Antibacterial Activity of Lotus Leaves (Nelumbo Nucifera) Against Food-Borne Pathogens

Corresponding Author: Yuanda Song School of Food Science and Technology, Jiangnan University, 1800 Lihu Rd, Wuxi, Jiangsu, China, 214122, China Email: cxt214@126.com Abstract: The antibacterial activity of different solvent extracts from lotus leaves against four food-borne bacteria (Escherichiacoli, Salmonellatyphimurium, Staphyloccocusaureus, Bacillussubtilis) was analyzed by agar diffusion method and the ethanol extract of the plant was found to have the highest antimicrobial activity with the diameter of inhibition zones ranging from 17.2 to 17.8 mm. Then the ethanol extract of lotus leaves were further separated by MCI-gel column and the antibacterial activity were investigated viamacrodilution broth method. Furthermore, preliminary phytochemical test were also carried out. Fraction 3 eluted from MCI-gel was found to have highest antibacterial activity with the minimum inhibitory concentration and minimum bactericide concentration values in the ranges of 0.0313~0.125 g mL −1 and 0.0626~0.25 g mL −1 , respectively. Antifungal activity analysis showed that ethanol extract of lotus leaves had a low inhibitory activity against all fungi. The result of the phytochemical analysis of the extract showed the presence of phenolic compounds, flavones and alkaloids which may be responsible for its antibacterial activity. Furthermore, lotus leaves extract was observed to have a better antiseptic capacity than sodium benzoate in apple juice (preservative used in food). Therefore, lotus leaves may be used as a botanical natural food preservative against food-borne pathogens.


Introduction
Spoilage of food products is due to chemical, enzymatic or microbial activities.One-fourth of the worlds food supply is lost through microbial activity alone (Ghaly et al., 2010). Although the chemical food additives have a good effect on microorganism control and have been widely used in food preservation, it is reported that synthetic chemicals like paraben, have been found to have harmful effects (Ishiwatari et al., 2007). Besides, a series of food safety issues has aroused consumers' increasing concerns and their wish of pursuing more natural and safer antimicrobial compounds. Some natural substances of plantorigin, phenolic compounds and secondary metabolites suchas flavonoids, tannins, alkaloids, organic acid and essential oils, have been intensively reportedto be biologically active, especially endowed with antimicrobial properties (Cowan, 1999).
Lotus (Nelumbo nucifera) is an angiosperm and classified in floating leaved plants among aquatic plants. It distributes widely in Hunan, Hubei, Zhejiang and Jiangsu Provinces of China. China is rich in lotus leaves resources. Lotus leaves has been included in the second batch of "both food and medicine" list by the Ministry of Health of the People's Republic of China in 1999, which is consideredassafe in traditional food processing. Many bioactive and pharmacologically important compounds have been found in Nymphaeas pecies and used in medicine. It has been reported that the quercetin extracted from lotus leaves may be a potential antibacterial agent for periodontitis (Li and Xu, 2008). Furthermore, the flavonoids, alkaloids and volatile oils from lotus leaves has been reported have strong inhibition on bacteria but no distinct inhibition on yeast and mold (Yihong, 2007). Besides the antibacterial activity, lotus leaves extracted by methanol and ethanol was reported to possess antioxidant activities (Choe et al., 2010). With the above information, the lotus leaves were tested for antibacterial activity against some food-borne pathogens. The aim of the research was to evaluate the antimicrobial activity and qualitative analysis phytochemicals of lotus leaves.

Reagents and Equipment
Lotus leaves were collected from local ponds at Wuxi, China in October, 2012. Petroleum ether, chloroform, ethyl acetate, n-butyl alcohol Dimethylsulphoxide (DMSO), sodium benzoate, oxford plant were purchased from Sino harm Chemical Reagent Co. Ltd. MCI-gel column (CHP20/P120) was purchased from Mitsubishi Chemical Holdings Corp.

Extract Preparation
Lotus leaves were dried at room temperatureandcrushed into powders with a grinder xinnuo,Co,Ltd,Shanghai,China). Thepowders (20 g) were extracted by maceration in different solvents, petroleum ether, chloroform, ethyl acetate, n-butyl alcohol, ethanol and aqueous successively for 12 h. Thereafter, the extracts were filtered through Whatman No. 1 filter paper and condensed by rotary evaporator at 45°C, the dried extractions were dissolved in 100% Dimethyl Sulfoxide (DMSO) to a final concentration of 1 g mL −1 (defined as 20 g lotus leaves/20 mL DMSO) and stored in 4°C for further analysis.

Inhibition Zone Test
The inhibition zones were determined by agar diffusion method (CLSI, 2012) with some modifications. Tested bacterial and fungi were cultured in nutrient broth at 37 or 28°C for 24 h, then 100 µL of suspension bacteria and fungi spores were coated on the plats. Holes (6 mm in diameter) were made by Oxford cup and impregnated with 100 µL of different solvent extracts. The DMSO was used as negative control. Sodium benzoate (100 mg mL −1 ) was used as positive control. All the plates were incubated at 37°C for 24 h. Each assay was replicated three times.

Determination of Minimum Inhibitory Concentration (MIC), Minimum Bactericide Concentration (MBC) and Minimum Fungicidal Concentration (MFC)
MIC is the lowest concentration that completely inhibits growth of the organism in the tubes as detected by the unaided eye. The MIC were determined by macrodilution broth method (Wikler, 2009). The tested lotus leaves extract added to tubes contains autoclaved nutrient broth to keep the final concentrations of extract in the range of 31.3~500 mg mL −1 . All the bacteria and yeast cultures were adjusted to 0.5 McFarland standard contain approximately 1 to 2×10 8 CFU mL −1 , then the adjusted in oculum suspension in broth was diluted to keep the final bacteria concentration of 5×10 5 CFU mL −1 . Hemocytometer was used to control the final fungi spores concentration of 0.4 to 5×10 4 cells mL −1 (CLSI, 2002). MIC values were recorded after incubated for 16 to 20 h.
The inoculated tubes showing no increases in turbidity were coated on plats. The plates were incubated for 24 h at 37°C for bacterial strains and for 24 to 72 h at 28°C for fungal strains. The corresponding concentration of the extracts on which the growth of the microorganism was totally inhibited was determined as the MBC or MFC values.

Phytochemical Screening
The antibacterial fractions isolated by MCI-gel column were subjected to phytochemical tests as described by (Morsy, 2014) to determine the presence of various chemical compounds.

Antiseptic Capacity in Apple Juice
The fresh apple juice was used to evaluate the antiseptic capacity of lotus leaves extract in food system. The ethanol extract of lotus leaves dissolved in water was added to the apple juice with the final concentration of 0.05% (w/v). The same concentration of sodium benzoate was used as the positive control. Then, the apple juice was stored at 37°C. The total counts of bacteria were determined by diluting the apple juice with water to a suitable concentration and 1 mL of the sample was coated on the surface of Luria-Bertaniagar culture medium. Colony counting after 24 h of incubation at 37°C wasperformed at 0, 3, 6, 9, 12 and 15 days. All procedures were performed under sterile conditions.

Statistical Analysis
Descriptive statistical analyses for calculating the means and the standard deviation of the mean were performed using the Statistical Package for Social Sciences (SPSS) version 19 program. A probability value at p<0.05 was considered statistically significant.

In Vitro Antibacterial Assay
The antibacterial activity of the plant extract can be judged according to the size of the inhibition zone. As shown in Table 1, the petroleum ether and aqueous extracts of lotus leaves were not effective against the growth of bacteria tested. Comparatively, the ethanol extract exhibited the highest inhibitory effect on four tested bacteria with the diameter of inhibition zones ranging from 17.2±0.6 to 17.8±0.3 mm.
The inhibitory effect of ethanol extract was higher than the positive control sodium benzoate, which is effective food preservative of bacteria spoilage. The inhibitory effects of other solvent extractions are relatively weak with the inhibition zone less than 17 mm.

In Vitro Antifungal Assay
The ethanol extract of lotus leaves was also tested for its antifungal activity, as shown in Table 2, the lotus extract exhibited comparative inhibitory effect to sodium benzoate against S.cerevisiae with inhibition zone of 13.4 mm, where as neither lotus leaves extract nor sodium benzoate had any antifungal activity (inhibition zone) against the myceliumof A.nigerand P.citrinum. Ethanolextract of lotusleaves showed MIC/MFC valuesof 0.25/0.25 (g mL −1 ) against S.cerevisiae, 0.5/1 (g mL −1 ) againstthe spore of A.niger and P.citrinum. Sodium benzoate showed MIC/MFC value of 0.0625/0.125 (g mL −1 ) against S. cerevisiae, 0.125/0.25 (g mL −1 ) against A.niger and P.citrinum.

Antibacterial Assay of MCI Gel Separated Samples
The antibacterial activity of five fractions separated by MCI-gel was assessed by the inhibition zones and MIC/MBC values. As shown in Fig. 1, all fractions except fraction 1 and 5 showed different degrees of antibacterial activity with the fraction 3 being found to be most effective against the tested bacteria with diameter of inhibition zones ranging from 13.5~15.3 mm, while as positive control the diameter of inhibition zones of sodium benzoate ranging from 12.7~14.1 mm. The result indicated that S.typhimurium was the most sensitive bacterium to the fraction 2, 3, 4 with the inhibition zone of 11.4, 15.3 and 14.2 mm, respectively.  NI a NI means no inhibition zone Each value is expressed as mean ± SD (n = 3) 100 mg mL −1 sodium benzoate was used as positive control DMSO was used as negative control a, b, c, d and e: Different letters within same column means significant different at p<0.05 Table 2. Antifungal activity of ethanol lotus leaves extract against food-borne pathogens Inhibition zone (mm) (Mycelium)  Table 3. Fraction 3 had the highest antibacterial activity which is consistent with the results of inhibition zones test, the MIC value of fraction 3 against the tested bacteria ranged from 0.0313 to 0.125 g mL −1 and MBC from 0.0626 to 0.25 g mL −1 , respectively. As a control, the solvent (10% DMSO) did not show any inhibition on the growth of the tested bacteria.

Phytochemical Screening
Results of the different phytochemicalscreening for the bioactive fractions of lotus leaves (Nelumbo nucifera) are shown in Table 4. Fraction 2, fraction 3, fraction 4 were positive for the presence of phenolic compounds and flavones. Fraction 3, but notfraction 2 and fraction 4, wasalso positive for alkaloids. In addition, all fractions were negative for tannins, organic acid, amino acids and proteins.

Antiseptic Capacity in Apple Juice
We used apple juice to evaluate the prevervation effect of lotus leaves extract by monitoring the total counts of bacteria. Since lotus leaves showed significant in vitro antibacterial activity, we proposed that lotus leaves might possess a potential antiseptic capacity in a food system. As shown in Fig. 2, lotus leaves extract had a better antiseptic capacity than sodium benzoate in apple juice.

Discussion
In vitro antibacterial assay results are in agreements with the findings of (Dubey et al., 2012) who reported that ethanol extract of most plants had effective antimicrobial activity against all the isolated multidrug resistant bacteria. Furthermore, the extracts (ethanol, n-butyl alcohol) of leaves showed significant activity against Gram-negative bacteria and Grampositive bacteria.While antifungal activity analysis showed that ethanol extract of lotus leaves had a low inhibitory activity against all fungi.
According to some reports the presence of secondary metabolites in plants such as alkaloids (Gurudeeban et al., 2013;Budeyri et al., 2012) and flavones (Islam et al., 2002;Li et al., 2012) had antimicrobial activities. This may explain why fraction 3 had the best antimicrobial activity, it indicated that the alkaloids and flavones may have synergistic effect against bacteria growth. While some alkaloids such as colchicine, aconitine, scopolamine, strychnine are toxic even they are isolated from natural product, there is no reports about the toxicological evaluation of lotus leaves alkaloids. It is indispensable for performing toxicological evaluation of lotus leaves alkaloids in the future for its safety applied in food.

Conclusion
Based on this study it may be concluded that the antibacterial activity of the lotus extract could be related to the presence of alkaloids and flavones components. Results of present study suggest that the lotus leaves extract possess antibacterial compounds that may be used as a botanical natural food preservative against food-borne pathogens.