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Effect of Escherichia coli and Lactobacillus casei on Luteolin Found in Simulated Human Digestion System

Seung-Jae Lee, Seung Yuan Lee, Sun Jin Hur*

Department of Animal Science and Technology, Chung-Ang University, 4726 Seodong-daero, Daedeok-myeon, Anseong-si, Gyeonggi 450-756, Republic of Korea

Abstract: This study was conducted to investigate the effects of in vitro human digestion and enterobacteria (Escherichia coli and Lactobacillus casei) on the digestibility and structure of luteolin. Luteolin was passed through an in vitro digestion system that simulates the composition of the human mouth, stomach, small intestine, and large intestine and contains enterobacteria. The luteolin content was not altered by mouth or stomach digestion, but it was decreased by small intestine digestion. Large intestine digestion by enterobacteria also decreased the luteolin content; L. casei reduced the luteolin content more than E. coli. Moreover, digestion in the large intestine by a combination of E. coli and L. casei reduced the luteolin content more than digestion by individual enterobacteria. This study will provide insight into how enterobacteria influence the digestibility and structure of phytochemicals.

Keywords: In vitro human digestion, luteolin, enterobacteria, escherichia coli, lactobacillus casei

Received April 10, 2015; Revised May 12, 2015; Accepted May 19, 2015

Citation: Seung-Jae Lee, Seung Yuan Lee, Sun Jin Hur, Effect of Escherichia coli and Lactobacillus casei on Luteolin Found in Simulated Human Digestion System. Journal of Food and Nutrition Research, 3 (5). 311-316. January 2015. doi:10.12691/jfnr-3-5-4

1. Introduction

Phytochemicals are well known for their various pharmacological properties, such as antioxidant and disease preventive/protective effects. Polyphenol luteolin (a yellow flavone) is a common dietary form of luteolin, which is present in various fruits, herbs, and vegetables (Lin, et al., 2008; Mencherini, et al., 2007). Luteolin has been shown to have stronger antioxidant activity and lower potential prooxidant activity than many common flavonoids, such as quercetin and myricetin, and to show potential health benefits in humans (Seelinger, et al., 2008). Numerous studies have shown that luteolin exhibits a number of health-promoting functions, including anticancer (Lee, et al., 2001; Ong, et al., 2010), anti-inflammatory (Seelinger et al., 2008; Xagorari et al., 2001), and cardioprotective (Liao, et al., 2011) properties. Therefore, luteolin is regarded as one of the most important bioactive phytochemicals, with potential applications in a wide range of pharmaceutical, nutraceutical, and functional food products.

In nutritional studies in both animals and humans, in vivo feeding methods usually provide the most precise results. However, they are time-intensive and expensive, which is why much effort has been dedicated to the development of in vitro processes (Boisen & Eggum, 1991). In vitro human digestion provides a useful alternative to in vivo models for rapidly screening food materials. The ideal in vitro human digestion model would provide accurate results in a short time and could thus serve as a tool for rapid screening of foods and delivery systems with different compositions and structures (Coles, et al., 2005; Fuller, 1991). A review by Hur et al. (2011) (S. J. Hur, Lim, et al., 2011) reported that during the last few decades, researchers have utilized a number of in vitro human digestion models to test the structural and chemical variation in different foods under simulated gastrointestinal conditions, although the reliability of these models has not yet been widely accepted. However, the effect of enterobacteria on phytochemicals during in vitro human digestion has not yet been evaluated. Therefore, development of new technologies in search for form changes of phytochemical by another factor is opportunities for the research database.

2. Materials and Methods

2.1. Materials

Bicarbonate, potassium thiocyanate, sodium phosphate dibasic, sodium phosphate monobasic, sodium chloride, calcium chloride, ammonium chloride, urea, a-amylase, uric acid, mucin, bovine serum albumin, pepsin, pancreatin, lipase, bile salt extraction were purchased from Sigmae Aldrich chemical company (St Louis, MO, USA). All other reagents were of the highest grade commercially available.

2.2. In vitro Human Digestion Model

A digestion model that simulates the mouth, stomach, and intestine was used in this research, modified from those described by previous studies (Versantvoort, et al., 2005; Hur et al., 2009; Hur et al., 2011). To simulate digestion in the mouth, a 5 mL sample of luteolin in DMSO was mixed with 5 mL of saliva fluid (pH 6.8) and stirred for 5 min. Approximately 10 mL of simulated gastric fluid (pH 1.3) was added and the mixture was stirred with a magnetic stirrer for 2 h. Finally, 10 mL of duodenal juice (pH 8.1) and 5 mL of bile juice (pH 8.2) were added and the mixture was stirred for 2 h. All digestive juices were heated to 37°C. The composition of the simulated saliva, gastric, duodenal, and bile fluids are listed in Table 1. During in vitro simulation of human digestion, the large intestine step requires enterobacteria such as E. coli and L. casei. Following the small intestine step, 35 mL of E. coli and L. casei were applied to the sample and incubated for 4 h at 37°C. A schematic diagram of the in vitro model of human digestion of luteolin using enterobacteria is shown in Figure 1.

Table 1. Constituents and concentrations of the various synthetic juices used in the in vitro human digestion model

Figure 1. Schematic diagram of the in vitro human digestion of luteolin and subsequent chemical analysis

2.3. Preparation of Microorganisms

E. coli was obtained from the American Type Culture Collection (ATCC), and L. casei MCL was isolated from feces collected from healthy adults. The E. coli and L. casei were stored at -80°C until they were used. The E. coli was cultured in Luria-Bertani (LB) medium (DifcoTM LB broth, Miller, MD, USA) and the L. casei was cultured in MRS medium (DifcoTM Lactobacilli MRS Broth, Miller, MD, USA) in a shaker-incubator at 37°C and 150 rpm. After incubation, the final colony counts for E. coli and L. casei were in the range of log 10⁸–10¹⁰.

2.4. Measurement of Luteolin by HPLC

Luteolin, digested luteolin, and luteolin digested by intestinal microorganisms were analyzed using high-performance liquid chromatography (HPLC, HP Agilent 1100, Hewlett Packard Co) on a Fortis H₂O column (250 mm × 4.6 mm, 3 μm) using a water : tetrahydrofuran: trifluoroacetic acid gradient (97.9 : 2 : 0.1, v/v/v) and acetonitrile at a flow rate of 1.2 mL/min. The volume of sample injected for analysis was 20 μL, and the detection wavelength was set at 350 nm. All solutions were passed through a 0.45-μm Whatman membrane filter before injection onto the HPLC column.

3. Results and Discussion

The results showed the changes of luteolin contents at different concentrations (0.5, 1.0, and 2.0 mg/mL) during in vitro human digestion (Table 2). Changes in luteolin content were not observed following mouth or stomach digestion (data not shown), but luteolin content was reduced after small intestine and large intestine digestion. Thus, luteolin is stable under the digestion conditions found in the mouth and stomach, but is unstable under the digestion conditions found in the small intestine. A possible mechanism for the changes in luteolin content observed during in vitro human digestion is pH fluctuation. Boyer et al. (2005) found that flavonoids are stable at lower storage pH and less stable at higher pH (Boyer, et al., 2005). Bermudez-Soto et al. (2007) also reported that polyphenols are largely stable during gastric digestion (under acidic conditions), but that they are quite sensitive to mildly alkaline conditions, as found in the small intestine. In the model of digestion used in this study, the pH shifts dramatically between the stomach and the small intestine, from pH 1.5 to pH 7.5, mainly because bile salt has a higher pH. This change in pH is the primary factor involved in the irreversible breakdown of luteolin. Therefore, we hypothesize that in vitro digestion in the small intestine of humans may affect the structure of luteolin.

Figure 2. Effect of in vitro human digestion and enterobacteria on the digestion/absorption rate of luteolin. A; 0.5, B; 1.0, C; 2.0 mg/mL of undigested luteolin. D; 0.5, E; 1.0, F; 2.0 mg/mL of digested luteolin. Luteolin following in vitro digestion with E. coli (G; 1.0, H; 2.0 mg/mL). Luteolin following in vitro digestion with L. casei (I; 1.0, J; 2.0 mg/mL), Luteolin following in vitro digestion with mixed E. coli and L. casei (K; 1.0, L; 2.0 mg/mL)

Figure 3. Effect of in vitro human digestion and enterobacteria on the mass spectrum of luteolin. A: luteolin, B: luteolin following mouth digestion, C: luteolin following stomach digestion, D: luteolin following small intestine digestion, E: luteolin following large intestine digestion with E. coli, F: luteolin following large intestine digestion with L. casei, G: luteolin following large intestine digestion with E. coli and L. casei

Table 2. Effect of in vitro human digestion and enterobacteria on the digestibility and structure of luteolin

E. coli is known to be found in the large intestines of humans and animals, and to aid in the processes of digestion and absorption. Lactobacillus spp. can be found in materials isolated from plants and the gastrointestinal tracts of humans and animals (Walter, 2008). These enterobacteria affected the luteolin content during in vitro digestion in humans. In this study, the luteolin content was decreased during large intestine digestion (Figure 2 G–L). This result may be due to the effects of enterobacteria (E. coli and L. casei) during large intestine digestion. Luteolin contents determined that the antagonism of microorganism or flavonoid influenced by pH. Shimoi et al. (1998) investigated the intestinal absorption of luteolin7-ο-β-glucoside and luteolin in rats and humans by HPLC (Shimoi, et al., 1998). They found that luteolin 7-ο-β-glucoside was absorbed after its hydrolysis to luteolin by intestinal microbes, which can hydrolyze glucosides of flavonoids. Braune et al. (2001) also reported that luteolin was first degraded to eriodictyol and then to 3-(3,4-dihydroxyphenyl) propionic acid and phloroglucinol by reduction of the double bond in the 2,3-position, which was observed following fermentation by the human intestinal tract bacterium Eubacterium ramulus. Our previous survey found that lactic acid bacteria possess various active enzymes such as amylase, dehydrogenases, glucosidase, phenolic acid decarboxylases, and phenol reductase (Hur et al., 2014). Luteolin contains four hydrogen bond donors and six hydrogen bond acceptors, and these hydrogen bonds could be cleaved by the hydrolysis enzymes possessed by the lactic acid bacteria used in this study. In Figure 2 (G–J), the luteolin content was higher following digestion with E. coli than following digestion with L. casei, during large intestine digestion. Thus, L. casei may influence the luteolin content more strongly than E. coli during in vitro human digestion. L. casei may have a stronger effect due to its production of lactic acid; the consequent pH fluctuations could accelerate hydrolysis of luteolin during digestion in the large intestine. Furthermore, the luteolin content was found to be lower following digestion with both E. coli and L. casei than following digestion by individual enterobacteria in the large intestine. Thus, digestion by individual enterobacteria more strongly affects the structure of luteolin than digestion by enterobacteria in combination, in the large intestine (Figure 3). This result may be due to antagonism between E. coli and L. casei during in vitro human digestion. The mechanism of the antagonism remains unclear, but we hypothesize that lactic acid production by L. casei and the consequent pH decline might inhibit the growth of E. coli. Lactic acid can act on the cell membrane of E. coli, changing the fatty acid composition and affecting the H⁺ and Na⁺ ion exchange or H⁺/ATPase activity of the plasma membrane. In this study, we found that the population of E. coli was smaller than that of L. casei during in vitro human digestion, although the initial populations were the same (data are not shown). In an earlier study, Reid et al. (1988) found that lactobacilli were coaggregated with E. coli. These results indicate that L. casei are antagonistic to E. coli in the gastrointestinal tract; this antagonistic activity might have influenced the digestibility and structure of luteolin during in vitro human digestion.

4. Conclusions

The results of in vitro human digestion models often differ from those of in vivo models because of the difficulties involved in accurately simulating the highly complex physicochemical and physiological events that occur in human digestive tracts. However, this paper is the first to investigate the relationship between enterobacteria and changes in luteolin during in vitro human digestion. This study will provide insight into how enterobacteria influence the digestion and structure of phytochemicals.

Acknowledgements

This investigation was supported by Chung-Ang University.

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