Lactococcus gasseri C6a2 and application thereof

Abstract

The invention discloses lactococcus gasseri C6a2 and application thereof. The preservation number of the lactococcus gasseri C6a2 is CGMCC (China General Microbiological Culture Collection Center) NO: 31024; the 16S rRNA (ribosomal Ribonucleic Acid) gene sequence of the lactococcus gasseri C6a2 is as shown in SEQ ID NO: 1. The bacterial strain is a macrobrachium rosenbergii intestinal absolute dominant bacterium, has a typical probiotic function, is high in growth speed and activity, can quickly enter a logarithmic phase, has relatively high acid production and acid resistance, can secrete lipase, has relatively high bacteriostatic activity on staphylococcus aureus, aeromonas hydrophila and aeromonas veronii, and can be used for preparing the macrobrachium rosenbergii. The bacterium can be used for preparing a special probiotic preparation for macrobrachium rosenbergii, so that the sustainable development of the macrobrachium rosenbergii industry in China is promoted.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Lactococcus gasseri C6a2 and applications thereof. Background Art

[0002] The development and utilization of probiotics is an important technical means to implement healthy aquaculture. In the field of shrimp farming in my country, the effect of the use of probiotics is not satisfactory. There are mixed probiotic preparations in my country, and most products do not have strain numbers. Many probiotic preparations purchased by farmers not only have no strain numbers (unclear source), but also lack clinical trials. There are neither industry standards nor national standards for probiotic products for aquaculture in my country, from production raw materials to end products. Many businesses have taken advantage of loopholes in laws and regulations to use probiotics for human medicine, health foods, and livestock and poultry farming in aquaculture. The incorrect use of probiotics has led to shrimp farmers' lack of confidence in probiotic products. The probiotic industry has suffered a huge crisis of trust. It is urgent to strengthen the research and development and promotion of probiotics for aquaculture.

[0003] Intestinal bacteria are species specific. There are significant differences in the structure and characteristics of the intestinal flora of shrimps of different species. Even the intestinal flora structure of shrimps of the same species but in different breeding environments is significantly different. The probiotic products sold on the market may not be suitable for current aquatic animal breeding, and may even have adverse effects on the host. Therefore, domestic experts and scholars have proposed the idea of ​​using homologous bacteria (probiotics screened from the intestine), that is, isolating bacteria directly from the intestines of aquatic animals and identifying their probiotic functions. The use of homologous bacteria can not only ensure that probiotics are harmless to the host, but also enable probiotics to effectively settle in the host's intestines and optimize the flora structure, thereby promoting intestinal health.

[0004] The screening and characterization of probiotics is a long and scientific process, and should be developed and utilized based on a scientific and rigorous screening and identification process. The research and development of probiotics must be targeted at a certain species, evaluate the probiotic properties of the strain, analyze the biochemical characteristics of the strain, and then evaluate the application effect, and finally identify the probiotics and build a strain library. This requires long-term investment in manpower, material resources, and financial resources, and the continuous accumulation of strain data results, in order to develop effective probiotic preparations.

[0005] Although my country's probiotics industry has developed rapidly, there are still many problems such as unclear strain sources, unknown functions, and improper use, which lead to potential hidden dangers and risks in the healthy development of my country's aquatic products. In particular, most of the aquaculture industries at home and abroad use general probiotics, seriously ignoring the typical characteristics of probiotic species specificity, which has resulted in probiotics being unable to effectively serve the aquaculture industry. The two extreme views of "probiotics are omnipotent" and "probiotics are useless" have prevailed in the aquatic industry. The reasons for this drawback can be summarized into two aspects:

[0006] First, the threshold of the probiotics industry is low, it is easy to cultivate and use, and it is cheap, which leads to widespread abuse of probiotics;

[0007] Secondly, there are many types of aquatic animals, and it is difficult to develop new probiotic products for a specific species. In addition, the research and development of probiotic preparations specifically for aquatic animals requires sufficient financial support, and most manufacturers cannot and are unwilling to invest in research and development.

[0008] my country is a major Macrobrachium rosenbergii industry country, with an average annual output of more than 130,000 tons, accounting for more than half of the global output, and the market has always been in short supply, with huge market potential. Driven by my country's "Healthy China" strategy, it is a general trend to construct a probiotic preparation exclusively for Macrobrachium rosenbergii. Therefore, the present invention conducts intestinal sampling for Macrobrachium rosenbergii, uses bacterial isolation and culture technology to deeply analyze the structure of culturable bacteria in the intestinal flora, and focuses on the physiological characteristics and genetic evolution relationship of the dominant bacteria, and comprehensively evaluates the activity of potential probiotics in inhibiting pathogens, bile, pH and temperature tolerance, antibiotic sensitivity, enzyme production ability and activity, aiming to prepare a microecological preparation exclusively for Macrobrachium rosenbergii, thereby promoting the sustainable development of my country's Macrobrachium rosenbergii industry. Summary of the invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Lactococcus gasseri C6a2 and its application. The present invention isolates a strain of Lactococcus gasseri C6a2 from the intestinal tract of Macrobrachium rosenbergii. The strain is an absolute dominant bacterium in the intestinal tract of Macrobrachium rosenbergii and has typical probiotic characteristics. It can be used to prepare a probiotic preparation exclusive to Macrobrachium rosenbergii, thereby promoting the sustainable development of my country's Macrobrachium rosenbergii industry.

[0010] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0011] The invention provides a Lactococcus garvieae C6a2, whose preservation number is CGMCC NO:31024.

[0012] Furthermore, the 16S rRNA gene sequence of Lactococcus garvei C6a2 is shown in SEQ ID NO: 1.

[0013] The present invention also provides a use of the Lactococcus gasseri C6a2 in antibacterial treatment.

[0014] Furthermore, the antibacterial strain is a pathogenic bacteria.

[0015] Furthermore, the pathogenic bacteria are Staphylococcus aureus, Aeromonas hydrophila and Aeromonas veronii.

[0016] The present invention also provides a Lactococcus gasseri fermentation agent, wherein the fermentation agent comprises the Lactococcus gasseri C6a2 at a concentration of 1×10 8 CFU / mL.

[0017] The present invention also provides a method for preparing the Lactococcus gasseri fermentation agent, which comprises adjusting the activated Lactococcus gasseri C6a2 bacterial solution to an OD of 625 =1.0, inoculated in TSB liquid culture medium at 1% inoculation amount, and fermented for 48 hours at 28°C to obtain a Lactococcus gasseri fermentation agent, wherein the concentration of the Lactococcus gasseri fermentation agent is 1×10 8 CFU / mL.

[0018] The present invention also provides an application of the Lactococcus gasseri C6a2 in producing lipase.

[0019] The present invention also provides an application of the Lactococcus gasseri C6a2 in preparing a special intestinal probiotic preparation for Macrobrachium rosenbergii.

[0020] The present invention also provides a probiotic preparation for the intestinal tract of Macrobrachium rosenbergii, wherein the probiotic preparation comprises the Lactococcus gasseri C6a2 at a concentration of 1×10 8 CFU / mL.

[0021] The above-mentioned Lactococcus garvei C6a2 has the following probiotic functions:

[0022] (1) Antibacterial function

[0023] (2) Fast growth and strong vitality

[0024] (3) Has acid-producing function

[0025] (4) Has enzyme production function

[0026] (5) Acid-resistant.

[0027] Beneficial effects of the present invention:

[0028] The present invention separates 367 strains of bacteria from the intestinal contents of Macrobrachium rosenbergii, of which 234 strains of Lactococcus garvieae account for 63.76% of the total bacteria, and are absolutely dominant bacteria. Lactococcus garvieae C6a2 has a fast growth rate, can quickly enter the logarithmic growth phase, has strong acid production and acid resistance, can secrete lipase, and has strong antibacterial activity against Staphylococcus aureus, Aeromonas hydrophila and Aeromonas vernix, which indicates that Lactococcus garvieae can be used to prepare an intestinal probiotic preparation exclusive to Macrobrachium rosenbergii. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is an agarose gel electrophoresis analysis of genomic DNA of culturable bacteria in the intestine of Macrobrachium rosenbergii;

[0030] In the figure, M: DNA Marker (DL2 000); Bands 1-18: bacterial DNA samples;

[0031] Figure 2 This is an agarose gel electrophoresis analysis of the 16S rRNA gene PCR products of culturable bacteria in the intestine of Macrobrachium rosenbergii;

[0032] In the figure, M: DNA Marker (DL2 000); Bands 1-18: PCR products;

[0033] Figure 3 This is the result diagram of the number of isolated intestinal bacteria of Macrobrachium rosenbergii;

[0034] Figure 4 This is the result diagram of the separation ratio of culturable bacteria in the intestine of Macrobrachium rosenbergii;

[0035] Figure 5 The phylogenetic tree of Lactococcus garrisonii-related species constructed based on 16S rRNA sequences;

[0036] Figure 6 This is the macroscopic morphology of Lactococcus garvei C6a2;

[0037] Figure 7 This is the result of the inhibition of Lactococcus garvei C6a2 on the main harmful bacteria in the animal intestine;

[0038] In the figure, the pathogenic bacteria are A: Staphylococcus aureus; B: Aeromonas hydrophila; C: Aeromonas vermifuge;

[0039] Figure 8 is the growth curve of Lactococcus garvieae C6a2 at 28°C;

[0040] Fig. 9 This is a graph showing the acid production ability of Lactococcus garvieae C6a2 at 28°C;

[0041] Fig.10 This is a diagram of enzyme production by Lactococcus garvei C6a2;

[0042] In the figure, A: protease screening medium; B: lipase screening medium; C: amylase screening medium; D: cellulase medium;

[0043] Fig.11 This is the plate count chart of the acid resistance test of Lactococcus garvieae C6a2;

[0044] In the figure, the labels under the picture represent pH value, treatment time and bacterial solution dilution multiple;

[0045] Fig.12 This is a graph showing the results of the acid resistance test of Lactococcus garvieae C6a2;

[0046] In the figure, the survival rate of Lactococcus garvieae C6a2 after incubation in pH = 5, pH = 4 and pH = 3 environments for 2 h, 4 h and 8 h; the data are expressed as the average of the results of three repeated experiments, and the error bars represent SD;

[0047] Fig.13 This is the plate count chart of the high temperature resistance test of Lactococcus garvieae C6a2;

[0048] In the figure, the labels under the picture represent the water bath temperature, treatment time and bacterial solution dilution multiple;

[0049] Fig.14 This is a diagram showing the results of a high temperature resistance test of Lactococcus garvieae C6a2;

[0050] In the figure, the survival rate of Lactococcus garvei C6a2 after being incubated in a water bath at 50°C, 55°C and 60°C for 10 min, 20 min and 30 min; the data are expressed as the average of the results of three repeated experiments, and the error bars represent SD. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0052] Example 1 Isolation and Identification of Lactococcus garvieae C6a2

[0053] 1. Isolation and purification of intestinal flora of Macrobrachium rosenbergii

[0054] (1) Methods

[0055] Use 75% ethanol to disinfect the surface of healthy shrimps, and cut the intestines in a clean bench. Dissolve the intestinal samples in 1 mL of PBS solution and homogenize them. Use the stepwise dilution method to obtain bacterial suspensions of different concentrations. Take 10 -3 ~10 -6 100 μL of the dilution of the dilution gradient was spread on LB solid medium and cultured in a 28°C incubator for 24-48 hours, with three replicates for each gradient. A single colony was picked and placed in LB liquid medium and cultured in a shaker at 28°C and 180 r / min for 48-72 hours.

[0056] (2) Conclusion

[0057] A total of 367 bacterial strains were isolated from the intestine of Macrobrachium rosenbergii. The bacterial genomic DNA was extracted and the results of agarose gel electrophoresis of the genomic DNA were as follows: Figure 1 shown.

[0058] 2. Extraction of bacterial genome and identification of bacterial species

[0059] (1) Methods

[0060] The bacteria were collected by centrifugation, the bacterial wall was broken with lysozyme, and then the bacterial genome was extracted according to the kit instructions. The DNA quality was tested by 1% agarose gel electrophoresis. The PCR product was detected by 1% agarose gel electrophoresis and then 16S rRNA sequencing was performed. The sequencing results were compared with BLAST on the NCBI website to identify the classification of the strain. The phylogenetic tree was constructed using MEGA 11.0 software.

[0061] (2) Conclusion

[0062] a. Using the genomic DNA of 367 bacterial strains as templates and universal primers for PCR amplification, we can obtain a band of about 1300 bp, and the band is single and bright. The results are as follows: Figure 2 shown.

[0063] b. Use the Blast tool in NCBI to compare the 16S rRNA sequences of each strain. The results are as follows Figure 3 and Figure 4 As shown, there were 234 strains of Lactococcus garvieae, accounting for 63.76% of the total bacteria, which was an absolute dominant bacteria; followed by 14 strains of Citrobacter freundii, 8 strains of Lactococcus lactis, and 8 strains of Acinetobacter johnsonii, accounting for 3.81%, 2.18% and 2.18% of the total bacteria, respectively.

[0064] c. Select a strain of Lactococcus garrisonii (C6a2), collect the 16S rRNA gene sequences of the reference strain with the highest similarity to this strain and other strains with high similarity, and construct the NJ phylogenetic tree.

[0065] The results are as follows Figure 5 As shown, the results showed that the sequence of isolate C6a2 was homologous to that of Lactococcus garvei (GenBank accession number: MW135263.1), with a similarity of 100%, and it can be determined that the strain is Lactococcus garvei; Lactococcus garvei has a high homology with Lactococcus lactis and Lactococcus taiwanensis.

[0066] 3. Physiological and biochemical analysis of Lactococcus garvei C6a2

[0067] (1) Methods

[0068] According to Bergey's Manual of Bacterial Identification (8th edition), glucose oxidation and fermentation test, nitrate reduction test, gelatin liquefaction test, starch hydrolysis test, hydrogen sulfide production test, oxidase test, indole test, methyl red test and citrate test were conducted on Lactococcus garvei.

[0069] (2) Conclusion

[0070] The colonies of Lactococcus garvei C6a2 are white, round and convex in macroscopic morphology ( Figure 6 ), the microscopic morphology is spherical.

[0071] The results of physiological and biochemical identification are shown in Table 1: nitrate reduction and methyl red reactions were positive, while glucose, gelatin, starch, hydrogen sulfide, oxidase, indole and citrate reactions were negative.

[0072] Table 1 Physiological and biochemical identification

[0073]

[0074] Note: “+” represents a positive result; “-” represents a negative result.

[0075] Based on the above morphological, physiological and biochemical analysis and molecular biological analysis: the isolate C6a2 was named Lactococcus garvieae C6a2, and its preservation number is CGMCC NO: 31024. It was deposited in the General Microbiology Center of China National Culture Collection Administration on June 20, 2024, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0076] Example 2 Antibacterial test of Lactococcus garvei C6a2

[0077] 1. Methods

[0078] Pathogenic bacteria Staphylococcus aureus, Aeromonas hydrophila, Aeromonas veronii, Citrobacter freundii, Vibrio parahaemolyticus and Escherichia coli were selected as indicator bacteria for antibacterial test. The pathogenic bacteria culture solution was diluted to 1×10 8 After measuring CFU / mL, 100 μL was taken and spread on TSB plate, and 10 μL of Lactococcus garvieae solution was added to each filter paper by filter paper method, and PBS buffer was used as blank control. Each test was repeated three times. Cultured at 28℃ for 24h, and the diameter of the inhibition zone (mm) was measured.

[0079] 2. Conclusion

[0080] The results of the inhibitory activity of Lactococcus garvei C6a2 against pathogenic bacteria are shown in Tables 2 and Figure 7: Obvious inhibition zones can be observed on the plates of Staphylococcus aureus, Aeromonas hydrophila and Aeromonas vermifuge, and the inhibition zones are 22.63±0.85mm, 19.99±1.30mm and 13.63±1.03mm, respectively;

[0081] The results showed that Lactococcus gasseri C6a2 had strong antibacterial activity against Staphylococcus aureus, Aeromonas hydrophila and Aeromonas vermifuge. No inhibition zone was observed on the plates of Citrobacter freundii, Vibrio parahaemolyticus, Escherichia coli and Vibrio anguillarum, indicating that Lactococcus gasseri C6a2 had no significant antibacterial activity against these four pathogens.

[0082] Table 2 Inhibitory effect of pathogenic bacteria

[0083]

[0084] Example 3 Growth conditions and acid production capacity detection of Lactococcus garvei C6a2

[0085] 1. Methods

[0086] The activated Lactococcus garvieae C6a2 bacterial solution was adjusted to OD 625 =1.0, inoculated in TSB liquid medium at 1% inoculum, cultured at 28°C with shaking at 180r / min, and measured OD625 after 3h, and then measured every 1h, with three replicates. At the same time, the pH value of the bacterial solution was measured at 4, 8, 16, 24, 48 and 72h using a pH meter, with three replicates.

[0087] 2. Conclusion

[0088] a. The results are as follows Figure 8 As shown: Lactococcus garvieae C6a2 grows faster. When the inoculation amount is 1%, it enters the logarithmic growth phase within 3 hours, enters the plateau phase after 8 hours, and reaches the maximum growth density (OD625=1.32) after 12 hours.

[0089] b. Fig. 9 As shown in the figure, the suitable temperature range of Lactococcus gasseri C6a2 is relatively wide, and it grows well between 28 and 37°C. The pH of the culture medium decreases with the increase of culture time, indicating that Lactococcus gasseri C6a2 has the characteristic of acid production, reaching the maximum acid production at 13h, pH = 6.01, and thereafter the pH value no longer has a downward trend.

[0090] Example 4 Test on enzyme production ability of Lactococcus garvei C6a2

[0091] 1. Methods

[0092] Lactococcus garvieae C6a2 strain was inoculated in solid screening medium for protease, lipase, amylase and cellulase, with 3 replicates for each strain. Incubate at 28℃ for 24h. Stain the screening medium for amylase and cellulase, wherein 2mL of Lugol's solution was added to the amylase screening medium and stained for 1min; 0.2% Congo red solution was added to the cellulase screening medium and stained for 5min, and then washed twice with 5% NaCl solution. Observe whether the colony changes color or a transparent circle appears around it. The enzyme-producing bacteria with enzyme production ability were inoculated in 100mL of fermentation medium and cultured for 24h, centrifuged at 4 000×g for 10min, and the supernatant was taken. The activity of the corresponding enzyme was determined using an enzyme activity detection kit.

[0093] 2. Conclusion

[0094] The results are as follows Fig.10 As shown: Olive oil and indicator pigment neutral red were added to the lipase screening medium, the bacteria produced lipase to decompose the olive oil, and the surrounding color changed from orange to red, indicating that Lactococcus garvieae C6a2 produced lipase;

[0095] The colonies grown in the protease, amylase and cellulase screening medium had no transparent circle generated and no color change after staining, indicating that Lactococcus garvieae C6a2 did not produce protease, amylase and cellulase.

[0096] Lactococcus gasseri C6a2 was inoculated into lipase fermentation medium and cultured for 24 hours. The supernatant of the fermentation broth was detected. The results showed that the lipase activity of Lactococcus gasseri C6a2 was 22.09±0.92 U / mL.

[0097] Example 5 Acid and high temperature resistance test of Lactococcus garvieae C6a2

[0098] 1. Acid resistance test of Lactococcus garvei C6a2

[0099] (1) Methods

[0100] The activated Lactococcus garvieae C6a2 was inoculated into TSB medium and cultured at 28°C and 180 r / min for 48 h. The cells were collected by centrifugation at 3000 × g for 5 min and diluted to 1 × 10 8 CFU / mL was used as a control, and 5 mL of the dilution was inoculated into 100 mL of TSB culture medium with pH = 3, pH = 4 and pH = 5. Samples were taken at 2h, 4h and 8h, and the plates were plated after gradient dilution. Each dilution was repeated 3 times, and the survival rate of the strain was calculated.

[0101] (2) Conclusion

[0102] The results are as follows Fig.11 and 12As shown: the survival rates of Lactococcus gasseri C6a2 in pH = 5 medium were 79.78%, 88.18%, and 92.63% respectively; the survival rates in pH = 4 medium were 69.94%, 71.90%, and 74.23% respectively; and the survival rates in pH = 3 medium were all 0%. The research results show that Lactococcus gasseri C6a2 has a strong acid resistance.

[0103] 2. High temperature resistance test of Lactococcus garvei C6a2

[0104] (1) Methods

[0105] The activated Lactococcus garvieae C6a2 was inoculated into TSB medium and cultured at 28°C and 180 r / min for 48 h. The cells were collected by centrifugation at 3 000 × g for 5 min and diluted to 1 × 10 8 CFU / mL was used as a control, and 5 mL of the dilution was treated in a water bath at 50℃, 55℃ and 60℃ for 10min, 20min and 30min, respectively. After gradient dilution, the plates were coated with the solution, and each dilution was repeated 3 times to calculate the survival rate of the strain.

[0106] (2) Conclusion

[0107] The results are as follows Fig.13 and 14 As shown, at 50℃, the survival rates were 99.81%, 99.94%, and 98.77% respectively; at 55℃, the survival rates were 85.13%, 53.78%, and 43.99% respectively; at 60℃, the survival rate was 39.63% after 10 minutes of treatment, and the survival rates were 0% after 20 minutes and 30 minutes of treatment. The results showed that Lactococcus gasseri C6a2 is not resistant to high temperatures.

[0108] Example 6 Preparation of Lactococcus garvieae fermentation agent

[0109] The activated Lactococcus garvieae C6a2 bacterial solution was adjusted to OD 625 =1.0, inoculated in TSB liquid culture medium at 1% inoculation amount, fermented and cultured for 48h at 28°C to obtain Lactococcus gasseri fermentation agent, wherein the concentration of Lactococcus gasseri fermentation agent is 1×10 8 CFU / mL.

[0110] Example 7 Macrobrachium rosenbergii exclusive intestinal probiotic preparation

[0111] A probiotic preparation for the intestinal tract of Macrobrachium rosenbergii contains the above-mentioned Lactococcus gasseri C6a2, and its concentration is 1×10 8 CFU / mL.

[0112] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A Lactococcus garvieae C6a2, whose deposit number is CGMCC NO: 31024.

2. The Lactococcus garvei C6a2 according to claim 1, characterized in that: The 16S rRNA gene sequence of Lactococcus garvei C6a2 is shown in SEQ ID NO:

1.

3. Use of Lactococcus gasseri C6a2 as claimed in claim 1 in producing lipase.

4. A use of Lactococcus garvieae C6a2 as claimed in claim 1 in antibacterial treatment.

5. The application according to claim 4, characterized in that: The antibacterial strain is a pathogenic bacteria.

6. The application according to claim 5, characterized in that: The pathogenic bacteria are Staphylococcus aureus, Aeromonas hydrophila and Aeromonas veronii.

7. A Lactococcus garvei fermentation agent, characterized in that: The fermentation agent comprises the Lactococcus garvieae C6a2 as claimed in claim 1, and its concentration is 1×10 8 CFU / mL.

8. A method for preparing the Lactococcus garvei fermentation agent according to claim 7, characterized in that: The method is to adjust the activated Lactococcus garvieae C6a2 bacterial solution to an OD 625 =1.0, inoculated in TSB liquid culture medium at 1% inoculation amount, and fermented for 48 hours at 28°C to obtain a Lactococcus gasseri fermentation agent, wherein the concentration of the Lactococcus gasseri fermentation agent is 1×10 8 CFU / mL.

9. Use of Lactococcus gasseri C6a2 as claimed in claim 1 in preparing a special intestinal probiotic preparation for Macrobrachium rosenbergii.

10. A probiotic preparation specifically for the intestinal tract of Macrobrachium rosenbergii, characterized in that: The probiotic preparation comprises the Lactococcus garvieae C6a2 as claimed in claim 1, and the concentration thereof is 1×10 8 CFU / mL.

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