AU2016305391B2

AU2016305391B2 - Novel Lactobacillus sp. microorganisms, and composition for animal feed comprising same

Info

Publication number: AU2016305391B2
Application number: AU2016305391A
Authority: AU
Inventors: Gi Duk Bae; Seok Woo Chee; Sung Hun Kim; Eun Kyung Lee; Joong Su Lee
Original assignee: CJ CheilJedang Corp
Current assignee: CJ CheilJedang Corp
Priority date: 2015-08-12
Filing date: 2016-06-03
Publication date: 2019-01-17
Anticipated expiration: 2036-06-03
Also published as: MY188407A; KR101873898B1; AU2016305391A1; KR20170020215A; PH12018500069A1; WO2017026635A1; CN107949633B; CN107949633A

Abstract

The present invention relates to a composition for an animal feed comprising a

Description

NOVEL LACTOBACILLUS SP. MICROORGANISMS, AND

COMPOSITION FOR ANIMAL FEED COMPRISING SAME [Technical Field] [1] The present invention relates to a novel Lactobacillus species and an animal feed composition comprising the same.

[Background Art] [2] Lactobacillus species are homo-fermentative or hetero-fermentative lactic acid bacteria that can be commonly found in the intestinal tracts of animals, including humans, and during fermentation of dairy products and vegetables. Lactobacillus species maintain the acidic pH of the intestine to inhibit the proliferation of harmful bacteria such as E. coli and Clostridium species and to ameliorate diarrhea and constipation. Lactobacillus species are known to play a role in vitamin synthesis, anticancer activity, lowering serum cholesterol, and the like.

[3] Studies have been researched on the probiotics as feed additives according to the aforementioned properties of the Lactobacillus sp. microorganism. Bacterial diarrhea in livestock results in reduction of a growth rate and survival rate. Therefore, in order to increase livestock productivity, various antibiotics have been added to animal diet at a pharmaceutical dose. In recent years, however, the problem of antibiotic resistance and harmful effect of residual antibiotic substance in meat product have been discussed in worldwide because of its excessive use. Accordingly, governments in many countries have started to limit the usage of the antibiotics in animal feed and think the organic way of rearing livestock animals (Korean Patent Laid-Open Publication No. 10-199878358) (McEwen and Fedorka-Cray, Antimicrobial use and resistance in animals, Clinical infectious Diseases, Volume 34, June 2002, pages S93-S106).

[Disclosure] [Technical Problem] [4] The present inventors have found that heat-killed or dead (hereinafter, called as inactive cells) cells of Lactobacillus rhamnosus CJLR1505 belonging to the genus Lactobacillus have the following effects. First, inactive cells of Lactobacillus rhamnosus CJLR1505 have the ability to inhibit competitive adherence of harmful enterobacteria to intestinal epithelial cells, contributing to the formation of enteric bacterial flora. Inactive cells of Lactobacillus rhamnosus CJLR1505 produce lipoteichoic acid that prevents harmful enterobacteria from settling on the intestinal mucous membrane. Lipoteichoic acid is a cell wall constituent eluted from the inactive bacterial cells whose cell walls are destroyed in the small intestine. As a result, harmful enterobacteria such as E. coli and Salmonella species are not adsorbed to the surface of the intestinal mucous membrane in the enteric environment and excreted by intestinal secretions and intestinal peristalsis. In addition, the use of inactive cells of Lactobacillus rhamnosus CJLR1505 in animal feeds is effective in increasing the rate of weight gain of animals and leads to an increase in feed efficiency due to their ability to degrade triglycerides. Furthermore, intake of feeds including inactive cells of Lactobacillus rhamnosus CJLR1505 is effective in enhancing the immunity of animals.

[5] The present invention provides Lactobacillus rhamnosus CJLR1505 as a novel Lactobacillus species and an animal feed composition including inactive cells of the Lactobacillus species and is aimed at improving the rate of weight gain of animals and immunity of animals against diseases when the animals ingest a feed including the animal feed composition.

[Technical Solution] [6] One embodiment of the present invention provides Lactobacillus rhamnosus CJLR1505 (KCCM11721P).

[7] Another embodiment of the present invention provides an animal feed composition comprising Lactobacillus rhamnosus CJLR1505 (KCCM11721P) or inactive cells of the strain.

[8] A further embodiment of the present invention provides a method for producing inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P), comprising culturing Lactobacillus rhamnosus CJLR1505 (KCCM11721P) to prepare a culture solution, indirectly heating the culture solution at a temperature ranging from 70°C to 160 °C using a heat exchanger, rapidly cooling the indirectly heated culture solution to a temperature ranging from 10°C to 60 °C at a rate of 10 to 100 L/min, and isolating inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM1172IP) from the rapidly cooled culture solution.

[Advantageous Effects] [9] Inactive cells of Lactobacillus rhamnosus CJLR1505 according to the present invention have the ability to degrade triglycerides, adsorb endotoxins, inhibit the growth of pathogenic bacteria, and activate digestive enzymes. Due to this ability, an animal feed comprising inactive cells of Lactobacillus rhamnosus CJLR1505 can improve the rate of daily weight gain of animals and the immunity of animals against diseases.

[Description of Drawings] [10] FIG. 1 shows electron microscopy images of Lactobacillus rhamnosus CJFR1505.

[11] FIG. 2 shows a phylogenetic tree of Lactobacillus rhamnosus CJFR1505.

[12] FIG. 3 shows electron microscopy images showing the number of inactive bacterial cells of Lactobacillus rhamnosus CJFR1505 and Lactobacillus rhamnosus KCCM 32450 as a standard strain adherent to intestinal epithelial cells after the ability of the strains to adhere to intestinal epithelial cells was evaluated.

[Best Mode] [13] The present invention will be now described in more detail. Disclosures that are not included herein will be readily recognized and appreciated by those skilled in the art, and thus their description is omitted.

[14] One embodiment of the present invention is directed to Lactobacillus rhamnosus CJLR1505 (KCCM11721P).

[15] The present inventors sampled the distal end of the small intestine of a piglet, washed the sample with sterile distilled water, and plated the washed sample on an MRS medium supplemented with 0.001% bromcresol purple (BCP). After anaerobic culture at 37 °C, 47 lactic acid producing strains were screened. The screened strains were subcultured and 23 species of them were secondarily isolated by a morphological method. The antibacterial activities and digestive enzymatic activities of the 23 species were compared. Of these species, 11 species were tertiarily isolated. The isolated 11 lactic acid producing strains were measured for bile tolerance, acid tolerance, and adsorptivity to cells in the intestinal wall of animals. One strain with the best characteristics in terms of sugar fermentation, the ability to inhibit the growth of pathogenic bacteria, digestive enzymatic activity, and the ability to degrade triglycerides was finally isolated.

[16] The finally isolated lactic acid producing strain was named Lactobacillus rhamnosus CJLR1505 and was deposited with the Korean Culture Center of

Microorganisms (KCCM) on July 8, 2015 (accession number KCCM11721P).

[17] The morphological and physiological properties of Lactobacillus rhamnosus CJLR1505 are shown in Table 1.

[18] [Table 1]

1.Morphological properties (Morphologies when grown in MRS solid media)
- Cell shape	Bacillus
- Cell polymorphism	-
- Motility	-
- Spore formation	-
2. Physiological properties
- Gram staining	Gram positive
- Catalase	Negative
- Oxidase	Negative
- Growth temperature and time	37 °C, 18-48 h
- Oxygen demand	Facultative anaerobic

[19] As shown in FIG. 1, Lactobacillus rhamnosus CJFR1505 is bacilliform and does not form spores. The live cells and inactive cells of the strain are distinguished from each other by the activity of glycoprotein on the cell wall surface.

[20] Lactobacillus rhamnosus CJFR1505 was analyzed using an API kit for biochemical assay. As a result, Lactobacillus rhamnosus CJFR1505 was identified as having similar sugar availability to Lactobacillus rhamnosus, as shown in Table 2. 16s rRNA sequence of Lactobacillus rhamnosus CJLR1505 was requested to Macrogen. As a result, the molecular biological properties of Lactobacillus rhamnosus CJLR1505 were found to be similar (99%) to those of Lactobacillus rhamnosus strains, as shown in FIG. 2.

[21] [Table 2] Analysis of sugar availability of Lactobacillus rhamnosus CJLR1505

No.	Sugar	Lactobacillus rhamnosus CJLR1505
0	Temoin	-
1	Glycerol	-
2	Erythritol	-
3	D-arabinose	+
4	L-arabinose	-
5	D-ribose	+
6	D-xylose	-
7	L-xylose	-
8	D-adonitol	-
9	Methyl-aD-xylopyranoside	-
10	D-galactose	+
11	D-glucose	+
12	D-fructose	+
13	D-mannose	+
14	L-sorbose	-
15	L-rhamnose	+
16	D-ulcitol	+
17	Inositol	±
18	D-mannitol	+
19	D-sorbitol	+
20	Methyl-aD-mannopyranoside	-
21	Methyl-aD-glucopyranoside	-
22	N-acetylglucosamine	+
23	Amygdalin	+
24	Arbutin	+
25	Aesculin	+
26	Salicin	+
27	D-cellobiose	+
28	D-maltose	-
29	D-lactose	+
30	D-melibiose	-
31	D-saccharose	+
32	D-trehalose	+
33	Inulin	-
34	D-melezitose	+
35	D-raffinose	+
36	Amidon	-
37	Glycogen	-
38	Xylitol	-
39	Gentiobiose	+
40	D-turanose	-
41	D-lyxose	-
42	D-tagatose	+
43	D-fucose	-
44	L-fucose	+
45	D-arabitol	-
46	L-arabitol	-
47	Gluconate	+
48	2-ketogluconate	-
49	5-ketogluconate	-

[22] Lactobacillus rhamnosus CJLR1505 (KCCM11721P) is excellent in acid tolerance, bile tolerance, antibacterial activity, digestive enzymatic activity, and ability to degrade triglycerides. Due to these advantages, an animal feed comprising Lactobacillus rhamnosus CJLR1505 (KCCM11721P) has high efficiency and is effective in improving the rate of weight gain of animals.

[23] Inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) have the ability to inhibit competitive adherence of harmful enterobacteria to intestinal epithelial cells, contributing to the formation of enteric bacterial flora. Inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) produce lipoteichoic acid that prevents harmful enterobacteria from settling on the intestinal mucous membrane. Lipoteichoic acid is a cell wall constituent eluted from the inactive bacterial cells whose cell walls are destroyed in the small intestine. In addition, inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) can adhere to pathogenic microbes and endotoxins to enhance the immunity of animals against diseases because of their higher hydrophobicity and stronger tendency to aggregate than live cells of the strain.

[24] A further embodiment of the present invention is directed to an animal feed composition comprising Lactobacillus rhamnosus CJLR1505 (KCCM11721P) or inactive cells of the strain.

[25] The animal feed composition may further comprise a carrier. The carrier is not particularly limited and may be any of those known in the art.

[26] Examples of suitable carriers comprise saccharides (e.g., lactose, Dmannitol, D-sorbitol and sucrose), starches (e.g., corn starch and potato starch), and inorganic salts (e.g., calcium phosphate, calcium sulfate, and precipitated calcium carbonate). The animal feed composition may comprise 1,0^x 10⁸ cfu to 1.0^χ10¹⁰ cfu of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) or inactive cells of the strain per gram. Specifically, the animal feed composition may comprise 1.0^χ10⁸cfu to 3.0^χ10⁹ cfu of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) or inactive cells of the strain per gram. For example, the animal feed composition may comprise 5^χ10⁸ cfu of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) or inactive cells of the strain per gram.

[27] When the content of the inactive bacterial cells is in the range defined above, the ability of the animal feed composition to degrade triglycerides, adsorb endotoxins, inhibit the growth of pathogenic bacteria, and activate digestive enzymes can be maximized.

[28] In a further embodiment, there is provided an animal feed prepared using the animal feed composition. The animal feed may be prepared by mixing the animal feed composition with a general feed. The general feed may comprise typical ingredients, for example, corn, soybean, soybean oil, and amino acids. According to a further embodiment, the animal feed may be prepared by mixing the animal feed composition with another animal feed composition.

[29] The animal feed is not particularly limited so long as it is fed to livestock such as cows, pigs, and horses. For example, the animal feed may be a pig feed.

[30] Another embodiment of the present invention is directed to a method for producing inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P), comprising culturing Lactobacillus rhamnosus CJLR1505 (KCCM11721P) to prepare a culture solution, indirectly heating the culture solution at a temperature of ranging from 70°C to 160 °C using a heat exchanger, rapidly cooling the indirectly heated culture solution to a temperature of ranging from 10°C to 60 °C at a rate of ranging from 10 L/min to 100 L/min, and isolating inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) from the rapidly cooled culture solution.

[31] Specifically, inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) may be prepared by the following procedure. First, Lactobacillus rhamnosus CJFR1505 (KCCM11721P) is cultured in an MRS agar medium to prepare a culture solution. In one embodiment, Lactobacillus rhamnosus CJFR1505 (KCCM11721P) may be plated on an MRS agar medium using a loop and cultured at 37 °C for 24 hours to prepare a culture solution.

[32] Then, the culture solution is indirectly heated at a temperature of ranging from 70°C to 160 °C using a heat exchanger. Specifically, the culture solution may be indirectly heated at the temperature of ranging from 80 °C to 150 °C, more specifically at a temperature of ranging from 90 °C to 120 °C.

[33] The indirectly heated culture solution is rapidly cooled to a temperature ranging from 10°C to 60 °C, specifically a temperature ranging from 20 °C to 50°C, for example, 4 °C, at a rate of ranging from 10 L/min to 100 L/min. Then, inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) are isolated from the rapidly cooled culture solution.

[34] The method may further comprise mixing a protective agent with the isolated inactive cells, followed by spray drying.

[35] The protective agent is not particularly limited and may be, for example, selected from the group consisting of yeast extract, dextrose, raw sugar, and mixtures thereof. The mixing with the protective agent and the subsequent spray drying prevent the inactive cells from being contaminated by external environmental factors and facilitate the distribution of the inactive cells.

[36] The spray drying refers to a process in which a liquid is sprayed at one time under a stream of hot air to obtain a dry liquid product. Examples of suitable spray drying processes comprise, but are not limited to, centrifugal spraying using a rotating disc and press spraying using a pressure nozzle.

[37] More specifically, the inactive cells are mixed with yeast extract, dextrose, and raw sugar, and the resulting mixture is spray dried to obtain a powder. For example, the inactive cells may be mixed with sugar (e.g., raw sugar) and/or starch (e.g., dextrose), suspended in water (e.g., distilled water), and spray dried to obtain a powder. During the spray drying, hot air enters through an inlet at a temperature of ranging from 120°C to 200 °C, preferably ranging from 130°C to 170 °C, and escapes through an outlet at a temperature of ranging from 30°C to 150 °C, preferably ranging from 50°C to 100 °C. However, the spray drying is not limited to these conditions.

[38] The yeast extract is added in an amount of ranging from 0.04 to 50 parts by weight, more preferably ranging from 0.1 to 10 parts by weight, based on 100 parts by weight of the mixture. The dextrose is added in an amount of ranging from 1 to 100 parts by weight, more specifically, ranging from 10 to 50 parts by weight, based on 100 parts by weight of the mixture. The raw sugar is added in an amount of ranging from 0.2 to 50 parts by weight, more specifically, ranging from 0.4 to 10 parts by weight, based on 100 parts by weight of the mixture.

[39] After spray drying, the powder of the dead bacterial cells may be mixed with an inorganic salt (e.g., calcium phosphate, calcium sulfate or calcium carbonate). The inorganic salt (e.g., calcium carbonate) is advantageous in controlling the moisture content of the powder of the inactive cells due to hygroscopicity thereof.

[40] More specifically, the powder of the inactive cells may be used in an amount of ranging from 0.05 to 50% (w/w), more specifically ranging from 0.5 to 30% (w/w), more specifically ranging from 0.5 to 5% (w/w), and the inorganic salt (e.g., calcium carbonate) may be used in an amount of ranging from 1 to 80% (w/w), specifically ranging from 5 to 50% (w/w), more specifically ranging from 5 to 20% (w/w), based on the total weight of the animal feed.

[41] The animal feed composition contains at least 10^s cfu, preferably ranging from 10^s to 10¹⁰ cfu, more preferably ranging from 10⁹ to 10¹⁰ cfu of the inactive cells per gram. The composition optionally further contains ranging from 5 to 99% (w/w), preferably ranging from 50 to 90% (w/w), more preferably ranging from 1 to 10% (w/w) of an inorganic material.

[Mode for Invention] [42] The present invention will be described in more detail with reference to the following examples. However, it should be understood that these examples are provided for illustration only and are not to be construed in any way as limiting the present invention.

[43] Description of details apparent to those skilled in the art will be omitted.

[44] EXAMPLES [45] Experimental Examples 1-6 [46] Lactobacillus rhamnosus CJLR1505 (KCCM11721P) was evaluated in terms of safety, acid tolerance, bile tolerance, antibacterial activity, digestive enzymatic activity, and the ability to degrade triglycerides. Known Lactobacillus rhamnosus KCCM 32450 was used as a comparative strain.

[47] Experimental Example 1: Evaluation of safety of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) [48] The safety of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) was evaluated by hemolysis, gelatin liquefaction, and phenylalanine deaminase tests in accordance with the standard test method for safety provided by the Korea Bioventure Association. A test was conducted to investigate whether harmful metabolites (e.g., ammonia) were produced. The results are shown in Table 3.

[49] [Table 3]

Strain	Tests
	Gelatin liquefaction	Phenylalanine deaminase	Hemolysis	Ammonia production
Lactobacillus rhamnosus CJLR1505	Negative	Negative	α-hemolysis, Safe	Negative

[50] As can be seen from the results in Table 3, Lactobacillus rhamnosus CJLR1505 (KCCM11721P) was negative in all tests, comprising gelatin liquefaction and phenylalanine deaminase tests. Lactobacillus rhamnosus CJLR1505 (KCCM11721P) did not produce ammonia. Lactobacillus rhamnosus CJLR1505 (KCCM1172IP) turned out to be safe in the hemolysis test.

[51] Experimental Example 2: Evaluation of acid tolerance of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) [52] The strain Lactobacillus rhamnosus CJLR1505 and the comparative strain 15 Lactobacillus rhamnosus KCCM 32450 were precultured in MRS media. The two strains were diluted 10‘⁶ fold with MRS broths whose pH values were adjusted to 2, 3, and 7. The dilute bacterial solutions were cultured at 37 °C, plated on MRS agar media at predetermined time points, and anaerobically cultured for 48 h. After culture, the numbers of colonies were counted. The results of the acid tolerance experiment are shown in Table 4.

[53] [Table 4]

Strains	MRS broth (pH7)	MRS broth (pH2)	MRS broth (pH3)
	Number of live cells (CFU/mL)	Number of live cells (CFU/mL)	Number of live cells (CFU/mL)
Oh	1 h	3 h	Oh	1 h	3 h	Oh	1 h	3 h
Lactobacillus rhamnosus CJLR1505	5.8x10^s	6.0x10^s	6.2x10^s	5.8x10^s	2.3x10^s	2.0x10^s	5.8x10^s	3.5x10^s	2.0x10^s
Lactobacillus rhamnosus KCCM 32450	6.4^10⁸	6.8x10^s	8.6x10^s	6.4x10^s	2.Οχ 10²	2.0xl0²	6.4x10^s	1.0x10^s	5.0x10

[54] As can be seen from the results in Table 4, the number of live cells of Lactobacillus rhamnosus CJLR1505 was less reduced compared to that of the comparative strain Lactobacillus rhamnosus KCCM 32450, indicating better acid tolerance of Lactobacillus rhamnosus CJLR1505.

[55] Experimental Example 3: Evaluation of bile tolerance of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) [56] The strain Lactobacillus rhamnosus CJLR1505 and the comparative strain Lactobacillus rhamnosus KCCM 32450 were precultured in MRS media. The two strain cultures were adjusted to pH 4. A bile acid solution (Oxgall) was added to the cultures such that the concentrations were 0%, 0.3%, and 1%. The mixtures were diluted 10‘⁶ fold with MRS broths. The dilute bacterial solutions were cultured at 37 °C, plated on MRS agar media at predetermined time points, and anaerobically cultured for 48 h. After culture, the numbers of colonies were counted. The results are summarized in Table 5.

[57] [Table 5]

Strains	MRS broth (pH4)	MRS broth (pH4 and Oxgall 0.3%)	MRS broth (pH4 and Oxgall 1%)
	Number of live cells (CFU/mL)	Number of live cells (CFU/mL)	Number of live cells (CFU/mL)
Oh	1 h	3 h	Oh	1 h	3 h	Oh	1 h	3 h
Lactobacillus rhamnosus CJLR1505	1.8x10’	4.2x10’	1.9xl0⁸	1.8x10’	1.0x10’	6.8x10	1.8x10’	7.2x10	2.9xl0⁴
Lactobacillus rhamnosus KCCM 32450	1.6x10’	4.6x10’	6.8x10’	1.6x10’	6.0x10	4.2xl0⁴	1.6x10’	4.6xl0⁴	2.0x10’

[58] As can be seen from the results in Table 5, no significant decreases in the number of viable cells of Lactobacillus rhamnosus CJLR1505 were found in both 0.3% and 1% Oxgall solutions at pH 4. In addition, the number of live cells of Lactobacillus rhamnosus CJLR1505 was less reduced compared to that of the standard strain, indicating that Lactobacillus rhamnosus CJLR1505 can grow even in the presence of bile acid in animals.

[59] Experimental Example 4: Evaluation of antibacterial activity of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) [60] Four species of pathogenic bacteria (E. coll K88, E. coll ATCC 25922, Salmonella typhimurium KCCM 25922, and Salmonella cholerasuis KCCM 10709) were liquid cultured for 24 h. The cultures (10⁵‘⁶ cfu each) were plated with sterile cotton swabs on tryptic soy broth (TSB) media (BD, ETSA).

[61] After plating, Lactobacillus rhamnosus CJLR1505 and Lactobacillus rhamnosus KCCM 32450 were diluted to 10⁹ cfu/mL with PBS buffer. The dilute bacterial solutions (50 pi each) were plated on paper discs (diameter 4 mm) and allowed to stand at room temperature until they permeated sufficiently into the paper discs, followed by aerobic culture at 37 °C for 18 h. The culture solutions were centrifuged at 3,000 x g for 10 min. The supernatants were washed three times with PBS buffer to isolate only the bacterial cells. The size of inhibition zones was determined as a difference between the diameter of the entire clear zone and the diameter of the agar wells. The results are shown in Table 6.

[62] [Table 6]

Strains	E. coli K88	E. coli ATCC 25922	Salmonella typhimurium KCCM 25922	Salmonella cholerasuis KCCM 10709
Lactobacillus rhamnosus CJLR1505	++	+++	+++	++
Lactobacillus rhamnosus KCCM 32450	++	++	++	++

10-15m: +, 15-20 mm: ++, 21-25 mm: +++ [63] As can be seen from the results in Table 6, both the strain Lactobacillus rhamnosus CJLR1505 and the standard strain Lactobacillus rhamnosus KCCM 32450 showed an inhibitory effect on the proliferation of pathogenic microbes but the strain Lactobacillus rhamnosus CJLR1505 was found to have better antibacterial activity than the standard strain Lactobacillus rhamnosus KCCM 324501.

[64] These results are associated with the inherent effects of the strain Lactobacillus rhamnosus CJLR1505 rather than the effects of metabolites produced from the strain, demonstrating that the strain can inhibit the proliferation of harmful microbes when fed to animals.

[65] Experimental Example 5: Evaluation of digestive enzymatic activity of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) [66] An experiment was conducted to determine whether the strain Lactobacillus rhamnosus CJLR1505 (KCCM11721P) has enzymes capable of degrading carbohydrates, proteins, and phosphorus.

[67] 0 .5% of skim milk was added to MRS agar media to investigate whether the strain has protease activity. 0.2% of methyl cellulose was added to MRS agar media to investigate whether the strain has cellulase activity. 0.2% (w/v) of corn starch was added to MRS agar media to investigate whether the strain has a-amylase activity, and media supplemented with 0.5% Ca-phytate were prepared to investigate whether the strain has phytase activity. The isolated strains were streaked onto the prepared media, cultured for 24 h, and observed.

[68] The culture media were treated with 2% Congo red reagent (Sigma, USA) and washed with 1 M sodium chloride (NaCI). The α-amylase and cellulase activities were determined by observing color changes. The protease and phytase activities were determined depending on whether clear zones were formed. The results are described in Table 7.

[69] [Table 7]

Strains	Protease activity	Phytase activity	Lipase activity	Cellulase activity	Amylase activity
Lactobacillus rhamnosus CJLR1505	+++	+++	++++	++++	++++
Lactobacillus rhamnosus KCCM 32450	++	++	+++	+++	+++

l-10m: +, 11-20 mm: ++, 21-30 mm: +++, 31-35 mm: ++++ [70] As can be seen from the results in Table 7, the strain Lactobacillus rhamnosus CJLR1505 had higher digestive enzymatic activities than the standard strain Lactobacillus rhamnosus KCCM 32450.

[71] These results demonstrate that Lactobacillus rhamnosus CJLR1505 can improve feed efficiency when fed to livestock animals.

[72] Experimental Example 6: Evaluation of ability of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) to activate triglyceride degradation [73] The bile salt hydrolase (BSH) activity of Lactobacillus rhamnosus CJLR1505 was investigated. As a result, a white precipitate was formed in an MRS solid medium supplemented with 2 mM taurodeoxycholate hydrate (TDCA, Sigma,

USA), demonstrating the enzymatic activity of the strain. Lactobacillus rhamnosus CJLR1505 showed a similar precipitation profile to the standard strain Lactobacillus rhamnosus KCCM 32450 as a TDCA positive control. However, no precipitation was observed in a medium supplemented with 2 mM sodium glycodeoxycholate (GDCA, Sigma, USA) and the growth of the bacteria was limited. The results are described in Table 8.

[74] [Table 8]

Conjugated bile acid/strain name	Lactobacillus rhamnosus CJLR1505	Lactobacillus rhamnosus KCCM 32450
TDCA	+	+
GDCA	+	-

+: grown, -: not grown [75] As can be seen from the results in Table 8, Lactobacillus rhamnosus CJLR1505 had high bile tolerance and produced BSH capable of degrading triglycerides. These results indicate that Lactobacillus rhamnosus CJLR1505 is a functional strain that can affect the daily weight gain of animals when fed to the animals.

[76] Experimental Examples 7-9 [77] Each of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) and Lactobacillus rhamnosus KCCM 32450 as a standard strain was plated on an MRS agar medium using a loop and cultured at 37 °C for 48 hours.

[78] Then, the culture solution was indirectly heated at a temperature of 100 °C using a heat exchanger and rapidly cooled to 10 °C at a rate of ranging from 30 L/min to 100 L/min. Inactive cells of the strain was isolated from the rapidly cooled culture solution.

[79] The ability of the inactive cells to adhere to intestinal epithelial cells was tested. The hydrophobicity of inactive cells of Lactobacillus rhamnosus CJLR1505 and the tendency of inactive cells of the strain to aggregate were compared with those of live cells of the strain. The daily weight gain and the feed conversion ratio of common feed comprising inactive cells of Lactobacillus rhamnosus CJLR1505 were compared with those of common feed without inactive cells of the strain.

[80] Experimental Example 7: Evaluation of ability of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) to adhere to intestinal epithelial cells [81] HT-29 cells as animal cells were furnished from the Korea Cell Line Bank (KCLB) and the ability of the Lactobacillus rhamnosus CJLR1505 (KCCM11721P) to adhere to the intestinal epithelial cells was tested in accordance with the method of Kim et al. (Kim et. al., Probiotic properties of Lactobacillus and Bifidobacterium strains isolated from porcine gastrointestinal tract, Applied Microbiology and Biotechnology, Volume 74, April 2007, pages 1103-1111) and Hirano et al. (Hirano et. al., The effect of Lactobacillus rhamnosus on enterohemorrhagic Escherichia coli infection of human intestinal cells in vitro, Microbiology and Immunology, Volume 47, 2003, pages 405-109). The monolayer-forming HT-29 cells were washed three times with PBS buffer and 0.5 mL of antibiotic-free RPMI1640 medium was added thereto.

[82] Inactive cells of Lactobacillus rhamnosus CJLR1505 and inactive cells of Lactobacillus rhamnosus KCCM 32450 at concentrations of ~10⁹ cells/mL were suspended in RPMI, inoculated into well plates, and cultured at 37 °C for 2 h. After completion of the culture, each culture was washed three times with PBS buffer to remove non-adherent inactive cells of the strain and confirm the ability of the strain to adhere to the intestinal epithelial cells after washing. After gram staining, the numbers of inactive cells were countered under a microscope to evaluate the ability to adhere to the intestinal epithelial cells. The experimental results are shown in FIG.

3.

[83] The experimental results show that the number of the inactive bacterial cells of Lactobacillus rhamnosus CJLR1505 after 2 h was confirmed to be 5.4x 10^scells/mL, which was larger than the number (3.2 xlO⁶ cells/mL) of the inactive cells of the standard strain Lactobacillus rhamnosus KCCM 32450, confirming better ability of Lactobacillus rhamnosus CJLR1505 to adhere to intestinal epithelial cells.

[84] Experimental Example 8: Evaluation of hydrophobicity of live cells and inactive cells of Lactobacillus rhamnosus CJLR1505 and tendency of live cells and inactive cells of the strain to aggregate [85] Self-aggregation and simultaneous aggregation were evaluated to confirm differences in hydrophobicity and tendency to aggregate between live cells and inactive cells of Lactobacillus rhamnosus CJLR1505.

[86] Self-aggregation was evaluated by the following procedure. First, live cells or inactive cells of Lactobacillus rhamnosus CJLR1505 were diluted to an OD₆oo of 0.5. 1 mL of toluene was added to 3 mL of the dilute strain, followed by vortexing for 90 sec. Thereafter, the mixture was allowed to stand in a water bath at 37 °C for 1 h. After removal of the toluene, the OD₆oo of the aqueous layer was measured.

[87] Simultaneous aggregation was evaluated by the following procedure. First, live cells or inactive cells of Lactobacillus rhamnosus CJLR1505 were mixed with the same amount of E. coli K88, Salmonella typhimurium KCCM 25922 or Salmonella cholerasuis KCCM 10709 as a pathogenic microbe (i.e. pathogenic microbe : live cells or inactive cells = 1 : 1 (1.5 ml each). 1 mL of toluene was added to 3 mL of the mixture, followed by vortexing for 90 sec. Thereafter, the resulting mixture was allowed to stand in a water bath at 37 °C for 1 h. After removal of the toluene, the OD₆oo of the aqueous layer was measured.

[88] Hydrophobicity was calculated by 100 x (initial OD₆oo ~ OD₆oo after 1 h)/initial ODaoo· [89] The results of the self-aggregation and simultaneous aggregation are described in Table 9.

[90] [Table 9]

Pathogenic strains	Lactobacillus rhamnosus CJLR1505
Live cells	inactive cells
Self-aggregation	11%	35%
Simultaneous aggregation	E. coli K88	45%	87%
Salmonella typhimurium KCCM 25922	30%	65%
Salmonella cholerasuis KCCM 10709	15%	45%

[91] As can be seen from the results in Table 9, the self-aggregation and simultaneous aggregation of inactive cells of Lactobacillus rhamnosus CJLR1505 were 2-3 times higher than live cells of the strain. The extracellular hydrophobicity of a microbial species and the tendency of a microbial species to aggregate are affected by the characteristics and surface structure of cell surface protein. The extracellular hydrophobicity of inactive cells of Lactobacillus rhamnosus CJLR1505 and the tendency of inactive cells of Lactobacillus rhamnosus CJLR1505 to aggregate increased because the surface protein structures of the inactive cells of the strain were different from those of live cells of the strain, as shown in FIG. 1. Therefore, Lactobacillus rhamnosus CJLR1505 is expected to be available as a functional strain that adheres to endotoxins, affecting disease resistance.

[92] Experimental Example 9: Comparison of feed comprising inactive cells of

Lactobacillus rhamnosus CJLR1505 with general common feed [93] The final mixture containing inactive cells of Lactobacillus rhamnosus CJLR1505 was added to common feed in dose of 0.2% (w/w) to prepare experimental feed in weaning piglet diet. Common feed for weaning piglets was used as a control feed. The feeds were fed to weaning piglets for 28 days. The initial body weights (kg), final body weights (kg), daily weight gains (g), daily feed intakes (g), and feed conversion ratios of the animals were measured. The results are shown in Table 10.

[94] [Table 10]

	Group administered inactive cells of Lactobacillus rhamnosus CJLR1505	Control group
Number of head of animals tested	24	24
Initial weight (kg)	6.98	7.00
Final weight (kg)	18.86’	17.76
Daily weight gain (g)	425’	383
Daily feed intake (g)	528	526
Feed conversion ratio	1.25	1.38

Significantly(statistically) different from those of the control group counterpart.

[95] As can be seen from the results in Table 10, the group administered inactive cells of Lactobacillus rhamnosus CJLR1505 showed significantly better results in both daily weight gain and feed conversion ratio than the control group.

Claims

[CLAIMS] [Claim 1 ]

Lactobacillus rhamnosus CJLR1505 (KCCM11721P).
[Claim 2]

An animal feed composition comprising Lactobacillus rhamnosus CJLR1505 (KCCM11721P) or inactive cells of the strain.
[Claim 3]

The animal feed composition according to claim 2, further comprising a carrier.
[Claim 4]

The animal feed composition according to claim 2 or 3, wherein the animal feed composition comprises the inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) of ranging from 1.0^χ10⁸ cfuto 1.0^χ10¹⁰ cfu per gram.
[Claim 5]

An animal feed comprising the composition according to any one of claims

2 to 4.
[Claim 6]

A method for producing inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P), comprising culturing Lactobacillus rhamnosus CJLR1505 (KCCM11721P) to prepare a culture solution, indirectly heating the culture solution at a temperature of ranging from 70 °C to 160 °C using a heat exchanger, rapidly cooling the indirectly heated culture solution to a temperature of ranging from 10 °C to 60 °C at a rate of ranging from 10 L/min to 100 L/min, and isolating inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P) from the rapidly cooled culture solution.
[Claim 7]

The method according to claim 6, further comprising mixing a protective agent with the isolated inactive cells of Lactobacillus rhamnosus CJLR1505 (KCCM11721P), followed by spray drying the mixture.

5 [Claim 8]

The method according to claim 7, wherein the protective agent is selected from the group consisting of yeast extract, dextrose, raw sugar, and mixtures thereof.