Antibacterial activity and mechanism of B-type oligomeric procyanidins from lotus seedpod on enterotoxigenic Escherichia coli

Journal of Functional Foods 38 (2017) 454–463

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Antibacterial activity and mechanism of B-type oligomeric procyanidins from lotus seedpod on enterotoxigenic Escherichia coli Cuie Tang a,b, Bijun Xie a, Zhida Sun a,⇑ a b

College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China Key Laboratory of Environment Correlative Dietology, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 28 March 2017 Received in revised form 4 September 2017 Accepted 14 September 2017

Keywords: Oligomeric procyanidins Antibacterial activity Membrane damage Enterotoxigenic Escherichia coli

a b s t r a c t This study aims to evaluate the antimicrobial activity and mechanism of B-type oligomeric procyanidins from lotus seedpod (LSPC) against two Enterotoxigenic Escherichia coli (ETEC) strains, and the effects of LSPC on the growth of six different beneficial bacteria were also investigated. The antimicrobial activity assay showed LSPC exerted its action on ETEC strains in a dose-manner, in which the growth of ETEC strains were promoted at low concentration but inhibited at the concentration higher than 0.8 mg/mL and 1.2 mg/mL respectively. In comparison, the tolerance of beneficial bacteria on LSPC was significant higher than ETEC strains, LSPC promoted the growth of 5 Lactobacillus strains at the concentration of 0.8 mg/mL. Cell morphology, cell permeability and cell integrity assays suggested that the mechanism of LSPC against ETEC strains was by disturbing cell membrane structure and function. The current results demonstrated the potential benefit of LSPC to the gut microbial regulation. Ó 2017 Published by Elsevier Ltd.

1. Introduction Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. There are many factors, including bacterial, viral and parasitic pathogen can cause the diarrheal (WHO, 2006). Enteric bacteria have been reported to be the causative agent for significant infectious diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals, in the meantime could decrease growth rate of weaned piglets (Holland, 1990). Escherichia coli strains are one of the main etiological agents involved in severe dehydrating diarrhea. In addition, enterotoxigenic Escherichia coli (ETEC) is one of diarrheagenic E. coli strains that would cause travelers’ diarrhea in adults and acute secretory diarrhea disease in young children and animals (Gomes et al., 2016). Moreover, diarrhea infections induced by ETEC is also an important cause of morbidity and mortality in

Abbreviations: ETEC, Enterotoxigenic Escherichia coli; E. coli, Escherichia coli; LSPC (B-OPC), oligomeric procyanidins of lotus seedpod; LPOPC (A-OPC), oligomeric procyanidins of litchi pericarp; DP, degree of polymerization; MIC, minimal inhibitory concentration; MBC, minimum bactericidal concentration; SEM, scanning electron microscopy; TEM, transmission electron microscopy; LB, LuriaBertani; MRS, de Man Rogosa and Sharpe. ⇑ Corresponding author. E-mail address: [email protected] (Z. Sun). https://doi.org/10.1016/j.jff.2017.09.046 1756-4646/Ó 2017 Published by Elsevier Ltd.

weaned piglets (Grange, Mouricout, Levery, Francis, & Erickson, 2002). ETEC colonizes the small intestine and produces one or two types of enterotoxins, including heat-labile enterotoxin (LT) and heat-stable enterotoxins (ST) (Fleckenstein et al., 2010). Annually, over 200 million people, in which mainly infant and young children involved, infect diarrhea induced by ETEC strains and lead to approximately 6000,000 deaths in developing countries (Walker, 2015). The intake of contaminated food and water remain the major cause of ETEC infection. Currently, fluid replacement therapy and antibiotics therapy are two major therapies for diarrhea (Dubreuil, 2013). Unfortunately, the uncontrolled using of antibiotics for killing the bacteria has caused resistant effect that will bring a huge safe threat to the whole public health (Zhang, Wu, Zhang, Lai, & Zhu, 2016). Furthermore, the increasing restriction on the using of antibiotics in the Europe has led to a pressing demand for alternatives. Dietary specific antibodies against ETEC fimbriae and/or against LT from eggs (Henning-Pauka, Stelljes, & Waldmann, 2003), and plasma (Niewold et al., 2007) were proven successful but are relatively costly (Harmsen et al., 2005). Therefore, a growing interest has been paid in natural therapeutic agents such as plant polyphenol as an effective alternatives, which could reduce diarrhea infections in animals for showing high antimicrobial activities to pathogen bacteria and the ability to mediate composition of microbial community (Dubreuil, 2013).

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The beneficial effects of polyphenol on human health by modulating gut microbiotic composition have been widely reviewed (Cardona, Andres-Lacueva, Tulipani, Tinahones, & Queipo-Ortuno, 2013; Hervert-Hernández & Goñi, 2011), numerous studies have also demonstrated that polyphenol showed not only antimicrobial action on selective pathogenic bacterial (Bansal et al., 2013; Cushnie & Lamb, 2011; Das, Islam, Marcone, Warriner, & Diarra, 2017; Govardhan Singh, Negi, & Radha, 2013; Pertuzatti et al., 2016; Singh et al., 2016; Taguri, Tanaka, & Kouno, 2004), but also stimulation effect on the growth of beneficial bacteria (Cueva et al., 2010; Hervert-Hernandez, Pintado, Rotger, & Goni, 2009; Parkar, Redgate, McGhie, & Hurst, 2014). Furthermore, the potential of its antidiarrheal function on ETEC have also been demonstrated. It is reported that specific polyphenol can inactivate the heat-labile toxin of ETEC by forming LT–polyphenol aggregates (Verhelst, Schroyen, Buys, & Niewold, 2013). And Bruins et al. showed black teat extract can reduce diarrhea infected by ETEC in pig model (Bruins et al., 2006, 2011). Many other polyphenol extracts from plant have also been claimed to be effective on antidiarrheal (Lacombe, Tadepalli, Hwang, & Wu, 2013), however, few of them have been extensively studied (Dubreuil, 2013). Therefore, the selection of potential active polyphenol from different sources will be a promising agent to cure diarrheal. Procyanidins are complex polyphenols composed of flavan-3-ol units, which are widespread in plants and daily foods such as cinnamon, sorghum bran, lotus seed pod, litchi pericarp, berries, fruits, tea, dark chocolate, and wine (Li et al., 2015; Mena, Calani, Bruni, & Rio, 2014). Procyanidins can be classified into B-type or A-type based on different characteristic linkages between structure units (Mendoza-Wilson et al., 2016; Ou & Gu, 2014). Literatures showed that procyanidins exhibited high antibacterial activity on many different pathogenic bacteria, and they were the effective component on Escherichia coli (E. coli) strains in the polyphenols. Bhattacharya et al. concluded that catechin was one of the major antibacterial compounds against E. coli O157:H7 and E. coli (ATCC 25922) in the polyphenolic fractions of Kombucha (Bhattacharya et al., 2016). Karioti et al. showed that procyanidin B3 and prodelphinidin C exhibited lower minimum inhibitory (MIC) and bactericidal concentration (MBC) on E. coli (ATCC 35210) in the comparison with the other 12 phenolics from Quercus ilex leaves (Karioti et al., 2011). Moreover, oligomeric procyanidins fraction have been reported that they displayed higher antibacterial activity on E. coli (ATCC 25922) as compared to its monomer (Mayer et al., 2008). So, oligomeric procyanidins might be a promising effective antibacterial agent to pathogenic E. coli. Generally, compounds which showed active on the inhibition of other pathogenic E. coli, would most probably exhibit the same effect on ETEC strains (Dubreuil, 2013). However, the action of procyanidins against ETEC strains still only had barely reported, and the antibacterial mechanism is also unclear. As a traditional medicine, lotus seedpod had been recorded that it was able to cure bloody flux and the splenic diarrhea (Ni, 2005), and it was also used to prepare the disinfectant in clinical department (Lu, 2015). However, the functional composition involved in lotus seedpod for antidiarrheal and antibacterial hadn’t been investigated yet. Previous researches of our laboratory indicated that the oligomeric procyanidins from lotus seedpod (LSPC) displayed high antioxidant activity (Ling, Xie, & Yang, 2005), and played a plenty of beneficial effects on human health, such as immunityenhancing, modulation of the gut flora and memory-enhancing (Li, Sui, Wu, Xie, & Sun, 2017; Xiao et al., 2015; Zhang, Duan, Deng, Yang, & Xie, 2005; Zhang, Cheng, Luo, & Duan, 2016). And the potential antibacterial of LSPC on staphylococcus aureus had also been demonstrated (Wang, Qi, Xie, Shi, & Cai, 2004). Therefore,

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the objective of this research was to evaluate the antibacterial activity of oligomeric procyanidins from lotus seedpod on two ETEC strains, and its effect on the growth of six different beneficial bacteria were also investigated. In addition, the action mechanism of LSPC on ETEC strains was illustrated by different analysis, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), and flow cytometry analysis. 2. Materials and methods 2.1. Chemicals and materials HPLC grade (+)-catechin, ( )-epicatechin and propidium iodide were obtained from Sigma–Aldrich (St. Louis, MO, USA). LuriaBertani (LB) broth, LB agar, MacConkey Agar, de Man Rogosa and Sharpe (MRS) broth and MRS agar medium were purchased from Hope Bio-technology (Qingdao, China). All other reagents used in this research were analytical grade and water was Milli-Q quality. Fresh lotus seedpod was obtained from Honghu Lantian (Hubei, China), and fresh litchi (Litchi chinensis Baila) was purchased from Guangzhou to collect litchi pericarp. These two-plant materials were stored at 18 °C before using. 2.2. Extraction and sample preparations A-type and B-type oligomeric procyanidins were extracted from fresh lotus seedpod and litchi pericarp, the extraction and analysis methods were based on our laboratory methods (Li et al., 2012; Xiao et al., 2012). As comparing to commercial grape seed procyanidins, the purity of B-type oligomeric procyanidins from lotus seedpod (LSPC) and A-type oligomeric procyanidins from litchi pericarp (LPOPC) measured by Butanol-HCl assay were 98.65 ± 0.53% and 99.56 ± 1.2% respectively, and their main compositions reported by our laboratory were shown in Table 1 (Li, Sui, Li, Xie, & Sun, 2016; Li et al., 2012). 2.3. Bacterial strains and culture conditions Two ETEC strains used in the present research were K88ac (C83715), which was obtained from the China Institute of Veterinary Drug Control, and F18ac (2134P), which was donated by Prof. Pengjian from College of Animal Science in Huazhong Agricultural University. Both strains were stored in 15% (v/v) glycerol stocks at 80 °C and sub-cultured monthly before used. Lactobacillus rhamnosus HN001 (ATCC SD5675), Lactobacillus plantarum LP-115 (SD5209), Lactobacillus paracasei LPC-37 (ATCC SD5275), Streptococcus thermophilus ST-21 (ATCC SD5207); Bifidobacterium breve BB-03 (ATCC SD5206) were provided by Ò DuPontTM DaniscoÒ (Shanghai, China), and Bifidobacterium BB-12 Ò was purchased from HANSEN (BB-12 , Chr. Hansen, Hónsholm, Denmark). All the probiotics were prepared from freeze-dried powder stocks ( 80 °C). K88ac and F18ac were cultured 4–6 h in LB broth at 37 °C with shaking at 200 rpm routinely to obtain cells in the exponential phase of growth. The cultures were washed three times, resuspended in 0.9%NaCl and then assayed for cell numbers. All the Lactobacillus powders, except Bifidobacterium breve BB-03 and BB12 that cultured under an-aerobically condition (85%N2-10% H2-5%CO2) with 0.05% cysteine supplement, were incubated in MRS agar plates at 37 °C for 24 h, and then re-cultured overnight in the fresh MRS broth at the same conditions. These bacteria were harvested by centrifugation (4500g, 5 min), washed three times in 0.9%NaCl and assayed for cell numbers.

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Table 1 The composition of LSPC and LPOPC. Components (%) a

LSPC LPOPCb

(+)-Catechin

( )-Epicatechin

Dimers

Trimers

Tetramers

Total

10.9 2.7

9.1 37.92

53.6 28.3

19.5 19.8

1.9 1.6

95.0 90.32

Date resource from Li et al., 2016. a LSPC contains B-type dimers, trimers and tetramers. b LPOPC contains A-type dimers, trimers and tetramers.

2.4. Antimicrobial activity

2.7. Transmission electron microscopy (TEM) analysis

2.4.1. Concentration effects of procyanidins The minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) of procyanidins against ETEC were determined by the broth micro-dilution method as described by CLSI (CLSI, 2012). Briefly, procyanidins were dissolved in LB medium with concentration of 6 mg/mL, and then serially diluted twofold in the same medium containing approximately 5  105 CFU/ mL of ETEC strains. The mixture incubated into 96-microtiter plates with concentrations ranged from 6 to 0.0047 mg/mL at 37 °C for 15 h and all the pH of growth medium had not detected significant change. The MIC was defined as the lowest concentration that inhibited the growth of strains, and the MBC was determined as the lowest concentration that led to no colony appearing on the plate after 20 h incubation. After MBC test, the effect of procyanidins against ETEC strains from 0 to the concentration of MBC value were monitored by counting viable cells on LB agar plate at the same culture condition. All these assays were performed in triplicate.

The intracellular organization changes treated with or without procyanidins examined by TEM. The sample preparation was the same as SEM. The ETEC were treated with 2  MBC and 4  MBC concentration of procyanidins for 12 h. After overnight fixing with 2.5% (v/v) glutaraldehyde, the bacterial cells were treated as described by Cao et al. (2011). After post fixation in 1% osmium tetroxide for 2 h at room temperature, the cells were stained in 5% uranyl acetate (w/v) in 50% (v/v) of ethanol for 1 h. After dehydration, the cells were embedded in SPI-812 and polymerized at 60 °C for 12 h. For TEM examination, 50–60 nm thick sections were cut by ultra-microtome, mounted onto copper grids and then stained with 2% uranyl acetate, and observed with H-7650 transmission electron microscope (Hitachi Instruments Inc., Tokyo, Japan) at an operating voltage of 80 kV. Images were recorded with 832 ORIUS camera (Gatan, Pleasanton, CA, USA).

2.5. Effects of procyanidins on probiotics strains The effects of procyanidins on the growth curve of six different probiotics were carried in the MRS liquid media. Procyanidins were dissolved in MRS media to give concentrations of MIC and MBC of the K88ac. After filtration with 0.2 lm sterile membrane, 100 lL sample cultures were added to the 96 well micro-plate with approximately 107CFU/mL of probiotics. All the cultures except Bifidobacterium breve BB-03 and Bifidobacterium BB-12Ò that cultured under an-aerobically condition (85%N2-10%H2-5%CO2) with 0.05% cysteine supplement, were incubated statically at 37 °C for 24 h. The growth of these bacteria were followed by measuring the A600nm of the cultures every 2–3 h during the incubation period. All the growth experiments were repeated at two separated occasions in triplicate. 2.6. Scanning electron microscopy (SEM) analysis The morphological changes of ETEC strains with or without LSPC treatment were observed by SEM as described by Lv et al. (2014) with some modifications. Logarithmic growth phase cells of two ETEC strains were harvested by centrifugation (4500g, 5 min, 4 °C) and re-suspended (OD600  1.0) in 0.85% sterile saline. Procyanidins were added to obtain concentration of 2  MBC and 4  MBC, and then the control (without procyanidins) and samples were incubated at 37 °C for 7 h. Control and treated cell suspension were harvested by centrifugation (4500g, 5 min) and rinsed twice with 0.1 Mphosphate buffer solution (PBS, pH 7.2). The cells were re-suspended and fixed overnight at 4 °C with 2.5% (v/v) glutaraldehyde in PBS. After centrifugation, the cells were dehydrated using a graded ethanol (30%, 50%, 70%, 90% and 100%) and 100% acetone series. Finally, the samples were placed on SEM support, and then coated with gold-palladium under vacuum for 5 min. The examination was carried out with Su-8010 scanning electron microscope (Hitachi Instruments Inc., Tokyo, Japan).

2.8. Effect of procyanidin on cell membrane permeability The extracellular K+ concentration was measured to assess the damage on cell membrane permeability by procyanidins. The sample was treated as the same as SEM, the ETEC were treated with0, 1, 2 and 3  MBC concentration of procyanidins for 19 h, 2 mL control and treated cell suspensions were taken at various time. After centrifugation (4500g, 5 min, 4 °C), the supernatants were filtrated by 0.2 lm membrane, and the K+ content of the supernatants were determined by 6300 atomic absorption spectroscopy (SHIMADZU, Co. Ltd. Suzhou, China) 2.9. Effect of procyanidins on cell membrane integrity The membrane integrity damage of two ETEC strains treated by procyanidins was evaluated as previously described by Otto et al. with some modifications (Otto, Cunningham, Hansen, & Haydel, 2010). Logarithmic growth phase cells of two ETEC strains were washed and re-suspended to approximately 108 CFU/mL in 0.85% sterile saline. Procyanidins were added to obtain concentration of 2MBC and 4MBC, and then the control (without procyanidins) and samples were incubated at 37 °C for 2 h. After centrifugation, the cell pellet was washed twice and re-suspended in the same saline. The cells were stained with 20 lM propidium iodide (PI) in the dark for 20 min. The cell suspensions were then washed and examined by BD FACS Verse flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 15 mW, 488 nm argon ion laser, the FL2 channel was used to analysis the red fluorescence of PI-stained cells and FlowJo X 10.0 software was used for the data analysis. Furthermore, 5 lL of cell suspensions were fixed on a microscope slide then examined by fluorescence microscopy (DM3000, Leica, Germany) at 640 nm. 2.10. Statistical analyses Statistical analysis was carried out on SPSS software, and data were recorded as mean values of three replicates along with their standard deviation were performed in triplicate. The significant

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differences in means were tested by the method of One-way analysis of variance (ANOVA) with significant level a = 0.05. 3. Results and discussion 3.1. Antimicrobial activity 3.1.1. Antimicrobial activity of LSPC on ETEC strains In this study, we first compared the antibacterial action of oligomeric procyanidins of lotus seedpod (LSPC), oligomeric procyanidins of litchi pericarp (LPOPC) and their monomer against K88ac (C83715) and F18ac (2134P) ETEC strains by MIC and MBC assays (Table 2). LSPC and its monomer catechin exerted higher antibacterial activity (MIC 0.8–1.25 mg/mL and MBC 1.25–1.5 mg/mL) than LPOPC (MIC 2.5–3.5 mg/mL and MBC 3.5–4.5 mg/mL). And epicatechin, the monomer of LPOPC, hardly exhibited any activity against two test ETEC strains. It can be seen that procyanidins tested showed significantly different activities on different bacterial strains. This results was consistent with the previous research, in which the difference in the source, composition and the structure of the procyanidins may play a significant role in the antibacterial action (Xie, Yang, Tang, Chen, & Ren, 2014). To the best of our knowledge, no studies had reported the antibacterial activity of procyanidins against ETEC strains, but the similar effects on other E. coli as our results were observed. The same MIC value (1.25 mg/mL) of catechin on E. coli 8099 was reported by Zeng, Huang, Chi-Ling, and Liu (2009), and no inhibition effect of epicatechin against E. coli K 12 was also observed at 1.0 mg/mL (Amarowicz, Pegg, & Bautista, 2000). Our results indicated that the antibacterial activity of B-type oligomeric procyanidins (LSPC) was higher than A-type oligomeric procyanidins (LPOPC), MBC value of the former was almost 2 times lower than the latter, and the same effect was also observed between B-type and Atype procyanidins against C. albicans and C. neoformans (Kolodzeij, Kayser, Latte, & Ferreira, 1999). This variation of antibacterial activity between LSPC and LPOPC may be attributed to their linking method and their monomeric unit difference, in which LSPC was mainly composed by catechin, while the structure unit of LPOPC was epicatechin. So in this paper, we chose B-type procyanidins LSPC for further investigation. In addition to the inhibition effect of procyanidins against Escherichia coli, the simulation effect had also been reported, and the tested concentration range for a given procyanidins contributed to this totally different action. In order to better understand the dose responses of LSPC against ETEC strains, the concentration from 0 to 1.5 mg/mL (MBC value) was examined by plate count method on K88ac and F18ac. Results showed that LSPC exhibited similar concentration effect on both two tested ETEC strains, it inhibited the growth of ETEC strains at the concentration higher than 0.8 mg/mL, but enhanced their growth at the concentration lower than 0.8 mg/mL (Fig. 1). This antibacterial action of LSPC with dose-manner on ETEC strains may more accurately explain why catechin increased the growth of E. coli from 0 to 0.2 mg/mL (Freestone, Walton, Haigh, & Lyte, 2007; Tzounis et al., 2008), but inhibition effect was noticed from 0 to 1.2 mg/ mL (Shan, Cai, Brooks, & Corke, 2007; Vaquero, Alberto, & de

Fig. 1. The inhibition effect of LSPC concentration on two ETECs.

Nadra, 2007). The similar dose-dependent bacteriostatic effects of catechin against E. coli O157:H7 and E. coli had also reported (Dai, Wang, Kong, Peng, & Xiao, 2010; Freestone et al., 2007). All the reported data suggested that procyanidins may stimulate the growth of bacteria by providing extra carbon sources (Mena et al., 2014), providing essential iron under ion-restrictive condition for their iron-chelating ability (Freestone et al., 2007), or being metabolized to the phenolic acids metabolites that increased the growth of E. coli (Tzounis et al., 2008). And the inhibition mechanism of LSPC against the ETEC strains will be confirmed in the following sections. 3.1.2. Effect of LSPC on beneficial bacteria In order to better understand the effect of LSPC on the other beneficial bacteria in the gut, the effect of LSPC on the growth curve of six different Lactobacillus strains were evaluated at the concentration of MIC (0.8 mg/mL) and MBC (1.5 mg/mL) value of K88ac. The growth curves of the tested Lactobacillus strains incubated with LSPC were shown in Fig. 2. From Fig. 2, the data indicated that LSPC affected the growth of Lactobacillus strains tested in dose and strain-dependent manners. Compared with the control samples, the proliferation of Lactobacillus paracasei LPC-37 (ATCC SD5275) and Bifidobacterium BB-12Ò were significantly increased during the whole incubation time in the exposure of 0.8 mg/mL LSPC. Lactobacillus rhamnosus HN001 (ATCC SD5675), Lactobacillus plantarum LP-115 (ATCC SD5209) and Streptococcus thermophilus ST21 (ATCC SD5207) were slightly increased at the end of incubation, while Bifidobacterium breve BB-03 (ATCC SD5206) were slightly inhibited. However, when the treated concentration increased to 1.5 mg/mL, the growth of Bifidobacterium breve BB-03 (ATCC SD5206) and Lactobacillus plantarum LP-115 (SD5209) were slightly inhibited, while the other four species were significantly inhibited, which was agree with previous reports on Bifidobacterium spp (Tzounis et al., 2008; Viveros et al., 2011), Lactobacillus rhamnosus (Parkar, Stevenson, & Skinner, 2008), Lactobacillus

Table 2 Minimal inhibitory concentration (MICs) and minimal bactericidal concentration (MBCs) of procyanidin against two ETEC strains. Strains

K88ac F18ac a

LSPC

LPOPC

Catechin

Epicatechin

MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL)

0.80 ± 0.00 1.20 ± 0.00

1.50 ± 0.00 1.50 ± 0.00

2.50 ± 0.00 3.50 ± 0.00

3.00 ± 0.00 4.50 ± 0.00

1.25 ± 0.00 1.25 ± 0.00

1.25 ± 0.00 1.25 ± 0.00

>5.00 >5.00

nda nd1

nd, not detected.

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Fig. 2. Effect of LSPC on the growth curves of the probiotics. (A) Lactobacillus paracasei LPC-37. (B) Lactobacillus rhamnosus HN001. (C) Lactobacillus plantarum LP-115. (D) Streptococcus thermophilus ST-21. (E) Bifidobacterium BB-12Ò (BB-12Ò). (F) Bifidobacterium breve BB-03.

plantarum (Rodriguez, de las Rivas, Gomez-Cordoves, & Munoz, 2008) and other lactobacillus spp. In addition, the antibacterial activities of LSPC on the tested Lactobacillus strains were also checked by MIC assay, which revealed that all the beneficial bacteria still grew well at the concentration of 6 mg/mL (data not shown). The data showed that LSPC could enhance the proliferation of Lactobacillus paracasei, Bifidobacterium BB-12Ò, Lactobacillus rhamnosus, Lactobacillus plantarum and Streptococcus thermophilus at the concentration of MIC value of K88ac, implying it may act as a promoting factor to stimulate the growth of Lactobacillus strains by providing extra energy source or as a chelating agent just like the other polyphenols (Freestone et al., 2007; Hervert-Hernández & Goñi, 2011). Most of Lactobacillus strains had been proved that they could modulate the gut microbiota and play a beneficial role in human health (Bernardeau, Vernoux, Henri-Dubernet, & Gueguen, 2008). Specially, the Bifidobacteria, as a part of intestinal microflora, could affect the immune system network regulation, and then play a beneficial role in the gut ecology and host health (Mian et al., 2016; Parkar, Trower, & Stevenson, 2013). Notwithstanding, LSPC appeared to be beneficial in regulating the microbial growth through selectively inhibiting the growth of ETEC strains but promoting the growth of Lactobacillus at certain range of concentration. These in vitro action of LSPC in turn supported its potential effects to maintain the gastrointestinal health in vivo system, and it could be as a kind of promising therapeutic polyphenol to modulate the gut disorder problem infected by ETEC strain. 3.2. The antibacterial mechanism of LSPC on ETEC strains 3.2.1. SEM analyses SEM and TEM analyses were used to observe the morphological changes of K88ac and F18ac cells before and after treatment with LSPC. Visible bacterial membrane damage of ETEC strains following treatment with LSPC was obtained by SEM (magnification of 40,000). Fig. 3 showed SEM images of K88ac and F18ac cells treated with LSPC at its 2  MBC and 4  MBC for 12 h respectively. The control had regular morphology, a dump and smooth cell surface

(Fig. 3A and D), but treatment with LSPC induced significant membrane damage for both tested ETEC strains. The membrane surface of K88ac and F18ac cells treated with LSPC at 2  MBC became deflated and displayed irregularly wrinkles and small holes (Fig. 3B and E), but became completely distorted and displayed deep wrinkles and bigger holes by treatment with LSPC at 4  MBC (Fig. 3C and F). All the changes indicated that LSPC may induce alterations in the external structures of this two ETEC strains, causing leaking of cytoplasmic components such as proteins, nucleic acids, and K+. 3.2.2. TEM analyses The intracellular organization changes of ETEC strains with or without LSPC treatment were also visualized by TEM (Fig. 4). TEM images (Fig. 4A, Fig. 4D) displayed the intracellular structures for control K88ac and F18ac cells. Those two bacterial cells showed normal and even distribution of the cytoplasm and compact cell surface. On the contrary, K88ac and F18ac cells treated with LSPC were clearly damaged. After 12 h treatment with LSPC at 2  MBC, the separation of cytoplasmic membrane from the cell wall and the leakage of cytoplasmic content were observed (Fig. 4B and E). Furthermore, when treated with LSPC at 4  MBC, severely damages like membrane contraction, lower density, vacuolization and distortion were noticed for both tested ETEC cells treated (Fig. 4C and F). These changes observed in the TEM images at different treatment concentrations were in consistent with foregoing observed results by SEM, which further demonstrated that LSPC could induce seriously changes of intracellular organization of ETEC strains and showed their antibacterial action by the disruption of cell membrane. Similar observations for procyanidins or polyphenols against the other Escherichia coli and other bacteria were also reported (Polewski et al., 2016; Yi, Zhu, Fu, & Li, 2010; Zhao, Chen, Zhao, & Yu, 2015) 3.2.3. Effect of LSPC on cell membrane permeability Based on the results of SEM and TEM, LSPC showed the ability to disrupt membrane and structure of ETEC cells. However, the infor-

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Fig. 3. Scanning electron microphotographs of K88ac (A-C) and F18ac (D-F) treated with LSPC. (A) (D): untreated cell; (B) (E): cells treated with LSPC at 2  MBC level; (C) (F): cells treated with LSPC at 4  MBC level. Wrinkles, distortion (arrows #1) and holes (arrows #2) are visible.

Fig. 4. Transmission electron micrographs of K88ac (A–C) and F18ac (D–F) treated with LSPC for 7 h. (A) (D): untreated cell; (B) (E): cells treated with LSPC at 2  MBC level; (C) (F): cells treated with LSPC at 4  MBC level. The separation of cytoplasmic membrane from the cell wall and the leakage of cytoplasmic content (arrows #1), vacuolization (arrows #2) and distortion of the cell (arrows #3) were visible.

mation of images from SEM and TEM were limited, which didn’t explain how and why affected by LSPC. Hence, to better understand how dose of LSPC affects the cell membrane permeability of ETEC strains, the extracellular K+ ion concentrations of K88ac and F18ac with or without LSPC treatment were measured at different concentrations for various time (Fig. 5). As compared to the control cells, the ETEC cells treated with LSPC can cause significant leakage of intracellular K+ ions. In the exposure to LSPC for 1 h, the efflux of K+ ions for both K88ac and F18ac cells increased up to 2.5 mg/mL and 2.0 mg/mL respectively as the

treatment concentration increasing to 3  MBC. The extracellular K+ ion concentration for both ETEC cells treated with LSPC increased significantly during the first 6 h treatment, and the maximum K+ concentration reached to 4.5 mg/mL and 4.0 mg/mL for K88ac and F18ac, respectively. Moreover, F18ac seemed to be more resistant to LSPC at low concentration as compare to K88ac, which was agree with results of MIC assay. The extracellular K+ concentration increased demonstrated that LSPC could change the membrane permeability of ETEC cell in a dose-dependent and time-dependent manner.

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Fig. 5. Extracellular K+ level in K88ac (A) and F18ac (B) treated with LSPC at concentration of 0, 1, 2 and 3  MBC values.

3.2.4. Effect of LSPC on cell membrane integrity The red fluorescent probe propidium iodide (PI) was used to stain the nucleic acid of bacteria cells suffered with membrane integrity disruption (Chikindas et al., 1993; Joshi et al., 2010), and the fluorescence intensity of PI was investigated by flow cytometry and fluorescence microscopy. Results of flow cytometry analysis were presented in the histograms (Fig. 6). The quantity of bacterial cells disrupted in the bacterial population was displayed in a percentage, which was stained by PI. In the absence of LSPC, 97.8% of K88ac cells and 95.3% of F18ac cells presented no PI fluorescent signal, implying that the cell membranes were integrated.

However, when treated with the LSPC at the concentration of 2  MBC, cells stained increased up to 35.4% and 35.2% for K88ac cells and F18ac respectively. Almost all the cells (95.4% and 95.6%) were stained by PI after treating with LSPC at the concentration of 4  MBC. This dose-dependent assay indicated that LSPC possessed the ability to damage the cell cytoplasmic membrane of two test ETEC strains, and resulted in cell death. The membranes damaged of ETEC cells were observed by fluorescence microscopy, as showed in Fig. 7. The cells stained with red PI indicated the permeabilized cells that were dead. Quantitatively, for untreated K88ac and F18ac cells, only a few cell were stained

Fig. 6. Flow cytometric histograms of K88ac (A–C) and F18ac (D–F) treated with LSPC. (A), (D): untreated cell; (B), (E): cells treated with LSPC at 2  MBC level; (C), (F): cells treated with LSPC at 4  MBC level. Data acquisition was set to 10,000 events for each experiment. FL3-H channel represents the red fluorescence of PI.

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Fig. 7. Fluorescent images of bacterial cells of K88ac (A–C) and F18ac (D–F) treated with LSPC for 12 h. (A) (D): untreated cell; (B) (E): cells treated with LSPC at 2  MBC level; (C) (F): cells treated with LSPC at 4  MBC level.

with PI (Fig. 7A and D), and the number of rod-shaped PI-stained cells increased gradually for both tested ETEC strains (Fig. 7B, C, E and F) as treatment concentration of LSPC increasing. These results were further confirmed with the forward results observed by flow cytometry analysis. With the aid of extracellular K+ ion concentration and flow cytometry analysis, we investigated how LSPC exerted its action against ETEC strains. The exposure of ETEC strains to LSPC caused the leakage of K+ ions in dose-dependent and time-response manner, which would result in an osmotic imbalance across cell membrane (Garcia, Manas, Gomez, Raso, & Pagan, 2006; Kang, Mauter, & Elimelech, 2008). As the extracellular K+ ion concentration increased, the irreparable damage of the bacterial may occur. As shown in flow cytometry analysis, the damage of bacterial cell may lead to pore formation, resulting in high PI fluorescence intensity, and then caused the disruption of cell membrane integrity. This was followed by the distortion and pore formation of the cell morphology, as displayed by the images of SEM and TEM, which ascribed to cell death or even cell lysis. And then cell lysis would cause the leakage of cytoplasmic components. The results of our studies demonstrated that LSPC exerted its antimicrobial activity by a mechanism that acted on the cell membrane, which was consistent with the reported antibacterial mechanism of flavan-3-ol on the other pathogen bacteria (Ikigai, Nakae, Hara, & Shimamura, 1993; Nakayama et al., 2013; Yi et al., 2010; Zhao et al., 2015). Generally, bacteria cell membrane was composed mainly by hydroxylated phospholipids, which protected the membrane from damage by antibacterial compounds. And catechin, the monomeric units of LSPC, had been proved that it was able to act on cytoplasmic membrane lipid bilayer by aggregation or reduction in lipsomer membrane fluidity (Stapleton, Shah, Ehlert, Hara, & Taylor, 2007; Tsuchiya, 1999), which was agree with the observation of our study. Therefore, the proposed mechanism by which LSPC exerted the antimicrobial action on ETEC strains was due to the interaction between LSPC and lipid bilayer of cell membrane, so

leading to reduction in lipsomer membrane fluidity and then cause cell membrane contraction. In the exposure of treatment of LSPC, the affected membrane resulted in pore formation and allowed the leakage of cytoplasmic component, which eventually led to cell death or cell lysis. The interaction between LSPC and cell outer membrane was further supported by the observations of TEM, in which the seriously separation of cytoplasmic membrane from the cell wall, cell membrane contraction was clearly observed for ETEC cells at high treatment concentration of LSPC. Besides the damage of cytoplasmic membrane function mechanism, the inhibition of nucleic acid synthesis and energy metabolism were also reported as other antibacterial mechanism (Gradisar, Pristovsek, Plaper, & Jerala, 2007; Xie et al., 2014). These different antibacterial mechanisms reported for polyphenol were regarded as existing in ‘cause’ and ‘effect’ relationship, in which one action would cause the others, so further investigation was indeed to confirm the other possible action of LSPC on ETEC strains (Cushnie & Lamb, 2011). 4. Conclusions For the first time, the present work demonstrated that B-type oligomeric procyanidins from lotus seedpod selectively simulated the growth of certain probiotic organisms but inhibited the growth of ETEC strains at certain concentration. The Important significance of this study is developed a new sources of functional foods for against ETEC infection. The big difference of MIC and MBC values between LSPC and LPOPC against ETEC strains indicated that the antibacterial action of procyanidins were strict structuredependent. Moreover, the inhibition and promotion effects of LSPC at different concentration range on ETEC strains and different beneficial bacteria demonstrated that the antibacterial activities of LSPC were also in a dose-dependent and strain-dependent manner. In addition, the mechanism of LSPC on ETEC strains was by disruption of cell membrane structure and function. Certainly, these in vitro findings will be required future confirmed in animal models, but our study supports the potential ability of procyanidins as a

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