Antibacterial Effect of Black Pepper Petroleum Ether Extract against Listeria monocytogenes and Salmonella typhimurium

e aim of the present study was to evaluate the antibacterial eect of black pepper petroleum ether extract (BPPE) against Listeria monocytogenes ATCC 19115 and Salmonella typhimurium ATCC 14028. e results showed that the BPPE had a strong antimicrobial activity against L. monocytogenes and S. typhimurium, and 2-methylene-4,8,8-trimethyl-4-vinyl-bicyclo[5.2.0]nonane (9.36%) and caryophyllene oxide (4.85%) were identied as the two primary components of BPPE. e ability of cells to break down hyperoxide was decreased, and the activities of POD and CATwere inhibited. e activities of key metabolic enzymes shed some light on the biochemical mechanism of aglycon cell growth inhibition, indicating that the energetic metabolism of L. monocytogenes and S. typhimurium was markedly inuenced by the BPPE. e contents of key organic acids varied signicantly, resulting in remarkable abnormalities in the energetic metabolism of L. monocytogenes and S. typhimurium. us, the consecution of energetic metabolism was destroyed by the BPPE, which contributed to metabolic dysfunction, the suppression of gene transcription, and cell death.


Introduction
With the development of food processing techniques, various chemical preservatives have been added to our food for daily sustenance [1]. However, food safety remains a very important public health issue, especially for the application of food preservatives in food processes. erefore, a new methodology for reducing or eliminating food-borne pathogens has become a pressing issue [2].
Antibiotics, since their introduction over 60 years ago, quickly became the main strategy for controlling bacterial infections in clinical medicine. However, the increase in antibiotic use led to increased bacterial resistance, which is encouraging the search for new active compounds against pathogens. Researchers found that some natural products from plant sources (black pepper, ginger, garlic, onion, etc.) [3][4][5], animal sources (nucleoprotamine, antibacterial peptides, and chitosan) [6,7], and microbial sources (kojic acid, ε-poly-L-lysine, and nisin) have signi cant e ects on food-borne pathogens [8][9][10][11]. All of these natural products showed little in uence on the health of the human body.
Since ancient times, black pepper has been commonly used as a spice in cooking. Moreover, this spice is highly valued as a folk medicine because of its antibacterial properties and other physiological bene ts, particularly in treating pain, the u, muscle aches, and rheumatism [12]. Recent studies have shown that black pepper extract can inhibit food spoilage and food pathogens [13][14][15][16]. e structure, preservatives, and antibacterial e ect of the pepper extracts were reported. We previously explored the optimum extraction process as well as the inhibition and minimal inhibitory concentration (MIC) of the black pepper petroleum ether extract (BPPE) on L. monocytogenes and S. typhimurium. However, the antibacterial mechanisms contributing to the inhibition of these strains remain unclear.
Hence, the objective of this study was to investigate the inhibitory effect of the BPPE on Listeria monocytogenes and Salmonella typhimurium by determining the activities of key metabolic enzymes, the contents of organic acids involved in aerobic respiratory metabolism, and their possible lysis activity following antioxidant enzyme release in the broth medium.

Materials and Chemicals.
e black pepper (Piper nigrum L., confirmed by Prof. Weimin Zhang, Hainan University) used in this study was purchased from Nanguo Supermarket (Haikou, China), grown in Wanning, Hainan Province, China, and picked in June 2017 (nine months after flowering). Black pepper was purchased from the Nanguo Supermarket (Haikou, China). e malate dehydrogenase (MDH) assay kit, succinate dehydrogenase (SDH) assay kit, peroxidase (POD) assay kit, and hydrogen peroxidase (CAT) assay kit were acquired from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). KH 2 PO 4 and nicotinamide adenine dinucleotide (NADP + ) were purchased from Amresco (Solon, OH, USA). Brain heart infusion (BHI) broth was purchased from the Guangdong HuanKai Microbial Tech Co., Ltd., and nutrient broth (NB) was acquired from the Beijing Solarbio Science & Technology Co., Ltd. All other chemicals were of analytical grade. BPPE was prepared according to our previous report (80% ethanol, 50°C, and 12 h) [17]. (ATCC 14028) and Listeria monocytogenes (ATCC 19115) were purchased from Guangdong Microbiology Culture Center (Guangzhou, China). L. monocytogenes was grown and maintained on slants of brain-heart infusion agar (Huankai Microbial Sci. & Tech, Co., Ltd., Guangdong, China). S. typhimurium and L. monocytogenes were cultured at 37°C for 24 h in the master slant culture tubes. e bacteria were washed twice with sterile NaCl water (0.9%, w/w) and then resuspended in sterilized saline water at a concentration of 1-2 × 10 7 CFU/mL as measured by the Maxwell turbidimetry. One milliliter of S. typhimurium or L. monocytogenes was added into sterile Erlenmeyer flasks with 49 mL of fresh liquid culture bath. e BPPE was dissolved in ethanol (80%, v/v) and then added into the treated group to yield the minimum inhibitory concentration (MIC), and the MIC tests have been conducted previously [18]. e negative control group had the same volume of ethanol but without BPPE, and the positive control group had no ethanol and BPPE. All of the groups were agitated at 130 rpm in an environmental incubator shaker at 37°C.

Time-Kill Curve.
e antimicrobial activity of BPPE was determined in triplicate using a plate colony-counting method [19]. In brief, Salmonella typhimurium and Listeria monocytogenes were cultured with shaking at 37°C for 24 h, washed twice with sterile NaCl water (0.9%, w/w), and resuspended. Subsequently, the BPPE was dissolved in ethanol and added to two test tubes containing Salmonella typhimurium and Listeria monocytogenes to make the concentration of the antibacterial 1.25 mg/mL and 0.625 mg/mL, respectively. en, the antibacterial effects of the BPPE on the two bacteria at 0, 0.5, 1, 2, 4, 6, and 8 h were observed. Ten milliliters of the suspension were serial diluted by sterile NaCl (0.9%, w/w), coated, and cultured at 37°C for 24 h. Finally, the bacterial population was counted.

Determination of the Antioxidant Enzyme Assay.
Peroxidase reduces hydrogen peroxide and oxidizes a wide number of compounds including thiocyanate ions and fatty acids [20]. Peroxidase plays a role in the extracellular defense against stress, biosynthesis and degradation of lignin, intracellular removal of hydrogen peroxide, and oxidation of toxic reductants [21]. e effects of the BPPE on the activities of POD and CAT were determined at different concentrations. Concentrations of 1/4 MIC, 1/2 MIC, MIC, 2 MIC, and 4 MIC of the BPPE were added to the bacterial suspension (1.0 × 10 7 cfu/mL), and the mixture was cultured at 37°C in an incubator shaker for 24 h. en, 10 mL of the suspension was broken by an ultrasonic liquid processor (UP200S, Hielscher Ultrasonics GmbH, Teltow, Germany) in an ice bath for 6 min (550 W, working for 10 s with an interval of 5 s). e cell debris was removed, and the supernatants were collected by centrifugation at 5600 g for 12 min at 4°C. e activities of POD and CAT were explored by a POD assay kit and CAT assay kit, respectively. e content of the protein was determined by the Bradford method at 595 nm, and the standard curve was made using bovine serum albumin [22].

Dehydrogenase Activity Measurements and Total Protein
Content.
e bacteria strains grown in liquid nutrient medium were agitated at 146 g in an environmental incubator shaker at 37°C overnight, and the concentration was diluted to approximately 1-2 × 10 7 CFU/mL with sterile nutrient broth. en, the bacteria were collected by centrifugation at 8960 g for 15 min at 4°C, washed twice with sterile phosphate buffered saline (PBS, pH � 7.4), and resuspended in 5 mL of sterile PBS. e bacterial suspensions were treated by an ultrasonic liquid processor in an ice bath for 6 min (550 W, working for 10 s with an interval of 5 s), and the supernatants were collected by centrifugation at 10,000 g for 15 min at 4°C, followed by storage in an ice bath [17]. e activities of malate dehydrogenase (MDH), succinate dehydrogenase (SDH), and kinase pyruvate (PK) were determined by the operation of an assay kit according to the manufacturer's instructions [23,24].

Pyruvic Acid Determination.
e content of pyruvic acid was determined according to the method of Spoel and Dong [25]. At the designated time intervals, 5 mL of bacterial suspension was centrifuged at 6000 g for 15 min, and the supernatant was collected and stored at 4°C. e 2,4-dinitrophenol (1 mL), trichloroacetic acid (0.3 mL, 8.0%), and the supernatant (0.1 mL) were transferred to a test tube; the mixture was placed in a water bath at 37°C for 10 min; and then 10 mL of sodium hydroxide (0.4 mol/L) was added. e absorbance of the mixture was read at 520 nm, and the content of pyruvic acid was calculated using a pyruvic acid calibration curve.

Content of ATP.
Adenosine 5′-triphosphate is the basic carrier of energy in organisms. e variation in the ATP content is directly related to the energy metabolism of organisms [26]. e content of ATP was determined by an ATP assay kit. Ten milliliters of culture suspension were washed and broken by ultrasonic treatment in the manner described above. en, 30 µL of the supernatant was used to determine the content of ATP according to the instructions of the assay kit.

GC-MS Analysis.
e chemical composition of BPCE was determined by gas chromatography-mass spectrometry (GCMS). GC-MS experiments were performed on an Agilent Technologies 7890A gas chromatograph (Santa Clara, CA) and an Agilent 7683B autoinjector coupled with a 240 Agilent Ion Trap mass spectrometer (MS/MS). e mass spectral scan rate was 2.86 scans/s. e GC was operated at a helium (ultrahigh purity) flow rate of 0.7 mL/min under a head pressure of 10 psi, and the injection volume was 1 µL.
e MS was operated in the electron ionization (EI) mode using an ionization voltage of 70 eV and a source temperature of 230°C. e scan type used was the automated method development function (AMD), and the optimum MS/MS excitation amplitude was 1.20 V. Relative percentages of the primary components were calculated by integrating the registered peaks.

Statistical Analysis.
e data of enzyme activity and bacteria count were statistically analyzed using IBM SPSS statistical software (version 21.0, SPSS Inc., Chicago, IL, USA). e differences between means were assessed by analysis of variance (ANOVA) with Duncan's test using a significance level of p < 0.05. e results showed that the BPPE at the MIC exerted strong bactericidal activity, as indicated by the significant reduction in microbial counts, which indicated that the BPPE has a perfect antibacterial activity against L. monocytogenes and S. typhimurium. erefore, the BPPE can be regarded as a natural and efficient antiseptic of pathogens. Similar to our findings, the major components and essential oils of black and red pepper showed good antimicrobial activity for Escherichia coli O157:H7 and Staphylococcus aureus [27,28].

3.2.
e Antibacterial Effect on the Peroxidase Enzyme. e analysis of key enzymes provided further interesting information on the antimicrobial activity of the BPPE. As shown in Figure 2, the activities of POD and CAT were remarkably influenced by the BPPE. e activities of the enzymes decreased with increases of the BPPE concentration. In particular, POD of L. monocytogenes and S. typhimurium showed 80-85% activity inhibition in the presence of 4 MIC of the BPPE. e BPPE inhibits L. monocytogenes and S. typhimurium CATactivity by decreasing it to 70-50%, making cells more susceptible to oxidative injuries. e mechanisms that enabled microorganisms to survive after the release of POD and CAT include the degradation of H 2 O 2 and the inhibition of the ROS-mediated cell death. e BPPE inhibits the activities of POD and CAT, thereby making bacterial cells more susceptible to oxidative injury and reducing their capability to eliminate H 2 O 2 [29].

Influence of the BPPE for EMP.
Pyruvate kinase (PK) could promote the production of pyruvate from glycolysis and is helpful with regard to energy metabolism. Additionally, pyruvic acid is the raw material to produce acetyl-CoA, which is the initial reactant in the Krebs cycle, one of the most important metabolic pathways for the generation of energy and metabolites in living organisms [30]. Pyruvic acid, an important intermediate metabolite, is associated with many metabolic pathways in microorganism. For example, pyruvic acid connects EMP, TCA, and HMP. If pyruvic acid is accumulated, normal physiological metabolism would be inhibited, especially the TCA pathway of bacteria [31]. e activity of the tested bacteria's dehydrogenase enzyme was markedly influenced by the presence of the BPPE. In detail, the activity of PK for the L. monocytogenes has been significantly decreased by the addition of the BPPE for 6 h before the test. Additionally, the activity of PK for L. monocytogenes was chaotic compared with that of PK for the control group. e content of pyruvic acid can be controlled by the activity of PK. e activity of PK for S. typhimurium was remarkably inhibited by the presence of the BPPE, which resulted in the content of pyruvic acid decreasing.
us, the tricarboxylic acid cycle was inhibited when there is BPPE in the culture. e content of pyruvic acid in the culture solution is shown in Figure 3. roughout the entire incubation process of L. monocytogenes, the pyruvic acid contents in the control groups slightly decreased within 0-6 h, and then they increased from 0.1193 g/L to the nal concentration of 0.1602 g/L ( Figure 4). However, the concentration of pyruvic acid signi cantly increased during the incubation process in the culture, and the nal concentration value was 0.1795 g/L. e value of the treated group for S. typhimurium increased within 6-24 h, and the value was above that of the control group during the incubation process. After 24 h, the concentration of pyruvic acid in the treated group was 0.0931 g/ L, whereas those in the control group and alcohol group were 0.0747 g/L and 0.0763 g/L, respectively.

Dehydrogenase of TCA.
MDH and SDH are the catalyzing enzymes in the Krebs cycle, which is conducive to providing ATP. e SDH could catalyze succinic acid to fumaric acid, and the MDH catalyzes malic acid to oxaloacetate [32]. us, the succinic acid reaction and malic acid reaction were the important points for producing energy by the Krebs cycle. As is shown in Figure 3, there were signi cant di erences in the activities of the test enzymes between the BPPE and blank groups.
As is shown in the picture, the activity of SDH in the BPPE was higher than that of the control group; however, the MDH was inhibited for L. monocytogenes by the BPPE. e inhibition of MDH indicates a decrease in its capability to utilize malic acid to restore NAD + , which is essential for the reaction of glyceraldehyde 3-phosphate dehydrogenase during glycolysis.
In particular, SDH showed a 100-300% activity increase in the presence of 1.25 mg/mL and 0.625 mg/mL BPPE for L. monocytogenes and S. typhimurium. e promotion of the key enzyme in the aerobic energetic metabolism of the bacteria, which exists in the mitochondrial membrane, indicates an increase in its capability to utilize succinic acid to restore FADH 2 , an essential reaction for biological oxidation.
e BPPE also in uences the activity of another fundamental enzyme for energy metabolism, malate dehydrogenase (MDH). is enzyme is also the key regulation point of the Krebs cycle, one of the most important metabolic pathways for the generation of energy and metabolites in living organisms. In particular, MDH showed remarkable inhibition in the presence of the BPPE for L. monocytogenes, indicating that malic acid was accumulated, the content of oxaloacetate was decreased, and NAD + accepting H + to form NADH was blocked. However, the activity of MDH increased at 12 h before, and then the activity of MDH was inhibited by the presence of the BPPE. ese results were consistent with the ndings of Hu et al. [33].

ATP.
e ATP levels of L. monocytogenes and S. typhimurium are shown in Figure 5. In the control group, the ATP level was increased in rst 6 h and 3 h for L. monocytogenes and S. typhimurium, respectively, which may be due to growth and reproduction. After 24 h, the concentration of ATP in the treated group was signi cantly lower than that in the control group, which could be due to that the ATP was rapidly degraded when the cells died. is result indicated that the BPPE could change the respiratory metabolism of the two bacteria. e biological function of ATP synthase is dependent on the membrane potential [34]. erefore, we could further investigate the e ects of the BPPE on the cytoplasmic membrane potential of L. monocytogenes and S. typhimurium [7], in which ATP production had been inhibited.

GC-MS Analysis of the Chemical Composition of BPPE.
e GC-MS spectrum of BPPE is presented in Figure 6, in which 125 chemical constituents were identi ed. And the 30 Journal of Food Quality primary substances are presented in Table 1.
In addition, natural products and naturally derived compounds from black pepper may have applications in controlling pathogens in foods.
e challenge is to isolate, purify, stabilize, and incorporate natural antimicrobials into foods without adversely a ecting sensory, nutritional, and safety characteristics [42]. And the components analysis of GC-MS and the MIC could ensure the food security of BPPE, whereas the impact of BPPE on organoleptic properties of food requires further studies.

Conclusion
is study describes the antimicrobial e ect of organic compounds in black pepper on the energy metabolism of e BPPE is particularly active against L. monocytogenes and S. typhimurium. First, the BPPE could rapidly destroy the permeability of the cell wall and membrane, and this phenomenon resulted in the substantial rapid loss of cell contents and freedom from the separation of macromolecular substances from intracellular substances. e POD and CAT were markedly influenced by the BPPE with the increase of its concentration. e active ingredients of the BPPE entered the cells and interacted with key enzymes in the glycolytic pathway, eventually hindering and disordering cell metabolism. Furthermore, the accumulation of pyruvic acid indicated that the ability to expend reducing sugars decreased and the ability of cells to produce energy was reduced. e activity of MDH was remarkably influenced by the BPPE, and this proved that the additive could influence cellular respiration by disrupting the TCA pathway, which could result in abnormal physiological metabolism in the cell. e reduction of ATP proved that the BPPE could destroy bacterial respiratory metabolism and lead to free access to intracellular material, which could result in cell death. e result of GC-MS revealed that 2-methylene-4,8,8-trimethyl-4-vinyl-bicyclo [5.2.0]nonane (9.36%) and caryophyllene oxide (4.85%) were the two primary potential antimicrobial components of BPPE. Overall, the current investigation would facilitate the development of antibacterial agents targeting energy metabolism. is experimental result provides an approach for developing convenient and efficient antimicrobial agents in the food and pharmaceutical industries, which is of great significance to food security. Data Availability e data used to support the findings of this study are included within the article. Disclosure e authors alone are responsible for the content and writing of the paper.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Wenxue Chen and Haiming Chen conceived and designed the experiments; Hui Tang performed the experiments; Yueying Hu, Qiuping Zhong, and Weijun Chen analyzed the data; Wenxue Chen and Ningxin Jiang wrote the original and revised manuscript.