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Prebiotic effectiveness of inulin extracted from edible burdock
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Anaerobe 14 (2008) 29–34 www.elsevier.com/locate/anaerobe
Food microbiology
Prebiotic effectiveness of inulin extracted from edible burdock Dandan Li, Jin M. Kim, Zhengyu Jin, Jie Zhou Food Science and Technology, Jiang Nan University, Wuxi, Jiangsu 214122, China Received 29 June 2007; received in revised form 16 October 2007; accepted 18 October 2007 Available online 26 November 2007
Abstract To investigate the prebiotic potential of burdock inulin (B-INU), the in vitro and in vivo effects of B-INU on bacterial growth were studied. B-INU significantly stimulated the growth of bifidobacteria in Man–Rogosa–Sharp (MRS) medium, anaerobically. Compared with chicory inulin (C-INU), long-chain inulin (L-INU) and fructooligosaccharides (FOS), 1% (w/v) B-INU promoted the specific growth rate of beneficial bacteria. The decreases of media pH with B-INU were almost the same as that with C-INU and FOS. In vivo, B-INU significantly increased the number of lactobacilli and bifidobacteria (Po0.05) in cecal content. Mice fed with B-INU, C-INU and FOS for 14 days had greater number of cecal beneficial bacteria population than those fed with L-INU for 14 days. In addition, all fructans did not cause any side effects, such as eructation and bloating. Results indicated that inulin extracted from edible burdock showed prebiotic properties that could promote health. r 2007 Elsevier Ltd. All rights reserved. Keywords: Burdock; Inulin; Fructooligosaccharides; Prebiotic; Cecal microbiota
1. Introduction Prebiotics are compounds, usually carbohydrates, which are resistant to direct metabolism by the host and reach the caeco-colon where they are preferentially utilized by selected groups of beneficial bacteria [1]. Modulation of the human gut flora by prebiotic oligosaccharides has the potential benefit to human health by enhancing levels of ‘beneficial’ gut bacteria [2]. The term prebiotic has been defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one, or a limited number of bacteria [3]. The prebiotics (carbohydrates) are resistant to enzymatic hydrolysis in the upper gastrointestinal tract and reach the caeco-colon intact. There they are completely fermented. This fermentation converts inulin into biomass, shortchain fatty acids, lactate and gases [4]. Intestinal microbiological ecosystem has been associated with human Abbreviations: B-INU, burdock inulin; C-INU, chicory inulin; L-INU, long-chain inulin; FOS, fructooligosaccharides; DP, degree of polymerization; G, glucose; F, fructose; GF, sucrose; MRS, Man–Ragosa–Sharp. Corresponding author. Tel.: +86 510 85919189; fax: +86 510 85919189. E-mail address: [email protected] (Z. Jin). 1075-9964/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2007.10.002
health. In infants, bifidobacteria are the predominant bacteria, but studies show that there is a substantial decrease in their population as human age. There are definite advantages to establish and maintain proper bifidobacteria levels [5]. Beneficial flora including the genera of bifidobacteria or lactobacilli improves the caeco-colon environment by suppressing the growth of pathogenic bacteria and production of carcinogenic materials and thus improving immunity [6]. Burdock is a dull, pale green, stems about 3–4 ft and branched, rising from a biennial root. The burdock roots are fleshy, wrinkled, crowned with a tuft of whitish, soft, hairy leaf-stalks, gray-brown externally, whitish internally, with a somewhat thick bark, about a quarter of the diameter of the root, and soft wood tissues, with a radiate structure. As a rule, they are 12 in or more in length and about 1 in thick, sometimes, however, they extend 2–3 ft, making it necessary to dig by hand. Burdock inulin (B-INU), chicory inulin (C-INU), long-chain inulin (L-INU) and fructooligosaccharides (FOS) are among the fructans, which are undigestible in the upper gastrointestinal tract. Inulin is a linear, highly polymerized fructan of average degree of polymerization (DP) 12, produced by extraction from chicory roots or burdock
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roots. It consists of a linear chain of fructose with b-(2-1) linkages with a terminal glucose unit. L-INU has a higher average DP 22 than that of B-INU or C-INU. FOS with average DP 4.5 can be produced by chemical degradation or controlled enzymatic hydrolysis of inulin using endoglycosidases. Furthermore, FOS can also be produced on a commercial scale, from sucrose, using a fungal enzyme from either Aureobasidium sp. or Aspergillus niger [7]. DP is the value that corresponds to the total number of saccharide units (G and F units) in a given inulin sample divided by the total number of inulin molecules, without taking into account the monosaccharides, glucose (G) and fructose (F), and the disaccharide, sucrose (GF), which are possibly present in the sample. By calculating the fructoseto-glucose ratio (number of fructose units per number of glucose units), the average DP is obtained: DP ¼ number of F units per G units+1 G unit. The objective of this study was to evaluate the prebiotic properties of inulin extracted from edible burdock in vitro and in vivo. In addition, prebiotic properties of B-INU, C-INU, L-INU or FOS with different DP were also evaluated. 2. Materials and methods 2.1. Prebiotics Commercially available prebiotic oligosaccharides were used: B-INU (inulinX96.5%, mono+disacch.p2%); C-INU (inulinX92.2%, mono+disacch.p2.0%); L-INU (inulinX94.6%, mono+disacch.p4%) and FOS (FOSX 91.0%, sucrose+monosacch.p5.0%). B-INU was extracted in our laboratory [8]. C-INU was purchased from Wede Biologicals Co., Ltd. (Beijing, China). L-INU was purchased from Tianmen Hylae inulin R&D Co., Ltd. (Hubei, China). FOS was purchased from Baolingbao Biotechnology Co., Ltd. (Shandong, China). Basal MRS (Man–Ragosa–Sharp), BBL agar, LBS agar, EMB agar and EC agar were purchased from Kaiyang Biologicals Co., Ltd. (Shanghai, China). Oxoid Anaerobic System was purchased from Oxoid GmbH (Wesel, Germany). All other chemicals and media preparations used in this investigation were purchased from Sigma (Shanghai, China). Bifidobacterium adolescentis ATCC 15703 was obtained from Medical College of Fudan University (Shanghai, China). 2.2. Prebiotic effect of B-INU in vitro After the standard strains of B. adolescentis ATCC 15703 had been incubated anaerobically (73% N2:20% CO2:7% H2) for 48 h at 37 1C, the stock cultures were stored in 10% glycerol at 70 1C until needed. MRS broth supplemented with 0.5 g/L L-cysteine hydrochloride, 0.2 g/L of sodium thioglycolate, and 0.1 g/L of CaCl2 2H2O was used as the stock culture medium and as the basal medium. Basal MRS broth supplemented with a final concentration
of 0.5%, 1%, 2%, 3% or 4% (w/v) of B-INU was divided into five test tubes (5 mL in each), inoculated with 50 mL overnight culture of the beneficial bacteria, and incubated at 37 1C in anaerobic jars—Oxoid Anaerobic System with Gas Pak H2+CO2. To examine the effects of B-INU on cell growth, the bacteria were cultivated overnight in the basal medium at 37 1C, and diluted to 106 cells/mL, 50 mL of this was then inoculated into 5 mL of basal medium containing fructans (B-INU, C-INU, L-INU and FOS). The bacteria were then cultured under anaerobic conditions for 24 h at 37 1C. After the culture broth had been centrifuged for 15 min with 2500 g, the precipitate was washed twice with PBS (0.1 M phosphate buffer pH 7.4, 0.9% saline), and diluted one- to five-fold with PBS. The diluted cells were mixed well and then turbidity was measured at 600 nm. All measurements were performed in quadruplicate parallels and repeated at least twice. 2.3. Prebiotic effect of B-INU in vivo 2.3.1. Animals and diets Female Kunming mice (2071 g body weight, 6 weeks old) were purchased from Chinese Academy of Medical Sciences and housed in a room at 22 1C and 55% relative humidity, with a 12-h light/dark cycle. Mice were fed the control diet, containing (g/kg diet): pregelatinized cornstarch 646, soluble casein 204, DL-methionine 3, corn oil 97, a mineral mixture 45 [providing (g/kg diet): Ca2(PO4)3 16.6, K2(PO4)3 10.5, CaCO3 8.0, NaCl 3.1, MgSO4 3.9, FeSO4 0.3, ZnSO4 0.2, MnSO4 0.2, CuSO4 0.04, CoSO4 4 104, KHSO4 4 104, (NH4)2SO4 4 104, MgO 0.9], a vitamin mixture 5 [providing (mg/kg diet): allrac-a-tocopherol (500 UI/g) 150, all-trans-retinylacetate (500,000 UI/g) 8, cholecalciferol (400,000 UI/g) 2.5, nicotinic acid 45, calcium pantothenate 15, thiamin hydrochloride 5, riboflavin 9, pyridoxine (PN) hydrochloride 5, ascorbic acid 113, folic acid 2, p-aminobenzoic acid 113, vitamin B-12 (1 g/kg) 67.8, biotin 0.4, phylloquinone 2, myo-inositol 113]. All experiments were in accordance with National Research Council Guidelines of China for the care and use of laboratory animals. After a week’s acclimation period, mice were randomly divided into five groups (eight for each group) and fed one of the following five diets: control diet alone (control group), control diet+5% B-INU (B-INU group), control diet+5% C-INU (C-INU group), control diet+5% L-INU (L-INU group) and control diet+5% FOS (FOS group). Fructans were used to partially replace the corn starch component of the control diet. The dietary treatments were lasted for 14 days. The body weights of the rats were recorded weekly. Mice were observed for any sign of eructation or bloating during 14 days of diet treatment. 2.3.2. Cecal sample preparation On the 15th day of dietary treatment, the mice were euthanized using CO2 asphyxiation and the cecum was
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2.4. Statistical analysis The results were analyzed using one-way ANOVA, and significant differences between groups were determined by the Duncan’s multiple range test [9]. All statistical analyses were performed using the SPSS package program version 10.0.1 and differences were considered significant at Po0.05. 3. Results
Growth (turbidity at 600 nm)
2.3.3. Bacteriological analysis of cecal contents Medium used for the detection of bifidobacteria from cecal contents was BBL agar. Lactobaccilli were enumerated on LBS agar. Enterobacteria, and enterococci were enumerated on EMB agar, and EC agar. For cultivation of bacteria, cecal content (0.1 g) was dispersed in 2 mL of 5% cysteine using glass beads (3 mm diameter). Further dilutions were made using 5% cysteine solution anaerobically. One hundred microliters of each dilution was spread on the plates of various media. Plates for bifidobacteria and lactobacilli were incubated anaerobically (73% N2:20% CO2:7% H2) at 37 1C for 48 h. Plates for enterobacteria, and enterococci were incubated aerobically at 37 1C for 72 h. Plates were counted after 24–48 h inoculation and reported log CFU/g.
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then excised (3 cm). The contents from cecum were collected into plastic tubes. The tubes were snap frozen in liquid nitrogen and stored at 70 1C. The bacteria were enumerated from cecal content.
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Concentration (%) of burdock inulin in the medium Fig. 1. Effect of burdock inulin concentration on the (a) population growth of bifidobacteria and (b) decrease in pH of the medium by bifidobacteria. Results are given as means7S.D. of quadruplicate samples.
3.1. Prebiotic effect of B-INU in vitro 3.1.1. Effect of B-INU on the growth of bifidobacteria in vitro As shown in Fig. 1a, 1% (w/v) B-INU had a slight stimulatory effect on bifidobacterial growth compared with the controlled medium. The pH values of the media dropped with the increase of the mass of bifidobacteria (Fig. 1b). Supplemented with 1% (w/v) B-INU was found to have better effect on stimulating the growth of B. adolescentis ATCC 15703 compared with that without B-INU (Fig. 2a). The growth curves of B. adolescentis ATCC 15703 reached plateau phase after 24 h cultivation with B-INU, whereas it took 32 h to reach plateau phase without B-INU. Therefore, the growth rate of the bacteria in B-INU-containing medium was faster than that in the medium without B-INU. Moreover, the final bacterial mass in the medium with B-INU was greater than that in the medium without B-INU. The metabolism of fructans was always accompanied by a progressive fall of the pH of the culture medium, which indicates that the sugar might be fermented by bifidobacteria producing lactic acid, and thus decreases pH values [10]. As shown in Fig. 2b, pH values of the media dropped
along with the increases of beneficial bacterial populations. The decrease of pH values after incubation with B-INU suggests that the bifidobacteria were able to utilize B-INU. These results indicated that B-INU potently stimulated the growth of bifidobacteria, which supported the potential prebiotic effect of B-INU. 3.1.2. Effect of fructans of different DP on the growth of bifidobacteria in vitro As shown in Fig. 3, the majority of bifidobacteria strains studied utilized FOS and B-INU as well as C-INU. L-INU does not enhance the growth of bifidobacteria significantly (Fig. 3a). However, B-INU, C-INU and FOS significantly stimulate the growth of bifidobacteria. The strains stimulated by FOS and B-INU, as well as C-INU, did not ferment L-INU. The pattern of pH decrease in the media containing inulin was similar to that in the media containing FOS. The prebiotics (carbohydrates) are fermented by anaerobic bacteria leading to the production of lactic acid, short-chain fatty acids (acetate, propionate, and butyrate), and gases (H2, CO2, and CH4) [4]. Although it was a relatively poor indicator of bacterial growth, pH change suggested that bifidobacteria were able to use B-INU and C-INU as well as FOS. The results indicated that
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Fig. 2. Effect of burdock inulin on the (a) population growth of bifidobacteria and (b) decrease in pH of the medium by bifidobacteria at different cultivation times (’: in the medium without burdock inulin and m: in the medium with burdock inulin). Results are given as means7S.D. of quadruplicate samples.
utilization of inulins by bifidobacteria depended on the DP of fructo-oligomeric chains. It indicated that bifidobacteria utilized short-chain FOS and inulin as well as short chain, but not highly polymerized inulins. In vitro studies on the effect of fructans as a prebiotic on pure culture bacteria were followed by in vivo studies on the selective stimulation of bifidobacterial growth in the presence of complex intestinal bacterial populations. In this way, we can prove the indicative of prebiotic character of the fructans or their preparations. 3.2. Prebiotic effect of B-INU in vivo The prebiotic effect of inulin on the growth of bifidobacteria and lactobacilli has been well proven [3,11]. Dramatic increase in the number of the beneficial microflora has been shown in vivo animal studies at a level between 1% and 5% [12,13]. Therefore, in this study highest level (5%) of various fructans was evaluated for its prebiotic effect. B-INU stimulated the proliferation of cecal bifidobacteria and lactobacilli (Fig. 4). On the 27th day, cecal bifidobacteria and lactobacilli of the B-INU group were more abundant than that of the control (Po0.05).
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Fig. 3. Effect of fructans of different degree of polymerization on the (a) population growth of bifidobacteria and (b) decrease in pH of the medium by bifidobacteria. Counts different from the control at the significance level at 0.05 is indicated by asterisk (*). Results are given as means7S.D. of quadruplicate samples.
Bacterial counts for enterobacteria and enterococci were not significantly affected by the ingestion of B-INU. The data also suggested that the composition of the cecal microbiota was influenced by the type of prebiotic (Fig. 4). Feeding of a diet containing a prebiotic resulted in the stimulation on the growth of various bacterial groups, in particular acidogenic bacteria such as bifidobacteria (Fig. 4a) and lactobacilli (Fig. 4b). Enterobacteria (Fig. 4c) and Enterococci counts (Fig. 4d) were not significantly affected by the different diets. FOS is not digested by intestinal enzymes or by enteric bacteria [14]. However, bifidobacteria have a relatively high amount of b-fructosidase [15], which enables bifidobacteria to use FOS or inulin as a nutrient. Bacterial counts for cecal bifidobacteria and lactobacilli of the prebiotic group were also increased during the 2 weeks of fructans supplementation (Po0.05). These variations in the microbial populations were observed with no significant alterations in body weight and the amount of food eaten. L-INU was not degraded by bifidobacteria, as we have shown in vitro (Fig. 3). The results of in vivo studies on mice confirmed the prebiotic effectiveness of B-INU, C-INU and FOS, indicating selective increase in cecal bifidobacteria count. The effect of L-INU was more diverse and
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Fig. 4. Effect of fructans (5%) in the diet of different degree of polymerization on the intestinal (a) bifidobacteria, (b) lactobaccilli, (c) enterobacteria and (d) enterococci of mice. Counts different from the control at the significance level at 0.05 is indicated by asterisk (*). Results are given as means7S.D. of quadruplicate samples.
seemed to be dependent on the presence and ability of other bacteria to initiate degradation, followed by the possible subsequent stimulation of bifidobacterial growth. Body weights of mice increased steadily throughout the course of the experiment. After 7 days of acclimation and 14 days of diet treatment, mean weights of the mice were 3271.3 g for the mice fed on the control diet, 2972.8 g for those fed on the diet containing B-INU, 2971.7 g for those fed on the diet containing C-INU, 2872.2 g for those fed on the diet containing L-INU and 2971.5 g for those fed on the diet containing FOS group before killing. Mice appeared healthy, and their coat was velvet-like. In addition, consumption of B-INU did not cause intestinal discomfort such as eructation or bloating. 4. Discussion In this study, we demonstrated that B-INU was a potential novel source of prebiotics both in vitro and in vivo. On the basis of the data obtained through in vitro fermentations, FOS and inulin (B-INU or C-INU) exhibited a potential to be used as an effective source of prebiotics by increasing the populations of bifidobacteria.
FOS and short-chain inulin (B-INU or C-INU) caused greater in vitro growth rates of bifidobacteria than did L-INU, these data were consistent with those reported by Roberfroid and Van Loo [11], implying that the DP of fructan was the key factor that decides the accessibility of fructan to bifidobacteria. According to Biedrzycka and Bielecka [16], the main factors responsible for susceptibility of saccharides to fermentation are: chemical structure, composition of monomer units, DP and possible linear or branched structure, as well as water solubility. Although prebiotics offer one rational approach to the probiotic concept, the health consequences have not yet been defined. Bifidobacteria and lactobacilli are the most widely studied probiotic strains and have been shown to exert a wide range of health benefits [17,18]. Bifidobacteria and/or lactobacilli are good target organisms. Many lactobacilli and bifidobacteria are able to produce natural antibiotics, which can have a broad spectrum of activity against various intestinal pathogens [19,20]. Discovered at the start of the last century, bifidobacteria are considered as a key commensal in human–microbe interactions, and are believed to play a pivotal role in maintaining a healthy gastrointestinal tract [21]. Lactobacilli and bifidobacteria
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are non-pathogenic bacteria of the bowel tract. These microorganisms may increase resistance to disease by retarding the growth of pathogenic and putrefactive bacteria and by producing inhibitory substances, as well as competing directly for substrates and mucosal attachment sites [22–24]. Furthermore, these microorganisms have immunomodulatory effects influencing the interactions between lactic acid bacterial cell wall components and immune cells, and it is believed that prebiotics may have a similar effect [25]. Great number of bifidobacteria in the cecum of mice resulting from the ingestion of short-chain inulin in this study may promote beneficial effects within the gastrointestinal tract. The results of our in vivo study with mice confirmed the prebiotic effectiveness of inulin extracted from edible burdock. Further work is needed to investigate its effects on human subjects. Acknowledgments This study was supported by the Shandong Laboratory Animal Sciences. We are grateful to Professor Jin for his technical support and collaboration. References [1] Ziemer C, Gibson G. An overview of probiotics, prebiotics and synbiotics in the functional food concept: perspectives and future strategies. Int Dairy J 1998;8:473–9. [2] Gibson GR. Dietary modulation of the human gut microflora using prebiotics. Br J Nutr 1998;80:S209–12. [3] Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 1995;125:1401–12. [4] Roberfroid MB. Dietary fiber, inulin, and oligofructose: a review comparing their physiological effects. Crit Rev Food Sci 1993;33: 103–48. [5] Silva RF. Use of inulin as a natural texture modifier. Cereal Foods World 1996;41:792–4. [6] Tancrede C. Role of human microflora in health and disease. Eur J Clin Microbiol Infect Dis 1992;11:1012–5. [7] Corradini C, Bianchi F, Matteuzzi D, Amoretti A, Rossic M, Zanoni S. High-performance anion-exchange chromatography coupled with pulsed amperometric detection and capillary zone electrophoresis with indirect ultra violet detection as powerful tools to evaluate prebiotic properties of fructooligosaccharides and inulin. J Chromatogr A 2004;1054:165–73.
[8] Kardosˇ ova´ A, Ebringerova´ A, Alfo¨ldi J, Nosa´l’ova´ G, Franˇova´ S, Hrˇ ı´ balova´ V. A biologically active fructan from the roots of Arctium lappa L., var. Herkules. Int J Biol Macromol 2003;33:135–40. [9] Duncan DB. Multiple range and multiple F test. Biometrics 1985;11: 1–42. [10] Rastall RA, Maitin V. Prebiotics and synbiotics: towards the next generation. Curr Opin Biotechnol 2002;13:490–6. [11] Roberfroid MB, Van Loo JAE. A review of the bifidogenic nature of chicory inulin and its hydrolysis products. J Nutr 1998;128:11–9. [12] Santos A, San Mauro M, Marquina Dı´ az D. Prebiotics and their long-term influence on the microbial populations of the mouse bowel. Food Microbiol 2006;23:498–503. [13] Hu B, Gong Q, Wang Y, Ma Y, Li J, Yu W. Prebiotic effects of neoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe 2006;12:260–6. [14] Hidaka H, Hirayama M, Tokunaga T, Eida T. The effects of undigestible fructooligosaccharides on intestinal microflora and various physiological functions on human health. Adv Exp Med Biol 1990; 270:105–7. [15] De Vries W, Gerbrandy SJ, Stouthamer AH. Carbohydrate metabolism in Bifidobacterium bifidium. Biochim Biophys Acta 1967;136:415–25. [16] Biedrzycka E, Bielecka M. Prebiotic effectiveness of fructans of different degrees of polymerization. Trends Food Sci Technol 2004; 15:170–5. [17] Gismondo MR, Drado L, Lombardi A. Review of probiotics available to modify gastrointestinal flora. Int J Antimicrob Agents 1999;12:287–92. [18] Simmering R, Blaut M. Pro- and prebiotics—the tasty guardian angels? Appl Microbiol Biotechnol 2001;55:19–28. [19] Gibson GR, Wang X. Regulatory effects of bifidobacteria the growth of other colonic bacteria. J Appl Bacteriol 1994;77:412–20. [20] Shiba T, Aiba Y, Ishikawa H, Ushiyama A, Takagi A, Mine T, et al. The suppressive effect of bifidobacteria on Bacteroides vulgatus, a putative pathogenic microbe in inflammatory bowel disease. Microbiol Immunol 2003;47:371–8. [21] Leahy SC, Higgins DG, Fitzgerald GF, van Sinderen D. Getting better with bifidobacteria. J Appl Microbiol 2005;98:1303–15. [22] Coconier M-H, Lievin V, Hemery E, Servin AL. Antagonistic activity against Helicobacter infection in vitro and in vivo by the human Lactobacillus acidophilus strain LB. Appl Environ Microbiol 1998;64: 4573–80. [23] Finlay BB, Falkow S. Common themes in microbial pathogenicity. Microbiol Rev 1989;53:210–30. [24] Jacobsen CN, Nielsen VR, Hayford AE, Moller PL, Michaelsen KF, Paerregaard A, et al. Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Appl Environ Microbiol 1999;65:4949–56. [25] Schiffrin EJ, Rochat F, Link-Amster H, Aeschlimann JM, DonnetHugues A. Immune system stimulation by probiotics. J Dairy Sci 1995;78:1597–606.
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Food microbiology
Prebiotic effectiveness of inulin extracted from edible burdock Dandan Li, Jin M. Kim, Zhengyu Jin, Jie Zhou Food Science and Technology, Jiang Nan University, Wuxi, Jiangsu 214122, China Received 29 June 2007; received in revised form 16 October 2007; accepted 18 October 2007 Available online 26 November 2007
Abstract To investigate the prebiotic potential of burdock inulin (B-INU), the in vitro and in vivo effects of B-INU on bacterial growth were studied. B-INU significantly stimulated the growth of bifidobacteria in Man–Rogosa–Sharp (MRS) medium, anaerobically. Compared with chicory inulin (C-INU), long-chain inulin (L-INU) and fructooligosaccharides (FOS), 1% (w/v) B-INU promoted the specific growth rate of beneficial bacteria. The decreases of media pH with B-INU were almost the same as that with C-INU and FOS. In vivo, B-INU significantly increased the number of lactobacilli and bifidobacteria (Po0.05) in cecal content. Mice fed with B-INU, C-INU and FOS for 14 days had greater number of cecal beneficial bacteria population than those fed with L-INU for 14 days. In addition, all fructans did not cause any side effects, such as eructation and bloating. Results indicated that inulin extracted from edible burdock showed prebiotic properties that could promote health. r 2007 Elsevier Ltd. All rights reserved. Keywords: Burdock; Inulin; Fructooligosaccharides; Prebiotic; Cecal microbiota
1. Introduction Prebiotics are compounds, usually carbohydrates, which are resistant to direct metabolism by the host and reach the caeco-colon where they are preferentially utilized by selected groups of beneficial bacteria [1]. Modulation of the human gut flora by prebiotic oligosaccharides has the potential benefit to human health by enhancing levels of ‘beneficial’ gut bacteria [2]. The term prebiotic has been defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one, or a limited number of bacteria [3]. The prebiotics (carbohydrates) are resistant to enzymatic hydrolysis in the upper gastrointestinal tract and reach the caeco-colon intact. There they are completely fermented. This fermentation converts inulin into biomass, shortchain fatty acids, lactate and gases [4]. Intestinal microbiological ecosystem has been associated with human Abbreviations: B-INU, burdock inulin; C-INU, chicory inulin; L-INU, long-chain inulin; FOS, fructooligosaccharides; DP, degree of polymerization; G, glucose; F, fructose; GF, sucrose; MRS, Man–Ragosa–Sharp. Corresponding author. Tel.: +86 510 85919189; fax: +86 510 85919189. E-mail address: [email protected] (Z. Jin). 1075-9964/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2007.10.002
health. In infants, bifidobacteria are the predominant bacteria, but studies show that there is a substantial decrease in their population as human age. There are definite advantages to establish and maintain proper bifidobacteria levels [5]. Beneficial flora including the genera of bifidobacteria or lactobacilli improves the caeco-colon environment by suppressing the growth of pathogenic bacteria and production of carcinogenic materials and thus improving immunity [6]. Burdock is a dull, pale green, stems about 3–4 ft and branched, rising from a biennial root. The burdock roots are fleshy, wrinkled, crowned with a tuft of whitish, soft, hairy leaf-stalks, gray-brown externally, whitish internally, with a somewhat thick bark, about a quarter of the diameter of the root, and soft wood tissues, with a radiate structure. As a rule, they are 12 in or more in length and about 1 in thick, sometimes, however, they extend 2–3 ft, making it necessary to dig by hand. Burdock inulin (B-INU), chicory inulin (C-INU), long-chain inulin (L-INU) and fructooligosaccharides (FOS) are among the fructans, which are undigestible in the upper gastrointestinal tract. Inulin is a linear, highly polymerized fructan of average degree of polymerization (DP) 12, produced by extraction from chicory roots or burdock
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roots. It consists of a linear chain of fructose with b-(2-1) linkages with a terminal glucose unit. L-INU has a higher average DP 22 than that of B-INU or C-INU. FOS with average DP 4.5 can be produced by chemical degradation or controlled enzymatic hydrolysis of inulin using endoglycosidases. Furthermore, FOS can also be produced on a commercial scale, from sucrose, using a fungal enzyme from either Aureobasidium sp. or Aspergillus niger [7]. DP is the value that corresponds to the total number of saccharide units (G and F units) in a given inulin sample divided by the total number of inulin molecules, without taking into account the monosaccharides, glucose (G) and fructose (F), and the disaccharide, sucrose (GF), which are possibly present in the sample. By calculating the fructoseto-glucose ratio (number of fructose units per number of glucose units), the average DP is obtained: DP ¼ number of F units per G units+1 G unit. The objective of this study was to evaluate the prebiotic properties of inulin extracted from edible burdock in vitro and in vivo. In addition, prebiotic properties of B-INU, C-INU, L-INU or FOS with different DP were also evaluated. 2. Materials and methods 2.1. Prebiotics Commercially available prebiotic oligosaccharides were used: B-INU (inulinX96.5%, mono+disacch.p2%); C-INU (inulinX92.2%, mono+disacch.p2.0%); L-INU (inulinX94.6%, mono+disacch.p4%) and FOS (FOSX 91.0%, sucrose+monosacch.p5.0%). B-INU was extracted in our laboratory [8]. C-INU was purchased from Wede Biologicals Co., Ltd. (Beijing, China). L-INU was purchased from Tianmen Hylae inulin R&D Co., Ltd. (Hubei, China). FOS was purchased from Baolingbao Biotechnology Co., Ltd. (Shandong, China). Basal MRS (Man–Ragosa–Sharp), BBL agar, LBS agar, EMB agar and EC agar were purchased from Kaiyang Biologicals Co., Ltd. (Shanghai, China). Oxoid Anaerobic System was purchased from Oxoid GmbH (Wesel, Germany). All other chemicals and media preparations used in this investigation were purchased from Sigma (Shanghai, China). Bifidobacterium adolescentis ATCC 15703 was obtained from Medical College of Fudan University (Shanghai, China). 2.2. Prebiotic effect of B-INU in vitro After the standard strains of B. adolescentis ATCC 15703 had been incubated anaerobically (73% N2:20% CO2:7% H2) for 48 h at 37 1C, the stock cultures were stored in 10% glycerol at 70 1C until needed. MRS broth supplemented with 0.5 g/L L-cysteine hydrochloride, 0.2 g/L of sodium thioglycolate, and 0.1 g/L of CaCl2 2H2O was used as the stock culture medium and as the basal medium. Basal MRS broth supplemented with a final concentration
of 0.5%, 1%, 2%, 3% or 4% (w/v) of B-INU was divided into five test tubes (5 mL in each), inoculated with 50 mL overnight culture of the beneficial bacteria, and incubated at 37 1C in anaerobic jars—Oxoid Anaerobic System with Gas Pak H2+CO2. To examine the effects of B-INU on cell growth, the bacteria were cultivated overnight in the basal medium at 37 1C, and diluted to 106 cells/mL, 50 mL of this was then inoculated into 5 mL of basal medium containing fructans (B-INU, C-INU, L-INU and FOS). The bacteria were then cultured under anaerobic conditions for 24 h at 37 1C. After the culture broth had been centrifuged for 15 min with 2500 g, the precipitate was washed twice with PBS (0.1 M phosphate buffer pH 7.4, 0.9% saline), and diluted one- to five-fold with PBS. The diluted cells were mixed well and then turbidity was measured at 600 nm. All measurements were performed in quadruplicate parallels and repeated at least twice. 2.3. Prebiotic effect of B-INU in vivo 2.3.1. Animals and diets Female Kunming mice (2071 g body weight, 6 weeks old) were purchased from Chinese Academy of Medical Sciences and housed in a room at 22 1C and 55% relative humidity, with a 12-h light/dark cycle. Mice were fed the control diet, containing (g/kg diet): pregelatinized cornstarch 646, soluble casein 204, DL-methionine 3, corn oil 97, a mineral mixture 45 [providing (g/kg diet): Ca2(PO4)3 16.6, K2(PO4)3 10.5, CaCO3 8.0, NaCl 3.1, MgSO4 3.9, FeSO4 0.3, ZnSO4 0.2, MnSO4 0.2, CuSO4 0.04, CoSO4 4 104, KHSO4 4 104, (NH4)2SO4 4 104, MgO 0.9], a vitamin mixture 5 [providing (mg/kg diet): allrac-a-tocopherol (500 UI/g) 150, all-trans-retinylacetate (500,000 UI/g) 8, cholecalciferol (400,000 UI/g) 2.5, nicotinic acid 45, calcium pantothenate 15, thiamin hydrochloride 5, riboflavin 9, pyridoxine (PN) hydrochloride 5, ascorbic acid 113, folic acid 2, p-aminobenzoic acid 113, vitamin B-12 (1 g/kg) 67.8, biotin 0.4, phylloquinone 2, myo-inositol 113]. All experiments were in accordance with National Research Council Guidelines of China for the care and use of laboratory animals. After a week’s acclimation period, mice were randomly divided into five groups (eight for each group) and fed one of the following five diets: control diet alone (control group), control diet+5% B-INU (B-INU group), control diet+5% C-INU (C-INU group), control diet+5% L-INU (L-INU group) and control diet+5% FOS (FOS group). Fructans were used to partially replace the corn starch component of the control diet. The dietary treatments were lasted for 14 days. The body weights of the rats were recorded weekly. Mice were observed for any sign of eructation or bloating during 14 days of diet treatment. 2.3.2. Cecal sample preparation On the 15th day of dietary treatment, the mice were euthanized using CO2 asphyxiation and the cecum was
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2.4. Statistical analysis The results were analyzed using one-way ANOVA, and significant differences between groups were determined by the Duncan’s multiple range test [9]. All statistical analyses were performed using the SPSS package program version 10.0.1 and differences were considered significant at Po0.05. 3. Results
Growth (turbidity at 600 nm)
2.3.3. Bacteriological analysis of cecal contents Medium used for the detection of bifidobacteria from cecal contents was BBL agar. Lactobaccilli were enumerated on LBS agar. Enterobacteria, and enterococci were enumerated on EMB agar, and EC agar. For cultivation of bacteria, cecal content (0.1 g) was dispersed in 2 mL of 5% cysteine using glass beads (3 mm diameter). Further dilutions were made using 5% cysteine solution anaerobically. One hundred microliters of each dilution was spread on the plates of various media. Plates for bifidobacteria and lactobacilli were incubated anaerobically (73% N2:20% CO2:7% H2) at 37 1C for 48 h. Plates for enterobacteria, and enterococci were incubated aerobically at 37 1C for 72 h. Plates were counted after 24–48 h inoculation and reported log CFU/g.
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then excised (3 cm). The contents from cecum were collected into plastic tubes. The tubes were snap frozen in liquid nitrogen and stored at 70 1C. The bacteria were enumerated from cecal content.
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Concentration (%) of burdock inulin in the medium Fig. 1. Effect of burdock inulin concentration on the (a) population growth of bifidobacteria and (b) decrease in pH of the medium by bifidobacteria. Results are given as means7S.D. of quadruplicate samples.
3.1. Prebiotic effect of B-INU in vitro 3.1.1. Effect of B-INU on the growth of bifidobacteria in vitro As shown in Fig. 1a, 1% (w/v) B-INU had a slight stimulatory effect on bifidobacterial growth compared with the controlled medium. The pH values of the media dropped with the increase of the mass of bifidobacteria (Fig. 1b). Supplemented with 1% (w/v) B-INU was found to have better effect on stimulating the growth of B. adolescentis ATCC 15703 compared with that without B-INU (Fig. 2a). The growth curves of B. adolescentis ATCC 15703 reached plateau phase after 24 h cultivation with B-INU, whereas it took 32 h to reach plateau phase without B-INU. Therefore, the growth rate of the bacteria in B-INU-containing medium was faster than that in the medium without B-INU. Moreover, the final bacterial mass in the medium with B-INU was greater than that in the medium without B-INU. The metabolism of fructans was always accompanied by a progressive fall of the pH of the culture medium, which indicates that the sugar might be fermented by bifidobacteria producing lactic acid, and thus decreases pH values [10]. As shown in Fig. 2b, pH values of the media dropped
along with the increases of beneficial bacterial populations. The decrease of pH values after incubation with B-INU suggests that the bifidobacteria were able to utilize B-INU. These results indicated that B-INU potently stimulated the growth of bifidobacteria, which supported the potential prebiotic effect of B-INU. 3.1.2. Effect of fructans of different DP on the growth of bifidobacteria in vitro As shown in Fig. 3, the majority of bifidobacteria strains studied utilized FOS and B-INU as well as C-INU. L-INU does not enhance the growth of bifidobacteria significantly (Fig. 3a). However, B-INU, C-INU and FOS significantly stimulate the growth of bifidobacteria. The strains stimulated by FOS and B-INU, as well as C-INU, did not ferment L-INU. The pattern of pH decrease in the media containing inulin was similar to that in the media containing FOS. The prebiotics (carbohydrates) are fermented by anaerobic bacteria leading to the production of lactic acid, short-chain fatty acids (acetate, propionate, and butyrate), and gases (H2, CO2, and CH4) [4]. Although it was a relatively poor indicator of bacterial growth, pH change suggested that bifidobacteria were able to use B-INU and C-INU as well as FOS. The results indicated that
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Fig. 2. Effect of burdock inulin on the (a) population growth of bifidobacteria and (b) decrease in pH of the medium by bifidobacteria at different cultivation times (’: in the medium without burdock inulin and m: in the medium with burdock inulin). Results are given as means7S.D. of quadruplicate samples.
utilization of inulins by bifidobacteria depended on the DP of fructo-oligomeric chains. It indicated that bifidobacteria utilized short-chain FOS and inulin as well as short chain, but not highly polymerized inulins. In vitro studies on the effect of fructans as a prebiotic on pure culture bacteria were followed by in vivo studies on the selective stimulation of bifidobacterial growth in the presence of complex intestinal bacterial populations. In this way, we can prove the indicative of prebiotic character of the fructans or their preparations. 3.2. Prebiotic effect of B-INU in vivo The prebiotic effect of inulin on the growth of bifidobacteria and lactobacilli has been well proven [3,11]. Dramatic increase in the number of the beneficial microflora has been shown in vivo animal studies at a level between 1% and 5% [12,13]. Therefore, in this study highest level (5%) of various fructans was evaluated for its prebiotic effect. B-INU stimulated the proliferation of cecal bifidobacteria and lactobacilli (Fig. 4). On the 27th day, cecal bifidobacteria and lactobacilli of the B-INU group were more abundant than that of the control (Po0.05).
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Fig. 3. Effect of fructans of different degree of polymerization on the (a) population growth of bifidobacteria and (b) decrease in pH of the medium by bifidobacteria. Counts different from the control at the significance level at 0.05 is indicated by asterisk (*). Results are given as means7S.D. of quadruplicate samples.
Bacterial counts for enterobacteria and enterococci were not significantly affected by the ingestion of B-INU. The data also suggested that the composition of the cecal microbiota was influenced by the type of prebiotic (Fig. 4). Feeding of a diet containing a prebiotic resulted in the stimulation on the growth of various bacterial groups, in particular acidogenic bacteria such as bifidobacteria (Fig. 4a) and lactobacilli (Fig. 4b). Enterobacteria (Fig. 4c) and Enterococci counts (Fig. 4d) were not significantly affected by the different diets. FOS is not digested by intestinal enzymes or by enteric bacteria [14]. However, bifidobacteria have a relatively high amount of b-fructosidase [15], which enables bifidobacteria to use FOS or inulin as a nutrient. Bacterial counts for cecal bifidobacteria and lactobacilli of the prebiotic group were also increased during the 2 weeks of fructans supplementation (Po0.05). These variations in the microbial populations were observed with no significant alterations in body weight and the amount of food eaten. L-INU was not degraded by bifidobacteria, as we have shown in vitro (Fig. 3). The results of in vivo studies on mice confirmed the prebiotic effectiveness of B-INU, C-INU and FOS, indicating selective increase in cecal bifidobacteria count. The effect of L-INU was more diverse and
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Fig. 4. Effect of fructans (5%) in the diet of different degree of polymerization on the intestinal (a) bifidobacteria, (b) lactobaccilli, (c) enterobacteria and (d) enterococci of mice. Counts different from the control at the significance level at 0.05 is indicated by asterisk (*). Results are given as means7S.D. of quadruplicate samples.
seemed to be dependent on the presence and ability of other bacteria to initiate degradation, followed by the possible subsequent stimulation of bifidobacterial growth. Body weights of mice increased steadily throughout the course of the experiment. After 7 days of acclimation and 14 days of diet treatment, mean weights of the mice were 3271.3 g for the mice fed on the control diet, 2972.8 g for those fed on the diet containing B-INU, 2971.7 g for those fed on the diet containing C-INU, 2872.2 g for those fed on the diet containing L-INU and 2971.5 g for those fed on the diet containing FOS group before killing. Mice appeared healthy, and their coat was velvet-like. In addition, consumption of B-INU did not cause intestinal discomfort such as eructation or bloating. 4. Discussion In this study, we demonstrated that B-INU was a potential novel source of prebiotics both in vitro and in vivo. On the basis of the data obtained through in vitro fermentations, FOS and inulin (B-INU or C-INU) exhibited a potential to be used as an effective source of prebiotics by increasing the populations of bifidobacteria.
FOS and short-chain inulin (B-INU or C-INU) caused greater in vitro growth rates of bifidobacteria than did L-INU, these data were consistent with those reported by Roberfroid and Van Loo [11], implying that the DP of fructan was the key factor that decides the accessibility of fructan to bifidobacteria. According to Biedrzycka and Bielecka [16], the main factors responsible for susceptibility of saccharides to fermentation are: chemical structure, composition of monomer units, DP and possible linear or branched structure, as well as water solubility. Although prebiotics offer one rational approach to the probiotic concept, the health consequences have not yet been defined. Bifidobacteria and lactobacilli are the most widely studied probiotic strains and have been shown to exert a wide range of health benefits [17,18]. Bifidobacteria and/or lactobacilli are good target organisms. Many lactobacilli and bifidobacteria are able to produce natural antibiotics, which can have a broad spectrum of activity against various intestinal pathogens [19,20]. Discovered at the start of the last century, bifidobacteria are considered as a key commensal in human–microbe interactions, and are believed to play a pivotal role in maintaining a healthy gastrointestinal tract [21]. Lactobacilli and bifidobacteria
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are non-pathogenic bacteria of the bowel tract. These microorganisms may increase resistance to disease by retarding the growth of pathogenic and putrefactive bacteria and by producing inhibitory substances, as well as competing directly for substrates and mucosal attachment sites [22–24]. Furthermore, these microorganisms have immunomodulatory effects influencing the interactions between lactic acid bacterial cell wall components and immune cells, and it is believed that prebiotics may have a similar effect [25]. Great number of bifidobacteria in the cecum of mice resulting from the ingestion of short-chain inulin in this study may promote beneficial effects within the gastrointestinal tract. The results of our in vivo study with mice confirmed the prebiotic effectiveness of inulin extracted from edible burdock. Further work is needed to investigate its effects on human subjects. Acknowledgments This study was supported by the Shandong Laboratory Animal Sciences. We are grateful to Professor Jin for his technical support and collaboration. References [1] Ziemer C, Gibson G. An overview of probiotics, prebiotics and synbiotics in the functional food concept: perspectives and future strategies. Int Dairy J 1998;8:473–9. [2] Gibson GR. Dietary modulation of the human gut microflora using prebiotics. Br J Nutr 1998;80:S209–12. [3] Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 1995;125:1401–12. [4] Roberfroid MB. Dietary fiber, inulin, and oligofructose: a review comparing their physiological effects. Crit Rev Food Sci 1993;33: 103–48. [5] Silva RF. Use of inulin as a natural texture modifier. Cereal Foods World 1996;41:792–4. [6] Tancrede C. Role of human microflora in health and disease. Eur J Clin Microbiol Infect Dis 1992;11:1012–5. [7] Corradini C, Bianchi F, Matteuzzi D, Amoretti A, Rossic M, Zanoni S. High-performance anion-exchange chromatography coupled with pulsed amperometric detection and capillary zone electrophoresis with indirect ultra violet detection as powerful tools to evaluate prebiotic properties of fructooligosaccharides and inulin. J Chromatogr A 2004;1054:165–73.
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