JP2006158396A - Novel lactic acid bacterium strain for producing cactus lactic acid fermentation product and method for producing cactus fermentation product by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel lactic acid bacterium strain obtained from fruit of cactuses, particularly Opuntia ficus-indica, a method for producing cactus fermentation product by using the same and a method for growing flatfish by using the fermentation product. <P>SOLUTION: The method for producing cactus fermentation product comprises at least a step where cactus is fermented by using Lactobacillus plantarum CNU001 (KCTC18105P). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel lactic acid bacterium strain obtained from cactus, in particular, prickly pear fruit, a method for producing a lactic acid bacterium fermentation product of cactus using the same, and a flounder breeding method using a lactic acid bacterium fermentation product produced by the production method About.

  Prickly pear (Opuntia ficus-indica), also called a perennial in Korea, is a native plant of Jeju. Since it has the property of growing well even in relatively barren soil, it is widely cultivated in wasteland that is difficult to open and contributes as a small income source for farmers.

  As typical effects of prickly pear cactus, antibacterial and antiviral are known, and in recent years, an increase in immune function and an antidiabetic effect have also been reported. Such an effect can be obtained by ingesting prickly pear fruit as it is or processing prickly pear as it is. In addition, prickly pear cactus is disadvantageous in terms of efficient intake of active ingredients because the active ingredient is covered with mucus, and it is unpleasant for ordinary humans because of its unique odor and high viscosity when taken. Is invited. As a method for solving this problem, a functional beverage has been developed in which an extract is extracted from prickly pear fruits and stems and fructose is added thereto.

  However, when extracting the extract, its high viscosity not only makes it difficult to mix evenly when added as an additive to functional materials for other uses such as aquaculture feed, but also the recovery rate is low. However, there is a drawback that the active ingredient contained in the fiber is lost.

  In order to solve such problems, the stage of selecting and washing the prickly pear fruit, the stage of grinding the cactus fruit with a mixer to obtain a homogeneous liquid, and the homogenous liquid such as molasses or syrup A step of kneading an effective microorganism preparation such as a fermentation accelerator and lactic acid bacteria, a step of performing fermentation at room temperature for 2 weeks or more to obtain a fermentation broth, and a step of freeze-drying the fermentation broth to form a powder And a method for producing a fermented cactus product and a method for cultivating flounder using the same (for example, see Patent Document 1 below).

However, effective microorganisms used in the above technology are not excellent in fermentation performance, and as a result, there has been an increasing demand in the art for effective microorganisms having superior fermentation ability.
Korean Patent No. 2003-94879

  Therefore, the present inventors are further excellent in the performance of fermenting cactus, in particular, prickly pear cactus, and in raising flounder using the obtained fermented cactus, a novel that can further promote the growth of flounder We conducted intensive research to find new microorganisms. As a result, the present inventor produced a fermented product of cactus, in particular, prickly pear cactus, using Lactobacillus plantarum CNU001 taken from the fruit of the cactus, and raised flounder using the produced fermented product as a feed additive. Then, it was found that the growth of flounder was so high that it was astonishing, and the present invention was completed.

  Accordingly, an object of the present invention is to provide a novel lactic acid bacteria strain taken from cacti, in particular, prickly pear fruit.

  Furthermore, another object of the present invention is to provide a method for producing a fermented lactic acid bacterium of cactus using the novel lactic acid bacterium strain.

  Still another object of the present invention is to provide a method for breeding flounder using a fermented lactic acid bacterium produced using the novel lactic acid bacterium strain.

  In order to achieve the above-mentioned objects, the present invention provides a novel microorganism having excellent performance for fermenting cacti, particularly prickly pear cactus, and Lactobacillus plantarum CNU001 (KCTC18105P) taken from the fruit of cactus. provide.

  When microorganisms are used to ferment cactus, especially prickly pear cactus to produce a cactus fermented product and this is used to raise flounder, it is possible to further promote the growth of flounder. It can be used as an agent.

  Lactobacillus plantarum CNU001, a novel microorganism discovered by the present inventors, was established in the Korea Biotechnology Research Institute's Biological Resource Center located at No. 52, Goseong-dong, Daegu-gu, Daejeon, Korea, October 2004 The strain was deposited on the 14th and was given the deposit number of the strain KCTC18105P.

  By using Lactobacillus plantarum CNU001 taken from cactus fruit according to the present invention as a strain, producing a fermented product of cactus, particularly prickly pear cactus, and breeding flounder using the produced fermented product, Since the growth of Japanese flounder is large enough to keep an eye out, the cactus fermented extract using the strain of the present invention can be used as a feed additive for various animals.

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

  Lactobacillus plantarum CNU001 as a novel microorganism developed by the present inventors can be cultured in an MRS liquid medium or agar medium for Lactobacillus well known in the art. Regarding conditions and methods for culturing microorganisms according to the present invention, culture conditions and methods well known in the art for culturing Lactobacillus plantarum can be used. For example, Lactobacillus plantarum CNU001, which is a microorganism of the present invention, can be cultured based on the method described in Example 1.

  Further, Lactobacillus plantarum CNU001, which is a microorganism according to the present invention, is a Neisseria gonorrhoeae and is stained by Gram positive and exhibits a catalase negative reaction. Furthermore, the microorganism of the present invention exhibits sugar fermentability having 99.9% homology with Lactobacillus plantarum (ATCC 14917). More specifically, the microorganism CNU001 according to the present invention includes L-arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, sorbitol, α-methyl-D-mannoside, N-acetyl-glucosamine, amygdalin, arbutin, Sugar sources such as esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose, raffinose, and D-turanose can be used.

  Furthermore, the strain according to the present invention has 99% similarity in the sequences of Lactobacillus plantarum DSM20205 and 16S rRNA.

  However, although the microorganism CNU001 according to the present invention has considerable similarity as compared with the Lactobacillus plantarum species, it is compared with the conventionally known Lactobacillus plantarum species or many other lactic acid bacteria. Thus, it exhibits extremely excellent performance in fermentability of cacti, particularly prickly pear cactus, and also shows a considerable improvement effect from the aspect of growth effect of Japanese flounder using the produced cactus fermented product. The effect of improving the fermentability of microorganisms and the growth of Japanese flounder has not been reported to the industry so far.

  The cactus using the microorganism Lactobacillus plantarum CNU001 according to the present invention, in particular the prickly pear cactus, is almost the same as the conventional method for producing a cactus using a lactic acid bacterium, particularly a prickly pear cactus, for example, It can be produced in the same manner as the method described in Patent Document 1. For example, a step of selecting and washing the prickly pear fruit, a step of grinding the cactus fruit with a mixer to obtain a homogeneous liquid, a fermentation accelerator such as molasses or syrup and the like according to the present invention A step of mixing Lactobacillus plantarum CNU001 as a strain, a step of performing fermentation at room temperature for 1 minute to 2 weeks or more to obtain a fermentation broth, and freeze-drying the fermentation broth A method comprising the step of powdering is used for the production of the cactus fermentation. In the production, fermentation of cactus lactic acid bacteria, such as the amount of Lactobacillus plantarum CNU001 used as a strain for fermentation, the amount of use of a fermentation accelerator such as molasses or syrup that can be used, the fermentation conditions, the method for producing freeze-dried powder, etc. Various conditions for obtaining a product can be referred to from the above-mentioned Patent Document 1, or can be obtained through a normal experiment.

  On the other hand, according to the present invention, as a problem that occurs when cactus, particularly prickly pear cactus, is extracted and used, there are inconveniences in use or storage due to high viscosity, and functional substances for other uses such as aquaculture feed. Examples include uneven mixing when used as an additive, a low recovery rate of the active ingredient in the cactus, and disappearance of the active ingredient in the cactus. These problems can be avoided by using Lactobacillus plantarum CNU001 as a lactic acid bacterium according to the present invention.

  Furthermore, according to the present invention, Lactobacillus plantarum CNU001 as a strain according to the present invention has extremely high viability and is extremely excellent in intestinal colonization ability. In this case, the strain CNU001 according to the present invention is present in the fermented product in a living state. Further, in the Japanese flounder ingested with such a cactus fermented product, the strain CNU001 according to the present invention can be transmitted to the intestine in a live state and can be established. Change the microflora. As a result, their beneficial effects can improve the intestinal health of the flounder, which results in a significant improvement in flounder breeding efficiency.

  On the other hand, the cactus fermented product produced using the strain according to the present invention can be usually used as a feed additive for flounder culture.

  When the cactus fermented product according to the present invention is used as a feed additive, it exhibits a high antibacterial effect against various microorganisms that should avoid infection, such as streptococci, Edwards, and Vibrio, when cultivating Japanese flounder. By using such a cactus fermented product according to the present invention, it is possible to improve the flounder breeding environment and obtain a high breeding effect.

  Further, when the cactus fermented product is used as an additive for feed, the cactus fermented product exhibits an antiviral action such as inhibition of the growth of EHV virus and osmotic gastrointestinal virus.

  Due to the above advantages, the cactus fermented product according to the present invention produced using Lactobacillus plantarum CNU001 as a novel strain is useful as a feed additive for Japanese flounder.

  On the other hand, a technique for producing a feed using the fermented cactus according to the present invention as an additive for feed includes a method known in the art for producing a feed by adding an additive known in the art to the feed. It can be manufactured in the same manner.

  When flounder is cultivated using a feed containing the cactus fermented product according to the present invention as a flounder feed additive, the flounder shows a considerable growth effect in both the fry stage and the adult stage.

  The amount of the cactus fermented product added to the feed varies depending on the production method of the feed, the animal to be reared, the health condition of the animal, and the like. Those skilled in the art will recognize appropriate addition amounts by repeating experiments.

  The cactus fermented product according to the present invention can be used as an additive for flounder feed. In addition to flounder, pets such as dogs and cats, aquatic animals such as various fishes, various reptiles and various amphibians It can also be used as an additive for various animal feeds including various insects, and these are also included within the scope of the present invention.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in full detail, it cannot be overemphasized that this invention is not limited by this.

  In the following examples, cactus means prickly pear cactus unless otherwise specified.

Example 1. Separation and identification of novel lactic acid bacteria 1) Isolation of bacteria Aseptically homogenized and squeezed naturally ripened cactus fruit, stepped with the following sterilized physiological saline (0.9% NaCl) And then smeared on MRS medium supplemented with 0.5% CaCO ₃ and cultured at 30 ° C. for 3 days. After 3 days of culture, the strain with the clearest ring formed around the strain was taken. The strain thus obtained was subcultured in the same medium to obtain a single strain. This strain was confirmed to be of the genus Lactobacillus plantarum through subsequent experiments, and this strain was named Lactobacillus plantarum CNU001 (hereinafter also abbreviated as “CNU001”). The strain was deposited on October 14th, 2004 at the Korea Biotechnology Research Institute Biological Resource Center, 52, Uodong-dong, Daegung-gu, Daejeon, South Korea. As a result, the deposit number of strain KCTC18105P was deposited. Was given.

2) Culture of CNU001 strain Lactobacillus MRS liquid medium and Lactobacillus MRS agar medium (Difco Laboratories, MD, USA) were used for culturing the isolated strain CNU001. In the case of a liquid medium, 55 g of a medium was placed in 1 L of distilled water and boiled for 2 minutes to completely dissolve all the compounds, and then sterilized at 121 ° C. for 15 minutes. In the case of a solid medium, 70 g of the medium was placed in 1 L of distilled water and boiled for 2 minutes to completely dissolve all the compounds, and then sterilized at 121 ° C. for 15 minutes. The culture was prepared by inoculating a 500 mL Erlenmeyer flask with a single colony of the isolated strain in a liquid medium separated by 100 mL and culturing in shaking water at 30 ° C. and 150 rpm for 2 to 3 days. .

3) Identification of isolated strain CNU001-Bacterial shape characteristics Using a Lactobacillus MRS liquid medium, a portion of a liquid culture cultured at 30 ° C for 2 to 3 days was examined for shape characteristics using a microscope. However, it became clear that it was a koji mold. In addition, as a result of Gram staining, it was revealed that it was a Gram positive bacterium. Furthermore, as a result of examining the presence or absence of catalase production, a catalase negative reaction was shown.

-Physiological characteristics of bacteria and utilization of carbon source In order to investigate the physiological characteristics of strains and the availability of carbon sources, the API 50 CHL (Biomerieux, France) system was used, and the sugar fermentation characteristics were examined according to the manufacturer's guidelines. I tried. The isolated strain is cultured at 30 ° C. for 3 days, and then inoculated into the substrate of the Lactobacillus kit. The resulting sugar fermentation characteristics of the isolated strain are shown in Table 1 below. As shown in Table 1 below, the CNU001 strain was found to have 99.9% homology with Lactobacillus plantarum. In Table 1, ATCC14917 species are Lactobacillus plantarum species.

-Base sequence analysis of fungal 16S rRNA The isolated strain was inoculated into Lactobacillus MRS liquid medium (Difco, USA), cultured at 30 ° C for 18 hours, and then centrifuged at 10,000 xg for 10 minutes. Got. Thereafter, genomic DNA was isolated using NucleoSpin Tissue Kit (ClonTech Lab., Inc.) according to the manufacturer's guidelines. PCR was performed using 27f (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1522r (5'-AAAGAGGTGATCCARCCGCA-3') primers to amplify 16S rDNA from the separated total DNA. The conditions for PCR are as follows. After denaturation at 95 ° C. for 5 minutes, the mixture was circulated 33 times at a cycle of 95 ° C.-1 minute, 55 ° C.-1 minute, 72 ° C.-1 minute, and finally further reacted at 72 ° C. for 10 minutes. . The amplified PCR product was confirmed by electrophoresis on a 1% agarose gel. Check PCR products after ligated to the QIAEX ^R II Gel Extraction was recovered and purified using a kit pGEM-T Easy Vector (Promega , Madison, WI), it was transformed into E. coli JM109. Then, after culturing at 37 ° C. for 18 hours using 40 μg / mL X-gal and IPTG and 50 μg / mL ampicillin-containing LB agar medium, white colonies were selected. Subsequently, the gene sequence was analyzed using SP6 and T7 primers. The result of the analyzed gene sequence is shown in FIG.

  Analysis of the gene sequence was performed with BigDye Cycle Sequencing kit (PE Applied Biosystems, Foster City, CA) and ABI Prism 310 automatic sequencer (PE Applied Biosystems, Foster City, CA). At this time, the base sequence was analyzed using DNA starprogram and the blast search database of Gene Bank. In order to clarify the phylogenetic relationship of bases, analysis was performed using Lasergene's Megalign program. The method used at this time was a Clustal method. The results of identification by analysis of the 16S rDNA base sequence are shown in FIG. As shown in FIG. 1, it was revealed that the CNU001 strain has 99% homology with Lactobacillus plantarum DSM20205.

Example 2 Characteristics of Isolated Lactic Acid Bacteria CNU001 1) Verification of Cactus Fermentation Ability of Isolated Strain After removing the cactus seeds, they were washed with water. Next, 1 L of distilled water was added to 10 kg of cactus fruit that had been thorned and boiled at 90 ° C., and then squeezed to remove solids to obtain fruit juice for fermentation.

  The fruit juice for fermentation of the cactus thus obtained was fermented using Lactobacillus plantarum CNU001 as the strain of the present invention. That is, 100 mL of sterilized MRS liquid medium was inoculated with 1 mL of the strain solution and cultured at 30 ° C. for 20 hours. This culture broth was inoculated 0.2% at a time into cactus fruit juice and dispensed into 2 L culture bottles and sealed. Subsequently, while culturing at 25 ° C. for 7 days, pH, sugar content, proper acidity, the number of lactic acid bacteria, and the like were examined for each fermentation period.

-Sugar content by fermentation period The sugar content by fermentation period was measured with respect to the obtained fermentation broth using a refractometer (Hand refractometer, No. 507-1, NOW, Japan). The result is shown in FIG.

  As shown in FIG. 2, the sugar content during the culture period showed only a slight change from 4.0 Brix to 3.2 to 3.4 Brix, and no significant change was observed.

-Measurement of pH and total acidity of fermentation broth according to fermentation period The pH was measured with a pH meter by taking a certain amount of the sample. In addition, after adding 5 mL of sterilized water to 5 mL of fermentation liquid, the total acidity was dripped to pH8.3 with 0.1N NaOH, and was calculated based on the following formula. The result is shown in FIG.

  Total acidity = (0.1N NaOH consumption (mL) × F × 0.009 / sample volume) × 100

  As shown in FIG. 3, when fermentation was in progress, the pH decreased rapidly from the first day of culture, and the pH at the end of fermentation was pH 3.50. The change in total acidity produced acid at high speed as the fermentation progressed, and the acidity was 0.85% at the end of the fermentation.

-Measurement of the number of lactic acid bacteria 0.5 mL of the fermented liquid thus obtained was suspended in 4.5 mL of a 0.9% NaCl solution and diluted to 10-fold units. Thereafter, the cells were cultured for 3 days at 30 ° C. by a pour plating method using Lactobacillus MRS agar medium containing 0.5% CaCO ₃ , and the colonies were bright and relatively clear (clear zone). The number of things that formed was measured. The same experiment was repeated three times to calculate the average value, and the result is shown in FIG.

  As shown in FIG. 4, the change in the number of lactic acid bacteria was measured logarithmically while inoculating CNU001 strain into cactus fruit juice and fermenting at 25 ° C. Furthermore, when the growth characteristics according to the fermentation period were examined under the conditions of each inoculation amount of 6.1 to 6.9 CFU / mL, it significantly increased in one day of culture and reached 8.4 CFU / mL. From the first day to the end of fermentation, the level was almost similar.

-Analysis of sugar and organic acid of fermentation broth After centrifuging the fermentation broth thus obtained, the supernatant was taken and passed through a 0.2 µm millipore membrane filter. With respect to the filtrate, saccharides in the fermentation broth were analyzed and quantified using HPLC. The conditions at this time are as follows.
・ HPLC: Water-carbohydrate column (Water carbohydrate column)
・ Detector: ELSD2000
- eluent: _{acetonitrile:} H 2 O (75:25)
・ Flow rate: 1.0 mL / min ・ Injection volume: 10 μL

  The organic acid in the fermentation broth was analyzed using a Supelcogel TMC-610H column. Furthermore, the detector was UV (210 nm), the eluent was 0.1% phosphoric acid, the flow rate was 0.5 mL / min, and the injection volume was 5 μL. The results are shown in FIGS.

  As shown in FIG. 5, when the change in the sugar content with the fermentation period was analyzed, the glucose content rapidly decreased from 4.6 mg / mL to 1.0 mg / mL until the third day of fermentation. Since then, it has decreased gradually. Fructose tended to decrease gradually as fermentation progressed. And the content of sucrose was almost unchanged.

  Furthermore, as shown in FIG. 6, citric acid, malic acid, lactic acid, acetic acid and the like were mainly separated according to the change in the content of organic acid in the cactus squeezed juice fermented with Lactobacillus plantarum CNU001. . Citric acid was about 1.8 mg / mL before fermentation, but there was little change during the fermentation process. In the case of lactic acid, it was not detected before fermentation, but increased as fermentation progressed. In particular, after the end of fermentation, the lactic acid content was 3.87 mg / mL.

Example 3 Production of Cactus Fermented Product Using CNU001 Fermentation was carried out over 3 to 4 days after inoculating lactic acid bacteria after adjusting the cactus fruit juice to pH 6.6 before fermentation. The maltose was added to the fermentation broth to a concentration of 10% and then homogenized. Next, skim milk was added to the fermentation broth as a cryoprotectant at a concentration of 10%, and then mixed and homogenized. Subsequently, it was frozen at −70 ° C. for 3 days and dried in a freeze dryer. The dried sample was pulverized into a powder.

Example 4 Component analysis of cactus fermented product obtained using CNU001 In order to confirm the fermentation efficacy of the strain of the present invention, the contents of various components before and after fermentation of cactus using the strain CNU001 according to the present invention were analyzed.

  Using Lactobacillus plantarum CNU001 strain as a fermenting strain, content analysis of all components of cactus before fermentation and after fermentation, amino acid components and lipid components was performed, and the results are shown in Tables 2, 3 and 4 below, respectively.

  As is clear from the results shown in the table below, it was found that the fermentation using the strain of the present invention significantly reduced the content of mucous components such as fibers in cactus, which had been regarded as a problem in the past. It was.

  Tables 2-2, 3-2 and 4-2 show the contents of all components of cactus, components of amino acids and lipid components analyzed after lyophilization after fermentation of cacti, respectively.

Embodiment 5 FIG. Distribution of intestinal bacterial flora in the administration group in which the cactus fermented broth produced using the CNU001 strain was administered to Japanese flounder and in the control group in which this was not, this example uses Lactobacillus plantarum CNU001 as the strain of the present invention. The cactus fermented product was manufactured according to Example 3, and the manufactured fermented product was added to EP feed (Nisshin Marubeni Feed, Japan) at a concentration of 1% by weight, and this was then ingested by Japanese flounder for about 3 months. This was performed to confirm how much the strain of the present invention was established in the intestine of the flounder when the strain was administered to the flounder.

The result is shown in FIG. That is, as shown in FIG. 7, after the cactus fermented product fermented with Lactobacillus plantarum CNU001 strain was continuously administered to Japanese flounder, the change in the intestinal bacterial flora of the Japanese flounder was measured by the viable count. In the control group, only normal intestinal bacteria were mainstream, but in the case of the test group to which the fermented product was administered, the number of intestinal bacteria was reduced by about 23.6% compared to the control group, Lactic acid bacteria were present at a rate of 3.3 Log ₁₀ CFU / g intestine.

Example 6 Antibacterial Effect of Fermented Cactus According to the Present Invention In this example, an experiment was conducted to confirm the antibacterial effect observed in Japanese flounder (Paralichthys olivaceus) when the flounder was ingested with the cactus fermented product produced according to Example 3. It was. As bacteria for which the antibacterial effect is confirmed, streptococci, Edward bacteria, and Vibrio bacteria, which are required to be sterilized during the cultivation of Japanese flounder, were used.

1) Streptococcus In order to examine the effect of the cactus fermented product on the growth of Streptococcus, the growth rate of the bacteria in the liquid medium was comparatively analyzed. First, the freeze-dried cactus fermentation product produced according to Example 3 was suspended in BHI (Brain Heat Infusion, Difco) so that the final concentration was 2% (w / v), and then at room temperature. Mix for 30 minutes, then adjust the pH to 7.0. This mixture was put in an ice box, and sonication for 15 seconds and standing for 15 seconds were repeated 8 times. After sonication, insoluble substances were removed with filter paper, and then passed through a syringe filtration sterilizer (0.45 μm pore size), and the results were used as an experimental group. BHI to which no cactus fermented product was added was used as a control group.

The experimental group and the control group were inoculated with streptococci previously cultured in BHI for 18 hours to a level of 1.2 × 10 ⁴ CFU / mL, then inoculated with standing at 22 ° C., then 0, 2, 4 The number of streptococci was measured at 8, 12, and 24 hours. In the experimental group and the control group, 100 μL of the culture solution was diluted in sterilized physiological saline at each measurement period, and then 2% NaCl was added to TSA (Tryptic Soy agar, Difco). ). Each experiment was repeated twice, and the results are shown in Table 5 below and FIG. As shown in the following Table 5 and FIG. 8 (A), an extremely high bacterial growth inhibitory effect was recognized as compared with the control group. That is, in the case of the control group to which no cactus was added, the bacterial count of the original 4.1 log ₁₀ CFU / mL grew to 9.2 log ₁₀ CFU / mL after 24 hours, whereas the cactus fermentation product In the added experimental group, the number of bacteria was 4.7 log ₁₀ CFU / mL after 24 hours of culture, and no bacterial growth was observed.

  Moreover, the pathogenicity experiment of streptococci was conducted as follows. The flounder used for the experiment is 6cm in size, and is bred with 1.0% cactus fermented feed for 3 months in the FRP round water tank (φ74cm × 80cm) set up at the Institute of Marine Fisheries Resources in Jeju What was done was used. During the breeding period, the breeding water temperature is 17.4 to 21.6 ° C., the salt concentration is 33.0 to 35.4 ‰, DO 5.84 to 7.12 mg / L, and the pH is 7.57 to 8.04. It was in range.

Specific experimental methods are described below. That is, flounder fed with feed containing fermented cactus (EP feed containing cactus fermented at a concentration of 1% by weight) and flounder fed with EP feed were divided into 5 experimental groups and 5 control groups, respectively. Separately arranged. Each group consists of 4 flounder. After culturing the activated streptococci in BHI liquid medium, 9.8, 9.8 × 10 ² , 9.8 × 10 ⁴ , 9.8 × 10 ⁶ per mL. Then, 0.1 mL of 9.8 × 10 ⁸ streptococci was inoculated into the peritoneal cavity. While bred for 14 days after inoculation, the presence or absence of drowning and appearance symptoms were confirmed daily. Dying and expression fish were dissected and observed for lesions, and bacteria were cultured in the liver and kidney. The results are shown in Table 6 below.

As shown in Table 6 above, in the case of Japanese flounder that did not take the feed with the cactus fermented product, 78% of the control group inoculated with 1 × 10 ⁹ viable cells (streptococci) died in 9 days. The control group inoculated with 1 × 10 ³ viable cells died 50% on day 12, and the control group inoculated intraperitoneally with 1 × 10 ¹ viable cells died 25% on 10 days. Moreover, in the control group inoculated with 1 × 10 ⁷ viable cells, although there was no moribund one, ascites and eyeball protrusion were seen as clinical symptoms, and causative bacteria such as inoculum from liver, kidney, etc. It was detected.

However, in the case of Japanese flounder fed with cactus fermented feed, the experimental group inoculated with 1 × 10 ⁹ viable cells was killed by 50% in 8 days, and the experimental group inoculated with 1 × 10 ⁷ viable cells Only 25% drowned in 13 days.

  Thereby, it became clear that the cactus fermented extract according to the present invention has an antibacterial effect against cocci.

2) Edward bacteria In order to examine the effect of the cactus fermented product on the growth of Edward bacteria, the growth rate of bacteria in a liquid medium was comparatively analyzed. First, the freeze-dried cactus fermented product produced according to the method of Example 3 was suspended in BHI so that the final concentration was 2% (w / v), and then mixed at room temperature for 30 minutes to adjust the pH to 7 Adjusted to 0.0. This mixture was put in an ice box, and sonication for 15 seconds and standing for 15 seconds were repeated 8 times. After sonication, insoluble substances were removed with filter paper, and then passed through a syringe filtration sterilizer (0.45 μm pore size), and the results were used as an experimental group. BHI to which cactus was not added was used as a control group.

The experimental group and the control group were inoculated with streptococci previously cultured in BHI for 18 hours at a level of 1.2 × 10 ⁴ CFU / mL, then allowed to stand at 22 ° C., and after inoculation, 0, 2, 4, The number of streptococci was measured at 8, 12 and 24 hours. The number of bacteria is measured in the experimental group and the control group by diluting 100 μL of the culture solution into sterilized physiological saline at each measurement time, and then smearing it on a BHI agar medium supplemented with 2% NaCl. It was broken. Each experiment was repeated twice, and the results are shown in Table 7 below and FIG. 8 (B).

As shown in Table 7 and FIG. 8 (B), the case of the control group, after culturing 12 hours 8.4log ₁₀ CFU / mL, 24 hours later showed the number of bacteria 11.0log ₁₀ CFU / mL, prickly pear fruit for fermentation powder was added experimental group showed 9.5log ₁₀ CFU / mL count bacteria 4.5log ₁₀ CFU / mL, 24 hours after 12 hours of culture. From this, it was confirmed that the growth-inhibiting effect of Edward bacteria lasts up to 12 hours.

  In addition, Edward's pathogenicity experiment was conducted as follows. The flounder used for the experiment is 6cm in size, and is bred with feed containing 1.0% cactus fermented product for 20 days in the FRP round water tank (φ74cm × 80cm) set up at the Institute of Marine Fisheries Resources in Jeju What was done was used. During the breeding period, the breeding water temperature is 17.4 to 21.6 ° C., the salt concentration is 33.0 to 35.4 ‰, DO 5.84 to 7.12 mg / L, and the pH is in the range of 7.57 to 8.04. Met.

As a specific experimental method, flounder fed with feed containing fermented cactus (EP feed containing cactus fermented product at a concentration of 1% by weight) and flounder fed with only EP feed were each subjected to 5 experiments. They were divided into groups and 5 control groups. Each group consists of 4 flounder, and after cultivating activated Edward bacteria in a BHI liquid medium, the grown strain is suspended in sterile physiological saline, and 9.8, 9.8 × 10 ² , 9 per mL. 0.1 mL of 0.8 × 10 ⁴ , 9.8 × 10 ⁶ , and 9.8 × 10 ⁸ Edward bacteria were inoculated into the peritoneal cavity. While bred for 20 days after inoculation, the presence of drowning and appearance symptoms were checked daily. The moribund and expression fish were dissected and observed for lesions, and bacteria were cultured in the liver and kidney. The results are shown in Table 7-2 below.

As shown in Table 7-2, in the case of Japanese flounder that did not ingest cactus fermented feed, 100% died in 9 days in the control group inoculated with 1 × 10 ⁹ viable cells, and 1 × 10 ³ In the control group inoculated with ˜1 × 10 ⁷ viable cells, 25% died at 16 days and in the control group injected with 1 × 10 ¹ viable cells 50% died at 12 days . In contrast, in the case of Japanese flounder fed with cactus fermented feed, 100% died in 8-14 days in both the powder inoculated with 1 × 10 ⁹ viable cells and the liquid experimental group. The experimental group inoculated with 1 × 10 ⁷ viable cells and the control group showed almost the same mortality rate, and no significant difference was observed.

  On the other hand, experimentally, flounder infected with Edward bacteria was seen as the main symptoms in terms of appearance, such as blackening of the colouration, deenteration, protrusion of the eyeball and cloudiness, mucus on the body surface. Anatomically, there was a large amount of general pale mucous ascites, hepatic congestion and kidney and spleen enlargement, similar to those of naturally infected fish. From the kidney tissue of Japanese flounder infected with Edward bacteria, detachment and necrosis of tubular epithelial cells and a marked decrease in tubulars were observed. From the liver of some individuals, necrotic cells and their cell masses were observed in the connective tissue region adjacent to the hepatopancreas.

3) Vibrio bacteria In order to examine the effect of the cactus fermented product on the growth of Vibrio bacteria, the growth rate of the bacteria in a liquid medium was comparatively analyzed. First, the freeze-dried cactus fermentation product produced according to the method of Example 3 was suspended in BHI to a final concentration of 2% (w / v), and then mixed at room temperature for 30 minutes to adjust the pH. Adjusted to 7.0. This mixture was put in an ice box, and sonication for 15 seconds and standing for 15 seconds were repeated 8 times. After sonication, insoluble substances were removed with filter paper, and then passed through a syringe filtration sterilizer (0.45 μm pore size), and the results were used as an experimental group. BHI without added cactus was used as a control group.

After inoculating Vibrio bacteria previously cultured for 18 hours in BHI in the experimental group and the control group at a level of 1.2 × 10 ⁴ CFU / mL, the mixture was allowed to stand at 22 ° C., and then 0, 2, 4, Vibrio bacteria counts at 8, 12 and 24 hours were measured. The number of bacteria is measured in the experimental group and the control group by diluting 100 μL of the culture solution into sterilized physiological saline at each measurement time, and then smearing it on a BHI agar medium supplemented with 2% NaCl. It was broken. Note that each experiment was repeated twice, and the results are shown in FIG.

As shown in FIG. 8 (C), the control group, indicates the number of bacteria 11.1log ₁₀ CFU / mL after 12 hours culture 9.2log ₁₀ CFU / mL, 24 hours after, the actual fermentation powder prickly pear for the applied experimental groups showed 6.4log ₁₀ CFU / mL count bacteria 3.6log ₁₀ CFU / mL, 24 hours after 12 hours of culture. Thereby, it was confirmed that the growth inhibitory effect of Vibrio bacteria lasts up to 24 hours.

  Furthermore, an experiment on the pathogenicity of Vibrio was performed as follows. The flounder used in the experiment is 6cm in size, and is fed with 1.0% cactus fermented feed for one month in the FRP round water tank (φ74cm × 80cm) set up at the Institute of Marine Fisheries Resources in Jeju. The reared one was used. During the breeding period, the breeding water temperature is 17.4 to 21.6 ° C., the salt concentration is 33.0 to 35.4 ‰, DO 5.84 to 7.12 mg / L, and the pH is in the range of 7.57 to 8.04. there were.

The specific experimental method is as follows. That is, each group consists of 4 flounder, and after cultivating activated Vibrio bacteria in a BHI liquid medium, the grown strain is suspended in sterile physiological saline, 1 × 10, 1 × 10 ³ , 1 per mL 0.1 mL each of × 10 ⁵ , 1 × 10 ⁷ , and 1 × 10 ⁹ vibrio bacteria was inoculated into the peritoneal cavity. While bred for 31 days after inoculation, the presence or absence of drowning and appearance symptoms were checked daily. The moribund and expression fish were dissected and observed for lesions, and bacteria were cultured in the liver and kidney. The results are shown in Table 8 below.

As a result, as shown in Table 8, in the flounder that did not feed the lactic acid bacteria-containing feed of the present invention, when inoculated with 1 × 10 ⁹ viable cells, 100% died in 1 day, and 1 × 10 ^When inoculated with ⁷ viable cells, 75% died in 3 days, and when inoculated with 1 × 10 ⁵ viable cells, 25% died in 8 days.

Moreover, as shown in Table 8, in the Japanese flounder fed with the feed containing the cactus fermented product, when 1 × 10 ⁹ viable cells were inoculated, 100% died in 1 day and 1 × 10 ⁷ viable When cells were inoculated, 50% died in 4 days. Ascites and eyeball protrusion were seen as clinical symptoms, and causative bacteria such as inoculum were detected from the liver and kidney.

  On the other hand, flounder infected with Vibrio bacteria was seen as the main symptoms in terms of its appearance, such as blackening of coloration, deenteration, protrusion of the eyeball and cloudiness, and mucus on the body surface. Anatomically, there was a large amount of general pale mucous ascites, hepatic congestion and kidney and spleen enlargement, similar to those of naturally infected fish. From the kidney tissue of Japanese flounder infected with Vibrio spp., Tubule epithelial cell detachment and drowning, and a marked decrease in tubules were observed. From the livers of some individuals, dying cells and cell masses were observed in the connective tissue region adjacent to the hepatopancreatic fistula.

Example 7 Antiviral effect of fermented cactus according to the present invention 1) Growth inhibitory effect of fermented cactus against equine herpesvirus (EHV) Equine herpesvirus (EHV) is a DNA virus belonging to the family Herpesviridae. When infected, it is a transient fever at the initial infection of the mare and rhinopneumonia, mainly nasal discharge. If a pregnant horse is infected after the middle of pregnancy, fetal infection occurs, resulting in abortion Is known to be caused. This experiment was conducted in order to confirm whether the cactus fermented product according to the present invention has a growth inhibitory effect on EHV.

  The specific experimental method is as follows. That is, MDBK cells were grown in a monolayer on a 96-well microtiter plate. After removing the cell culture medium, each well was infected with 50 PFU / 50 μL of EHV virus for 1 hour, and 100 μL of the cactus fermented product prepared according to Example 3 diluted in 2-fold stages was dispensed into each well. Thereafter, the presence or absence of virus growth was observed by the ratio of the number of plaques in the well of the test group to the control group well. Each experiment was performed 4 times, and then the average value was calculated.

  As a result, it was confirmed that in the plate treated with the cactus fermented product produced according to Example 3, no virus was propagated and no plaque was produced, so that a 100% suppression effect appeared.

  Furthermore, in order to confirm whether the cactus fermentation product has a preventive effect against EHV virus, the following experiment was performed. That is, MDBK cells were grown in a monolayer on a 96-well microtiter plate. After removing the cell culture solution, 50 μL of the cactus fermented product prepared according to Example 3 diluted to a 2-fold level was dispensed into each well and incubated for 1 hour. After removing the diluted cactus fermentation in each well, each well was infected with 50 PFU / 50 μL of EHV virus for 1 hour. The presence or absence of virus growth was observed by the ratio of the number of plaques in the wells of the test group to the number of plaques in the control group wells without cactus extract. Each experiment was performed 4 times, and then the average value was calculated.

  As a result, in the plate which processed the cactus fermented material manufactured according to Example 3, the virus was not propagated and the plaque was not produced at all, so that it was confirmed that the protective effect was 100%.

2) Growth inhibitory effect of cactus fermented product against bovine herpesvirus (BHV) Bovine herpesvirus (BHV) is caused by infection with DNA virus belonging to the family Herpesviridae and is an infectious disease of cattle respiratory and genital types . It is known to cause high fever and acute inflammatory mortality and miscarriage of the respiratory system, and contagious pustular vulvovaginitis and glans foreskinitis, sometimes causing encephalitis in calves. This experiment was conducted to confirm whether the cactus fermented product according to the present invention has a growth-inhibiting effect on BHV.

  The specific experimental method is as follows. That is, MDBK cells were grown in a monolayer on a 96-well microtiter plate. After removing the cell culture medium, each well was infected with 50 PFU / 50 μL of BHV virus for 1 hour, and 100 μL of the cactus fermented product prepared according to Example 3 diluted in 2-fold stages was dispensed into each well. Thereafter, the presence or absence of virus growth was observed by the ratio of the number of plaques in the wells of the test group to the number of plaques in the wells of the control group. Each experiment was performed 4 times, and then the average value was calculated.

  As a result, it was confirmed that in the plate treated with the cactus fermented product manufactured according to Example 3, no virus was propagated and no plaque was produced, so that a 100% suppression effect was obtained.

  Furthermore, in order to confirm whether the cactus fermented product has a preventive effect against the BHV virus, the following experiment was performed. That is, MDBK cells were grown in a monolayer on a 96-well microtiter plate. After removing the cell culture solution, 50 μL of the cactus fermented product prepared according to Example 3 diluted to a 2-fold level was dispensed into each well and cultured for 1 hour. After removing the diluted cactus fermentation from each well, each well was infected with 50 PFU / 50 μL of EHV virus for 1 hour. The presence or absence of virus growth was observed by the ratio of the number of plaques in the test group wells to the number of plaques in the control group wells without cactus extract. Each experiment was performed 4 times, and then the average value was calculated.

  As a result, in the plate treated with the cactus fermented product produced according to Example 3, no virus was propagated and no plaque was produced, so it was confirmed that a 100% preventive effect was obtained.

Example 8 FIG. Breeding flounder using fermented cactus according to the present invention In this example, to confirm that the fermented cactus according to the present invention has an excellent growth effect on flounder bred when used as a feed additive. It was conducted.

  In the larval season of flounder, using PP small water tank (1 ton), 1% by weight of cactus fermentation broth was adsorbed to EP feed (Nisshin Marubeni Feed, Japan) against the feed weight, and experimented twice a day. Sufficient supply was provided until the fish stopped feeding. This was continued for 3 months. The length, weight and survival rate of flounder before and after the experiment were examined, and the results are shown in Table 9 below.

  On the other hand, for adult flounder fish, a PP small water tank (1 ton) is used, and freeze-dried cactus fermented powder is mixed with MP feed (a feed produced by the inventor, 90% maji and 10% fish meal. Adsorbed at various concentrations from 0.02 to 0.16% by weight based on the weight ratio) until the experimental fish no longer feeds twice a day. Supplied. This was continued for 8 months. The length, weight and survival rate of flounder before and after the experiment were examined, and the results are shown in Table 10 below.

Example 9 Breeding effect of Japanese flounder in comparison with feed additive according to the present invention and feed additive containing fermented cactus extract produced using other microorganisms EM (useful complex microorganism) as a fermentation strain used for cactus fermentation We selected five types of photosynthetic bacteria and lactic acid bacteria, and examined the effect of each fermented liquid on the growth of Japanese flounder. Next, in the same manner as in Example 8, the growth effects of Japanese flounder bred using each feed additive were compared, and the results are shown in Table 11 below.

  As shown in Table 11 below, it was found that the rearing effect was maximized in the case of Lactobacillus plantarum CNU001.

It is a result obtained by analysis of the base sequence of 16S rRNA, and is a diagram showing to which line a new lactic acid bacterium CNU001 strain taken from cactus fruit belongs. It is a figure which shows the change of the sugar content according to fermentation period in the case of fermenting cactus using the novel lactic acid bacteria CNU001 strain based on this invention. It is a figure which shows the change of the acidity according to fermentation period in the case of fermenting cactus using the novel lactic acid bacteria CNU001 strain based on this invention. It is a figure which shows the change of the number of lactic acid bacteria according to the fermentation period obtained when inoculating the novel lactic acid bacterium CNU001 strain which concerns on this invention in the extract of a cactus fruit and fermenting at 25 degreeC. It is a figure which shows the result of the content analysis of the saccharide | sugar according to the fermentation period obtained when fermenting the novel lactic acid bacteria CNU001 strain based on this invention. It is a figure which shows the change of the content of the organic acid in the extract of the cactus fruit fermented with the novel lactic acid bacteria CNU001 strain based on this invention. It is a figure which shows how much the strain of this invention was settled in the intestine of Japanese flounder when the novel lactic acid bacterium CNU001 strain according to the present invention was administered to Japanese flounder. It is a figure which shows the antibacterial effect of the novel lactic acid bacteria CNU001 strain based on this invention. (A) is an effect on streptococci, (B) is an effect on Edward, and (C) is an effect on Vibrio. It is a figure which shows 16S rRNA base sequence of Lactobacillus plantarum CNU001.

Claims (7)

  A method for producing a fermented cactus lactic acid bacterium comprising a step of fermenting cactus with Lactobacillus plantarum CNU001 (KCTC18105P) as a lactic acid bacterium. Selecting cactus fruits and washing them with water;
Grinding the cactus fruit with a mixer to obtain a homogeneous liquid;
Mixing a fermentation accelerator such as molasses or syrup with the homogeneous liquid and Lactobacillus plantarum CNU001 as a strain according to the present invention;
Performing fermentation at room temperature for 1 minute to 2 weeks or more to obtain a fermentation broth;
Freeze-drying the fermentation broth to a powder,
The method for producing a fermented cactus lactic acid bacterium according to claim 1, comprising:   The method for producing a fermented cactus lactic acid bacterium according to claim 2, wherein the cactus is a prickly pear cactus.   A cactus fermented lactic acid bacterium prepared by the method according to claim 1.   The fermented lactic acid bacterium of cactus according to claim 4, wherein the fermented lactic acid bacterium of cactus is used as an additive for flounder feed.   A flounder breeding method comprising the step of feeding flounder as a flounder feed additive, a flounder breeding feed containing a fermented cactus lactic acid bacterium prepared by the method according to claim 1. Lactobacillus plantarum CNU001 (KCTC18105P) as a lactic acid bacteria strain.