Allium Tuberosum) Fermented by Leuconostoc Mesenteroides

Home , Allium, Allium ampeloprasum, Allium tuberosum, Chives

Appl Biol Chem (2016) 59(3):349–357 Online ISSN 2468-0842 DOI 10.1007/s13765-016-0166-7 Print ISSN 2468-0834

ARTICLE

Characteristics of Chinese chives (Allium tuberosum) fermented by Leuconostoc mesenteroides

Heeok Hong1 . Kai-Min Niu2 . Jun-Hyeong Lee2 . Sangbuem Cho3 . Sung-Gu Han4 . Soo-Ki Kim2

Received: 19 November 2015 / Accepted: 12 January 2016 / Published online: 1 February 2016 Ó The Korean Society for Applied Biological Chemistry 2016

Abstract This study was conducted to investigate the Haemophilus parasuis, Salmonella gallinarum, Escher- characteristics of bioactive compounds in Chinese chives ichia coli O157, and Burkholderia. sp. Although LO also juice (FC) fermented with Leuconostoc mesenteroides showed antibacterial effects against the above-mentioned SK1962, a lactic acid bacteria isolated from Kim-chi. L. pathogenic bacteria, its antibacterial activities were gen- mesenteroides SK1962 only (LO) was used as comparison. erally lower compared with those of FC. The results show The pH of FC gradually decreased from 6.21 to 4.23 during that the antioxidant and antibacterial activities in Chinese the 48-h incubation period, which was similar to that of chives was induced by fermentation with L. mesenteroides LO. The growth of L. mesenteroides SK1962 in FC was SK 1962. In conclusion, fermentation may lead to an higher compared with that in LO for various incubation increase in bioactive compounds including total polyphe- times, with the exception of the 8-h incubation. Total nol and ﬂavonoid. polyphenol and ﬂavonoid contents in FC were higher as compared with those in LO during incubation, leading to Keywords Antimicrobial activity Á Antioxidative increased antioxidant activity in FC at different incubation activity Á Chinese chives juice Á Fermentation Á times. Moreover, FC was more effective than LO in Leuconostoc mesenteroides reduction of superoxide free radical production in primary bovine mammary alveolar cells. In addition, FC demonstrated antibacterial properties against pathogenic bacteria such as Listeria monocytogenes, Pantoea agglomerans, Introduction

Historically, mankind has used various plants and their extracts for the treatment of diseases (Cowan 1999). Approximately 250,000–500,000 plant species have been & Soo-Ki Kim found on Earth, however, only 1 % of those plants have [email protected] been researched for their pharmaceutical potentials 1 Department of Medical Science, School of Medicine, Konkuk (Meleńdez and Capriles 2006). Plants and their extracts University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, possess numerous health benefits, and their functions can Republic of Korea be classified into three categories: direct antimicrobial, 2 Department of Animal Sciences and Technology, Konkuk immunomodulating, and growth promoting (Hernańdez University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, et al. 1984). These functions can be applied in the main- Republic of Korea 3 tenance and promotion of human health. The Chinese chive Animal Resources Research Center, Konkuk University, 120 (Allium tuberosum) is a monocotyledonous plant belonging Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea to the Allium genus. The Allium genus also includes garlics and onions, which have been recognized as rich sources of 4 Department of Food Science of Animal Resource, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, secondary metabolites such as polyphenols, flavonoids, and Republic of Korea sulfides (Havey et al. 2004; Wouters et al. 2013). In 123 350 Appl Biol Chem (2016) 59(3):349–357 particular, garlic chives have abundant vitamin A, vitamin Materials and methods C, minerals, and dietary fibers (Park et al. 2002). For many years, it has been utilized as foodstuffs and traditional Preparation of Chinese chives juice herbs due to its antioxidant, detoxification, and anticancer and fermentation medium effects. It has been reported that the strong antioxidant effects of Chinese chives are due to the presence of Allium tuberosum was purchased from the local market polyphenols and antioxidant vitamins (Lundega˚rdh et al. (Seoul, Korea). For the preparation of Chinese chives juice 2008; Bernaert et al. 2012). Furthermore, its sulfur-con- medium, the juice was prepared. Briefly, the tubers of A. taining compounds have also been shown to demonstrate tuberosum were cleaned through tap water and deskinned. antibacterial (Mostafa et al. 2000; Seo et al. 2001; Lun- The juice was prepared by grinding with juicer (Angel- dega˚rdh et al. 2008) and anticancer (Park et al. 2002; Xiao juicer, Busan, Korea), and solid particles were separated by et al. 2005) activities. Fermentation is known as a filtering using 4-layer gauzes. Then liquid fraction (filtrate) promising procedure for increasing the nutritional quality was used for medium preparation. For the fermentation of of plant foods (Frias et al. 2005), and improving biological chives juice, the filtrate was added to 29 MRS (de Man, activities of plant compounds including antioxidant and Rogosa and Sharpe) at a 1:1 (v/v) ratio, and pH was antimicrobial activities (Lee et al. 2004; Wu et al. 2011). In adjusted to 7.0. This media was named CMRS. addition, several products of microbial fermentation are incorporated into food as additives and supplements such Bacterial isolation for the fermentation of Chinese as antioxidants, anticancer agents, sweeteners, and preser- chives juice vatives (Couto and Sanromań 2006). Microorganisms play an important role in the production of a wide range of For the purpose of isolating bacteria involved in the fer- primary and secondary metabolites (Wijesinghe et al. mentation of Chinese chives without any inoculation, the 2012). For example, Leuconostoc sp., lactic acid bacteria, culture was incubated for 3 days as mentioned above, and is used in a large variety of dairy and food fermentations was spread on MRS agar media. Among the major bacteria (Kleerebezem et al. 1997). It is also used in the production that overcame the antibacterial activity of Chinese chives of several biologically useful materials such as polyphe- juice, one of the isolates was selected, and identified by nolic compounds and ginsenosides, which also possess molecular phylogenetic analysis based on the 16S rRNA antioxidative properties (Park et al. 2012; Yang et al. 2012; gene sequence. Polymerase chain reaction (PCR) was Bernaert et al. 2013). Therefore, there is increasing inter- employed for the analysis of 16S rRNA gene sequence ests in the physiochemical properties in the fermented using universal primers (27F, 50-AGA GTT TGA TCC vegetables. Several studies have tried to characterize the TGG CTC AG-30; 1492R, 50-GGT TAC CTT GTT ACG fermentation process of Allium plants. Bernaert et al. ACT T-30) (Weisburg et al. 1991). The sequences of PCR (2013) reported that spontaneous fermentation process was products were determined by the DNA analyzer (ABI highly related to the enhancement of biological activities PRISM 3730XL, Applied Biosystem, Franklin Lakes, NJ, such as antioxidant activity and profiles of flavonoids and USA). Identities of the sequences from isolated strains polyphenol. Another study concerning leek fermentation were searched at GenBank using BLAST (Basic local could be found in the fermentation or ripening of Kim-chi, alignment search tool). Multiple sequence comparisons Korean traditional fermented cabbage. Over ripening via were performed using the CLUSTRAL_W program rapid growth of some lactic acid bacteria in Kim-chi is a (Thompson et al. 1994). Calculation of evolutional distance factor of low quality cause of strong acidic tastes. With the and construction of phylogenetic tree were conducted to reason, Lee and Kim (1999) tried to supplement chive in identify the isolated strain by the Maximum Composite Kim-chi to suppress the growth of lactic acid bacteria Likelihood method using the MEGA 4 program (Tamura Leuconostoc involved in over ripening. Very recently, et al. 2004, 2007). Yang et al. (2014) have reported the isolation of lactic acid bacteria from leek Kim-chi and suggested Weissella con- Determination of total polyphenols fusa LK4 as a starter culture for leek fermentation. The present study was conducted to isolate a specific Total polyphenol content in the extracts was determined by bacterial strain that demonstrate good viability during the the modified Folin–Ciocalteu method (Wolfe et al. 2003). fermentation of the Chinese chives juice. We analyzed the Aliquot of each extracted sample (0.1 mL) was added to fermentative characteristics and biological activities of 2mL2%Na2CO3. Samples were vortexed for 10 min at Chinese chives juice fermented by Leuconostoc mesen- room temperature, and 0.1 mL 50 % Folin–Ciocalteu teroides SK1962. reagent was added. The mixture was vortexed for 15 s, and

123 Appl Biol Chem (2016) 59(3):349–357 351 allowed to react for 30 min at 40 °C. Absorbance of each added into the well plate. Then cells were incubated with a sample was measured at 700 nm using the Shimadzu UV- ﬁnal concentration of 1 lM dihydroethidium (DHE) or

1601 spectrophotometer. Samples of extracts were evalu- dimethyl sulfoxide (blank) in a 5 % CO2 incubator for ated at a final concentration of 1 mg/mL. Total polyphenol 30 min. Cells were washed with 3 9 PBS, fixed with 4 % content in gallic (25–500 ppm) was calculated using the formaldehyde, and washed again with 3 9 PBS. Slides following equation based on the calibration curve: were mounted with ProLong Gold Antifade reagent con- y = 0.0261x, R2 = 0.9897, where x was the absorbance, taining 40,6-diamidino-2-phenylindole (DAPI) (Invitrogen, and y was the gallic equivalent (mM/mL). Carlsbad, CA) to visualize the nuclei. Slides were evaluated under an Olympus IX71 fluorescence microscope and Determination of total flavonoids the images were captured digitally using an Olympus DP71 camera and DP controller software (Olympus Optical Co. Total flavonoids were estimated using previously published Ltd, Tokyo, Japan). The images shown are representatives methods (Ordonez et al. 2006). Samples (0.5 mL) were of three independent experiments. mixed with 1.5 mL 95 % ethanol, 0.1 mL 10 % aluminum chloride, 0.1 mL 1 M potassium acetate, and 2.8 mL dis- Antibacterial activity assay tilled water. After the tubes were mixed for 30 min at room temperature, the absorbance was measured at 415 nm. Antibacterial activities of fermented Chinese chives were Sample extracts were evaluated at a final concentration of tested by the paper disk diffusion method, as described 1 mg/mL. Total flavonoid content was calculated in quer- elsewhere (Valsaraj et al. 1997). The above media cetin (5–100 ppm) using the following equation based on including Chinese chives juice was fermented for 3 days the calibration curve: y = 0.0752x, R2 = 0.9996, where x with 1 % L. mesenteroides SK1962 at 30 °C. Aliquots (100 was the absorbance, and y was quercetin equivalents in mg/ lL) of the Chinese chive culture were dropped onto ster- mL. ilized paper disks 6 mm in diameter. The paper disk was dried for 30 min and placed on plates inoculated with DPPH assay pathogenic bacteria. The plate was incubated at 30 °C for 18 h. The diameter of clear zone was measured. The Free radical scavenging activities were determined in vitro pathogenic bacteria used are as follows: Listeria monocy- using the 2-diphenyl-2-picrylhydrazyl (DPPH) photometric togenes KACC 10550, Staphylococcus aureus assay, as described previously (Tadic et al. 2008) with KCCM40405, Burkholderia sp., Pantoea agglomerans, some modification. Fermented Chinese chives (100 lL) Haemophilus parasuis, H. somnus ATCC 9184, Salmonella were mixed with 900 lL 0.04 mg/mL DPPH in methanol gallinarum, and Escherichia coli O157. Some of these solution. The mixture was allowed to react for 30 min with pathogenic bacteria were obtained from the National gentle agitation. Reduction of violet color was induced by Veterinary Research Quarantine Service in South Korea. free radical scavenging activity, and absorbance was measured with a spectrophotometer at 517 nm. Each sol- vent used in the extraction was used as a negative control, Results and discussion and tert-butyl-hydroxytoluene was used as a positive control. The percentage inhibition was calculated by compar- Isolation of bacteria involved in fermentation ing sample absorbance with negative controls, and was of Chinese chives juice plotted against sample concentrations. Among the major bacteria types that were able to survive Determination of superoxide radical scavenging or overcome the antibacterial activity of Chinese chives activity in MAC-T cells juice, one of isolates was selected and sequenced for the 16S RNA gene. Based on the results of the molecular Superoxide radical scavenging activity was determined in phylogenetic analysis, it was determined that the isolate bovine mammary epithelial cells (MAC-T) by the method was closely related to L. mesenteroides LMG8159 (Ac- (Han et al. 2013). Samples were prepared by using only cession no HM443957), and was designated as L. mesen- Chinese chives juice (CO), supernatants of fermented MRS teroides SK1962 (Fig. 1). L. mesenteroides is known as a broth only (LO) and Chinese chives (FC) mixing with MRS heterofermentative lactic acid bacterium, and it is reported medium as 1:1 (v/v) by L. mesenteroides for 24 h. MAC-T to be involved in the fermentation of various fruits and cells were grown to confluence on cover glass in 6-well vegetables with other lactic acid bacteria in the Lacto- plate. Final concentration of each sample (50 lL/mL) was bacillus and Pediococcus genera (Cho et al. 2006).

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Fig. 1 Phylogenetic tree based on the 16S rRNA gene sequence of isolate SK1962 and other strains belonging to the Leuconostoc genus. Bootstrap values (based on 1000 trials and only values [50 %) are shown at the nodes. Bar denotes 0.01 substitutions per nucleotide position

Effect of Chinese chives juice on various lactic acid and CMRS. Only Lactobacillus lactis was sensitive to both bacteria CMRS and CO. L. lactis SK2083 was more sensitive to Chinese chives juice as compared with L. lactis SK2085. Because Chinese chives juice is known to have an Therefore, CMRS was reconﬁrmed to be good media for antibacterial activity, it is questionable whether various the fermentation of Chinese chives juice by L. mesen- lactic acid bacteria including the isolated strain L. mesenteroides SK1962. teroides can grow normally on CMRS media. As shown in Table 1, CMRS did not show any antibacterial activity pH changes and cell growth of L. mesenteroides against L. mesenteroides SK1962, whereas Chinese chives SK1962 at varying concentrations of Chinese chive juice only (CO) cultures demonstrated antibacterial activity juice (11.3 mm). Weissella sp. also displayed similar patterns as the L. mesenteroides SK1962. However, Lactobacillus The change of pH and growth of L. mesenteroides SK1962 brevis, Lactobacillus plantarum, Lactobacillus sakei, and at different concentrations of Chinese chive juice were Lactobacillus reuteri showed strong resistance to both CO estimated. As shown in Fig. 2A, the pH was 6.5 in LO (L. mesenteroides SK1962 only cultured on MRS broth), while it was 7.0 in FC (Chinese chives juice fermented by L. Table 1 Antibacterial activity of Chinese chives juice against vari- mesenteroides SK1962) at the start of the incubation per- ous lactic acid bacteria iod. The pH fell sharply after 8 h of incubation, and Strain Clear zone diameter (mm) gradually decreased at different incubation times in both CMRS CO treatments. After 48 h of incubation, the pH was 4.16 in LO and 4.15–4.33 in FC. One of the characteristic features Lactobacillus brevis SK1304 ND ND of fermentation by lactic acid bacteria is the resultant Lactobacillus sakei SK1958 ND ND decrease in pH due to the production of organic acids Leuconostoc mesenteroides SK1962 ND 11.3 ± 0.6 during fermentation (Wu et al. 2011). In the presence of Lactobacillus lactis SK2083 19.7 ± 0.6 25.3 ± 2.1 Chinese chives, pH was higher than the control until 24 h Lactobacillus lactis SK2085 17.0 ± 1.0 21.3 ± 1.5 of incubation. After 24 h, no remarkable difference was Lactobacillus reuteri SK2636 ND ND detected between the pH of FC and LO. Weissella sp. SK3493. ND 8.0 ± 0.0 Even though Chinese chive is well known for its Lactobacillus plantarum SK3494 ND ND antibacterial activity (Lee et al. 2004), L. mesenteroides CMRS Chives juice mixed with 2 X MRS broth at a 1:1 (v/v) ratio; SK1962 showed normal growth in 10–50 % Chinese CO Chives juice only chives juice during incubation (Fig. 2B). However, its Hole diameter was 6 mm growth in LO dropped sharply after 24 h incubation. ND not detected Results indicate that addition of Chinese chives juice 123 Appl Biol Chem (2016) 59(3):349–357 353

(CO, 2.63 mM). Previous studies by Bernaert et al. (2012) showed that total phenolic acid content varied between 5 and 15 mg GAE per gram of dry weight in 30 leek culti- vars. In addition, fermentation of leek resulted in an increase in polyphenolic compounds as compared with that of fresh leeks. In addition, the polyphenol content of FC was 53–89 %, which was higher than that of LO. Speciﬁ- cally, the polyphenol content of FC was 83 and 89 % higher as compared to that of LO after 16 and 24 h of incubation, respectively. It was possible that total polyphenol content decreased due to biodegradation polyphenol compounds in Chinese chives during the process of fermentation. Similarly, Othman et al. (2009) also reported the loss of antioxidant phenolic compounds during fermentation of Chetoui olives.

Effect on total ﬂavonoid content during the fermentation of Chinese chives juice

Total flavonoid content in FC was maintained at 1.04–1.14 mg/mL during fermentation (Fig. 3B); flavonoid content in FC was the highest at 24 h, at 1.14 mg/mL. However, flavonoid content of FC was less than that of Chinese chives juice only (CO), which was measured to be 1.17 mg/mL. This suggests that the total flavonoid content was slightly decreased by fermentation. On the other hand, the total flavonoid content of FC was two times that of LO, Fig. 2 Changes in pH (A) and cell growth (B) as a function of which slightly increased from 0.38 mg/mL to 0.44 mg/mL Chinese chive juice concentration during the fermentation. No agreements have been reached in previous studies promoted the growth of L. mesenteroides SK1962 as regarding the total flavonoid content in fermented and non- compared with control, suggesting that L. mesenteroides fermented plants. The flavonoid content was increased in SK1962 can be used to ferment Chinese chives, and is not Lactobacillus-fermented Graptopetalum paraguayense E. inhibited by its antibacterial activity. Furthermore, it was walther,asobservedbyWuetal.(2011). It is possible that the shown that Chinese chives juice could be used as a good variability in flavonoid content may be due to specific features nutrient source for the growth of L. mesenteroides SK 1962 of biodegradation of plant compounds during fermentation. cells. Therefore, maximum 50 % addition of Chinese chives juice was used for the fermentation in further study. DPPH-radical scavenging activity during fermentation of Chinese chives juice Effect on total polyphenol content during the fermentation of Chinese chives juice Figure 3C shows antioxidant activities in LO and FC during the fermentation. DPPH-radical scavenging activity The effect of Chinese chives juice on polyphenol content of CO was 2.74 mM, while that of FC was a little higher was represented in Fig. 3(A). Total polyphenol content was than CO at different fermentation times. Antioxidant initially measured to be 5.67 mM prior to incubation, and activity of FC were 2.78–2.97 mM during 24 h of incu- this dropped following the 8-h incubation in FC. After 16- bation. Antioxidant activities in FC and LO were main- and 24-h incubations, polyphenol content transiently tained for the first 24 h, and then decreased to 2.25 mg/mL increased before decreasing to 3.44 mM. In LO, total in FC and 1.93 mg/mL in LO at 48 h. This is because polyphenol gradually decreased from 3.50 to 2.25 mM polyphenol and flavonoid contents also decreased at 48 h. It during the 48-h incubation. Total polyphenol content was also observed that antioxidant activities of FC were increased 31–87 % at various incubation times in the higher as compared to those of LO at different fermentation Chinese chives juice fermented by L. mesenteroides times. This may account for the higher total polyphenol SK1962 (FC) as compared with Chinese chives juice only and flavonoid contents in FC compared to those of LO. 123 354 Appl Biol Chem (2016) 59(3):349–357

Fig. 3 Changes in biological activities of Chinese chives juice during fermentation by L. mesenteroides SK1962. (A) Total polyphenol content. (B) Total ﬂavonoid content. (C) DPPH-radical scavenging activity. Filled and empty circles mean L. mesenteroides only in medium and fermented Chinese chives juice media, respectively. FC Chinese chives juice fermented by L. mesenteroides SK1962

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Fig. 4 Superoxide free radical scavenging activity in MAC-T cells (E) Addition of low concentration of fermented chives into treated during fermentation of Chinese chives juice by Leuconostoc mesen- MAC-T cells with DHE. (F) Addition of high concentration of teroides SK1962. (A) MAC-T cells without treatment of DHE. fermented chives into treated MAC-T cells with DHE. (G) Addition (B) Addition of DW into treated MAC-T cells with DHE. (C) Addi- of L. mesenteroides culture supernatant into treated MAC-T cells with tion of MRS medium only into treated MAC-T cells with DHE. DHE (D) Addition of chives only into treated MAC-T cells with DHE. Kusznierewicz et al. (2004) reported an increase in the observed increased DPPH-radical scavenging activity in antioxidant activity of cabbage following a 2-week fer- carrot juice after 2 days of fermentation with Lactobacillus mentation process. Bernaert et al. (2013) also reported that delbrueckii subsp. bulgaricus. fermentation of green leaves of leek for 21 days resulted in It is known that polyphenol acts as an antioxidant by a 62 % increase in antioxidant capacity as compared with scavenging free radicals and preventing ROS damage activity of fresh leek. The antioxidant activity increased (Perron and Brumaghim 2009). The present study showed with increasing phenolic acid content. Nazzaro et al. (2008) that enhanced antioxidant effect of fermented Chinese

123 356 Appl Biol Chem (2016) 59(3):349–357 chives juice originated from the increased polyphenol and Table 2 Antibacterial activities against pathogenic bacteria during ﬂavonoid contents. fermentation by L. mesenteroides SK1962 Pathogenic bacteria Clear zone diameter, mm Superoxide radical scavenging activity in MAC-T Cultured media CO cells during the fermentation of Chinese chives juice MRS CMRS

Superoxide free radical scavenging activity was estimated Pantoea agglomerans 10.0 ± 0.0 9.7 ± 1.2 ND by the intensity of red fluorescence in MAC-T cells. The Haemophilus parasuis 11.7 ± 0.6 9.7 ± 0.6 ND fluorescent dye DHE is sensitive to reactive oxygen species Salmonella gallinarum 10.7 ± 0.6 10.7 ± 1.5 ND such as superoxide through oxidation of the dye which Escherichia coli O157 11.0 ± 1.7 13.0 ± 1.0 ND results in staining cells with a bright fluorescent red. The Burkholderia. sp. 10.7 ± 1.2 12.3 ± 0.6 10.3 ± 2.1 red areas in the cells represent oxidized DHE representing Listeria monocytogenes 18.0 ± 1.0 18.3 ± 2.1 18.3 ± 1.5 the production of superoxide. The nuclei were stained with Antibacterial activities: 8 mm = no inhibition; 8 mm B very mild DAPI presenting blue color. As shown in Fig. 4, both the inhibition \ 12 mm); 12 mm B moderate inhibition \ 15 mm; culture supernatant of L. mesenteroides (LO) and non-fer- 15 mm B strong inhibition mented chives (CO) decreased superoxide production in L. mesenteroides SK1962 was cultured for 24 h MAC-T cells at 12-h post-treatment. However, fermented ND Not detected chives juice by L. mesenteroides was more effective in reducing superoxide production whether using low (25 lL/ mL) or high (50 lL/mL) concentration. Thus far, lactic acid bacteria is widely known for its antioxidant effect. Wu inhibition effect against L. monocytogenes and moderate et al. (2014) reported a strong antioxidant property on inhibitory effects against H. parasuis. Overall, antibacterial scavenging superoxide anion free radicals of different activities of FC were higher than those of LO. fractions of Lactobacillus, which could protect the Caco-2 Alzoreky and Nakahara (2003) reported weak antibac- cell from oxidative stress. Chinese chives have traditionally terial activity in extracts from some edible plants com- been used as nutraceutical foods containing abundant monly consumed in Asia. The antibacterial activity of antioxidant compounds. In the study, the enhancing scav- ginger extract exhibited no inhibitory effects on E. coli enging activity properly involved with the mechanism of (Chand 2013). Similarly, antibacterial activity of fresh fermentation. This may be caused by an increase in the Chinese chives had no inhibitory effect on E. coli O157 in amount of phenolic compounds and flavonoids from the this study. The diameter of the clear zone on E. coli for the degradation of Chinese chives during microbial fermenta- garlic extract was 9.67 mm in the study by Chand (2013), tion. It may also result from the liberation or synthesis of a while this study showed that the clear zone in fermented variety of antioxidant compounds inducing by the struc- Chinese chives was 13.00 mm. Therefore, it is possible that tural breakdown of cell walls of Chinese chives as sug- fermented Chinese chives have increased antibacterial gested in Hur et al. (2014). activity against E. coli as compared to fresh plants belonging to the Allium genus such as ginger, garlic, and leek. Therefore, our results showed that fermented Chinese Antibacterial activities during the fermentation chives with L. mesenteroides SK1962 could strongly of Chinese chives juice inhibit bacterial growth. According to the above results, it could be concluded Antibacterial activities were determined by measuring the that fermentation increases various bioactive compounds size of clear zone against the pathogenic bacteria: L. including polyphenols and flavonoid, which increases monocytogenes, P. agglomerans, H. parasuis, S. galli- antioxidant and antibacterial properties in L. mesenteroides narum, E. coli O157, and Burkholderia sp. As shown in SK1962-fermented Chinese chives. Table 2, antibacterial activities of CO showed the strongest inhibitory effect against L. monocytogenes and very mild Acknowledgments This research was supported by the Konkuk inhibitory effect against Burkholderia. sp. However, it had University. no inhibitory effect on P. agglomerans, H. parasuis, S. gallinarum, and E. coli O157. FC demonstrated antibac- References terial effects against all tested bacteria, exerted the strongest inhibitory effects against L. monocytogenes and E. coli Alzoreky NS, Nakahara K (2003) Antibacterial activity of extracts O157. In addition, LO also showed antibacterial effects from some edible plants commonly consumed in Asia. Int J Food against all pathogenic bacteria. LO demonstrated strong Microbiol 80:223–230 123 Appl Biol Chem (2016) 59(3):349–357 357

Bernaert N, De Paepe D, Bouten C, De Clercq H, Stewart D, Van Othman NB, Roblain D, Chammen N, Thonart P, Hamdi M (2009) Bockstaele E, De Loose M, Van Droogenbroeck B (2012) Antioxidant phenolic compounds loss during the fermentation of Antioxidant capacity, total phenolic and ascorbate content as a Chetoui olives. Food Chem 116:662–669 function of the genetic diversity of leek. Food Chem Park YJ, Kim MH, Bae SJ (2002) Anticarcinogenic effects of Allium 134:669–677 tuberosum on human cancer cells. Korean J Food Sci Technol Bernaert N, Wouters D, De Vuyst L, De Paepe D, De Clercq H, Van 34:688–694 Bockstaele E, De Loose M, Van Droogenbroeck B (2013) Park BG, Jung HJ, Cho YW, Lim HW, Lim CJ (2012) Potentiation of Antioxidant changes of leek (Allium ampeloprasum var. porrum) antioxidative and anti-inflammatory properties of cultured wild during spontaneous fermentation of the white shaft and green ginseng root extract through probiotic fermentation. J Pharm leaves. J Sci Food Agric 93:2146–2153 Pharmacol 65:457–464 Chand B (2013) Antibacterial effect of garlic (Allium sativum) and Perron NR, Brumaghim JL (2009) A review of the antioxidant ginger (Zingiber officinale) against Staphylococcus aureus, metabolisms of polyphenol compounds related to iron binding. Salmonella typhi, Escherichla coli and Bacillus cereus. J Micro- Cell Biochem Biophys 53:75–100 biol Biotechnol Food Sci 2:2481–2491 Seo KI, Moon YH, Choi SU, Park KH (2001) Antibacterial activity of Cho J, Lee D, Yang C, Jeon J, Kim J, Han H (2006) Microbial S-methyl methanethiosulfinate and S-methyl 2-propene-1-thio- population dynamics of kimchi, a fermented cabbage product. sulfinate from Chinese chive toward Escherichia coli O157: h7. FEMS Microbiol Lett 257:262–267 Biosci Biotechnol Biochem 65:966–968 Couto SR, Sanromań MA (2006) Application of solid-state fermen- Tadic´ VM, Dobric´ S, Markovic´ GM, Dordevic´ SM, Arsic´ IA, tation to food industry: a review. J Food Eng 76:291–302 Menkovic´ NR, Stevic´ T (2008) Anti-inflammatory, gastropro- Cowan MM (1999) Plants products as antimicrobial agents. Clin tective, free-radical-scavenging, and antimicrobial activities of Microbiol Rev 14:564–584 Hawthorn berries ethanol extract. J Agric Food Chem Frias J, Miranda ML, Doblado R, Vidal-Valverde C (2005) Effect of 56:7700–7709 germination and fermentation on the antioxidant vitamin content Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large and antioxidant capacity of Lupinus albus L. var. Multolupa. phylogenies by using the neighbor-joining method. Proc Natl Food Chem 92:211–220 Acad Sci USA 101:11030–11035 Han SG, Newsome B, Hennig B (2013) Titanium dioxide nanopar- Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular ticles increase inflammatory responses in vascular endothelial evolutionary genetics analysis (MEGA) software version 4.0. cells. Toxicology 306:1–8 Mol Biol Evol 24:1596–1599 Havey MJ, Galmarini CR, Go¨kce AF, Henson C (2004) QTL Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: affecting soluble carbohydrate concentrations in stored onion improving the sensitivity of progressive multiple sequence align- bulbs and their association with flavor and health-enhancing ment through sequence weighting, position-specific gap penalties attributes. Genome 47:463–468 and weight matrix choice. Nucleic Acids Res 22:4673–4680 Hernańdez L, Munõz RA, Miro´ G, Martıńez M, Silva-Parra J, Cha´vez Valsaraj R, Pushpangadan P, Smitt UW, Adsersen A, Nyman U PI (1984) Use of medicinal plants by ambulatory patients in (1997) Antimicrobial screening of selected medicinal plant from Puerto Rico. Am J Hosp Pharm 41:2060–2064 India. J Ethnopharmacol 58:75–83 Hur SJ, Lee SY, Kim YC, Choi IW, Kim GB (2014) Effect of Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S fermentation on the antioxidant activity in plant-based foods. ribosomal DNA amplification for phylogenetic study. J Bacteriol Food Chem 160:346–356 173:697–703 Kleerebezem M, Beerthuyzen MM, Vaughan EE, de Vos WM, Wijesinghe WAJP, Lee WW, Kim YM, Kim YT, Kim SK, Jeon BT, Kuipers OP (1997) Controlled gene expression systems for lactic Kim JS, Heu MS, Jung WK, Ahn G, Lee KW, Jeon YJ (2012) acid bacteria: transferable nisin-inducible expression cassettes Value-added fermentation of Ecklonia cava processing by- for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl product and its antioxidant effect. J Appl Phycol 24:201–209 Environ Microbiol 63:4581–4584 Wolfe K, Wu X, Liu RH (2003) Antioxidant activity of apple peels. Kusznierewicz B, S´miechowska A, Bartoszek A, Namiesńik J (2004) J Agric Food Chem 51:609–614 The effect of heating and fermenting on antioxidant properties of Wouters D, Bernaert N, Conjaerts W, Van Droogenbroeck B, De white cabbage. Food Chem 108:853–861 Loose M, De Vuyst L (2013) Species diversity, community Lee GC, Kim YK (1999) Changes in fermentation characteristics of dynamics, and metabolite kinetics of spontaneous leek fermen- kimchi added with leek. J Korean Soc Food Sci Nutr 28:780–785 tations. Food Microbiol 33:185–196 Lee CF, Han CK, Tsau JL (2004) In vitro inhibitory activity of Wu SC, Su YS, Cheng HY (2011) Antioxidant properties of Chinese leek extract against Campylobacter species. Int J Food Lactobacillus-fermented and non-fermented Graptopetalum Microbiol 94:169–174 paraguayense E. Walther at different stages of maturity. Food Lundega˚rdh B, Botek P, Schulzov V, Hajslov J, Stro¨mberg A, Chem 129:804–809 Andersson HC (2008) Impact of different green manures on the Wu D, Sun MZ, Zhang C, Xin Y (2014) Antioxidant properties of content of S-alk(en)yl-L-cysteine sulfoxides and L-ascorbic acid Lactobacillus and its protecting effects to oxidative stress in leek (Allium porrum). J Agric Food Chem 56:2102–2111 CACO-2 cells. J Anim Plant Sci 24:1766–1771 Meleńdez PA, Capriles VA (2006) Antibacterial properties of tropical Xiao D, Pinto JT, Gundersen GG, Weinsein IB (2005) Effects of a series plants from Puerto Rico. Phytomedicine 13:272–276 of organosulfur compounds on mitotic arrest and induction of Mostafa MG, Mima T, Ohnishi ST, Mori K (2000) S-alkylcysteine apoptosis in colon cancer cells. Mol Cancer Ther 4:1388–1398 ameliorates doxorubicin toxicity in the heart and liver in mice. Yang EJ, Si Kim, Park SY, Bang HY, Jeong JH, So JH, Rhee IK, Planta Med 66:148–151 Song KS (2012) Fermentation enhances the in vitro antioxidative Nazzaro F, Fratianni F, Sada A, Orlando P (2008) Synbiotic potential effect of onion (Allium cepa) via an increase in quercetin of carrot juice supplemented with Lactobacillus spp. and inulin content. Food Chem Toxicol 50:2042–2048 or fructooligosaccharides. J Sci Food Agric 88:2271–2276 Yang J, Ji Y, Park H, Lee J, Park S, Yeo S, Shin H, Holzapfel WH Ordonez AAL, Gomez JD, Vattuone MA, Isla MI (2006) Antioxidant (2014) Selection of functional lactic acid bacteria as starter activities of Sechiumedule (Jacq.) Swart extracts. Food Chem cultures for the fermentation of Korean leek (Allium tuberosum 97:452–458 Rottler ex Sprengel. Int J Food Microbiol 191:164–171

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