WO2011055927A2 - Composition of mixed microorganisms growing in super-highly acidic conditions, and use thereof - Google Patents

Abstract

The present invention relates to a composition of mixed microorganisms growing in super-highly acidic conditions, and to the use thereof. The mixed composition according to the present invention comprises lactobacillus and yeast which can grow in super-highly acidic conditions. When added to feed, the microorganisms of the present invention can reach the inside of the intestine without being killed by the stomach acid of an animal. The microorganisms of the present invention have antibacterial effects against enteric bacteria, and protect animals from becoming infected with pathogenic microorganisms, and thus promote the growth of the animals. In addition, the mixed microorganisms can reduce offensive odors from food waste, livestock muck, or the like.

Description

Acetic Acid Growth Mixed Microbial Compositions and Uses thereof

The present invention relates to a microbial preparation containing acetic acid lactic acid bacteria and acetic acid yeast, which is capable of growing in acetic acid and is not killed by strong gastric acid and can be reached in the intestine. It provides a composition of natural "cocktail" consisting of, intrinsic organic acid and intrinsic bacteriocin, to a mixed microbial agent that can promote the growth of the animal.

Pathogenic bacterial infections in the stomach and intestines of animals such as chicken, poultry and shrimp, as well as pigs, are a major problem for consumers as well as farmers. Even if farmers adhere to very strict hygiene standards in their homes, it is very difficult to prevent these infections. The onset of severe diarrhea in suckling piglets depends on the immunological factors in the relationship between natural E. coli and lactic acid in the intestine. Frequent use of antibiotics to fight infection results in a supply of concentrated antibiotics to humans using the livestock for food by bioaccumulation, and continued exposure of antibiotics aids the survival of antibiotic resistant strains. This is a big problem that occurs. Some of these bacteria become resistant to many antibiotics, resulting in the death of animals even if treated with antibiotics. Once pathogenic bacteria cause intestinal flora to be imbalanced, food digestibility is reduced, so normal animal weight gain cannot be achieved. In addition to the obvious economic loss of these breeders, there are many cases where the quality of meat is degraded, and there is a risk that consumers of this meat may be infected. A group of animals that are very susceptible to bacterial infection are piglets, which frequently get infected from sows. When piglets are isolated from sows by weaning and begin to feed solids, they present a serious problem in their intestines. This problem is often severe enough for piglets to die. In addition, these infections cause large economic problems for farmers, as well as the death of several piglets as well as the excessively long breeding period of infected animals. Salmonella and campylobacter infections in birds are very common, causing significant economic losses for farmers and severe or fatal infections for chicken consumers. Salmonella infections in chicken rearing are developing around the world in an incurable state as Salmonella bacteria become highly resistant to all kinds of antibiotics.

Theoretically, the best way to solve these problems is perhaps to find a way to prevent pathogenic bacteria from adhering to the mucous membrane surface, or, for animals already suffering from imbalance of the bacterial flora, to restore normal flora. To find out.

Lactobacillus sp. Microorganisms are lactic acid bacilli that are homozygous or heterozygous and are commonly found in the intestines of animals including humans and in the fermentation of dairy products or vegetables. Lactobacillus microorganisms maintain acidic intestinal pH to inhibit the growth of harmful bacteria such as E. coli and Clostridium , improve diarrhea and constipation, as well as improve immune function, vitamin synthesis, anticancer activity, Serum cholesterol lowers. Acidophillin produced by lactic acid bacilli is known to inhibit the growth of dysentery, Salmonella, Staphylococcus and Escherichia coli. It also acts to stop diarrhea by inhibiting the growth of diarrhea causative bacteria and normalizing the intestinal flora.

Bacterial diarrhea in livestock causes reduced weight gain and mortality. Therefore, in order to prevent this and increase the productivity of livestock, the addition of antibiotics to the feed has generally been widely practiced. However, because of the emergence of resistant bacteria against antibiotics and the remaining antibiotics in livestock products, policies are being pushed towards regulating the use of antibiotics in feeds, and the organic livestock breeding law is emphasized. Currently, the use of probiotics is highly recommended as an alternative to antibiotic use. Recently, researches to develop probiotics and livestock feeds have been actively conducted using the above characteristics of Lactobacillus microorganisms.

European Patent No. 0861905 discloses a novel Lactobacillus strain and pharmaceutical compositions and dairy products for the treatment of gastrointestinal diseases including the same, and International Patent No. 99/29833 describes probiotics and natural medicines applicable to foods. A useful strain, Lactobacillus paracasei, has been disclosed.

Korean Patent Publication No. 1998-78353 discloses a novel acid-resistant Lactobacillus genus microorganism having harmful microbial inhibitory activity and a probiotic active agent for livestock containing the same.

Korean Patent Application No. 10-2001-0085666 discloses Lactobacillus paracasei , a novel Lactobacillus microorganism isolated from the fermentation broth of kimchi and its use as a probiotic, feed, deodorant and food additive. have.

Ugarte MB et al., Lactobacillus perolens , Lactobacillus casei , Lactobacillus casei from Argentine cheese in J. Food Prot. 69, pp. 2983-91, 2006. Lactobacillus plantatum , Lactobacillus rhamnosus , Lactobacillus qubatus and Lactobacillus permentum were isolated and all species were resistant to 25 ppm lysozyme. It was reported that it was resistant to% bile acid, and the pH of the gastric acid solution of pH 2.0 decreased from 3.2 to 7 log, and showed antimicrobial activity against pathogenic bacteria.

Deck Mosquera Brewer selenium cis acetonitrile in a hygienically Kinoko tea drink in a short time As the invention using the yeast Japanese Patent Publication No. 3084348 (filed 06/02/1995) relates to a method for producing industrially bakteo wave Ste Uriah Taunus (Acetobacter pasteurianus) acetonitrile bakteo Oh Shetty (Acetobacter aceti), a My process serenity busy with saccharide (Saccharomycesce cerevisiae), Brett Gaetano My process Brewer selenium sheath (Brettanomyces bruxellensis), and Xi Kosaka Roman access mobile are selected, one or two from (Zygosaccharomycesce bailii) It describes a method for producing black tea mushroom beverage producing bacteria and black tea mushroom beverage, characterized in that the selection of more than one species.

U.S. Patent Publication No. 5512465 filed May 23, 1994 filed Dekkera bruxellensis as a method for producing optically active 1.3-butanediol.

In 2006, L. Conterno et al. (Am. J. Enol. Vitic 57 (2)) studied the genetic and physiological characteristics of Brettanomyces bruxellensis strains. Rain, while presenting in 139-147. All 47 strains of B. bruxellensis were resistant to 10% alcohol concentrations, and all strains were able to grow at pH 2.5 (94% of them viable at pH 2.0), one third of the strains. Reported that they were able to grow at 10 ° C and the other 1/3 survived at 37 ° C.

In addition, PCT / SE1997 / 00252 filed Feb. 4, 1997, Pediococcus pentosaceus, Pediococcus acidylactici, Pichia parinosa, Deckera brucellensis, Bacillus, Streptococcus, Staphylo There have been reports of non-ruminant feed additives comprising three or more of the microorganisms of the Caucasus. Said document said that the microorganism showed resistance to gastric acid, was toxic to humans, and had a bactericidal effect and an immunity improving effect.

However, there have been no reports on the antimicrobial, animal growth, and odor removal effects of a mixture of Deckera brucellensis, Lactobacillus casei, and Lactobacillus peronance. The present inventors have confirmed that the mixture of Dec. Brusselenssis that can grow at ultra low acidity and lactic acid bacteria that can grow at ultra low acid has an antibacterial effect, promotes animal growth, and removes odor, and has completed the present invention.

It is an object of the present invention to provide a microbial preparation comprising Deckera brucellenssis and Lactobacillus casei, Lactobacillus peronence, which has an antibacterial effect, an animal growth promoting effect, and an odor removing effect.

Lactobacillus casei WS-1 (KCCM10896P), Lactobacillus casei W-1 (KCCM10897P), Lactobacillus ferrence W-3 (KCCM10898P), Lactobacillus casei W-4 (KCCM10899P) and Lactobacillus casei W- 6 (KCCM10900P) and Decera Brucelensis WY-1 (KCCM10901P) are provided.

The present invention acetic acid growth mixed microbial composition is composed of lactic acid bacteria and yeast and can be grown under acetic acid conditions. When the microorganism according to the present invention is added to the feed, it is not killed by gastric acid and arrives well in the intestine, and has an antibacterial effect on the intestinal bacteria, thereby preventing the infection of the animal from pathogenic microorganisms and promoting the growth of the animal. In addition, the mixed microorganism effectively removes odor from food waste or livestock manure. Therefore, it is possible to replace antibiotics added to animal feed and contribute to human health, and effective in removing odors, and thus can be effectively used to improve the human living environment.

FIG. 1 shows colony and scanning electron microscopy (SEM) cell morphology of isolated Lactobacillus strains formed on Lactobacilli MRS Broth ( MRS , Difco) plates containing bromophenol blue.

Figure 2 shows the scanning electron microscope (SEM) cell morphology of the isolate strain WY-1 (yeast).

FIG. 3 shows the results of identification of the isolated strains using random amplified polymorphic DNA (RAPD).

Figure 4 shows a schematic through 16S rDNA sequencing of the isolate strain WS-1.

Figure 5 shows a schematic diagram through 16S rDNA sequencing of the isolate strain W-1.

Figure 6 shows a schematic diagram through 16S rDNA sequencing of the isolate strain W-3.

Figure 7 shows a schematic diagram through 16S rDNA sequencing of the isolate strain W-4.

8 shows a schematic diagram of 16S rDNA sequencing of the isolated strain W-6.

9 shows a schematic diagram of 16S rDNA sequencing of the isolated strain WY-1.

Figure 10 shows the results of measuring the number of live bacteria in FeedFree ™ in the medium for each pH by fluorescence biostaining method.

11 shows the results of the antimicrobial test of FeedFree ™ against microorganisms causing fish disease.

The present invention is Lactobacillus casei WS-1 (KCCM10896P), Lactobacillus casei W-1 (KCCM10897P), Lactobacillus ferrence W-3 (KCCM10898P), Lactobacillus casei W-4 (KCCM10899P) and Lactobacillus ka It provides an antimicrobial microbial composition comprising J-W-6 (KCCM10900P) and Decera Brucelensis WY-1 (KCCM10901P). The microbial composition may further include additional materials such as bacteriocin and organic acids. The organic acid may include one or more combinations selected from the group consisting of lactic acid, fish acid, vinegar acid, succinic acid, tartaric acid, maleic acid, malic acid and citric acid.

On the other hand, the antimicrobial composition according to the present invention can be formulated into powders and granules according to methods that can be easily carried out by those skilled in the art. For example, in the case of an oral solution, pharmaceutically acceptable carriers include distilled water, oleic acid ethyl, ethanol, propylene glycol, glycerin, and the like; Ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, tocopherol, etc .; Phenyl mercury nitrate is prepared using chimerosal, benzalkonium chloride, phenol, cresol, paraoxybenzoic acid methyl, benzyl alcohol, and the like as a preservative. Animal powders and granules are prepared by containing commonly used sugars such as vitamins, glucose, lactose, starch, powder, vermiculite or various powders and liquid enzymes in the range of usage.

In addition, the administration of the complex antimicrobial agent according to the present invention is directly administered orally or mixed with negative water, and the powder and granules are administered mixed with negative and feed.

The second aspect of the present invention is Lactobacillus casei WS-1 (KCCM10896P), Lactobacillus casei W-1 (KCCM10897P), Lactobacillus ferrence W-3 (KCCM10898P), Lactobacillus casei W-4 (KCCM10899P) And Lactobacillus casei W-6 (KCCM10900P) and Decera Brucelensis WY-1 (KCCM10901P) provides a microbial composition for animal feed comprising a. Although not limited thereto, preferably the animal is a pig or a chicken.

The microbial composition may further include additional materials such as bacteriocin and organic acids. The organic acid may include one or more combinations selected from the group consisting of lactic acid, fish acid, vinegar acid, succinic acid, tartaric acid, maleic acid, malic acid and citric acid.

The feed includes a pulverized product obtained by pulverizing any one or a mixture of corn, wheat, wheat, soybean wheat, soybean, rice, and the like, and processed by-products such as rice bran, wheat bran, and barley bran obtained during the processing thereof. Can be used.

The third aspect of the present invention is Lactobacillus casei WS-1 (KCCM10896P), Lactobacillus casei W-1 (KCCM10897P), Lactobacillus ferrence W-3 (KCCM10898P), Lactobacillus casei W-4 (KCCM10899P ) And Lactobacillus casei W-6 (KCCM10900P) and Decera Brucelensis WY-1 (KCCM10901P).

The microbial composition may further include additional materials such as bacteriocin and organic acids. The organic acid may include one or more combinations selected from the group consisting of lactic acid, fish acid, vinegar acid, succinic acid, tartaric acid, maleic acid, malic acid and citric acid.

The food composition of the present invention may be formulated in a convenient form for ingestion of powders, capsules, pills, tablets and drinks according to conventional methods.

The compositions can also be added to food or beverages. Foods that can be added for food development include, for example, various foods, meats, drinks, chocolates, snacks, sweets, pizzas, ramen noodles, other noodles, gums, ice creams, alcoholic beverages, vitamin complexes, and health supplements. Etc.

The fourth aspect of the present invention is Lactobacillus casei WS-1 (KCCM10896P), Lactobacillus casei W-1 (KCCM10897P), Lactobacillus ferrence W-3 (KCCM10898P), Lactobacillus casei W-4 (KCCM10899P) And Lactobacillus casei W-6 (KCCM10900P) and Decera Brucelensis WY-1 (KCCM10901P) provides a deodorant microbial composition comprising.

The microbial composition may further include additional materials such as bacteriocin and organic acids. The organic acid may include one or more combinations selected from the group consisting of lactic acid, fish acid, vinegar acid, succinic acid, tartaric acid, maleic acid, malic acid and citric acid.

As the carrier for the deodorant composition, one or more supported materials selected from the group consisting of powdered clay, activated carbon, coke, steel mill slag, volcanic ash, and combustion materials may be used. As clays, zeolite, vermiculite, diatomaceous earth, etc. may be used. Kaolin, Onggi, Feldspar, Charged Earth, Talc can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Analysis of Microorganism Types in Microbial Formulation FeedFree ™

1. Experimental method

1) Isolation of Lactobacillus Strains

In order to separate lactic acid bacteria from Meteorite Food Co., Ltd.'s FeedFree ™ microbial agent, white colonies were selected and isolated from strains formed on MRS plate medium.

(1) Colony form observation of isolate

Strains isolated from the microbial agent were inoculated in MRS medium containing 0.002% bromophenol blue ( BPB ) and incubated at 37 ° C for 24 hours, and then the color and shape of the colonies were analyzed under a dissecting microscope (Zoom 2000, Leica). Observed.

(2) Gram dyeing of isolated strain

① Blot the bacteria on the slide and heat fix, and then stain them with crystal violet solution for 1 minute.

② Stain for 1 minute with Iodine solution.

③ Bleach for 30 seconds in ethanol.

④ Gram dyeability is determined by observing with an optical microscope (Nikon, Eclipse 80i).

(3) Morphology observation using scanning electron microscope (SEM)

① Cultivation of strains

: Incubate in Lactobacillus selective medium MRS

② Glutaraldehyde (fixed glutaraldehyde)

: Fix cultured lactic acid bacteria in 2.5% Glutaraldehyde for 24 hours.

③ Dehydration

: 50% and 70%, 80%, 90% and 100% concentration of ethanol is treated sequentially.

④ dry

Treat with 50% and 100% isoamyl acetate.

⑤ Apply gold coating.

⑥ Observe with a scanning electron microscope (XL30ESEM, philips).

2) Isolation of Yeast Strains

Yeast in Feedfree ™ genus was cultured for 3 days at 28 ° C. in yeast broth PDA (Potato Dextrose Agar, Difco) medium, and then observed with a scanning electron microscope (XL30ESEM, philips).

3) Sequence analysis of isolated strains isolated from microbial preparations

(1) Sequence analysis of bacteria

Purely isolated isolates were shaken at 150 rpm in trypticase Soy Brith Agar ( TSA , Difco). DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN). Primers used for PCR amplification of 16S rDNA are shown in Table 1. PCR amplification was performed at 95 ° C. for 2 minutes, 30 cycles at 94 ° C., 30 seconds at 58 ° C., 45 cycles at 72 ° C., and reaction at 72 ° C. for 5 minutes.

Table 1 Primers used for PCR amplification of 16S rDNA

F-primer	R-primer
27F	342R
261F	517 R
530F	787R
802F	956 R
1114F	1100R
	1492R

The amplified product of 16S rDNA was purified using a PCR product purification kit (Qiagen). Genetic analysis was performed using Genetic Analyzer (Genetic analyzer 310A, Applied Biosystems). Data analysis was performed using the database of DDBJ / NCBI / Genebank. (Thompson et al ., 1994) and PHYLIP program (Felsenstein, 1993) were used to confirm the systematic location.

(2) Identification of Yeast Strains

DNA was extracted according to the benzyl chloride method, and the 26S rDNA region was amplified by PCR. Primers used for amplification were YF (5'-GCATATCAATAAGCGGAGGAAAAG-3'-SEQ ID NO: 1), YR (5'-GGTCCGTGTTTCAAGACG-3'-SEQ ID NO: 2). PCR conditions were 2 minutes at 95 ° C; 30 seconds at 94 ℃, 30 seconds at 55 ℃, 45 seconds at 72 ℃ was repeated 30 times, held at 75 ℃ 5 minutes and then maintained at 16 ℃. The partial sequence of the amplified 26S rDNA was purified using a PCR product purification kit (Qiagen), and the PCR purified product was analyzed using the Genetic Analyzer 310A (Applied Biosystems).

4) Identification using Random Amplified Polymorphic DNA (RAPD)

(1) Fine identification of Lactobacillus

Random amplified polymorphic DNA (RAPD) is used when genetic flexibility between species, subspecies, or mutant strains is difficult to identify through 16 rRNA analysis and DNA-DNA homology experiments. In the RAPD method, genetic variation between strains can be confirmed by comparing and amplifying DNA band patterns by amplifying DNA using random primers for strains having very similar gene sequences. The strains used were strains of Lactobacillus sp. WS-1, W-1, W-4, and W-6, which were not effectively identified by rDNA analysis alone. As a standard strain, Lactobacillus cassia subtype Tolerance ATCC was used. 25599, Lactobacillus casei ATCC 393, Lactobacillus paracasei subspecies Paracasei ATCC 27216 and Lactobacillus paracasei subspecies Paracasei ATCC 25302 were used.

Primers used were OPL-05 (ACGCAGGCAC-SEQ ID NO: 3), OPL-06 (CCCGTCAGCA-SEQ ID NO: 4), PCR conditions were 5 minutes at 94 ℃; After 35 cycles of 1 min at 94 ° C., 2 min at 32 ° C., and 2 min at 72 ° C .; Hold at 72 ° C. for 5 minutes and then at 4 ° C.

2. Results

(1) Colony morphology of isolated strain WS-1

The isolated strain WS-1 colonies showed circular and convex in MRS medium containing bromophenol blue ( BPB ), and dark blue nuclei were observed in the center of the colonies. Indicated. Gram staining of the isolated strain WS-1 and observation with an optical microscope (Nikon, Eclipse 80i × 1,000) showed that the isolated strain WS-1 showed a gram-positive short bacilli. As a result of observing the cell morphology of the isolated strain WS-1 using a scanning electron microscope (SEM), it showed a bacilli of the size of 0.8 × 1.0 ㎛ ~ 1.0 × 2.0 ㎛ ( Fig. 1 ).

(2) Colony morphology of isolated strain W-1

Colonies of the isolated strain W-1 showed a rounded shape (circular, convex) in the MRS medium to which bromophenol blue ( BPB ) was added. In addition, a dark blue nucleus was observed in the center of the colony, indicating the morphological characteristics of the lactic acid bacteria colony. Gram staining of the isolated strain W-1 was observed with an optical microscope (Nikon, Eclipse 80i × 1,000). As a result, the isolated strain W-1 showed a gram-positive form of short bacilli. As a result of observing the cell morphology of the isolated strain W-1 using a scanning electron microscope (SEM), it showed a bacillus form of 0.7 × 1.7 ㎛ ~ 0.7 × 3.3 ㎛ size ( Fig. 1 ).

(3) Colony form characteristics of isolate strain W-3

Colonies of the isolated strain W-3 showed irregular morphology (irregular, umbonate) in MRS medium to which bromophenol blue ( BPB ) was added. The colonies of lactic acid bacteria colonies showed morphological characteristics. The isolated strain W-3 was gram stained and observed with an optical microscope (Nikon, Eclipse 80i × 1,000). As a result, the isolated strain W-3 showed a gram-positive form of short bacilli. As a result of observing the cell morphology of the isolated strain W-3 using a scanning electron microscope (SEM), it showed a bacilli of the size of 0.7 × 1.3 ㎛ ~ 0.7 × 2.0 ㎛ ( Fig. 1 ).

(4) Colony morphology of isolated strain W-4

Colonies of the isolated strain W-4 showed a circular shape (circular, convex) in the MRS medium to which bromophenol blue ( BPB ) was added. In addition, a dark blue nucleus was observed in the center of the colony, indicating the morphological characteristics of the lactic acid bacteria colony. The isolated strain W-4 was gram stained and observed with an optical microscope (Nikon, Eclipse 80i × 1,000). As a result, the isolated strain W-4 showed a gram-positive form of short bacilli. As a result of observing the cell morphology of the isolated strain W-4 using a scanning electron microscope (SEM), it showed a bacilli of the size of 0.6 × 0.8 ㎛ ~ 0.5 × 1.0 ㎛ ( Fig. 1 ).

(5) Colony morphology of isolated strain W-6

Colonies of the isolated strain W-6 showed a circular (convex, convex) in the MRS medium added with bromophenol blue ( BPB ), and was generally blue. In addition, a dark blue nucleus was observed in the center of the colony, indicating the morphological characteristics of the lactic acid bacteria colony. Gram staining of the isolated strain W-6 was observed with an optical microscope (Nikon, Eclipse 80i × 1,000). As a result, the isolated strain W-6 showed a gram-positive form of short bacilli. As a result of observing the cell morphology of the isolated strain W-6 by using a scanning electron microscope (SEM), it showed a bacilli of the size of 0.5 × 1.0 ㎛ ~ 0.8 × 2.9 ㎛ ( Fig. 1 ).

(6) Colony morphology of isolated strain WY-1

As a result of observing the cell morphology of the isolated strain WY-1 using a scanning electron microscope (SEM), it showed a monobacterium form of the size of 0.8 × 1.0 ㎛ ~ 1.2 × 1.0 ㎛ ( Fig. 2 ).

(7) Sequence analysis result

① The isolated strain WS-1 strain is a strain belonging to a lineage group including a species of the genus Lactobacillus as a result of molecular analysis based on the 16S rDNA nucleotide sequence (1,417bp, SEQ ID NO: 5), Lactobacillus casei ) strain ( Lactobacillus casei ATCC 334; Accession No. = CP000423.1) and 99.99% (identities = 1415/1417, Gaps = 1/1415) and showed a flexible relationship, and Lactobacillus paracasei strain ( Lactobacillus paracasei strain DJ1; Accession No. = DQ462440) and 99.99% (identities = 1415/1417, Gaps = 1/1415) was confirmed to show a flexible relationship. As a result of phylogeny with other Lactobacillus strains, the Lactobacillus paracase or Lactobacillus casei had a high degree of flexibility ( FIG. 4 ).

② The isolated strain W-1 strain is a strain belonging to a lineage group including a species of the genus Lactobacillus as a result of molecular analysis based on the entire sequence of 16S rDNA (1,410bp, SEQ ID NO: 6). Lactobacillus casei ) strain ( Lactobacillus casei ATCC 334; Accession No. = CP000423.1) and 99.36% (identities = 1407/1416, Gaps = 9/1416) showed a flexible relationship, and Lactobacillus paracasei It was confirmed that the strain ( Lactobacillus paracasei strain DJ1; Accession No. = DQ462440) and 99.36% (identities = 1407/1417, Gaps = 9/1416). As a result of phylogeny with other Lactobacillus strains, Lactobacillus casei and Lactobacillus paracasei had a high degree of flexibility ( FIG. 5 ).

③ The isolated strain W-3 strain is a strain belonging to a lineage group including a species of the genus Lactobacillus as a result of molecular analysis based on the entire sequence of 16S rDNA (1,417bp, SEQ ID NO: 7). Lactobacillus perolens ) strain ( Lactobacillus perolens L534; Accession No. = Y19168.1) and 99.07% (identities = 1397/1410, Gaps = 8/1410) showed a flexible relationship, and phylogenetic classification with other Lactobacillus strains was performed. As a result, the soft relationship with the Lactobacillus perforation was high ( FIG. 6 ).

④ The isolated strain W-4 strain is a strain belonging to the phylogenetic group including the species of the genus Lactobacillus as a result of molecular analysis based on the entire sequence of 16S rDNA (1,390bp, SEQ ID NO: 8), Lactobacillus casei ( Lactobacillus casei ) strain ( Lactobacillus casei ATCC 334; Accession No. = CP000423.1) and 98.91% (identities = 1378/1393, Gaps = 13/1393) showed a flexible relationship, and Lactobacillus paracasei The strain ( Lactobacillus paracasei ; Accession No. = DQ462440.1) and 98.91% (identities = 1378/1393, Gaps = 13/1393) showed a flexible relationship. As a result of phylogeny with other Lactobacillus strains, there was a high degree of flexibility in Lactobacillus casei and Lactobacillus paracasei ( FIG. 7 ).

⑤ The isolated strain W-6 strain is a strain belonging to the phylogenetic group including the species of the genus Lactobacillus as a result of molecular analysis based on the entire sequence of 16S rDNA (1,414bp, SEQ ID NO: 9). Lactobacillus casei ) strain ( Lactobacillus casei ATCC 334; Accession No. = CP000423.1) and 99.43% (identities = 1409/1417, Gaps = 8/1417) showed a flexible relationship, Lactobacillus paracasei The strain ( Lactobacillus paracasei ; Accession No. = DQ462440.1) and 99.43% (identities = 1409/1417, Gaps = 8/1417) showed a flexible relationship. As a result of phylogenetic classification with other Lactobacillus strains, the Lactobacillus casei and Lactobacillus paracasei had a high degree of flexibility ( FIG. 8 ).

(8) Identification result using random amplified polymorphic DNA (RAPD)

The result amplified by PCR was confirmed after electrophoresis at 1.5% agarose gel, 50V. The result of RAPD with OPL-5 primer is shown in FIG. 3 . All isolate strains (strain WS-1, W-1, W-4 and W-6 exhibited the same DNA bands as those of the typical 1A 2A, 3A and 4A bands in the Lactobacillus casei groups ATCC 25599 and ATCC 393). Strains of species WS-1, W-1, W-4, W-6 have been identified as Lactobacillus cascai.

Therefore, the isolated strain WS-1 was named Lactobacillus casei WS-1. The isolated strain W-1 was named Lactobacillus casei W-1. The isolated strain W-3 was named Lactobacillus perolens W-3. The isolated strain W-4 was named Lactobacillus casei W-4. Therefore, isolated strain W-6 was named Lactobacillus casei W-6.

(9) Identification of Yeast Strains

The analyzed base sequence is shown in SEQ ID NO: 10.

The base sequence was searched for homology with DDBJ / NCBI / Genebank, and the phylogenetic location was confirmed using Cluster X program (Thompson et al, 1994) and PHYLIP program (Felsenstein). Molecular phylogenetic analysis based on the 26S rDNA sequence of the WY-1 strain confirmed that the strain belonged to the systematic group including the species of the genus Dekkra , and showed a 99% flexibility with the Dekkra bruxellensis strain. It became. Thus, yeast representative strain WY-1 was identified as Dekkra bruxellensis ( Fig. 9 ). Dekkra bruxellensis was named WY-1.

Therefore, Lactobacillus strains and yeast strains were deposited on December 7, 2007, respectively, with the accession numbers KCCM10896P, KCCM10897P, KCCM10898P, KCCM10899P, KCCM10900P, and KCCM10901P, respectively, to the Korean spawn association.

Example 2 Analysis of Organic Acid in Microbial Feedfree ™

Meteorite Food Feed ™ microorganisms are removed by infiltration using a 0.02 μm filter, inoculated and incubated at 28 ° C. for 15 days. Analyze by ion chromatography DX-500 (Ion Choromatograph DX-500, Dionex, USA) and quantify with Ion Pac ASII program.

TABLE 2 Analytical Organic Acids

number	Analytical Organic Acid Class
One	Oxalic Acid (OA)
2	Maleic acid (MEA)
3	Tartaric acid (TA)
4	Citric acid (CA)
5	Pyruvic acid (PA)
6	Acetic acid (AA)
7	Malic acid (MA)
8	Lactic acid (LA)
9	Succinic acid (SA)
10	Formic acid (FA)

<Result>

As a result of qualitative and quantitative analysis of 10 types of organic acid standards in microbial cultures, large amounts of lactic acid (4940.074ppm) and acetic acid (2030.97ppm) were detected in microbial preparations, and others (266.119ppm) and superpoic acid were detected. (216.79 ppm), succinic acid (105.148 ppm) and tartaric acid (49.831 ppm) were detected. In addition, maleic acid (0.604ppm), malic acid (21.125ppm) and citric acid (2.214ppm) were analyzed in very small amounts. Formic acid (formic aicd, FA) was not detected.

Example 3 Investigation of Growth Capacity at Acidic pH of Microorganisms in FeedFree ™

(1) Growth Performance at Acidic pH of Lactobacillus

The pH of Lactobacilli MRS Broth ( MRS , Difco), a lactic acid bacteria selection medium, was adjusted to pH 2.5, pH 3.0, pH 3.5, pH 4.0, pH 4.5, pH 5.0. 1 ml of the microbial agent was inoculated into the pH adjusted MRS medium. Stationary culture at 28 ℃, OD was measured at 600nm in 12 hours.

<Result>

After adjusting the MRS medium to pH 2.5 ~ 5.0, inoculation of microorganisms to measure the growth of each pH, the rapid increase in OD after 48 hours incubation at pH 4.0, pH 4.5 and pH 5.0 can be observed. there was. In addition, at pH 2.5, pH 3.0 and pH 3.5, the growth seemed to proceed slowly after 72 hours of incubation, but the OD value was too low to confirm the growth.

Therefore, fluorescence biostaining was used to selectively stain live cells only. The fluorescence biostaining method can count microorganisms quickly and accurately using fluoro fluorescein diacetate ( CFDA ), a fluorescent dye.

As a result of measuring the number of viable bacteria in each medium by fluorescence biostaining, lactic acid bacteria and yeast were observed even in pH 2.5 medium, which was acetic acid condition, and it was confirmed that the number of lactic acid bacteria increased about 100 times ( FIG. 10 ).

(2) Growth Performance at Acidic pH of Decera Brucelensis WY-1 (KCCM10901P)

After incubating for 3 days at 28 ℃ in the PDA plate medium purified purely separated Decera Brucelensis WY-1 to pH 2.0 ~ pH 4.5, and the number of viable cells is as follows. pH 2.0 PDA medium: 8.8 ± 4.8 × 10 ⁴ cfu / ml; pH 2.5 PDA medium: 1.14 ± 9.7 × 10 ⁵ cfu / ml; pH 3.0 PDA medium: 7.0 ± 7.5 × 10 ⁴ cfu / ml; pH 3.5 PDA medium: 4.6 ± 4.3 × 10 ⁴ cfu / ml; pH 4.0 PDA medium: 1.2 ± 2.2 × 10 ⁴ cfu / ml; pH 4.5 PDA medium was 7.5 ± 3.3 × 10 ³ cfu / ml. After inoculation into the PDB liquid medium adjusted to pH 2.0 ~ pH 4.5, the result of measuring the viable cell count of yeast using carboxy fluorescein diacetate ( CFDA ) fluorescence staining method is as follows. pH 2.0 PDA medium: 1.06 ± 0.2 × 10 ⁷ cells / ml: pH 2.5 PDA medium: 1.92 ± 0.2 × 10 ⁷ cells / ml; pH 3.0 PDA medium: 9.52 ± 0.2 × 10 ⁶ cells / ml; pH 3.5 PDA medium: 5.48 ± 0.1 × 10 ⁶ cells / ml; pH 4.0 PDA medium: 4.0 ± 0.1 × 10 ⁶ cells / ml; pH 4.5 PDA medium: 3.92 ± 0.1 × 10 ⁶ cells / ml. Therefore, it was shown that Decera brucellensis WY-1 (KCCM10901P) can be grown in pH 2.0 medium, which is acetic acid.

Example 4 FeedFree ™ Antimicrobial Activity

(1) Antimicrobial activity against microorganisms causing fish disease

To test the antimicrobial activity of FeedFree ™, Feedfree ™ was filtered twice with a 0.45 μm membrane filter at 0.2 μm and the filtrate was used for testing while refrigerated. The bacterium used for the test is a vibrio orchard (Vibro ordalii), Edward Siela Tarda (Edwardsiella tarda), Tenashiba coolum species (Tenacibaculumsp.) and Streptococcus inaeae (Streptococcus iniae) Was used. The antibacterial test was performed by Vibrio Ordari (Vibro ordalii), Edward Siela Tarda (Edwardsiella tarda) And Streptococcus inaeStreptococcus iniae) Was used Tryptic soy agar (TSA) and Tryptic soy broth added 1.5% NaCl as a culture medium, Tenashiba coolum species (Tenacibaculumsp.) used Cytophaga agar (CA) and Cytophaga broth (CB) with 1.5% NaCl. The bacterial cultures shaken and cultured in each liquid medium were vibrio ordari, Edward ciella tarda, and Gram-positive bacteria Streptococcus inae.⁷ In CFU / ml, tenash baculum species (Tenacibaculumsp.) is about 10⁶ The medium suspension test bacteria were prepared by suspending each liquid medium at a concentration of CFU / ml. Mix each medium suspension test solution well with a stirrer and dispense 5ml each into a sterile test tube, add Feedfree ™ to 1% (v / v), and incubate for 0, 3, 5, 10, 30 minutes, The bacterial counts were measured as absorbance values at 540 nm every hour. As a control, 1% (v / v) of each medium was added to the culture medium suspension without feedfree ™, and the measurement was performed in the same manner. Three repetitions were performed. The antimicrobial activity of FeedFree ™ was different for each species. In the Streptococcus erythrophilic test zone, as shown in FIG. 11, it began to show a significant inhibitory effect for 3 minutes after the start of the reaction, and the effect was shown to last for the test period. Therefore, the filtrate showed a significantly higher antimicrobial effect than the control (11).

(2) Antimicrobial activity against intestinal pathogenic microorganisms

In order to confirm whether FeedFree ™ has bactericidal activity (inhibition ability) in pathogenic microorganisms, bactericidal test was performed on bacteria, a representative pathogenic strain.

Sample condition: The sterilization power was tested using 1% solution of 100% diluted feed-free stock solution.

Test strain: Escherichia coli ATCC 25922

Escherichia coliO157 ATCC 43895

Staphylococcus aureus ATCC 25923

Vibrio parahaemolyticus KCTC 2471

Salmonella typhi KCTC 2424

Pseudomonas aeruginosa KCTC 1636

Candida albicans KCTC 7965

Test Method: Escherichia coli, Escherichia coli O157, Staphylococcus, Vibrio, Pseudomonas aeruginosa, and Salmoreella were enriched in the liquid medium (Brain l leart lnfusion Broth) and used for the test. Candida applied Sabouraud Dextrose Broth .

After inoculating the bacteria in sterilized physiological saline to measure the initial bacterial count, the sample was added to 1% and left at room temperature. After 30 seconds, after 2 minutes, after 5 minutes, after 24 hours to determine the reduction rate for the initial bacteria by measuring the number of bacteria.

Test result :

As shown in Table 3 , the bactericidal activity of bacteria was confirmed even under the condition of 1% solution prepared by diluting the stock solution 100 times.

TABLE 3 Antimicrobial Test Results for Pathogenic Microorganisms

time	Early	30 seconds later	2 minutes later	5 minutes later	24 hours later
E. coli	3.1 X 10 5	2.8X10 5	2.6 X 10 5	2.3 X 10 5	67.7% Suppression
E. coli 0157	2.8X10 5	2.5 X 10 5	2.4 X 10 5	2.1 X 10 5	71.2% inhibition
S. aureus	2.7 X 10 5	2.2 X 10 5	1.4 X 10 5	1.0X10 5	70.5% Suppression
V. parahaemolyticus	1.1 X 10 5	7.5X10 5	2.4 X 10 5	4.8 X 10 5	Not detected
S. typhi	3.2 X 10 5	2.8X10 5	2.1 X 10 5	1.0X10 5	90.7% Suppression
P. aeruginosa	4.8 X 10 5	4.6 X 10 5	4.2 X 10 5	3.8X10 5	70.8% Suppression
C. albicans	3.4 X 10 5	3.4 X 10 5	3.4 X 10 5	3.4 X 10 5	0%

Example 5 Growth Promotion of Chicken and Pork by Feedfree ™

1. Effect of Feedfree ™ Supplementation on Pig Productivity

Table 4 shows the effect on feed productivity by feeding FeedFree ™ to piglets added. The treatments were negatively fed control and non-feedfree ™ 0.1 and 0.2% diets, with 12 males and 6 females per treatment. At the start, body weight was about 7kg and there was no difference between treatments. The survey items were weight gain, feed intake, feed requirement, and intestinal microorganisms. After four weeks of feeding Freefree ™, Feedfree ™ 0.1% treatment gained 0.59 kg higher than that of control and feed rate improved by 0.035. However, the 0.2% negative diet was higher than the optimum level because feed rate and weight gain were not improved compared to the control.

Table 4 Effect of Feed-Free ™ Supplementation on Productivity of Weaning Pigs

Treatment Zone (%)	Weight gain (Kg)	Feed Intake (Kg)	Feed rate
Control	13.85	22.35	1.614
FeedFree ™ 0.1	14.44	22.81	1.579
FeedFree ™ 0.2	14.35	22.76	1.692

Feed feed ™ supplemented at about 22-23 kg body weight tended to improve feed demand and weight gain in the 0.1% -0.2% treatment group compared to the control group. Feeding rate of 0.2% was higher than that of control (Table 5).

Table 5 Effects of Feed-Free ™ Supplementation on Productivity of Middle-aged Pigs

Treatment District (%)	Weight (Kg)	Weight gain (Kg)	Feed Intake (Kg)	Feed requirement
Control	46.48	24.72	53.13	2.150
FeedFree ™ 0.1	47.50	24.88	52.67	2.117
FeedFree ™ 0.2	45.07	23.32	51.05	2.189

2. Influence of Feedfree ™ / Feed on Feed Product Performance on Weaned Pigs

This study was conducted to investigate the effect of FeedFree ™ on LHD ternary hogs and weight of young pigs weighing about 11 kg. The treatments were treated without feedfree ™, ie, control, commercial antibiotic 0.1% treatment, feedfree ™ 0.1, 0.2, 0.4% treatment. The trial period was eight weeks from June 17, 2000, with unlimited water and feed. In addition, feedfree ™ 0.2% added to the commercial feed, the last treatment, was placed at about 23Kg in weight similar to the existing treatments from July 15, and the same experiment was conducted until shipment.

Body weight gained from 28 days of age (approximately 11 kg) to about 56 days (4 weeks after initiation) showed an increase in antibiotics and Feedfree ™ 0.1 and 0.2% in the control group compared to the control group, but 0.4% in the control group compared to the control group. Indicated. Feed intake tended to increase overall in Feedfree ™ diets, but feed intake tended to decrease with increasing levels. At about 84 days of age, eight weeks after the start of the experiment, the gain was higher in the Feedfree ™ 0.1% diet compared to the other treatments, and the feed demand was also improved. In this experiment, Feed Free ™ showed a tendency to improve feed demand as compared to the control group, but the feed group higher than 0.2% had lower weight gain than the 0.1% group, so it was about 11kg weaning pig to 40kg. Swine pigs suggest that additions / payments between 0.1 and 0.2% are required.

Table 6 Effect of Feed / Fed on Feedfree ™ Feed on Productivity of Weaning Pigs Approximately 12 kg (June 15, 2000)

Treatment District (%)	Weight gain (Kg)	Feed Intake (Kg)	Feed rate
Control	30.67	65.82	2.146
Antibiotic 0.1	31.04	62.46	2.012
FeedFree ™ 0.1	31.79	63.77	2.006
FeedFree ™ 0.2	30.67	62.73	2.045
FeedFree ™ 0.4	29.74	62.06	2.087
Commercial Feed + Feedfree ™ 0.2%	21.12	37.62	1.781

In Table 6, at the weight of about 40 Kg at the start of the experiment, the 0.1% FeedFree ™ treatment group showed a tendency to increase the weight gain and feed requirement compared to the control group.

3. Increased productivity of broilers by Feedfree ™

From the local hatchery, a young broiler with a high yield of 45 days of hatching is obtained. All broilers were vaccinated with New Castle and Bronchitis sprays in the hatcheries and no other vaccinations were made. Each cage had 30 feather-sexed broilers, totaling 15 cages (1.5 x 4.3 M). Each cage was equipped with large rice (sawdust) on a concrete floor and a single tube feed and nipple drinking water supply line to provide ad libitum to feed and drinking water access. The room temperature was controlled by raising and lowering the heat provided by the incubator lamp and the site curtain. The broiler was fed with corn-bean feed using the Meth method (Table 7).

TABLE 7 Composition of basic feed for broilers

Ingredients and Composition	For 0-20 days	22-49 days for growth
	%
corn	56.12	60.79
Soybean meal, 48	37.50	32.61
Fat, poultry	3.07	3.43
Limestone	0.73	0.78
Defluorinated Phosphate	1.75	1.56
saline	0.29	0.32
Vitamin Premix	0.25	0.25
Minor Premix	0.07	0.08
DL-98 Methionine	0.20	0.17
Calculated composition ratio
Crude protein,%	22.50	20.50
Metabolic energy, kcal / kg	3,080	3,150
Lee Sin, %	1.26	1.12
calcium, %	0.95	0.990
Phosphoric Acid,%	0.45	0.41

Vitamin premix contains vitamin A 6614 IU / kg feed; Cholecalciferol 705 IU; Vitamin E, 13 IU; Riboflavin, 6.6 mg; Ca panthothenate, 12 mg, nicotinic acid, 39 mg, vitamin B ₆ , 1.9 mg; Minadione, 1.3 mg; Folic acid, 0.72 mg; d-biotin, 0.055 mg; Thiamine, 1.1 mg; Ethoxyquin, 125 mg.

Trace minor premixes include 60 mg of Mn, 50 mg of Zn, 30 mg of Fe, 5 mg of Cu, and 1.5 mg of I per kg of feed.

Formulate a basic feed that meets or exceeds the requirements for initiation (1 to 21 days) and growth (22 to 49 days). Young broilers randomly designate one of three feed treatments. The classification of feed is as follows. Excludes basic feed containing 750 g / mtons of coban60 (insecticide), and basic feed (control) containing 62.6 g / mton of bacitracin (antibiotic), or coban60 (insecticide) and bacitracin (antibiotic) And feed supplemented with 0.1% or 0.2% by weight of Feedfree ™.

The experimental broilers were observed twice daily and the dead were removed and the body weight was recorded. The uneconomics with severe abnormalities in the legs were removed, weight recorded and euthanized. Death and leg abnormalities were recorded and recorded at the end of the study to determine mortality and leg abnormalities. Body weights were measured on days 21 and 42 for broilers and weight gain and feed conversions were calculated. Feed conversion is determined by the difference between the weight put into the feeder and the remaining weight at 21 and 42 days. Body weight checks for mortality and mortality of broilers were used to control feed consumption.

Body weight, weight gain, feed intake, weight gain to feed ratio, mortality, broiler and leg dysfunction rates from 1 to 42 days are shown in Table 8 (Feedfree ™ 's effect on weight gain in young broilers).

[Revision under Rule 26 03.01.2011]
Table 8

The body weights of broilers fed feed supplemented with 0.1% or 0.2% by weight of live bacteria between 1 and 42 days showed no difference compared to the control (group fed with insecticide and antibiotics) (P> 0.05). The same results show that feed free ™ supplements are more effective for broiler chicken growth than supplemented with basic insecticides and antibiotics. Feedfree ™ therefore replaced antibiotics to promote chicken growth.

<Example 6> Deodorization effect by FeedFree ™

Odors generated from livestock wastewater and livestock waste, phosphorus, industrial and household organic waste, wastewater treatment plant and sewage treatment plant are mainly fatty acid-based odors and VOCs such as ammonia and amines. It was confirmed whether FeedFree ™ removes odors in the same manner as a known test method (see Korean Patent Application No. 10-2001-0085666, etc.), and the results are shown in Table 9.

Table 9 Reduction of odor components after Feedfree ™ treatment

Test Items	Test result
	Early	0.5 hours	1 hours	3 hours	6 hours
Trimethylamine	60.0 ppm	24.0 ppm	13.0 ppm	7.0 ppm	3.0 ppm
ammonia	60.0 ppm	18.0 ppm	12.0 ppm	5.0 ppm	2.0 ppm

As shown in Table 9, FeedFree ™ reduced trimethylamine by 95% in 6 hours and ammonia by 97%.

Claims (4)

1. Lactobacillus casei WS-1 (KCCM10896P), Lactobacillus casei W-1 (KCCM10897P), Lactobacillus ferrence W-3 (KCCM10898P), Lactobacillus casei W-4 (KCCM10899P), Lactobacillus casei W- An acid resistant animal growth promoting and antimicrobial active composition consisting of 6 (KCCM10900P) and Decera Brucelensis WY-1 (KCCM10901P).
2. Lactobacillus casei WS-1 (KCCM10896P), Lactobacillus casei W-1 (KCCM10897P), Lactobacillus ferrence W-3 (KCCM10898P), Lactobacillus casei W-4 (KCCM10899P), Lactobacillus casei W- 6 (KCCM10900P) and Dekera Brucelensis WY-1 (KCCM10901P) is composed of essential components, acid-resistant animal growth promoting and antimicrobial active composition.
3. The acid resistant animal growth promoting and antimicrobial active composition according to any one of claims 1 to 3, wherein the animal is a pig or a chicken.
4. Lactobacillus casei WS-1 (KCCM10896P), Lactobacillus casei W-1 (KCCM10897P), Lactobacillus ferrence W-3 (KCCM10898P), Lactobacillus casei W-4 (KCCM10899P) and Lactobacillus casei W- Acid-resistant animal growth promoting and antimicrobial activity feed, comprising 1 to 4 prokaryotic and eukaryotic Decera brucellensis WY-1 (KCCM10901P) selected from the group consisting of 5 (KCCM10900P). additive.

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