KR101633909B1 - Lactobacillus plantarum mz-1 strain with antimicrobial activity against harmful bacteria to hatchery fish - Google Patents
Lactobacillus plantarum mz-1 strain with antimicrobial activity against harmful bacteria to hatchery fish Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
- A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
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Abstract
More particularly, the present invention relates to a method for culturing Lactobacillus plantarum mz-1 having antimicrobial activity against aquaculture and harmful bacteria, and more particularly, to a method for producing Lactobacillus plantarum mz- A microorganism preparation containing the strain and a culture solution thereof as an active ingredient, and a treatment method for harmful bacteria. In accordance with the present invention, it is possible to provide a microorganism which shows antibacterial activity against the harmful bacteria Edwardsiella tarda , Streptococcus iniae or Vibrio ichthyoenteri , The Lactobacillus plantarum mz-1 strain was newly isolated, and it is expected to be useful as a cultured antimicrobial agent which can be utilized in aquaculture environment in an environmentally friendly manner.
Description
More particularly, the present invention relates to a method for culturing Lactobacillus plantarum mz-1 having antimicrobial activity against aquaculture and harmful bacteria, and more particularly, to a method for producing Lactobacillus plantarum mz- A microorganism preparation containing the strain and a culture solution thereof as an active ingredient, and a treatment method for harmful bacteria.
The domestic aquaculture industry is exposed to the danger of rapid deterioration of immunity and rapid spread of infection due to deterioration of the water quality environment because there are many forms of breeding at high density within a limited area. This causes enormous damage to the farming fish of the aquaculture species, and astronomical economic damage is caused by the increase of diseases such as bacteria and virus infection and spread of infectious diseases.
Currently, various kinds of antibiotics are used alone or in combination in order to prevent the damage as described above. Antibiotic resistant microorganisms are increasing due to the continuous use of such antibiotics. Antimicrobial resistant microorganisms can infect humans, destroying cultured germs and destroying biological equilibrium, causing abnormalities in defense mechanisms against infection, ultimately facilitating the invasion of pathogenic microorganisms, , And may have a bad influence on the use of cultured fish as food.
Although there are many kinds of pathogenic microorganisms in cultured fishes, Edward 's disease, Streptococcus, and Vibriosis are the most common diseases that have been suffering from domestic diseases. Edward's disease is characterized by anemic fever, abdominal distension, ulceration, and abscess formation in a variety of aquaculture species such as eel, tilapia, red sea bream, maggie, and flounder. Streptococcus is caused by darkening of body color, ocular hemorrhage, Fever, farms, and ulcers, and major organ hemorrhages occur. In addition, it is a representative disease that causes continuous damage to aquatic farms by causing hemorrhage, muscle necrosis, hemorrhage, ocular hemorrhage and protrusion in the onset of vibriosis, petechial hemorrhage and inflammation in the organ. These diseases are causing continuous damage in various high-density farms depending on the season, and it is urgent to take measures against them.
In order to solve the above-mentioned problems, the present invention provides a method for preventing and / or treating a harmful bacterium such as Edwardsiella tarda , Streptococcus iniae or Vibrio ichthyoenteri , To improve the feed efficiency and to greatly improve the growth rate of cultured fish.
The present invention relates to a lactobacillus plantarum mz-1 strain (accession number: KCTC-18401P) having antimicrobial activity against cultured fish harmful bacteria.
The present invention also relates to a microbial preparation for antimicrobial activity against a cultured fish harmful bacterium comprising the strain and a culture solution thereof as an active ingredient.
In the present invention, the cultured bacterium can be, but is not limited to, Edwardsiella tarda , Streptococcus iniae or Vibrio ichthyoenteri .
In the present invention, the composition may be in the form of a food, a food additive, a feed additive or a medicament, though not limited thereto.
In the present invention, the food is not limited but may be a milk product, fermented milk, drink, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, ≪ / RTI >
The present invention also relates to a probiotic composition comprising the strain or a culture thereof as an active ingredient.
The present invention also relates to a method for producing a microbial preparation for antifungal agents against harmful bacteria comprising the step of culturing the strain.
The present invention also relates to a method for controlling harmful bacteria by treating the strain or a culture thereof with harmful bacteria.
In accordance with the present invention, it is possible to provide a microorganism which shows antibacterial activity against the harmful bacteria Edwardsiella tarda , Streptococcus iniae or Vibrio ichthyoenteri , The Lactobacillus plantarum mz-1 strain was newly isolated, and it is expected to be useful as a cultured antimicrobial agent which can be utilized in aquaculture environment in an environmentally friendly manner.
Fig. 1 relates to selection of an antimicrobial active microorganism for controlling a pathogenic bacterium of a fish.
Figure 2 relates to the ability of the Lactobacillus plantarum mz-1 to control the phytopathogenic bacterium Edwardsiella tarda .
Figure 3 relates to the ability of Lactobacillus plantarum mz-1 to control against the pathogenic bacterium Streptococcus iniae .
Figure 4 relates to the ability of Lactobacillus plantarum mz-1 to control the phytopathogenic bacterium Vibrio ichthyoenteri .
Figure 5 shows the results of measurement of lysozyme activity, which is a nonspecific immune response in epithelial tissues.
FIG. 6 shows the results of measurement of lysozyme activity as a nonspecific immune response in the stomach tissue.
Fig. 7 shows the results of measurement of lysozyme activity, which is a nonspecific immune response in intestinal tissues.
The present invention relates to a Lactobacillus plantarum mz-1 strain (accession number: KCTC-18401P) having antimicrobial activity against aquaculture harmful bacteria.
The new strains were able to isolate and identify new strains by analyzing the biological sequence similarity of molecular systems in traditional fermented foods, shrimp embryos and inland water farm samples, and named this as Lactobacillus plantarum mz-1 (KCTC) on September 1, 2015 at the Korea Research Institute of Bioscience and Biotechnology.
The present invention also relates to a microbial preparation for antimicrobial activity against a cultured fish harmful bacterium comprising the strain and a culture solution thereof as an active ingredient.
In the present invention, the cultured bacterium is not limited to a strain belonging to the genus Edwardsiella sp., Streptococcus sp. Or Vibrio sp. And preferred examples according to an embodiment of the present invention may be Edwardsiella tarda , Streptococcus iniae or Vibrio ichthyoenteri .
Edward C. Ella Tarda, Edward W., Streptococcus infection, Streptococcus, and Vibriochitiotei are bacteria that cause vibriosis. Using the lactic acid bacteria isolated and identified as described above, they can be used as a microbial agent The optimal conditions for the production of physiologically active substances and metabolites have been established for development.
In the present invention, the composition may be in the form of a food, a food additive, a feed additive or a medicament, though not limited thereto.
In the present invention, the food is not limited but may be a milk product, fermented milk, drink, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, ≪ / RTI >
The present invention also relates to a probiotic composition comprising the strain or a culture thereof as an active ingredient.
The present invention also relates to a method for producing a microbial preparation for antifungal agents against harmful bacteria comprising the step of culturing the strain.
The present invention also relates to a method for controlling harmful bacteria by treating the strain or a culture thereof with harmful bacteria.
Hereinafter, the present invention will be described in more detail by way of examples. However, these embodiments are provided to aid understanding of the present invention, and the scope of the present invention is not limited to these examples in any sense.
[Example 1] Isolation and identification of microorganisms for controlling harmful bacteria
For the isolation of lactic acid bacteria and useful microorganisms, 1 mL of each sample was taken in a conventional fermented food such as sesame leaf chestnut, Chinese cabbage kimchi, Dongchimi, miso, fermented seafood, 7.2) and then serially diluted with a decidual dilution method. 100 μL of the supernatant of each of the diluted samples was evenly spread on MRS (DIFCO, USA), a culture medium for lactic acid bacteria isolation and culture, and TSA (DIFCO, USA), a general bacterial culture medium. The plated plates were incubated at 37 ° C. for 60 hours. A number of microorganisms including lactic acid bacteria were isolated. The isolated strains were stored in a culture medium supplemented with 20% glycerol.
Screening of microorganisms with bactericidal ability of pathogenic bacteria in fish
In order to isolate microorganisms capable of controlling against pathogenic bacteria, Edwardsiella tarda , Streptococcus iniae or Vibrio ichthyoenteri , Edwardsiella tarda FP5060, Vibrio ichthyoenteri FP4004, and Streptococcus iniae FP5228 isolated from flounder were purchased from the National Institute of Fisheries Science and Technology and cultured at 30 ℃ and 30 ℃ using TSB (DIFCO, USA) and MB (DIFCO, USA) After the cells were cultured at 100 rpm for 24 hours, the cells were serially diluted by declining with PBS to 10 5 to 10 7 cells / mL of live cells. Then, the cells were plated on TSB (DIFCO, USA) Respectively.
The separated bacteria were cultured for 24 hours at 30 ° C with a 1-cm-long line, and strains showing a clear zone were selected and selected as microorganisms having antimicrobial activity against fish pathogenic bacteria (FIG. 1).
Through the above process, six species of microorganisms capable of controlling against the pathogenic bacterium Edwardsiella tarda , Streptococcus iniae or Vibrio ichthyoenteri were obtained. MM2-5, MS13, MK-1, MK-3, MK-8 and MO-7 - were selected.
16S rRNA gene sequence analysis
For the identification of the strain, 16S rRNA gene sequencing was used. Homology analysis was performed using the NCBI BLAST (http://www.ncbi.nlm.nih.gov/blast/) for six isolates (Table 1). One of the strains isolated from these strains was identified by genetic analysis and named Lactobacillus plantarum mz-1 and deposited with KCTC on September 1, 2015.
[Table 1]
[Example 2] Culture of Lactobacillus plantarum mz-1
The medium composition included 40 g glucose, 5 g soybean peptone, 20 g yeast extract, 1 g magnesium sulfate, 1 g sodium chloride and 6 g sodium hydrogen phosphate. MRS medium was activated for the system, and the seeds were cultured in a 100 mL flask. The seedlings were cultured at 30 ° C and 135 rpm for 24 hours, and then cultured under the same conditions for 36 hours to obtain 3 L of culture.
For the cultivation of Lactobacillus plantarum mz-1, a 300 L large-scale culture was carried out using a 500 L fermenter. The seeds for culturing were inoculated with 1% of the culture inoculation rate of 3 L obtained above. The seeds were inoculated on the medium and incubated for 60 hours. The cells were recovered by a centrifugal centrifuge at 15,000 rpm at 1000 ml / min. The collected cells were lyophilized for 80 hours using 20% lactose (DIFCO, USA) as an excipient.
The dried cells were homogenized using a pin mill, and then 1 g was diluted by decidualization in PBS. The cells were plated on MRS (DIFCO, USA) medium and cultured at 30 ° C for 48 hours. Respectively.
As a result, the viable cell count of the formulated microorganism preparation was 2.5 x 10 10 cfu / g.
Example 3 Lactobacillus Planta room (Lactobacillus plantarum) the fish when pathogenic bacteria Edward Ella tar mz-1 (Edwardsiella tarda), streptococcus am Ke (Streptococcus iniae) or Vibrio Yeats thio yen Terry (Vibrio ichthyoenteri) Protection against
In order to compare the effect of Lactobacillus plantarum mz-1 powder formulated through Example 2 on fish pathogenic bacterium, an indoor rearing tank similar to the actual farm was installed to measure the dissolved oxygen amount and the water temperature After maintaining the optimum conditions of eel culture at 6.5 mg / L and 25 to 29 ℃, pathogen abatement concentration and mortality of eel fish were measured according to the timing of each hospital bacterium.
Edwardsiella tarda and Streptococcus iniae strains were cultured in TSB medium and Vibrio ichthyoenteri strains were cultured in MB medium respectively and then subjected to PBS decidual dilution method , And the inoculated samples were prepared so that the number of viable cells was 10 8 to 10 9 cells / mL, and the concentration of pathogenic bacteria in the tank was in the range of 10 4 to 10 5 cells / mL (FIGS. 2 to 6 4).
1 g of feed was administered once a day to each experimental group and the formulated Lactobacillus plantarum mz-1 powder was mixed at a weight ratio of 100 times of the above feed, orally administered to the eel's fry, Negative control without administration of the microbial agent of the present invention, and the reduction rate of the mortality and the pathogenic bacteria were confirmed.
As a result, as shown in the following Table 2, the kill rate of the eel crabs was remarkably reduced for the three pathogenic bacteria, and the pathogenic bacteria also showed a high abatement effect.
[Table 2]
[Example 4] Improvement of nonspecific immune response of Lactobacillus plantarum mz-1
In order to improve the growth rate of Lactobacillus plantarum mz-1 and to confirm the efficacy of the feed, two experimental groups according to the administration of Lactobacillus plantarum mz-1 powder to the eel farm located in Buan- , And the fish were stocked with only one experimental group, and the fry of more than 3 pieces were selected from 42 to 148 days after the first stocking, and the epithelial tissues, stomachs and intestines were extracted to perform lysozyme activity analysis.
The weights of the epithelium, stomach and intestine were separately measured, and 0.005 M sodium phosphate buffer (pH 7.4) corresponding to 5 times the weight (v / w) was added thereto, followed by grinding with an organic homogenizer. Thereafter, the supernatant was taken by centrifugation at 4 ° C., and then freeze-dried microcococcus lysodeikticus (Sigma, USA) cells were suspended in the buffer at a concentration of 0.1 mg / mL and used as a substrate. The activity value of the spectrophotometer at the elapse of 0.5 minutes and 5.5 minutes after the start of the experiment was measured at A600 to give an absorbance reduction of 0.001 per minute (Fig. 5 to Fig. 7).
As a result, in the experimental group to which the powder was administered, the lysozyme distribution was 1 to 1.8 in epithelium, 1.36 in average, 1.47 to 6.53 in stomach tissue, 3.91 in average and 8.93 to 17.8 in average and 13.46 in average, The activity of lysozyme was high, which was as high as 16.67 in the viscera (Fig. 7).
From the above, it can be concluded that Lactobacillus plantarum mz-1 preparation has effects such as improvement of growth rate, improvement of digestibility, improvement of immune mechanism, etc., and it is expected that the effect of the increase of immunity is increased.
The present invention relates to a Lactobacillus plantarum mz-1 strain having antimicrobial activity against harmful fishes, a microorganism preparation for antibiosis and a microorganism preparation for growth improvement comprising the same, wherein the microorganism and the microorganism The present inventors have confirmed that the present invention can provide an effective control effect on cultured and harmful bacteria and a remarkable improvement in growth rate and immunity against bacteria through the use of the preparation.
Institution name: Korea Biotechnology Research Institute
Access number: KCTC18401P
Funding date: 20150901
Claims (8)
Wherein the microbial agent is in the form of a food or a food additive.
The food is selected from the group consisting of milk products, fermented milk, drink, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, Wherein the microbial agent is an antimicrobial agent.
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| KR101372200B1 (en) | 2010-12-21 | 2014-03-14 | 한국생명공학연구원 | Lactobacillus plantarum SY99 and Use Thereof |
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| JP2009159955A (en) * | 2007-12-12 | 2009-07-23 | Kyushu Medical:Kk | Probiotics lactic acid bacterium separated from inside of prawn intestine |
| KR20120064416A (en) * | 2010-12-09 | 2012-06-19 | 주식회사 제일바이오 | Lactobacillus plantarum clp-1 strain having anti-virus and anti-bacterial activity and direct-fed microorganisms comprising the same |
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