The Journal of Nutrition

Effects of Probiotics and Prebiotics

Effects of a Fermented Milk Drink

Containing Lactobacillus casei Strain Shirota
on the Human NK-Cell Activity1,2
Kazuyoshi Takeda and Ko Okumura*
Department of Immunlogy, Juntendo University, School of Medicine, Tokyo 113-8421, Japan

Abstract
Nine healthy middle-aged and 10 elderly volunteers drank fermented milk containing 4 3 1010 live cells of Lactobacillus
casei strain Shirota daily for 3 wk, and their natural killer (NK) activity and other immunological functions were examined. In
the experiments with middle-aged volunteers, NK activity significantly increased (P , 0.01) 3 wk after the start of intake,
individuals. In the experiments with elderly volunteers, NK activity significantly decreased (P , 0.01) in the control group
3 wk after the start of intake; however, the intake of Lactobacillus casei strain Shirota maintained the NK activity.
These results suggest that daily intake of Lactobacillus casei strain Shirota provides a positive effect on NK-cell
activity. J. Nutr. 137: 791S793S, 2007.

Introduction
Lactic acid bacteria are commonly used for the manufacture of
fermented milk products and are known to have excellent nutritional and flavoring effects. Recent studies have revealed that
some strains of lactic acid bacteria have health-promoting effects
through improvement of the intestinal microflora and modulation of the immune system (1,2). Lactobacillus casei strain
Shirota (LcS)3 has been shown to have potent antitumor effects
in rodents (3,4), and immunomodulatory effects of this bacterium also have been well documented in animal models (5). In
humans, an LcS preparation has been shown to prevent the
recurrence of superficial bladder cancer, and a possible effect of
LcS on the immune system has been suggested (6,7). Furthermore, habitual intake of lactic acid bacteria containing LcS has
recently been shown to reduce the risk of bladder cancer, and an
effect on the immune system of healthy people has also been
1
Published as a supplement to The Journal of Nutrition. The articles included in
this supplement are derived from presentations and discussions at the World
Dairy Summit 2003 of the International Dairy Federation (IDF) in a joint
IDF/FAO symposium entitled Effects of Probiotics and Prebiotics on Health
MaintenanceCritical Evaluation of the Evidence, held in Bruges, Belgium. The
articles in this publication were revised in April 2006 to include additional relevant
and timely information, including citations to recent research on the topics
discussed. The guest editors for the supplement publication are Michael de
Vrese and J. Schrezenmeir. Guest Editor disclosure: M. de Vrese and
J. Schrezenmeir have no conflict of interest in terms of finances or current
grants received from the IDF. J. Schrezenmeir is the IDF observer for Codex
Alimentarius without financial interest. The editors have received grants or
compensation for services, such as lectures, from the following companies that
market pro- and prebiotics: Bauer, Danone, Danisco, Ch. Hansen, Merck, Muller
Milch, Morinaga, Nestec, Nutricia, Orafti, Valio, and Yakult.
2
Author disclosure: no relationships to disclose.
3
IFN, interferon; LcS, Lactobacillus casei strain Shirota; NK, natural killer.
* To whom correspondence should be addressed. E-mail: kokumura@med.
Juntendo.ac.jp.

0022-3166/07 $8.00 2007 American Society for Nutrition.

suggested (8); however, the precise mechanisms of action remain

to be discovered (9). In this study, we examined the effects of
drinking fermented milk containing LcS on the immune system
of healthy individuals, especially the effect on the NK-cell
activity of peripheral blood mononuclear cells to investigate the
mechanisms underlying the antitumor effect of LcS.

Materials and Methods

Subjects. Nine healthy middle-aged (3045 y) and 10 elderly (5575 y)
volunteers who had relatively low levels of NK-cell activity, below 45%
cytotoxicity (10), were adopted as subjects. The subjects were separated
into 2 groups, an experimental group and a control group, and the
experiments were done 2 times alternating groups. The experimental
group drank 1 bottle of Yakult 400 (containing 2.5 g of nonfat dry milk
solids, 14.4 g of sugars, flavors, and at least 4 3 1010 live LcS cells per
bottle) after lunch every day for 3 wk, and the control group drank the
same amount of unfermented milk, which had a basic composition similar
to that of Yakult 400 except for the bacteria, after lunch every day for 3
wk. Blood samples were collected at 5 times: before any ingestion of the
milk product, 1 wk and 3 wk after the start of intake, and 3 wk and 2 mo
after the period of intake. The data from serological analysis clearly
indicated that the health condition of all volunteers remained well during
all experimental periods. All experiments were done in accordance with
the guidelines of the Helsinki Declaration and the ethical committee for
clinical experiments of Juntendo University School of Medicine.
NK-cell activity. NK-cell activity against K562 target cells at an E/T
ratio 20 was measured by the 51Cr-release assay or the Eu-release assay
as described previously (11,12).
Flow cytometry. The frequency of T cells and CD32, CD161, and
CD561 NK cells among the peripheral blood mononuclear cells was
analyzed by 3-color flow cytometry as described (11). The cells were
incubated with a saturating amount of FITC-conjugated anti-human
791S

Downloaded from jn.nutrition.org by guest on October 17, 2016

elevated NK cell activity remained for the next 3 wk, and this effect was particularly prominent in the low-NK-activity

CD16 mAb, phycoerythin (PE)-conjugated anti-human CD56 mAb, and

Cy-chrome-conjugated anti-human CD3 mAb or FITC-conjugated antihuman CD4 mAb and PE-conjugated anti-human CD8 mAb. All
staining reagents were obtained from BD Bioscience (San Jose, CA).
The stained cells were then analyzed on FACSCalibur (BD Bioscience),
and the data were processed by the Cell Quest program (BD Bioscience).
ELISA. Serum IFN-a and IFN-g were analyzed by enzyme-linked immunosorbent assay (Bender MedSystms, Vienna, Austria).
Statistical analysis. Statistical analysis was done by a paired t test in all
experiments. The corelation between the magnitude of the increase in
NK-cell activity and NK-cell activity before intake was determined by
Pearsons method, setting statistical significance at P , 0.05.

Results

Figure 1 Change in NK-cell activity during the study period. Each of the
volunteers drank a bottle of Yakult 400 (d) or unfermented (s) milk daily for 3
wk, and NK-cell activity was measured 5 times against K562 target cells at an E/
T ratio of 20 by the Eu release assay. Data are shown as mean 6 SE for 9
individuals in the experimental group and 8 individuals in the control group. **P
, 0.01, *P , 0.05 as compared with the values before intake.

792S

Supplement

Figure 2 The relation between the magnitude of the increase in NK-cell

activity 3 wk after the start of intake and levels of NK-cell activity before intake.
Increase in NK-cell activity 3 wk after intake from that before intake was pointed
with NK-cell activity before intake for each individual in the experimental group.
The correlation was determined by Pearsons method. Statistical significance
was set at P , 0.05.

Experiments with the elderly subjects. In the experimental

group, NK-cell activity was not significantly augmented by the
intake of LcS (Fig. 3). On the other hand, NK-cell activity in the
control group was significantly decreased (P , 0.01) 3 wk after
intake. The frequency and the cell numbers of CD32 CD161
CD561 NK cells, CD41 cells, or CD81 cells were not significantly changed in either group. These findings suggest that the
continuous intake of the fermented milk containing LcS inhibited the decrease of NK-cell activity in elder individuals by some
mechanism, perhaps maintenance of the cytotoxic activity per
NK cell rather than an actual increase in the number of NK cells.

Discussion
In this study, we demonstrated that habitual intake of lactic acid
bacteria containing LcS positively influenced NK-cell activity,
which resulted in the augmentation of NK-cell activity in middleaged sunjects and inhibition of the decrease of NK-cell activity in
elderly individuals. Despite the augmentation of NK-cell activity, the number of NK cells was not significantly changed. Thus,

Figure 3 Changes in NK-cell activity against K562 target cells at an E/T ratio of
20 of elderly subjects 3 wk after the start of intake. Subject drank either Yakult
400 or control unfermented milk; the changes in NK-cell activity in each elderly
subject (s) and mean 6 SE of all individuals (d) are shown.

Downloaded from jn.nutrition.org by guest on October 17, 2016

Experiments with the middle-aged subjects. In the experimental group, NK-cell activity was significantly increased 1 wk
(P 0.0598) and 3 wk (P 0.0050) after the start of intake of
the fermented milk drink, compared with the NK-cell activity
before intake (Fig. 1). The NK-cell activity remained elevated for
the next 3 wk (P 0.0221). Two months after the period of
intake, NK-cell activity was found to have returned to almost the
same level as that before intake. On the other hand, the level of
NK-cell activity in the control group did not change significantly
during the entire experimental period. Moreover, the magnitude
of the increase in NK-cell activity induced by intake of the
fermented milk drink was inversely correlated with the levels of
NK-cell activity before intake (P 0.0163) (Fig. 2). These
findings suggest that the continuous intake of the fermented milk
containing LcS is effective to augment NK-cell activity, particularly prominent in the individuals who have low levels of NKcell activity.
The frequencies and the cell numbers of CD32 CD161
CD561 NK cells, CD41 cells, and CD81 cells were not significantly changed in either group (data not shown), the concentration of serum IFN-a did not change, and IFN-g could not be
detected in serum at all (data not shown).

it seems that the augmentation of NK-cell activity by intake of

LcS would be mediated by an increase in cytotoxic activity per
NK cell rather than an increase in the number of NK cells. Although we could not detect an increasing serum IFN-a or IFN-g
caused by the intake of LcS, augmentation of NK-cell activity
may have been mediated by IL-12 and IFN-g because it has been
reported that LcS activates macrophages to induce IL-12 and
that this cytokine in turn activates T cells to secrete IFN-g
(13,14).
It is known that certain lifestyle factors, including smoking
and mental stress, exert a negative influence on NK-cell activity,
but balanced nutrition, physical exercise, and other positive
lifestyle choices are associated with high levels of NK-cell
activity (15,16). It has been reported that NK cells play a critical
role in immune surveillance against tumor development and
viral infections (17,18) and that intestinal microflora can modulate the NK-cell activity (1921). Therefore, our results strongly
suggest that drinking fermented milk containing LcS can
augment NK-cell activity, which results in the maintenance of
a healthy life and prevention of diseases.

Literature Cited
1.
2.
3.

4.

5.
6.

Gill HS. Stimulation of the immune system by lactic cultures. Int Dairy
J. 1998;8:53544.
Salminen S, Ouwehand AC, Isolauri E. Clinical applications of probiotic bacteria. Int Dairy J. 1998;8:56372.
Kato I, Endo K, Yokokura T. Effects of oral administration of
Lactobacillus casei on antitumor responses induced by tumor resection
in mice. Int J Immunopharmacol. 1994;16:2936.
Takagi A, Matsuzaki T, Sato M, Nomoto K, Morotomi M, Yokokura T.
Inhibitory effect of oral administrartion of Lactobacillus casei on 3methylcholanthrene-induced carcinogenesis in mice. Med Microbiol
Immunol (Berl). 1999;188:1116.
Matsuzaki T. Immunomodulation by treatment with Lactobacillus casei
strain Shirota. Int J Food Microbiol. 1998;41:13340.
Aso Y, Akaza H, the BLP Study Group. Prophylactic effect of a
Lactobacillus casei preparation on the recurrence of superficial bladder
cancer. Urol Int. 1992;49:1259.

8.

9.

10.

11.

12.

13.

14.

15.
16.

17.
18.
19.

20.

21.

Aso Y, Akaza H, Kotake T, Tsukamoto T, Imai K, Naito S, the BLP

Study Group. Preventive effect of a Lactobacillus casei preparation on
the recurrence of superficial bladder cancer in a double-blind trial. Eur
Urol. 1995;27:1049.
Ohashi Y, Nakai S, Tsukamoto T, Masumori N, Akaza H, Miyanaga N,
Kitamura T, Kawabe K, Kotake T, et al. Habitual intake of lactic acid
bacteria and risk reduction of bladder cancer. Urol Int. 2002;68:
27380.
Spanhaak S, Havenaar R, Schaafsma G. The effect of consumption of
milk fermented by Lactobacillus casei strain Shirota on the intestinal
microflora and immune paraments in humans. Eur J Clin Nutr. 1998;
52:899907.
Nagao F, Yabe T, Xu M, Okumura K. Phenotypical and functional
analysis of natural killer cells form low NK activity individuals among
healthy and patient populations. Nat Immun. 1995;14:22533.
Takeda K, Hayakawa Y, Kaer LV, Matsuda H, Yagita H, Okumura K.
Critical contribution of liver natural killer T cells to a murine model of
hepatitis. Proc Natl Acad Sci USA. 2000;97:5498503.
Nagao F, Yabe T, Xu M, Yokyama K, Saito K, Okumura K. Application
of non-radioactive europium (Eu1) release assay to a measurement of
human natural killer activity of healthy and patient populations.
Immunol Investig. 1996;25:50718.
Shida K, Makino K, Morishita A, Takamizawa K, Hachimura S,
Ametani A, Sato T, Kumagai Y, Kaminogawa S. Lactobacillus casei
inhibits antigen-induced IgE serection through regulation of cytokine
production in murine splenocyte cultures. Int Arch Allergy Immunol.
1998;115:27887.
Kato I, Tanaka K, Yokokura T. Lactic acid bacterium potently induces
the production of interleukin-12 and interferon-g by mouse splenocytes.
Int J Immunopharmacol. 1999;21:12131.
Hoffman-Goetz L, Pedersen BK. Exercise and the immune system: a
model of the stress response. Immunol Today. 1994;15:3827.
Nakachi K, Imai K. Enviromental and physiological influences on
human natural killer cell activity in relation to good health practices.
Jpn J Cancer Res. 1992;83:798805.
Herberman RB, Oltaldo JR. Natural killer cells: Their roles in defences
against diseases. Science. 1981;214:2430.
Talmadge JE, Meyers KM, Prieur DJ, Starkey JR. Role of NK cells in
tumor growth and metastasis in beige mice. Nature. 1980;284:6224.
Bartizal KF, Salkowski C, Batish E. The influence of a gastrointestinal
microflora on natural killer cell activity. J Reticuloendothel Soc. 1983;
33:38190.
Bartizal KF, Salkowski C, Pleasants JR, Balish E. The effect of microbial
flora, diet, and age on the tumoricidal activity of natural killer cells. J
Leukoc Biol. 1984;36:73950.
Cebra JJ, Periwal SB, Lee G, Lee F, Shroff KE. Development and
maintenance of the gut-associated lymphoid tissue (GALT): the role of
enteric bacteria and viruses. Dev Immunol. 1998;6:138.

L. casei and NK cells

793S

Downloaded from jn.nutrition.org by guest on October 17, 2016

Acknowledgment
The authors thank Yakult Co. for the supply of the L. casei
strain Shirota fermented milk product, Yakult 400, and the
placebo used for this study.

7.