Antibacterial Effect of Noni Juice In Vitro and Applied to Fresh-Cut Papaya to Control Escherichia coli O157 :H7

Unidad de Tecnoloǵıa de Alimentos, Secretaŕıa de Investigación y Posgrado, Universidad Autónoma de Nayarit, Ciudad de la Cultura S/N, Colonia Centro, C.P. 63000, Tepic, Nayarit, Mexico Programa de Doctorado en Ciencias Biológico Agropecuarias, Universidad Autónoma de Nayarit. Km. 9, Carretera Tepic-Compostela. C. P. 63780, Xalisco, Nayarit, Mexico Unidad Académica de Agricultura, Universidad Autónoma de Nayarit. Km. 9, Carretera Tepic-Compostela. C. P. 63780, Xalisco, Nayarit, Mexico


Introduction
e demand of consumers for safer, fresh, higher-quality foods with long shelf life has increased [1]. is has led to the development of preservation technologies through minimal processing techniques such as applying natural additives (such as polyphenols, organic acids, essential oils, and plant extracts) and physical methods (ozone-based treatment, ultraviolet radiation, pulsed light, cold plasma, ultrasound, and novel packing practices) [1][2][3]. Consequently, this kind of processing has become a subject of special care due to the presence of certain pathogens such as Escherichia coli O157 : H7, which is considered by the WHO [4] as one of the 14 pathogens causing severe gastrointestinal diseases. Nonetheless, there is little information about the behavior of these pathogens in precut fruits such as papaya [5].
Papaya is mostly consumed due to its sensory and functional proprieties [6]; nevertheless, this tropical climacteric fruit presents a short shelf life due to a rapid pulp softening and microbial contamination (less than eight days of storage) [7,8]. e food processing industry uses conservatives of synthetic origin to assure the quality and innocuousness of this fruit and others, which become a danger to the health of consumers and the development of resistance of microorganisms [9]. erefore, the search for natural conservatives with effective antibacterial properties that preserve the quality of a fresh-cut product and do not cause deterioration in the product's sensorial attributes has emerged [10]. In this regard, noni juice (NJ) has demonstrated an efficient activity for preserving sensory proprieties and microbiological control over fresh-cut fruits such as mango. e soaking of mango cubes in NJ had an antimicrobial effect for 15 days, reducing the count of aerobic mesophyllic, fungi, and yeast; besides, it maintained the color (CIE L * a * b * , Chroma, and Hue angle) during 12 days of storage [11].

Noni Juice Preparation.
Noni fruits (Morinda citrifolia L.) in the maturity of consumption were harvested in San Blas, Nayarit, Mexico. Fruits were carried to the laboratory of the Unidad de Tecnología de Alimentos of the Universidad Autónoma de Nayarit, in Tepic, Nayarit, Mexico. Fruits were washed with tap water; then, the fruit was placed in sealed plastic jars and left in natural fermentation into the dark for 60 days at 28 ± 3°C [11,18]. Fermented juice was filtered and centrifuged for 15 min at 3,050 xg (Centrifuge 5804 R, Eppendorf AG, Germany). e supernatant was decanted and stored in dark jars at 25°C until its use. e NJ yield was 84% w/w (juice/original weight of fruit). e physicochemical characteristics of the NJ were pH 3.46 ± 0.01, titratable acidity of 0.1 ± 0.011% of citric acid, and 9.9 ± 0.2°Brix.

Papaya Preparation.
Fruits of papaya (Carica papaya L.) var. Maradol at consumption maturity (edible condition) were acquired in a local market. Fruits were disinfected by immersion in a sodium hypochlorite solution at 200 ppm for 1 min [19]. After that, papaya fruits were rinsed with water, peeled, and then cut into cubes of 8 cm 3 .

Inhibitory Evaluation of NJ In Vitro.
e inhibitory effect of NJ was determined in liquid culture and measured with a microplate reader (Multiskan GO model 1510, ermo Fisher Scientific Oy, Vantaa, Finland) using 96 well plates. Each well (sample) contained 150 µL of TSB at concentrations of 10 4 , 10 5 , and 10 6 cells/mL of E. coli and 50 µL of NJ using a TSB with strain as a control. Plates were incubated for 24 h at 37°C. e cell density was determined by measuring the absorbance at 600 nm [22]. e pH of the medium after NJ addition was 5.2 ± 0.1. e treatments were performed six times; each experimental unit was a well. e experiment was repeated three times. e minimum inhibitory concentration (MIC) was considered the lowest concentration of NJ that inhibits the growth of E. coli [23]. Each well of the microplate contained 95 µL of nutrient broth, 5 µL of the inoculum (E. coli at 1 × 10 7 cells/mL), and 100 µL of NJ at concentrations of 50, 40, 30, 20, 10, 5, and 2.5%. Subsequently, the plates were incubated for 24 h at 37°C, shaking for 20 s each h. e bacterial growth was determined by measuring absorbance at 600 nm using a microplate reader (Multiskan GO model 1510, ermo Fisher Scientific Oy, Vantaa, Finland). e treatments were performed six times, and each experimental unit was a well. e experiment was repeated three times.

Antibacterial Evaluation of Noni Juice In Vivo.
e experiment was divided into two stages. In the first stage, 200 g of fresh-cut papaya fruit were immersed in an E. coli suspension (1 × 10 5 CFU/mL) for one minute. en, the remaining suspension was drained out. en, fresh-cut papaya fruits were immersed in pure NJ, evaluating three treatments (immersion in NJ for 0, 2.5, and 5 min) and a control (immersion in sterile distilled water). In the second stage, the NJ effect was evaluated on total coliforms natives of the papaya using the same treatments of immersion as previously described but with no immersion in pathogen suspension.
e treatments were stored in polyethylene domes at 4°C and monitored for 18 days, with a sampling every third day to evaluate the presence of total coliforms. Fresh-cut papaya cubes were homogenized in a stomacher (Stomacher 400, AbaTec, México). en, serial dilutions were prepared. e counting plate method was used to determine the number of CFU of either E. coli or total coliforms (CFU/g) using VRBA. E. coli was determined in papaya cubes that were inoculated, and total coliforms were determined in papaya that were not inoculated [24].

Physicochemical Analysis of Papaya.
For 18 days, physicochemical parameters (pH, titratable acidity, and total soluble solids (TSS)) were evaluated according to the Official Methods of Analysis of AOAC [25]. e pH was measured in a Denver pH-meter model 250 (Denver Instrument Company, Denver, CO, USA); TSS was measured by refractometer Auto Abbe model 10500 (Reichert-Jung, Leica Inc., Buffalo, NY, USA). Titratable acidity was determined by NaOH 0.1 N neutralization. e results were expressed in % of citric acid (AOAC, 2000.939.05).

Statistical
Analysis. Data were analyzed by analysis of variance (ANOVA) with a statistical significance of 5%. e Tukey test (P ≤ 0.05) was used when ANOVA found statistical differences. Statistical analyses were performed using STATISTICA 10 software.

In Vitro Treatments.
e NJ inhibition effect against E. coli O157 : H7 at concentrations of 10 4 , 10 5 , and 10 6 cells/mL is shown in Figure 1. As can be seen, all the concentrations treated with NJ maintained a static growth behavior compared to control. e control treatments stop growing between 14 and 17 h of incubation because, during this period, they reach the stationary phase. Besides, the maximum growth was different for each inoculum concentration. ese results may be due to bacteria in the stationary phase accumulating toxic catabolic products in the environment in a short time, resulting in a decrease in the number of viable cells, also known as the death phase [26]. e MIC was determined using seven NJ concentrations starting from an E. coli concentration of 1 × 10 7 cells/mL. Figure 2 represents the growth behavior of the bacteria. e lowest NJ concentrations (10, 5, and 2.5%) showed no inhibitory effect on E. coli, in contrast with higher concentrations (50, 40, 30, and 20%). is result suggests a static effect, which was corroborated with the maximum rate value (µmax). Table 1 shows significant differences among the (µmax) of the treatments (P < 0.05). e lowest concentration able to decrease the microbial growth was NJ 20% (µmax -0.0066 h −1 ), while bacteria control (E. coli suspension of 1 × 10 7 CFU/mL) had a µmax 0.36175 h −1 . Also, Table 1 shows the values of phase lag, although there was not a relationship between the concentration of NJ and the time of the lag phase. e antibacterial activity of noni fruit and noni juice has been related to the diversity of bioactive compounds they contain. Deng et al. [26] identified iridoids (deacetylasperulosidic and asperulosidic acids) from noni fruit with antibacterial activity against E. coli, Candida albicans, and Staphylococcus aureus. Anugweje [27] reported secondary   metabolites like phenols, steroids, terpenoids, alkaloids, tannins, flavonoids, saponins, glycosides, reducer sugars, and acid compounds. Wall et al. [28] found organic acids in NJ such as acetic, ascorbic, butyric, citric, dehydroascorbic, galacturonic, malonic, succinic, shikimic, and tartaric acids. ese organic acids are responsible for NJ's pH, which was 3.16 ± 0.05 [11]. Negi [10] summed up some natural antimicrobial action mechanisms, describing that terpenoids and phenols can cause cell membrane disruption. Besides, phenols and flavonoids can chelate metals. Also, the authors mentioned that coumarins and alkaloids could affect the genetic material producing microbial inhibition. NJ could be considered as one of the fermented juices with higher antimicrobial activity. Sripraya and Trachoo [29] found that fermented juices of lime, orange, kaffir lime, pineapple, avocado, star fruit, and sugar cane had antimicrobial activity. ese fermented juices showed pH values between 3.1 and 3.4; however, unlike NJ, these juices' antimicrobial activity was related to acidity.
On the other hand, some authors found differences in the MIC to control E. coli. Natheer et al. [30] reported inhibition of E. coli due to the application of NJ at 25 mg/mL. Tintino et al. [31] elucidated the inhibitory capacity from ethanolic extract of noni fruit at 50-100 mg of dry extract/ mL, testing in vitro against Pseudomonas aeruginosa and Escherichia coli. ese variations in MIC may be related to the tolerance capacity of the bacteria to noni juice. In this study, the MIC of NJ to the bacteria was 20% (200 mg/mL), a high concentration compared to those reported by Natheer et al. [30] and Tintino et al. [31]. A possible explanation is that the bacteria studied in this work could present similar resistance mechanisms to those observed with the antibiotic rifampicin [20,21].

Antibacterial Evaluation of NJ In Vivo.
In the first stage of treatments applied to fresh-cut papaya fruit (Figure 3(a)), i.e. samples were immersed for one min. In E. coli suspension, the bacterial population was constant on papaya control (without NJ immersion).
ere were no significant statistical differences throughout the storage days (P > 0.05), keeping a concentration between 2 and 3 log 10 CFU/g of fresh-cut papaya fruit. Singh and Shalani [32] mentioned that pH, water activity, and temperature act as barriers against the growth of microorganisms. However, Abadias et al. [33] and O'Beirne et al. [34] reported similar behavior to those presented in this work. ese authors, as well as Strawn and Danyluk [5], mentioned that at 4 ± 1°C E. coli O157 : H7 could survive due to papaya's intrinsic factors such as pH, which could have influenced the E. coli population to remain stable. As seen in the later section, the papaya cubes treated with NJ and E. coli maintained a pH of between 4.88 and 5.72 throughout the experiment.
Concerning monitoring the papaya treated with noni juice, in treatment 3 (immersion in NJ for 5 min), logarithmic decreases of E. coli were observed. On day 0, a reduction of 3.7 log units regarding the initial inoculum was observed; however, an increase of 0.3 log units concerning control was found on day zero. On the other hand, a reduction of 1.6 log 10 UFC/g of fresh-cut papaya fruit was found on the sixth day of storage regarding control. Furthermore, there was a reduction of 3.4 log 10 UFC/g of fresh-cut papaya fruit on the 12 th day of storage regarding control (Figure 3(a)). Likewise, in treatment 2 (immersion in NJ for 2.5 min), there was a decrease of 2.96 log 10 CFU/g of the population of E. coli on the 9 th day of sampling relative to control. In treatment 1 (immersion for 0 min), we found a difference of about 1 log10 CFU/g relative to the control during the experiment, which shows an antibacterial effect even when the exposure time to NJ is minimal.
Although the NJ had a bacteriostatic effect on E. coli in the in vitro treatments, it had a bactericidal effect in vivo treatments.
is may be due to differences in the composition of the culture medium used in vitro treatment and the composition of papaya. Zampieri et al. [35] mention that differences between carbon sources can affect the resistance to antimicrobials due to changes in energy metabolism and intracellular and extracellular putative metabolite profiles. e bacteriostatic effect in vitro and the bactericide effect in vivo of the NJ could be related to its metabolites. e presence of compounds such as coumarins, flavonoids, phenolic acids, and iridoids has been reported in NJ [36]. Kumar et al. [17] mention that the antibacterial effect could be due to iridoids or other antimicrobial compounds in noni fruit. Atkinson [37] reported that the antimicrobial effect of noni fruit is due to the presence of phenolic compounds such as L-asperuloside, acubin, scopoletin, alizarin, and other anthraquinones. Duncan et al. [38] showed that scopoletin inhibits E. coli. Likewise, Sina et al. [16] reported the phytochemical composition of M. citrifolia fruit juice, finding chemical compounds such as alkaloids, flavonoids, tannins, leuco-anthocyanins, saponosids, reducing compounds, and O-heterosids in fresh and fermented juice. Kang and Song [39] mention that the hydroalcoholic extract of noni fruit causes cell damage and increases the cell membrane permeability of Listeria monocytogenes, which could cause the death of the cell. Nagalingam et al. [40] conducted extractions with ethanol and methanol of noni fruit, identifying the presence of steroids, flavonoids, tannins, and saponins, which are also attributed to antimicrobial properties.
In the second stage (Figure 3(b)), papaya control (no treated with E. coli), the total coliforms population was kept at 3 log 10 CFU/g approximately. On the other hand, in fresh-cut papaya fruit immersed between 0 and 2.5 min in NJ, the total coliform population was kept between 0 and 1 log 10 CFU/g for nine days. In contrast, for the freshcut papaya fruit immersed for 5 min in NJ, total coliforms were maintained between -1 and 1 log 10 CFU/g until the 18 th day. Differences between samples treated and nontreated with E. coli can be due to the initial inoculum concentration and that the total coliforms could be more sensitive than E. coli. e strain of E. coli used in this study is resistant to the antibiotic rifampicin [20]. Martinez [41] mentions that bacteria can present diverse antibiotic resistance mechanisms, which could help E. coli resist the effects of the noni juice metabolites. is resistance of E. coli to NJ is not very efficient in vivo treatments. It could be due to NJ having several metabolites with different action mechanisms [36], the carbon sources available on the papaya cubes [35], and the intrinsic factors of the papaya such as pH and acidity [5]. However, the immersion time could be an important factor in the inhibition of E. coli and total coliforms. Alam et al. [42] mention that the immersion time in osmotic processes is related to solute gain. In this sense, with the longer immersion time, the metabolites present in NJ could migrate into the papaya cubes, making the inhibitory effect of E. coli and total coliforms more effective.

Total Soluble Solids.
e TSS results of fresh-cut papaya fruit during storage at 4 ± 1°C, control, and treated with E. coli by the effect of the immersion time in NJ are shown in Figure 4(a) and Figure 4(b), respectively. On day 18, the samples, either without inoculum and treated by NJ immersion or with inoculum and treated by NJ immersion, had a significant decrease in TSS content (P > 0.05). However, the magnitude of treatments varied from 3.16% to control and 14.9% to the treatment of 0 min of immersion.
is is similar to the report by Riviera-López et al. [43] on papaya Maradol stored in refrigeration. ey reported that the product stored at 5°C had lower respiration rates and showed a small decrease in TSS content.
is indicates that NJ and E. coli did not significantly affect the TSS of the fresh-cut papaya fruit (P > 0.05).

pH Values.
e behavior of pH in fresh-cut papaya during their storage at 4 ± 1°C, not inoculated, and inoculated with E. coli, for the effect of the immersion time in NJ is shown in Figures 5(a) and 5(b), respectively. e fresh-cut papaya treated with NJ has a lower pH than those treated with water (control); this may be due to the organic acids present in the NJ. e treatment control, either in the presence or absence of E. coli, showed a significant decrease in the pH values (P < 0.05). ese changes in the pH values during the storage period may be associated with the growth of microorganisms and the subsequent production of organic acids [44]. In general, the papaya fruit immersed in NJ kept the pH values constant, with a slight increase at the end of the storage period.
Fajar-Falah et al. [45] found that organic acid content will decrease during the ripening process, but TSS and pH will increase; however, acidity increases (a pH decrease) at the end of the shelf life of the fresh produce as an indicator of fresh product for the consumer. is means that the freshcut product is not acceptable to eat. e abovementioned indicates that the papaya cubes untreated with NJ lose their quality quickly, and they are not acceptable for consumption on day 3.

Titratable Acidity.
e papaya acidity tended to increase in both treatments (immersion in E. coli and not immersion in E. coli treatment) at the end of the experiment. Besides, NJ treatment for 0, 2.5, and 5.0 min ( Figure 6) showed that the longer the immersion time in NJ, the acidity value is higher.
In the treatments without the addition of E. coli, a decrease in the acidity is shown during the first 12 days of storage, to then increase significantly at day 15 (P < 0.05) and end with a decrease in acidity, that is, normal behavior in precut fruits [45].
However, the fresh-cut papaya was immersed in a suspension of E. coli and (Figure 6(b)) showed a constant increase in acidity throughout the storage period. is behavior has been observed in papaya cubes by edible coating and stored at 5°C for 12 days [46]. NJ could contain polysaccharides [13][14][15][16], which could form an edible coating [47].

Conclusions
Based on the results obtained in this study, NJ showed a bacteriostatic effect on E. coli in vitro. Besides, the application of NJ to fresh-cut papaya fruit showed bacterial inhibition in vivo, evidencing it as a possible bacterial control agent in the precut fruit industry. e immersion of fresh-cut papaya on NJ for 0 min is enough to control Escherichia coli O157 : H7; however, an immersion for at least 2.5 min is necessary to control others coliforms bacteria. e precut fruit immersion time in NJ is a critical factor in bacteria survival. However, further studies are needed regarding the conservation of the physicochemical and sensory properties of the fruit when applying the NJ.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of Interest.  Journal of Food Quality 7