Elsevier

Journal of Functional Foods

Volume 14, April 2015, Pages 513-518
Journal of Functional Foods

The prophylactic effect of probiotic Bacillus polyfermenticus KU3 against cancer cells

https://doi.org/10.1016/j.jff.2015.02.019 Get rights and content

Highlights

  • B. polyfermenticus KU3 strongly inhibited the proliferation of cancer cells.

  • B. polyfermenticus KU3 attenuate inflammatory activity under stimulatory conditions.

  • The safety was demonstrated using normal cells and enzyme production.

  • These results demonstrate the probiotic characteristics of B. polyfermenticus KU3.

Abstract

Bacillus polyfermenticus KU3 was isolated from kimchi, a Korean dish made from fermented vegetables and its potential probiotic characteristics were investigated. The spore cell of B. polyfermenticus KU3 was highly resistant to artificial gastric juice and survived for 24 h in artificial bile acid. B. polyfermenticus KU3 did not generate the carcinogenic enzymes, β-glucosidase, N-acetyl-β-glucosaminidase, and β-glucuronidase, and adhered strongly to HT-29 human intestinal epithelial cell lines. Using the [3–4,5-dimethylthiazol-2-yl]-2,5-diphenyletrazolium bromide assay, we found that B. polyfermenticus KU3 strongly inhibited the proliferation of cancer cells such as HeLa, LoVo, HT-29, AGS, and MCF-7 cells. The supernatant of B. polyfermenticus KU3 had an anticancer effect against HeLa and LoVo cells. Conversely, the proliferation of normal MRC-5 cells was not inhibited. We also demonstrated the anti-inflammatory activity of B. polyfermenticus KU3 under inflammatory conditions, as shown by the reduction in nitric oxide and proinflammatory cytokines (TNF-α, IL-10, TGF-β2, and COX-2). These results demonstrate the probiotic characteristics of B. polyfermenticus KU3 and provide evidence for the effect of this bacterium against various cancer cells.

Introduction

The market value of probiotic products in 2012 was valued at $26,126 million and is estimated to grow at an annual growth rate of 6.2%. It has been forecasted that the probiotics market will be worth as much as $1733 million by 2019 (Markets & Markets, 2014). Probiotics are defined as living or dead microorganisms that exert a beneficial effect on the health of the host organism, when ingested in sufficient amounts (O'Sullivan, 2001). To date, studies suggest that probiotics may have a clinical prophylactic application in various therapies such as anticancer (Chen & Khismatullin, 2014), antioxidant, anti-allergy (Lee, Kim, Han, Eom, & Paik, 2014), cholesterol-lowering (Guo, Zhang, Yuan, Yue, & Li, 2015) and antidiabetic treatments (Chen et al, 2014, Giraffa, 2012). The prophylactic effects observed were dependent on the type of probiotic strain studied. It has been suggested that probiotics may exert an anticancer effect by decreasing the influence of chemical carcinogens by the following means: (1) the detoxification of ingested carcinogens; (2) the alteration of the environment of the intestine, which decreases the populations or metabolic activities of bacteria that may generate carcinogenic compounds; (3) the induction of apoptosis via the production of metabolic products such as butyrate; (4) the production of compounds that inhibit the growth of tumor cells; and (5) the stimulation of the immune system (Lee et al, 2011, Parvez et al, 2006, Uccello et al, 2012).

Because of the involvement of inflammation in carcinogenesis, the use of anti-inflammatory drugs have been developed as preventative strategies against many types of cancers (Commane et al, 2005, Westbrook et al, 2010). Cytokine-expressing inflammatory cells produce large amounts of nitric oxide (NO), prostaglandin E2 (PGE2), cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Nitric oxide and PGE2 are important proinflammatory mediators produced by inducible NO synthase and cyclooxygenase-2 (COX-2), respectively (Otte et al., 2009). Nitric oxide can exert a positive (i.e., tumoricidal) and negative (i.e., tumor growth and proliferation) effect on cancer depending on its concentration in the blood (Korde Choudhari, Chaudhary, Bagde, Gadbail, & Joshi, 2013). Lipopolysaccharide (LPS) induces cancer metastasis by (1) forming an attachment between the endothelium and cancer cells, and (2) triggering the release of TNF-α that leads to a strong expression of endothelial cell adhesion molecules (Chen & Khismatullin, 2014).

The Bacillus species has been used in various probiotic products for at least 50 years (Duc, Hong, Barbosa, Henriques, & Cutting, 2004). The species that have been most extensively examined include Bacillus subtilis, Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, and Bacillus polyfermenticus (Hong, Duc, & Cutting, 2005). The spores of Bacillus spp., being heat-stable, have a number of advantages over species that do not produce spores, such as Lactobacillus spp., given that they can be stored at room temperature in a desiccated form without any deleterious effect on viability.

Bacillus polyfermenticus SCD, commercially termed as Bispan strain in Japan and Korea, has been used in the treatment of long-term intestinal disorders (Chang et al, 2007, Im et al, 2009). This bacterial species has a cholesterol-reducing activity, exerts antioxidative and antimutagenic effects, has strong adherent properties in the colon, and demonstrates anticarcinogenic effects in vitro and in vivo. B. polyfermenticus is currently registered and is a “generally recognized as safe” (GRAS) organism by the Korea Food and Drug Administration (KFDA). Furthermore, B. polyfermenticus has been added to animal and aquatic feed as a protective measure against aquatic pathogenesis and fungal contamination (Kim, Kim, Kim, Park, & Kang, 2009). Novel isolation and characterization of B. polyfermenticus has been reported, and the isolation source of B. polyfermenticus has been known as air and Meju (Korean soy paste) (Jung et al, 2012, Yang et al, 2012).

The aim of this study was to investigate the probiotic effect of B. polyfermenticus KU3 isolated from kimchi, a Korean dish made from fermented vegetables. In addition, the prophylactic anticancer and anti-inflammatory effects of Bacillus strains were demonstrated in vitro.

Section snippets

Bacterial strain and culture conditions

B. polyfermenticus KU3 was isolated from kimchi using spread plating method, and this strain was maintained at −70 °C in tryptic soy broth (TSB; Becton Dickinson, San Diego, CA, USA) containing 20% (v/v) glycerol. B. polyfermenticus SCD isolated from Bispan (Binex Co. Ltd., Busan, South Korea) was used as a control strain. Working cultures were propagated in TSB medium, with shaking at 150 rpm for 12 h at 37 °C.

Vegetative cells

Bacillus strains were cultivated in TSB medium with shaking at 150 rpm for 12 h at

Tolerance to artificial gastric juice and artificial bile acid

The survival of vegetative and spore cells of B. polyfermenticus KU3 and B. polyfermenticus SCD in artificial gastric juice (pH 2.5, 0.1% pepsin, for 2 h) and artificial bile (0.3% oxgall, for 24 h) was assessed. The vegetative cells of B. polyfermenticus KU3 (57.3% survival rate) and B. polyfermenticus SCD (64.83% survival rate) were weakly maintained as in artificial gastric juice and bile (Table 1). Conversely, the spore cells of B. polyfermenticus KU3 and B. polyfermenticus SCD were

Conclusion

B. polyfermenticus KU3, isolated from kimchi, did not generate any detectable carcinogenic enzymes, and the spore cell was able to survive under gastric conditions compared to B. polyfermenticus SCD. Our data also demonstrated that B. polyfermenticus KU3 was a broader cytotoxic against various cancer cells than B. polyfermenticus SCD. The anti-inflammatory effect of B. polyfermenticus KU3 was demonstrated by measuring a decrease in NO production and proinflammatory cytokines. From our results

Acknowledgements

This paper was supported by Konkuk University in 2013.

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