KR101306662B1 - Substitutes of immunity reinforcement agent and antibiotics and the method thereof - Google Patents

Abstract

The present invention relates to an adjuvant and an antibiotic replacement material and a method for preparing the same, and more particularly, after adding sugar to a mixture (hereinafter, referred to as juice) of tangerine, banana, pineapple, and soybean meal, known aspergillus bacteria After inoculation with Aspergillus nicer NRRL 2322 (ATCC 10577), primary culture was carried out for 5 to 10 days at 32-35 ° C. for fermentation to produce fermented organic acids including citric acid. (Scoria / Jeju volcanic stone / aka Matsutake) and Lactobacillus plantarum lactobacillus seed inoculated and fermented organically produced by primary fermentation in 2nd fermentation at 28 ~ 35 ℃ for 5-7 days. Lactobacillus Lactobacillus plantarium L-5 and L-6 Lactobacillus in Lactobacillus, which are excellent in acid resistance and strongly inhibit harmful bacterial growth, are developed, Lactobacillus fermentation composition and amino acid chelated complex ion by organic acid having stable and mature quality of amino acid chelated complex ion mineral are produced by combining with amino acids fermented and separated from dissolved cluster ionic mineral and soybean meal The present invention relates to an adjuvant and an antibiotic substitute for producing a mineral and a method for producing the same.
The fermentation product of the mixture, such as fruit, of the present invention contains various fermented organic acids, lactic acid bacteria and sugars, and produces amino acid chelated complex ionic minerals. It can be usefully used as a feed composition, it is safe for animals or humans, excellent acid resistance, and by using a composition for preparing a composition that strongly inhibits harmful bacterial growth, it is possible to manufacture an immunopotentiator and antibiotic replacement material.

Description

Immunopotentiators and antibiotic substitutes and methods for their preparation {SUBSTITUTES OF IMMUNITY REINFORCEMENT AGENT AND ANTIBIOTICS AND THE METHOD THEREOF}

The present invention relates to an immunostimulating agent and an antibiotic replacement material and a method for preparing the same, and more specifically, after adding sugar to a mixture (hereinafter, referred to as juice) of tangerine, banana, pineapple, and soybean meal, known bacteria Aspergillus Aspergillus nicer was inoculated with NRRL 2322 (ATCC 10577) and cultured for 1 to 5 days at 32-35 ° C. for 1-10 days to ferment and mature to produce fermented organic acids including citric acid. Inoculated with Scoria / Jeju volcanic stone / aka Songi) and Lactobacillus plantarum lactobacillus, followed by secondary fermentation at 28-35 ° C for 5-7 days sequentially to produce fermented organic acid. Lactobacillus Lactobacillus Plantanium L-5 and L-6 Lactobacillus in Lactobacillus, which are excellent in acid resistance and strongly inhibit the growth of harmful bacteria, are developed. Lactobacillus fermentation composition and amino acid chelated complex ion mineral by organic acid having a certain standard quality which is stable and matured by combining with amino acids fermented and separated from ionic minerals and soybean meal It relates to an adjuvant and an antibiotic replacement material for producing the same and a method for producing the same.

As a technique for producing a natural fermented organic acid, there have been proposals for preparing organic acids using tangerine gourd as a fermentation substrate. For example, Sakamoto et al. Theoretical evidence for the use as a carbon source for fermentation production of glycol, citric acid and lactic acid has been suggested (Sakamoto R., I.Shooro and M.Arai: Journal of Fermentation Engineering, Japan. 60.327 [1982]), Kuma Guy et al. Reported the production of citric acid by Aspergillus niga. [Kumagai.K., S. Usami and S. Hattori: Journal of Fermentation Engineering, Japan. 59 (5). 461--464 (1981)

Lactic acid bacteria are classified into homofermentative bacteria and heterofermentative bacteria, and homofermentative bacteria mainly produce lactic acid using hexose, and heterofermented bacteria produce other substances such as acetic acid, carbon dioxide, and ethanol together with lactic acid. It has various functions such as inhibiting pathogenic bacteria, antimutagenicity, antitumor activity, immune enhancement, and cholesterol lowering as a beneficial effect of human body. (Sanders, ME 2000.Considerations for use of probiotic bacteria to modulate) human health.J. Nutr. 130: 384S-390S.)

Lactic acid bacteria are beneficial microorganisms that produce lactic acid and do not produce harmful substances such as indole, phenol, amine, and ammonia. Products using lactic acid bacteria have been developed in various ways such as probiotics, fermenting agents, seasoning deodorants, organic acid drinks, health foods, etc. as a function of various metabolites and microorganisms themselves, and the development of technology is increasing for the purpose of health promotion. In addition, as an element showing the antimicrobial effect produced by lactic acid bacteria, low pH, organic acids, bacteriocin, ethanol, diacetyl, low reduction potential and the like exhibits antibacterial activity.

Many harmful bacteria are present in feeds and foods and can sometimes cause disease in livestock or humans. Salmonella is well known as a foodborne bacterium among foodborne pathogenic microorganisms, and Salmonella typhimurium, a representative pathogenic bacterium, is infected from contaminated foods, especially chicken and eggs, and gastroenteritis, nausea, vomiting, abdominal pain and diarrhea. And sometimes fever (Salyers, A. A and DD Whitt. 2002. Salmonella species, PP. 381-397. Bacterial pathogensis: A Molecular Approach. ASM press. Washington, DC).

Escherichia coli is mostly harmless, but E. coli, which is mainly O and H serotypes, causes diarrhea (Lior, H. 1994. Classification of Escherichia coli, pp 31-72, In C.). Gyles (ed.), Escherichia coli in Domestic Animal and Humans, CAB International, Wallingford, United Kingdom.

Staphylococcus aureus (Staphylococcus aureus) causes skin infections and toxin-mediated food poisoning.

Kimchi fermentation, fruit juice, vegetable juice fermentation has a unique characteristic that the fermentation proceeds as a variety of microorganisms such as Bacillus, yeast, lactic acid bacteria coexist rather than a single fermentation. Therefore, the predominant microorganisms vary depending on the culture time, conditions, and fermentation state, and thus, the properties of the fermentation product vary. In particular, fermented products using fruit juice is a fermentation using natural products, the Republic of Korea Patent Publication (2001-0037013; Publication No.) has been disclosed a method for preparing a fruit juice fermentation broth, the extract, the method of using the extract for seasoning and the method of using the extract as a deodorant .

In the novel Lactobacillus microorganism, it is very difficult to select a specific microorganism in fermentation using fruit juice and incubate for a certain period of time to produce a composition capable of suppressing effective organic acid components and harmful bacteria, Depending on the kind of microorganisms predominantly changed by the type of vegetable or composition, the components of the final fermentation composition may vary greatly, and there is a problem of irregular quality, such as a difference in efficacy effect. In addition, by selecting a microorganism that strongly inhibits harmful pathogenic bacteria in the final fruit fermentation product and using it as a seed, a method of preparing a product of a certain standard and enhancing the efficacy of the composition is required.

In addition, minerals are inorganic components of living organisms except for three elements such as carbon, hydrogen and oxygen among the elements that make up living organisms. Also called minerals, it refers to one of the five nutrients along with protein fatty carbohydrate vitamins. For example, it is well known that the interest in minerals is increasing and the development of mineral extraction and various products using the minerals are released.

These minerals are composed of calcium (Ca), phosphorus (P), magnesium (Mg), potassium (K), sodium (Na), zinc (Zn), liver (Mn), iron (Fe), and copper (Cu). ), Cobalt (Co), and many other elements, especially in humans, although deficiency does not occur but is indispensable to animals such as copper (Cu), cobalt (Co), manganese (Mn), zinc (Zn) At the same time contains.

In other words, the deficiency of copper (Cu) and cobalt (Co) among the nutrients required by the animal causes a decrease in hemoglobin production, resulting in anemia, and cobalt (Co) has recently been considered important as a member of vitamin B12. Mn) deficiency causes animals to lose fertility, and zinc (Zn) deficiency leads to poor growth.

As described above, chelated minerals having excellent absorption rate and firm buffering capacity have not been disclosed as domestic or foreign amino acid-mineral chelate products, and the products claimed to be chelated in the prior art as described above are assured. Since it has no characteristics as a binding structure and the discontinuous amino acid chelate, organic minerals with high absorption rate in the body cannot be developed. In other words, the end-of-life amino acid chelate mineral products that are commercially available until now are produced by evaporation-concentration drying.

Immunity enhancers and antibiotic substitutes and methods for preparing the same according to the present invention have been devised to solve the conventional problems as described above, and have the following problems.

First, inoculate a certain seed to a mixture of tangerine, banana, pineapple, soybean meal, molasses and the like to produce fermented organic acid, excellent acid resistance under the atmosphere of the organic acid and natural minerals, excellent inhibitory ability to harmful bacteria, and improve the quality of the final fermentation composition As a novel microbial strain with improved efficacy, the present invention is to provide a method for producing lactic acid bacteria for immunostimulants and antibiotic substitutes and lactic acid bacteria for immunostimulants and antibiotic substitutes prepared thereby.

Second, in the atmosphere of the fermented soybean meal and organic acids and natural minerals to maintain the quality of the final fermentation composition using the amino acid chelated complex ion minerals to produce lactic acid bacteria for improving the efficacy and immunosuppressive agent and antibiotic replacement material and It is to provide a juice fermentation broth for the immunopotentiator and antibiotic substitute prepared thereby.

Third, using the juice fermentation broth for immunosuppressants and antibiotic substitutes can be used as a disinfectant replacement and odor generating inhibitors of the barn, and prepared by the feed additives in liquid or powder form, and a method for producing an immune enhancer and antibiotic substitutes To provide an adjuvant and an antibiotic substitute prepared by

The solution of the present invention is not limited to those mentioned above, and other solutions not mentioned can be clearly understood by those skilled in the art from the following description.

Method for producing lactic acid bacteria for immunosuppressive agents and antibiotic substitutes according to the present invention is 35 to 45 parts by weight of water, 15 to 25 parts by weight of tangerine crushed syrup, 3 to 8 parts by weight of banana crushed syrup, 3 to 8 parts by weight of pineapple crushed syrup, S1 step of preparing a mixture A of 15 to 25 parts by weight of soybean meal and 5 to 15 parts by weight of molasses;

Inoculate 3 to 5 parts by weight of known bacteria Aspergillus nicer NRRL 2322 (ATCC 10577) to 100 parts by weight of the mixture A, and fermented at 32-35 ° C. for 5 to 10 days to prepare an organic acid as a primary fermentation product. Step S2;

S3 step of mixing and stirring 5-10 parts by weight of SukKorea to 100 parts by weight of the first fermentation product to prepare a stirring product containing ionic minerals;

S4 to inoculate 3 to 5 parts by weight of the known bacterium Lactobacillus plantarum lactobacillus spawn and fermented at 28 to 35 ° C. for 5 to 7 days to prepare an amino acid chelated complex ion mineral as a secondary fermentation product. step;

Filtering the secondary fermentation to produce a filtrate with a liquid immunopotentiator and an antibiotic substitute (hereinafter referred to as S-5);

20 to 30 parts by weight of the liquid product is mixed with 70 to 80 parts by weight of SKOREA, dried, and crushed to form a powdered immunopotentiator and antibiotic substitute material (hereinafter referred to as S-6);

In addition, the immunopotentiator and antibiotic replacement material according to the present invention is characterized in that it is prepared by the preparation method.

According to the immunostimulant and antibiotic substitute according to the present invention, and a method for producing the same, sugars are added to a mixture of tangerine, banana, pineapple, soybean meal, molasses, etc. It is produced by fermented organic acid, develops new lactic acid bacteria which are excellent in acid resistance under the atmosphere of organic acid and natural minerals, and strongly inhibits harmful bacterial growth, and amino acid chelated complex ion mineral is produced, Provides quality adjuvant and antibiotic substitutes.

In addition, according to the present invention, an immunostimulant and an antibiotic substitute material and a method for preparing the same, antimicrobial activity and probiotic effect by probiotics and antimicrobial agents, antibiotic substitute foods, and immunostimulant feed compositions can be usefully used in animals and humans. It is safe, has excellent acid resistance, strongly inhibits the growth of harmful bacteria, and when used as a drink, has the effect of preventing and treating pathogen infections and providing great help for health promotion.

In addition, according to the immune enhancing agent and antibiotic replacement material according to the present invention and a method for producing the same, it can also be used as a disinfectant replacement and odor generation inhibitor of the barn using the fruit fermentation broth, and by producing a feed additive in the form of liquid or powder And effects that can replace antibiotics.

The effects of the present invention are not limited to those mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

1 is a process chart showing a method for preparing an immunostimulant and antibiotic replacement material according to the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings and the contents described below. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the embodiments disclosed herein are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

1 is a process chart showing an immunopotentiator and an antibiotic substitute material according to the present invention and a method of manufacturing the same.

Method for producing lactic acid bacteria and amino acid chelated complex ion minerals for immunosuppressants and antibiotic replacement substances according to the present invention is 35 to 45 parts by weight of water, 15 to 25 parts by weight of tangerine crushed syrup, 3 to 8 parts by weight of banana crushed syrup, pineapple S1 step of preparing a mixture A of 3 to 8 parts by weight of crushed syrup, 15 to 25 parts by weight of soybean meal, and 5 to 15 parts by weight of molasses;

Inoculate 3 to 5 parts by weight of known bacteria Aspergillus nicer NRRL 2322 (ATCC 10577) to 100 parts by weight of the mixture A, and fermented at 32-35 ° C. for 5 to 10 days to prepare an organic acid as a primary fermentation product. Step S2;

S3 step of mixing and stirring 5-10 parts by weight of SukKorea to 100 parts by weight of the first fermentation product to prepare a stirring product containing ionic minerals;

Inoculate 3-5 parts by weight of the known bacterium Lactobacillus plantarum lactobacillus strain and ferment at 28-35 ° C. for 5-7 days to produce a novel lactic acid bacterium and an amino acid chelated complex ion mineral as a secondary fermentation product. S4 step of preparing a;

Filtering the secondary fermentation product to produce a filtrate with a liquid immunopotentiator and an antibiotic substitute material;

20 to 30 parts by weight of the liquid product is mixed with 70 to 80 parts by weight of SKOREA, dried, and crushed to form a powdered immunopotentiator and antibiotic substitute material S6 step; includes.

Specifically, 35 to 45 parts by weight of water, 15 to 25 parts by weight of tangerine crushed syrup, 3 to 8 parts by weight of banana crushed syrup, 3 to 8 parts by weight of pineapple crushed syrup, 15 to 25 parts by weight of soybean meal, 5 to 15 parts by molasses Inoculate 3 to 5 parts by weight of the known bacterium Aspergillus nicer NRRL 2322 (ATCC 10577) to 100 parts by weight of the mixture A, which was sterilized, and fermented at 32 to 35 ° C. for 5 to 10 days. The fermented organic fermentation is stabilized by fermentation to produce fermented organic acids, including citric acid, 5-10 parts by weight of SKOREA and known bacteria Lactobacillus plantarum (Latobacillus plantarum) Lactobacillus 3 From the secondary fermentation product inoculated at -5 parts by weight and fermented at 28-35 ° C. for 5-7 days, lactic acid bacteria A could be obtained as a microorganism with strong acid resistance even under acidic pH of 2.5-3.5.

In the present invention, the agitated substance is dissolved by ionizing the mineral of the metal oxide state contained in natural minerals Korea by fermentation organic acid to produce ionic minerals, the resulting ionic minerals are fermented by soybean meal and amino acid produced by decomposition Produces chelate ion minerals can be useful as antimicrobial activity and probiotics by probiotics and antimicrobial agents, antibiotic replacement foods and immunopotentiator compositions.

Lactobacillus A was isolated from the secondary fermentation and identified through sequencing of 16s rRNA (ribosomal RNA). As a novel lactic acid bacterium, Lactobacillus Plantarum L-5 and Lactobacillus sp. ) L-6 was obtained.

The novel lactic acid bacteria, Lactobacillus plantanium L-5 and Lactobacillus genus L-6, obtained by the method for preparing lactic acid bacteria for immunosuppressive agents and antibiotic replacement substances according to the present invention, were in November 2006 KACC (Korea Agricultural Culture Collection). (KACC-91284P) and (KACC-91285P), respectively.

Furthermore, L-6 in Lactobacillus has 97% similarity to Lactobacillus satsumensis through 16s rRNA sequencing and classification, further highlighting its novelty.

In the present invention, the novel lactic acid bacteria Lactobacillus Plantarum (Lactobacillus Plantarum) L-5 and Lactobacillus sp. (Lactobacillus sp.) L-6 including the L-6 is safe for animals and humans, acid resistance It is excellent and has the effect of strongly inhibiting harmful bacterial growth, it is possible to manufacture a substance that can replace the immune enhancers and antibiotics using this.

Specifically, the juice fermentation broth fermented using the novel Lactobacillus Plantarum L-5 and Lactobacillus sp. L-6 in the present invention is threatened from various harmful bacteria. Livestock can be provided as a feed additive.

Lactobacillus Plantarum (Lactobacillus Plantarum) L-5 and Lactobacillus sp. L-6 fermented to the juice fermentation broth fermented using the novel Lactic acid bacteria Lactobacillus Plantarum (Lactobacillus Plantarum) L-6 in liquid phase It can be used as a more useful additive for animals infected with harmful bacteria by making it used as a feed additive for the drinking water, it can also be prepared as an additive in the form of powder by adsorption drying using, for example.

In addition, the juice fermentation broth can be utilized for the purpose of reducing the odor of livestock farms, there is a feature that can reduce the odor of the farmhouse to 40 to 90% level, another object is to filter the juice fermentation broth It may also be prepared as a beverage composition for the purpose of health promotion.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples illustrate the present invention in detail, and the scope of the present invention is not limited by the following examples.

Step S1 of preparing a mixture A by mixing tangerine, banana, pineapple, soybean meal, molasses and water, and step S2 of preparing an organic acid as a primary fermentation product.

After preparing a medium consisting of 50 parts by weight of orange juice, 10 parts by weight of banana, 10 parts by weight of pineapple, 10 parts by weight of soybean meal, 20 parts by weight of molasses, and 100 parts by weight of water, the known strain Aspergillus nicer NRRL 2322 ( ATCC 10577) was inoculated into a sealed container and fermented at 35 ° C. for 8 days.

S3 step of preparing a stirred solution containing ionic minerals and S4 step of preparing an amino acid chelated complex ion mineral which is a secondary fermentation product

Sealed by inoculating 15 to 30 parts by weight of S. Korea (Scoria / Jeju volcanic stone / aka: pine) and 3 parts by weight of lactic acid bacterium Lactobacillus Plantarum prepared in 90 parts by weight of the primary fermented product prepared in Example 1 After fermentation at 35 ° C. for 6 days in a container, a liquid fermentation product (hereinafter referred to as S-5) containing amino acid chelated ion minerals was prepared by filtration.

Test Example 1 Analysis of Fermented Organic Acid

As can be seen in [Table 1], S-5 produced and filtered by primary organic acid fermentation and secondary lactic acid fermentation, the organic acid content of the fermented product was 7.8%, of which almost 80% Citric acid produced about 20% of the remaining organic acids, including other lactic acids. It is believed that their production ratios were mostly generated during the primary organic acid fermentation.

Organic acid type

content(%)
Citric acid 78.4 Natural acetic acid 7.4 Natural Lactic Acid 6.7 Natural malic acid 4.6 Natural Succinic acid 2.9

Test Example 2 Mineral Content Analysis

Mineral components of S Korea (Song powder) and the secondary fermentation product of Example 2 were analyzed. As can be seen in [Table 2] in the analysis results, the level of the secondary fermentation product of Example 2 is higher than the raw stone is expected to be mixed with the minerals contained in the fruits used as raw materials, rather than the raw stone Low levels are believed to prevent oxidized minerals from dissolving in organic acids.

S Korea (Songs powder)
(PPM) Secondary Fermentation of Example 2
(PPM) Al (Aluminum) 231 9.5 Ca (calcium) 247.4 10 Fe (iron oxide) 241.6 8 K (potassium) 33.32 1.5 Mg (magnesium) 317 9.3 Na (sodium) 64.04 3.3 S (sulfur) 10.824 0.3 Ba (barium) 2.186 0.04 Cd (Cadmium) Not detected Not detected Co (cobalt) 0.7419 0.001 Cr (divalent chromium) 1.089 0.03 Cu (copper) Not detected 0.0003 Mn (manganese) 21.14 0.2 Mo (molybdenum) Not detected Not detected Ni (nickel) 2.959 0.033 Pb (lead) Not detected Not detected Sr (strontium) 1.095 0.09 Ti (titanium) 2.604 1.5 V (vanadium) 3.308 0.024 Zn (zinc) 3.025 0.028

Isolation of Microorganisms (Lactobacillus A)

Lactic acid bacteria A was isolated from the secondary fermentation product (S-5) of Example 2. First, as a lactic acid bacterium separation medium, Mann Rogosa Sharpe (MRS: BD, Becton, Dickinson and Company, France) agar medium containing 10% by weight of S-5 of Example 2 was used, and anaerobic anaerobic culture at 37 ° C. for 3 days. Six kinds of lactic acid bacteria, L-1, L-2, L-3, L-4, L-5, L-6, were isolated using random selection method to separate the lactic acid bacteria by culturing colony type and color. It was.

[Test Example 3] antibacterial bactericidal capacity verification

Ten isolated lactic acid bacteria A were tested for their antibacterial activity. The harmful bacteria used in the test were Salmonella typhimurium, Salmonella gallinarum, Staphylococcus aureus and Escherichia coli.

Hazardous bacteria were incubated in TSB medium [Tryptone 1.7%, Soytone 0.3%, Salt 0.5%] for 1 day, followed by Tryptic Soy agar (TSA: Tryptone 1.7) containing 100% and 1.5% agar. %, Soytone 0.3%, NaCl 0.5%) Mix 100 ml culture medium and penicylinder (Fisher Scientific, Cat. No. 07-907-5, USA, capacity 300, ID 6 mm, OD 8 mm) on 20 ml aliquots of agar plate , H 10mm) was flame sterilized and placed at regular intervals, and then the culture solution of each lactic acid bacterium was dropped by 100 volumes, left at room temperature for about 20 minutes, then transferred to an incubator and cultured at 37 ° C. for 3 days, and the diameter of the inhibited ring appeared. Comparative agar diffusion methods were used. As can be seen in Table 3, the isolated lactic acid bacteria A had excellent inhibition ability against harmful bacteria.

Strain

Diameter of Inhibitory Ring (mm)
S. typhimurium S. gallinarium S. aureus E. coli Control group (MRS) 0 0 0 0 L-1 25 23 25 26 L-2 23 23 23 25 L-3 24 20 22 20 L-4 27 26 26 27 L-5 28 28 29 29 L-6 29 29 27 28

Test Example 4 Flame Resistance Test

MRS agar medium was inoculated with 7 species of lactic acid bacteria isolated on a plate medium prepared by adjusting the salt concentration to 0%, 2%, 4%, and 6% by weight, respectively, and then grown at 37 ° C for 7 days. Was tested to confirm the resistance to salt. As can be seen in Table 4, L-1, L-2 and L-4 strains were grown at 4 wt% salt concentration, but L-3 was 2 wt%, L-5 and L-6 were 6 It also grew up to wt% and showed excellent growth even in high saline.

Strain

Salt (NaCl) concentration
0% 2% 4% 6% Control group + - - - L-1 + + + - L-2 + + + - L-3 + + - - L-4 + + + - L-5 + + + + L-6 + + + +

Test Example 5 Acid Resistance Test

MRS liquid medium was prepared by dividing into six groups of pH 2.5, 3.0, 3.5, and unadjusted, and then inoculated with each of the six lactic acid bacteria isolated and incubated at 37 ° C. for 5 days to observe growth. As a result, as can be seen in Table 5, L-1 and L-2 grew to pH 3.5, and L-3, L-4 L-5 and L-6 grew to pH 2.5. Therefore, L-5 and L-6 bacteria were determined as lactic acid bacteria capable of growing at low pH.

Strain

MRS

pH3.5

pH3.0

pH2.5
Control group +++ - - - L-1 +++ + - - L-2 +++ ++ - - L-3 +++ + + + L-4 +++ ++ ++ ++ L-5 +++ ++ ++ ++ L-6 +++ +++ ++ ++

Test Example 6 Identification of Lactic Acid Bacteria A

As a result of identification through the nucleotide sequence of the 16S rDNA, the types of lactic acid bacteria contained in the fruit fermented milk were as follows.

Lactobacillus acidophilus / L-1,

Lactobacillus mali / L-2,

Lactobacillus hilgardii / L-3,

Lactobacillus plantarum / L-4,

And as a new lactic acid bacterium

Lactobacillus Plantarum / L-5, and

Lactobacillus sp./L-6

Looking at L-5 and L-6 in detail as the novel lactic acid bacteria, L-5, L-6 strains selected for 16S rRNA sequencing for strain identification were produced after culturing on MRS agar plate medium Single colonies (Colony) were taken and genomic DNA was isolated using AccuPrep Genomic DNA Extraction Kit (BIONEER CO., Ltd, Korea).

After confirming the genomic DNA by electrophoresis, PCR (GeneAmp PCR System 2700, USA) was subjected to Primer 27F (5AGAGTTTGATCMTGGCTCAG>) and 1492R (5 TACGGYTACCTTGTTACGACTT) under conditions of Denaturation 95/30 sec, Annealing 55/1 min, Extension 72/1 min. After a total of 35 cycles using>), 16S rRNA of about 1500 bp was confirmed by electrophoresis, and the amplified 16S rRNA was purified using AccuPrep PCR Purification kit. The nucleotide sequence was analyzed using an Automated DNA Sequencer (ABI 3100, Applied Biosystem Inc., USA), and primers were used in addition to the above 27F and 1492R. .

Subsequently, the sequences are combined using Clustal X software to use Advanced Blast search (Altschul et al ., 1997) provided by The National Center for Biotechnology Information (NCBI) (http: //www.ncbi.nlm.nih). After comparing with GenBank sequence, Dendrogram was finally created by neighbor-joining analysis.

The 16S rDNA sequences of the novel lactic acid bacteria L-5 and L-6 are as described below, respectively, L-5 shows 99% similarity to Lactobacillus Plantarum (Lactobacillus Plantarum) Lactobacillus Plantarum (L-5), L-6 was identified as 97% similar to Lactobacillus satsumensis, L. nagelii (L. nagelii), but the significance was different. According to the bacterium of the genus Lactobacillus.

<16S rDNA sequence of L-5>

ATGAAGTTGA GTGCTTGCAT TTAACTGATT TGACATTAAG ACGAGTGGCG 50

AACTGGTGAG TAACACGTGG GTAACTTGCC CCGAAGCGGG GGATAACATT

TGGAAACAGG TGCTAATACC GCATAACAAC AAAAACCACA TGGTTTTTGT

TTAAAAGATG GTTTCGGCTA TCACTTTGGG ATGGACCCGC GGCGTATTAG

CTAGTTGGTG AGGTAATGGC TCACCAAGGC AATGATACGT AGCCGACCTG

AGAGGGTAAT CGGCCACATT GGGACTGAGA CACGGCCCAA ACTCCTACGG

GAGGCAGCAG TAGGGAATCT TCCACAATGG ACGAAAGTCT GATGGAGCAA

CGCCGCGTGA GTGATGAAGG GTTTCGGCTC GTAAAACTCT GTTGTTGGAG

AAGAACGGGT GTCAGAGTAA CTGTTGACAT CGTGACGGTA TCCAACCAGA

AAGCCACGGC TAACTACGTG CCAGCAGCCG CGGTAATACG TAGGTGGCAA 500

GCGTTGTCCG GATTTATTGG GCGTAAAGCG AGCGCAGGCG GTTTTTTAGG

TCTGATGTGA AAGCCTTCGG CTTAACCGGA GAAGTGCATC GGAAACCGGG

AGACTTGAGT GCAGAAGAGG ACAGTGGAAC TCCATGTGTA GCGGTGAAAT

GCGTAGATAT ATGGAAGAAC ACCAGTGGCG AAGGCGGCTG TCTGGTCTGC

AACTGACGCT GAGGCTCGAA AGCATGGGTA GCGAACAGGA TTAGATACCC

TGGTAGTCCA TGCCGTAAAC GATGAGTGCT AAGTGTTGGA GGGTTTCCGC

CCTTCAGTGC TGCAGCTAAC GCATTAAGCA CTCCGCCTGG GGAGTACGAC

CGCAAGGTTG AAACTCAAAG GAATTGACGG GGGCCCGCAC AAGCGGTGGA

GCATGTGGTT TAATTCGATG CTACGCGAAG AACCTTACCA GGTCTTGACA

TCTTCTGCTA ACCTAAGAGA TTAGGCGTTC CCTTCGGGGA CGGAATGACA 1000

GGTGGTGCAT GGTTGTCGTC AGCTCGTGTC GTGAGATGTT GGGTTAAGTC

CCGCAACGAG CGCAACCCTT ATTGTCAGTT GCCAGCATTT AGTTGGGCAC

TCTGGCGAGA CTGCCGGTGA CAAACCGGAG GAAGGTGGGG ATGACGTCAA

ATCATCATGC CCCTTATGAC CTGGGCTACA CACGTGCTAC AATGGACGGT

ACAACGAGTC GCGAAACCGC GAGGTCAAGC TAATCTCTTA AAGCCGTTCT

CAGTTCGGAT TGTAGGCTGC AACTCGCCTA CATGAAGTTG GAATCGCTAG

TAATCGTGGA TCAGCATGCC ACGGTGAATA CGTTCCCGGG CCTTGTACAC

ACCGCCCGTC ACACCATGAG AGTTTCAACA CCCAAAGCCG GTGAGGTAAC

CTTCGGGGGC CAGCCGTC 1418

<16S rDNA sequence of L-6>

GATGCTTGCA TCTTTTAAAA AGTTGAGTGG CGAACGGGTG AGTAACACGT 50

GGGTAACCTG CCTTAAAGTG GGGGATAACA CTTGGAAACA GGTGCTAATA

CCGCATAACC ATCAAAACCG CCTGGTTTTG ATGTTAAAGA TGGTTCTGCT

ATCGCTTTAA GATGGACCCG CGGCGTATTA GCTAGTTGGT GAGGTAACGG

CTTACCAAGG CAATGATACG TAGCCGAACT GAGAGGTTGA TCGGCCACAT

TGGGACTGAG ACACGGCCCA AACNCCTACG GGAGGCAGCA GTAGGGAATC

TTCCACAATG GACGAAAGTC TGATGGAGCA ACGCCGCGTG AGTGAAGAAG

GTTTTCGGAT CGTAAAACTC TGTTGTCAGA GAAGAACGTG TGTGAGAGTA

ACTGTTCACG CAGTGACGGT ATCTGACCAG AAAGTCACGG CTAACTACGT

GCCAGCAGCC GCGGTAATAC GTAGGTGGCA AGCGTTGTCC GGATTTATTG 500

GGCGTAAAGG GAACGCAGGC GGTCTTTTAA GTCTGATGTG AAAGCCTTCG

GCTTAACCGA AGTCGGGCAT TGGAAACTGG GAGACTTGAG TGCAGAAGAG

GAGAGTGGAA CTCCATGTGT AGCGGTGAAA TGCGTAGATA TATGGAAGAA

CACCAGTGGC GAAAGCGGCT CTCTGGTCTG TAACTGACGC TGAGGTTCGA

AAGCGTGGGT AGCAAACAGG ATTAGATACC CTGGTAGTCC ACGCCGTAAA

CGATGAATGC TAAGTGTTGG AGGGTTTCCG CCCTTCAGTG CCGCAGCTAA

CGCATTAAGC ATTCCGCCTG GGGAGTACGA TCGCAAGATT GAAACTCAAA

GGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAA

GCAACGCGAA GAACCTTACC AGGTCTTGAC ATCTTTTGCT AACCTGAGAG

ATCAGGTGTT CCCTTCGGGG ACAAAATGAC AGGTGGTGCA TGGTTGTCGT 1000

CAGCTCGTGT CGTGAGATGT TGGGTTAAGT CCCGCAACGA GCGCAACCCT

TATTGTTAGT TGCCAGCATT TAGTTGGGCA CTCTAACGAG ACTGCCGGTG

ACAAACCGGA GGAAGGTGGG GATGACGTCA AATCATCATG CCCCTTATGA

CCTGGGCTAC ACACGTGCTA CAATGGACGG TACAACGAGT CGCAAGACCG

CGAGGTCAAG CTAATCTCTG AAAACCGTTC TCAGTTCGGA TTGCAGGCTG

CAACTCGCCT GCATGAAGTC GGAATCGCTA GTAATCGCGG ATCAGCATGC

CGCGGTGAAT ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGA

GAGTTTGTAA CACCCAAAGC C 1371

Test Example 7 Organic Acidity and pH Test

In step S-2 of preparing the organic acid of Example 1, a certain amount of the sample was taken every two days, and the organic acidity and pH were measured. As a result, the results as shown in [Table 6] were obtained. The organic acid was steadily produced according to the culture period, and the organic acid production was excellent in the later stages of culture.

Strain 0 days 2 days 4 days 6 days 8 days S-2 pH 6.87 5.39 4.11 3.30 2.35 Organic acidity 0.02% 1.10% 2.86% 4.89% 7.83%

Preparation of Liquid and Powdered Feed Additives Using S-5

100 parts by weight of S-5, a liquid fermentation product containing amino acid chelated ion minerals prepared in Example 2, was diluted with 5 times of water, and then sugar was added at a concentration of 5 parts by weight, and 2 parts by weight of Sukko powder was prepared first. The mixture was allowed to stand for one month and then cultured to prepare a feed additive for liquid drinking water. In addition, 25 wt% of liquid feed additives were added by mixing 75 wt% of SKOREA powders in a second step and then dried at 42 ° C. for 2 days to prepare a powdery feed additive S-6.

[Test Example 8] piglet productivity improvement effect

Powdered feed additive S-6 of Example 4 was fed by adding 2 kg to 1 ton of pig feed compounded feed.

As a result, the total weight gain was about 10% from 13.87Kg to 15.25Kg in the control, and the diarrhea index decreased by 86% from 61.3% to 8.6% in the control, and the mortality rate was about 66% (from 9.13% to 3.08%). Decreased.

[Test Example 9] Improvement effect of broiler productivity

Powdered feed additive S-6 of Example 4 was fed by adding 2 kg to 1 ton of broiler feed.

As a result, the growth rate improved by 3.9% from 95.8% to 99.7% in the control, and the average weight increased by about 14% from 1.69kg to 1.93% in the control.

Test Example 10 Effect of Intestinal Microorganisms

Powdered feed additive S-6 of Example 4 was fed by adding 2 kg to 1 ton of broiler feed.

As a result, the intestinal harmful microorganisms can be confirmed that the number of E. coli and Salmonella in the small intestine and caecum more reduced than the control as compared to the control group as shown in the [Table 7].

(Unit: log ₁₀ cfu)

division

Escherichia coli

Salmonella Control Test section Control Test section Intestine 6.36 6.21 5.77 4.94 Caecum 7.40 7.31 5.07 4.71

[Test Example 11] egg laying rate improvement effect

The powdery feed additive S-6 of Example 4 was added to 1 ton of brown laying hens, and the feed was fed from 36 weeks old to 42 weeks old.

As can be seen in [Table 8], the egg production rate was initially decreased from 93.0% to 92.2% in the control group, but it increased steadily from 37 weeks of age, and showed an improvement of 5.3% from 90.0% to 94.8% at 42 weeks of age.

Week 36 37 38 39 40 41 42 Egg production (%) Control 93.0 92.0 92.0 91.0 91.0 91.0 90.0 S-6 92.2 93.2 93.9 93.2 93.5 94.2 94.8 % Increase -0.9 +1.3 +2.1 +2.4 +2.7 +3.5 +5.3

Experimental Example 12 Effect of Declining for Flatfish Mortality

In the liquid feed additive of Example 11, 4Kg of 4 tons of seawater was added to an aquarium operated with an oxygen supply device, and 100 flounders weighing about 1Kg were cultured for 30 days without feeding to test mortality.

As a result, control flounder died 67% (horses) after 3 days, and 100% (100 horses) died after 5 days. In contrast, 98% (98) survived after 30 days of feeding in the feed additive for drinking water.

The embodiments described herein and the accompanying drawings are merely illustrative of some of the technical ideas included in the present invention. Therefore, since the embodiments disclosed herein are not intended to limit the technical spirit of the present invention but to explain, it is obvious that the scope of the technical spirit of the present invention is not limited by these embodiments. Modifications and specific embodiments that can be easily inferred by those skilled in the art within the scope of the technical spirit included in the specification and drawings of the present invention should be construed as being included in the scope of the present invention.

Claims (8)

35 to 45 parts by weight of water, 15 to 25 parts by weight of tangerine crushed syrup, 3 to 8 parts by weight of banana crushed syrup, 3 to 8 parts by weight of pineapple crushed syrup, 15 to 25 parts by weight of soybean meal, molasses 5 to 15 parts by weight S1 step of preparing A;
Inoculate 3 to 5 parts by weight of known bacteria Aspergillus nicer NRRL 2322 (ATCC 10577) to 100 parts by weight of the mixture A, and fermented at 32-35 ° C. for 5 to 10 days to prepare an organic acid as a primary fermentation product. Step S2;
S3 step of mixing and stirring 5-10 parts by weight of SukKorea to 100 parts by weight of the first fermentation product to prepare a stirring product containing ionic minerals; And
Inoculate 3-5 parts by weight of known bacteria Lactobacillus platarum lactobacillus spawn in the agitated and fermented at 28-35 ℃ for 5-7 days to prepare a secondary fermented lactic acid bacteria and amino acid chelated complex ion mineral S4 step; to produce a fermentation product containing a lactic acid bacteria and amino acid chelated complex ion minerals comprising a.
A fermentation product comprising a lactic acid bacterium and an amino acid chelated complex ion mineral, which is prepared by the method of claim 1.
delete Among the lactic acid bacteria isolated and identified in claim 2, 3-7 parts by weight of Lactobacillus plantanium L-5 (KACC-91284P) and L-6 (KACC-91285P) genus Lactobacillus, respectively, or inoculated in a juice and mixed at 28-35 ℃ A fermentation product containing a lactic acid bacterium and an amino acid chelated complex ion mineral, characterized in that the fermentation broth is produced by fermentation for 5 to 7 days.
Preparation of a feed additive comprising a lactic acid bacteria and amino acid chelated complex ion mineral, characterized in that the liquid feed additive is prepared in a fermentation product containing the lactic acid bacteria and amino acid chelated complex ion mineral prepared by the method of claim 4 Way.
The method according to claim 5,
20 to 30 parts by weight of the liquid feed additives mixed with 70 ~ 80 parts by weight of Sukkorea dried and then pulverized to prepare a powdered feed additive, characterized in that for producing a feed additive.
A feed additive, which is prepared by the method of claim 5 or 6.
According to claim 2, The lactic acid bacteria Lactobacillus plantarium L-5 (Accession No. / KACC-91284P) and Lactobacillus genus L-6 (Accession No. / KACC-91285P) Lactobacillus and amino acid chelated, characterized in that Fermented product containing complex ion mineral.

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