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© 2007 The American Society for Nutrition J. Nutr. 137:791S-793S, March 2007

Supplement: 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

* To whom correspondence should be addressed. E-mail: kokumura{at}med.Juntendo.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Nine healthy middle-aged and 10 elderly volunteers drank fermented milk containing 4 x 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, elevated NK cell activity remained for the next 3 wk, and this effect was particularly prominent in the low-NK-activity 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.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
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 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
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Subjects. Nine healthy middle-aged (30–45 y) and 10 elderly (55–75 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 x 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 CD3–, CD16+, and CD56+ 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 CD16 mAb, phycoerythin (PE)-conjugated anti-human CD56 mAb, and Cy-chrome-conjugated anti-human CD3 mAb or FITC-conjugated anti-human 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-{alpha} and IFN-{gamma} 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 Pearson's method, setting statistical significance at P < 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    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 NK-cell activity.


Figure 1
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  		Figure 1  Change in NK-cell activity during the study period. Each of the volunteers drank a bottle of Yakult 400 (•) or unfermented ({circ}) 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 ± 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.

 

Figure 2
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  		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 Pearson's method. Statistical significance was set at P < 0.05.

 
The frequencies and the cell numbers of CD3– CD16+ CD56+ NK cells, CD4+ cells, and CD8+ cells were not significantly changed in either group (data not shown), the concentration of serum IFN-{alpha} did not change, and IFN-{gamma} could not be detected in serum at all (data not shown).

    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 CD3– CD16+ CD56+ NK cells, CD4+ cells, or CD8+ 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.


Figure 3
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  		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 ({circ}) and mean ± SE of all individuals (•) are shown.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
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 middle-aged 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, 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-{alpha} or IFN-{gamma} caused by the intake of LcS, augmentation of NK-cell activity may have been mediated by IL-12 and IFN-{gamma} 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-{gamma} (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 (19–21). 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.


   ACKNOWLEDGMENTS
 
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.


   FOOTNOTES
 
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 Maintenance—Critical 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, Müller Milch, Morinaga, Nestec, Nutricia, Orafti, Valio, and Yakult. Back

2 Author disclosure: no relationships to disclose. Back

3 IFN, interferon; LcS, Lactobacillus casei strain Shirota; NK, natural killer. Back


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

1. Gill HS. Stimulation of the immune system by lactic cultures. Int Dairy J. 1998;8:535–44.

2. Salminen S, Ouwehand AC, Isolauri E. Clinical applications of probiotic bacteria. Int Dairy J. 1998;8:563–72.

3. 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;16:29–36.[Medline]

4. Takagi A, Matsuzaki T, Sato M, Nomoto K, Morotomi M, Yokokura T. Inhibitory effect of oral administrartion of Lactobacillus casei on 3-methylcholanthrene-induced carcinogenesis in mice. Med Microbiol Immunol (Berl). 1999;188:111–6.[Medline]

5. Matsuzaki T. Immunomodulation by treatment with Lactobacillus casei strain Shirota. Int J Food Microbiol. 1998;41:133–40.[Medline]

6. 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;49:125–9.[Medline]

7. 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;27:104–9.[Medline]

8. 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;68:273–80.[Medline]

9. 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;52:899–907.[Medline]

10. 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;14:225–33.[Medline]

11. 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;97:5498–503.[Abstract/Free Full Text]

12. Nagao F, Yabe T, Xu M, Yokyama K, Saito K, Okumura K. Application of non-radioactive europium (Eu+) release assay to a measurement of human natural killer activity of healthy and patient populations. Immunol Investig. 1996;25:507–18.[Medline]

13. 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;115:278–87.[Medline]

14. Kato I, Tanaka K, Yokokura T. Lactic acid bacterium potently induces the production of interleukin-12 and interferon-{gamma} by mouse splenocytes. Int J Immunopharmacol. 1999;21:121–31.[Medline]

15. Hoffman-Goetz L, Pedersen BK. Exercise and the immune system: a model of the stress response. Immunol Today. 1994;15:382–7.[Medline]

16. 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;83:798–805.[Medline]

17. Herberman RB, Oltaldo JR. Natural killer cells: Their roles in defences against diseases. Science. 1981;214:24–30.[Abstract/Free Full Text]

18. Talmadge JE, Meyers KM, Prieur DJ, Starkey JR. Role of NK cells in tumor growth and metastasis in beige mice. Nature. 1980;284:622–4.[Medline]

19. Bartizal KF, Salkowski C, Batish E. The influence of a gastrointestinal microflora on natural killer cell activity. J Reticuloendothel Soc. 1983;33:381–90.[Medline]

20. 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;36:739–50.[Abstract]

21. 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;6:13–8.[Medline]





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