CN108660097B

CN108660097B - Screening and application of fish-source enterococcus faecium R8

Info

Publication number: CN108660097B
Application number: CN201810499241.3A
Authority: CN
Inventors: 孙敬锋; 毛晴
Original assignee: Tianjin Agricultural University
Current assignee: Tianjin Agricultural University
Priority date: 2018-05-23
Filing date: 2018-05-23
Publication date: 2021-11-09
Anticipated expiration: 2038-05-23
Also published as: CN108660097A

Abstract

The invention provides a safe and probiotic Enterococcus faecium R8 strain which is screened and separated from crucian intestinal tracts and is preserved in China general microbiological culture collection center with the preservation number of CGMCC NO.15229 and the preservation date of 2018, 1 month and 17 days. The strain has the advantages of strong heat resistance, wide acid and alkali resistance, high stress resistance and more remarkable probiotics, has a more obvious inhibiting effect on common aquatic pathogenic bacteria such as aeromonas veronii, staphylococcus haemolyticus, vibrio parahaemolyticus and vibrio vulnificus, and can be widely applied to aquaculture as a feed additive.

Description

Screening and application of fish-source enterococcus faecium R8

Technical Field

The invention belongs to the technical field of microbial additives for aquatic products, and relates to separation and identification of Enterococcus faecium (Enterococcus faecium) strains, safety tests and identification and application of stress resistance of Enterococcus faecium strains, which have obvious inhibition effects on aquatic product common pathogenic bacteria, namely aeromonas veronii, staphylococcus haemolyticus, vibrio parahaemolyticus and vibrio vulnificus.

Background

At present, with the gradual acceptance of people to microecological products and the increasingly stricter use limitation of governments to antibiotics in recent years, people look more to green, environment-friendly and efficient microecological products when seeking substitutes for antibiotic products. Lactic acid bacteria are important probiotics and important dominant flora in animal intestinal tracts, and are increasingly becoming the popular research field of feed additives due to the characteristics of no toxicity, no residue, no side effect and the like. However, since the lactic acid bacteria are anaerobic bacteria and have low tolerance to high temperature, it provides obstacles for the application and development of lactic acid bacteria products. The enterococcus faecium has the unique stress resistance of lactic acid bacteria and has unique advantages in production and processing. Therefore, enterococcus faecium is favored in the application technology of microecological preparation, in particular biological feed additive, in the field of animal husbandry.

In 2008, the ministry of agriculture in China issued announcements clearly stipulate the types of microbial additives for feeding in the feed additive variety catalog, and the enterococcus faecium is listed as a common lactobacillus strain for the microecological preparation. A large number of facts prove that the enterococcus faecium preparation has good effects on the aspects of increasing weight gain of young livestock and poultry, improving immunity of animals, regulating intestinal microecological balance, improving feed return, reducing diarrhea rate, reducing death rate and the like.

Although enterococcus faecium is widely used, relatively few reports have been made on the aquaculture industry to screen aquatic animals for probiotics. Many probiotics used in aquaculture are not screened from the aquatic animal or living environment, but are derived from some terrestrial animals, which results in the probiotic bacterial strains not being colonized or being unstable. Only indigenous microorganisms screened from the host can better adapt to the environment and inhibit pathogenic bacteria more efficiently, while probiotic strains which are not isolated from the fish digestive tract often cannot stably colonize the fish digestive tract.

Disclosure of Invention

The invention aims to provide a safe probiotic which is screened and separated from the intestinal tract of crucian and has probiotic characteristics, aiming at the defects of the prior art. The strain has the advantages of strong heat resistance, wide acid and alkali resistance, high stress resistance and more remarkable probiotics, and has a more obvious inhibiting effect on common aquatic pathogenic bacteria such as aeromonas veronii, staphylococcus haemolyticus, vibrio parahaemolyticus and vibrio vulnificus. The 16S rDNA method is used for identifying the strain as Enterococcus faecium (Enterococcus faecium), and related tests are carried out on the safety, stress resistance and application of the strain.

The Enterococcus faecium R8 and the bacterial strain Enterococcus faecalis are preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.15229 and the preservation date of 2018, 1 month and 17 days.

Enterococcus faecium R8 is obtained by separating and screening fish intestinal tracts, and crucian is adopted in the invention.

The enterococcus faecium R8 can be applied to aquaculture, effectively colonizes in intestinal tracts to play a probiotic role, remarkably improves the survival rate and the weight gain rate of aquatic organisms, and has obvious inhibiting effect on common aquatic pathogenic bacteria, namely aeromonas veronii, staphylococcus haemolyticus, vibrio parahaemolyticus and vibrio vulnificus.

Enterococcus faecium of the present inventionR8 can be used in the form of feed additive, preferably in an amount of 2 w%, and has a bacterial liquid concentration of 1.0 × 10⁸cfu/mL。

The invention has the following advantages:

(1) the strain is derived from intestinal contents of common fishes (crucian), is obtained by separation and screening and is named as enterococcus faecium R8. Has obvious inhibition effect on common pathogenic bacteria (aeromonas veronii, staphylococcus haemolyticus, vibrio parahaemolyticus and vibrio vulnificus) of aquatic products, and provides a favorable basis for replacing antibiotic additives.

(2) The strain can tolerate gastric acid and the environment of high bile salt of small intestine, and can colonize in intestinal tract to exert probiotic effect.

(3) The strain has strong high-temperature tolerance, and can tolerate the survival rate of 60 ℃, 30min, 70 ℃ and 5min as high as 90%. The strain lays a foundation for being used as a feed additive and being used for actual production.

(4) Animal experiments prove that the strain has no toxic or harmful effect on original hosts and can be used as a feed additive.

The strain is prepared into a probiotic preparation, and when the probiotic preparation is applied to a feed additive, the influence of antibiotics in aquatic feeds on water environment pollution and human health can be greatly reduced. Reducing the generation of drug resistance of bacteria and realizing the sustainable development of aquaculture industry.

Drawings

FIG. 1: and separating the growth state of the screened enterococcus faecium strain R8 on the MRS solid culture medium.

FIG. 2: gram-positive reaction of enterococcus faecium R8 (. times.1000)

FIG. 3: and (3) PCR amplification detection results of the 16S rDNA gene.

Lanes in the figure: m: marker; 1: strain R8

FIG. 4 construction of phylogenetic trees based on 16S rDNA sequences

FIG. 5: results of bile salt resistance test

FIG. 6: results of acid resistance test

FIG. 7: results of high temperature resistance test

FIG. 8: enterococcus faecium R8 growth curve

Detailed Description

The present invention is further explained below. For convenience of description, the apparatus of the present invention omits necessary or conventional operation steps or conditions, and those skilled in the art can make any adjustments according to the needs of the reaction.

Example 1: isolation and identification of probiotic strains

First, isolation of the Strain

The sample is taken from healthy crucian purchased in the market, after the body surface is disinfected by 75% medical alcohol, the crucian is dissected aseptically and the intestinal tract is taken out, precooled sterilized normal saline is added according to the proportion of 1: 10, the sample is fully homogenized by a glass homogenizer, the homogenate is diluted according to 10 times series and then is coated by an MRS culture medium plate, and the sample is cultured for 24-36h at the constant temperature of 37 ℃ and then is observed. Selecting bacterial colonies with clear edges, dispersion and the size of about 1mm for pure culture. And (3) carrying out primary judgment on the pure culture through morphological observation and gram staining, and carrying out subculture on the gram-positive cocci with single, paired or chain bacteria. The growth characteristics of the strain of the invention on MRS agar culture are shown in figure 1, and the gram staining characteristics are shown in figure 2.

The bacterial strain belongs to gram-positive cocci, the colony morphology on MRS solid agar medium is circular, the bacterial colony is arranged in pairs or short chains, the bacterial colony is milk white, the surface is smooth, the edge is neat, and the diameter of the thallus is about 0.5-1.0 mm. Under the microscope oil microscope observation, the strain cell is spherical, singly generates or pairs, and has less opposite generation and chain. No spore and no flagellum.

Second, preliminary identification of the isolated plants

The isolated strain was named as R8 and identified by a physiological and biochemical identification tube (Hangzhou Bing and microbial agents Co., Ltd.), comprising: glucose, xylose, sucrose, lactose, sorbitol, arabinose, raffinose, mannitol, arginine double hydrolase and bile esculin hydrolysis test. The bile esculin hydrolysis test of the strain is positive, arabinose, fermented sorbitol and the like can be hydrolyzed, the identification result (shown in table 1) is compared with a common bacteria system identification manual (Dongxuzhu and the like, 2001), the description of the strain and the enterococcus faecium species is basically consistent, and the isolate R8 is preliminarily determined to be the enterococcus faecium.

TABLE 1 Biochemical identification of enterococcus faecium R8

Third, confirmation of R8 isolates

On the basis of the identification, 16S rDNA gene sequence detection is carried out on the strain R8, and the strain is further confirmed by constructing phylogenetic tree analysis.

Firstly, extracting the genome of the isolate: the total DNA of the isolate was extracted by boiling (Chengli, 2015).

(II) primer design for amplifying 16S rDNA gene: the amplification primer adopts a bacterial universal primer,

a forward primer F: 5'-AGAGTTTGATCATGGCTCAG-3' the flow of the air in the air conditioner,

reverse primer R: 5'-GGTTACCTTGTTACGACTT-3' are provided.

(III) PCR amplification of 16S rDNA Gene

The 16S rDNA genome of the strain R8 genome was amplified using the above primers, and the reaction system is shown in Table 2. The PCR reaction program is: 10min at 95 ℃, 5min at 94 ℃, 40s at 55 ℃, 40s at 72 ℃, 30 cycles, 10min at 72 ℃ and finishing the reaction at 4 ℃. Taking 4L of PCR product, carrying out electrophoresis detection on 1.5% agarose gel, observing the result by a gel electrophoresis image analysis system, and handing the product to Shanghai bioengineering technology company for purification and clone sequencing of the PCR product.

TABLE 2 PCR reaction System for R816S rDNA

(IV) construction of phylogenetic trees based on 16S rDNA sequences

A band with 1523bp size is detected by a gel electrophoresis test (see figure 3); blast alignment shows that the sequence homology with Enterococcus faecalis model strain DSM20477(AJ276355.1) is 99.87%, the sequencing result is submitted to GenBank (https:// www.ncbi.nlm.nih.gov), the accession number (MF928076) is obtained, 18 bacteria of the same genus with higher 16S rDNA homology with strain R8 are selected through Blast, MEGA 5.2 software is used for carrying out homology alignment on the 19 sequences, a phylogenetic tree is constructed by a Neighbor-join method, analysis shows that the strain naturally gathers one branch with Enterococcus faecalis (see figure 4), and R8a is further judged to be Enterococcus faecium.

Example 2: bacteriostatic test of enterococcus faecium strain R8

The indicator bacteria are aeromonas veronii, staphylococcus haemolyticus, vibrio parahaemolyticus and vibrio vulnificus (provided by aquatic animal diseases and immunology laboratories of the academy of Tianjin agriculture). The method for primary screening by a dibbling method (Smith P, 1993) and secondary screening by an Oxford cup method (Liudongmei, 2006) is adopted for carrying out the bacteriostasis test, and the specific steps are as follows: adjusting the concentration of the indicator fungus solution to 10⁶cfu/mL, 0.1mL is aspirated and spread on LB agar medium, then a single strain to be tested is picked by an inoculating loop and spotted on a plate. Culturing at 37 deg.C for 24h, and observing the zone of inhibition.

And re-screening by adopting an Oxford cup method. Adjusting the concentration of the activated strain to be detected to 10 h⁷cfu/mL, the concentration of the indicator bacterium liquid is adjusted to 10⁶cfu/mL, 0.1mL pipetted onto LB agar medium. And (3) placing the sterilized Oxford cup on a flat plate coated with the indicator bacteria by using a sterile forceps, and slightly pressing to ensure that the bottom of the Oxford cup is tightly attached to the culture medium. 200 mu L of primarily screened probiotic bacteria liquid is added into each Oxford cup, then the plate is put into a constant temperature box at 37 ℃ for culturing for 24h, and the size of the inhibition zone is measured. The results are shown in Table 3.

TABLE 3 enterococcus faecium R8 in vitro bacteriostasis test

As can be seen from table 3, the strain is effective against common aquatic pathogenic bacteria: the aeromonas veronii, the staphylococcus haemolyticus, the vibrio parahaemolyticus and the vibrio vulnificus have obvious inhibiting effects.

Example 3: evaluation of safety of enterococcus faecium Strain

First, hemolysis test

The R8 bacterial liquid cultured for 24h is inoculated on a rabbit blood plate, the rabbit blood plate is cultured for 24h at 37 ℃, and the generation of hemolysin is judged according to the formation of a transparent ring around a bacterial colony. As a result, it was observed that no clear circles were formed and there was no hemolytic reaction.

Second, animal test

Inoculating R8a strain on LB agar medium, culturing at 37 deg.C for 24 hr, eluting with 0.75% sterile normal saline to obtain bacterial suspension, diluting bacterial suspension by 10 times, injecting bacterial suspension of different dilution into abdominal cavity of crucian by sterile injector to obtain bacterial suspension with concentration of 1.0 × 10⁸cfu/mL, 12 injections per group were used as experimental groups. The same volume of sterile saline was injected as a control group. And (4) putting the crucian carp after injection into the aquarium to continue breeding, recording death situation every day, and continuously observing for 7 d. The test result shows that the crucian carp in the experimental group does not show abnormality compared with the control group. The R8 strain has no toxic effect on crucian.

Example 4: stress resistance and growth characteristics of enterococcus faecium R8

Stress resistance test

(I) acid resistance test

The activated enterococcus faecium R8 suspension was inoculated into pH 2.0, 3.0, and 4.0 at an inoculum size of 5%, respectively, incubated at a constant temperature of 37 deg.C, and the solution with a pH of 7.0 was set as a control. After 2h, 4h and 8h, the OD600 values of the bacterial solutions were measured. Each experiment was repeated three times and averaged.

As shown in fig. 5, the growth rates of the treated groups at pH 2.0 and pH 3.0 were significantly reduced in the acidic environment of strain R8, and the growth rate of the treated group at pH 4.0 was lower than that of the control group at pH 7.0, but was not significantly changed. As the treatment time was prolonged, viable bacteria were detected in each experimental group and showed a tendency of growth, wherein the treatment group at pH 4 grew better than the treatment groups at pH 2 and pH 3. This indicates that the R8 strain has better tolerance ability to low pH value, is beneficial to resisting gastric acid environment and colonizes intestinal tract.

(II) bile salt tolerance test

The activated R8 bacterial liquid is respectively inoculated into solutions with the cholate concentration of 0.2%, 0.4%, 0.6%, 0.8%, 1.0% and 1.2% according to the inoculum concentration of 5%, and the solutions are cultured at the constant temperature of 37 ℃, and the cholate concentration of 0% is set as a control group. After 24h, the OD of the bacterial liquid was measured at a wavelength of 600nm, and the measurement was repeated three times for each experiment and averaged.

The results are shown in fig. 6, the growth state of the strain R8 was good under the conditions of bile salt concentration of 0.20% and 0.40%, and there was no significant difference compared with the control group; in the environment with bile salt concentration higher than 0.60%, the growth of the strain R8 is inhibited. Meanwhile, when the bacterial strain is subjected to a bile salt concentration limit test, the bacterial strain is found to have the existence of live bacteria in 3 parallel experiments under the conditions of 1.00 percent and 1.20 percent of bile salt concentration. This indicates that the strain R8 has a prominent advantage in bile salt tolerance, and is beneficial for colonizing the intestinal tract and exerting a probiotic effect.

(III) high temperature resistance test

The R8 bacterial liquid after shaking culture at 35 ℃ for 4h is inoculated into LB liquid culture medium according to the inoculum size of 5%. Culturing at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, and 90 deg.C for 2min, 5min, 10min, 20min, and 30min respectively, and measuring OD600 value of the bacteria liquid. Meanwhile, the culture was carried out in a 37 ℃ water bath as a control group. Each experiment was repeated three times and the average was taken.

As shown in FIG. 7, the growth of enterococcus faecium strain began to decrease after 5min treatment at 50 ℃ as compared with the control group (37 ℃), but showed a slow increase as compared with the control group before treatment; the viable count is reduced at 60 deg.C, 70 deg.C, 80 deg.C, and 90 deg.C. After 2min of treatment at 60 ℃, the strain shows a growth trend, and then the number of viable bacteria is reduced but is not obvious; after heat treatment for 30min at 70 ℃ and 80 ℃, the loss of the number of bacteria is large, and viable bacteria still exist; the viable count of the experimental group at 90 ℃ is linearly reduced along with the extension of the heat treatment time, and the viable count is hardly detected within 30 min. The result shows that the enterococcus faecium R8a shows certain tolerance capability to heat treatment below 80 ℃, and lays a foundation for the strain to be used as a feed additive and applied to actual production.

Second, measurement of growth Curve

Inoculating the bacterial liquid of the strain R8 into a fresh LB liquid culture medium according to the inoculation amount of 5%, culturing in a shaking table at constant temperature under the conditions that the pH is 7.0, the temperature is 37 ℃ and 30 per thousand, and measuring the OD value of the bacterial liquid every 2h at the wavelength of 600nm for 28 h.

The growth curve (as shown in figure 8) of the strain R8 has four stages, wherein 0-4 h is a latency period, a logarithmic growth period is started after 4h and lasts for 16h, and the maximum thallus number is 1.81 multiplied by 10 when 16h is reached⁹cfu/mL, followed by stationary phase, and 24h later, decay phase. The enterococcus faecium has the great advantages of high growth speed and short proliferation period as a probiotic preparation, and lays a foundation for the development and utilization of the enterococcus faecium as a probiotic of aquatic animals.

Example 5: application of enterococcus faecium R8 in penaeus vannamei culture

Enterococcus faecium R8 was applied in the standard crude stage of Penaeus vannamei, and 2 greenhouse-covered Penaeus vannamei crude soil ponds were selected in Tianjin, each pond having a size of 0.6 mu. Feeding prawn slices and starter in one pond, and mixing with bacterial solution R8 (10) at a ratio of 2% per day⁸CFU/ml); application was continued for 21 days. The other pond is fed with shrimp flakes and hatch feed only. The result shows that the survival rate of the standard coarse shrimp seeds mixed with the enterococcus faecium is improved by 16 percent and the weight gain rate is improved by 13 percent compared with the standard coarse shrimp seeds only fed with the prawn slices and the opening material.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. An Enterococcus faecium (Enterococcus faecium) R8 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.15229 and the preservation date of 2018, 1 month and 17 days.

2. Use of Enterococcus faecium (Enterococcus faecium) R8 according to claim 1 in the preparation of an aquaculture preparation.