KR20020013670A - Probiotic preparations for aquacultured fish and its production method - Google Patents

Abstract

PURPOSE: A microbial preparation useful as an additive to feeds for fish farming and a producing method thereof are provided, thereby improving the tolerance to diseases and decreasing lethality of fish. CONSTITUTION: The microbial preparation MGD100(KFCC-11189) useful as an additive to feeds for fish farming consists of Lactobacillus plantarum, Lactobacillus brevis and Enterococcus faecium in a ratio of 1:1:1. The microbial preparation MGD100(KFCC-11189) has antimicrobial activity to Staphylococcus aureus (ATCC6538), Vibrio cholerae (ATCC 14547), Escherichia coli (ATCC 29522), Salmonella enteritidis (IFO 3313), and Pseudomonas aeruginosa (IFO 3080), and is viable under conditions of the temperature of 15 to 25 deg.C and the salt concentration of 1 to 6%. The microbial preparation MGD100(KFCC-11189) is produced by mixing freeze-dried Lactobacillus plantarum, Lactobacillus brevis and Enterococcus faecium.

Description

Probiotic preparations for aquacultured fish and its production method

[Industrial use]

The present invention relates to a microbial preparation for feed additives and a method of manufacturing the same, and more particularly, to promote the anti-histories and promote the development and digestion of fish and to prevent the expression of antibiotic resistant bacteria by using microbial agents instead of antibiotics. And it relates to a method for producing the microbial preparation to establish a culture process to enable high-density cultivation of the separated microorganisms used in the microbial preparation and to use the culture filtrate dried of the microorganisms as a feed additive.

[Private Technology]

Recently, demand and supply of microbial preparations are increasing due to tightening regulations on the use of antibiotics in feed additives. However, the microbial preparations used in feed for fish farms are not suitable for farmed fish growing at 15 ℃ to 25 ℃ by using microorganisms with optimal development temperature of 37 ℃ for human or livestock, and especially in seawater farmed fish. It is impossible.

The development of microbial products that can be grown at low temperature and high salt can contribute to the improvement of fish income level by promoting the anti-medical history, development and digestion of fish, and it can prevent the abuse of antibiotics due to the substitution of antibiotics. Its importance is very important. In addition, by formulating a dry powder without discharging the filtrate and using it as a feed additive, the cost for waste water purification will be reduced and an economical production and manufacturing method will be established.

An object of the present invention is to develop a beneficial microflora in the intestines of fish as part of the development of microbial preparations that can suppress and replace antibiotics in aquaculture, promote growth, as well as increase immunity and disease prevention and water pollution. It is to provide a microbial agent for performing such functions.

1 is a flow chart of a formulation method according to the invention

In order to achieve the object of the present invention as described above, the present invention has been deposited as deposit No. KFCC-11189, used as a feed additive of fish Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus brevis (Lactobacillus brevis), Provided is a microbial preparation MDG100 containing Enterococcus faecium and dried culture filtrate.

The present invention is seeded culture of Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fascium in the culture medium for lactobacillus, and the seed cultured Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fascium are inoculated in the fermenter. Producing the Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fascium by main culture; Centrifuging each of the cultured microbial cultures to obtain a microbial concentrate, and lyophilizing by adding 10% skim milk and 1% maltose; It provides a method for producing the microbial preparation comprising the step of mixing the lyophilized Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fasium and the dried product of the culture filtrate.

Hereinafter, the present invention will be described in more detail.

Considering the growth environment of fish, we developed a microbial preparation for feed additive using microorganisms isolated from low-temperature ripening kimchi. As strains for feed additives, Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fascium were isolated and selected. A culture process was established in which a large amount of isolated microorganisms to be used in the microbial preparations were cultured in high concentrations and the culture filtrate was used.

As a source of low-temperature ripening kimchi as a separation source, 200 single colonies (yellow) forming a yellow ring were first isolated from a solid plate count agar containing Bremocresol Purple. The strain was grown for 2 days at 15 ℃ using MRS medium containing 6.0% NaCl was selected for the second time. The secondary screened strains were cultured in MRS liquid medium for 25 hours at 48 ° C., and then compared to growth inhibitory activity against Escherichia coli using supernatant. Finally, Lactobacillus plantarum, Lactobacillus brevis and Three strains of Enterococcus fascium were finally selected.

Based on the culture conditions and physiological characteristics of each strain selected for the preparation of microorganisms, experiments were conducted to establish a culture process technology for cultivating microorganisms in large quantities.

The composition and culture conditions of the selected medium in consideration of the classification and physiological characteristics of the isolated strain are shown in Table 1 below.

Table 1

In order to know the history of the microorganisms selected as microbial agents (hereinafter, deposited strains), antimicrobial activity was investigated by agar diffusion using a circular filter. Instructions bacteria on antibacterial activity of the culture is Staphylococcus Cocos brother Julius (Staphylococcusaureus) ATCC6538, Vibrio cholera (Vibrio cholerae) ATCC14547, Escherichia coli (Escherichia coli) ATCC29522, Salmonella Entebbe utility disk (Salmonella enteritidis) IFO3313, Pseudomonas Ke Ryuji Pseudomonas aeruginosa IFO3080 was used. After solidification by dividing the tryptone and soy flour a solid medium (Tryptic soy agar) containing 10ml of the culture plate to the section assignment ion medium 24 hours the culture broth as indicated at 25-30 ℃ (LB medium) thereon; ⁵ Layer 2 ml of solid nutrient medium (containing agar 0.4%) added to -10 ⁶ / ml of bacteria, and then put a circular filter (diameter 8mm) on which the culture solution and supernatant preparation of the deposited strain were buried. The incubation width of the indicator was measured when incubated for 24 hours at ℃ and compared with 0.1% amoxicillin as a control antibiotic. The results of measuring the antimicrobial activity of Lactobacillus brevis showed that the antimicrobial activity of Staphylococcus aureus (S.aureus) in the culture, culture supernatant, and supernatant neutralizing samples was greater than the 50 μl drop effect of the control drug. In the case of biprio cholera , the culture sample showed the same effect as the drop amount of 30 μl of the reference drug, and the control drug was excellent in the overall Escherichia coli (E.coli) . In the case of Salmonella enteritidis, 50 μl of the culture sample was equivalent to that of 10 μl of the control drug, and P. aeruginosa showed the same antimicrobial activity as 50 μl of the culture sample.

Enterococcus hwesi antimicrobial results compared to the control drug of measuring the activity of the Titanium For star fatigue Cocos brother Julius showed a culture solution and culture supernatant and the supernatant was neutralized sample including both the control drug 10㎕ antimicrobial activity than one antimicrobial activity represents the amount of drip V. cholera In case of all three samples, the effect of 30µl drop of the reference drug was similar. In the case of Salmonella enteritidis, all three samples were similar to the effect of 10 μl drop of the reference drug. For Pseudomonas aeruginosa, 50 μl of the culture sample was equivalent to the effect of 50 μl of the control drug.

As a result of measuring the antimicrobial activity of Lactobacillus plantarum, in the case of Staphylococcus aurius , the culture sample was equivalent to the 30 μl drop of the reference drug and the supernatant showed the same result as the 10 μl drop of the control drug. . In the case of Vibrio cholera , the culture sample was equivalent to the drop amount of 30 µl of the reference drug. In the case of Escherichia coli , the control drug was excellent overall. In the case of Salmonella enteritidis, the effect of the culture sample was similar to that of the control drug 30 μl. In the case of Pseudomonas aeruginosa, the culture and supernatant samples showed an equivalent effect to the effect of the 50 μl drop of the control drug.

Deposited strains were cultured in MRS medium for Vibrio cholera bacteria for 24 hours in 2.5g NaCl-containing saengdangbuion medium (LB medium), and then the test group was placed in MRS medium containing 2.5% NaCl and 10 strains of Vibrio cholera bacteria. ⁶ / ml of the bacteria were inoculated and mixed, and the control group was inoculated with only Vibrio cholera bacteria in the lyse-coated ions medium (LB medium) containing 2.5% NaCl. Each inoculated medium was cultured at 25 ° C., and cultured at 6 hour intervals and diluted with sterile saline solution. Incubated for 48 hours at ℃. After incubation, the number of Vibrio cholera bacteria was measured from the colonies shown on the lactose liquid plate medium, and the viable cell counts of the deposited strains were measured from bromocresol purple (BCP) medium.

The growth inhibition and bactericidal activity of the Vibrio cholera strains of Lactobacillus brevis strains were normal, but the growth of the vibrio cholera alone was normal. After 30 hours, the number was drastically reduced to 1 × 10 ² / ml. However, Lactobacillus brevis strain showed a viable cell number of 9 × 10 ⁸ / ml.

The growth inhibition and bactericidal activity of the Vitroo cholera strains of Enterococcus fascium strains were normal, but the Vibrio cholera cultures showed normal growth. At 48 hours, the viable cell number was decreased to 80 / ml, but Enterococcus fascium maintained the viable cell count of 9.5 × 10 ⁸ / ml.

Growth inhibition and bactericidal activity of the Vibrio cholera bacteria of Lactobacillus plantarum strains were normal, but growth was normal in the Vibrio cholera alone culture but up to 18 hours after inoculation with Lactobacillus plantarum. After 18 hours, the number of bacteria began to decrease rapidly, and after 48 hours, no Vibrio cholera bacteria appeared, but the Lactobacillus plantarum bacteria maintained a viable cell count of 6.3 × 10 ⁸ / ml.

The microorganisms and Lactobacillus acidophilus selected as the microbial preparations were MRS medium, Lactobacillus, in comparison with the type of microorganism used as a livestock probiotics in the environment of low temperature and high salt of deposited bacteria. Lactobacillus sporogenes is bromocresol purple (BCP) agar, Lactobacillus bifidus is BL agar, Clostridium butyricum is fluid thioglycolate medium, Bacillus subtilis observed growth in starch medium. The results are shown in Table 2 below.

The deposited strains were incubated in a 6 L fermentation tank (NBS) using an optimal production medium and then centrifuged to recover the cells. The recovered cells were homogenized by mixing with a lyophilized protective agent consisting of skim milk (10% skim milk) and maltose (1%), and then frozen at -40 ° C and dried in a lyophilizer (IL Shin Lab). The dried cells were pulverized, powdered, diluted in sterile physiological saline, plated in bromocresol purple medium, incubated at 25 ° C. for 48 hours, and viable cells were measured for viability before freeze-drying. Considering the number 100%, the survival rate after lyophilization was determined to be 60% or more.

Artificial gastric juice was used by adding 1% pepsin to the MRS liquid medium adjusted to pH2.5 using HCl according to the method of Kobayashi et al. 0.1g of lyophilized cell powder was placed in 10ml of artificial gastric juice and left at 25 ° C for 1 hour. The cell powder treated in artificial gastric juice was diluted in sterile physiological saline solution and plated in bromocresol purple medium, and the number of colonies generated after incubation at 25 ° C for 48 hours was measured and compared with the number of viable cells before gastric juice treatment. The survival rate was over 60% when the survival rate was 100%.

The formulation method according to the invention is shown in FIG. 1.

EXAMPLE

Preferred examples and comparative examples of the present invention are described. However, the following examples are merely examples of the present invention showing the constitution and effects of the present invention, and the present invention is not limited to the following examples.

Experiments were performed to administer the microbial preparations according to the present invention to the flounder, and the growth rate and mortality rate of the fish were investigated. Aquaculture experiments were conducted on flounder at the seawater farm. 316 flounder were bred for 50 days at water temperature of 15 ° C-25 ° C.

In this example, the microbial preparation was mixed daily with 1 g of feed per kg.

The same practice was followed except that the microbial agent was not fed in this example.

In the Examples and Comparative Examples, the weight gain of the flounder according to the passage of time was examined, and the results are shown in Table 3.

As shown in the table, an average weight gain of 17.9% was observed in the comparative example for 50 days, while in the example, a 27.0% increase was observed, and an increase of 50% was observed in the example. That is, the weight gain of the fish of the embodiment reaches 1.5 times higher than the weight gain of the fish of the comparative example. This is because the administered microorganisms and cultured dry foods enhance intestinal function and increase digestibility.

In the Examples and Comparative Examples, the mortality of flounder over time was examined and the results are shown in Table 4 below.

As shown in the above table, the mortality rate of 5% was shown in the comparative example while the mortality rate of 0% was shown in the example, and the survival rate was excellent in the example. This may be because the microorganisms administered were excellent in antimicrobial activity against the bacteria causing fish disease and the anti-history was enhanced and the fish did not cause disease.

The microbial preparation of the present invention is economical for promoting growth and replacement of antibiotics by enhancing the anti-pathology of fish and improving digestion efficiency.

Claims (4)

Microbial agent MGD100, deposited under No. KFCC-11189, consisting of a 1: 1: 1 mixture of Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fascium, used as feed additives for fish. Staphylococcus aureus ATCC6538, Vibirio cholerae ATCC14547, Escherichia coli ATCC29522, Salmonella enteritidis IFO3313, Pseudomonas aeroinosa auginosa 30 Microbial agent MDG100 and culture medium dried having antimicrobial activity against. Microbial preparation MDG100 capable of growing under environmental conditions of 15 ℃ -25 ℃ and 1-6% salinity. Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fascium were cultured in a culture medium for lactobacillus culture, and the seed cultured Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fascium were inoculated into a fermenter and incubated. Producing the Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fascium; Centrifuging each of the cultured microbial cultures to obtain a microbial concentrate, and lyophilizing by adding 10% skim milk and 1% maltose; Mixing the lyophilized Lactobacillus plantarum, Lactobacillus brevis and Enterococcus fascium with the culture filtrate dried;