KR101420836B1 - Livestock feed additive with the culture medium of Latobacillus plantarum - Google Patents

Abstract

The present invention relates to an antibiotic substitute feed additive comprising a culture of Lactobacillus plantarum as an antimicrobial agent. When used as a feed additive, it can be used as a feed additive to prevent immune enhancement, growth promotion and disease prevention effects of livestock without addition of antibiotics. It has the advantage of bringing economic benefits through increased productivity of livestock farming farmers, and development of livestock industry and organic farming (eco-friendly farming).

Description

(Livestock feed additive with the culture medium of Latobacillus plantarum) containing the culture liquid of Lactobacillus plantarum as an antimicrobial agent,

The present invention relates to a livestock feed additive, and more particularly, to an antibiotic substitute livestock feed additive containing a culture solution of Lactobacillus plantarum ( Lactobacillus plantarum ) as an antimicrobial agent.

In the intestines of livestock, many kinds of microorganisms coexist with each other and form an ecosystem. Intestinal bacteria are anaerobically or fermented by using continuously supplied feeds. The physical and chemical environments such as temperature, pH, osmotic pressure, and oxidation and reduction are almost constant in the body of livestock, The metabolism activity is constant.

However, environmental stress such as extreme climate, emotion, and hyperacidosis caused by excessive intake of concentrated feed destroys intestinal homeostasis of livestock, resulting in a decrease in immunity, resulting in a significant decrease in productivity of slaughtered animals .

As a preventive and therapeutic means for this, medicines such as antibiotics and chemotherapy have been successfully used. Over the past 40 years, the feed industry has been able to achieve high productivity gains through the use of antibiotics, the highest scientific discovery of the century. By using antibiotics, the growth of pathogens, which are the primary uses of these antibiotics, was suppressed and the growth of livestock could be promoted, and the productivity of livestock such as meat, milk, and eggs could be improved. Indeed, antibiotic-promoting mechanisms have shown better growth and improved productivity in harsh environments. For this reason, the addition of antibiotics to livestock feeds has improved nutrient utilization and productivity, and has been achieved for the prevention and treatment of various diseases. In addition, recently, it has been used for the purpose of growth of livestock, promotion of fattening, improvement of feed efficiency, etc., and it is recognized as a core preparation necessary for improving productivity of livestock.

However, there have been many problems with the use of antibiotics in recent years, and in some countries, the use of antibiotics has been limited, as it restricts the import of livestock meat that has been fed antibiotics. The reason for regulating the use of antibiotics is that first, the number of effective microorganisms in the intestines is reduced, second, microorganisms resistant to antibiotics are increased due to abuse, and third, the antibiotics used in livestock are milk, And fourth, antibiotics consumed through livestock products increase resistance and cause resistance to antibiotics (eg, the birth of super bacteria), and the like.

Already in 2006, the European Union (EU) has introduced zinc bacitracin, spiramycin, and tylosin phosphate, which are antibiotics that have been allowed to be used as feed additives based on the slogan of animal welfare and organic livestock farming ), And the use of virginiamycin. In Korea, on July 1, 2011, all antibiotics were banned. These prohibition measures are used to promote the growth of livestock bacterium resistant to antibiotics used in the human body to parasitic bacterium to transmit the resistance to the chemicals used in the human body is resistant to treatment of human disease difficult It was carried out under the premise that there is a risk of harming health.

Under this circumstance, it can be used for the production of livestock products in domestic academia and agriculture and livestock industry. However, it is not persistent in meat, it is safe for the health of the human being ingested but also promotes the growth of livestock and can prevent and cure bacterial diseases like antibiotics Efforts to find non-antibiotic biological tools have continued. However, we have not found a clear replacement candidate.

Probiotics, probiotics, physiologically active substances, immunostimulants, organic and inorganic acids and mineral agents are currently being considered as candidates for antibiotic substitute candidates. It is known that more than 300 kinds of probiotics for nursery poultry are circulated in the untested state in only one of the probiotics. However, the use of unexamined additives is accompanied by the growth stagnation due to the prohibition of antibiotics use, Which is a major factor in increasing the production cost of the product.

Korean Patent Laid-Open Publication No. 10-2001-0035412 (2001. 05.07) discloses a composition for a livestock feed composition for the replacement of antibiotics, which comprises adding a germanium biotite instead of an antibiotic to a conventional preparation feed used in the livestock industry , It is possible to produce livestock with excellent growth rate and immunity, such as when a large amount of antibiotics are blended, and to avoid all kinds of adult diseases by excluding all or part of the use of antibiotics harmful to the human body Quot ;, which is incorporated in a basic feed comprising corn, soybean meal, molasses, salt, vitamin premix, and mineral premix in a livestock feed composition with 36 ppm germanium and biotite , Muscovite, feldspar, tourmaline, zircon, garnet, apatite, opaque minerals, and 0.93% in the far-infrared wavelength range of 5 to 20 μm , And 0.1% to 3.0% of germanium biotite exhibiting an emissivity and a radiant energy of 4.31 x 102 W / m < 2 > 占 퐉.

In the present invention, a new antibiotic substitute livestock feed additive capable of substituting antibiotics forbidden for use in livestock feeds as described above is provided.

The present invention relates to a method for producing Lactobacillus plantarum , comprising the steps of: culturing Lactobacillus plantarum ; After the above-mentioned cultivation, centrifuging the obtained cell culture medium to remove the cells and recovering the supernatant; And removing the remaining microbial cells by filtration of the supernatant recovered in the above step. The present invention also provides an antibiotic substitute animal feed additive characterized by comprising the culture liquid obtained from the above step as an active ingredient.

The invention of the number of lactic acid bacteria Lactobacillus Planta room there is a feature in using a culture medium of (Latobacillus plantarum), to be the case in the experiment of the present invention, the Lactobacillus genus Lactobacillus of several lactic acid bacteria Bacillus Planta room (Latobacillus plantarum ) showed the best antibacterial activity.

The term 'active ingredient' in the present invention means that the culture solution obtained above is added to a livestock feed additive so as to serve as an antibiotic, and it may include other excipients besides the culture liquid. The antibiotic-substituted livestock feed additive developed in the present invention is added to the feed as an antibiotic substitute, thereby contributing to the enhancement of the immunity of the livestock.

On the other hand, in the animal feed additive for an antibiotic substitute according to the present invention, the medium is preferably Yeast Extract; Sodium acetate; NaCl; Na ₂ HPO ₄ ; K ₂ HPO ₄ ; KH ₂ PO ₄ ; (NH ₄₎ Cl; Glucose; MgSO _4; And a manganese sulphate. Such a medium composition is more economical than the conventional MRS medium used for culturing lactic acid bacteria.

Meanwhile, in the antibiotic-substituted livestock feed additive of the present invention, the antibiotic-substituted livestock feed additive may further include CGR (Chlorella Growth Factor). As a result of the test of the present invention, it was confirmed that CGF helps to proliferate the lactic acid bacteria. As a result, the feed additive of the present invention can be commercialized by adding liquid CGF. Further, when CGF is consumed by animal animals, As well as the growth of lactic acid bacteria.

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

In the present invention, showed a Lactobacillus bacteria (Lactobacillus) Comparison of the antibacterial activity against the antibiotic culture medium obtained by selecting the culture the four lactic acid bacteria state from among the genus Lactobacillus, Lactobacillus Planta room (Latobacillus plantarum) the one with the highest broth culture antibacterial .

In order to test the antibacterial activity of Lactobacillus plantarum ( Lactobacillus plantarum ), the antimicrobial activity of each strain was examined by using 32 times dilution (disk zone method), 5 ~ 10% ), And it was confirmed that the temperature stability of the antimicrobial culture liquid was stable to heat at 100 ° C or higher and the antimicrobial activity was stable up to pH 5 or less. In addition, in order to confirm the stability against intestinal lytic enzyme, the stability test for various lytic enzymes showed that the antimicrobial activity was stable in all the lytic enzymes.

The antimicrobial activity of Lactobacillus plantarum ( Latobacillus plantarum ) was compared with that of antibiotics (Apramycin, Tetracycline, Vancomycin) used as a conventional feed additive. Compared with conventional antibiotics, apramycin (1:15) was superior to antibiotics.

In order to confirm the storage stability of the antimicrobial culture solution, the antimicrobial activity test was carried out after storage at 4 ° C., 4 ° C., 25 ° C. and 37 ° C., The antimicrobial activity was predicted to be stable even for more than 16 weeks.

Meanwhile, in the present invention, the production cost of the antimicrobial substance is increased by 1/100% by using the medium developed in the present invention in place of the expensive MRS medium in the mass production of the antibacterial culture material by culturing the Lactobacillus plantarum ( Latobacillus plantarum ) 12 and as a feed additive, it was able to secure price competitiveness with existing antibiotics. In addition, CGF was added to a large amount of cultured antimicrobial culture material, and it was confirmed that it could be helpful to inhibit the growth of lactic acid bacteria and to prevent the growth of harmful bacteria in the intestines when it was developed as a feed additive.

On the other hand, the prototype manufactured using the antimicrobial culture solution of the present invention was confirmed to have an antibiotic substitute by confirming the growth effect and the immunity-enhancing effect by adding to the feed of the needy piglets.

At a time when the need for technology to develop natural antimicrobial and natural functional materials due to prohibition of the addition of antibiotics in the feed is very important, it is necessary to improve the HACCP requirements of the feed market, When the culture of Lactobacillus plantarum ( Lactobacillus plantarum ), which is developed by the present invention, is used as a feed additive, it has the effect of enhancing the productivity of the livestock farming farm by obtaining the immunity enhancement, growth promotion and disease prevention effect of the livestock, The development of animal husbandry industry and organic agriculture (environment friendly agriculture) can be the basis of.

In addition, it is possible to reduce the burden on the livestock producers by providing the natural antibiotic, which is inexpensive by the mass production and formulation technique developed in the present invention, to the livestock farmer.

1 is a graph showing the antimicrobial activity of the antimicrobial culture solution according to the lactic acid bacteria.
Fig. 2 is a result of the antibacterial activity test by the disc zone method in which the culture solution (antimicrobial culture solution) of Lactobacillus plantarum of the present invention was diluted with distilled water twice in order.
Fig. 3 is a result (5 hours of shaking culture) of a culture solution (antimicrobial culture solution) of Lactobacillus plantarum according to the present invention by performing an antibacterial activity test by an absorption measuring method.
Fig. 4 shows the result of confirming the temperature stability of the culture (antimicrobial culture solution) of Lactobacillus plantarum ( Latobacillus plantarum ) according to the present invention by disk zone method.
FIG. 5 is a result (5 hours of shaking culture) of the culture solution (antimicrobial culture solution) of Lactobacillus plantarum according to the present invention as determined by the light absorption measurement method.
FIG. 6 is a graph showing the results of the present invention, plantarum ) was determined by the disk zone method.
FIG. 7 is a graph showing the effect of the present invention on Lactobacillus plantarum plantarum ) was determined by absorption spectrophotometry.
FIG. 8 is a graph showing the results obtained by the method of the present invention showing that Lactobacillus plantarum plantarum ) was determined by disk zone method.
FIG. 9 is a graph showing the effect of the present invention on the activity of Lactobacillus plantarum plantarum ) was determined by absorption spectrophotometry.
FIG. 10 is a graph showing the effect of the present invention on Lactobacillus plantarum plantarum ) against apramycin was investigated.
Fig. 11 is a graph showing the effect of the present invention on the activity of Lactobacillus plantarum plantarum ) to the tetracycline (Tetracycline).
FIG. 12 is a graph showing the effect of the present invention on the activity of Lactobacillus plantarum plantarum ) to the vancomycin.
FIG. 13 is a graph showing the activity of the Lactobacillus plantarum plantarum ) for the long term storage stability.
Fig. 14 shows the difference in the antibacterial activity according to the culture time (MRS medium and LPCM medium).
Fig. 15 shows the effect of addition of CGF on the antibacterial activity and the growth of lactic acid bacteria.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Example  1: Verification of antibacterial activity by lactic acid bacteria]

Various Lactobacillus LP (Latobacillus plantarum) and LB ( Latobacillus brevis ), LC ( Latobacillus casei ), LA ( Latobacillus acidophilus ) in the culture medium.

L.P (Latobacillus plantarum), L.B (Latobacillus brevis), L.CLatobacillus casei), L.ALatobacillus acidophilus) Was cultured in MRS medium at 37 ° C for 72 hours. The resulting culture mixture was centrifuged at 8,000 × g for 20 minutes to remove the cells, and the supernatant was filtered through a 0.22 μm filter to completely remove the remaining cells. Respectively. Thereafter, the obtained pure culture broth was prepared by lyophilization using a freeze dryer.

For the antibacterial test,The harmful bacteria Salmonella Taipimorumium (Salmonella typhimurium) (SL1344) and E. coliE. coli) (K12),Propionibacterium acnes (Propionbacterium acne), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas Alejino (Pseudomonas aeruginosa) Was cultured at 37 ° C for 15 hours in a culture medium of LB and the corresponding pathogenic microorganism, and incubated in a new medium for 5 hours at 37 ° C with shaking. The absorbance was measured and the cell concentration was 1 × 10⁷lt; RTI ID = 0.0 > cfu / ml. < / RTI > To test the antibacterial activity, a test sample for antimicrobial activity was added to a conical tube (15 ml) for each concentration.⁷cfu / ml of noxious bacteria was added to the final 1x10 <⁵cfu / ml, and the medium was filled to a final volume of 2 ml. After incubation for 15 hours (Overnight) at 37 占 폚, OD₆₂₀And the antimicrobial activity (%) was measured by the degree of inhibition of the proliferation of the harmful bacteria.

As shown in FIG. 1, in addition to the LP strain, LB ( Latobacillus brevis ), LC ( Latobacillus casei ), LA ( Latobacillus acidophilus , but the antimicrobial effect was lower than that of LP lactic acid bacteria.

Therefore, LP ( Latobacillus plantarum) strain having the highest antibacterial activity among various lactic acid bacteria was selected as the strain of the present invention, and the following test was conducted.

[ Example  2: Lactobacillus Flora Room ( Latobacillus plantarum Analysis and Characterization of Antimicrobial Activity by Culture Concentration]

 1. Verification of antibacterial activity by concentration

Lactobacillus plantarum was cultivated in MRS medium at 37 ° C for 72 hours, and the resulting culture mixture was centrifuged at 8,000 × g for 20 minutes to remove the cells. The supernatant was filtered through a 0.22 μm filter to completely remove the remaining cells To prepare a pure culture solution (antibacterial culture solution). On the other hand, a pure culture medium in which the cells were completely removed was prepared by freeze-drying. Hereinafter, this sample is also referred to as a " frozen-concentrated antibacterial sample ".

The antimicrobial activity test of the antimicrobial culture broth or the frozen concentrated antimicrobial sample had gram-positive and gram-negative harmful strains respectively and proceeded by the following two methods.

1) Disc-zone method: Micrococcus leteus , a gram- (Nutrient broth agar plate) containing 4% of Nutrient broth (culture at 30 ° C, Nutrient broth, 72 hours) was prepared and grooves having a diameter of 8 mm were made in the agar plate to drip the antibacterial test sample. In the grooves, a sample of antibacterial activity test (antimicrobial culture solution) was added at a rate of 100 μl / well and cultured at 30 ° C. for 24 hours, and the size of the resulting circular inhibitory circle was confirmed. As a result of the experiment, it was confirmed that the antimicrobial activity was up to 32 times the diluted concentration of the antimicrobial culture liquid as shown in FIG.

2) Absorption measurement method: Gram positive salmonella Salmonella typhimurium (Salmonella typhimurium) (Strain SL1344) was cultured at 37 ° C in an LB medium for 15 hours, and cultured in a new LB medium at 37 ° C for 5 hours with shaking. The absorbance was measured and the cell concentration was 1 × 10⁷cfu / ml. < / RTI > To test the antibacterial activity, 0 to 25% (0 to 500 μl / 2 ml) of the antibacterial activity test sample (antibacterial culture solution) required for the experiment was added to a conical tube of 15 ml,⁷cfu / ml of noxious bacteria was added so as to be 1%, and the LB medium was filled to a final volume of 2 ml. After incubation at 37 ° C for 5 to 15 hours, OD₆₂₀, And the antimicrobial activity (%) was measured to the extent of inhibiting the growth of the noxious bacteria. As a result of the experiment, it was confirmed that when the culture was shaken for 5 hours, the increase in the amount of the antimicrobial culture solution as shown in FIG. 3 was inhibited.

On the other hand, at the addition amount of 10% (200 μl / 2 ml) of the antimicrobial culture solution, it was confirmed that the proliferation of the harmful bacteria was completely inhibited even after 15 hours of culture for 5 hours and then the antibacterial activity was maintained (not shown).

 2. Characterization

 end. Thermal stability test

1 ml of the antimicrobial culture prepared in Example 2 was treated at 37 to 121 ° C for 4 hours each, diluted 4 times with distilled water, and tested for antibacterial activity by disk zone method. As a result, as shown in FIG. 4, the size of the inhibitory rings formed at all the temperatures was the same, and the size thereof was the same as that of the 1/4 diluted plate of the antimicrobial culture liquid in the concentration-dependent antimicrobial activity test. Therefore, it was confirmed that the antimicrobial culture solution obtained from the culture solution of Lactobacillus plantarum was stable to heat.

On the other hand, the antibacterial activity of the antimicrobial culture solution prepared in Example 2 was measured by an absorption measuring method. The antimicrobial culture solution was added with 25% (500 μl / 2 ml culture) (1/4 dilution effect of the initial antimicrobial culture solution), and the absorbance was measured after shaking culture for 5 hours. As shown in FIG. 5, when the antimicrobial activity of the antimicrobial culture solution after the heat treatment at each temperature was tested, the proliferation of the harmful bacteria was completely inhibited. Even after heat treatment by 100 ° C and 121 ° C sterilization, I could confirm that it remained. Therefore, the antimicrobial culture solution obtained from the culture solution of Lactobacillus plantarum of the present invention was once confirmed to be stable to heat.

  I. Verification of Stability by Degradation Enzyme

500 μl of the antimicrobial culture solution prepared in Example 2, 100 μl of each of the digesting enzyme solutions (10 mg / ml) and pH adjusting solution A (1N NaOH) shown in FIG. 6 were added to a total of 900 μl (pH 7.0) For 6 hours. The pH of the antimicrobial culture solution was adjusted to the initial pH by adding 100 쨉 l of pH adjusting solution B (1N HCl), and then 1 ml of the solution was lyophilized. The lyophilized, frozen and concentrated antimicrobial samples were completely dissolved in 2 ml of distilled water (1/4 dilution of the antimicrobial culture solution), and then subjected to the antimicrobial activity test by disc method. As shown in FIG. 6, the inhibition ring sizes of the respective protease-treated samples were the same as those of the control (CTL) without the protease treatment. Therefore, it was confirmed that the antimicrobial culture solution obtained from the culture medium of Lactobacillus plantarum was stable to various degrading enzymes.

On the other hand, the antimicrobial culture solution prepared in Example 2 was subjected to a stability test for each enzyme by an absorption measuring method. 500 μl of the antimicrobial culture solution prepared in Example 2, 100 μl of each of the digestion enzyme solutions (10 mg / ml) and pH control solution A (1 N NaOH) were added to a total of 900 μl (pH 7.0) . 100 쨉 l of pH control solution B (1N HCl) was added, and the pH of the culture solution was again adjusted to the initial pH, and then 1 ml was lyophilized. The lyophilized, frozen and concentrated antimicrobial samples were completely dissolved in 500 μl of distilled water and then tested for their antibacterial activity by absorbance measurement. The dissolved frozen concentrated antimicrobial solution was added with 25% (500 μl / 2 ml culture) (1/4 dilution effect of the initial antimicrobial culture solution) and the absorbance was measured after shaking culture for 15 hours. As a result, as shown in FIG. 7, it was confirmed that the proliferation of the harmful bacteria was inhibited regardless of whether or not the digestive enzymes were treated, as compared with the case where no antimicrobial sample was added (NC). Therefore, the antimicrobial culture solution obtained from the culture solution of Lactobacillus plantarum was confirmed to be stable to various degrading enzymes.

  All. pH  Stability verification

500 占 퐇 of the antimicrobial culture solution prepared in Example 2 and pH-adjusted solution A (1N NaOH) were added to prepare 1 ml of a sample from pH 4 to pH 10, followed by lyophilization. The lyophilized, freeze-dried concentrated antimicrobial samples were completely dissolved in 500 μl of distilled water (effect of the original antimicrobial culture broth solution), and then tested for antimicrobial activity by disk zone method. As a result of the experiment, it was confirmed that the antimicrobial activity was up to pH 6 as compared with Control (CTL) without pH control as shown in FIG. Therefore, it was confirmed that the antimicrobial culture solution obtained from the culture solution of Lactobacillus plantarum was stable up to pH 5.

On the other hand, the antimicrobial culture solution prepared in Example 2 was subjected to the stability test by pH with the light absorption method. 500 μl of the antimicrobial culture solution prepared in Example 2 and 1 ml of pH adjusting solution A (1N NaOH) were added to prepare 1 ml of a sample from pH 4 to pH 10, followed by lyophilization. The lyophilized, concentrated, frozen and concentrated antimicrobial samples were completely dissolved in 100 μl of distilled water, and then tested for their antibacterial activity by an absorption measuring method. The dissolved frozen concentrated antimicrobial solution was added with 25% (500 μl / 2 ml culture) (1/4 dilution effect of the initial antimicrobial culture solution) and the absorbance was measured after shaking culture for 5 hours. As a result, as shown in FIG. 9, it was confirmed that the proliferation of the harmful bacteria was inhibited up to pH 5, and the antibacterial activity was decreased from pH 6 or higher, compared with the case where no antimicrobial sample was added (NC). Therefore, it was confirmed that the antimicrobial culture solution obtained from the culture solution of Lactobacillus plantarum was stable up to pH 5.

[ Example  3: Lactobacillus Flora Room ( Latobacillus plantarum ) Comparison of the antimicrobial activity of the culture medium and existing antibiotics]

1. Test Method

end. Paper disk for antibacterial test ( paper disk ) Produce

The frozen concentrated antimicrobial sample (30 g) prepared in Example 2 was completely dissolved by using 40 ml of PBS buffer to prepare a concentrated antimicrobial liquid (60 ml). For antibacterial test, 100 μl of each solution sample containing 10%, 30%, and 50% (diluted with PBS) of concentrated antimicrobial solution was dispensed on a paper disk, followed by drying in a 50 ° C. dryer for 1 hour. Apramycin, tetracycline, and vancomycin were diluted to a concentration of 0.01 to 10 mg / ml as comparative antibiotics, and 100 μl each was dispensed on a paper disk. Lt; / RTI > for 1 hour.

I. Preparation of harmful bacteria

Salmonella typhimurium and E. coli K99 , Gram-positive bacteria, Staphylococcus aureus , Gram-positive bacteria, Staphylococcus aureus, aureus 5, 8, and 336 strains were used. Plate inoculation was performed at 1 × 10 ⁷ cfu / 100 μl. After dispensing, the dried paper disks were inoculated and cultured at 37 ° C for 12 hours, and the sizes of the inhibitory rings were measured and compared.

2. Test results

end. To Apramycin  Comparison of antibacterial activity against

As shown in Table 1 and Fig. 10 below, apramycin was expressed in E. coli , The concentrated antimicrobial solution exhibited an antibacterial activity against E. coli K99 at a concentration of 50% (freezing-concentrated antimicrobial sample (Antimicrobial activity in the same manner as in Example 1 ) was measured in Salmonella typhimurium at a concentration of 30% (15 μg of the frozen concentrated antimicrobial sample and 0.48 ml of the initial antimicrobial culture) in 1 μg of apramycin Respectively.

Comparison of antibacterial activity against apramycin clean zone Content (100 μl) E. coli K99 S. typhimurium Apramycin 1 g 1.5 cm 1.3 cm 10 g 2 cm 2 cm 100 μg 2.6 cm 2.7 cm Frozen-concentrated antimicrobial sample 10% 5 g 0 0 30% 15 g 1 cm 1.4 cm 50% 25 ㎍ 1.4 cm 1.6 cm

I. Tetracycline ( Tetracycline) Comparison of antimicrobial activity against

As shown in the following Table 2 and FIG. 11, tetracycline exhibited antibacterial activity against E. coli K99 at a concentration of 1000 μg (inhibitory size> 1 cm) and S. typhimurium It does not show antibacterial activity. On the other hand The concentrated antimicrobial solution was prepared from E. coli , In K99 , 30% concentration (15 μg of the frozen concentrated antimicrobial sample, 0.48 ml of the initial antimicrobial culture) was added to Salmonella typhimurium in a concentration of 50% (25 μg of the frozen concentrated antimicrobial sample and 0.8 ml of the initial antimicrobial culture) Showed antibacterial activity.

Comparison of antimicrobial activity against tetracycline clean zone Content (100 μl) E. coli K99 S. typhimurium Tetracycline 10 g 0 0 100 μg 1 cm 0 1000 g 1.2 cm 0 Frozen-concentrated antimicrobial sample 10% 5 g 0 0 30% 15 g 1 cm 1.4 cm 50% 25 ㎍ 1.4 cm 1.6 cm

All. Vancomycin ( Vancomycin )

As shown in Table 3 and FIG. 12, vancomycin exhibited antibacterial activity (inhibitory size> 1 cm) at a concentration of 30 μg in S. aureus type 5, type 8, type 336, The concentrated antimicrobial solution showed antibacterial activity in S. aureus type 5, type 8, type 336 at 30% concentration (15 μg of frozen concentrated antimicrobial sample, 0.48 ml of initial antimicrobial culture).

Comparison of antibacterial activity against vancomycin clean zone Content (100 μl) S. aureus type5 S. aureus type8 S. aureus type336 Vancomycin 30 ㎍ 1.3 cm 1.5 cm 1.4 cm Frozen-concentrated antimicrobial sample 10% 5 g 0 cm 0 cm 0 cm 30% 15 g 1.2 cm 1.5 cm 1.2 cm 50% 25 ㎍ 1.4 cm 1.7 cm 1.6 cm

[ Example  4: Lactobacillus Flora Room ( Latobacillus plantarum ) Stability verification of long-term storage of culture medium]

1. Test Method

end. Preparation of antimicrobial culture liquid

Lactobacillus plantarum was cultivated in MRS medium at 37 ° C for 72 hours. The obtained mixed culture broth was centrifuged at 8,000 × g for 20 minutes to remove cells, and the supernatant was filtered through a 0.22 μm filter to remove remaining cells A pure culture medium was prepared.

I. Set trial period

The prepared antimicrobial culture solution was fully sealed and stored at 4 ° C, 25 ° C, and 37 ° C for 16 weeks. The samples were collected every 2 weeks and tested for antibacterial activity.

All. Antimicrobial activity test

The harmful bacteria Salmonella Salmonella typhimurium ) (Strain SL1344) was cultured at 37 ° C in LB medium for 15 hours, shake cultured in new LB medium at 37 ° C for 5 hours, and the absorbance was measured and diluted with LB medium to a cell concentration of 1 × 10 ⁷ cfu / Respectively. To test the antibacterial activity, 400 μl of a test sample for antibacterial activity was added to a conical tube of 15 ml, and 1 × 10 ⁷ cfu / ml of the prepared microorganism was added to the conical tube to a final volume of 4 ml. LB medium was filled. After incubation at 37 ° C for 4 hours to 15 hours (Overnight), the absorbance was measured at OD ₆₂₀ and the antimicrobial activity (%) was measured to the extent of inhibiting the proliferation of harmful bacteria.

2. Test results

Lactobacillus planta ( Latobacillus plantarum ) for 16 weeks. The results showed that there was no difference in the antibacterial activity according to the storage temperature and storage period. It was confirmed that there was also no change in properties depending on each storage temperature and storage period, and during the test period (16 weeks), Lactobacillus planta ( Latobacillus plantarum ) lactic acid bacteria were found to be stable.

[ Example  5: Lactobacillus Flora Room ( Latobacillus plantarum ) Development of medium composition for increasing antimicrobial activity in culture medium]

1. Test Method

end. Mass culture medium composition

The LPCM medium developed in the present invention was used for culturing Lactobacillus plantarum ( Latboracillus plantarum ), to obtain a MRS medium and a high concentration of antimicrobial culture medium, which are widely used. The composition of the LPCM medium developed in the present invention is shown in Table 4 below.

LPCM Reagent name 1 L composition Yeast extract
(Yeast Extract) 2.0 g Sodium acetate
(Sodium acetate) 5.0 g NaCl 5.0 g Na ₂ HPO ₄ 3.6 g K ₂ HPO ₄ 0.8 g KH ₂ PO ₄ 1.8 g (NH ₄₎ Cl 1.0 g glucose
(Glucose) 40 g TMS
(trace metal solution) 5 ml water Remaining balance  At this time, the TMS has the following composition. water Based on 100ml MgSO ₄ 4 g Manganese sulfate
(Manganese sulphate) 0.5 g

I. Mass culture method

MRS and LPCM media were each prepared in 10 L plastic bottles capable of sterilization and then sterilized. 10% glycerol Lactobacillus planta ( Latobacillus < RTI ID = 0.0 > plantarum ) The seed stock was inoculated into a sterile 250 ml glass bottle containing 100 ml of MRS medium, incubated in a 37 ° C incubator for 24 hours, and then inoculated to 1% of each sterilized 10 L medium. The culture conditions were incubated in a 37 ° C. incubator for 24 to 72 hours.

All. Recovery of antimicrobial culture solution

In order to recover the culture supernatant, the bacterial culture broth obtained after the final culture was centrifuged at 8,000 × g for 20 minutes to remove the cells, and the supernatant was filtered through 0.22 μm to completely remove the remaining cells, thereby recovering the pure antibacterial culture.

la. Antimicrobial activity test

The harmful bacteria Salmonella Salmonella typhimurium (Strain SL1344) was cultured at 37 ° C in LB medium for 15 hours, shake cultured in new LB medium at 37 ° C for 5 hours, and the absorbance was measured to determine the cell concentration to 1 × 10 ⁷ cfu / Lt; / RTI > For the antimicrobial activity test, a 15 ml conical tube was added with a test sample for antimicrobial activity, and 1 × 10 ⁷ cfu / ml of a harmful microorganism was added to the mixture in an amount of 1%, and the mixture was filled with LB medium to a final volume of 4 ml . After incubation at 37 ° C for 20 hours with shaking, the absorbance was measured at OD ₆₂₀ and the antimicrobial activity (%) was measured to the extent of inhibiting the proliferation of harmful bacteria.

2. Test results

end. Difference in Antimicrobial Activity according to Culture Time

As shown in FIG. 14, when the Lactobacillus plantarum was cultured in the MRS medium, the antibacterial activity was 100% for 24 hours from about 50% after 48 hours, and cultured in LPCM medium The antimicrobial activity started to increase from about 50% for 24 hours to 100% after 72 hours (when the antimicrobial culture was added to the total culture volume of 4 ml of the antimicrobial activity test)

The growth of lactic acid bacteria was higher in the MRS medium than in the LPCM medium. However, there was no difference in the antibacterial activity after 72 hours, and it was confirmed that there was no correlation between the growth of lactic acid bacteria and the antibacterial activity. However, the LPCM medium may be suitable for mass production of optimal antimicrobial materials considering the antimicrobial activity, culture time, and medium unit cost, although the 100% antimicrobial activity may be different in that the culture time is 24 hours in the MRS medium.

I. CGF  Effect of addition on antibacterial activity and lactobacillus growth

CGF (Chlorella Growth Factor) is a chemical substance that promotes lactic acid bacteria growth, and is a biologically active substance existing only in chlorella. Nucleotides are a major component and are a kind of DNA and RNA nucleic acids. It is a component that revitalizes cells. It has been found to have effects such as growth promotion of animals and plants, immunity and anti-cancer, and enhancement of antibacterial activity. In recent years, the National Livestock Academy has reported on the effect of chlorella powder in broiler diets on oil productivity and blood characteristics. Chlorella added to feeds have higher productivity than broiler chicks raised with antibiotics, Studies have also shown that microbial counts show higher lactic acid bacteria content.

Therefore, in order to investigate the effect of addition of CGF on the antibacterial activity and lactic acid bacteria growth in the mass production process of the present invention, liquid culture CGF was added to LPCM medium as a mass culture medium for 0 to 30% The absorbances were compared.

As a result of the test, as shown in Fig. 15, the antibacterial activity of the antibacterial culture broth cultured with the addition of CGF was evaluated as follows. When the antibacterial activity test was carried out by adding 400 μl of the antibacterial culture solution to the total culture volume of 4 ml of the harmful bacteria, , And the antibacterial activity was slightly increased according to the concentration of CGF when 300 .mu.l of the antibacterial culture was added (1 / 13.3 dilution effect). The growth of lactic acid bacteria also tended to increase with the addition of CGF, indicating that the culture with 30% CGF increased by 75% compared with the culture without CGF.

In conclusion, although the difference in antibacterial activity due to the addition of CGF was not significant in the mass culture process, the effect of lactic acid bacteria growth was confirmed. Therefore, CGF was added as a feed additive rather than CGF in a mass culture process, It is anticipated that the antimicrobial activity will be maintained and the effect of enhancing the proliferation of intestinal lactic acid bacteria by CGF addition can be expected.

All. Characteristics of optimized culture medium

In general, lactic acid bacteria are cultured through expensive MRS medium. The main purpose of the MRS medium is to produce lactic acid bacteria, and the high unit price of this medium has been a cause of the increase of the production unit price and the feed additive price in the commercialization of the lactic acid bacteria-containing feed composition.

On the other hand, the object of the present invention is not to produce expensive lactic acid bacteria, but to determine whether it is possible to mass-produce the antibacterial culture liquid produced in the lactic acid bacteria culture process at a lower cost, and therefore it is inappropriate to use expensive MRS medium.

The LPCM medium developed in the present invention is suitable for mass production of an antibacterial culture solution by culturing Lactobacillus plantarum , and the medium cost is about 240 won per liter, which is higher than the expensive MRS (2,900 won per liter) medium Since it is about 12 times (about 92%) cheaper, it is possible to lower the total production cost of the antibacterial culture liquid by lowering the unit cost of the culture medium.

As a result of the test, it was confirmed that CGF was helpful for the growth of lactic acid bacteria. As a result, it is possible to produce CGF as a feed additive by adding liquid CGF to an antibacterial culture produced by an inexpensive LPCM medium, and furthermore, It is possible to achieve the effect of the one-pounding which can help the proliferation of lactic acid bacteria in addition to the inhibition of proliferation.

[ Example  6: Preparation of antibiotic substitute feed additive of the present invention]

1. Production process

end. Preparation of antimicrobial culture liquid

Lactobacillus plantarum was cultured for 72 hours with shaking at 37 DEG C using LPCM medium developed by the present invention. The resulting culture mixture was centrifuged at 8,000 x g for 20 minutes to remove cells, and the supernatant was centrifuged And the remaining cells were completely removed by 0.22 탆 filter to prepare 100 L of a pure antibacterial culture liquid.

I. Mixing process

Mixed grains and pure antimicrobial culture were mixed at a ratio of 1: 1, and the mixed grains were mixed with corn and soybean meal pulverized powder in a mixer, and mixed with pure antibacterial culture solution once more.

All. Adsorption drying process

The mixed powder containing the pure antibacterial culture was transferred to a dryer and air dried at 55 ° C for 24 hours to adsorb the culture liquid on the surface of the mixed grain. At this time, to uniformly adsorb the culture liquid, the mixed powders were evenly mixed while being dried.

la. Finished production

The final antimicrobial feed additive after the adsorption drying process was pale brown coarse powder, which was used for the following specification tests after confirming the degree of contamination through dry state, property and microorganism test.

[ Example  7: Specification test - As an antibiotic substitute, the antimicrobial culture solution of the present invention Weaned  Growth abilities and effects on immune responses]

 1. Purpose of Testing

The antimicrobial culture preparation (the prototype prepared in Example 6) obtained by culturing Lactobacillus plantarum was added to the nursery pellets to verify the growth effect of the piglet piglets and the immunity enhancement effect. Respectively.

 2. Test method

For the test material, a culture supernatant obtained by culturing Lactobacillus plantarum ( Lactobacillus plantarum ) using LPCM medium was used as an adsorbent and a dried product.

  end. Experimental Animal and Specification Experiment

Specimens were tested for 5 weeks at Seoul National University 's attached experimental farm in Taedong, Gwon Seon - gu, Suwon. (Landrace × Yorkshire × Duroc) with an average body weight of 7.01 ± 0.91 kg, which were weaned at 28 ± 3 days of age, were given. Randomized Complete Block Design (RCBD). Experimental diets were fed in pigs with free feed and water temperature control and easy to control temperature. In order to measure daily gain, daily dietary intake and feed efficiency, And feed intake.

  I. Experimental design and experimental feed

Experimental treatments consisted of treatments with antibiotics and treatments with the prototypes prepared in Example 6. 2) Positive control (basal diet + Neomycin 0.1%) 3) A (basal diet + 0.1% prototype) 4) B (basal diet + 0.2% prototype) basal diet + 0.5% prototype). Experimental diets were prepared by replacing corn - soybean meal with barley - based diets by adding the same amount of additives to corn or soybean oil. Specimens were classified into two stages (Phase 1 0 ~ 2 weeks; Phase 2 3 ~ 5 weeks).

  All. Diarrhea frequency Diarrhea Occurence )

There are various ways to investigate the frequency and degree of diarrhea, but the most commonly used method is to judge the color, the year (moisture content) and the state of the experiment money by dividing them into three levels or five levels, There is a risk that the intention of the experimenter may be reflected or not objectively. Therefore, for the diarrhea frequency measurement, the experiment was conducted at 8:00 am every day after the start of the experiment. In the piglets of four pigs in each money room, individuals with watery diarrhea traces around the anus And the frequency of diarrhea was measured. The frequency of diarrhea was measured as zero in the absence of hydronephrotic diarrhea, and the diarrheal frequency was measured as 42 during the experimental period. Measurements of all diarrhea indexes were performed by one representative to maintain maximum objectivity.

  la. Blood characteristics Blood Profile )

Blood was collected from the jugular vein in six of the treatment groups at each time point and collected from the white blood cell (WBC), neutrophil (NE), neutrophil, lymphocyte (LY, Lymphocyte), monocyte (MO, monocyte) , Eosinophil), basophil (BA, Basophil), IL-2 (Interleukin-2 lymphokine) and γ-IFN (Interferon gamma) in the blood were analyzed. WBC, NE, LY, MO, EO and BA were analyzed by Flow Cytometry method and the remaining items were expressed as a ratio to the amount of analyzed WBC. The flow cytometry was able to analyze the difference in the size and internal composition of the nuclei when the nucleated cells passed the detection region in the flow state and all the detection was performed at the single- , And quantified by using several fluorescent materials simultaneously in each cell. IL-2 and γ-IFN were analyzed by whole blood on the day of blood collection. The remaining blood samples were collected in BD Vacutainer and centrifuged at 3,000 rpm at 4 ° C for 15 minutes. Respectively. After that, only serum was separated into a micro tube storage container and stored at -20 ° C. until analysis.

  hemp. Minute, microorganism in the minute

After feeding the experimental diets, 6 specimens were randomly selected for each treatment stage at the beginning of the experiment and each weight and feed intake level for each step of the specimens. The specimens were collected by an anal stimulation method and the microorganisms ( Salmonella , E. coli , Lactobacillus and Streptococus ). The specimens of the collected specimens were photographed, and the specimens were examined. The specimens were collected on a cotton swab for disease examination and used for disease examination. For microbial analysis, 6-minute samples for each treatment were diluted by mixing 1 g each with 9 ml of distilled water Respectively. One milliliter of the diluted stock solution was extracted and diluted with 9 ml of distilled water. In this way, analysis was performed by diluting to a level of 1,000,000 times (1 × 10 ⁶ ). 1 ml of the diluted sample was taken out, and the microbial medium (Sanita, LTS Korea) was covered with the film cover and the solution was dispensed into the medium. Again, the film cover was slightly covered and then cultured in an incubator at 35 ° C for 24 hours. Streptococcus for 48 hours in consideration of the characteristics of the strain for kusu (Streptococus) were incubated. After incubation, the number of microorganisms developed on the microbial culture medium was calculated.

  bar. Chemical analysis and statistical analysis

Statistical analysis was carried out by using the general linear model (GLM) of SAS (2004), and the significance test was performed for the collected data. In the specification experiment, the money room was used as the experimental unit. In the metabolic experiment and blood test, (LSD) multiple test method. P <0.05 was considered significant difference, and P <0.01 was considered significant difference.

 2. Test results

  end. Growth Grade

At 5th week of piglet termination, significant difference was found in body weight. PC treated with neomycin as an antibiotic showed higher body weight than all treatments except 0.5% of prototype. There were no statistically significant differences in the weight of piglets compared to the NC treatments in all four treatments supplemented with prototype. The average daily gain was significantly higher in the treatments supplemented with antibiotics during the 5 weeks of the experiment than in the other treatments. Daily dietary intakes were also significantly higher in treatments with antibiotics at 2 to 5 weeks and 5 weeks, but no significant differences were found between the other treatments. Feed efficiency was significantly improved in the 0-2 week period compared to the treatments containing 0.1% and 0.2% of the treatments supplemented with 0.5% of the prototype and 0.1% and 0.2% of the treatments added antibiotics And lower feed efficiency than that of one treat. As a result, the addition of the prototype did not show enough amount of germicide, feed intake and feed efficiency to replace antibiotics. However, the 0.5% added diet showed numerically higher feed weight and feed efficiency than the NC treatment, And also showed the best growth performance.

As a result of the specification results of the treatment with 0.5% of the prototype, the use of antibiotics in the feed was totally forbidden. At present, the intestinal microflora can be corrected and the beneficial bacteria of the beneficial bacteria and the epithelial cells of the beneficial bacteria can be recovered, It seems to be.

  I. Diarrhea

Influence of the addition of protozoa on the diarrhea of piglets was investigated. Generally, diarrhea occurs at the beginning of the reason due to sudden environmental change after the reason, transportation stress, nutritional stress due to feed change, and the like. The causes of sudden diarrhea and microbial pathogens caused by sudden changes in feed and environmental conditions lead to serious diarrhea. The cause of pigs is the change in the structure and function of digestive organs, which is caused by the pathogenic microorganism E. coli Diarrhea is caused by an infection in the digestive tract.

In this experiment, there was no significant difference in the diarrheal frequency during the entire experimental period, except that the treatment with antibiotics had significantly improved diarrheal incidence compared to the other treatments, There was also no. However, the treatment with 0.5% of the prototype showed lower diarrhea than the NC treatment in 0 ~ 2 weeks. As with the growth, 0.5% of the treatments seemed to reduce diarrhea most.

  All. Blood characteristics Blood Profile )

Blood analysis at the beginning of the experiment and at the end of each step showed no significant difference between the treatment periods in leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Overall, the treatment with antimicrobials did not show a negative trend compared to the NC and PC treatments, suggesting the possibility of replacing certain antibiotics. This implies that the addition of the prototype can be used to predict the effect of the immunity enhancement that prevents the entry of pathogens or viruses from the outside.

  la. Minute

As a result of observing the samples taken at the treatment start date and the end date of each specimen stage, the treated specimens containing 0.5% of the prototype at the end of Phase 1 and the end of Phase 2 showed less water than the other specimens containing prototype , Which is thought to have influenced the low outcome in diarrhea.

  hemp. Minute microorganism

The effect of the addition of the prototype on the microbes of piglets was examined. As a result of the experiment, there was no significant difference in the treatment period in the results of the microorganisms analyzed at the end of phase 1. However, the treatment with antibiotics showed the lowest numbers of salmonella formation compared to the other treatments, and Escherichia coli was found to be numerically less than the antibiotic-added treatments in all three treatments with prototype, It was thought that there was an effect that can inhibit colony formation of E. coli. The diarrhea frequency of the diarrhea indicated in the previous section was statistically similar to that of the antibiotics supplemented with 0.5% of the prototype, suggesting that the addition of the prototype was helpful in preventing diarrhea of the piglet . The results of the microbial analysis in the intramuscular administration at the end of the experiment showed that there was a significant difference between the treatments in the Lactobacillus strains. Except for the treatment with 0.2% of the prototype, all of the treatments with Lactobacillus were more beneficial than the NC treatments, indicating that the addition of prototype had a positive effect on the formation of beneficial bacteria in the intestines.

 3. Conclusion

This experiment was carried out to evaluate the antibiotics substitution of the prototype when added to nursery pellets, objective evaluation based on specifications, diarrhea frequency, blood analysis, fecundity and microorganisms. Compared to the NC treatment, the feed efficiency of 0.5% prototype was significantly higher than that of 0.1% and 0.2%, and the feed efficiency was higher than that of NC treatment. This prohibition is expected to have a positive effect when used in a corn-soybean concentrate feed. In diarrhea, the diarrhea frequency of 0 ~ 2 weeks of 0.5% addition of prototype was lower than that of NC treatment in numerical value as well as in growth rate, and it was found that addition of prototype also contributed to reduce diarrhea occurrence. As a result of blood analysis, there was no significant difference in all analysis items. This means that all of the treatments with prototypes did not show any deterioration compared to the treatments with antibiotics. For this reason, the addition of appropriate prototypes is interpreted as having the potential to replace antibiotics. In summary, the results of the analysis of intramuscular microorganisms showed that the number of E. coli was smaller than that of the treatment with antibiotics in all three treatments using the prototype at the end of phase 1. In addition, except for the treatments containing 0.2% of the prototype product, all of the treatments using the prototype showed a significant increase in the lactobacilli, which are beneficial bacteria, compared to the NC treatments, and the addition of the prototype had a positive effect on the formation of the beneficial bacteria in the intestines It was revealed.

Piglets that have been largely shaken by the intestinal microflora due to changes in the breeding environment and nutritional pattern immediately after the induction of the disease and the various stresses have fallen into a state of temporary indigestion in the process of changing from a liquid state milk in which the digestive absorption is good to a solid feed in which the vegetable protein is the main component do. In addition, heat / humidity, struggle, and transportation stress add to the digestion and absorption of nutrients, which causes abnormal growth of the intestinal flora using undigested nutrients, causing diarrhea. In the case of bacterial diarrhea, which is the cause of diarrhea after weaning, we can consider the anomaly of intestinal flora rather than the disease itself. If the harmful bacteria have a long-term dominance at this point, they will suffer from persistent diarrhea as well as anorexia and dehydration, which will greatly affect productivity. The addition of appropriate prototypes to the infant stage would correct the shaken intestinal microflora and help restore the superiority of the beneficial bacteria and the epithelial cells of the intestinal canal. Also, in the situation where the immune substance received from breast milk needs to be solved on its own, the prototype will be helpful, will help digestion and absorption of the feed, and will affect the synthesis of other nutrients.

In conclusion, the prototype was found to have an improvement in the specimen grade, diarrhea frequency, and microbial microflora.

Claims (3)

Culturing a Lactobacillus plantarum ; After the above-mentioned cultivation, centrifuging the obtained cell culture medium to remove the cells and recovering the supernatant; And filtering the supernatant recovered from the above to completely remove the remaining microbial cells. The culture solution and the CGF (Chlorella Growth Factor)
An antibiotic substitute livestock feed additive characterized by the effect of lactic acid bacteria growth.
The method according to claim 1,
The above-
Yeast Extract; Sodium acetate; NaCl; Na ₂ HPO ₄ ; K ₂ HPO ₄ ; KH ₂ PO ₄ ; (NH ₄₎ Cl; Glucose; MgSO _4; Wherein the antimicrobial agent is constituted by a manganese sulphate.
delete

Images