CN114657081A

CN114657081A - Ternary composite bacterium preparation and application thereof

Info

Publication number: CN114657081A
Application number: CN202011523972.0A
Authority: CN
Inventors: 喻凌寒; 林诗琪; 章英; 陈衍学; 林林; 邓文芬
Original assignee: Hubei Lutiandi Biological Engineering Co ltd
Current assignee: Hubei Lutiandi Biological Engineering Co ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-06-24

Abstract

The invention discloses a ternary composite bacterium preparation and application thereof, wherein ternary composite bacteria in the preparation are prepared by mixing enterococcus faecium, bacillus licheniformis and lactobacillus plantarum according to the viable bacteria content ratio of 0.5-1: 0.5-1, the ternary composite bacterium is prepared by mixing enterococcus faecium, bacillus licheniformis and lactobacillus plantarum, and can be used as a nitrite degradation agent in aquaculture to effectively remove nitrite in water, the lowest use viable bacteria concentration of the ternary composite bacterium for removing nitrite in water is 0.288 hundred million/mL, and the removal rate is up to more than 90%.

Description

Ternary composite bacterium preparation and application thereof

Technical Field

The invention relates to the technical field of microecological preparations, in particular to a ternary complex bacterial preparation and application thereof as a nitrite degradation agent for removing nitrite in water.

Background

The probiotics are screened in natural environment or animal intestinal tracts, and are safe, free of residue and free of drug resistance. Experimental studies have shown that probiotics are considered as potential replacements for antibiotics in large-scale farming. In 2001, the food and agriculture organization/world health organization (FAO/WHO) of the united nations defined probiotics as "active microorganisms that produced beneficial effects on the body when ingested in sufficient quantities". The american FDA and American Association for Feed Control Officials (AAFCO) published 2009 to allow 46 microbial species to be used as feed additives. The microbial feed additive approved to be used in China at present comprises 16 varieties of bacillus, bifidobacterium, enterococcus faecalis, enterococcus lactis, lactobacillus and the like, wherein most of the varieties belong to lactic acid bacteria.

Although some reports about the effect of bacillus on degrading nitrite nitrogen exist at present, most of the effects are not ideal due to the influence of factors such as complexity and changeability of the culture water environment, insufficient nitrogen reduction stability of strains and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a ternary composite bacterium preparation capable of effectively removing nitrite in a water body, and solves the problem of accumulation of nitrite in an aquaculture water body under an aerobic condition.

In order to achieve the purpose, the invention adopts the technical scheme that:

a ternary composite bacteria preparation is prepared by mixing enterococcus faecium, Bacillus licheniformis and Lactobacillus plantarum at viable bacteria content ratio of 0.5-1: 0.5-1.

The application of the ternary composite bacterium preparation is as a nitrite degradation agent, can effectively remove nitrite in water, and the minimum use viable bacteria concentration of the ternary composite bacterium for removing nitrite in water is 0.288 hundred million/mL.

Compared with the prior art, the method adopts the enterococcus faecium, the bacillus licheniformis and the lactobacillus plantarum to mix and prepare the ternary complex bacteria which are used as the nitrite degrading agent in aquaculture and can effectively remove the nitrite in the water body, the lowest use viable bacteria concentration of the ternary complex bacteria for removing the nitrite in the water body is 0.288 hundred million/mL, and the removal rate is up to more than 90%.

Detailed Description

The present invention will be further described with reference to the following examples for facilitating understanding of those skilled in the art, and the description of the embodiments is not intended to limit the present invention.

Example 1

0.5kg of enterococcus faecium powder, 1kg of bacillus licheniformis powder and 0.5kg of lactobacillus plantarum powder are taken and evenly mixed to prepare the ternary composite bacterial preparation.

Example 2

0.5kg of enterococcus faecium powder, 1kg of bacillus licheniformis powder and 1kg of lactobacillus plantarum powder are taken and evenly mixed to prepare the ternary composite bacteria preparation.

Example 3

Taking 1kg of enterococcus faecium powder, 1kg of bacillus licheniformis powder and 1kg of lactobacillus plantarum powder, and uniformly mixing to prepare the ternary composite bacteria preparation.

Example 4

1kg of enterococcus faecium powder, 1kg of bacillus licheniformis powder and 0.5kg of lactobacillus plantarum powder are taken and evenly mixed to prepare the ternary composite bacterial preparation.

Example 5

0.8kg of enterococcus faecium powder, 1kg of bacillus licheniformis powder and 0.8kg of lactobacillus plantarum powder are taken and evenly mixed to prepare the ternary composite bacterial preparation.

Example 6

The embodiment is a detection test for removing nitrite in water by using ternary complex bacteria, and the detection method refers to the following steps:

1. measuring the viable count of the enterococcus faecium powder by using a pouring method: placing the plate in an incubator, and culturing for 24 hours at 36 ℃; counting the culture medium: 10g/L of beef extract, 5g/L of yeast extract, 10g/L of peptone, 20g/L of glucose, 2g/L of dipotassium phosphate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate, 80g/L of tween, 2g/L of triammonium citrate and 15g/L of agar, wherein the pH value before sterilization is 6.2-6.6.

2. Measuring the viable count of the bacillus licheniformis powder by a pouring method: placing the plate in an incubator, and culturing for 24 hours at 36 ℃; counting the culture medium: the beef extract is 5g/L, the peptone is 10g/L, the glucose is 5g/L, the agar is 15g/L, and the pH value before sterilization is 7.0-7.5.

3. Measuring the viable count of the lactobacillus plantarum bacterial powder by using a pouring method: placing the plate in an incubator, and culturing for 24 hours at 36 ℃; counting the culture medium: 10g/L of beef extract, 5g/L of yeast extract, 10g/L of peptone, 20g/L of glucose, 2g/L of dipotassium phosphate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate, 80g/L of tween, 2g/L of triammonium citrate and 15g/L of agar, wherein the pH value is 6.2-6.6 before sterilization.

4. Preparing 1mg/L sodium nitrite 0.2g/L sucrose detection solution, taking 100mL in a triangular flask as CK, and repeating for three times.

5. Adding 99mL of sodium nitrite detection solution into 1g of enterococcus faecium powder, placing the enterococcus faecium powder in a glass bead shaking bottle, placing the shaking bottle in a shaking table, shaking for 30 minutes at 200r/min at 20 ℃, and fully and uniformly mixing.

6. Adding 99mL of sodium nitrite detection solution into 1g of bacillus licheniformis powder, placing the mixture into a glass bead shaking bottle, placing the glass bead shaking bottle into a shaking table, shaking the glass bead shaking bottle for 30 minutes at the temperature of 20 ℃ at the speed of 200r/min, and fully and uniformly mixing.

7. Adding 99mL of sodium nitrite detection solution into 1g of lactobacillus plantarum powder, placing the mixture in a glass bead shaking bottle, placing the shaking bottle in a shaking table, shaking for 30 minutes at the temperature of 20 ℃ at a speed of 200r/min, and fully and uniformly mixing.

8. Respectively and uniformly mixing enterococcus faecium, bacillus licheniformis and lactobacillus plantarum according to the proportion described in the embodiment 1-5, adding 99mL of sodium nitrite detection solution into 1g of composite bacterial powder respectively, putting the mixture into a glass bead shaking bottle, putting the glass bead shaking bottle into a shaking table, shaking the glass bead shaking bottle for 30 minutes at the temperature of 20 ℃ and at the speed of 200r/min, and fully and uniformly mixing.

9. The four bacterial liquids are respectively taken 10mL, 5mL, 2mL, 1mL, 0.5mL, 0.2mL and 0.1mL, the sodium nitrite detection liquid is added to the four bacterial liquids to be constant volume of 100mL, and the three steps are respectively carried out.

10. Placing the CK in the step 4 and the sample to be tested in the step 9 into an incubator for static culture at 32 ℃ for 24 hours.

11. And taking 25mL of the sample to be detected in a 50mL centrifuge tube the next day, centrifuging for 10 minutes at 5000r/min, and taking the supernatant.

12. And respectively taking 10mL of CK and the sample supernatant to be detected in a test tube, respectively adding five drops of a nitrite detection reagent I, shaking and uniformly mixing, standing for 5 minutes, then adding five drops of a nitrite detection reagent II, shaking and uniformly mixing, standing for 10 minutes for color development, and determining the nitrite content by using a water quality detector.

13. Calculating the nitrite removal rate of different concentrations of the four action methods to obtain the lowest concentration of the nitrite added by the four methods when the nitrite removal rate reaches more than 85%, and calculating the viable bacteria content of the bacteria powder with the lowest concentration of the four methods by combining the viable bacteria number measured in the steps 1, 2 and 3, wherein the specific results are shown in the following table 1:

TABLE 1

	Minimum use viable bacteria concentration (hundred million/mL)	Nitrite removal rate
			Enterococcus faecium	0.325	90.1%
Bacillus licheniformis	0.48	88.9%
			Lactobacillus plantarum	0.3	92.5%
Ternary composite bacterium	0.288	92.8%

Finally, the lowest addition amount of the enterococcus faecium is 0.325 hundred million/mL, the lowest addition amount of the bacillus licheniformis is 0.48 hundred million/mL, the lowest addition amount of the lactobacillus plantarum is 0.3 hundred million/mL, the lowest addition amount of the ternary complex bacteria is 0.288 hundred million/mL, the usage amount of the ternary complex bacteria is the lowest in the four methods, and the effect is optimal.

Claims

1. A ternary complex bacterium preparation is characterized in that: the ternary complex bacteria in the preparation are mixed by enterococcus faecium, Bacillus licheniformis and Lactobacillus plantarum according to the viable bacteria content ratio of 0.5-1: 0.5-1.

2. The use of the ternary complex bacterial formulation of claim 1, wherein: the ternary complex bacterium preparation is used as a nitrite degradation agent and is used for removing nitrite in a water body.

3. The use of the ternary complex bacteria preparation according to claim 2, characterized in that: the lowest use viable bacteria concentration of the ternary complex bacteria for removing the nitrite in the water body is 0.288 hundred million/mL.