Composition containing three lactic acid bacteria strains and application of composition to prevention of EHP of white shrimps

Abstract

The invention discloses a composition containing three lactic acid bacteria strains and an application of the composition in prevention of EHP (Ethylene Hypothionyl Phthalate) of white shrimps. The three lactic acid bacteria strains provided by the invention are respectively enterococcus faecium EF08, lactobacillus plantarum LP28 and lactobacillus acidophilus LA1063. The mass ratio of the enterococcus faecium EF08 to the lactobacillus plantarum LP28 to the lactobacillus acidophilus LA1063 is 1 to 3 to 0.01. Experiments show that the P1 and P2 combined probiotic strain has the application of preventing and reducing EHP disease infection of the white shrimps. Therefore, the composition disclosed by the invention can be used for preparing a medicine for preventing microsporidia EHP infection.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a composition containing three lactic acid bacteria and use of the composition in preventing EHP in white shrimp. Background Art

[0002] Hepatopancreatic microsporidiosis (HPM) is one of the most serious diseases in shrimp farming in recent years. It first occurred in grass shrimp farming in Thailand in 2004, and the pathogen was confirmed to be Enterocytozoon hepatopenaei (EHP) in 2009 (Tourtip S, S. et al. Enterocytozoon hepatopenae isp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships [J]. J. Invertebr. Pathol, 2009, 102, 21-29). Subsequently, farmed white shrimp, blue shrimp, etc. were also infected with EHP (Tang KFJ. et al. Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP), amicrosporidian parasite infecting penaeid shrimp[J].Invetebr.Pathol.2015,130,37-41).

[0003] Microsporidiasis of shrimp is caused by a type of microsporidia that infects the hepatopancreas of shrimp. The infection mainly occurs in the cells of the hepatopancreas, causing problems such as growth retardation and shrimp eating feed but not growing, resulting in significant economic losses.

[0004] Recently, genomic taxonomy analysis confirmed that EHP is similar to fungi (Newman SG. Microsporidian Impacts shrimp production-industry efforts address control, not eradication [J]. Glob. Aquac. Advocate, 2015, 16-17). Currently, there is no treatment for shrimp microsporidiosis, and most people use preventive measures to reduce the impact of the disease, especially disinfection before breeding and stocking shrimp fry without EHP infection. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a composition containing three lactic acid strains and its use in preventing EHP in white shrimp. The present invention screened out three lactic acid strains, combined them in a specific ratio, and evaluated the feasibility of white shrimp ingesting the combined probiotic strains to prevent microsporidia EHP infection. The present invention found that the combined probiotic strains have the use of preventing and reducing EHP disease infection in white shrimp.

[0006] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0007] The invention provides a composition containing three lactic acid strains, wherein the three lactic acid strains are respectively Enterococcus faecium EF08, Lactobacillus plantarum LP28 and Lactobacillus acidophilus LA1063.

[0008] A preferred implementation manner of the present invention is that the mass ratio of Enterococcus faecium EF08, Lactobacillus plantarum LP28 and Lactobacillus acidophilus LA106 is 1:3:0.01.

[0009] Preferably, the bacterial count of the composition is not less than 1.5×10 10 CFU / g.

[0010] Preferably, the effective amount of the composition to be applied is 1×10 5 CFU / g / feed.

[0011] Preferably, the effective amount of the composition to be applied is 1×10 6 CFU / g / feed.

[0012] The present invention provides two groups of compositions containing three lactic acid bacteria:

[0013] The first group is EF08, LP28 and LA1063 with a mass ratio of 1:3:0.01, 1×10 6 CFU / g / feed, recorded as P1;

[0014] The second group is EF08, LP28 and LA1063 with a mass ratio of 1:3:0.01, 1×10 5 CFU / g / feed, recorded as P2.

[0015] The present invention also provides use of the composition in preventing microsporidia EHP infection.

[0016] The composition significantly reduces the EHP concentration in the hepatopancreas of white shrimp.

[0017] The present invention also provides use of the composition in preparing a drug for preventing microsporidia EHP infection.

[0018] Beneficial effects:

[0019] Currently, there is no treatment for microsporidiosis in shrimp, and only preventive measures can be used, such as stocking shrimp fry without EHP infection and disinfecting the pond to reduce the chance of infection. The present invention found that the combined probiotic strains of P1 and P2 have the use of preventing and reducing the infection of white shrimp EHP disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of detecting EHP in shrimp hepatopancreas by polymerase chain reaction.

[0021] Wherein, M is a marker; P is a positive result; N is a negative result; Lanes 1 to 5: samples from the control group (CON), P1 group and P2 group. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0023] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0025] Experimental strains of the present invention:

[0026] The control group CON refers to the white shrimp feed without adding any probiotics.

[0027] Probiotic 1 (P1 group): EF08, LP28, LA1063 (1×106 CFU of probiotics)

[0028] Probiotic 2 (P2 group): EF08, LP28, LA1063 (1×10 5 CFU of probiotics)

[0029] Example 1 Preparation of Probiotic Composition Probiotic 1

[0030] EF08, LP28 and LA1063 were mixed evenly in a mass ratio of 1:3:0.01 to obtain a probiotic composition; the probiotic composition was added to the white shrimp feed so that each gram of the feed contained 1×10 6 CFU of probiotics.

[0031] Example 2 Preparation of Probiotic Composition Probiotic 2

[0032] EF08, LP28 and LA1063 were mixed evenly in a mass ratio of 1:3:0.01 to obtain a probiotic composition; the probiotic composition was added to the white shrimp feed so that each gram of the feed contained 1×10 5 CFU of probiotics.

[0033] Example 3 Microsporidia infection experiment

[0034] The test groups prepared in Example 1 and Example 2 were fed to white shrimp for 8 weeks and then orally infected with microsporidia to understand the potential of probiotics in preventing microsporidia infection.

[0035] Before the infection experiment, the shrimps were kept in a 60L aquarium containing 40L seawater (25psu) at a water temperature of 27±1℃ and a continuous oxygen supply device. Each group was set up with 3 replicates, 10 shrimps in each replicate group, and a total of 30 shrimps.

[0036] Take microsporidia-positive shrimp (white shrimp that is tested for the presence of microsporidia in the hepatopancreas by polymerase chain reaction) as the microsporidia for infection, and take the fresh hepatopancreas and feed it directly to the experimental shrimp for feeding, once in the morning and once in the afternoon, for a total of 2 times. Before infection, cut the hepatopancreas containing microsporidia for infection into small pieces and mix them to avoid the problem of inconsistent infection amount. The amount of hepatopancreas fed each time is 10% of the shrimp body weight, and the amount of microsporidia in the hepatopancreas is confirmed by real-time PCR to determine the infection concentration. The concentration of microsporidia in the hepatopancreas used to infect the shrimp is 2.85±1.77×10 5 copies / ng DNA.

[0037] In addition, 30 shrimps from the control group were fed with the hepatopancreas of microsporidia-negative shrimps (negative shrimps were defined as shrimps in which no microsporidia were detected) as negative controls, once in the morning and once in the afternoon, for a total of 2 times.

[0038] After the infection experiment, the experimental feed was continuously fed twice a day for 1 week, and then 5 shrimps from each replicate group were taken for microsporidia detection to evaluate the protective effect of probiotics on white shrimp infection with microsporidia.

[0039] Example 4 Microsporidia Detection

[0040] All experimental instruments were sterilized in advance to avoid contamination that could affect the results.

[0041] During the test, 20 mg of tissue was collected from the hepatopancreas of white shrimp for DNA extraction, followed by purity and concentration determination and storage at -20°C for future use.

[0042] The primer sequences for microsporidia detection were designed according to the literature method (Wang L. et al. Integrated qPCR and staining methods for detection and quantification of Enterocytozoon hepatopenaei in shrimp Litopenaeus vannamei [J]. Microorganisms, 2020, 8, 1366).

[0043] EHP-F: GCAGCACTCAAGGAATGGC;

[0044] EHP-R: TTTCGTTAGGCTTACCCTGTGA.

[0045] The amplified fragment is approximately 238bp, which can be used for PCR testing to understand the infection situation first.

[0046] PCR reaction conditions:

[0047] Step 1: 98°C, 5 minutes;

[0048] Step 2 was 98°C for 30 seconds, 56°C for 30 seconds, and 72°C for 10 seconds, repeated for 35 cycles;

[0049] Step 3 is 72°C for 10 minutes.

[0050] Figure 1 The results of detecting EHP in shrimp hepatopancreas by polymerase chain reaction.

[0051] Figure 1The results showed that serious microsporidia infection was detected in all five white shrimp samples in the control group, while mild microsporidia infection was observed in the P1 and P2 groups.

[0052] from Figure 1 The EHP infection rate of white shrimp can be understood in the figure. The brighter the image is, the more serious the infection is.

[0053] After initially understanding the infection rate of shrimps in each group, the present invention further performed microsporidia infection concentration analysis using real-time PCR.

[0054] Before analysis, pGEM-T vector (Promega, A3600) containing a microsporidian DNA fragment (Nucleotide sequence: PQ351408) was taken to determine the plastid DNA concentration. The known concentration of DNA standard was converted into gene copy number (refer to Loffler EE. et al. Enrichment, cultivation, and detection of reductively dechlorinating bacteria [J]. Methods Enzymol, 2005, 397, 77-111), which was used as the standard stock solution for this experiment and stored at -20°C for future use.

[0055] Gene copy number = (DNA ng / μl) × (1μl / rxn) × (6.02 × 10 23 ) / (plasmid size [bp] × 660 × 10 9 ).

[0056] When preparing the standard curve of microsporidia gene copy number, the prepared microsporidia plastid DNA standard was diluted 10 times in a row (10 1 -10 8 ), each sample was repeated three times, and a set of non-template control (NTC) without template was added to confirm that the operation process was contamination-free, followed by real-time PCR analysis.

[0057] The sample real-time PCR reaction system is as follows:

[0058] 1 μl template (standard plasmid DNA), 10 μl 2x qPCRBIO SyGreen Blue Mix Hi-ROX (PB20.12, PCRBIOSYSTEM, London, UK), 0.2 μM EHP-F, 0.2 μM EHP-R, and finally the total reaction volume was made up to 20 μl with DEPC water.

[0059] Subsequently, the relative gene expression analysis was performed using the ABIPRISM 7900 Sequence Detection System (Applied Biosystems). Reaction conditions: Step 1 was 95°C for 5 minutes; Step 2 was 95°C for 10 seconds, 60°C for 30 seconds, repeated for 40 cycles; Step 3 was 95°C for 15 seconds.

[0060] After analysis, the copy number of the plasmid DNA standard and the Ct value are plotted into a standard curve, and the increase calculation formula is E slop =10 (-1 / slope) -1. The Ct value of the sample to be tested after real-time PCR analysis can be converted into the microsporidia infection concentration through the standard curve.

[0061] The experimental results are shown in Table 1:

[0062] Table 1 Effect of probiotic composition on microsporidia concentration in the hepatopancreas of white shrimp 7 days after oral challenge with EHP

[0063]

[0064] The data are expressed as (mean ± standard error). Variables with different superscript letters represent significant differences (p<0.05).

[0065] It can be seen from Table 1 that after the white shrimp was challenged with EHP, the P1 and P2 groups could significantly reduce the EHP concentration in the hepatopancreas of the white shrimp. Therefore, the probiotic strains combined with P1 and P2 have the use of preventing and reducing the infection of EHP disease in white shrimp.

[0066] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A composition containing three lactic acid bacteria, characterized in that: The three lactic acid strains are respectively Enterococcus faecium EF08, Lactobacillus plantarum LP28 and Lactobacillus acidophilus LA1063.

2. The composition according to claim 1, characterized in that The mass ratio of the Enterococcus faecium EF08, Lactobacillus plantarum LP28 and Lactobacillus acidophilus LA1063 is 1:3:0.

01.

3. The composition according to claim 1, characterized in that The bacterial count of the composition is not less than 1.5×10 10 CFU / g.

4. The composition according to claim 1, characterized in that The effective amount of bacteria used in the composition is 1×10 5 CFU / g / feed.

5. The composition according to claim 1, characterized in that The effective amount of bacteria used in the composition is 1×10 6 CFU / g / feed.

6. Use of the composition according to any one of claims 1 to 5 in preventing microsporidia EHP infection.

7. The use according to claim 6, characterized in that The composition significantly reduces the EHP concentration in the hepatopancreas of white shrimp.

8. Use of the composition according to any one of claims 1 to 5 in the preparation of a medicament for preventing microsporidia EHP infection.

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