Anticancer effect of the five surfactin isomers produced during cheonggukjang fermentation by Bacillus pumilus HY1 isolated from Korean traditional fermented soy sauce

1 Food Science Research Institute, SBT Business Division, Kolmar BNH Inc., Sejong 30003, Republic of Korea 2 Industry Academy Cooperation Foundation, Andong National University, Andong, 36729, Republic of Korea 3 Department of Life Resources Industry, Dong-A University, Busan 49315, Republic of Korea 4 Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 52725, Republic of Korea

In a previous study, we reported the production of surfactin from a potential probiotic, Bacillus subtilis CSY191, and the purified surfactin exhibited anticancer activity against human breast cancer MCF-7 cells [4]. The identification and separation of the various beneficial components in chenoggukjang enables a demonstration of their health benefits and promotes the development of distinct functional products for the food industry.
In the present study, surfactin was purified from strain B. pumilus HY1, a bacterial strain commonly detected in traditional Korean fermented soy sauce (kanjang). The surfactin was fractionated through TLC and RP-HPLC and characterized using mass spectrometry.

Isolation and purification of surfactin
Surfactin was isolated from strain HY1 according to Lee et al. [4].

Mass spectrometric analysis of surfactin isoforms
The purified CLP was analyzed using matrix-assisted laser desorption ionization time-

Amino acid and mineral analysis
The amino acid and mineral composition analysis of the purified CLP wines were determined according to methods previously described by Cho et al. [22]. The amino acid of the purified CLP was sequenced using electrospray ionization tandem mass spectrometry (ESI-MS/MS; Finnigan-MAT TSQ 700, San Jose, USA). The sample was dissolved in 50% aqueous methanol containing 1% formic acid prior to injection into the mass spectrometer.
ESI-MS/MS spectra were acquired after inducing collisions between precursor ions and nitrogen collision gas at acceleration voltages of 50 V.

Extraction and analysis of surfactin
The extraction and analysis of surfactin were performed according to Lee et al. [4] methods. Ten grams of ground cheonggukjang was extracted using 30 mL of methanol and adjusted to pH 2.0 with concentrated HCl by shaking (160 rpm

Isolation of surfactin from B. pumilus HY1
The surfactin was produced during the fermentation of high producer B. pumilus size marker, yielding a molecular mass of 1,036. Reverse phase HPLC was used to analyze surfactin, followed by purification at 214 nm ( Fig. 1).

Mass spectrum of surfactin by B. pumilus HY1
The species. These peaks contained essentially pure C12 to C16 surfactin species, respectively. The purified surfactin was analyzed using ICP MS, and potassium and calcium ions were detected (Table 1)

Effect of SEC on the growth of cancer cells
We used the well-characterized MTT assay to assess the metabolic activity of cells, to determine whether surfactin could inhibit the growth of MCF-7 and Caco-2 cells, two cancer cell lines. MCF-7 and Caco-2 cells were incubated with SEC at concentrations ranging from 0 to 120 μg/μL of surfactin, as shown in Fig. 5. The SEC inhibited the growth of cells in a concentration-dependent manner. Morphological changes provide the most direct criteria for recognizing the apoptotic process. As shown in Fig. 6, apoptotic cells were observed at 24 h after exposure to SEC (100 μg/μL of surfactin).

Discussion
In the present study, we investigated the purification of surfactin from B. pumilus HY1, the CLP (such as iturin and surfactin) producing strain isolated from kanjang [21]. The In previous, the biological activities of surfactin occur via interactions with cellular membranes and it may have detergent-like effect on cell membranes [4,10]. The many researches have penetrated that hydrophobic residues of the peptide moiety permeate into the membrane interface, leading to membrane permeabilization but not surface effect [4,24]. At higher concentrations, this detergent-like activity could stabilize leaks due to the compound's ability to generate multiple-structured polymers [24]. Although it is not yet clear whether a sub-optimal concentration (IC50 100 μg/μL) of surfactin affects the cell morphology (Fig. 6), surfactin-treated cells appeared to be leaky and/or lysed. Meta et al. [25]. proposed that surfactin may disturb a biochemical reaction(s) that occurs at a specific membrane site perhaps via the long chain fatty acid, acting as a pseudosubstrate (similar to PIP3 or a farnesyl group) for either the PI3K/Akt or farnesyltransferase/Ras/ERK pathways, which promote apoptosis. In a previous study, Cao et al. [26] suggested that surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway and the surfactin has notable anti-tumor effects on MCF-7 cells; however, there was no obvious cytotoxicity on normal cells.
Carbohydrate and lipoprotein extracts from edible mushrooms and several bacterial cells were reported for their growth inhibition towards malignant cancer cells [27]. Wakamatsu et al. [27] discovered that lipoproteins induce neuronal differentiation in PC12 cells and provided the groundwork for the use of microbial extracellular CLP as a novel reagent for the treatment of cancer cells. Wang et al. [30] reported that a new CLP purified The content of surfactin increased from 0.3 mg/kg on day 0 to a final content of 51.2 mg/kg, corresponding to the cell concentration increases of 3.0-11.7 log CFU/g during cheonggukjang fermentation (Fig. 4). Several authors have studied the production of surfactin from solid-state fermentation [4,10,32,33]. This process is associated with the production of a higher concentration of surfactin in solid-state fermentation, which occurs in the manufacturing of soybean-fermented food [10]. In particular, Slvinski et al. [34] studied the production of surfactin through B. pumilus UFPEDA 448 in solid-state fermentation, using a medium based on okara with the addition of sugarcane bagasse as a bulking agent.
This result shows that the concentration of surfactin is proportional to the concentration of cells because surfactin is produced from Bacillus sp.
The cheonggukjang is reported to have anticancer, blood pressure reduction, hypocholesterolemic, and fibrinolytic properties [4,19]. However, although these effects of cheonggukjang have been extensively studied, little is known about its potential antitumoral activities. In addition, we previously reported that the surfactin content, during cheonggukjang fermentation with B. subtilis CSY191, increased from 0.3 to 48.2 mg/kg over 60 h of fermentation, while the level of anticancer activity increased from 2.6-to 5.1-fold [4].
These findings will improve the quality of Korean traditional soybean fermented foods as one of the best functional foods because of their wide variety of antitumor, antimicrobial, antifungal, and antiviral activities.
In conclusions, we identified five potential isoforms of surfactin from B. pumilus HY1.
Surfactin is a