CN112322539B

CN112322539B - Enterococcus faecium R-NTR-1 from ocean and screening method and application thereof

Info

Publication number: CN112322539B
Application number: CN202011273381.2A
Authority: CN
Inventors: 马桂珍; 李霁虹; 李文进; 许建合; 董霆; 周荣翔
Original assignee: Jiangsu Ocean University
Current assignee: Jiangsu Ocean University
Priority date: 2020-11-14
Filing date: 2020-11-14
Publication date: 2022-05-20
Anticipated expiration: 2040-11-14
Also published as: CN112322539A

Abstract

The invention relates to an Enterococcus faecium (Enterococcus faecalis) R-NTR-1 from sea, the preservation number of which is CCTCC NO: M2020654. The invention also discloses a screening method and application of the lactobacillus faecium (Enterococcus faecalis) R-NTR-1 strain. The bacterial strain is obtained by screening sea mud in Nantong city such as Dongxian county, has stronger effect of degrading nitrite and ammonia nitrogen, has the degradation rate of more than 90 percent for the nitrite with the content of 125mg/L and the degradation rate of more than 90 percent for the ammonia nitrogen with the content of 50mg/L within about 56 hours for Enterococcus faecium (Enterococcus faecium) R-NTR-1, and has better application prospect.

Description

Enterococcus faecium R-NTR-1 from ocean and screening method and application thereof

Technical Field

The invention relates to a new bacterial strain, in particular to enterococcus faecium R-NTR-1 from sea; the invention also relates to a screening method and application of the strain.

Background

Along with the prevalence of intensive culture modes with high density and high bait casting, organic matters, animal wastes and residual baits in water are accumulated continuously, so that the content of ammonia nitrogen gradually exceeds the bearing capacity of a water body, in addition, exogenous ammonia nitrogen entering the water body in the modes of sewage discharge, surface runoff, atmospheric sedimentation, pesticide residue and the like is increased day by day, and the content of nitrite is increased under the action of flora such as nitrobacteria and the like in the water body. The nitrite and ammonia nitrogen in the water body are too high in content and cannot be degraded, so that the problems of water body oxygen deficiency and the like can be caused, the physiological function of aquatic animals is disturbed and the growth of the aquatic animals is influenced due to the mild condition, and the animals cannot breathe and die in the severe condition. At present, the pollution of nitrite and ammonia nitrogen in the aquaculture water body is mainly controlled by three methods, namely a physical method, a chemical method and a biological method, the physical method has complex treatment steps and higher cost, and is only suitable for degrading low-concentration nitrite and ammonia nitrogen; the chemical method for degrading nitrite and ammonia nitrogen needs the help of related chemical substances, the required reaction time is long, and the problems of secondary pollution and the like are easy to occur; the method for repairing the aquaculture water body by using the microorganisms is increasingly emphasized due to the advantages of no toxic or side effect, short action time, no pollution and the like.

Lactic acid bacteria (lactic acid bacteria) are a generic term for a group of gram-positive bacilli or cocci that ferment lactose or glucose to produce large amounts of lactic acid, most anaerobic or facultative anaerobic, spore-free, acid tolerant. Lactic acid bacteria are widely distributed in the natural world and are widely varied, and there are about 40 or 300 genera of lactic acid bacteria found in the natural world, including Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium), Enterococcus (Enterococcus), Streptococcus (Streptococcus), Lactococcus (Lactobacillus), Pediococcus (Pediococcus), Leuconostoc (Leuconostoc), kiwium (atobium), bacillus (sporobacter), gemfibrobacter (Gemella), Saccharococcus (Saccharococcus) and cycloserine (brochotrix).

The application of lactic acid bacteria has a long history, and with the progress of scientific technology and production level and the attention of people on health, the lactic acid bacteria play an increasingly important role in the fields of livestock and poultry breeding, food production, medicine and the like. In livestock and poultry breeding, the abuse of antibiotics causes the problems of bacterial drug resistance, antibiotic residue, superinfection and the like. As a potential antibiotic substitute, the lactic acid bacteria have wide application prospect. In animal bodies, lactic acid bacteria can reduce the pH value of gastrointestinal tracts, maintain the balance of intestinal flora, enhance the immunity of organisms and improve the production performance of animals, so that the lactic acid bacteria are continuously put into use and made into commercial preparations as a novel feed additive. The influence of a selenium-enriched lactobacillus Chinese medicinal preparation (LSTCM) on the immunity and the oxidation resistance of the fattening pigs is explored by Gao Chang et al, and the results show that the selenium-enriched lactobacillus Chinese medicinal preparation can remarkably improve the immunity and the oxidation resistance of serum and tissues of the fattening pigs. Experiments on enteritis prevention and production performance influence of the longhongyan on piglets of 20 days old show that the compound lactobacillus preparation can effectively improve the humoral immunity and cellular immunity resistance of the piglets, has good promotion effect on piglet enteritis prevention and has good promotion effect on piglet growth and development.

The lactobacillus has the effects of regulating the balance of intestinal flora and the like, and can improve the flavor and the nutritive value of food when being applied to the food. Sera Jung et al explored the effect of lactic acid bacteria on the production of phenyllactic acid in kimchi, and the results showed that the addition of lactic acid bacteria to kimchi can increase the yield of phenyllactic acid during fermentation, and effectively inhibit the activity of infectious microbes in kimchi. The result of the process that hawthorn pectin oligogalacturonic acid (PGA) is added in the fermentation process of the yoghourt by xylonite and the like is optimized shows that the addition of PGA can inhibit the activity of lactic acid bacteria, thereby reducing the acidity of the yoghourt, effectively prolonging the shelf life of the yoghourt and improving the flavor of the yoghourt.

Probiotic lactic acid bacteria have antibacterial and anti-infectious effects, and Shepasan et al invent food, oral cleaning and pharmaceutical compositions containing Lactobacillus strains such as Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus paracasei, Lactobacillus rhamnosus, etc., for inhibiting oral pathogenic bacteria. Probiotic lactic acid bacteria also have cholesterol-lowering effect, and giardia and the like find some special enzyme systems with cholesterol-lowering effect in bifidobacteria. Guo Jing et al explored the influence of the lactic acid bacteria separated and screened from naturally fermented soybeans on serum cholesterol in rats, and the results showed that the lactic acid bacteria can help reduce the prevalence of cardiovascular diseases such as atherosclerosis, and have a certain protective effect on the liver. In addition, the probiotic lactic acid bacteria also have an anti-tumor effect. Bacteriocin nisin has been used as a substitute for bovine breast antibiotic in the 40's of the 20 th century, showing its potential for treating breast cancer.

The lactic acid bacteria can produce nitrite reductase and have strong degradation effect on nitrite, and simultaneously, the lactic acid bacteria can produce lactic acid and can inhibit the growth of nitrite reducing bacteria, thereby inhibiting the regeneration of nitrite. Zhang Qing Fang et al deduced that the degradation of nitrite by lactic acid bacteria was divided into 2 stages of NiR degradation and acid degradation. In the early stage of fermentation, when the pH of a culture solution is more than 4.5, degrading nitrite by lactic acid bacteria mainly through NiR degradation; in the later stage of fermentation, acid generated by lactic acid bacteria reduces the pH value of the culture solution, and the degradation of nitrite is mainly acid degradation after the pH value is less than 4.0. The lactic acid bacteria also have a certain degradation effect on ammonia nitrogen, the degradation may be that the lactic acid bacteria indirectly promote the nitrification by utilizing nitrite, and the degradation may also be that an enzyme system generated by the bacterial strains utilizes a nitrogen source. At present, researches on degrading nitrite and ammonia nitrogen by utilizing lactic acid bacteria have been reported, for example, Guo Shihua and other people separate two lactic acid bacteria with the capacity of resisting bile salt and degrading nitrite from pickles, and provide excellent fermentation strains for producing low-nitrite pickles; the sturgeon and the like research the degradation of the total nitrogen and the total phosphorus of the pond sediment by the potassium hydrogen persulfate composite salts and the beneficial viable bacteria (photosynthetic bacteria, bacillus and lactic acid bacteria), and the results show that the increase range of the total nitrogen and the total phosphorus is slowed down after the composite potassium hydrogen persulfate particles and the beneficial viable bacteria are comprehensively regulated, so that more nutrient substances are released into a water body, and the nutrient substances are supplied to phytoplankton for utilization and improve assimilation. At present, the degradation of nitrite and ammonia nitrogen by utilizing lactic acid bacteria is mostly concentrated in the field of food, and reports of the single use of lactic acid bacteria in aquaculture are rare.

Therefore, the research on obtaining the marine lactobacillus which can be used for water pollution treatment has important significance.

Disclosure of Invention

The invention aims to solve the technical problem of providing a novel enterococcus faecium R-NTR-1 from the sea aiming at the defects of the prior art.

The invention aims to solve another technical problem of providing a screening method of enterococcus faecium R-NTR-1.

The invention aims to solve another technical problem and provides application of enterococcus faecium R-NTR-1.

The technical problem to be solved by the present invention is achieved by the following technical means. The invention relates to an Enterococcus faecium (Enterococcus faecium) R-NTR-1 from sea, which is characterized in that: the preservation number is CCTCC NO: M2020654.

The Enterococcus faecium (Enterococcus faecium) R-NTR-1 strain related by the invention is preserved in China Center for Type Culture Collection (CCTCC) in 10 and 29 months in 2020, and the preservation number is as follows: CCTCC NO: M2020654, Phone: (027)87682319 fax (027)87883833E-mail: cctcc @ whu. edu. cn address: wuhan, Wuhan university; and E, postcode: 430072.

the invention also discloses a screening method of the Enterococcus faecium (Enterococcus faecium) R-NTR-1, which is characterized by comprising the following steps:

(1) separation and purification: taking a proper amount of collected sea mud and seawater samples, putting the sea mud and seawater samples into a 50ml centrifugal tube filled with 45ml of enrichment medium, adding 5ml of sterile liquid paraffin, and standing and culturing at 28 ℃ for 24 hours; after the culture medium is turbid, samples are taken for gradient dilution, 10 are respectively taken^-5、10^-6、10^-7 Coating 100 mu L of 3 diluted concentration samples on an MRS plate with the diameter of 9cm, culturing at the constant temperature of 28 ℃ for 24h, selecting bacterial colonies with calcium-dissolving rings, and carrying out three-region streak purification on the MRS plate to obtain a single bacterial colony to obtain a plurality of lactobacillus strains;

(2) screening of nitrite degrading lactic acid bacteria

Suspending different strains of lactobacillus in 5 × 10⁷Inoculating the CFU/mL into a 50mL centrifuge tube filled with 25mL nitrite degrading bacteria screening culture medium according to the inoculation amount of 5% V/V, and standing and culturing at 28 ℃ for 24 hours; centrifuging the fermentation liquid at 4 deg.C and 8000r/min for 10min, and measuring OD of supernatant of different strains by naphthyl ethylenediamine hydrochloride spectrophotometry₅₃₈(ii) a Calculating the nitrite concentration in the fermentation liquor of different strains according to a nitrite standard curve, and calculating the nitrite degradation rate of different strains by taking a blank culture medium without inoculated bacterial liquid as a reference; each strain was treated one, inoculated into 3 centrifuge tubes for 3 replicates; screening to obtain a bacterial strain R-NTR-1 with the highest nitrite degradation rate and 70.54 percent of degradation rate;

(3) determination of ammonia nitrogen degradation

Suspending different strains of lactobacillus in 5 × 10⁷Inoculating the CFU/mL into a 50mL centrifuge tube filled with 25mL nitrite degrading bacteria screening culture medium according to the inoculation amount of 5% V/V, and standing and culturing at 28 ℃ for 24 hours; centrifuging the fermentation liquid at 4 deg.C and 8000r/min for 10min, collectingDetermination of OD of supernatants of different strains by indophenol blue spectrophotometry₆₃₇Calculating the ammonia nitrogen concentration in the fermentation liquor of different strains according to a standard curve, and calculating the ammonia nitrogen degradation rate of different strains by taking an uninoculated screening culture medium as a reference; each strain was treated one, inoculated into 3 centrifuge tubes for 3 replicates; determining that the strain with the highest ammonia nitrogen degradation rate is R-NTR-1 and the degradation rate is 34.95%;

(4) the lactobacillus R-NTR-1 strain is identified to accord with the characteristics of Enterococcus (Enterococcus), the 16S rRNA gene sequence of the R-NTR-1 is compared with the sequence in NCBI, the similarity of the 16S rRNA of the R-NTR-1 strain and the 16S rRNA gene sequence of the Enterococcus faecium is 99.86 percent, and the R-NTR-1 strain is determined to be the Enterococcus faecium (Enterococcus faecium).

The invention also discloses an application of the enterococcus faecium R-NTR-1, wherein the enterococcus faecium R-NTR-1 is used as a nitrite and/or ammonia nitrogen degrading microbial inoculum.

Compared with the prior art, the invention has the following beneficial effects:

the invention screens and obtains Enterococcus faecium (Enterococcus faecium) R-NTR-1 with stronger effect of degrading nitrite and ammonia nitrogen from the sea mud of southeast county, the thallus density in the fermentation liquor is gradually increased along with the prolonging of the culture time, the nitrite and ammonia nitrogen concentration in the fermentation liquor is gradually reduced, the nitrite and ammonia nitrogen concentration is rapidly reduced along with the increasing of the thallus density, the nitrite and ammonia nitrogen concentration is reduced to 42.56mg/L and 17.21mg/L from the initial 120.35mg/L and 49.67mg/L in 24 hours, the degradation rate reaches 64.64 percent and 65.35 percent, the ammonia nitrogen concentration in the fermentation liquor is continuously reduced along with the continuous growth of the bacterial strain, the nitrite and ammonia nitrogen content in the fermentation liquor are respectively 8.34mg/L and 4.56(mg/L, the degradation rate respectively reaches 93.0 percent and 90.82 percent) in 56 hours, which indicates that the bacterial strain has stronger nitrite and ammonia nitrogen degradation capability, and the degradation effect is continuously stable. Has good application prospect.

Drawings

FIG. 1 is a graph showing the degradation rate of nitrite by 9 lactic acid bacteria;

FIG. 2 is a graph showing the degradation rate of 9 lactic acid bacteria to ammonia nitrogen;

FIG. 3 is a colony morphology feature map of strain R-NTR-1;

FIG. 4 is a diagram showing the morphology of the bacterial cells of the strain R-NTR-1 at 1000-fold magnification;

FIG. 5 is a phylogenetic tree diagram of the 16S rRNA sequence of R-NTR-1 strain.

The Enterococcus faecium (Enterococcus faecium) R-NTR-1 strain related by the invention is preserved in China Center for Type Culture Collection (CCTCC) in 10 and 29 months in 2020, and the preservation number is as follows: CCTCC NO: M2020654.

Detailed Description

The following further describes particular embodiments of the present invention to facilitate further understanding of the present invention by those skilled in the art, and does not constitute a limitation to the right thereof.

Example 1, Enterococcus faecium (Enterococcus faecium) R-NTR-1 Strain experiment:

in the following experiments, lactic acid bacteria with the function of efficiently degrading nitrite and ammonia nitrogen are separated and screened from different marine samples, and the strains are subjected to species identification, so that excellent microbial strains are provided for aquaculture water pollution treatment.

1 materials and methods

1.1 test materials

1.1.1 culture Medium

(1) Lactic acid bacteria enrichment medium: MRS liquid culture medium;

(2) lactic acid bacteria screening culture medium: MRS solid culture medium;

(3) nitrite-degrading bacteria screening culture medium: supplementing 125mg/L NaNO in MRS liquid culture medium₂。

(4) Screening culture medium for ammonia nitrogen degrading bacteria: supplementing 235.8mg/L (NH4) in MRS liquid medium₂SO₄。

1.1.2 reagents and their formulation

(1) Preparing a sulfanilic acid solution: dissolving 5.0g of sulfanilic acid in 50mL of concentrated hydrochloric acid, adding water to dilute to 300mL, cooling, transferring to a 500mL volumetric flask, adding water to the marked line, and shaking up. Stored at 4 ℃ in brown reagent bottles.

(2) Preparing a naphthyl ethylenediamine hydrochloride solution: 0.5g of naphthyl ethylenediamine hydrochloride is dissolved in 100mL of water, transferred to a 500mL volumetric flask, added with water to the marked line and shaken up. Stored at 4 ℃ in brown reagent bottles.

(3) Preparation of solution A: 0.3622g of sodium nitroferricyanide containing 2 crystal waters are weighed, dissolved in water and made into a sodium nitroferricyanide solution with a mass concentration of 1.25% by volume to 25mL, and the solution is stored at 4 ℃ in a brown reagent bottle. 5.00g of phenol is weighed, dissolved in 400mL of water, added with 2.0mL of prepared sodium nitroferricyanide solution, and the solution is prepared to 500mL with constant volume and stored in a brown reagent bottle at 4 ℃.

(4) Preparation of solution B: 2.50g of NaOH, 2.0g of trisodium citrate and 3.5mL of NaClO are weighed, dissolved in 400mL of water, the volume is adjusted to 500mL to obtain solution B, and the solution B is stored in a brown reagent bottle at 4 ℃.

1.2 methods

1.2.1 isolation and purification of lactic acid bacteria

Collecting sea mud and seawater samples from different sea areas of Nantong and Linyuanchong, taking appropriate amount of sea mud and seawater samples, placing into 50ml centrifuge tube filled with 45ml enrichment medium, adding 5ml sterile liquid paraffin, and standing and culturing at 28 deg.C for 24 hr. After the culture medium is turbid, the sample is subjected to gradient dilution, 10 are respectively taken^-5、10^-6、10^-7And (3) coating 100 mu L of the 3 diluted concentration samples on an MRS plate with the diameter of 9cm, culturing at the constant temperature of 28 ℃ for 24h, picking colonies with calcium-dissolving rings, and performing three-zone streaking purification on the MRS plate to obtain single colonies.

1.2.2 screening of nitrite degrading lactic acid bacteria

1.2.2.1 determination of nitrite degradation by different Lactobacillus strains

Suspending different strains of lactic acid bacteria (5X 10)⁷CFU/mL) was inoculated at 5% (V/V) into a 50mL centrifuge tube containing 25mL nitrite-degrading bacteria screening medium and incubated at 28 ℃ for 24 hours. Centrifuging the fermentation liquid at 4 deg.C and 8000r/min for 10min, and measuring OD of supernatant of different strains by naphthyl ethylenediamine hydrochloride spectrophotometry₅₃₈. Calculating the nitrite concentration in the fermentation liquor of different strains according to a nitrite standard curve, and calculating the nitrite degradation rate of different strains by taking a blank culture medium without inoculated bacterial liquid as a reference.One treatment per strain, and 3 replicates of each strain inoculated into 3 centrifuge tubes.

Nitrite degradation rate (%) ([ control nitrite concentration (mg/L) — treated nitrite concentration (mg/L) ]/control nitrite concentration (mg/L) × 100%.

1.2.2.2 nitrite Standard working Curve is drawn

Weighing 125mg NaNO₂Dissolving in 500mL of water, fixing the volume to 1000mL, preparing a nitrite stock solution with nitrite concentration of 125mg/L, diluting the nitrite stock solution to prepare nitrite standard solutions with nitrite concentrations of 25, 50, 75, 100, 125, 150, 175 and 200mg/L, respectively sucking 100 muL of sodium nitrite standard solutions with different concentrations into 10mL centrifuge tubes, adding 4.0mL of sulfanilic acid solution, mixing, standing in the dark for 5min, adding 2.0mL of naphthyl ethylenediamine hydrochloride solution, mixing, standing in the dark for 15min, taking equal amount of distilled water and developing solution as blank control, and determining OD of nitrite solutions with different concentrations₅₃₈. Concentration of nitrite as abscissa, OD₅₃₈And drawing a standard curve for the ordinate, and calculating a regression equation and a correlation coefficient.

1.2.3 screening of Ammonia Nitrogen degrading lactic acid bacteria

1.2.3.1 determination of Ammonia Nitrogen degradation by different Lactobacillus strains

Suspending different strains of lactic acid bacteria (5X 10)⁷CFU/mL) was inoculated at 5% (V/V) into a 50mL centrifuge tube containing 25mL nitrite-degrading bacteria screening medium and incubated at 28 ℃ for 24 hours. Centrifuging the fermentation liquid at 4 deg.C and 8000r/min for 10min, and measuring OD of supernatant of different strains by indophenol blue spectrophotometry₆₃₇And calculating the ammonia nitrogen concentration in the fermentation liquor of different strains according to the standard curve, and calculating the ammonia nitrogen degradation rate of different strains by taking the non-inoculated screening culture medium as a reference. One treatment per strain, and 3 replicates of each strain inoculated into 3 centrifuge tubes.

Ammonia nitrogen degradation rate/% (control ammonia nitrogen concentration-treated ammonia nitrogen concentration)/control ammonia nitrogen concentration) x 100%

1.2.3.1 drawing of standard ammonia nitrogen working curve

Weigh 235.8mg (NH4)₂SO₄Dissolving in 500mL of water, diluting to 1000mL to obtain an ammonia nitrogen standard solution with an ammonia nitrogen concentration of 50mg/L, diluting the ammonia nitrogen stock solution to obtain ammonia nitrogen standard solutions with ammonia nitrogen concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50mg/L, adding 5mL of each of the solution A and the solution B into 100 μ L of ammonia nitrogen standard solutions with different concentrations, mixing completely, developing in a water bath at 37 ℃ for 20min, cooling to room temperature after taking out, measuring OD of the ammonia nitrogen standard solutions with different concentrations by taking equivalent distilled water and developing solution as a blank control, and measuring the OD of the ammonia nitrogen standard solutions with different concentrations₆₃₇. Using the concentration of ammonia nitrogen as the abscissa, OD₆₃₇And drawing a standard curve for the ordinate, and calculating a regression equation and a correlation coefficient.

1.2.4 identification of the type of lactic acid bacteria having nitrite and Ammonia Nitrogen degrading action

1.2.4.1 morphological observations

Inoculating the screened lactobacillus strain capable of efficiently degrading nitrite and ammonia nitrogen on an MRS culture medium, culturing for 24 hours at the temperature of 28 ℃, and observing and recording the size, the shape and the color change of a bacterial colony on the culture medium; picking single colony, observing the color and shape of thallus under microscope.

1.2.4.2 physiological and biochemical tests

Selecting 1-3 bacterial colonies of the bacterial strains to a centrifugal tube containing 2ml of sterile water, uniformly oscillating the bacterial strains to prepare uniform bacterial suspension with 0.5 McLeod turbidity, sucking 50-100 mu L of the bacterial suspension to inoculate the bacterial suspension to different biochemical tubes, culturing the bacterial suspension for corresponding time under different conditions according to a specification, observing a color development result of the reaction tube, and comparing the color development result with a result interpretation table to determine a reaction result.

According to the results of morphological observation and physiological and biochemical reactions, the comparison is "Bergey bacteria identification Manual" (ninth edition)^[25]And the primary identification of the strain in the lactic acid bacteria classification identification and experiment method.

1.2.4.316S rRNA sequence analysis

Samples of the lactic acid bacteria strains were sent to Shanghai Bioengineering services, Inc. to determine the 16S rRNA sequences of the strains. After the obtained gene sequences are completely spliced, BLAST comparison is carried out on the gene sequences and known sequences in a GenBank database, and MEGA7.0 software is adopted to construct a 16S rRNA gene sequence phylogenetic tree.

2 results and analysis

2.1 isolation of lactic acid bacteria

Using MRS culture medium, adopting plate marking method and calcium dissolving ring method, separating 30 sea water and sea mud samples to obtain 9 bacterial strains, carrying out gram staining and catalase reaction test on different bacterial strains, wherein the 9 bacterial strains have obvious calcium dissolving rings, the gram staining is positive, the catalase reaction is negative, the lactobacillus characteristics are met, and the 9 bacterial strains are preliminarily determined to be lactobacillus. Wherein 3 strains are isolated from sea mud of southeast City such as Dongxian county, and are numbered as R-NTR-1, R-2, and R-3; 1 strain isolated from seawater, numbered Q-1, from Qidong county, Nantong City, and 1 strain isolated from sea mud, numbered Q-2, from Qidong county; 1 strain is separated from seawater of the Gaogong island in the continuous cloud harbor, and the number is L-1, and 1 strain is separated from sea mud of the Gaogong island, and the number is L-2; 2 strains were isolated from seawater in Nelandia of Nelumbo Nucifera, numbered L-3 and L-4, respectively.

2.2 determination of nitrite degradation by different Lactobacillus strains

2.2.1 nitrite Standard working Curve

The prepared standard solution containing different nitrite concentrations is added into OD₅₃₈Then, the color is compared, the light absorption value is measured, the nitrite concentration is taken as the abscissa, and the OD is taken₅₃₈And drawing a nitrite standard working curve as a vertical coordinate. OD increases with nitrite concentration in the set concentration range₅₃₈Gradually increased to form positive correlation, the working curve equation is that y is 0.0092x +0.0124, R²The linear dependence is better at 0.990, and the nitrite concentration can be calculated.

2.2.2 determination of the nitrite degradation Effect of different Lactobacillus strains

Transferring the activated 9 lactic acid bacteria to a screening culture medium containing 125mg/L of nitrite, culturing for 24 hours at 28 ℃, determining the content of nitrite in the culture medium, and calculating the degradation amount and the degradation rate of the nitrite, wherein the results show that different lactic acid bacteria strains have certain effect of degrading the nitrite, the degradation rate of the nitrite of the strain R-NTR-1 is 70.54 percent at the highest, and the strains L-3 and L-4 are 53.42 percent and 50.58 percent respectively. The results are shown in Table 1 and FIG. 1.

TABLE 19 determination of nitrite degradation by lactic acid bacteria

Bacterial strains	OD₅₃₈	Nitrite content/mg	Nitrite degradation/mg
				R-NTR-1	0.338±0.0010j	35.391±0.1086j	89.609±0.1086a
R-2	0.659±0.0023g	70.319±0.2510g	54.681±0.2510d
				R-3	0.812±0.0006c	86.949±0.0627c	38.051±0.0627h
Q-1	0.973±0.0000b	104.413±0.0000b	20.587±0.0000i
				Q-2	0.753±0.0006d	80.536±0.0627d	44.464±0.0627g
L-1	0.689±0.0130e	73.543±1.4130e	51.457±1.4130f
				L-2	0.679±0.0006f	72.493±0.0627f	52.507±0.0627e
L-3	0.528±0.0006i	56.007±0.0627i	68.993±0.0627b
				L-4	0.560±0.0011h	59.486±0.1255h	65.514±0.1255c
CK	1.119±0.0006a	120.319±0.0627a	4.681±0.0627j

Note: in the table, letters represent the significant difference of data in the same column, different lower case letters in the same column represent significant difference, the same lower case letters in the same column represent insignificant difference, and p is less than or equal to 0.05.

2.3 determination of Ammonia Nitrogen degradation by different Lactobacillus strains

2.3.1 standard working curve for Ammonia Nitrogen

The prepared standard solution containing different ammonia nitrogen concentrations is placed in OD₆₃₇Carrying out color comparison, measuring light absorption value, taking ammonia nitrogen concentration as abscissa and OD₆₃₇And (5) making an ammonia nitrogen quasi-working curve as a vertical coordinate. In the set concentration range, OD is increased along with the increase of the concentration of ammonia nitrogen₆₃₇Gradually increased to form positive correlation, the working curve equation is that y is 0.0126x +0.0458, R²The linear correlation is better when the concentration is equal to 0.967, and the method can be used for calculating the ammonia nitrogen concentration.

2.3.2 determination of Ammonia Nitrogen degradation by different Lactobacillus strains

Transferring the activated 9 strains of lactic acid bacteria to a screening culture medium containing 50mg/L ammonia nitrogen, culturing at 28 ℃ for 24h, and measuring OD of fermentation liquor of different strains₆₃₇And calculating the ammonia nitrogen content and the degradation rate in the fermentation liquor. The results show that different strains of the separated lactobacillus have certain degradation effect in a culture medium with the ammonia nitrogen content of 50mg/L, wherein the degradation rate of the strain R-NTR-1 is the highest and is 34.95%, and the strains L-2 and R-3 are respectively 29.27% and 29.22%. The results are shown in Table 2 and FIG. 2.

Table 29 determination results of ammonia nitrogen degrading effect of lactobacillus strains

The result shows that the strain R-NTR-1 in the separated strain has stronger degradation effect on ammonia nitrogen and nitrite, and the strain is selected for identifying the strain.

2.4 identification of the types of lactic acid bacteria strains that efficiently degrade nitrite and ammonia nitrogen

2.4.1 morphological observations

The colony morphology of the strain R-NTR-1 on the MRS culture medium is round, convex, milky white and opaque; the cells are in the form of short rods with a length of 1.18-1.55 μm and a width of 0.74-0.82 μm, as shown in FIG. 3, and the cell morphology is shown in FIG. 4 when the magnification of the strain R-NTR-1 is 1000 times.

2.4.2 physiological and Biochemical Properties

The results of physiological and biochemical identification of the R-NTR-1 strain (Table 3) show that: after the indicator is dripped into the strain R-NTR-1, the color of the biochemical identification tube of maltose and glucose is changed into yellow, and the biochemical identification tube is positive, so that the strain has the capability of decomposing glucose and maltose, and cannot utilize arabinose and rhamnose as carbon sources; the urease test is positive, which shows that R-NTR-1 has the capability of decomposing urea to generate ammonia; arginine hydrolase, hydrogen sulfide test, indole test, nitrate reduction test were negative. Semi-solid agar test is positive, indicating that R-NTR-1 has motility.

TABLE 3 results of physiological and biochemical tests of lactic acid bacteria R-NTR-1

According to the morphological characteristics and the results of physiological and biochemical tests, refer to Bergey's Manual of bacteria identification (ninth edition)^[25]And related documents such as "lactic acid bacteria Classification and Experimental methods", it was preliminarily considered that the lactic acid bacteria R-NTR-1 strain conforms to the characteristics of Enterococcus (Enterococcus).

2.4.3 sequence analysis of 16S rRNA of R-NTR-1 Strain

The 16S rRNA gene sequence of R-NTR-1 was compared with the sequence in NCBI, and the similarity of the 16S rRNA of the R-NTR-1 strain and the 16S rRNA gene sequence of Enterococcus faecium (accession number NR _114742.1) was 99.86%.

The 16S rRNA sequences of different strains with higher homology similarity in the same genus are selected, a phylogenetic tree is constructed by adopting Mega7.0 software, and the result shows that R-NTR-1 and 2 strains of enterococcus faecium are positioned in the same branch and have the closest genetic relationship. The results are shown in FIG. 5. And further determining the R-NTR-1 strain as Enterococcus faecium (Enterococcus faecium) by combining morphological observation and physiological and biochemical reaction measurement results.

3 conclusion and discussion

The experiment screens and obtains a Enterococcus faecium (Enterococcus faecalis) R-NTR-1 with strong effect of degrading nitrite and ammonia nitrogen from the sea mud of southeast city, such as Dongxian county, wherein the degradation rate of the Enterococcus faecium R-NTR-1 to the nitrite with the content of 125mg/L is 70.54 percent within 24 hours, and the degradation rate of the Enterococcus faecium R-NTR-1 to the ammonia nitrogen with the content of 50mg/L is 34.95 percent.

Example 2, determination of the degradation of nitrite and Ammonia Nitrogen by Enterococcus faecium (Enterococcus faecalis) R-NTR-1 Strain:

inoculating R-NTR-1 strain to PDA slant, culturing at 28 deg.C for 18h, washing lawn on the slant with PD medium, adjusting the suspension concentration to 5 × 107CFU/mL as seed solution, inoculating 5% (V/V) of inoculum size to 60mL (NH) containing 250mg/L NaNO2 or 235.8mg/L₄)₂SO₄Sampling every 4h at 28 ℃ in a 250mL triangular flask of PD culture medium (with the ammonia nitrogen content of 50mg/L), determining the thallus density and the ammonia nitrogen concentration, calculating the ammonia nitrogen concentration and the nitrite concentration in fermentation liquor at different times and the degradation rate thereof according to a standard curve, and sampling 3 bottles each time for 3 times.

The results show that the cell density in the fermentation broth gradually increases and the nitrite and ammonia nitrogen concentrations in the fermentation broth gradually decrease with the increase of the culture time, the nitrite and ammonia nitrogen concentrations decrease rapidly with the increase of the cell density, the nitrite and ammonia nitrogen concentrations decrease from the initial 120.35mg/L and 49.67mg/L to 42.56mg/L and 17.21mg/L at 24h, the degradation rates reach 64.64% and 65.35%, the ammonia nitrogen concentrations in the fermentation broth continuously decrease with the continuous growth of the strain, and the nitrite and ammonia nitrogen contents in the fermentation broth are respectively 8.34mg/L and 4.56(mg/L, the degradation rates reach 93.0% and 90.82% respectively at 56h, which indicates that the strain has stronger nitrite and ammonia nitrogen degradation capability, and the degradation effect is continuously stable. the results are shown in Table 4:

TABLE 4 results of determination of degradation of nitrite and ammonia nitrogen by RNTR-1 strains at different times

Claims

1. The application of the Enterococcus faecium R-NTR-1 from the sea is characterized in that the Enterococcus faecium R-NTR-1 is used as a nitrite and/or ammonia nitrogen degrading microbial inoculum, and the preservation number of the Enterococcus faecium (Enterococcus faecium) R-NTR-1 is CCTCC NO: M2020654.