Elsevier

Food Chemistry

Volume 131, Issue 4, 15 April 2012, Pages 1347-1354
Food Chemistry

The production of surfactin during the fermentation of cheonggukjang by potential probiotic Bacillus subtilis CSY191 and the resultant growth suppression of MCF-7 human breast cancer cells

https://doi.org/10.1016/j.foodchem.2011.09.133Get rights and content

Abstract

Bacillus subtilis CSY191, the potential probiotics and surfactin-like compound producer, was isolated from doenjang (Korean traditional fermented soybean paste).The survival rate of this strain appeared to be the 58.3% under artificial gastric conditions after 3 h at pH 3.0. Surfactin was purified from the strain CSY191. Three potential surfactin isoforms were detected, with protonated masses of m/z 1030.7, 1044.7, and 1058.71. These different structures were detected in combination with Na+, K+ and Ca2+ ions by MALDI-TOF mass spectrometry. Upon 500 MHz 1H NMR analysis, the surfactin isoforms had identical amino acids (GLLVDLL) and hydroxy fatty acids (of 13–15 carbons in length). The MTT assay showed that surfactin inhibited growth of MCF-7 human breast cancer cells in a dose-dependent manner, with an IC50 of approximately 10 μg/ml at 24 h. Additionally, the surfactin contents, during cheonggukjang fermentation with strain CSY191, increased from 0.3 to 48.2 mg/kg over 48 h of fermentation, while the level of anticancer activity increased from 2.6- to 5.1-fold.

Highlights

Bacillus subtilis CSY191 was isolated from Korean soybean paste (doenjang). ► Surfactin identified three isoforms, seven amino acids and hydroxy fatty acids. ► Surfactin inhibited growth of MCF-7 cells (IC50: 10 μg/ml). ► Surfactin contents increased from 0.3 to 48.2 mg/kg during fermentation. ► During fermentation, the anticancer activity increased from 20.29% to 54.66%.

Introduction

Although not a new concept, probiosis has only recently begun to receive an increasing level of scientific interest. Probiotics are generally defined as live microbial feed supplements that can benefit the host by improving its intestinal balance (Fuller, 1991). Probiotics fall under two broad classifications: those for animal use and those for human use. Probiotics used in animal feed are considered alternatives to antibiotics (Hong, Duc, & Cutting, 2005). Probiotics for human use, on the other hand, are subjected to restrictions and come in many different forms. They are often sold in supermarkets as dairy-type products containing ‘live bacteria’ and in health food shops as capsules or tablets composed of lyophilised preparations of bacteria to promote ‘a healthy gut’. Many Bacillus products are used as ‘novel foods’ or as dietary supplements with various claims of ‘enhancing’ the well-being of the user by restoring the natural microflora of the gut (Fuller, 1991, Hong et al., 2005).

Surfactin, a biosurfactant produced by Bacillus subtilis, is a cyclic lipopeptide, built from a heptapeptide and a β-hydroxy fatty acid, with a variable chain of 13–15 carbon atoms. This biosurfactant is biodegradable, less toxic than chemical surfactants, and has many potential applications in various fields, including biomedicine (Singh & Cameotra, 2004). Surfactins have received much attention during the past two decades because they exhibit numerous pharmaceutical activities, including antimicrobial (Cho et al., 2003), anti-tumour (Cao et al., 2009, Cao et al., 2010), antiviral (Vollenbroich, Ozel, Vater, Kamp, & Pauli, 1997), anti-inflammatory (Kim et al., 2006), and immunosuppressive activities (Park & Kim, 2009). Surfactins are well known for multifaceted interactions with biological systems, resulting in a number of physiological and biochemical activities. Surfactins can be incorporated into the phospholipid bilayer and induce target cell permeabilisation and perturbation due to their amphipathic nature (Tang et al., 2010).

Fermented soybean foods, such as soybean paste (doenjang), soybean sauce (kanjang), and soybean cook (cheonggukjang) are examples of foods served as side dishes in Korea. These foods have been a major source of protein in the Korean diet for thousands of years. Here we focus on cheonggukjang, which is made from cooked whole soybeans fermented for two to three days, with microorganisms including Bacillus spp. (Cho et al., 2009). There have been several reports describing the antioxidant, antimicrobial, antigenotoxic, blood pressure-lowering and antidiabetic activities of cheonggukjang (Kim et al., 2008, Jung et al., 2006). Recently, we reported the isoflavone, flavanol and phenolic acid profiles observed during cheonggukjang fermentation (Cho et al., 2009). However, other functional components that are likely present in cheonggukjang remain unknown. The health food industry would benefit from the elucidation of the distinct functional products in cheonggukjang that could be separated and developed into novel foods or dietary supplements.

In this study, the potential probiotic and surfactin-producing B. subtilis strain, CSY191, was screened from Korean traditional fermented soybean paste (doenjang). The structure of surfactin produced from the CSY191 strain was identified by FAB, MALDI-TOF and NMR spectrometry. The surfactin was characterised by an anticancer activity test against human breast cancer cells and was then produced during the cheonggukjang fermentation by B. subtilis strain CSY191. To the best of our knowledge, this is the first report to provide the one guideline about the manufacture of functional soybean-containing foods using soybean and a main microbial fermenter, such as B. subtilis with functional abilities, potential probiotic and surfactin production properties.

Section snippets

Screening of potential probiotic Bacillus strains

For the screening of potential probiotic Bacillus strains, Korean traditional soybean paste (doenjang) samples were collected from a local market in Jinju, Korea. The collected samples were serially diluted with 0.85% saline solution (Daejung Chemical & Metals Co., Siheung, Korea) and then 0.1 ml of the diluted suspension was plated and incubated on TSB (Difco, Detroit, MI, USA) agar plates at 37 °C for 48 h. A distinctive Bacillus single colony was selected by a pure culture on each TSB agar

Identification of probiotic B. subtilis CSY191

Strain CSY191 was isolated from soybean paste (doenjang) and showed potential as a probiotics by tolerances to acid, artificial gastric acid, and bile acid as well as being a surfactin-like compound producer by screening on TSA with 5% defibrinated sheep blood. The survival rates of several Bacillus strains under acidic and artificial gastric conditions are shown in Fig. 1. In general, the survival rates of B. subtilis CSY191 decreased with time under acidic conditions. The B. subtilis CSY191

Discussion

In this paper, we have described the purification of surfactin from the potential probiotic B. subtilis CSY191, a surfactin-producing strain isolated from soybean paste (doenjang). The surfactins were fractionated by high resolution RP-HPLC and characterised by MALDI-TOF MS and 1H NMR spectra. In addition, surfactin concentration was determined during the fermentation of cheonggukjang by B. subtilis CSY191, and the antiproliferative activity of surfactin extract of cheonggukjang against the

Conclusion

We have reported the production of surfactin from a potential probiotic, B. subtilis CSY191. The purified surfactin of B. subtilis CSY191 exhibited anticancer activity against human breast cancer MCF-7 cells. In addition, the anticancer activity increased from 20.3% to 54.7% with SECs (100 μg/ml), depending on the surfactin contents (0.3–48.2 mg/kg) during fermentation. Further studies are necessary to define the in vivo anticancer effects of surfactin and to clarify its precise molecular

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant number 2010-0022508) and a grant (No. 08-C2-15-002) from the Academic-industrial Cooperative Research Program of the Small & Medium Business Administration (SMBA), Republic of Korea.

References (31)

  • P. Singh et al.

    Potential applications of microbial surfactants in biomedical sciences

    Trends Biotechnology

    (2004)
  • C.L. Su et al.

    Supernatant of bacterial fermented soybean induces apoptosis of human hepatocellular carcinoma Hep 3B cells via activiation of caspase 8 and mitochondria

    Food and Chemical Toxicology

    (2007)
  • D. Vollenbroich et al.

    Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis

    Biologicals

    (1997)
  • C.L. Wang et al.

    Induction of apoptosis in human leukemia K562 cells by cyclic lipopeptide from Bacillus subtilis natto T-2

    Peptides

    (2007)
  • X. Cao et al.

    Surfactin induces apoptosis and G2/M arrest in human breast cancer MCF-7 cells through cell cycle factor regulation

    Cell Biochemistry and Biophysics

    (2009)
  • Cited by (97)

    • Role of biosurfactants in improving target efficiency of drugs and designing novel drug delivery systems

      2023, Industrial Applications of Biosurfactants and Microorganisms: Green Technology Avenues from Lab to Commercialization
    View all citing articles on Scopus
    View full text