CN110408574B - Lactobacillus composite microbial agent and application thereof in resisting giant salamander iridovirus - Google Patents
Lactobacillus composite microbial agent and application thereof in resisting giant salamander iridovirus Download PDFInfo
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- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention relates to the technical field of aquatic product antiviral microecologics, and particularly relates to a lactobacillus composite microbial agent and application thereof in resisting carp herpesvirus II. The composite microbial inoculum of the invention has a preservation number of CCTCC NO: the lactobacillus rhamnosus, the lactobacillus plantarum and the lactobacillus paracasei of the M2019655 are mixed or fermented. The supernatant fermented by the composite microbial inoculum can reduce the infection activity of Giant Salamander Iridovirus (GSIV) on host cells, inhibit the infection of GSIV virus invasion on cells, has no toxic or side effect on the cells, and has better inhibition effect than that of lactobacillus rhamnosus YFI-6 used alone. The feed added with the composite microbial inoculum thalli can effectively reduce the death rate of giant salamander infected with GSIV, and can be used for preventing and treating giant salamander viral hemorrhagic disease.
Description
Technical Field
The invention relates to the technical field of aquatic product antiviral microecologics, and particularly relates to a lactobacillus composite microbial agent and application thereof in resisting giant salamander iridovirus.
Background
The traditional Chinese giant salamander (Andrias davidianus), commonly called giant salamander, belongs to the Amphibia (Amphiia), the Ceramiales (Caudata), the Cryptobranchidae (Cryptobranchidae) and the giant salamander (Andrias), and is the tailed amphibian with the largest individual and the longest life span. Chinese giant salamander is a rare and rare special product in China, belongs to the national secondary protection animal, and is recorded in appendix I of International trade convention on endangered wild animal and plant species. In recent years, as the giant salamander culture scale is gradually enlarged, the cognition of culture personnel on giant salamander diseases is shallow, so that various diseases of the giant salamander are gradually serious. The method not only causes the loss of precious giant salamander resources and culture economy, but also restricts the healthy development of the giant salamander intensive culture industry. The viral disease is the most serious disease to the giant salamander breeding, the pathogen transmission speed is high, the effective treatment cannot be obtained, and the death rate is over 90 percent. Gunn et al (2010) first reported that frog virus in artificially cultured giant salamanders can cause high morbidity and mortality. Subsequently, researchers isolate the giant salamander viral disease pathogen in sequence and confirm that the giant salamander viral disease pathogen is a member of Ranavirus (Ranavirus) in Iridoviridae (Iridoviridae). The main symptoms are: head swelling, bleeding in the back and abdomen, blood spots; swelling of limbs and ulcer. The stomach was found to be pale without food, with bleeding in the kidney and liver.
The prevention and treatment of diseases in aquaculture has long been an important problem that plagues the development of aquaculture. Currently, there are two main methods for controlling diseases of aquaculture animals, drug control and vaccine immunization. However, a series of problems such as side effects, drug residues, drug resistance, water pollution and the like caused by drug control are gradually paid attention by all social circles, and in addition, the treatment effect of the drug control on viral diseases is not very obvious; the development of the vaccine for fishing is influenced by the lack of sensitive cell lines, virus variation and inconvenient immune mode. Therefore, there is a continuing need for effective methods of controlling viral diseases, and probiotics are receiving widespread attention for their non-toxic, non-drug resistant, residue-free, antibacterial, antiviral, growth-promoting, green and safe advantages.
In recent years, a plurality of researchers think that lactic acid bacteria in the aquaculture process are expected to become a substitute of medicaments to a certain extent for preventing and treating various diseases, and a new strategy is provided for a disease prevention and control scheme. Yang Yong et al (2006) prove that the lactic acid bacteria metabolite has a very significant inhibition effect on the growth of Vibrio anguillarum, and the inhibition efficiency is over 90%. Gildberg et al (1998) feed cod fry with a feed containing lactic acid bacteria extracted from the viscera of Atlantic cod fry, and after 3 months, the fry was kept in an environment with strong pathogenic vibrio bacteria, and the disease resistance was improved. The existing data show that lactic acid bacteria not only have antibacterial activity, but also have antiviral activity. Ang et al (2016) found that Lactobacillus casei was able to control hand-foot-and-mouth disease by inhibiting infection by Coxsackie virus. At present, lactic acid bacteria are widely used in aquaculture processes, but research on inhibition of aquatic pathogenic bacteria and viruses by lactic acid bacteria is less.
At present, lactic acid bacteria are widely used in aquaculture processes, but research on inhibition of aquatic pathogenic bacteria and viruses by lactic acid bacteria is less. The giant salamander iridovirus which can inhibit the giant salamander iridovirus is screened out for the first time and is compounded with other lactic acid bacteria to form the lactic acid bacteria compound microbial inoculum, so that a new idea is provided for preventing and treating the virus.
Disclosure of Invention
The invention aims to provide a lactobacillus composite microbial inoculum, which comprises lactobacillus rhamnosus YFI-6, lactobacillus plantarum and lactobacillus paracasei, wherein the preservation number of the lactobacillus rhamnosus YFI-6 is as follows: CCTCC NO: M2019655.
The invention also aims to provide application of the lactobacillus composite microbial inoculum.
In order to achieve the purpose, the invention adopts the following technical measures:
separating a lactobacillus strain capable of resisting giant salamander iridovirus from a pond water sample in an aquaculture area, wherein the lactobacillus strain is named as YFI-6, the preservation number of the strain is CCTCC NO: M2019655, and the lactobacillus strain is compounded with lactobacillus plantarum and lactobacillus paracasei to obtain a lactobacillus composite microbial inoculum; commercially available Lactobacillus plantarum and Lactobacillus paracasei are capable of carrying out the present invention.
The lactobacillus composite microbial inoculum can be prepared by directly mixing lactobacillus rhamnosus YFI-6, lactobacillus plantarum and lactobacillus paracasei according to the effective bacterium concentration of 1.5-3: 0.5-2:1-2, or seed liquid of lactobacillus rhamnosus YFI-6, lactobacillus plantarum and lactobacillus paracasei is mixed according to the effective bacteria concentration of 1.5-3: 0.5-2:1-2, mixing and fermenting.
The application of the lactobacillus composite microbial inoculum comprises preparing a medicament for treating or preventing giant salamander viral hemorrhagic disease by using the lactobacillus composite microbial inoculum, or preparing a medicament for treating or preventing diseases caused by giant salamander iridovirus infection, or preparing a giant salamander iridovirus antiviral agent, or preparing an aquatic animal feed additive.
Compared with the prior art, the invention has the following advantages:
the prepared composite microbial inoculum prepared from lactobacillus rhamnosus YFI-6 and other lactic acid bacteria can resist Giant Salamander Iridovirus (GSIV) infection, and compared with the traditional antiviral chemical drugs, the lactobacillus rhamnosus YFI-6 composite microbial inoculum serving as a potential antiviral microecological preparation has the following advantages:
1. no toxic side effect, no residue, antibiosis, antivirus, growth promotion, green and safety.
2. Has the advantages of convenient and safe use, no immune stress, high economic benefit and the like
3. Can enter the fish body by oral administration, inhibit the invasion and proliferation of Giant Salamander Iridovirus (GSIV), and effectively prevent and treat the viral hemorrhagic disease of the giant salamander.
4. The lactobacillus rhamnosus YFI-6, the lactobacillus plantarum and the lactobacillus paracasei of the invention are expressed as a ratio of 2:1:1 is inoculated and then fermented to obtain the composite microbial inoculum, and the GSIV inhibition effect of the composite microbial inoculum is superior to that of single strain lactobacillus rhamnosus YFI-6.
Detailed Description
The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.
Example 1: strain isolation and identification
1. Lactobacillus rhamnosus strain isolation
The lactobacillus rhamnosus is obtained by separating from a pond water sample in an aquaculture area, specifically diluting the water sample in the aquaculture pond by 10 times continuously by using 0.85% sterile normal saline for 6 times, sucking 100 mu L of solution from each concentration gradient diluent to a BHI solid plate by using a pipette, uniformly coating by using a coating rod, numbering and repeating for 3 times. And after the uniform coating, placing the mixture in a super-clean workbench for 5-10 min to ensure that the bacteria liquid on the surface of the culture medium is fully absorbed. Finally, the plate was inverted and incubated in a constant temperature incubator at 30 ℃ for 24 hours. Selecting bacterial colonies with different forms, inoculating the bacterial colonies on a common broth plate for separation and purification, and measuring the antiviral function of different bacteria to finally obtain a bacterial strain capable of resisting the giant salamander iridovirus, which is named as YFI-6.
2. YFI-6 Strain identification
1) Physiological and biochemical characteristics
Taking a pure cultured strain YFI-6 of an MRS solid culture medium, streaking and inoculating a single colony on a BUG identification plate, culturing for 16-24 h at 30 ℃, taking an inoculation liquid of a Biolog bacteria identification kit IF-A when the colony size is proper, wiping the outer wall of a tube, and putting the tube into a Biolog turbidity meter to adjust the reading to be 100% T; a proper amount of single colonies were dipped into the inoculum using a sterile cotton swab to read between 92% T and 98% T by a turbidimeter, the mixture was transferred to GEN III plates in a volume of 100. mu.L per well using an 8-well pipette, and the plates were loaded into a Biolog system for culture, which automatically read and output the results.
The physiological and biochemical characteristics are shown in Table 1.
TABLE 1 physiological and biochemical characteristics of Strain YFI-6
2) Molecular biological identification of Strain YFI-6
The gene of the amplified strain 16SrRNA was amplified using the universal primer, 16SF (27F): AGAGTTTGATCMTGGCTCAG, 16SR (1492R): ggttactctgttacgaactt, synthesized by shanghai biotechnology services ltd. The strain YFI-6 is identified as lactobacillus rhamnosus by physiological and biochemical characteristic determination and 16S rDNA sequence homology analysis.
Lactobacillus rhamnosus YFI-6 belongs to Lactobacillus, gram-positive anaerobe, and has no plasmid; lactose can not be utilized, but monosaccharide can be metabolized, the growth temperature is suitable for 37 ℃, and after being cultured for 48 hours by a lactic acid bacteria culture Medium (MRS), white and round colonies with smooth and moist surfaces, neat edges and bulges are formed.
The strain is delivered to China center for type culture Collection in 2019, 8, 19 and is classified and named: lactobacillus rhamnosus YFI-6, accession number: CCTCC NO: and M2019655.
Example 2:
antiviral spectrum detection method for fermentation supernatant of lactobacillus rhamnosus YFI-6
Selecting a single colony of the rejuvenated lactobacillus rhamnosus YFI-6, inoculating the single colony in 150ml of MRS liquid culture medium, culturing at 37 ℃ for 24h, centrifuging at 5000rpm for 10min, taking supernatant, filtering with a 0.22 mu m filter membrane, and storing in a sterile centrifuge tube at 4 ℃ for later use. The inhibition effect of lactobacillus rhamnosus YFI-6 fermentation supernatant on carp herpesvirus II (CyHV-2), Giant Salamander Iridovirus (GSIV), Grass Carp Reovirus (GCRV) and carp spring viremia virus (SVCV) is respectively detected.
The cells used in this example were a Carassius auratus brain tissue cell line (GiCB, CCTCC NO: C2013179), a grass carp kidney cell line (CIK), and a carp epithelial tumor cell line (EPC).
The volume of the fermentation supernatant of Lactobacillus rhamnosus YFI-6 added in this example was 100. mu.L per well, and 100TCID was added500.1ml of virus solution was the same volume as the fermentation supernatant.
Respectively inoculating lactobacillus rhamnosus YFI-6 fermentation supernatant and CyHV-2 into GiCB cells in a 96-well culture plate growing into a monolayer simultaneously, inoculating lactobacillus rhamnosus YFI-6 fermentation supernatant and GSIV into EPC cells in the 96-well culture plate growing into a monolayer, inoculating lactobacillus rhamnosus YFI-6 fermentation supernatant and GCRV into CIK cells in the 96-well culture plate growing into a monolayer, inoculating lactobacillus rhamnosus YFI-6 fermentation supernatant and SVCV into EPC cells in the 96-well culture plate growing into a monolayer, inoculating the same amount of viruses into a control group, culturing for 90min, discarding mixed liquid, washing with PBS, and adding cell maintenance liquid to continue culturing. Cytopathic effect (CPE) was observed under an optical inverted microscope, and the antiviral effect of the fermentation supernatant of Lactobacillus rhamnosus YFI-6 was judged according to the amount of CPE, as shown in Table 2, wherein + indicates resistance and-indicates no resistance.
TABLE 2 antiviral Profile of Lactobacillus rhamnosus YFI-6
Example 3:
application of lactobacillus rhamnosus YFI-6 fermented supernatant in resisting Giant Salamander Iridovirus (GSIV)
A single colony of rejuvenated lactobacillus rhamnosus YFI-6 was picked and inoculated into 150ml of MRS liquid medium, cultured at 37 ℃ for 24h, centrifuged at 5000rpm for 10min, the supernatant was filtered through a 0.22 μm filter membrane and stored in a sterile centrifuge tube at 4 ℃ for further use in this example and example 4.
The cell used in this example was the carp epithelial tumor cell line (EPC). The cell maintenance solution used was M199 medium of 10% FBS;
virus titer detection by adding 1 × 10 density 10 to 96-well cell culture plates4mu.L of EPC cells/well were cultured at 25 ℃ for 16-24 hours. When the cells grow to 80-90%, inoculating the cells with a dilution of 101～1010The virus solution of (2) was cultured in an incubator at 25 ℃ for 2 hours, with 8 parallel wells for each dilution, at 100. mu.L/well. After the incubation was completed, the virus solution was recovered, the cells in the wells were rinsed 2 times with M199 medium, and 100. mu.L of cell maintenance medium was added to continue the culture for 96 hours. Experiment setting 3 groups are parallel, observing and recording CPE phenomenon of each dilution monolayer cell every 24h, recording corresponding lesion hole number, and calculating half histiocyte infection of giant salamander iridovirus according to Reed-Muench methodQuantity (TCID) of tissue culture induced dose50)。
The experimental groups were as follows:
group 1: the group was pretreated by fermentation of the supernatant with lactobacillus rhamnosus YFI-6, followed by virus inoculation: inoculating fermentation supernatant of Lactobacillus rhamnosus YFI-6 into cells of 96-well culture plate grown in monolayer for co-culture for 2 hr, washing, and adding 100TCID500.1ml of GSIV infects monolayer cells, put in an incubator to adsorb for 90min, add cell maintenance liquid after washing, and continue culturing.
And 2, group: the lactobacillus rhamnosus YFI-6 fermentation supernatant and the GSIV are inoculated into cells at the same time: and 100TCID500.1ml GSIV is mixed in equal volume, added into the cells of a 96-well culture plate growing into a monolayer, cultured for 90min, the mixed solution is discarded and washed by PBS, and cell maintenance solution is added for continuous culture.
And 3, group: firstly, adding virus to infect cells, and then inoculating lactobacillus rhamnosus YFI-6 fermentation supernatant: at 100TCID50And/0.1 ml of GSIV infects cells growing into a monolayer of 96-well culture plate, adsorbing for 90min in an incubator, washing, adding lactobacillus rhamnosus YFI-6 fermentation supernatant, culturing for 90min in an incubator, washing with PBS, adding cell maintenance liquid, and continuing to culture.
4 groups are as follows: adding the supernatant of lactobacillus rhamnosus YFI-6 and the cells of the 96-well culture plate which grows into a monolayer for co-culture for 2h, and then adding cell maintenance liquid for continuous culture.
And 5, group: 100TCID500.1ml of GSIV infects cells growing into a monolayer in a 96-well culture plate, the cells are placed in an incubator to adsorb 90min, then washed with PBS, and added with cell maintenance solution to continue culturing.
6 groups are as follows: normal cell control.
Indirectly measuring the inhibition rate of the fermented supernatant of the lactobacillus rhamnosus YFI-6 on GSIV by an MTT method after 48 hours,
viral inhibitory rate (lactobacillus treatment group OD)490Viral control group OD490) V (cell control OD)490Viral control group OD490)×100％
Specific inhibition rates are shown in table 3:
TABLE 3 inhibition of GSIV by Lactobacillus rhamnosus YFI-6 fermentation supernatant
Example 4:
the application of lactobacillus rhamnosus YFI-6 in preparing the anti-giant salamander iridovirus preparation comprises the following steps:
1) before the experiment, the giant salamanders (20 +/-2 g) are temporarily cultured for 2 weeks under the laboratory condition, and fed at 8 and 18 points every day, wherein the feeding amount is 1 percent of the weight of the fish; the experimental water is aerated tap water, the water temperature is 25 +/-1 ℃, and the dissolved oxygen is more than 5mgL-1pH is 7.3. + -. 0.5; the experimental giant salamander is free from virus and bacterial infection through detection.
The average was divided into 8 groups of 60 tails each. The following treatments are respectively carried out:
group 1: giant salamander intraperitoneal injection of 100 μ l GSIV (10)7TCID50) Injecting 100 mu l of lactobacillus rhamnosus YFI-6 fermentation supernatant into abdominal cavity after 48 h;
and 2, group: injecting 100 μ l of Lactobacillus rhamnosus YFI-6 fermented supernatant into abdominal cavity of giant salamander, and injecting 100 μ l of GSIV (10 μ l) into abdominal cavity after 48 hr7T CID50)；
And 3, group: simultaneous intraperitoneal injection of 100 mu l of GSIV (10) on giant salamander7TCID50) And 100. mu.l of fermentation supernatant of Lactobacillus rhamnosus YFI-6;
4 groups are as follows: feeding feed containing Lactobacillus rhamnosus YFI-6 to giant salamander for 2 days (10 days)7cfu/g) is fed at a rate of 1% of fish weight, and 100. mu.l GSIV (10 μ l) is injected intraperitoneally7TCID50)；
And 5, group: giant salamander intraperitoneal injection of 100 μ l GSIV (10)7TCID50) And feeding feed (10) containing Lactobacillus rhamnosus YFI-6 after 48h7cfu/g) the feeding amount is 1 percent of the weight of the fish.
6 groups are as follows: giant salamander intraperitoneal injection of 100 μ l GSIV (10)7TCID50) Simultaneously, feeding of feed (10) containing Lactobacillus rhamnosus YFI-6 is started7cfu/g) the feeding amount is 1 percent of the weight of the fish.
7 groups of: giant salamander intraperitoneal injection of 100 μ l GSIV (10)7TCID50)；
And 8 groups: injecting 100 mul of lactobacillus rhamnosus YFI-6 into abdominal cavity of giant salamander, and fermenting supernatant.
9 groups of: 100 mul sterile PBS was injected intraperitoneally.
Mortality of each giant salamander group was recorded within 14 days of mortality from the day of group treatment (table 4). And detecting the related immunity indexes of the giant salamanders of each group on the 0 th day and the 14 th day: respiratory burst activity, serum lysozyme activity, serum complement C3 levels.
Protection rate calculation formula:
the protection ratio was (V '-V)/V' x 100%
V' mortality of giant salamanders after direct GSIV injection (7 groups);
v, mortality of lactobacillus treatment group.
A respiratory burst activity detection method comprises the following steps:
the determination of the respiratory burst force is carried out according to the method described by Anderson: NBT reduction (Anderson, Brubacher et al 1998).
Determination of serum lysozyme Activity
Measurement of serum lysozyme activity was determined by the turbidity method described by Ellis (Ellis 1988). One lysozyme activity unit is defined as: an amount of lysozyme that could decrease the absorbance by 0.001 at a wavelength of 530nm for 1 min.
Determination of serum complement C3
The level of serum complement C3 is determined by Nanjing as-built complement C3 determination kit (Nanjing as-built bioengineering institute). Results are expressed in mg/mL.
TABLE 4 Lactobacillus rhamnosus YFI-6 protection rate on giant salamander
Example 5:
preparing a complex microbial inoculum:
the compound microbial inoculum comprises: lactobacillus rhamnosus YFI-6, lactobacillus plantarum, lactobacillus paracasei;
the method comprises the following steps of (1) mixing lactobacillus rhamnosus YFI-6, lactobacillus plantarum and lactobacillus paracasei liquid according to the effective bacterium concentration of 2:1:1 mixing to obtain fermentationSeed liquid (effective bacteria concentration of fermentation seed liquid 10)8cfu/mL), and mixing and fermenting; the lactobacillus plantarum and the lactobacillus paracasei are purchased from Beijing Sorleibao science and technology limited.
The specific steps of the mixed fermentation comprise: mixing Lactobacillus rhamnosus YFI-6, Lactobacillus plantarum and Lactobacillus paracasei according to the bacterial number of 2:1:1 to obtain fermented seed liquid (10)8cfu/mL), inoculating the mixed fermented seed liquid into a fermentation tank filled with an MRS liquid culture medium in an inoculation amount of 10% in volume ratio, and culturing at 30 ℃ at a stirring speed of 180rmp and an aeration rate of 3V/min for 48 hours;
the complex microbial inoculum prepared by fermentation is centrifuged at 5000rpm for 10min, the supernatant is filtered by a 0.22 mu m filter membrane and stored in a sterile centrifuge tube at 4 ℃ for later use in example 6 and example 7. Thalli obtained after the centrifugation of the complex microbial inoculum is added into the giant salamander feed as a feed additive and is used in example 7.
Example 6:
the application of the supernatant obtained by fermenting the complex microbial inoculum in resisting Giant Salamander Iridovirus (GSIV) comprises the following steps:
the cells used in this example were the carp epithelial cell line (EPC). CCTCC NO: the cell maintenance solution used for GDC0174 is M199 culture medium of 10% FBS; the volume of the supernatant of the complex microbial inoculum added in the embodiment is 100 mu L per hole, and 100TCID is added500.1ml of GSIV virus solution has the same volume with the fermentation supernatant; (ii) a
Virus titer detection by adding 1 × 10 density 10 to 96-well cell culture plates4mu.L of EPC cells/well were cultured at 25 ℃ for 16-24 hours. When the cells grow to 80-90%, inoculating the cells with a dilution of 101～1010The virus solution of (2) was cultured in an incubator at 25 ℃ for 2 hours, with 8 parallel wells for each dilution, at 100. mu.L/well. After the incubation was completed, the virus solution was recovered, the cells in the wells were rinsed 2 times with M199 medium, and 100. mu.L of cell maintenance medium was added to continue the culture for 96 hours. Experiment setting 3 groups are parallel, CPE phenomenon of each dilution monolayer cell is observed and recorded every 24h, corresponding lesion hole number is recorded, and half tissue infection dose (tissue culture infection dose) of EPC is calculated according to Reed-Muench method，TCID50)。
The experimental groups were as follows:
group 1: fermenting the supernatant fluid pretreatment group by using a complex microbial inoculum, and then inoculating GSIV virus: inoculating the supernatant to cells of 96-well culture plate, culturing for 2 hr, washing, and adding 100TCID500.1ml of GSIV infects monolayer cells, put in an incubator to adsorb for 90min, add cell maintenance liquid after washing, and continue culturing.
And 2, group: simultaneously inoculating the composite microbial inoculum fermentation supernatant and the GSIV into cells: and 100TCID500.1ml GSIV is mixed in equal volume and added into a 96-well culture plate growing into a single layer, the culture is carried out for 90min, the mixed solution is discarded and washed by PBS, and cell maintenance solution is added for continuous culture.
And 3, group: respectively, firstly adding virus to infect cells, inoculating a complex microbial inoculum to ferment supernatant: at 100TCID500.1ml of GSIV infects cells growing into a monolayer 96-well culture plate, the cells are adsorbed in an incubator for 90min and then washed, then a composite microbial inoculum fermentation supernatant is added, the cells are placed in an incubator for 90min and then washed by PBS, and cell maintenance liquid is added for continuous culture.
4 groups are as follows: adding the composite microbial inoculum fermentation supernatant and the cells of the 96-well culture plate which grows into a single layer for co-culture for 2h, and then adding a cell maintenance solution for continuous culture.
And 5, group: as a virus control: 100TCID500.1ml of GSIV infects EPC cells grown in a monolayer in a 96-well culture plate, and after 90min of adsorption in an incubator, PBS is washed, and cell maintenance solution is added to continue the culture.
6 groups are as follows: is a normal cell control.
Indirectly measuring the inhibition rate of the fermented supernatant of the lactobacillus rhamnosus YFI-6 on GSIV by an MTT method after 48 hours,
viral inhibitory rate (Complex bacterium treatment group OD)490Viral control group OD490) V (cell control OD)490Viral control group OD490)×100％。
TABLE 5 inhibition of GSIV by complex bacterial agent fermentation supernatant
Example 7:
the application of the supernatant of the complex microbial inoculum in resisting giant salamander iridovirus:
1) before the experiment, the giant salamanders (20 +/-2 g) are temporarily cultured for 2 weeks under the laboratory condition, and fed at 8 and 18 points every day, wherein the feeding amount is 1 percent of the weight of the fish; the experimental water is aerated tap water, the water temperature is 25 +/-1 ℃, and the dissolved oxygen is more than 5mgL-1pH is 7.3. + -. 0.5; the experimental giant salamander is free from virus and bacterial infection through detection. The average was divided into 8 groups of 60 tails each. The following treatments are respectively carried out:
group 1: giant salamander intraperitoneal injection of 100 μ l GSIV (10)7TCID50) Injecting 100 mul of composite microbial inoculum fermentation supernatant into the abdominal cavity after 48 hours;
and 2, group: injecting 100 mul of composite microbial inoculum fermentation supernatant into abdominal cavity of giant salamander, and injecting 100 mul of GSIV (10) into abdominal cavity after 48h7TCID50)；
And 3, group: simultaneous intraperitoneal injection of 100 mu l GSIV (10)7TCID50) And 100 mul of the mixed solution of the composite microbial inoculum fermentation supernatant;
4 groups are as follows: giant salamander is fed with feed containing complex microbial inoculum for 2 days (10)7cfu/g) is fed at a rate of 1% of fish weight, and 100. mu.l GSIV (10 μ l) is injected intraperitoneally7TCID50)；
And 5, group: giant salamander intraperitoneal injection of 100 μ l GSIV (10)7TCID50) Then feeding the feed (10) containing the compound microbial inoculum7cfu/g) was fed at 1% of the weight of the fish for 2 days.
6 groups are as follows: giant salamander intraperitoneal injection of 100 μ l GSIV (10)7TCID50) Simultaneously, feeding of feed (10) containing the complex microbial inoculum is started7cfu/g) the feeding amount is 1 percent of the weight of the fish.
7 groups of: giant salamander intraperitoneal injection of 100 μ l GSIV (10)7TCID50)；
And 8 groups: injecting 100 mul of composite microbial inoculum into abdominal cavity of giant salamander, and fermenting supernatant.
9 groups of: 100 mul sterile PBS was injected intraperitoneally.
Mortality of each giant salamander group was recorded within 14 days of mortality from the day of group treatment (table 6). And detecting the related immunity indexes of the giant salamanders of each group on the 0 th day and the 14 th day: respiratory burst activity, serum lysozyme activity, serum complement C3 levels.
Protection rate calculation formula:
the protection ratio was (V '-V)/V' x 100%
V' mortality of giant salamanders after direct GSIV injection (7 groups);
v, mortality of lactobacillus treated group.
A respiratory burst activity detection method comprises the following steps:
the determination of the respiratory burst force is carried out according to the method described by Anderson: NBT reduction (Anderson, Brubacher et al 1998).
Determination of serum lysozyme Activity
Measurement of serum lysozyme activity was determined by the turbidity method described by Ellis (Ellis 1988). One lysozyme activity unit is defined as: an amount of lysozyme that could decrease the absorbance by 0.001 at a wavelength of 530nm for 1 min.
Determination of serum complement C3
The level of serum complement C3 is determined by Nanjing as-built complement C3 determination kit (Nanjing as-built bioengineering institute). Results are expressed in mg/mL.
TABLE 6 protective rate of complex inoculant on giant salamander
Claims (5)
1. The lactobacillus composite microbial inoculum comprises lactobacillus rhamnosus YFI-6, lactobacillus plantarum and lactobacillus paracasei, wherein the preservation number of the lactobacillus rhamnosus YFI-6 is as follows: CCTCC NO: M2019655; the lactobacillus composite microbial inoculum is prepared by mixing seed liquid of lactobacillus rhamnosus YFI-6, lactobacillus plantarum and lactobacillus paracasei according to the effective bacterium concentration of 2:1:1, mixing and fermenting.
2. Use of the supernatant of the lactic acid bacteria complex bacterial agent of claim 1 in the preparation of a medicament for treating or preventing diseases caused by giant salamander iridovirus infection.
3. The use according to claim 2, wherein the disease is giant salamander viral hemorrhagic disease.
4. Use of the supernatant of the lactic acid bacteria complex bacterial agent of claim 1 in the preparation of giant salamander iridovirus antiviral agents.
5. The use of the lactic acid bacteria complex bacterial agent of claim 1 in the preparation of an aquatic animal feed additive.
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| Probiotics as Means of Diseases Control in Aquaculture, a Review of Current Knowledge and Future Perspectives;Hoseinifar SH et al.;《Front. Microbiol》;20181012;第9卷;Ariticle 2429 * |
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