FMRPs were prepared with whey protein (Davisco Foods International Inc., Le Sueur, MN, USA), sugars (galactose or glucose), and lactic acid bacteria (Lactobacillus rhamnosus 4B15 or Lactobacillus gasseri 4M13) (Table 1). For Maillard reaction, 50 mg/g of whey protein isolate and 25 mg/g of sugar (galactose or glucose) were mixed in deionized water, followed by shaking at 60 rpm in a pilot-scale instrument controlled by a pilot-scale pasteurization unit (Powerpoint International, Tokyo, Japan) at 65°C for 24 h. These Maillard reacted galactose and glucose were then fermented by L. rhamnosus 4B15 or L. gasseri 4M13 at 6 Log colony-forming unit (CFU)/mL at 37°C for 48 h to prepare FMRPs (Gal-4B15, Gal-4M13, Glu-4B15, and Glu-4M13). The cultures were then centrifuged at 2,000×g for 30 min (Hitachi CR-21G rotor R20A, Hitachi Ltd., Tokyo, Japan). Subsequently, the supernatants were transferred into aluminum containers, followed by freeze-drying using a lyophilizer (OPERON FDUT-8608, Gimpo, Gyeonggi, Korea) for 72 h. The freeze-dried FMRPs were powdered and stored at 4°C until further use.

To evaluate antimicrobial effects of FMRPs on C. perfringens, 1 mL of C. perfringens strains NCCP15911, NCCP15912, NCCP10846, NCCP10858, NCCP10976, and NCCP10347 were inoculated in 10 mL cooked meat medium (Oxoid, Basingstoke, Hampshire, UK), and the cultures were placed in airtight containers with Anaerogen (Oxoid, UK), followed by incubation at 37°C for 24 h. Then, 0.1 mL aliquots of the cultures were inoculated into 9 mL fresh brain-heart infusion (BHI; BD Difco, Sparks, MD, USA), followed by anaerobic incubation at 37°C for 24 h. The subcultures were mixed, and then centrifuged at 1,912×g and 4°C for 15 min. The cell pellets were washed twice with phosphate buffered saline (PBS, pH 7.4; 0.2 g of KH₂PO₄, 1.5 g of Na₂HPO₄, 8.0 g of NaCl, and 0.2 g of KCl in 1 L of distilled water). The cell suspensions of C. perfringens were then diluted to OD₆₀₀ = 0.01 with PBS to obtain 5 to 6 Log CFU/mL of inoculum.

To evaluate the antimicrobial effects of FMRPs on C. perfringens, a hundred microliters of C. perfringens inoculum and 900 μL of FMRPs (100 mg/mL; Gal-4B15, Gal-4M13, Glc-4B15, and Glc-4M13) were mixed in a well of a 24-well plate (SPL Life Sciences, Gyeonggi, Korea), and the plate was placed in an airtight container, containing Anaerogen (Oxoid, UK), followed by anaerobic incubation at 37°C for 24 h. The samples were then serially diluted with buffered peptone water (BPW; BD Difco, USA), and 100 μL of the diluents were spread-plated on tryptose sulfite cycloserine agar (TSC; Oxoid, UK) to enumerate C. perfringens cells.

To evaluate the iron chelation activity by FMRPs, 100 μL of FMRPs (20 mg/mL), 600 μL of distilled water, and 100 μL of 0.2 mM iron (II) chloride tetrahydrate (FeCl₂·4H₂O, SAMCHUN chemical Co. Ltd., Seoul, Korea) were mixed, and the mixture reacted for 30 sec at room temperature. Subsequently, 200 μL of 0.1 mM Ferrozine (3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p’-disulfonic acid monosodium salt hydrate; Sigma Aldrich, St. Louis, MO, USA) were suspended in the mixture, and the samples reacted for 10 min at room temperature. Thereafter, turbidity of the samples was measured at 562 nm with a UV-spectrophotometer (OPTIZEN 2120UV, Mecasys Co., Ltd., Daejeon, Korea), and iron chelation activity was calculated as follows;

Absorbance of blank solution was measured with distilled water instead of with samples [15].

To observe the effects of FMRPs on bacterial cell permeability in short time, propidium iodide (PI) uptake assay for FMRPs-exposed C. perfringens was conducted according to the method described by Kim et al [16]. C. perfringens were cultured in cooked meat medium at 37°C for 24 h. The bacterial cultures were centrifuged at 10,000×g for 1 min at room temperature, and supernatants were discarded. One-milliliter portion of each FMRP sample (200 mg/mL) was transferred to the cell pellets and mixed, followed by incubation at 37°C for 60 min. Subsequently, 50 μL of C. perfringens incubated with FMRP were diluted with 950 μL of PBS in a FACS tube (BD Bioscience, San Jose, CA, USA), and 10 μL of PI solution (1 mg/mL) (Sigma Aldrich, USA) was then added to the diluted cultures. The mixtures were analyzed with FACSCanto II (BD Bioscience, USA), and PI activities for C. perfringens exposed to FMRPs were calculated, using CellQuest Pro software (BD Bioscience, USA). The percentages of FMRPs-treated bacteria figured in each histogram are relatively calculated to PBS-treated bacteria at a same designated point.

To elucidate the antimicrobial activity of FMRPs against C. perfringens, morphological observations were conducted, using field emission scanning electron microscope (FE-SEM; JEOL JSM-7600F, JEOL USA Inc., Peabody, MA, USA), according to the method described by Kim et al [16]. C. perfringens were cultured in 500 μL BHI containing the glass slides anaerobically at 37°C for 24 h with Anaerogen (Oxoid, UK). After incubation, the glass slides were removed from the cultures, and aseptically transferred to sterile 24-well flat-bottomed polystyrene plates (SPL, Korea). Thirty milliliters of 1.8% glutaraldehyde (Sigma-Aldrich Co., St. Louis, MO, USA) were carefully placed over the glass slides, and left for 30 min at room temperature for the reaction, followed by washing glass slides three times with 1 mL distilled water for 5 min. Subsequently, the glass slides were treated with 2% osmium tetroxide solution (Sigma Aldrich, USA) for 20 min at room temperature for a second fixation, followed by washing them three times with 1 mL distilled water for 5 min. The fixed bacterial cells on the glass slides were gradually dehydrated with 1 mL of 25%, 50%, 75%, 90%, and 100% ethyl alcohol for 5 min. Afterwards, the glass slides were thoroughly dried with 15 μL hexamethyldisilazane (Sigma-Aldrich, USA). The samples were eventually coated with a sputter coater (Cressington 108auto SEM sputter coater, Cressington Scientific Instruments Ltd., Watford, UK), and the bacterial cells were observed, using field emission scanning electron microscopy.

The bacterial cell counts (Log CFU/mL) and iron chelation activity were analyzed by SAS (version 9.2; SAS Institute Inc., Cary, NC, USA), using general linear model procedure. Significant differences of LS-means were analyzed with a pairwise t-test at α = 0.05.

C. perfringens cell counts in the FMRPs significantly decreased (p<0.05) at 37°C for 12 and 24 h (Figure 1), regardless of FMRPs used. Especially Gal-4B15 reduced C. perfringens cell counts below detection limit (10 CFU/mL) even after 12 h of incubation (Figure 1). This result indicates that FMRPs had significant antimicrobial effects against C. perfringens. The functional effects of FMRPs, such as antihypertension and prevention of cardiovascular disease, were found in other studies [10,11]. Taken together with the antimicrobial functions of FMRPs against C. perfringens, FMRPs may reduce intestinal C. perfringens and may be potential materials as health-promoting supplements. Although some fermented dairy products which show antimicrobial activity against C. perfringens, such as Kefir and probiotic yogurt, already exist, FMRPs are distinctive due to its antihypertensive and cardiovascular disease-preventive effects [17].

Theoretically, high iron chelation activity indicates that iron as an essential nutrient for microorganisms in the environment is unavailable to bacteria, which in turn suppresses bacterial growth and decreases bacterial cell counts [18,19]. Gal-4M13 (23.87%) significantly showed high iron chelation activity compared to Glc-4M13 (0.50%) (p<0.05) (Table 2). This suggests that Gal-4M13 may have better iron chelation activities and thus, they may play an important role in inhibiting the growth by limiting uptake of iron in bacterial growth environments. However, only iron chelation activity is not affecting the antimicrobial effects of Gal-4M13 in this study, because Glc-4B15 and Glc-4M13 showed similar antimicrobial effects on C. perfringens, although they showed difference in iron chelation activity.

In PI uptake assay, movement of peaks in a histogram, which represents the number of events detected at particular intensity, to the right indicates that the cell membrane is damaged. In this study, the movement of the peaks to right, and the change in the shape of the peaks and the number of the events were observed after the FMRPs treatment. Movement of fractions towards right X-axis indicates damages of bacterial cells via detection of wavelength of PI which binds to DNA leaked from damaged bacterial cells [20]. The events of treated C. perfringens increased from 24.0% to 40.2% by Gal-4B15 and from 18.9% to 20.3% by Gal-4M13. Glc-4B15 and Glc-4M13 treatments also increased the portions of C. perfringens from 37.5% to 51.2% and from 13.1% to 19.9%, respectively (Figure 2). This result indicates that FMRPs caused cell membrane damage, and it might be one of the modes to destroy C. perfringens cells. To date, physicochemical properties of dairy antimicrobial peptides, such as net charge, amphipathicity, hydrophobicity, etc., have shown effects on interaction with cell membranes and integration of the membranes with the peptides [21]. Therefore, it is assumed that peptides in FMRPs specifically interacted with the bacterial cell membranes and occurred the cell leakage.

The PI uptake assay showed that FMRP treatments caused cell membrane damages, which were confirmed visually by FE-SEM. FMRP-treated C. perfringens had an uneven cell membrane, and pores on the cell membrane surface (Figure 3). In addition, the lengths of the FMRP-treated bacteria increased from 2.0 μm to 12.0 μm in average and 19.1 μm in maximum for C. perfringens. Normally each bacteria species consistently maintains their classical bacterial shapes with their complex mechanisms genetically and biochemically [22]. Bacterial cells were found to elongate in order to adjust stressful environments [23]. Everis and Betts [23] also suggested that the length of Clostridium spp. increases when they are exposed to stress. When the bacterial cell is exposed to high solute and nutrition concentration, the osmotic forces change the mechanical integrity of bacterial cell and determine the shape [24]. Thus, the elongation of the bacterial cells in this study would be a result of the stressful environment by FMRPs. Taken together with the result of iron chelation activity and PI uptake assay, FMRPs would have antimicrobial activity as they occurred cell membrane damages and iron chelating properties simultaneously.