Isolation of Bile Salt Hydrolase and Uricase Producing Lactobacillus brevis SF121 from Pak Sian Dong (Fermented Spider Plant) for using as Probiotics

The interesting application of bile salt hydrolase enzyme is reduction of cholesterol in serum and amelioration lipid profile. While uricase enzyme can be applied to convert insoluble uric acid to be soluble form and excrete from the body. Probiotics are living organisms with generally know that they can provide beneficial effects to their host. Several reports show that probiotic bacteria with bile salt hydrolase and uricase can improve hypercholesterolemia and hyperuricemia patient. The novel isolate of Lactobacillus from Pak Sian Dong in this study is identified as L. brevis SF121 and probably use as probiotic bacteria in the future. However, this isolate still need further experiments to investigate and improve properties of probiotics. Moreover, this finding suggests that Pak Sian Dong or fermented spider plant can be designated as a good source for probiotic screening and also defines as health-


Isolation of Bile Salt Hydrolase and Uricase Producing
Lactobacillus brevis SF121 from Pak Sian Dong (Fermented Spider Plant) for using as Probiotics INTRODUCTION Lactobacillus is gram positive bacteria in order Lactobacillales and commonly are defined as lactic acid producing bacteria based on their major by-product. This bacteria normally can be found in the environment, dairy product, animal gastrointestinal tract and fermented food.
Pak Sian Dong is a kind of fermented vegetable in Thailand, which made from spider plant. Spider plants are affiliated with Cleomaceae family and designated the scientific name as Cleome gynandra L. These plants usually are observed worldwide, including Thailand. Fermentation can degrade toxic substance, called hydrocyanic which is defined as a neurotoxin, in spider plant then this kind of food will be safe to consume 14 .
Probiotics are assigned as living organism such as bacteria, mold and yeast, which provide advantages to their living host. The first screening criteria for probiotic bacteria are colonizing properties, which are able to attach the surface of the host and can survive in a gastrointestinal tract environment. The other criteria are beneficial properties that promote the health status of the host [15][16][17] .
Deconjugation of bile acid is occurred by catalytic enzyme calls bile salt hydrolase. Various bacteria have ability to produce this enzyme, especially lactic acid bacteria and Enterobacteria. The interesting application for bile salt hydrolase is cholesterol lowering. There are many reports that used bile salt hydrolase producing bacteria for decreasing of cholesterol level in blood of animal models and human volunteers [18][19][20] .
Gout is a disease which links to serum uric acid. Resulting of solubility of uric acid in blood is low, high level of this substance can cause precipitation of uric acid crystal leading to inflammation of joints. Therapeutic of this disease is probably aiming at; first, reduction of uric acid production by using allopurinol drug for xanthine oxidase activity inhibition; second, using probenecid to inhibit tubular reabsorption of uric acid and; third, using pegloticase for acting as uricase enzyme 21,22 . Uricase or urate oxidase enzyme has ability to transform insoluble uric acid to allantoin which is soluble substance and can be excreted by the kidney. Therefore, catalytic for uric acid activity, uricase is interested to be used for hyperuricemia and gout. Variety genus of bacteria have been screened and isolated with uricase activity, such as Bacillus 23,24 , Pseudomonas 25 and Lactobacillus 26 .
The objective of this study was the determination of bile salt hydrolase activity, uricase activity and colonizing properties in Lactobacillus brevis which was isolated from Pak Sian Dong in Pathum Thani, Thailand.

MATERIALS AND METHODS Strain isolation and cultivation conditions
Lactobacillus brevis SF121 was isolated from Pak Sian Dong. Pak Sian Dong were purchased from local area in Pathum Thani province, a gram of each sample was mixed with 9 ml of NaCl, 0.9% (w/v) or normal saline solution (NSS). The mixture was cultured on MRS agar which contained CaCO 3 0.5% (w/v) and the plates were incubated for 48 h at 37⁰C incubator. Clear zone from CaCO 3 degradation was observed around the colony and the colony was picked to determine for lactic acid bacteria characteristics. The characteristics were included gram reaction with positive results and catalase test with negative results. Blood hemolytic zone of the isolate was also observed after 37°C incubated at least 72 h. All bacterial isolates were named which including L. brevis SF121 and stored at -80°C in glycerol until used.

Bile salt hydrolase activity and uricase activity
For determination of bile salt hydrolase activity of the isolates, MRS medium with 0.5% (w/v) of glycodeoxycholic acid (GCDA) (SigmaAldrich, USA) or taurodeoxycholic acid (TDCA) (Sigma-Aldrich, USA) were prepared as described by Yasiri et al. 27 . The isolates were streaked on GDCA-or TDCA-MRS agar plate and then placed at 37°C for 48 -72 h. After incubation, colony with halozone was observed and recorded for ability to produce bile salt hydrolase.
To determine uricase activity, plate assay with modification was performed 23,25 . The isolate was streaked on MRS agar containing 0.5% (w/v) uric acid (Sigma-Aldrich, USA) and clear zone were observed after incubation for 48 -72 h at 37°C.

Strain identification
API50CHL (Biomerioux, France) was used to identify the strain. In addition, the 16s rRNA sequence analysis also was used to confirm species of the strain. The bacterial genomic DNA of the isolate was extracted with GF-1 bacterial DNA extraction kit (Vivantis, Malaysia) and amplification of 16s rRNA by PCR was performed. The forward universal primer F8 (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse universal primer R1492 (5'-GGTTACCTT GTTACGACTT-3') were used for amplification. After PCR amplification and agarose gel electrophoresis, Novel Juice (GeneDireX, USA) was mixed with the product and then observed the result on 0.8% (w/v) agarose (Invitrogen, USA). The PCR products were sequenced and the sequencing results were compared with a database (NCBI).

Gastric fluid and bile salt tolerance
To determine the tolerance of gastric fluid and bile salt, the procedure with slightly modified from Mota et al. was followed 28 . Synthetic gastric fluid was prepared as follows; weighed NaCl for 2 g and pepsin for 3.2 g and then mixed in 1000 ml of water. Added concentrated hydrochloric acid to solution to make the pH 2.5 and finally the solution was sterilized by filtration with 0.22 µm pore size membrane. To determine gastric fluid tolerance, bacterial suspension was prepared by washing and adjusting the overnight culture of L. brevis SF121 with NSS to McFarland standard no. 0.5. A hundred microliter of the L. brevis SF121 cell suspension was applied to 900 µl of synthetic gastric fluid and incubated for 3 h at 37°C. NSS was used instead of synthetic gastric fluid as a control group.  28 .

Antibiotic susceptibility
In this study, the MIC of antibiotics, including , ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid, penicillin, tetracycline and vancomycin were determined for antibiotic susceptibility following CLSI (Clinical and laboratory standards institute) guideline. In brief, contained antibiotic MRS broth was transferred to a microplate with a serial two-fold diluted from 512 µg/ml to 1 µg/ ml. The overnight culture of L. brevis SF121 was diluted with NSS to prepare the suspension equal turbidity to 0.5 McFarland. The prepared L. brevis SF121 suspension was diluted hundred-fold with MRS and transferred to a filled microplate. The MIC was observed and interpreted after overnight incubation at 37°C.

Antimicrobial activity
The well diffusion assay was used to assess antimicrobial activity of L. brevis SF121. The twenty milliliters of Mueller-Hinton agar plates were prepared and swabbed with the indicator organisms. The indicator organisms in this study, including gram positive bacteria (Bacillus cereus, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecium) gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica Typhimurium, Shigella sonnei, Vibrio cholerae) and yeast (Candida albicans). All organisms were cultivated in Mueller-Hinton broth at 37°C for overnight before spreading on agar plates. A well on swabbed plate that made from cork borer no.3 was filled with 100 µl of L. brevis SF121 overnight culture supernatant, which was prior adjusted the pH to 7.0 with 1 N NaOH and filtered by 0.22 µm pore size membrane for sterilization. The plates were measured for the inhibition zone after overnight incubation at 37°C.

Lactobacillus isolation and characterization
Gram positive rod shaped bacteria with catalase negative and no hemolytic activity was selected from MRS agar. Bile salt hydrolase activity was shown as precipitated halozone around the colony of L. brevis SF121 on GDCA-MRS agar plate ( Fig. 1) but no activity observed on TDCA-MRS. The isolate also exhibited ability to convert insoluble uric acid in MRS agar plate to soluble form which showed as the clear zone around colony in the Fig.  2.

Lactobacillus identification
The selected isolate showed 96.6% identity with Lactobacillus brevis by API 50 CHL. The identification was confirmed by 16s rRNA sequencing, which indicated coherent result with 99% identical to L. brevis in NCBI database. From isolation and identification, this isolate was designated as Lactobacillus brevis SF121.

Determination of probiotic properties
From general probiotic property characterization, gastric fluid tolerance of the isolate in this study was 67.07 % and bile salt tolerance was 89.62%. The L. brevis SF121 presented 21.44% hydrophobicity For antibiotic susceptibility test, the isolate demonstrated sensitivity to only erythromycin and presented resistance to remaining antibiotics in this study (Table 1). Furthermore, all test organisms in the experiment, including gram positive bacteria, gram negative bacteria and yeast, could not be inhibited the growth by L. brevis SF121.

DISCUSSION
From screening and isolation result, Lactobacillus species had been isolated from Pak Sian Dong. Previous study showed that the various genus of lactic acid bacteria can be found in Pak Sian Dong, including Lactobacillus sp. 29 , thus the one report reveals potential of lactobacilli from this source have capability to use as probiotic 27 . There was no hemolytic activity presented on blood agar plate which could be confirmed safety of the isolate for further used in animal and human 17 .
Determination of colonizing properties revealed that the isolate can resist simultaneous gastrointestinal environment. Both gastric fluid tolerance and bile salt tolerance are key important properties for survival of probiotic bacteria that appropriate for oral route use 30 . The properties confirm that this bacteria can survive and provide beneficial effects to the host at gastrointestinal tract region. For hydrophobicity, the isolate The antibiotics in this study consisted of ampicillin (Ampi), chloramphenicol (Chlo), ciprofloxacin (Cipr), erythromycin (Eryt), nalidixic acid (Nali), penicillin (Peni), tetracycline (Tetr) and vancomycin (Vanc). showed a little percentage, however the bacteria probably use other mechanisms to attach the epithelium cells such as aggregation property 31 .
The novel isolate has been identified as L. brevis, which commonly be found in milk, plants and fermented products. This bacteria can be applied to use in industrial and food technology and normally defined as GRAS (Generally Recognized As Safe) 32 . Some strain of L. brevis presented probiotic properties, e.g., antioxidant properties 33 , anti-caries activity 34 , immunomodulating activity 35 and synergistic with prebiotic for antimicrobial activity 36 .
From the result of bile salt hydrolase, the isolate showed the activity only on MRS agar containing GDCA. This finding conformed to previous study that lactobacilli from Pak Sian Dong have an ability to hydrolyze GDCA but not TDCA 27 . According to bile salt hydrolase activity, this isolate can probably be alternatively used for cholesterol lowering application. Several reports published that lactobacilli, such as L. plantarum [37][38][39] , L. gasseri 40 , L. johnsonii 41,42 and L. brevis 43 , could be proved for bile salt hydrolase production. In 2013, L. plantarum with bile salt hydrolase was fed in high-cholesterol diet-induced rat could improve lipid profile in serum and liver of the rat 44 . Another report revealed that lactobacilli with strong expression of bile salt hydrolase have the capability to ameliorate hypercholesterolemia in mouse model 20 . In accordance with previous evidences, the novel strain, L. brevis SF121, might be able to reduce cholesterol by bile salt hydrolase activity.
Because of uricase enzyme is related to hyperuricemia therapy, this isolate with uricase activity is possibly appropriated to apply in this therapeutic approach. In human, uricase cannot be produced because of the loss of this enzymatic activity by genetic mutation during Miocene epoch 45 . Therefore, the compensation of this enzyme with intestinal microbes might be an important point. The normal route of administration of uricase (Pegloticase) is intravenous but there is a report that oral uricase administration in animal showed elimination of uric acid 46,47 . Moreover, alteration of Lactobacillus genus show relation with hyperuricemia in animal model 48 so the adjustment of lactobacilli population probably a helpful for treatment.

CONCLUSION
L. brevis SF121, which isolated from fermented spider plant or Pak Sian Dong in this study, found ability to present bile salt hydrolase and uricase activity. This isolate is probably able to use for reducing of cholesterol and uric acid in person who is defined as hypercholesterolemia and hyperuricemia. This isolate exhibited resistance to gastrointestinal condition and survived in proper time for using as oral probiotic bacteria. Due to possibility to find the bacteria with bile salt hydrolase and uricase activity in Pak Sian Dong, this kind of fermented food would be established as a health beneficial food and also a good source for probiotic bacteria screening. However, there are several properties of probiotic needs to be conducted in future study for probiotic authentication of the L. brevis SF121 isolate.

ACKNOWLEDGMENTS
We would like to thank Chulabhorn International College of Medicine for all facilities.