Antimicrobial activity of fermented Maillard reaction products, novel milk-derived material, made by whey protein and Lactobacillus rhamnosus and Lactobacillus gasseri on Clostridium perfringens

Objective The objective of this study was to evaluate the antimicrobial effects of fermented Maillard reaction products made by milk proteins (FMRPs) on Clostridium perfringens (C. perfringens), and to elucidate antimicrobial modes of FMRPs on the bacteria, using physiological and morphological analyses. Methods Antimicrobial effects of FMRPs (whey protein plus galactose fermented by Lactobacillus rhamnosus [L. rhamnosus] 4B15 [Gal-4B15] or Lactobacillus gasseri 4M13 [Gal-4M13], and whey protein plus glucose fermented by L. rhamnosus 4B15 [Glc-4B15] or L. gasseri 4M13 [Glc-4M13]) on C. perfringens were tested by examining growth responses of the pathogen. Iron chelation activity analysis, propidium iodide uptake assay, and morphological analysis with field emission scanning electron microscope (FE-SEM) were conducted to elucidate the modes of antimicrobial activities of FMRPs. Results When C. perfringens were exposed to the FMRPs, C. perfringens cell counts were decreased (p<0.05) by the all tested FMRPs; iron chelation activities by FMRPs, except for Glc-4M13. Propidium iodide uptake assay indicate that bacterial cellular damage increased in all FMRPs-treated C. perfringens, and it was observed by FE-SEM. Conclusion These results indicate that the FMRPs can destroy C. perfringens by iron chelation and cell membrane damage. Thus, it could be used in dairy products, and controlling intestinal C. perfringens.


INTRODUCTION
Clostridium perfringens, grampositive and sporeforming anaerobe, is the third common cause of foodborne illness in United States [1]. Also, this bacterium is responsible for the symptoms, such as abdominal cramps and severe diarrhea, caused by ingestion of food which is contaminated in large number of C. perfringens, especially proteinaceous foods, meat, and poultry [2]. Besides, C. perfringens generally inhabits in a human gut, and its prevalence in the gut increases, as humans get old [3]. A significant proportion of this bacterium in gut microbiota may be associated with autism in children and neurological disorders, such as neuromyelitis optica [46]. The virulence of C. perfringens attributes to its toxins, especially C. perfringens enterotoxin (CPE) produced during its sporulation in intestines [7]. Due to its ability to bind to claudin receptors, which are junction proteins in intestines, it is known that CPE creates pore on the cell membrane and results in cell death, causing diarrhea and abdominal pain [7]. Thus, this bacterium needs to be controlled. However, there are no edible materials which are applicable to human for destroying C. perfringens specifically inhabits in intestines.
In previous studies, glycated milk proteins, such as whey protein glycated with galactose and glucose, were studied for antiinflammatory activities and their protective effects against oxidative stress [8,9]. Furthermore, fermented Maillard reac tion products (FMRPs), which were produced from glycated milk proteins, have recently been introduced for their physi ological functions, such as antioxidant, antihypertensive, antithrombotic, and hepatoprotective properties [10,11]. It was reported that antimicrobial activities of Maillard reac tion products vary according to the kinds of amino acids [12]. Moreover, Maillard reaction products, which were de rived from the foods, such as coffee, beer, and wine, showed different antimicrobial activities [13,14]. Meanwhile, the an timicrobial activity of FMRPs has never been investigated yet to date. Moreover, the effect of Maillard reaction prod ucts against the bacteria inhabiting gastrointestinal tract was not elucidated. In order to reduce incidence of diseases due to the pathogenicity of intestinal microbes, and risks such as antibiotics and stress tolerance, it is necessary to study FM RPs possessing physiological and antimicrobial activities. Therefore, the objective of this study was to evaluate the an timicrobial activity of FMRPs against C. perfringens and to elucidate the mode of FMRPs for antimicrobial activity.

Preparation of fermented Maillard reaction products
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 pilotscale instrument controlled by a pilotscale pasteurization unit (Powerpoint International, Tokyo, Japan) at 65°C for 24 h. These Maillard reacted galac tose and glucose were then fermented by L. rhamnosus 4B15 or L. gasseri 4M13 at 6 Log colonyforming unit (CFU)/mL at 37°C for 48 h to prepare FMRPs (Gal4B15, Gal4M13, Glu 4B15, and Glu4M13). The cultures were then centrifuged at 2,000×g for 30 min (Hitachi CR21G rotor R20A, Hitachi Ltd., Tokyo, Japan). Subsequently, the supernatants were trans ferred into aluminum containers, followed by freezedrying using a lyophilizer (OPERON FDUT8608, Gimpo, Gyeong gi, Korea) for 72 h. The freezedried FMRPs were powdered and stored at 4°C until further use.

Bacterial preparation
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 brainheart 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 KH 2 PO 4 , 1.5 g of Na 2 HPO 4 , 8.0 g of NaCl, and 0.2 g of KCl in 1 L of distilled water). The cell sus pensions of C. perfringens were then diluted to OD 600 = 0.01 with PBS to obtain 5 to 6 Log CFU/mL of inoculum.

Antimicrobial effect of fermented Maillard reaction products
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; Gal4B15, Gal4M13, Glc 4B15, and Glc4M13) were mixed in a well of a 24well 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 sam ples were then serially diluted with buffered peptone water (BPW; BD Difco, USA), and 100 μL of the diluents were spreadplated on tryptose sulfite cycloserine agar (TSC; Oxoid, UK) to enumerate C. perfringens cells.
Absorbance of blank solution was measured with distilled water instead of with samples [15].

Propidium iodide uptake assay
To observe the effects of FMRPs on bacterial cell permea bility in short time, propidium iodide (PI) uptake assay for FMRPsexposed 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 discard ed. Onemilliliter 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 calcu lated, using CellQuest Pro software (BD Bioscience, USA). The percentages of FMRPstreated bacteria figured in each histogram are relatively calculated to PBStreated bacteria at a same designated point.

Morphological observation
To elucidate the antimicrobial activity of FMRPs against C. perfringens, morphological observations were conducted, using field emission scanning electron microscope (FE SEM; JEOL JSM7600F, 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 flatbottomed polystyrene plates (SPL, Korea). Thirty milliliters of 1.8% glutaraldehyde (SigmaAldrich 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 dis tilled 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 hexamethyl disilazane (SigmaAldrich, USA). The samples were eventually coated with a sputter coater (Cressington 108auto SEM sputter coater, Cressington Scientific Instruments Ltd., Wat ford, UK), and the bacterial cells were observed, using field emission scanning electron microscopy.

Statistical analysis
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 LSmeans were analyzed with a pairwise ttest at α = 0.05.

RESULTS AND DISCUSSION
Antimicrobial activities of fermented Maillard reaction products 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 Gal4B15 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 func tions of FMRPs against C. perfringens, FMRPs may reduce intestinal C. perfringens and may be potential materials as healthpromoting 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 car diovascular diseasepreventive effects [17].

Iron chelation activity
Theoretically, high iron chelation activity indicates that iron as an essential nutrient for microorganisms in the environ ment is unavailable to bacteria, which in turn suppresses bacterial growth and decreases bacterial cell counts [18,19]. Gal4M13 (23.87%) significantly showed high iron chelation activity compared to Glc4M13 (0.50%) (p<0.05) ( Table 2). This suggests that Gal4M13 may have better iron chela tion 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 Gal4M13 in this study, because Glc4B15 and Glc4M13 showed similar anti microbial effects on C. perfringens, although they showed difference in iron chelation activity.  Propidium iodide uptake assay In PI uptake assay, movement of peaks in a histogram, which represents the number of events detected at particular inten sity, 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 Xaxis 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 Gal4B15 and from 18.9% to 20.3% by Gal4M13. Glc4B15 and Glc4M13 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 mem  brane 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, amphipa thicity, 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 mem branes and occurred the cell leakage.

Morphological analysis
The PI uptake assay showed that FMRP treatments caused cell membrane damages, which were confirmed visually by FESEM. FMRPtreated C. perfringens had an uneven cell membrane, and pores on the cell membrane surface ( Figure  3). In addition, the lengths of the FMRPtreated 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 biochemi cally [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 bac terial 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.

CONCLUSION
The FMRPs (Gal4B15, Gal4M13, Glc4B15, and Glc4M13), which are already known for the functional properties, showed significant antimicrobial activity against C. perfringens. The antimicrobial activity of FMRPs would be caused by iron che lation and bacterial cell membrane damage. Moreover, FMRPs contributed to cause stress to C. perfringens as its elonga tion shows. Thus, the FMRPs can be used in various food to control the pathogen. In addition, it can be expected that consumption of FMRPs may control intestinal C. perfringens as healthpromoting supplements.