Isolation and Identification of Quercetin Degrading Bacteria from Human Fecal Microbes

Quercetin has a wide range of biological properties. The gut microflora can often modulate its biological activity and their potential health effects. There still is a lack of information about gut bacteria involving in this process. The strains of gut microbes from human feces that can transform quercetin were isolated and identified by in vitro fermentation. The results showed that Escherichia coli, Stretococcus lutetiensis, Lactobacillus acidophilus, Weissella confusa, Enterococcus gilvus, Clostridium perfringens and Bacteroides fragilis have the various ability of degrading quercetin. Among them, C. perfringens and B. fragilis were discovered to have the strongest ability of degrading quercetin. Additionally, quercetin can't inhibit the growth of C. perfringens. In conclusion, many species of gut microbiota can degrade quercetin, but their ability are different.


Introduction
Quercetin (3,39, 49, 5, 7-pentahydroxyflavone) is a flavonoid widely distributed in plant foods and herbal medicines. It has a wide range of biological properties including anti-oxidization [1,2], anti-mutagenicity [3], anti-inflammatory effects [4] and the prevention or decrease of cardiovascular diseases [5]. These beneficial effects could be related to the native forms of quercetin present in vegetables and fresh fruit, or their metabolites.
In general, the absorption of polyphenols in the digestive tract starts in the ileum, where the more complex structures (esters, glycosides, or polymers) that cannot be absorbed in their native form are hydrolysed by intestinal enzymes or the colonic microflora [6]. The non-absorbed polyphenols in the ileum will reach the colon whole [7][8][9], and be transformed by the gut microbiota enzymes (esterase, glucosidase, demethylation, dehydroxylation and decarboxylation enzymatic activities) [10] into a wide range of low-molecular-weight phenolic acids [11]. For example, polyphenols can be transformed by colonic microflora into phenolic acids, such as phenylvaleric, phenylpropionic, phenylacetic, benzoic and hippuric acids [12]. Thus, the gut microflora can often modulate the biological activity of these dietary polyphenols [13,14], and their potential health effects by the degrading process.
Although it is known that the intestinal microflora participate in the metabolism of flavonoids [15], there is a lack of information about the species/strains that may involved in the process. The aim of this research was to isolate and identify the bacterial strains in the human gut that can transform quercetin (one of flavonoids) in an in vitro model. Furthermore, the fermentation properties of the isolated strain(s) with quercetin were evaluated.

Fecal sample collection and selective bacterial culture
Fecal bacteria were isolated from freshly collected human feces under anaerobic conditions by diluting the feces with saline peptone water (0.1% (w/v) peptone and 0.85% (w/v) NaCl in distilled water). 100 ul of fecal suspension were then plated on the various selective agar media with 1% quercetin (Qiyun Biological Science & Technology Limited Co.), These selective media (Qingdao Hope Biol-Technology Co, Ltd) included Bile Esculin Azide Agar (BEA), MRS Agar and Brain Heart Infusion Broth (BHI) with 1.5% agar. The selective plates were individually incubated at 37uC in different incubator for 16 h, such as BEA in aerobic incubator, MRS in an anaerobic CO 2 incubator inflated with air containing 5% CO 2 and BHI agar in an anaerobic incubator.

Isolation of strains
All different colonies were picked up from each plate according to their appearance of. All isolates were purified by streak plate method and then stored at 280uC in a freezing medium [16] until used.

Verification of strains
The ability of transforming quercetin of the isolates was further verified with the following procedure: the strains were individually inoculated into their corresponding broth with 1% quercetin for 16 h. Then the broths were centrifuged at 5000 rpm for 3 min and the absorbance of supernatant was measured under 415 nm with UV/VIS Spectrophotometer after reacted 10 min with equal volume solution of 2% AlCl 3 dissolved in methanol. The broth with quercetin but no strains was set as the negative control. All tests were parallelled three times.

Extraction and preparation of genomic DNA from isolates
Isolates were individually cultured for 12 h in 1.5 ml corresponding selective broth at 37uC and cells were individually collected by centrifugation at 5000 rpm for 3 min. Broth was removed -and genomic DNA was extracted from the cell pellets by Invitrogen TM genomic DNA extraction kits. The extracted genomic DNA from each isolate was checked by horizontal gel electrophoresis with 0.8% (w/v) agarose containing 0.5 mg/ml ethidium bromide in 0.5X TBE. The gel was visualized using an Image MasterH VDS (Amersham Plc., Buckinghamshire, UK). The DNA concentration and purity were also determined using a spectrophotometric method (UV/VIS spectrophotometer, Shimadzu Corp., Kyoto, Japan) described by Maniatis et al. [17]. The DNA preparations were stored in 40 ml nuclease-free water at 220uC.

Amplification of 16S rDNA of isolates
The 16S rDNA fragment of each isolate was amplified by PCR with the universal primer set of 27-f (59-AGT TTG ATC CTG GCT CAG-3 9) and 1492-r (59-GTT ACC TTG TTA CGA CTT C-39) according to Naomi et al. [18] using an iCycler Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, USA). All reagents used in PCR amplification were purchased from Fermentas International Inc., Ontario, Canada. The amplification was done in 50 ml reaction volumes as described by Hoefel et al. [19]. Each PCR reaction consisted of 200 mM of each dNTP, 1.0 mM of each primer, 2.5 mM MgCl 2 , 1X PCR buffer, 2.5 U of Taq DNA polymerase, and 50 ng DNA template. The thermal cycling included an initial denaturation step at 95uC for 10 min; 30 cycles of a denaturation step at 95uC for 30 s, an annealing step at 50uC for 1 min, an extension step at 72uC for 2 min; and a final extension at 72uC for 10 min. The PCR product was checked using 0.8% (w/v) agarose gel electrophoresis. The gel was visualized using an Image MasterH VDS.
2.6. 16S rDNA sequencing DNA sequence analysis was performed by Shanghai Sanggong Inc., Shanghai, China. Homology searches of the 16S rDNA sequences were performed in the GenBank with the Blast program.

Proof and quantitative test of identified strains metabolizing quercetin
All identified strains were further proved by the chromogenic reaction: The cells were centrifuged from their corresponding broth medium and sprayed with the methanol solution of 2% AlCl 3 . If the cells can transform quercetin into other substances, they show grayish white (their own color); and if quercetin is not transformed by the bacteria, it would be absorbed at the surface of cells and react with aluminium chloride, then present yellow. Then the remaining of quercetin in supernatant broth was colorimetrically tested and compared by the method described above (see 2.3).

Statistics
Results are expressed as mean values with their standard deviation (SD). Statistical analyses were conducted with the Statistical Package for Social Science (SPSS for Windows, version 8.0; SPSS Inc., Chicago, IL, USA) to determine if variables differed among treatment groups.

Strains transforming quercetin
All isolates were picked up from three kinds of media plate and the counts were present at

Molecular identification by 16S rDNA
The above strains were further identified by 16S rDNA sequencing and showed in Table 1. Eight strains from BEA plates were verified were allas Enterococcus gilvus (99% similarity); there were four species including Lactobacillus acidophilus, Weissella confusa, Stretococcus lutetiensis and Escherichia coli (.97% similarity)identified in the MRS plates -a medium mainly used to selectively cultivate Lactobacillus spp; there were two species identified in BHI plates, Bacteriodes fragilis and Clostridium perfringens. The phylogenetic tree of these strains was present in Figure 1

Proof and comparison of strains identified in metabolizing quercetin
In the chromogenic reaction, the strains of E. coli, S. lutetiensis, L. acidophilus, W. confuse and E. gilvus present more yellow (Figure 2-B) than their corresponding controls (Figure 2-A). While the color of B. fragilis and C. perfringens showed grey which were similar to their corresponding control (Figure 2-A). The results indicated that some quercetin had been absorbed in the surface of strains 1-5, but not in the surface of 6-7. The comparison of different bacteria transforming quercetin was further present in Figure 3 and Figure 4. Only a little of quercetin was metabolized by E. coli, S. lutetiensis, L. acidophilus, W. confuse and E. gilvus due to their broth color changed only a little compared to the negative control ( Figure 3, Tube 8); however, the broth of B. fragilis and C. perfringens present no color like the positive control (Tube 1). According to the quantitative test, B. fragilis and C. perfringens transformed 95.99% and 96.27%, respectively remaining 1.5860.14 mg/mL and 1.7160.07 mg/mL quercetin, which almost equals to the degrading rate (96.66%) of bacterial mixture (1.4260.07 mg of remaining quercetin). But E. coli, and W. confusa only broke down a little quercetin (6.19% and 9.60%)  L. acidophilus and S. lutetiensis also presented their capacity in degrading quercetin (19.06% and 33.33%, respectively). The different strains of E. gilvus also present different capacity of metabolizing quercetin (47.45%, 37.03% and 23.67%) (Figure 4).
In this paper, seven different bacteria that can transform quercetin were isolated from fresh human feces, including E. gilvus, S. lutetiensis, E. coli, L. acidophilus, W. confusa, C. perfringens and B. fragilis. The difference of metabolizing quercetin among these strains has also been presented in this paper. Among them, B. fragilis and C. perfringens have the highest capacity. However, the B. fragilis group is the least common Bacteroides present in fecal flora, comprising only 0.5% of the bacteria present in stool [25]. C. perfringens (formerly known as C. welchii), considered to be relative with several gut chronic diseases, is also less in health gut [26,27]. Hence, quercetin can't be totally degraded by the two species for their minor amount in human gut. Figure 4 showed that quercetin had been almost totally broken down by the fecal mixture bacteria, which might be attributed to some other strains which were not isolated in current study or to the broth suitable for the growth of the two species.
As reported, plant polyphenols exert significant effects on the intestinal environment by modulation of the intestinal bacterial population, probably by acting as metabolic prebiotics. The growth of certain pathogenic bacteria such as C. perfringens, C. difficile and Bacteroides spp. were significantly repressed by plant phenolics and their derivatives, while commensal anaerobes like Clostridium spp., Bifidobacterium spp. and probiotics such as Lactobacillus spp. were less severely affected [15]. However, C. perfringens were firstly verified to highly degrade quercetin in this paper, hence quercetin can't inhibit the growth of C. perfringens. B. fragilis was also discovered the ability of degrading quercetin as C. perfringens, and their ability of degrading quercetin is stronger than the others discovered in this paper.
This study screened the degrading ability of human gut bacteria on quercetin, and discovered that many species of gut microbiota can degrade quercetin, but their ability are different. Two new species with totally degrading quercetin were isolated and identified.

Conclusion
E. gilvus, S. lutetiensis, E. coli, L. acidophilus, W. confusa, C. perfringens and B. fragilis were first discovered to have the ability of degrading quercetin in this paper. And their degrading ability was different. Among them, C. perfringens and B. fragilis have the strongest ability of degrading quercetin. Quercetin can't inhibit the growth of C. perfringens.