Considering the beneficial properties of peanuts, we hypothesized that peanuts may potentially maintain or stimulate the growth of probiotic bacteria and also may have antimicrobial effects on foodborne bacterial pathogens (Tzounis and others 2011 ; Calatayud and others 2013 ). We were especially interested in examining the positive properties of peanut white kernel and peanut skin. The antimicrobial and prebiotic‐like effects of both parts of peanuts on the growth of probiotic Lactobacillus as well as on major foodborne pathogens including EHEC, S . Typhimurium, and L. monocytogenes were investigated.

In 2003 , the Food and Drug Administration (FDA) approved a qualified health claim that suggests consuming 1.5 ounces (42 g) of most nuts, including peanuts (approximately 0.5 to 1.0 g of peanut skin and 15 to 20 g of peanut flour), as part of a diet low in saturated fat and cholesterol may reduce the risk of heart disease. The benefits on reducing cardiovascular diseases (Isanga and Zhang 2007 ; Ozcan 2010 ; Bao and others 2013 ; Jones and others 2013 ) with daily intake of peanut or other nut products has been suggested to associate with their phenolic compound Resveratrol (3,4,5‐trihydroxystilbene) (Bubonja‐Sonje and others 2011 ; Wang and others 2011 ). Additionally, Resveratrol once reached large intestine may exert prebiotic‐like benefits in large intestine by promoting the growth of selective beneficial gut microflora, especially Lactobacillus (Tzounis and others 2011 ; Calatayud and others 2013 ).

The Center for Disease Control and Prevention (CDC) estimated that 48 million illnesses, 128000 hospitalizations, and 3000 deaths are caused by foodborne pathogens (CDC 2013 ). Among bacterial pathogens, Escherichia coli O157:H7 (EHEC), Salmonella , and Listeria monocytogenes are the leading causes of foodborne illness and deaths in the United States (Dussurget 2008 ; Teunis and others 2008 ; Cummings and others 2012 ). Consequently, their prevention and growth inhibition are of great concern to public health professionals, farmers, and food producers. Antibiotics are commonly used either orally through drinking water or as feed additives (Page and Gautier 2012 ) for control of zoonotic pathogen colonization in gut of farm animal including cattle, swine, and chicken (Scallan and others 2011 ). However, it was demonstrated to worsen the niche for beneficial gut microflora (Andersson and Hughes 2010 ) and accelerate the drug‐resistance in human pathogens (DeWaal and Grooters 2013 ). Therefore, there is increasing interest in use of dietary supplement, especially prebiotics, to modulate the composition of the colonic microflora. The promotion of the growth of probiotics by prebiotics, in turn, is hypothesized to inhibit or even exclude the harmful bacteria like foodborne bacterial pathogens (Gibson 2004 ).

Major probiotics are essential part for human gut flora and often found as resident bacteria in milk and other dairy products. They can be divided into 2 main categories—colonizing species, such as Lactobacillus, Lactococcus , and Enterococcus , and free floating noncolonizing species including Bacillus and Saccharomyces (Sharma and Devi 2014 ). These beneficial microbes colonizing in the gut are able to ameliorate the overall health of humans by restructuring the gut microbial balance (Nueno‐Palop and Narbad 2010 ). The possible characteristics of probiotic microorganisms include production of bacteriocins, propionic acid, and vitamin B12, and other various effects include increasing the villous length and nutrient absorption, immuno‐stimulatory activities, and competitive exclusion of pathogenic microorganisms (Christina and others 2009 ).

Cell adhesion and invasion assay was performed according to the method described previously with slight modification (Salaheen and others 2014 ). Briefly, the INT407 cells grown in 24‐well plate with 800 μL DMEM were pretreated with 100 μL DMEM (control), 0.5% peanut flour, 0.5% peanut skin extract, 2×10 8 CFUs L. casei , 2×10 8 CFUs L. casei plus 0.5% peanut flour, and 2×10 8 CFUs L. casei plus 0.5% peanut skin extract separately for 1 h, with each treatment in triplicate. A 100 μL aliquot of EHEC, S . Typhimurium, or L. monocytogenes with multiplicity of infection of about 10 (2×10 8 CFUs) were inoculated into triplicate wells. Infected cells were incubated at standard condition for 2 h, and then washed 3 times with DMEM. The monolayers were lysed by 0.1% Triton X‐100 for 15 min, serial diluted, and plated on specific agars for adhesive bacterial CFU counting. To measure cell invasive activity, the washed cell monolayers after 2 h bacterial infection were incubated in DMEM containing 10% FBS and 250 μg/mL gentamicin for 1 h, then followed by 3‐times washing, Triton X‐100 lysis, serial dilution, and finally plating on agars mentioned above.

Whole milk purchased from local Giant market (College Park, Md., U.S.A.) was sterilized with UV irradiation for 2 h. Similarly, 400 μL aliquot of Lactobacillus cells suspension with approximately 7 log CFU/mL was added into 3.6 mL MRS broth (control), whole milk, or whole milk with 0.5% peanut skin extract, and then incubated at 37 °C for different time points (0, 24, 48, and 72 h). Serial dilutions in PBS and plating on MRS agar plates in triplicate were performed and followed by incubation for 18 h at 37 °C for growth. Bacterial CFUs were counted and results were expressed as the average number of bacterial CFUs from triplicate assays.

Bacterial cells from agar plates were collected in 10 mL PBS (0.1 M, pH 7.2) using 10 μL sterile disposable loops (Thermo Fisher Scientific Inc.). The optical density (OD 600 ) of each concentrated bacterial suspension was adjusted using PBS and measured by a LAMBDA BIO/BIO+ spectrophotometer (PerkinElmer, Beaconsfield, Buckinghamshire, UK) to make it contain approximately 7 log CFU/mL. A 400 μL aliquot of each adjusted bacterial suspension was added to sterilized tubes containing 3.6 mL MRS broth (for Lactobacillus ) or LB broth (for foodborne pathogens) or MRS:LB = 1:1 (v/v) broth (for mixed culture) in the presence or absence of 0.5% peanut flour or 0.5% peanut skin extract and then incubated at 37 °C for different time points (0, 24, 48, and 72 h). After incubation, serial dilutions were performed in PBS, followed by plating on agar plates (MRS agar for Lactobacillus , MacConkey agar for EHEC, LB agar for S . Typhimurium, and BHI agar for L. monocytogenes ) in triplicate followed by incubation for 18 h at 37 °C for growth. Bacterial colony forming units (CFUs) were counted and results were expressed as the average number of bacterial CFUs from triplicate assays.

In shell Jumbo Virginia raw peanut was purchased from a local market and shelled by hand to isolate the kernel fractions. Peanut skin was removed by hand and the white kernel was ground to form peanut flour. Both peanut skin and peanut flour were defatted by 2 extractions with n‐hexane (n‐hexane: peanut portion = 10 mL/g) for 12 h at room temperature (25 °C). Peanut flour and peanut skin fraction suspensions were prepared in sterilized distilled water (pH adjusted to 8.0 with 1 N NaOH), mixed well and sterilized with UV irradiation for 2 h.

Human intestinal INT407 cells (ATCC CCL‐6) were cultured at standard condition (37 °C, 5% CO 2 , 95% humidity) in Dulbecco's Modified Eagle Medium (DMEM) (Corning cellgro, Manassas, Va., U.S.A.) supplemented with 10% heat‐inactivated Fetal Bovine Serum (FBS) (Corning cellgro) and 100 U/mL of gentamycin (Lonza, Walkersville, Md., U.S.A.). The INT‐407 cells were seeded in 24‐well culture plate (Greiner bio‐one Inc., Monroe, N.C., U.S.A.) at 2×10 6 cells/mL and then they were cultured at the standard condition mentioned above up to more than 85% confluence. The semiconfluent cultures were washed with phosphate‐buffered saline (PBS) by 3 times and immersed in serum‐free DMEM for cell adhesion and invasion assay.

Pretreatment with peanut fractions or in the presence of L. casei reduced significantly the adhesion to and invasion into INT‐407 cells by these enteric bacterial pathogens (Figure 5 ). We observed that pretreatment with 0.5% peanut flour significantly ( P < 0.05) reduced the adherence ability of EHEC, S . Typhimurium, and L. monocytogenes to INT‐407 cells by 89.9%, 86.5%, and 40.3%, respectively. Likewise, 0.5% peanut skin extract also significantly ( P < 0.05) reduced the adhesion abilities of these 3 foodborne pathogens by 90.1%, 84.7%, and 54.9%, respectively. Preincubation of L. casei showed attenuated inhibitive effects, which reduced the adhesive level of EHEC, S . Typhimurium, and L. monocytogenes by 51.5%, 66.7%, and 61.0%, respectively. However, the combine effect, pretreated with L. casei and 0.5% peanut flour, further enhanced the inhibitive capability on pathogens’ adhesion ability up to 94.2% for EHEC, 97.2% for S . Typhimurium, and 83.8% for L. monocytogenes . When compared with single effect of L. casei , the combined effect of L. casei and pretreatment of 0.5% peanut skin extract did not inhibit more cell adhesive activity at significant level of P = 0.05. In the same study, we also found that cells treated with 0.5% peanut flour or 0.5% peanut skin extract showed significant reduction (96.8% to 98.6%) in invasion ability by these foodborne pathogens (Figure 5 D, 5 E, and 5 F). We also found that combined effect of pretreatment with L. casei and 0.5% peanut flour or 0.5% peanut skin extract enhanced the inhibitory effect of these pathogens by 100% (below the limit of detection) for EHEC, 93.9% for S . Typhimurium, and 83.4% for L. monocytogenes .

Growth conditions of these foodborne pathogens were determined in mixed culture with L. casei in the presence and absence of 0.5% peanut flour or 0.5% peanut skin extract. We found that EHEC (Figure 4 A) and S . Typhimurium (Figure 4 B) were completely excluded (below the limit of detection) from the medium by L. casei after 48 h. In the same study, L. monocytogenes (Figure 4 C) was drastically inhibited by L. casei by >4 logs after 48 h of incubation in mixed culture with L. casei . In mixed culture with L. casei in the presence of 0.5% peanut flour, the same inhibitory patterns on the growth of these enteric bacterial pathogens were found, but peanut flour, by stimulating growth of L. casei , assisted in quickly reducing more EHEC (>0.5 log CFU/mL at 24 h) and S . Typhimurium (>1.0 log CFU/mL at 24 h). In contrast, in the presence of 0.5% peanut skin extract, L. casei with same amount of inocula only showed reduced inhibitory effects on growth of EHEC (Figure 4 A) and S . Typhimurium (Figure 4 B), whereas L. monocytogenes (Figure 4 C) was completely excluded (below the limit of detection) after 48 h of incubation.

The antimicrobial properties of 0.5% peanut flour and 0.5% peanut skin extract against selected foodborne bacterial pathogens (EHEC, S . Typhimurium, and L. monocytogenes ) were evaluated in liquid cultures. Among these selected 3 enteric bacterial pathogens, 0.5% peanut flour only showed inhibitory effect on EHEC (Figure 3 A). At 24, 48, and 72 h time point of incubation, the growth of EHEC in the presence of 0.5% peanut flour was significantly inhibited by a range of 0.5 to 1 log CFU/mL. However, 0.5% peanut flour exhibited no significant negative effect on the growth of S . Typhimurium (Figure 3 B) or L. monocytogenes (Figure 3 C) within 72 h. On the contrary, EHEC (Figure 3 A) and S . Typhimurium (Figure 3 B) were stimulated slightly by 0.5% peanut skin extract within 72 h. Peanut skin extract only showed inhibitory effect on L. monocytogenes (Figure 3 C).

In order to further investigate the growth inhibitive effect of 0.5% peanut skin extract, the growth of 3 beneficial Lactobacillus strains were also examined in whole milk which serves as a natural reservoir for Lactobacillus . Time‐dependent comparative growth performance of L. casei, L. rhamnosus, and L. plantarum in the presence of peanut skin extract between controls and treatments are shown in Figure 2 . Lactobacillus strains showed overall better and longer growth in whole milk compared to MRS broth after 48 h. However, the growth of all 3 strains of Lactobacillus was significantly inhibited by 0.5% peanut skin extract in whole milk. The growth of L. casei (Figure 2 A), L. rhamnosus (Figure 2 B), and L. plantarum (Figure 2 C) was inhibited by >2 log CFU/mL at 24, 48, and 72 h time points in the presence of 0.5% peanut skin extract in whole milk, compared to growth in whole milk alone.

Growth conditions of 3 beneficial Lactobacillus strains were evaluated in MRS broth in the presence and absence of 0.5% peanut flour or peanut skin extract. Comparative growth of L. casei, L. rhamnosus, and L. plantarum between control and treatments is shown in Figure 1 . In the presence of 0.5% peanut flour, out of the 3 strains of Lactobacillus , L. casei (Figure 1 A) and L. rhamnosus (Figure 1 B) were stimulated at 24, 48, and 72 h time points of incubation in MRS broth. Out of these 2 strains, the most intensive stimulatory effect was found on L. casei , whose growth was promoted by more than 1 log CFU/mL within 72 h. However, peanut flour had no significant effect on the growth of L. plantarum and it grew similarly as it did in MRS broth in absence of peanut flour (Figure 1 C). In the same study, it was found that L. casei, L. rhamnosus, and L. plantarum were significantly inhibited by >2 log CFU/mL at 24, 48, and 72 h time points in MRS broth supplemented with 0.5% peanut skin extract (Figure 1 ).

Discussion

Previous reports have demonstrated that phenolic compounds from plant products could inhibit the growth of several harmful bacteria such as Salmonella Typhimurium (Puupponen‐Pimia and others 2005) and EHEC (Bubonja‐Sonje and others 2011). Resveratrol, one of the known compound of polyphenol, has been shown to extend the doubling time of multiple bacterial pathogens including S. Typhimurium, S. Enteritidis, E. coli, and so on (Jung and others 2009). In our study, the growth of the Gram‐negative foodborne pathogen EHEC was inhibited by nearly 1 log CFU/mL within 48 h of incubation in the presence of peanut flour, and the Gram‐positive foodborne pathogen L. monocytogenes was inhibited by more than 1 log CFU/mL at 24 h by use of peanut skin extract. Moreover, significant growth inhibition against both EHEC and L. monocytogenes was found within 72 h. This study indicates that components of peanut flour and peanut skin exhibit both different scopes and efficacies of antimicrobial effects depending on bacterial species. However, the growth stimulatory effect of peanut skin extract on Salmonella Typhimurium might provide Salmonella opportunities to survive or multiply in peanut products especially unblanched peanut butter.

We also observed that the growth of L. casei and L. rhamnosus were continuously and significantly stimulated up to 72 h by 0.5% peanut flour. This result agreed with the previous research of Salaheen and others (2014), which suggested that the production of fatty acids, one growth factor for Lactobacillus, was promoted by water‐soluble peanut flour fractions. However, in our study, no significant stimulatory effect on L. plantarum was observed within 72 h incubation in MRS broth supplemented with peanut flour. The possible explanation might be based on padA gene, a substrate‐inducible gene responsible for phenolic acid decarboxylase enzyme (PadA) expression, which counteract the promotive effect on this special Lactobacillus (Gury and others 2004). In the same study, we also evaluated the growth of 3 Lactobacillus strains in both MRS broth and whole milk supplemented with 0.5% peanut skin extract. Our data indicated that the growth of all 3 Lactobacillus strains was drastically reduced by peanut skin extract within 72 h, no matter whether in broth or milk compared with in controls. Based on study in 2005, peanut skins are considered as having no adverse effects and would qualify as a GRAS (generally recognized as safe) product (Yu and others 2010). Beside safety, peanut skins extract possess even much greater in vitro antioxidant properties than vitamin C and vitamin E (Yu and others 2010). However, no previous study has examined the role of peanut skins play on growth of beneficial bacteria. In general, our finding indicates that components in peanut skin might impair the in vitro growth of Lactobacillus. Further investigation is needed to know how peanut skin extract and its compounds inhibit the growth of Lactobacillus.

Combined with L. casei and peanut flour showed a drastically inhibitory effect against 3 major foodborne bacterial pathogens. Two possible explanations for this might be that compounds in peanut flour either induced the number of L. casei or the antimicrobial metabolites production by L. casei, both of which contribute to the competitive exclusion of foodborne pathogens. As antimicrobial resistance among foodborne pathogens especially Salmonella, L. monocytogenes, and E. coli growing rapidly recently, FDA has paid close attention on this emergent issue. This study has investigated the potential of combining probiotics and prebiotics in inhibiting foodborne pathogens. Our findings may suggest to replace the chemical antimicrobial use with bio‐competitive inhibitors and natural growth promotive components such as peanut flour, as an alternative to minimize the spread of multidrug resistance bacteria as well as it may ensure food safety for human. The combinational use of L. casei and peanut skin extract only showed strong inhibition on growth of L. monocytogenes. The antimicrobial effect of peanut skin extract itself on L. monocytogenes might be one explanation of this, as a result of which, though the amount as well as the inhibitive effect of L. casei in the mixed culture were attenuated by peanut skin extract, L. monocytogenes still was not able to survive in the supplemented mixed culture.

In the same study, we also found that L. casei, peanut flour, and peanut skin extract can reduce foodborne pathogens colonization on human intestinal cells. In general, the enteric bacterial pathogens’ infection processes usually initiate from intestinal epithelial cell adhesion and then followed by cell invasion through site‐specific ligands. However, normal microflora colonization in gut competitively prevents foreign bacterial pathogens from attachment. Both Lactobacillus and enteropathogens are known to express cell surface proteins and displays carbohydrate‐binding specificities on human intestinal cells (Jean‐Richard and others 2000). In order to attach and colonize host's gut, foodborne pathogens have to compete with normal gut microflora, as a consequence of which, we hypothesize that pretreatment of L. casei reduce the adhesive and invasive activities of pathogens by occupying the intestinal cell surface receptors. In addition, bioactive components in peanuts such as phenolic acids and flavonoids might inhibit the colonization of pathogens by reducing their flagellin and adhesin level. Simultaneously, the increased activities of cell attachment by L. casei in supplement with peanut flour may explain why combinational pretreatment with L. casei and peanut flour could exhibit much stronger adhesion and invasion inhibitive effect on 3 pathogens.