JP2003088362A - Lactobacillus belonging to the genus lactococcus useful for immunoactivation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To isolate a useful species for immunoactivation from lactobacillus belonging to the genus Lactococcus, having a high safety and suitable for the production of milk products, and applying as a probiotics. SOLUTION: This lactobacillus is Lactococcus lactis subspiecies lactis G50 species (FERM P-18415) which has immunoactivation activity.

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lactic acid bacterium belonging to the genus Lactococcus which is useful for immunostimulation. P-18415)
About. [0002] Lactic acid bacteria play an important role in the production of cheese, pickles, etc., and are taken into the body in the form of fermented milk, etc., so that the intestinal regulating action, the serum cholesterol lowering action, etc. It is known to exhibit various physiological effects based on the functionality of lactic acid bacteria. In recent years, the concept of “probiotics” (living microorganisms that work beneficially for maintaining the health of the host) has been introduced and the interest of such lactic acid bacteria has attracted widespread interest reflecting the health orientation of consumers. Many studies have been conducted. The term “probiotic” was originally written as “a microbial additive that beneficially affects the host animal by improving the balance of the intestinal flora” (Fuller, R. 198
9: J. Appl. Bacteriol. 66, 365-378), but now it is often used in a broad sense as described above. [0004] Currently, microorganisms widely used worldwide as probiotics are limited to Lactobacillus genus lactic acid bacteria and bifidobacteria. On the other hand, for Lactococcus lactic acid bacteria,
Previously, it was said that it could not pass through the digestive tract alive (Teuber, M., Geis, A. and Neve, H.:1992, The ge
nus Lactococcus. In: Balows, A., Truber, HG, Dwo
rkin, M., Harder, W. and Schleifer, KH (eds.) The
Prokaryotes, Vol II, 2 ^nd edn. Springer Verlag, N
ew York, pp.1482-1501). For this reason, there are few researches as probiotics compared to the above-mentioned microorganisms such as Lactobacillus lactic acid bacteria and bifidobacteria. Recently, however, it has become clear that some Lactococcus lactic acid bacteria can pass through the digestive tract as viable bacteria (Grahn, E., Holm,
SE, Lilja, H. and Sellgren K .: 1994, Scandinavia
n Journal of Nutrition 38,2-4.), the application of Lactococcus lactic acid bacteria to probiotics has been considered. Lactic acid bacteria belonging to the genus Lactococcus are used as starters for producing dairy products because of their good growth in milk, and play an important role in dairy industry use. Therefore, if a strain having properties effective as probiotics is found from Lactococcus lactic acid bacteria, there is a high possibility that it can be applied to the production of dairy products using the same. [0006] The immunostimulatory action by lactic acid bacteria is attracting attention as one of the probiotic functions, and is considered to contribute greatly to various diseases including cancer. So far, immunostimulatory action has been reported for lactic acid bacteria and bacterial cell components (Tejada-Simon, MV and Pestka, JJ
(1999), J. Food Prot., 12, 1435-1444, Perdigon, Ge
t al. (1999), J. Dairy Sci., 82, 1108-1114). In these reports, as an index of immunostimulation, an increase in the proliferation ability of T cells, an increase in the production amount of immunoglobulin A (hereinafter sometimes abbreviated as IgA), and various functions that cells produce. An increase in the amount of cytokine, which is a peptide, is used. However, many of these are reports on Lactobacillus lactic acid bacteria and bifidobacteria. [0007] As a method for supplying a substance having a certain physiological effect into the body, there are a method of taking it in the form of food and a method of administering it in the form of a medicine. Of these, the method of ingesting in the form of food has a better impression on the body than using pharmaceuticals, and it is considered that there is less resistance if taken as a food for daily intake for disease prevention. Therefore, it can play an important role in the production of dairy products to be ingested as food, and among lactic acid bacteria, it can find a useful strain for immunostimulation from Lactobacillus genus Lactobacillus which is highly safe along with Lactobacillus genus. It was desired. The object of the present invention is to search for Lactococcus lactic acid bacteria which are highly safe among lactic acid bacteria and are suitable for producing dairy products, and select strains useful for immunostimulation from the lactic acid bacteria. It is to be applied as a tics. Therefore, as a result of intensive studies, the present inventors succeeded in selecting a strain useful for immunostimulation from Lactococcus lactic acid bacteria suitable for dairy production. That is, the present inventors have found that there are strains in Lactococcus lactic acid bacteria that induce certain cytokine production from macrophages that are one of the immunocompetent cells. Means for Solving the Problems The present invention according to claim 1 is characterized in that Lactococcus lactis sub-species Lactis G50 strain (FERM) having an immunostimulatory action.
P-18415). BEST MODE FOR CARRYING OUT THE INVENTION Lactococcus lactis sub-species lactis G50 strain (F
ERM P-18415) can be selected from Lactococcus lactic acid bacteria by the following method. That is, the Lactococcus lactic acid bacterium having the immunostimulatory function of the present invention is an indicator of the amount of cytokine produced by the macrophage strain when the test lactic acid bacterium and a macrophage (macrophage) strain that is one of the immunocompetent cells are co-cultured. Can be selected as. As a macrophage strain used in the present invention, a BALB / c mouse-derived J774.1 (ATCC
TIB-67) shares. Macrophage strains co-cultured with lactic acid bacteria include the J774.1 strain derived from BALB / c mice and R
AW264.7 and the like can be mentioned, and in particular, BALB / c
The strain J774.1 derived from a mouse is preferred. This macrophage strain was transferred to a 4.6 well plate at 4.6 × 10 ^5.
~ 5.1 × 10 ⁵ cells / well, preferably 5.0 ×
Seed to become 10 ⁵ cells / well. Specifically, macrophages prepared at 5.0 × 10 ⁵ cells per ml are seeded in one well. Plates seeded with macrophages were 37% in the presence of 5% CO _2.
Culturing is carried out at 2 ° C for 2-3 days, preferably 2 days. As a medium for seeding macrophages, RPM supplemented with serum is used.
I medium (Rosewell Park Memorial Institute medium) and DMEM medium (Dulbecco Modified Eagle medium, manufactured by Sigma) can be used. After completion of the culture, the medium is removed from the plate by pipetting or the like, and RPMI medium to which serum components are newly added is added. The serum component added to the medium at this time includes fetal calf serum (hereinafter sometimes abbreviated as FCS) and the like.
Is preferred. When FCS is used as a serum component, an addition concentration of 10% is appropriate. In addition to the RPMI medium, a DMEM medium or the like is also used. Lactic acid bacteria co-cultured with macrophage strains are:
In addition to the G50 strain selected by the inventors, an independent administrative agency
Agricultural Technology Research Organization Livestock Pasture Research Institute (Sekizaki-cho, Inashiki-gun, Ibaraki Prefecture) and those obtained from RIKEN (Wako City, Saitama Prefecture) can also be used. Lactic acid bacteria are cultured for one day in advance according to a conventional method. Next, the cultured lactic acid strain is washed with 0.85% saline 1-2 times, preferably twice, suspended in RPMI medium supplemented with serum, and then 1 × 10 ⁷ to 1 × 10 ⁹ cells / The plate is inoculated into a well, preferably 1 × 10 ⁹ cells / well, and co-cultured with a macrophage strain. The conditions for the co-culture are appropriate at 37 ° C. for 8 to 48 hours, preferably 24 hours in the presence of 5% CO ₂ . After completion of co-culture, the culture supernatant is separated by a conventional method such as centrifugation, and the amount of cytokine contained in the supernatant is measured by ELISA (solid phase enzyme immunoassay, ELISA). In the case of ELISA, interleukin-12 (hereinafter sometimes abbreviated as IL-12) is op.
tEIA mouse IL-12 measurement kit (Pharmin Dien)
In the case of TNF-α, an optEIA mouse TNF-α measurement kit (manufactured by Pharmingen) is used according to the protocol of these kits. Interleukin-6
(Hereinafter sometimes abbreviated as IL-6)
A combination of anti-mouse IL-6 (manufactured by Pharmingen) and biotin-labeled anti-mouse IL-6 (manufactured by Pharmingen) may be used. The cytokines produced by the above co-culture include IL-12 that induces the activation of cellular immunity that is thought to contribute to cancer prevention, and IgA in the gastrointestinal tract.
Examples include IL-6 involved in humoral immune activation such as production, or TNF-α which is a factor having a damaging activity against tumor cells. As shown in the examples described later, it was confirmed that cytokine production was promoted by co-culturing lactic acid bacteria and a macrophage strain. That is, it was found that 6 out of 16 lactic acid bacteria test strains promote cytokine production of macrophage strains. This 6
Among the strains, only one strain promoted the cytokine production of all of IL-12, IL-6 and TNF-α most. Lactococcus lactis subspices Lactococcus lactis subs
p. lactis) G50 strain. This bacterium is deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and its deposit number is FERM P-18415. When this Lactococcus lactis subspecies lactis G50 strain is orally administered to mice, the production of cytokines is promoted more than the non-administered group to which this strain was not administered. When administered orally, about 10 g per kg body weight of the mouse may be used as a guide. Moreover, since this strain has the characteristics of the genus Lactococcus and grows well in milk, it can be applied to the production of dairy products as a starter. Furthermore, this strain has the effect of promoting cytokine production of macrophage strains even when killed by heat treatment. In the following, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. Example 1 In this example, about 16 strains of lactic acid bacteria obtained from the stock raising laboratory and the RIKEN,
Co-culture with c strain mouse-derived macrophage J774.1 was performed, and lactic acid bacteria having the ability to promote cytokine production were selected. In addition, the culture | cultivation was similarly performed about the control which does not inoculate lactic acid bacteria as a control, and the thing using lipopolysaccharide (henceforth LPS) as positive control. LPS is a macrophage activator, and in this experiment, Escherichia coli-derived LPS (manufactured by Sigma) was added at 1 μg / well. First, macrophage J774.1 strain is
5 x 10 ⁵ ce in a 24-well plate (Falcon)
lls / well, in the presence of 5% CO ₂ ,
The cells were cultured at 37 ° C. for 2 days. As a medium for seeding macrophages, an RPMI medium supplemented with 10% FCS was used. After completion of the culture, the medium was removed from the plate by aspiration using a pipette, and 0.9 ml of RPMI medium (manufactured by Sigma) newly added with 10% FCS (manufactured by Sigma) as a serum component was added. The test lactic acid bacteria to be co-cultured with the macrophage strain were cultured in advance for a whole day and night according to a conventional method. Next, the cultured lactic acid strain was washed twice with 0.85% saline and suspended in RPMI medium at 1 × 10 ¹⁰ cells / well, and then 0.1 ml per well (lactic acid bacteria were 1 × 10 ⁹ cells) was added and co-cultured with the macrophage strain. The conditions for co-culture were ₂ at 37 ° C. under 5% CO ₂ condition.
4 hours. After completion of co-culture, the culture supernatant is centrifuged (1
(2000 rpm, 10 minutes, 4 ° C.).
The amount of cytokine contained in the supernatant was measured by ELISA. If the ELISA is IL-12,
In the case of TNF-α using optEIA mouse IL-12 measurement kit (Pharmin Diene), optEIA mouse TNF-α
A measurement kit (manufactured by Farming Dien) was used. Also,
In the case of IL-6, measurement was carried out using a combination of anti-mouse IL-6 (manufactured by Pharmingen) and biotin-labeled anti-mouse IL-6 (manufactured by Pharmingen). The measurement was performed according to each protocol. FIG. 1 shows values obtained by measuring the amount of cytokine produced by co-culture of a macrophage strain and lactic acid bacteria by ELISA. As can be seen from FIG. 1, the amount of cytokine produced varies depending on the type of lactic acid bacteria used for co-culture. Among the lactic acid bacteria tested, only one strain has the effect of promoting the production of all IL-12, IL-6, and TNF-α cytokines, and is comparable to or higher than the positive control LPS. Met. This strain is G50
Is a stock. Example 2 In this example, Lactococcus lactis subspecies lactis G50 selected in Example 1 was used.
Regarding the strains, the ability of macrophage strains to promote cytokine production was examined when dead cells were used. Lactococcus lactis subspecies Lactus G50 strain dead cells were prepared by subjecting live cells to heat treatment at 100 ° C. for 50 minutes. The conditions for co-culture, the measurement of the amount of cytokine contained in the culture supernatant after culture, and the like were performed in the same manner as in Example 1. In addition, the culture | cultivation was similarly performed about the thing which is not inoculated with lactic acid bacteria as a control. The results are shown in FIG. The figure shows a relative change in the amount of cytokine produced when live cells or dead cells are used for culturing as lactic acid bacteria. That is, it is calculated by dividing each measured value by the measured value of the control. As a result, it was clarified that the G50 strain promotes IL-12 and IL-6 production of the macrophage strain as in the case of the living cells even if the cells are dead cells. Example 3 In this example, the effect on the immune system when Lactococcus lactis subspecies lactis G50 strain was orally administered to a living body was examined. G5
Strain 0 was cultured overnight according to a conventional method. Next, the above lactic acid bacterial strain was washed twice with 0.85% saline,
It was suspended in 10% skim milk (Snow Brand Skimmed Milk, Snow Brand Milk Products Co., Ltd.) so as to be 1 × 10 ⁹ cells per l. A 10% skim milk suspension of G50 strain viable cells was
Three BALB / c mice (weight average 20 g) were forcibly and continuously administered for 7 days or 14 days. In the control group, only 10% nonfat milk was orally administered in the same manner. The dose of the G50 strain is 10 per kg of mouse body weight.
g was used as a standard. On day 8 or 15 after administration, Peyer's patch cells and spleen cells were collected from the test mice. The collected Peyer's patch cells and spleen cells were seeded as single cells in a 24-well plate and cultured at 37 ° C. for 72 hours in the presence of 5% CO ₂ using RPMI medium supplemented with 10% FCS. After completion of the culture, the culture supernatant was collected, and the amount of cytokine contained in the supernatant from which cells were removed by centrifugation was measured by ELISA in the same manner as in Example 1. The results are shown in FIG. Peyer's patch cells are tissues that play an important role in the digestive tract immune system. This cell is considered to be an indicator of local immunity. Moreover, since spleen cells play a role such as immune defense against antigens generated in the blood, they are considered to be indicators of systemic immunity. As is apparent from the figure, in the Peyer's patch cells, the IL-12 production was about 4 times higher in the group administered with the G50 strain for 7 days than in the control group. I
About L-6, the 7-day administration group of this strain showed a higher production amount than the control group. On the other hand, regarding the spleen cells, the IL-12 production amount was higher in the 14-day administration group of the strain than in the control group. However, regarding IL-6, regardless of the administration period, the group to which the strain was administered had a slightly lower production amount than the control group. From these things, G5
It was revealed that the 0 strain strongly induced IL-12 production of immunocompetent cells in vivo. EFFECT OF THE INVENTION According to the present invention, a strain useful for immunostimulation selected from Lactococcus lactic acid bacteria which is highly safe and suitable for dairy production among lactic acid bacteria, as well as Lactobacillus genus. Is provided. Therefore, by using the strain as a starter or the like, it can be applied to the production of dairy products and its probiotic function can be utilized.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B are views showing cytokine production amounts when a macrophage strain and various lactic acid bacteria in Example 1 are co-cultured. In the figure, 527, 712, H4
8, G50 and J53 are Lactococcus lactis
Subspecies lactis; HP, H61, ML and CH2-1 are Lactococcus lactis subsp. Cr
emoris); N7, DRC1, 8W, CVT3, B75-
1W and B75-2Y are Lactococcus lactis
Lactococcus lactis subsp. Lactis
biovar. diacetylactis); 1112 represents lactic acid bacteria belonging to Lactobacillus reuteri, respectively. FIG. 2 is a graph showing a relative change in the amount of cytokine production when using live and dead cells of Lactococcus lactis subspecies lactis G50 strain of Example 2. In the figure, A shows the results for IL-12, and B shows the results for IL-6. FIG. 3 is a graph showing the amount of cytokine produced when Lactococcus lactis subspecies lactis G50 strain was orally administered in Example 3. In the figure, A is IL-12 production in Peyer's patch cells, B
Represents IL-6 production in Peyer's patch cells, and C represents IL-12 production in spleen cells.

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[Procedure Amendment] [Submission Date] August 7, 2002 (2002.8.7) [Procedure Amendment 1] [Document Name to be Amended] Description [Item Name to be Amended] 0017 [Amendment Method] Change [Amendment] [0017] When this Lactococcus lactis sub-species Lactis G50 strain is orally administered to mice, the production of cytokines is promoted more than the non-administration group to which this strain was not administered. When administered orally, about 10 mg per kg body weight of the mouse may be used as a guide. Moreover, since this strain has the characteristics of the genus Lactococcus and grows well in milk, it can be applied to the production of dairy products as a starter. Furthermore, this strain has the effect of promoting cytokine production of macrophage strains even when killed by heat treatment. [Procedure Amendment 2] [Document Name to be Amended] Description [Item Name to be Amended] 0025 [Correction Method] Change [Details of Amendment] [0025]
Three BALB / c mice (weight average 20 g) were forcibly and continuously administered for 7 days or 14 days. In the control group, only 10% nonfat milk was orally administered in the same manner. The dose of the G50 strain is 10 per kg of mouse body weight.
The standard was mg .

──────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI Theme code (reference) C12R 1:01) (72) Inventor Junichi Kurisu ▲ Saki Jun 2-2-1 Kannondai, Tsukuba City, Ibaraki Pref. Institution of Agricultural and Bioresources (72) Inventor Takashi Okamoto Ikenodai, Sekizaki-cho, Inashiki-gun, Ibaraki Pref. Independent Administrative Law Human Agricultural Research Institute F-term in the Institute of Animal Husbandry (reference) 4B065 AA01X AC20 BB40 CA44 4C087 AA01 AA02 BC56 CA09 NA14 ZB09 ZB26

Claims (1)

What is claimed is: [Claim 1] Lactococcus having immunostimulatory action
Lactis Subspecies Lactis G50 strain (FERM P-18415).