Production, Purification and Fecal Fermentation of Fructooligosaccharide by Ftase from Jerusalem Artichoke

International Food Research Journal 24(1): 134-141 (February 2017) Journal homepage: http://www.ifrj.upm.edu.my Production, purification and fecal fermentation of fructooligosaccharide by FTase from Jerusalem artichoke 1*Wichienchot, S., 1Prakobpran, P., 2Ngampanya, B. and 2Jaturapiree, P. 1Interdisciplinary Graduate School of Nutraceutical and Functional Food, The Excellent Research Laboratory for Cancer Molecular Biology, Prince of Songkla University, Hat Yai, Songkhla, Thailand 90112 2Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakorn Pathom, Thailand 73000 Article history Abstract Received: 11 August 2015 Fructooligosaccharides (FOS) has been used as prebiotic that serves as a substrate for microflora Received in revised form: in the large intestine. FOS are produced by fructosyltransferase (FTase) derived from some 15 February 2016 Accepted: 17 March 2016 plants such as Jerusalem artichoke, chicory, asparagus, banana, dragon fruit and onion. It was found that Jerusalem artichoke cultured in tropical region for 3-5 months showed good source of FTase. It had the highest crude enzyme activity of 0.253±0.003 U/ml. Optimal conditions for purification of FTase by chromatography techniques with anion exchangers showed the Keywords highest specific activity which increased from 1.411 to 2.240 U/ml. Optimum conditions Fructooligosaccharide for production of FOS were 20% sucrose, reaction time of 96 h and 1 U/ml FTase. It was Jerusalem artichoke found that highest FOS (35%) consisted of 27.5% 1-kestose (DP 2) and 7.5% nystose (DP 3). Fecal fermentation Fructooligosaccharide was further purified by yeast fermentation using 2.5% Saccharomyces Prebiotic cerevisiae TISTR5019 for 36 h. It could decrease sucrose from 46.1% to 28.7%. The chemical composition of partially purified freeze-dried FOS was analyzed by HPLC. It consisted of 35 % FOS, 17.4% sucrose, 7.6% glucose and 40% fructose. The prebiotic property of the partially purified FOS was evaluated by cultivation of human fecal microflora in batch culture. It was found that the FOS had prebiotic property with PI value of 2.33. There was also stimulated growth of Lactobacillus and Bifidobacterium. It also produced lactic acid, acetic acid, propionic acid and butyric acid at concentration of 1287.92, 20.97, 10.34 and 18.67 µg/ml, respectively and vitamins B1 and folic acid were produced at concentration of 19.19 and 20.00 µg/ml, respectively. © All Rights Reserved Introduction Fastinger et al., 2008; Calame et al., 2008). FOS can be found in large quantity in natural food, such Fructooligosaccharides (FOS) are among the as asparagus, banana, garlic, onion, wheat and most extensively studied prebiotics and include a dragon fruit. In FOS production process, enzymes diverse family of fructose polymers which vary responsible for catalyzing this reaction is extracted in length and can either be derivatives of simple from microbial source, but in this present time fructose polymers or fructose moieties attached to an enzyme for FOS production is extracted from a sucrose molecule (Wichienchot and Chinachoti, plants such as Jerusalem artichoke tubers. Jerusalem 2011). Fructooligosaccharides consist mainly of artichoke is a famous plants that can produce high 1-kestose, nystose, and 1F-fructofuranosyl nystose FOS and they grow well in tropic regions. Jerusalem which have 1~3 units of fructose. FOS are one in non- artichoke is classified in the genus Helianthus in the digestible and fermentable group that are the most family Asteraceae (Aster or Daisy family), in the popular compounds which gained specific interest order Asterales. Species in the genus Helianthus because it had been reported to possess significant are native to North America, but have very different prebiotic properties such as galacto-oligosaccharides distributions, varying from restricted (e.g., H. (GOS), fructo-oligosaccharides (FOS), gentio- argophyllus and H. ludens) to widespread (e.g., H. oligosaccharides, gluco-oligosaccharides, isomalto- annuus and H. tuberosus). Jerusalem artichoke tubers oligosaccharides (IMO), mannan-oligosaccharides, contain little or no starch, virtually no fat, and have chito-oligosaccharides (COS), melibiose, pectic a relatively low calorific value. The small amount of oligosaccharides (POS), and xylo-oligosaccharides fat present, trace amounts of monounsaturated and (XOS) (Gibson et al., 2004; Roberfroid, 2007; polyunsaturated fatty acids have been reported, but *Corresponding author. Email: [email protected] 135 Wichienchot et al./IFRJ 24(1): 134-141 no saturated fatty acids (Whitney and Rolfes, 1999). Phetchaboon research station, hosted by Kasetsart The polyunsaturated fatty acids linoleic (18:2 cis, cis University, Thailand. Jerusalem artichoke was grown n-6) and α-linoleic acid (18:3 n-3) have been recorded in nursery tree bag and watered every 2 days. After a as present at 24 mg and 36 mg•100 g–1 of raw tuber, month, the plant was transferred to a tree pot. Then respectively (Fineli, 2004). The tubers are a good the tubers were harvested at 3.5, 4, 5 and 6 months source of dietary fiber, beca use of the presence of and stored at -20oC until use. The relationship fructooligosaccharides and inulin. The inulin content between tuberization stage of Jerusalem artichoke of tubers ranges from 8 and 21% of fresh weight and fructosyltransferase activity (FTase) were (van Loo et al., 1995) and fructooligosaccharides investigated during 3.5-6 months. content between 5-6 % of fresh weight. The total carbohydrate about 16.7 g, dietary fiber 0.6 g per Extraction of crude enzyme 100g fresh weight that was calculated by difference Ten gram of the tubers was homogenized in 10 (minus protein, fat, water, and ash). FOS and scFOS ml of 0.007 M potassium phosphate buffer (pH 7.0) are resistant to digestion in the stomach and small containing 0.5% Triton X-100 and 0.5% cysteine and intestine (Ellegard et al., 1997) and thus are able stored for 16 h at 4oC. The homogenate was filtered to reach the colon. In the human colon where they through a cheese cloth and centrifuged (12,000 x g, are selectively fermented by beneficial bacteria, the 20 min, 4oC) to remove insoluble matter. microbe that ferment carbohydrate in the human colon (Bouhnik et al., 1999; Le Blay et al., 1999) Determination of protein content has diverse microbial ecology which is an important Protein content of the sample was determined food source for the microbes. As the diet is passed using Bradford method (Bradford, 1976). Solution from mouth to the small intestine and large intestine, was prepared at dilution of 1:4 (dye: DI water) and respectively, the waste food remain about 200 grams solution was filtered with filter paper then stored in with around 60% of its dry weight as microbes. The a amble bottle before use. The calibration curve was cecum has low pH which is very good for feeding and prepared by pipette at concentrations of 0, 0.05, 0.1, enhances microbial growth, the colon is the longest 0.2, 0.3, 0.4 and 0.5 mg/ml BSA. Distilled water part of large intestine and its pH is neutral (Macfarlane was used for blank. Pipette 200 μl of Bradford dye et al., 1998; Egert et al., 2006). Subsequently there to each microtubes and mixed well by inversion or is a growth spurt among the populations of bifidus gentle vortex mixing. Avoid foaming, which will lead bacteria (such as bifidobacteria and lactic acid to poor reproducibility. Absorbance of the sample bacteria) that eventually outgrow the pathogenic and/ was measured at 595 nm and a graph of absorbance or the putrefactive bacteria (such as colon bacilli) versus protein content was plotted. (Gibson and Roberfroid, 1995). Therefore there is a decrease in toxic fermentation products. FOS also aid Determination of fructosyltransferase activity in lowering the cholesterol and blood sugar levels, Pipette 100 μl of clear supernatant solution to and enhance absorption of mineral elements. make reaction with 0.1 M potassium phosphate buffer (pH 5.4) in presence of 0.575 M sucrose Materials and Methods and incubated for 24 h at 34oC. The reaction was terminated by heating to 95oC, 2 min. Sample Chemicals and microorganism was stored at -20oC before it was analyzed with Reagents for purification of enzyme, production of Somogyi–Nelson and GOD-POD method. Mixed fructooligosaccharides, analysis of oligosaccharides Nelson reagent and diluted crude enzyme sample or and sugar fermentation were purchased from Sigma- distilled water for blank in microtubes. Microtube Aldrich Co. Ltd. Reagents for studying in artificial was inserted in boiling water for 10 min. Addition colon system for batch culture and evaluation of of 100 μl of arsenomolybdate reagent and allowed to prebiotic index were purchased from Sigma-Aldrich stand for at least 5 min at room temperature. Samples Co. Ltd and Panreac AppliChem Co Ltd. The and standards were transferred from microtubes microbes from fresh human feces was obtained from to microplate and measure absorbance at 500 nm. a healthy donor who has not received antibiotics, Protein determination by glucose oxidase-peroxidase food supplement and other health food such as dairy (GOD-POD) method was used. Pipette 20 μl standards mix probiotic or prebiotic in the last 3 months. glucose (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 mg/ml), diluted crude enzyme samples or distilled water (for Preparation of plant materials blank) were transferred to microplate and added 200 Jerusalem artichokes were obtained from μl GOD-POD solution then kept in the dark for 40 Wichienchot et al./IFRJ 24(1): 134-141 136 min. Absorbance of samples were measured at 596 Microbial colony counting with fluorescent in situ nm. hybridization (FISH) technique The sample (375 μl) was added by 1,125 μl of Purification of FTase enzyme 4% (w/v) paraformaldehyde solution (pH 7.2), mixed Crude enzyme sample was loaded though Q and kept at 4oC overnight to fix bacterial cells.

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