KR20040084285A - Lactobacillus casei HY7201 and the products thereof - Google Patents

Abstract

PURPOSE: A microorganism Lactobacillus casei HY7201 and use thereof are provided, thereby effectively preventing diarrhea and gastroenteritis caused by rotavirus. CONSTITUTION: The microorganism Lactobacillus casei HY7201(KACC 91036) inhibits rotavirus infection, wherein Lactobacillus casei HY7201(KACC 91036) is isolated from human. The fermented milk inhibiting rotavirus infection contains Lactobacillus casei HY7201(KACC 91036) as an effective component. The lactic acid bacteria powder having inhibiting effect on rotavirus infection contains Lactobacillus casei HY7201(KACC 91036) as an effective component. The probiotics having inhibiting effect on rotavirus infection contains Lactobacillus casei HY7201(KACC 91036) as an effective component.

Description

Lactobacillus casei HY7201 and the products of Lactobacillus casei HY7201 having its inhibitory effect on rotavirus infection

The present invention relates to Lactobacillus casei HY7201, a lactic acid bacterium preparation, and fermented milk using the same against rotavirus known to cause acute enteritis and diarrhea in infants.

Rotavirus, first discovered in 1973 by the Australian bishop, belongs to the Reoviridae family. It is a major cause of acute diarrhea and gastroenteritis in infants, and continues to develop in both developed and developing countries. Onset. In particular, the incidence is high in children aged 6 months to under 2 years old and mild or asymptomatic in newborns or adults, but is estimated to cause 1 million infant deaths annually worldwide [Ref. Kapikian, AZ, in Fields Virology 3rd ed. 1657-1708 (1996). Rotaviruses do not have envelopes and have outer capsids and cores. Rotavirus's body contains 11 ds RNA fragment genomes in three protein shells. The outermost shell consists of the glycoprotein VP7 (37kDa) and VP4 (88kDa), which is activated by trypsin. The middle shell is a thick layer of VP6, which contains VP1, VP3 and 11 genome fragments within the core that are believed to label the structural proteins VP2, RNA polymerase and guanylytransferase. Frequent exchange of these RNA fragments between rotavirus strains results in genetic diversity [Ref. Estes, M. K., Microbiol. Rev. 53 (4): 410-449. (1989).

Rotavirus infects the small intestinal chollocytes and proliferates in the cytoplasm of these cells, resulting in impaired transport mechanisms. Damaged cells fall into the intestinal tract and release a large amount of virus so that the virus is found in feces. Diarrhea caused by rotavirus is due to the impaired absorption of sodium and glucose caused by the replacement of damaged cells of the villi with immature crypt cells. The treatment of enteritis caused by rotavirus is possible by supplementing the electrolyte and water lost by vomiting and diarrhea [Ref .: Santosham, M., J. Pediatr. 118: S44-S51. (1991)] To date, no effective rotavirus vaccine has been developed.

Rotavirus is difficult to develop vaccines because it is caused by various serotypes [Ref. Conner, M. E. Curr. Top. Microbiol. Immunol. 105: 253. (1994)]. Previous studies on vaccine development have focused on live poison vaccines. After decades of research to induce active mucosal immunity, a rotavirus vaccine was developed, and in December 1997, the US Food and Drug Administration approved Wyeth Laboratories' oral tetravalent live venom vaccine, and in 1998 obtained a final license Started. This vaccine was a live poison vaccine intended to prevent type G by infecting monkey rotavirus and human rotavirus. The orally administered live venom vaccine is intended to prevent infection by reaching the small intestine mucosa and proliferating in the mucosal cells to form IgA antibodies in the small intestine mucosa to neutralize the virus invading the IgA antibody. However, the vaccine will be discontinued only three months after its start-up. Intestinal overlap was observed in 15 infants who received the vaccine, and Wyeth Laboratories eventually withdrew from July 1999, only 11 months after obtaining a license, for the rotavirus. There is no vaccine [Reference: MMWR Morb Mortal Wkly Rep. 48 (43): 1007. (1999).

Due to the difficulty in the manufacture of vaccines, vaccines are being produced that produce parts of rotavirus constructs in expression systems such as Escherichia coli and baculovirus using genetic recombination technology. have. Several studies have been reported to defend against rotavirus infection with food ingredients such as colostrum, egg yolk, green tea extract, lactoferrin and licorice. In addition, it has been reported that the drinking of lactic acid bacteria or lactic acid bacteria fermented milk which is known to be beneficial for health is effective in preventing or treating acute diarrhea in infants caused by rotavirus infection. Infants with Streptococcus thermophilus and Bifidobacterium bifidum reduced the number of diarrhea and the rotavirus excreted in feces [Saavedra, JM Lancet. 344: 1046-1049. (1994)]. See also Lactobacillus reuteri [Ref. Shornikova, AV, J. Pediatr. Gastroenterol. Nutr. 24 (4),: 399-404. (1997)] or diarrhea symptoms were observed in infants fed Lactobacillus strain GG [Ref. Isolauri, E. Pediatrics. 88 (1): 90-97. (1991)], drinking Lactobacillus casei fermented milk in lactating rats has been reported to enhance mucoasa integrity and reduce rotavirus infection by villi to protect against diarrhea [Guerin-Danan, C] , J. Nutr. 131 (1): 111-117. (2001). And Bifidobacterium breve K-110 has been reported to inhibit rotavirus tm infection [Bae, EA, J. Microbiol. Biotechnol. 12 (4): 553-556. (2002)].

The present invention provides an infant diarrhea and acute enteritis prevention agent for providing a lactic acid bacterium having the ability to inhibit infection with rotavirus known to cause acute enteritis.

Figures 1a and 1b is a diagram staining the rotavirus-infected MA-104 cells by mixing MA-104 cells infected with rotavirus and Lactobacillus casei HY7201, respectively.

Figure 2 is a graph showing the effect of inhibiting rotavirus according to the amount of reduced skim milk culture medium of Lactobacillus casei HY7201

Figure 3 is a graph showing the rotavirus inhibitory effect of the culture medium or cells of Lactobacillus casei HY7201

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

The present inventors selected lactic acid bacteria having anti-infective activity against rotavirus isolated from diarrhea in domestic infants among Lactobacillus genus isolated from humans, and named it Lactobacilus casei HY7201. . The novel microorganism of the present invention was deposited with KACC No. 91036 to Korea Agricultural Biotechnology Research Institute on March 10, 2003.

Strain characteristics

The physiological characteristics of Lactobacillus casei HY7201 when incubated for 18 hours at 37 ° C. in MRS medium are shown in Table 1 below.

Physiological Characteristics of Lactobacillus Casei HY7201 Physiological characteristics result Cell morphology Bacillus Gram dyeing positivity Colony size Round Diameter 0.5 ~ 2.0mm Milky White Effects of Milk Coagulant Catalase Beta-galactose Degrading Enzyme +-+-Breath anaerobic Sugar availability Party reaction Party reaction Erythritol ribose adonitol galactose glucose fructose mannose sorbose lamnosmannitol sorbitol acetylglucosamine amigdaline arbutin esculin -+-+++++-++++++ Salicycin Serobiose Maltose Lactose Mellibiose Sucrose Trehalose Inulin Melesitostrafinosamidon Turanoostagatose Arabbitol ++-+-+-+-++-

Rotavirus infection suppression effect

After inoculating 0.2% of Lactobacillus casei HY7201 in 10% reduced skim milk and incubating for 18 hours at 37 ° C, rotavirus infection inhibitory activity was measured. As a result, Lactobacillus casei HY7201 showed an infection inhibition effect of 85-92%, which was higher than other lactic acid bacteria. (See Example 1 to be described later.)

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the scope of the present invention, it is a matter of course that ordinary changes are possible by those skilled in the art within the scope of the technical idea of the present invention.

Example 1

Selection of Lactic Acid Bacteria with Rotavirus Infection Inhibitory Activity

Lactobacillus usable in fermented milk were compared with the infection inhibitory activities against rotavirus Wa strain (ATCC VR-2018) and KRV01 (KFCC 11313) isolated from Korean infants.

Lactic acid bacteria were used for the test after 18 hours incubated in 10% reduced skim milk or MRS medium. To prepare a rotavirus, trypsin was added to the rotavirus solution at a concentration of 5 µg / ml, activated at 37 ° C for 1 hour, and then inoculated into MA-104 (Marcus monkey kidney cell) monolayer cells and adsorbed for 1 hour.

After adding trypsin-containing minimum essential medium (MEM) and incubating at 37 ° C, when cytopathic effects (CPE) is more than 90%, recovery and freezing and thawing in liquid nitrogen are repeated three times. Frozen was used for the experiment.

MA-104 monolayer cells were prepared in 6-well cell culture plates and washed twice with phosphate buffered saline (PBS). The rotavirus prepared to have a final titer of 1 × 10 ⁵ pfu / ml and the lactic acid bacteria culture medium prepared to have a final addition of 5% were allowed to stand for 30 minutes, and then infected with MA-104 cells and infected with MA-104 cells for 18 hours at 37 ° C., CO ₂ Incubated in the incubator and washed twice with PBS. 10% formalin is treated for 30 minutes to fix and washed again with PBS.

Next, 0.5% Triton X-100 was treated for 5 minutes, washed, and then PBA with 1% BSA (bovine serum albumin) added to PBS in an anti-rotavirus antibody at a ratio of 1: 250, followed by 30 minutes at 37 ° C. It was political.

After washing three times with PBA and adding PBA added donkey anti-goat IgG (peroxidase) fused peroxidase in the ratio of 1: 500 and left for 30 minutes at 37 ℃. After washing three times with PBS, a solution of 4 mg of 3-amino-9-ethylcarbazole per ml of N, N-dimethylformamide, 0.05M sodium acetate buffer, and hydrogen peroxide were mixed at a rate of 1.5 ml, 3.5 ml, and 5 µl, respectively. The color solution was added to the fixed cells and allowed to react for 15 minutes. The chromogenic solution was removed, washed with PBS and the infected cells were counted by reversed phase microscopy. Inhibition rate was determined by the ratio of the number of infected cells reduced by the sample and the number of infected cells of the control group inoculated with rotavirus only.

As shown in Table 2 below, the inhibition rate of various rotavirus infections was shown for each Lactobacillus strain, and the inhibition rate was similar in the rotavirus Wa strain and KRV01. In case of Lactobacillus casei HY7201, the infection rate of Wa strain was 85% and KRV01 92%, showing the highest rotavirus infection inhibition ability (Fig. 1a, 1b).

Inhibitory Effect of Lactic Acid Bacteria on Rotavirus Infection Strains Rotavirus infection inhibition rate (%) Wa strain KRV01 Lactobacillus acidophilus HY7121 25 17 Lactobacillus acidophilus HY7141 27 22 Lactobacillus acidophilus HY7155 11 20 Lactobacillus casei HY7201 85 92 Lactobacillus casei HY7244 12 5 Lactobacillus rhamnosus HY7321 8 15 Lactobacillus bulgaricus HY7403 45 33 Lactobacillus plantarum HY7244 37 30 Lactobacillus plantarum HY7207 42 28 Lactobacillus fermentum HY7255 10 16 Lactobacillus brevis HY7505 15 23

Example 2

Inhibitory Effects of Lactobacillus Casei HY7201 Concentrations on Rotavirus Infection

Lactobacillus casei HY7201 was inoculated with 0.2% in 10% reduced skim milk and incubated at 37 ° C. for 18 hours. The number of viable cells of the fermentation broth was 5 × 10 ⁸ cfu / ml, and the effect of inhibiting rotavirus infection according to the amount of fermentation broth added at 0.5%, 1%, 2.5% 5%, and 10% was performed according to Example 1, and in FIG. 2. As can be seen, when the concentration of reduced skim milk cultured with Lactobacillus casei HY7201 was 5% or more, it showed the highest inhibitory effect on rotavirus infection.

Example 3

Inhibitory Effects of Lactobacillus Casei HY7201 Cells on Rotavirus Infection

After inoculating 0.2% of Lactobacillus casei HY7201 in MRS medium, the cells were incubated at 37 ° C for 18 hours. The supernatant obtained by centrifuging the MRS culture at 6,000 × g, the cells and the rotavirus inhibitory effect of the MRS culture was tested according to Example 1. As a result, as shown in FIG. 3, as a result of measuring the inhibitory effect of the culture supernatant and the Rotavirus infection of the cells, the inhibitory effect of the cells was higher as 61%.

Example 4

Inhibitory Effect of Lactobacillus Casei HY7201 on Rotavirus Diarrhea in Mice

Five 5-day-old balb / c mice were assigned to 12 mice in each experimental group. Rotavirus SA11 was administered orally to the infected and administered groups, and MEM medium was orally administered to the control group. Rotavirus infection was infected for 2 days with 0.5 ml (1.5 × 10 ⁷ PFU / horses) of rotavirus once daily. The control group was not infected with rotavirus but instead received the same amount of MEM medium. In addition, one administration group received lactobacillus casei HY7201 culture medium (5 × 10 ⁸ cfu / ml) incubated for 18 hours at 37 ° C. in 10% reduced skim milk, and 10% reduced skim milk in other administration groups daily from 2 days to 7 days of age. Ml was administered. Diarrhea incidence was determined by pressing the mouse's abdomen lightly yellowish green stool was confirmed, diarrhea caused by the diarrhea index (Diarrhea score, DS) was counted. Diarrhea was 2 points for severe, 1 point for weak and 0 point for no symptom, and then divided by the total number of mice.

As shown in Table 3 below, the rotavirus infection group had severe diarrhea in 10 of 12 mice, and the diarrhea index was 1.75 due to weak diarrhea in 2 mice. The diarrhea index was 1.83 even when administration of reducing skim milk showed no inhibitory effect of rotavirus on reducing skim milk. In contrast, in the group treated with Lactobacillus casei HY7201 cultured in 10% reduced skim milk, mild diarrhea occurred in only two animals, and the diarrhea index was significantly lowered to 0.17. Therefore, the inhibitory effect of Lactobacillus casei HY7201 on rotavirus infection was confirmed.

Inhibitory Effect of Lactobacillus Casei HY7201 on Rotavirus Diarrhea treatment Number of experiment mice Number of mice that caused diarrhea (horses) Diarrhea Index (D.S.) Control 12 0 0 Rotavirus Infection Group 12 12 1.75 Reduced skim milk administration group 12 12 1.83 Lb. casei HY7201 group 12 2 0.17

Example 5

Fermented milk manufacturer

Using skim milk powder, raw milk with the content of non-solid content adjusted to 8-20% was sterilized at 72-75 ° C. for 15 seconds. After the sterilized crude oil was cooled, Lactobacillus casei 7201 was inoculated at a concentration of 10 ⁶ cfu / ml, and then cultured to pH 4.5. A syrup made of 0.1-50% juice, 0.1-20% dietary fiber, 0.5-30% glucose, 0.1-15% oligosaccharide, 0.001-10% calcium, 0.0001-5% vitamins, etc. was prepared in rust. The prepared syrup was sterilized and cooled, mixed with the culture solution at a predetermined ratio, stirred and homogenized, and then packaged in a container to prepare fermented milk. The fermented milk thus prepared showed good results in terms of flavor, physical properties and overall taste.

Example 6

Preparation of Lactic Acid Bacteria Powder

Lactobacillus casei HY7201 was inoculated in MRS broth at a concentration of 10 ⁶ cfu / mL and pH-control fermentation was performed at 37 ° C for 30 hours. The pH was adjusted to pH 5.7 ± 0.2 using ammonia gas as a neutralizer. After incubation, cells were recovered by centrifugation at 6,000 × g.

The recovered cells were mixed with a protective agent containing 2.5% whey and 5% sucrose in 5% reduced skim milk and prepared in powder using a freeze dryer. The dry powder of the Lactobacillus casei HY7201 thus prepared showed a viable cell count of 1 × 10 ¹¹ cfu / g or more.

Example 7

Lactobacillus Formulation Preparation

The lactic acid bacteria formulations, such as lactic acid bacteria foods, formal preparations were prepared using the lactic acid bacteria powder prepared in Example 4. Lactobacillus casei HY7201 in 20% by weight of dry lactobacillus powder 10% by weight oligosaccharide, 20% by weight anhydrous glucose, 5% by weight fructose, 2% by weight vitamin C, 5% by weight fruit powder, 5% by weight aloe, dietary fiber 15 wt% was mixed and dispensed in a predetermined amount in a stick, bottle, or can and packed.

The lactic acid bacterium preparation thus prepared maintained the number of viable cells of 5 × 10 ⁸ cfu / g, and as a result of the sensory test, had no effect on off-flavor and had good results in overall taste.

As can be seen from the above embodiment, the Lactobacillus casei HY7201 according to the present invention is effective in inhibiting rotavirus infection known as a cause of diarrhea and gastroenteritis in infants, and furthermore, prepared by using such products as lactic acid bacteria foods, suits, etc. When ingested to prevent diarrhea and gastroenteritis caused by rotavirus infection.

Claims (4)

Novel Lactobacilus casei HY7201 strain (KACC 91036) with inhibitory effect on rotavirus infection. A fermented milk that is effective in inhibiting rotavirus infection using the Lactobacillus casei HY7201 strain of claim 1 as an active ingredient. Lactobacillus casei HY7201 strain of claim 1 as an active ingredient lactic acid bacteria powder effective for inhibiting rotavirus infection. Lactobacillus Kasei HY7201 strain of claim 1 as an active ingredient lactic acid bacteria formulations that are effective in inhibiting rotavirus infection.