable mixture is reduced, the rest of a large amount of simple nutrient components in the iron chelate complex are preferentially taken by the strains in the fermentation liquor and are consistent with the metabolic action of the simple nutrient substances taken by the blank group, and meanwhile, the redundant chelated ferrous iron loses the protection of the corresponding enzyme-based stable mixture, so that the chelated ferrous iron reduces NaNO ₂ Into gaseous nitride, thereby enabling later-stage NaNO ₂ The content showed a steady linear decrease, and example 3 group showed no after 36h ₂ The degradation rate was 87.7%.

As shown in FIG. 4, example 2 group for NaNO ₂ The degradation effect of the enzyme is obviously lower than that of the blank group, and the degradation effect is the best compared with the group of the example 1 and the group of the example 3, so the mixture ratio of the ferrous chelate and the enzyme-based stable mixture in the example 2 is the best.

Experimental example 2

In the measurement process of experimental example 1, the number of viable bacteria in the sample was measured at 0h, 4h, 8h and 12h, respectively. The counting method comprises the following steps:

samples (100 μ L) were taken and diluted in sterile water in a gradient, the diluted bacterial solution was counted by a hemocytometer, and the total number of probiotic colonies was counted, the results of which are shown in table 3 below.

TABLE 3 statistics of the number of colonies in the fermentation broth (units: one/mL) in samples at different time periods

Measuring time	Blank group	EXAMPLE 1 group	EXAMPLE 2 group	EXAMPLE 3 group
					0h	1.579×10 ⁴	1.72×10 ⁴	1.618×10 ⁴	1.6246×10 ⁴
4h	2.216×10 ⁶	2.191×10 ⁶	1.210×10 ⁶	1.674×10 ⁶
					8h	2.856×10 ⁸	2.078×10 ⁸	2.423×10 ⁸	3.69×10 ⁸
12h	8.56×10 ⁸	3.27×10 ⁹	3.441×10 ⁹	4.312×10 ⁹

Statistical results show that example 3 amplified 5-fold compared to the blank, example 2 amplified 4-fold compared to the blank, and example 1 amplified 3.8-fold compared to the blank. The main reason is that the ferrous chelate adopted in the invention contains various nutrient substances and has a promoting effect on the growth of the thalli, the ferrous chelate accounts for the largest proportion in the example 3, and the promoting effect on the proliferation of the thalli is the most obvious.

Experimental example 3

S1 preparation of crude enzyme solution: stirring and mixing lactobacillus plantarum, lactobacillus casei, bacillus megaterium and enterococcus faecium according to the weight ratio of 3:2 to obtain a colony mixture, then inoculating the colony mixture into four fermentation media according to the inoculation amount of 4% by volume, to one of the fermentation media, a simple medium was added in a volume ratio of 0.01% as a control group, and to the other three fermentation media, nitrite reductase promoters prepared in examples 1, 2 and 3 in a volume ratio of 0.01% were added as example 1 group, example 2 group and example 3 group, the control group, the group of example 1, the group of example 2 and the group of example 3 were activated under anaerobic conditions at 28 ℃, then shaking and culturing at 30 ℃ for 120r/min, and sampling for 2h, 4h, 6h, 8h and 12h respectively, wherein the OD of the thalli in the sampled samples is controlled each time. ₆₀₀ Centrifuging the sample at 4 deg.C for 10min at 6000r/min with the value of 0.8 + -0.05, collecting thallus, and removing supernatant; shaking with phosphate buffer (pH 7.2) to suspend thallus, adding lysozyme, ultrasonic disrupting cells at 30 deg.C for 24 hr, and centrifuging at 4 deg.C for 15min at 8000r/min to obtain supernatant as crude enzyme solution.

S2 nitrite degradation: the crude enzyme solutions of the control group and the groups of example 1, example 2 and example 3 were added to NaNO ₂ Treating in the solution at 37 deg.C for a period of time, determining nitrite reductase activity, determining in parallel for 3 times, and taking the average value.

The activity unit of the nitrite reductase is measured by the concentration of the obtained crude enzyme liquid for reducing nitrite per hour. The more nitrite is degraded in the same time, the greater the activity of the enzyme. The calculation formula is as follows:

Y＝(X ₁ -X ₂ )/t×100％：

in the formula: y is enzyme activity, U; x ₁ The nitrite concentration before treatment is mg/mL; x ₂ Is the nitrite concentration in mg/mL when measured; t is the enzyme catalysis time, h.

The calculation results are shown in the following tables 4 to 7 and FIGS. 5 to 8.

TABLE 4 blank group different time period enzyme activity change (unit: U)

Time of sampling	1#	2#	3#	AVG±SD
					2h	14.535	12.718	15.443	14.232±1.388
4h	16.806	17.714	15.443	16.655±1.143
					6h	19.077	17.866	17.260	18.068±0.925
8h	17.941	17.487	18.623	18.017±0.572
					12h	20.288	19.228	19.380	19.632±0.573

As can be seen from Table 4, the enzyme activity of the blank group increased by 37.94% after 12 hours.

TABLE 5 variation of enzyme activity (unit: U) in different time periods for the group of example 1

Time of sampling	1#	2#	3#	AVG±SD
					2h	14.941	14.625	17.669	15.746±1.674
4h	18.623	17.714	16.352	17.563±1.143
					6h	20.288	19.077	18.169	19.178±1.063
8h	22.484	21.575	21.121	21.727±0.694
					12h	23.668	23.165	22.559	23.821±0.555

As can be seen from Table 5, the enzyme activity of the group of example 1 was increased 51.28% after 12h of treatment with the nitrite reductase promoter of example 1. It can be seen from fig. 5 that the blank group has an obvious difference from the group of example 1 in the promotion effect of the enzyme activity since 6 hours later, which indicates that after 6 hours, the absorption and utilization of the chelated ferrous iron by the strains contained in the fermentation broth in the logarithmic growth phase are gradually enhanced, the activity of the nitrite reductase is gradually improved, and after 12 hours, the enzyme activity improvement rate of the group of example 1 reaches 1.35 times that of the blank group.

TABLE 6 variation of enzyme activity (unit: U) at different time periods for the group of example 2

Time of sampling	1#	2#	3#	AVG±SD
					2h	15.443	16.352	13.626	15.141±1.388
4h	19.531	19.985	18.169	19.228±0.945
					6h	20.288	20.591	19.380	20.086±0.630
8h	23.165	22.484	22.029	22.559±0.572
					12h	24.982	24.376	24.073	24.477±0.463

As can be seen from Table 6, the enzyme activity of group 2 of example 2 was increased 61.67% after 12h of treatment with the nitrite reductase promoter of example 2. As can be seen by combining the graph 6, after 3 hours, compared with the blank group, the enzyme activity of the group in the example 2 is obviously increased, which indicates that a small amount of chelated ferrous iron with a high content in the example 2 is utilized for the synthesis of nitrite reductase in the lag phase of the probiotics, and after 6 hours, the strains contained in the fermentation liquor reach the logarithmic phase, and the probiotics fully utilize the chelated ferrous iron, so that the promotion effect on the activity of the nitrite reductase is more obvious. Compared with the blank group, after 12h, the accelerating effect (enzyme activity increasing rate) of example 2 on nitrite reduction reaches 1.625 times.

TABLE 7 variation of enzyme activity (unit: U) at different time periods for the group of example 3

Time of sampling	1#	2#	3#	AVG±SD
					2h	17.562	15.745	16.653	16.655±0.909
4h	20.288	18.017	19.379	19.228±1.143
					6h	21.399	20.491	21.399	21.096±0.524
8h	22.405	21.951	22.405	22.938±0.262
					12h	25.537	25.083	24.932	25.184±0.315

As can be seen from Table 7, the enzyme activity of the group of example 3 was increased 51.21% after 12h of treatment with the nitrite reductase promoter of example 3. As can be seen from the combination of FIG. 7, compared with the blank group, the enzyme activity of the group in example 3 is increased in a stable range, the high-content chelated ferrous iron preferentially reduces nitrite, the nutrient content in the fermentation broth is enough for the growth and proliferation of the thalli, and the component proportion in example 3 may not achieve the purpose that the thalli utilize chelated ferrous iron.

As can be seen from fig. 8, compared with the blank group, the groups in examples 1 to 3 all have significant effects of promoting the activity of nitrite reductase, wherein the group in example 3 has the most significant effect of promoting nitrite reductase, and the group in example 2 has the highest rate of improving nitrite reductase, which reaches 61.67%.

In conclusion, the product can remarkably promote the field planting of beneficial bacteria such as lactobacillus plantarum and the like in a water body, and the counting result of a blood counting plate shows that the number of the beneficial bacteria added with the product is 4-5 times higher than that of a blank group only added with simple nutrient components. Further treating NaNO in water body by alpha-naphthylamine method ₂ The product can obviously reduce NaNO within 36h as shown by the result of real-time detection for 36h ₂ In a reduced amount of NaNO ₂ And (3) stacking. The above results show that the product of the invention can effectively and obviously reduce NaNO ₂ The water quality regulator can be used as a good water quality regulator.

Furthermore, when the results of the above experimental examples 1 to 3 are analyzed, the number of the probiotics colonized in the water body after the treatment with the nitrite reductase promoter of the example 2 is amplified by 4 times compared with the blank group, the enzyme activity improvement rate is 61.67%, the nitrite degradation rate reaches 91.68%, and the method is an implementation scheme with the optimal comprehensive effect in the three examples.

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention.

Claims

1. The nitrite reductase promoter is characterized by comprising 60-80 parts by weight of 0.1mmol/LFeCl ₂ Solution and 40-80 parts of an enzyme-based stabilizing mixture, wherein the enzyme-based stabilizing mixture is prepared by a method comprising the following steps: 25: 25: 2: 1 the mass ratio of glucose, laminarin, sodium hydrosulfite and MnSO ₄ ·H ₂ O and CaCl ₂ ·2H ₂ O and then dissolving the mixture with an equal mass of water.

2. The nitrite reductase promoter of claim 1, wherein the fermentation broth ferrous chelate is 75 parts by weight and the enzyme-based stabilizing mixture is 50 parts by weight.

3. Use of the nitrite reductase promoter according to claim 1, wherein the nitrite reductase promoter is used as a water quality controlling agent.

4. The application of claim 3, wherein the compound fermentation broth and nitrite reductase promoter are added into aquaculture water, and are incubated together at normal temperature to obtain ferrous fermentation broth chelate, and the ferrous fermentation broth chelate is sprinkled into aquaculture environment;

5. The use as claimed in claim 4, wherein the complex fermentation broth is prepared by inoculating 3-4 vol.% of a mixture of colonies of Lactobacillus plantarum, Lactobacillus casei, Bacillus megaterium, and enterococcus faecium into a fermentation medium having a pH of 6.0-6.8, anaerobically culturing the mixture of colonies at 28 ℃ for 3 days to a water content of 80-90%.

6. The use of claim 5, wherein the fermentation medium comprises 7.50g meat and bone meal, 24.38g wheat bran, MgSO ₄ ·7H ₂ O 0.313g、(NH ₄ ) ₂ SO ₄ 1.0g、KH ₂ PO ₄ 2.0g and 1000mL of deionized water, and the preparation method comprises sterilizing the fermentation medium components at 121 ℃ under high temperature and high pressure, keeping for 15min, standing, cooling to 28 ℃ to form the culture medium, wherein the pH value of the fermentation medium is 6.0-6.8.

7. The use of claim 4, wherein the ratio of nitrite reductase promoter to complex fermentation broth by volume is 1: 400.