Authored by Byungjin Min*
Abstract
Proper washing treatment of fresh produce plays a critical role to reduce or minimize microbial contaminations for safe consumption. This study investigated antimicrobial activities of plant-based ingredients as an alternative wash to substitute chemical wash. From the preliminary test, five solutions: 1) 0.5% white distilled vinegar (DV); 2) 25% crude lemon juice (LJ); 3) DV + 0.1% origanum oil (DVO); 4) LJ+ 0.1% origanum oil (LJO) and 5) DV+ LJ + 0.1% origanum oil (DVLJO) and sterile water (control) were selected. To evaluate antimicrobial activities against foodborne pathogens both L. monocytogenes and S. Typhimurium zone of inhibition (ZOI), minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were measured. In addition, model study in an aqueous solution was designed to determine effective washing time against tested microorganisms, and it was performed at 2, 5, 15, 20 and 25 min. MICs and MBCs of natural ingredients against L. monocytogenes and S. Typhimurium were 0.03 to 0.78% and 0.06 to 1.56%, respectively, but higher concentration was required for lemon juice extract (12.5%). The combined treatment DVLJO exhibited the least MIC (0.03%) as well as MBC (0.06 %) against L. monocytogenes. The results indicated that combination of wash solutions reduced bacterial populations by ~3 to 5 log CFU/mL at 25 min of agitation. However, there were no significant differences in bacterial reductions by washing time between 5 and 25 min (P>0.05). Based on the results, it is suggested that combinations of vinegar, lemon juice and essential oil might be suitable as an alternative antimicrobial wash solution for raw or minimally processed foods. It is thought that formulated wash solutions with natural ingredients are readily available and easy to use at the house-hold level. However, further study is recommended to validate and specify the effectiveness of wash solutions in a real food system.
Keywords: Vinegar; Essential oil; Natural antimicrobials
Abbreviations: ATCC: American Type Culture Collection; CFU: Colony-Forming Unit; DV: White Distilled Vinegar; DVO: DV + Origanum Oil; LJ: Crude Lemon Juice; DVLJO: DV+ LJ + Origanum Oil; EO: Essential Oils; LJO: LJ+ Origanum Oil; MBC: Minimum Bactericidal Concentration; MIC: Minimum Inhibitory Concentration; OD: Optical Density; TSA: Tryptic Soy Agar; ZOI: Zone of Inhibition
Highlights
Natural ingredients as potential antimicrobials substantially reduced microbial growth.
A combination of the natural solution was more effective than a single use of antimicrobial.
Natural wash could be used to decontaminate fresh produce.
Introduction
Various intervention technologies such as physical, chemical, and biological treatments have been employed to control foodborne pathogens [1,2]. Compared to generic chemical methods, plantbased decontamination methods gaining popularity because it is generally considered as safe in a food matrix [3,4]. Also, plant-based antimicrobials are readily available and easy-to-use as compared to physical treatments [5]. Over the past few years, interests of using essential oils (EOs) and organic acids have been increased due to their significant antimicrobial activities against a broad spectrum of foodborne pathogenic bacteria [6-9].
Organic acids such as citric acid, acetic acid and lactic acid are impregnated into foods to enhance food safety and meat quality in particular [10]. Organic acids are being used in the food industry as preservative due to their acidic properties since concentrations of hydrogen ions impact the growth and survival of microorganisms in foods [2,5]. Several types of conventional vinegars are available on the market, and these are rice vinegar, distilled vinegar, balsamic vinegar, wine vinegar, black vinegar, bamboo vinegar, etc. In general, vinegars from fermented carbohydrate contains 3.75 to 5 % of acetic acid as an active ingredient [9]. Vinegar can inhibit a wide range of bacteria and used as an active decontamination agent, especially for fresh produce [11,12]. Likewise, EOs are also considered as natural antimicrobials which are colorless, complex, and comprise of volatile aromatic compounds present in plants [13]. The antimicrobial activities of EOs are due to their hydrophobic nature, in which it disrupts the cell membrane and cell wall of the bacteria [7]. Correspondingly, lemon is considered one of major citrus fruit along with oranges and mandarins. The essential compounds in lemon juice are water, citric acid, and carboxylic acid. It is reported that raw or partially cooked beef meats marinated with lemon juice along with heat treatment reduced the risk of foodborne pathogens [10].
Microbial food safety of raw or minimally processed fresh produce is a challenge in the food supply chain because it increases the risk of illness and causes significant reduction in shelf-life [14]. Current chemical treatment such as chlorine, nitric oxide, peroxyacetic acid, hydrogen peroxide, quaternary ammonia, and ozone are effective in reducing the microbial load in wash water. However, they have limited efficacy for the bacterial inactivation on fresh produce, by causing food oxidation, and possible risk of releasing carcinogenic by- products [15-18]. On the other hand, plant-based antimicrobials are alternative as they are often devoid of the many side effects as compared to synthetic chemicals. A potential use of plant-based ingredients with optimized formulations in washing treatment might be an alternative way to reduce or replace harmful chemicals use. Therefore, this study was designed to evaluate antimicrobial activities of natural ingredients such as vinegar, lemon juice and essential oil, and to compare antibacterial effectiveness of cocktail combinations against foodborne pathogens.
Materials and Methods
Collection of natural antimicrobials
Fresh lemons and different brands of conventional vinegar: white distilled vinegar, seasoned rice vinegar, balsamic vinegar, apple cider vinegar, and red wine vinegar were purchased from local grocery stores in Alabama, USA. Lactic acid (L – (+)-) solution (88-92%), Origanum oil (100%) were purchased from Sigma- Aldrich Company (St. Louis, USA) and stored at 4 °C ±0.5 until use.
Screening of natural antimicrobials
Selected vinegars are diluted with sterile distilled water to prepare 4% of stock solution. For lemon juice extract (LJ), fresh lemons were washed and cut with a sterile knife. The household juicer was used to obtain fresh LJ. Extracted crude lemon juice was filtered using filter paper (Whatman® No. 4). Filtered LJ was dissolved into sterile water (1:1 V/V) to make 50% lemon juice.
Culture preparation
Antimicrobial activities of natural chemicals were tested against L. monocytogenes (ATCC 13912) and Salmonella enteric Typhimurium (ATCC 51812). Test bacteria were obtained from the stock culture collection of the Food Safety Laboratory, Department of Food and Nutritional Sciences, Tuskegee University. Stock cultures were stored in Tryptic Soy Broth (Fulka analytical 22091, Sigma-Aldrich) at below 4 °C. Cultures were streaked onto TSA plates and incubated at 37 °C for 18±1 h. Subsequently, a colony of L. monocytogenes and Salmonella Typhimurium was aseptically inoculated into 5 mL TSA broth and incubated at 37 °C for 18±1 h. To achieve a viable cell population of 8-9 Log CFU/mL, 100 μL of bacterial suspension was transfer into 5 mL TSA broth and incubated at 37 °C 18±1 h. Bacterial culture was harvested by centrifugation at 5,000 rpm (Brofuge 22R, Heraeus Instruments, Inc., USA) for 5 min at 4 °C. The supernatant was carefully discarded, and the pellet was resuspended in sterile peptone water (0.1%) and thoroughly mixed by vortexing. Centrifugation and experiments were performed at least twice throughout the study. The suspension of washed cells was serially diluted (1:10) up to 6 Log10 CFU/mL in sterile peptone water (0.1%) to obtain an appropriate cell concentration. Defined numbers of inocula were determined by colony counting from the 18 h culture grown on TSA from each diluent. Optical density (at 600nm) of bacterial culture solution was measured using a spectrophotometer (Genesys 10 UV, Thermo Electron Corporation) and adjusted to 0.87, approximately 106-7 CFU/mL of bacterial populations.
Determination of antimicrobial activities using agar diffusion assay
The agar diffusion assay was performed to screen and evaluate the antimicrobial activities of natural wash solutions. Tryptic Soy Agar (soft) was prepared in Petri dishes (90 mm diameter) with approximately 106-7 CFU/mL of bacterial concentration. Sterile cork borer was used to prepare wells in the agar plates. In each well, 40 μL of treatment was carefully dispensed and incubated at 37 °C for 24 h. Recorded zone of inhibition (ZOI) from the agar diffusion assay, measurements were taken from the clear zone around the well, including the well diameter. The test was performed in duplicate for confirmation.
Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC)
The MICs of treatments were determined by standard broth microdilution. A 96-well microplate was used to determine the MICs. TSA broth was used as diluents and growth medium for the bacteria. Initially, 150 μL of TSA broth was aseptically dispensed into micro-wells, and an equal volume of the treatment was added, making a total of 300 μL as final volume. Subsequently, the mixture of TSA broth and treatments were properly mixed, and two-fold serial micro-dilutions were performed. Standardized inocula of 6-7 Log CFU/mL in 10 μL was added into each well except positive control (without treatments) and incubated at 37 °C for 24 h. The results were confirmed through turbidity resulted from the bacterial growth. Turbidity in each well were measured after 24 h. The MICs were recorded as the lowest concentration of treatment by no growth observation which means no turbidity is measured. The MBCs of natural ingredients were determined by standard broth microdilution. All the microwells containing organisms and treatment along with positive and negative controls were sub-cultured onto TSA plate. Then each plate was divided and labeled into ten equal compartments and inoculated with 10 μL of suspension from microplate to respective compartments and incubated at 37 °C for 24 h. After incubation, each well was examined for bacterial growth and MBC. If bacterial growth is fully inhibited by natural solutions, it was recorded as MBC. The remained content of the 96 well plates used for MIC and MBC were further subjected to perform bacterial growth in the liquid medium. This test is performed by measuring the optical density (OD) of bacterial growth in each well using a Bio Teck plate reader (Bio Teck, USA) at 600 nm.
Preparation of antimicrobial natural wash solutions
Five different natural antimicrobial wash solutions were selected. Wash solutions were prepared in 100 mL of cultural bottles using sterile water. Natural antimicrobials used for wash solutions were 0.5% white distilled vinegar (DV), 25% crude lemon juice extract (LJ), and combination of both DV and LJ in origanum oil includes; DVO (DV + 0.1% origanum oil), LJO (LJ + 0.1% origanum oil), DVLJO (DV + LJ + 0.1% origanum oil), all the samples were in total Vol/Vol percentage. Wash solutions were prepared on the same day of the experiment and stored in the refrigerator at 4 °C ±0.5 until used. The pH of the wash solutions was measured by pH meter (Denver Instrument, Model 215, USA). The measurement was performed in triplicates at 23 °C±2.
A model study in the natural wash solution
In vivo study was performed to determine the antimicrobial activities of each wash solution (DV, LJ, DVO, LJO, and DVLJO) and sterile water (control). During the study, an inoculum of Salmonella spp. and L. monocytogenes were inoculated into wash solutions. The concentration of inoculum in the wash solution was 6-7 log CFU/mL. The wash solution contained test organisms continuously agitated in an automatic shaker in 100 rpm (Gyratory- Shaker Model G2, New Brunswick Scientific Co., USA) at room temperature (23 °C±2) for 25 min. The solution contained test organism was withdrawn (20 μL) at 2, 5, 10, 15, 20, and 25 min intervals and dispensed onto respective compartments of TSA plates and incubated for 24 h to examine the growth of inoculated organisms. Simultaneously, 100 μL of the solution was dispensed onto another set of TSA plates and spread homogenously, incubated for 24 h to determine the exact number of colonies. The process was replicated for the confirmation of the results.
Statistical analysis
Throughout the study, all experiments with assays were carried out in triplicates. Analysis of variance among the treatments from collected data was performed by repeated measurement analysis of variances (RM- ANOVA) of the general model procedure using a statistical system SAS 9.3 (SAS Inst., Cary, N.C., U.S.A.). Mean comparisons were calculated using Fisher’s Protected least significant difference. Plate count data for bacterial populations were converted to a logarithmic scale before statistical analysis. The level of significance was determined at P < 0.05.
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