3.MI-Rachel Budhisatria.cdr Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.11.2.3ISSN 1978-3477, eISSN 2087-8575 Vol 11, No 2, June 2017, p 55-61 *Corresponding author; Phone: +62-81932000311, Email: rbudhisatria@gmail.com Nutraceuticals and functional foods have become important tools for consumers to manage their health and well-being. Many food components, especially food oligosaccharides and polysaccharides (including dietary fiber), have been shown to exhibit prebiotic activity. However, not all dietary carbohydrates are prebiotics. Clear criteria of prebiotics have been established. These criteria are 1) resistance to gastric acidity, degree of hydrolysis by mammalian enzymes, and degree of gastrointestinal absorption; 2) degree of fermentation by intestinal microflora, and 3) selective stimulation on the growth and/or activity of the intestinal bacteria that contribute to health and well- In Vitro and In Vivo Prebiotic Activities of Purified Oligosaccharides Derived from Various Local Bananas (Musa sp.): Tanduk, Uli, Raja Sereh, and Cavendish Banana is a good source of prebiotic, and in Indonesia it is consumed as staple food. The aims of this research were to evaluate the activity of purified oligosaccharides (POS) as prebiotic from various different local bananas (Musa sp.): Tanduk (T), Uli (U), Raja Sereh (RS), and Cavendish (C); and to investigate their capacity in promoting the growth of Lactobacillus sp., in vivo. In vitro investigation including purification of oligosaccharides from various different local bananas were by 80% ethanol extraction. Subsequently, absolute ethanol was reconstituted before precipitation/centrifugation for glucose removal. Water was also removed by freeze drying. POS from the four bananas were analyzed by Thin Layer Chromatography (TLC). Prebiotic activity of POS was investigated by measurement of Prebiotic Activity Score (PAS). In vivo investigation was conducted as followed, Balb/c mice were grouped into 6 groups with different prebiotics supplementation: negative control (4 mice, standard feed), positive control (6 mice, 15 mg of inulin ), and samples (5 mice, -1 150 mg of T, U, RS, or C banana g day ) for 40 days. Following 40 days after treatment, fecal viable counts of -1 Lactobacillus sp. and Enterobacteriaceae of Balb/c mice was measured (CFU g ) and analysed. PAS value revealed a positive correlation between the oligosaccharides from bananas and Lactobacillus paracasei, with PAS value for T (0.05), RS (0.15), U (0.33), and C (0.77). Overall data suggest that fecal viable counts of Lactobacillus sp. increased after 25 days administration of U, RS, and C banana when compared to controls. Contrastingly, the fecal viable counts of Enterobacteriaceae decreased after 40 days administration of U, RS, and C banana compared to the control. Different types of local bananas demonstrate diverse prebiotic activities, U and C promote Lactobacillus sp. growth and reduce Enterobacteriaceae count. PAS value of U and C suggest potential prebiotic activity, whereas T and RS do not. Key words: banana, lactic acid bacteria, oligosaccharides, PAS, prebiotics Pisang merupakan sumber prebiotik, dan di Indonesia pisang dikonsumsi sebagai makanan pokok. Tujuan penelitian ini untuk mengevaluasi aktivitas dari oligosakarida yang sudah dipurifikasi (POS) sebagai prebiotik dari berbagai macam pisang lokal (Musa sp.): Tanduk (T), Uli (U), Raja Sereh (RS), dan Cavendish (C); dan untuk mengetahui kapasitas pisang dalam memicu pertumbuhan Lactobacillus sp., in vivo. Percobaan In vitro dilakukan sebagai berikut: Oligosakarida dari berbagai macam pisang lokal yang berbeda dipurifikasi dengan 80% etanol. Selanjutnya, etanol absolut ditambahkan sebelum presipitasi/sentrifugasi untuk menghilangkan glukosa. Air dihilangkan dengan metode freeze drying. POS dari empat pisang dianalisis dengan Thin Layer Chromatography (TLC). Aktivitas prebiotik POS diukur dari hasil Prebiotic Activity Score (PAS). Sedangkan percobaan In vivo sebagai berikut: Mencit Balb/c dibagi menjadi 6 kelompok, dengan pemberian suplemen prebiotik yang berbeda: kontrol negatif (4 mencit, pakan biasa), kontrol positif (6 mencit, 15 mg inulin/g/hari), -1 -1 dan sampel (5 mencit, 150 mg pisang T, U, RS, dan C g hari ) selama 40 hari. Setelah 40 hari perlakuan, dihitung -1 jumlah Lactobacillus sp. dan Enterobacteriaceae dari sampel feses mencit dalam satuan CFU g dan dianalisa. Nilai PAS menunjukkan korelasi positif antara oligosakarida dari pisang dan Lactobacillus paracasei, dengan nilai PAS T (0,05), RS (0,15), U (0,33), dan C (0,77). Secara keseluruhan data dari perhitungan sampel feses untuk Lactobacillus sp. meningkat setelah 25 hari pemberian pisang U, RS, dan C dibandingkan dengan kontrol. Sebaliknya, hasil perhitungan sampel feses untuk Enterobacteriaceae menurun setelah 40 hari pemberian pisang U, RS, dan C dibandingan dengan kontrol. Perbedaan jenis pisang lokal menunjukkan aktivitas prebiotik yang berbeda, U dan C memicu pertumbuhan Lactobacillus sp. dan menurunkan perhitungan Enterobacteriaceae. Berdasarkan nilai PAS dari U dan C diduga berpotensi memiliki aktivitas prebiotik, dimana T dan RS tidak. Kata kunci: bakteri asam laktat, oligosakarida, PAS, pisang, prebiotik -1 -1 g day -1 RACHEL BUDHISATRIA*, ROSARIA, LUCY JAP, AND TAN TJIE JAN Biology Department, Universitas Pelita Harapan, Jalan M.H Thamrin Boulevard, Tangerang, 15811, Indonesia being of host organism (Thammarutwasik et al. 2009; Gibson et al. 2004). Prebiotic was first defined by Roberfroid and Gibson (1995), indigestible food ingredients that favorably affect the host by selectively promote growth and/or activity of beneficial bacteria in the colon, and thus improving the host health. Indigestible oligosaccharides, particularly fructo- oligosaccharides, are prebiotics. Intake of prebiotics can significantly modulate the colonic microbiota by increasing the number and activities of specific bacteria, such as Lactobacilli and Bifidobacteria and inhibit the growth or activities of pathogenic bacteria such as Enterobacteriaceae causing change in the microbiome. Prebiotics are known to indirectly promote physiological support of the gastrointestinal tract’s. (Gibson and Wang 1994; Roberfroid 2007). Acccording to Kahlon and Smith (2007) and Gibson and Wang (1994), banana is a source of prebiotics. Indonesia is the leading banana producer in Asia, producing more than 50 % of Asia’s supply of bananas. Indonesia has approximately 200 different kinds of local bananas (Prabawati et al. 2009). This variety possesses the potential for development into prebiotics that can contribute to human health. However, studies of prebiotics in local bananas have been limited (Kahlon and Smith 2007; Gibson and Wang 1994b). Herein, prebiotic activity of purified oligosacchari- des from various local bananas: Tanduk, Uli, Raja Sereh, and Cavendish were studied. Tests were conducted to evaluate their capability to promote the growth of Lactobacillus paracasei through PAS value, which is a method to determine the ability of a given prebiotics to better promote the growth of probiotic microorganism relatively better than to other pathogenic microorganisms (Huebner et al. 2007); and in vivo study were carried out on Balb/c mice by feeding them with different types of local bananas. Prebiotic activities were determined through their ability to promote the growth of Lactobacillus sp. by measuring increase of the bacterial count in feces. MATERIAL AND METHODS Samples and Subjects. Approximately 1.5 - 2 kg local bananas: Tanduk (T), Uli (U), Raja Sereh (RS), and Cavendish (C) were used for oligosaccharides extraction, purification, and in vitro analysis. Approximately 3 - 4 kg of each fresh local bananas (T, U, RS, and C), and a total of 30 male, twenty weeks old Balb/c mice were used in the present in vivo study. All animal study were performed followed the 56 BUDHISATRIA ET AL. Microbiol Indones Guidelines for The Housing of Mice in Scientific Institutions, Animal Welfare Unit, New South Wales Department of Industry (Fawcett 2012). Oligosaccharides Extraction and Glucose Removal from Samples. Oligosaccharides extraction was performed according to method previously described by Livingston (1990). Eight hundred forty gram of each type of bananas (T, U, RS, and C) were blended with 1.1 L of 80% ethanol. Blended banana ethanol mixtures were filtered with a cotton filter to remove the coarse particles. All collected filtrate were then heated at 70 °C for 15 min to inactivate potential oligosaccharide degrading enzymes, such as invertase. The resulting thick banana pulps were blended twice for about 5 min with 100 mL of distilled water and then agitated at 200 rpm at room temperature for 1 h, to homogenized. All extracts were then centrifuge at 900 g, for 20 min at 10 °C. Supernatant were then collected and evaporated to remove residual ethanol. The thick extracts were mixed with absolute ethanol (1:1 v/v) and stored at -2 °C for 24 h. The extracts were further centrifuged at 900 g for 15 min at room temperature. Glucose fraction in the supernatant was discarded. Absolute ethanol was re-added into the remaining pellet, mixed and centrifuged at 900 g for 15 min at room temperature and the supernatant were decanted as above. The step was repeated for thirty times until glucose was not detected anymore through thin layer chromatography (TLC) with n-butanol/1-propanol/ace- tic acid/water (3:1:1:1) as eluent. After glucose removal, a thick milky suspension containing pure oligosaccharides was then subjected to freeze drying. Dried oligosaccharide samples were then weighed. Pure oligosaccharides were verified by TLC with 90% formic acid as eluent. Prebiotic Activity Score (PAS) from Purified Banana Oligosaccharides. PAS was performed according to the procedure as described by Huebner et al. (2007). PAS is defined as follow: Whereby, p is defined as probiotics (Lactobacillus paracasei/Lp), e is defined as enteric bacteria (E. coli/ Ec), P is total plate count of Lp culture, and Ec culture 24 in prebiotic added medium after 24 h incubation, respectively, P is total plate count of Lp culture, and Ec 0 culture in prebiotic added medium before 24 hours incubation, respectively, G is total plate count of Lp 24 culture, and Ec culture in glucose added medium after 24 h incubation, respectively, G is total plate count of 0 -PAS = P - P24 0 G - G24 0 P - P24 0 G - G24 0 Lp culture, and Ec culture in glucose added medium before 24 h incubation, respectively. In the formula above, the probiotic Lactobacillus paracasei (Lp) was cultured in 3 mL modified de ManRogosa Sharpe broth without carbon source, agitated at 100 rpm at 37 °C, microaerophilic for 24 h. No prebiotic was added to negative control. 1% of inulin was added to positive control. 1% of purified oligosaccharide from banana T, U, RS, or C was added as tested samples. Escherichia coli (Ec) was cultured in 3 mL M9 minimal medium broth without carbon source, agitated at 100 rpm at 37 °C for 24 h to serve as basal media colony plate count. No prebiotic was added to negative control. 1 % of inulin was added to positive control. 1% of purified oligosaccharide from banana T, U, RS, or C was added as tested samples. Additional control plate containing 1 % glucose on basal media was also prepared. The above PAS plate count was performed as follow. A 50 μL of serial dilluted samples were spread on MRS agar for L. paracasei count, and on MacConkey agar for E. coli count. Serial dilution was performed to obtain countable CFU plate count (30 - -1 300 CFU plate ). All plates were incubated at 37 °C for 24 h under microaerophilic condition for L. paracasei count and aerobic condition for E. coli count. Colony -1 count in each plate was expressed in log CFU mL . Quantitative Analysis of Lactobacillus sp. and Enterobacteriaceae in Mice Feces Treated with Bananas. Balb/c mice were grouped into negative control (4 mice, standard feed), positive control (6 -1 -1 mice, 15 mg of inulin g day ), and samples (5 mice, -1 -1 150 mg of T, U, RS, or C banana g day ). All supplemented prebiotics were measured in dry weight. Fecal samples were collected from each mice at day 0, 17, 20, 25, and 40. For bacteria count, samples were diluted in physiological salt solution (1:9 v/v) (Hartemink and Rombouts 1999), and then 50 μL of each diluted feces was spread on Rogosa agar for Lactobacillus sp. and MacConkey agar for Enterobacteriaceae, at 37 °C for 24 - 48 h (Adami and Cavazzoni 1996). Colony count in each plate (CFU -1 -1 mL ) was converted into log CFU g sample. The -1 difference percentage log CFU g relative to day 0 (D0) was calculated. RESULTS Purified Oligosaccharides (POS) from Banana. The purification of banana from glucose after serial ethanol suspension and centrifugation produce excellent result (Fig 1). The inclusion of glucose in oligosaccharide samples will interfere PAS value because glucose will be used as carbon source by both bacteria, L. paracasei and E. coli (Date et al. 2014; Kunova 2011). The appearance of extracted POS from each bananas were different in color and texture (Fig 2). In Vitro Analysis Prebiotic Activity. To ensure glucose was completely eliminated for the TLC analyses, oligosaccharides purification was conducted. Each banana POS was further used to determine PAS -1 value, as shown in (Tabel 1) in log CFU mL . Cavendish (C) banana display highest PAS value of 0.77, followed by Uli (U) banana sample with PAS value of 0.33, Raja Sereh (RS) with PAS value of 0.15 and lastly Tanduk (T) with PAS value of 0.15. The PAS value of Uli (U) is comparable to inulin PAS value (0.33). Negative control shows a negative PAS value (-0.11). Effect of Feeding Bananas on Lactobacillus sp. Growth in Feces. Balb/c mice were used to study the effect of banana feeding on Lactobacillus sp. growth in gastrointestinal tract. Treated mice were given T, U, -1 -1 RS, or C at 150 mg of banana g day for 40 d. As positive control inulin was given to animal feed at 15 -1 -1 mg of inulin g day for 40 d. Feces of mice were collected as described in the methods. Uli (U), Raja Sereh (RS), and Cavendish (C) banana fed on mice result in a higher of Lactobacillus sp. count than negative control. In contrast inulin gave an inconsistant result throughout time fed. Tanduk (T) banana did not support the growth of Lactobacillus sp. but Enterobacteriaceae. Other nutrient factors might play a part in promoting Lactobacillus sp. growth. Fig 4 showed that T and RS banana promote Enterobacteria- ceae growth, whereas U and C, and inulin as positive control did not promote growth. Result displayed on day 40 reveal more disperse result on the effect of -1 different banana sample to the percentage log CFU g relative to D0. PAS value of U and C indicates that these bananas promoted the growth of Lactobacillus sp. and reduced the growth of Enterobacteriaceae during in vivo study. DISCUSSION Purified oligosaccharides from each different banana yield different mass after purification. According to Livingston (1990), centrifugation and precipitation process at 900 g should successfully eliminate monosaccharide (glucose) and other simple Volume 11, 2017 Microbiol Indones 57 Microbiol Indones58 BUDHISATRIA ET AL. A C B D A B C D E F Fig 1 TLC result showing glucose elimination after repeated extraction. Glucose detection was used n butanol: propanol: acetic acid: water with ratio 3: 1: 1: 1 as eluent. Then, was dried and added with visualization solution and was dried again in oven until spots appeared. A: inulin standard (0,02 mg); B: glucose standard (0.02 mg); C: extract from T (10 µL); D: extract from U (10 µL); E: extract from RS (10 µL); and F: extract from C (10 µL). Fig 2 Purified oligosaccharides (POS) from banana samples. Purifified oligosaccharides after freeze drying A: oligosaccharides from T; B: oligosaccharides from U, C: oligosaccharides from RS, and D: oligosaccharidess from C. Fig 3 TLC result showing oligosaccharides detection after glucose elimination. Oligosaccharides detection from each various local bananas. Oligosaccharides detection was used 90 % of formic acid as eluent. Then, was dried and added with visualization solution and was dried again in oven until spots appeared. A: inulin standard (0,02 mg); B: glucose standard (0,02 mg); C: T oligosaccharides (20 µL); D: U oligosaccharides (20 µL); E: RS oligosaccharides (20 µL); F: C oligosaccharides (20 µL); G: maltose standard (0.02 mg); and H: raffinose standard (0.02 mg). Volume 11, 2017 Microbiol Indones 59 V t0 t24 t0 t24 t0 t24 t0 t24 1 Inulin 9.59 5058.25 22.49 8669.62 5.98 8.70 6.35 8.94 2 Negative control 13.00 128.82 26.18 831.76 6.11 7.11 6.42 7.92 3 Glucose 17.30 1527.57 14.45 3767.04 6.24 8.18 6.16 8.58 4 T 89.81 3950.63 58.43 4786.30 6.95 8.60 6.77 8.68 5 U 28.71 3908.41 38.90 2766.94 6.46 8.59 6.59 8.44 6 RS 78.70 4813.93 57.28 4004.05 6.90 8.68 6.76 8.60 7 C 30.48 3265.88 35.48 164.44 6.48 8.51 6.55 7.22 Lp Ec 2.72 2.59 1.00 1.50 1.95 2.42 1.64 1.91 2.13 1.85 1.79 1.84 2.03 0.67 Lp Ec 1.40 1.07 0.51 0.62 - - 0.84 0.79 1.10 0.77 0.92 0.76 1.04 0.28 0.33 -0.11 - 0.05 0.33 0.15 0.77 RGR IV SamplesNo PAS value III I II -1 ∆ log CFU mL log -1 CFU mL-1 5CFU mL (×10 ) EcLpEcLp Table 1 Average results of PAS value from T, U, RS, and C Fig 4 Progression of Log CFU percentage of Lactobacillus sp. per gram of fresh feces at day 20 (D20), day 25 (D25), and day 40 (D40) relative to day 0 (D0) in Balb/c mice fed by various local bananas. : negative control, : inulin, : tanduk, : Uli, : raja sereh, and : cavendish. Fig 5 Progression of Log CFU percentage of Enterobacteriaceae per gram of fresh feces at day 20 (D20), day 25 (D25), and day 40 (D40) relative to day 0 (D0) in Balb/c mice fed by various local bananas. : negative control, : inulin, : tanduk, : Uli, : raja sereh, and : cavendish. D0 D20 D25 D40 20 15 10 5 0 -5 % P ro g re ss io n l o g -1 C F U g o f L a ct o b a ci ll u s sp . fr o m d ay 0 Day - 15 10 5 0 -5 -10 -1 % P ro g re ss io n l o g C F U g o f E n te ro b a ct er ia ce a e sp . fr o m d ay 0 D0 D25 D40D20 Day - 5 This table shows results Average PAS value from Inulin, negative control, T, U, RS, and C. Columns I are CFU mL (x10 ); -1 Columns II are log from columns I; Columns III are are the difference (or Δlog CFU mL ) from column II, namely t -t for Lp 24 0 -1 and Ec, respectively.Columns IV are relative growth ratio (RGR) obtained from column III, namely Δlog CFU mL from -1 Inulin, T, U, RS, and C for Lp and Ec, which divided by glucose Δlog CFU mL for Lp and Ec, respectively. The last column V is PAS values which obtained from RGR (columns IV) Lp-Ec. -1 Microbiol Indones60 BUDHISATRIA ET AL. microbiota of the feces reflects that of the distal colon. Many diseases, such as colon cancer, diverticular disease, ulcerative colitis, and inflammatory bowel disease originates from this organ (MacFarlane et al. 1992). Modulation in the microbiota of distal colon, such as higher count of Lactobacillus sp. and a lower count of Enterobacteriaceae, was able to prevent the onset of those diseases (Roberfroid et al. 2010). CFU of Lactobacillus sp. and Enterobacteriaceae in the feces were quantified in selective media for mini- mizing the growth of contaminant microorganisms. Lactobacillus sp. was quantified in Rogosa medium which has been proven to be selective against Lactobacillus sp. according to Gram staining, catalase and endospora assays. Meanwhile, MacConkey medium was used to quantify Enterobacteriaceae and its selectivity against this group of bacteria has been tested with Gram staining method. This study was done to prove the possible positive health effect of fresh bananas on gastrointestinal tract by measuring the increasing number of Lactobacillus in feces in comparison to Enterobacteriaceae. In feces of the treated Balb/c mice fed with the same bananas, the results showed that all bananas but T banana were able to promote Lactobacillus sp. growth, while T and RS, but not U or C, were able to promote growth Enterobacteriaceae. The contradicting result in vivo and in vitro could arise from different sample treatment. Glucose is not omitted in in vivo assay which could result in glucose interference towards colon bacteria yield. It is recommended that glucose is removed from all samples before administration to animal model. However, attention of the limitation on technique, that result in significant sample loss, used for purification of POS from banana sample used in this paper need to be consider. ACKNOWLEDGMENT The publication of this paper was supported by grant from Lembaga Penelitian dan Pengabdian kepada Masyarakat Universitas Pelita Harapan (LPPM UPH). We are greateful for the collaboration with Laboratorium Biologi Dasar (B202) and Laboratorium Biologi Lanjutan (B407) Universitas Pelita Harapan for providing us laboratory instruments. REFERENCES Adami A and Cavazzoni V. 1996. A standard procedure for assessing the faecal microflora of swine. Annali di glucose that have low degree of polymerization range (DP 3 - 5) from the extract which. (Roberfroid 2007; Slavin 2013). Thus, considerable amount of oligosacc- harides prebiotics were expected to be lost during purification. This is accountable to the minimum PAS value measurement. Banana POS from T, U, RS, and C were detected on TLC by using 90% of formic acid as eluent (Fig 3) which possesses very high polarity or strong acid. This suggest that POS with high DP might be undetected on the TLC performed (Holmes and O’Brien 1979; Zhang et al. 2007). Eluent of 100% acetic acid, and another of 1-butanol: formic acid: aquades, 4: 8: 1, did not produce good result. Appearances of purified POS from each banana were different. RS purified POS obtained as white color granule or powder. Similar appearance was described by Oliveira et al (2011) with agglomerate texture and difficult to ravel due to the strong glycosidic bond between the polisaccharides. T, U, and C purified POS appeared as a mixture of white and dark color granule and powder (Oliveira et al. 2011). The positive values of PAS for inulin, T, U, RS, and C, signifies that prebiotics or purified POS bananas are able to promote the growth of L. paracasei relatively better than E. coli. According to Rubel et al. (2014), the RGR value >1 means that tested samples (T, U, RS, and C), prebiotics/oligosaccharides addition as growth media could support the growth of probiotics such as Lactobacillus sp than using glucose as growth media (Rubel et al. 2014). Data reflected in Table 1 showed average results of PAS from in vitro analysis prebiotic activity. RGR value of prebiotic inulin and purified POS of U and C are >1, while T and RS are < 1 which indicates that T and RS could not promote L. paracasei growth well. As many types of oligosaccharides are present in fruits and plants, T and RS may have different oligosaccharide content than U and C (Roberfroid 2007). Other species of Lactobacillus should be studied to examine its capability to metabolize the oligoaccharides. C has much higher PAS value of 0.77 compare to inulin as control positive 0.33 (Tabel 1) which calls for further investigation. The PAS values showed that only two purified banana oligosaccharides, U and C, were capable of promoting the growth of L. paracasei with RGR >1, while the other two purified banana oligosaccharides T and RS were inconclusive, RGR <1. In vivo experiment was analyzed by using Balb/c mice to study the effect of banana feeding on Lactobacillus sp. growth in gastrointestinal tract. The Volume 11, 2017 Microbiol Indones 61 MacFarlane GT, Gibson GR, Cummings, JH. 1992. Comparison of fermentation reactions in different regions of the human colon. J Appl Bacteriol. 72(1):57- 64. doi:10.1111/j.1365-2672.1992.tb04882.x. Oliveira AJB, Goncalves RAC, Chierrito TPC, Santos MM, Souza LM, Gorin PAJ, Sassaki G, Lacomini M. 2011. Structure and degree of polymerization of fructooligosaccharides present in roots and leaves of Stevia rebaudiana (Bert.) Bertoni. Food Chem. 129(2): 305-311. doi:10.1016/j.foodchem.2011.04.057. Prabawati S, Suyanti, Setyabudi, D. A. 2009. Teknologi pasca panen dan pengolahan buah pisang. Bogor: Balai Besar Penelitian dan Pengembangan Pasca Panen Pertanian. Roberfroid M. 2007. Prebiotics: The Concept Revisited. J Nutr. 137(3 suppl 2): 830S-837S. Roberfroid M, Gibson GR, Hoyles L, McCartney AL, Rastall R, Rowland I, Wolvers D, Watzl B, Szajewska H, Stahl B, Guarner F, Respondek F, Whelan K, Coxam V, Davicco MJ, Léotoing L, Wittrant Y, Delzenne NM, Cani PD, Neyrinck AM, Meheust A. 2010. Prebiotic effects: metabolic and health benefits. Br J Nutr. 104 (Suppl. 2), S1e63. Rubel IA, Pérez EE, Genovese DB, Manrique GD. 2014. In vitro prebiotic activity of inulin-rich carbohydrates extracted from Jerusalem artichoke (Helianthus tuberosus L.) tubers at different storage times by Lactobacillus paracasei. Food Res Int. 62:59-65. doi:10.1016/j.foodres.2014.02.024. Slavin J. 2013. Fiber and prebiotics: mechanisms and health benefits. J Nutr. 5(4):1417-1435. doi:10.3 390/nu5041417. Thammarutwasik P, Hongpattarakere T, Chantachum S, Kongkarn K, Itharat A, Reanmongkol W, Tewtrakul S, Ooraikul B. 2009. Prebiotics-A review. Songklanakarin J Sci Technol. 31(4):401-408. Zhang Z, Xie J, Zhang F, Linhardt, R. J. 2007. Thin layer chromatography for the analysis of glycosaminoglycan oligosaccharides. Anal Biochem. 371(1):118-120. doi:10.1016/j.ab.2007.07.003. Microbiologia e Enzimologia. 46:1-9. Archibold HK. 1940. Fructosans in the monocotyledons a review. New Phytol. 39(2):185-219. doi:10.1111/j.1469- 8137.1940.tb07132.x. Date Y, Nakanishi Y, Fukuda S, Nuijima Y, Kato T, Umehara M, Ohno H, Kikuchi, J. 2014. In vitro evaluation method for screening of candidate prebiotic foods. Food Chem. 152: 251-260. doi:10.1016/j.foodchem.2013.11.126. Fawcett A. 2012. Guidilines for The Housing of Mice in Scientific Instutions. Retrieved from: http://www.ani- malethics.org.au/__data/assets/pdf_file/0004/249898/ Guideline-22-mouse-housing.pdf (4 November 2016). Gibson GR and Roberfroid MB. 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 125(6):1401-1412. Gibson GR and Wang X. 1994. Regulatory effects of bifidobacteria on the growth of other colonic bacteria. J Appl Bacteriol. 77:412-420. doi:10.1111/j.1365- 2672.1994.tb03443.x. Gibson GR, Probert HM, Van Loo JAE, Roberfroid MB. 2004. Dietary modulation of the human colonic microbiota: updating the concept of prebiotics. Nutr Res Rev. 17(2): 259-275. doi:10.1079/NRR200479. Hartemink R and Rombouts FM. 1999. Comparison of media for the detection of bifidobacteria, lactobacilli and total anaerobes from faecal samples. J Microbiol Methods. 36(3):181-192. doi:10.1016/S0167-7012(99) 00031-7. Holmes EW and O’Brien JS. 1979. Separation of glycoprotein-derived oligosaccharides by thin-layer chromatography. Anal Biochem. 93:167-170. doi:10.1016/S0003-2697(79)80131-1. Huebner J, Wehling RL and Hutkins RW. 2007. Functional activity of commercial prebiotics. Int Dairy J. 17(7):770-775. doi:10.1016/j.idairyj.2006.10.006 . Kahlon TS and Smith GE. 2007. In vitro binding of bile acids by bananas, peaches, pineapple, grapes, pears, apricots and nectarines. Food Chem. 101(3):1046-1051. doi:10.1016/j.foodchem.2006.02.059. Kunova G, Rada V, Lisova I, Rockova S, Vlkova, E. 2011. In vitro fermentability of prebiotic oligosaccharides by Lactobacilli. Czech J Food Sci. 29:S49-S54. Livingston DP. 1990. Fructan precipitation from a water/ethanol extract of Oats and Barley. Plant Physiol. 92(3):767-769. doi:10.1104/pp.92.3.767. 1: 55 2: 56 3: 57 4: 58 5: 59 6: 60 7: 61