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Prebiotics and their long-term influence on the microbial populations of the mouse bowel
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 23 (2006) 498–503 www.elsevier.com/locate/fm
Short communication
Prebiotics and their long-term influence on the microbial populations of the mouse bowel A. Santos, M. San Mauro, D. Marquina Dı´ az Department of Microbiology III, Biology Faculty, Complutense University of Madrid, Jose´ Antonio Nova´is, 2, 28040 Madrid, Spain Received 1 September 2004; received in revised form 1 April 2005; accepted 1 July 2005 Available online 10 October 2005
Abstract Various prebiotics were orally administered to mice and the evolution of different microbial populations was studied. The administration of prebiotics significantly increased lactobacilli and bifidobacteria in the large bowel content. Ingestion of prebiotics specifically lowered microbial populations of sulphite-reducing clostridia. Xylo-oligosaccharides (XOS) increased lactobacilli by 10 fold and produced the highest counts of bifidobacteria. In XOS-treated mice, levels of sulphite-reducing clostridia decreased significantly. Prebiotics slightly reduced the amount of aerobic bacteria and significantly increased the number of anaerobes in both the small and the large bowel. These effects of prebiotics were reverted by the basal diet. r 2005 Elsevier Ltd. All rights reserved. Keywords: Prebiotics; Mice; Microbial populations; Small bowel; Large bowel
1. Introduction The functional effect of prebiotics and probiotics is a growing area of research. The gastrointestinal tract contains a large variety of bacteria, which are either beneficial (Lactobacillus, Bifidobacterium, etc.) or detrimental (Clostridium, Shigella, etc.) to the host’s health. Species of the lactic acid bacteria Bifidobacterium and Lactobacillus are the most widely studied probiotic strains and have been shown to exert a wide number of health benefits (Gismondo et al., 1999; Simmering and Blaut, 2001). It is known that Lactobacillus and Bifidobacterium have low activities of enzymes involved in the formation of mutagens and carcinogens (b-glucuronidase, b-glucosidase, nitrate reductase, urease, azoreductase) compared with other anaerobes of the gastrointestinal tract (Pool-Zobel et al., 1993). Lactobacillus and Bifidobacterium strains have been shown to possess inhibitory activity towards the growth of pathogenic bacteria, the ability to adhere to the intestinal epithelial cells and other positive effects on the host health (Greene and Klaenhammer, 1994). Increased Corresponding author. Tel./fax: +34 91 394 49 64.
E-mail address: [email protected] (D.M. Dı´ az). 0740-0020/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2005.07.004
counts of these lactic acid bacteria in the bowel may be achieved by consumption of substances that are known to stimulate probiotic growth (Marquina et al., 2002; Santos et al., 2002). These substances are named as prebiotics. The premise is based on the fact that the large gut contains bacteria that are beneficial or detrimental to health. It has become clear over the last years that the group of nondigestible oligosaccharides (NDO) is likely to play an important nutritional role (Cummings and Macfarlane, 1997). Currently, food components that are seen to exert the best prebiotic effects are inulin-type fructans (Apajalahti et al., 2002; Kleessen et al., 2001). Although differing in their chemical characteristics, all the NDO resist digestion in the small intestine and they are potential substrates for bacteria of the large intestine. Simple stimulation of a particular micro-organism is not sufficient to demonstrate a true prebiotic effect. In vivo, the micro-organisms that survive in the mice bowel have been studied over the recent decades (Marquina et al., 2002; Tannock, 1979; Tannock et al., 1988). The mouse therefore provides an excellent model to determine the effect of a diet on the microbial populations of the gastrointestinal tract. The aims of the present work were to study the influence of the administration of prebiotics on the microbial ecology
ARTICLE IN PRESS A. Santos et al. / Food Microbiology 23 (2006) 498–503
of the gastrointestinal tract of mice and to evaluate the changes on these microbial populations. Furthermore, the persistence of the observed effects was studied.
2. Materials and methods 2.1. Prebiotics The following commercial preparations of prebiotic oligosaccharides were used: fructo-oligosaccharides (FOS, Raftilose P95, Orafti, Tienen, Belgium), long chain inulin (INU; Raftiline HP, Orafti), lactulose (LAC; Sigma Chemical Co., St. Louis, Missouri), xylo-oligosaccharides (XOS; Suntory, Osaka, Japan), galacto-oligosaccharides (GOS; Oligomate55, Yakult Institute, Tokyo, Japan), transgalactosylated-oligosaccharides (TOS; Yakult Institute), isomalto-oligosaccharides (IMO; Isomalt900, Sweetener Industry) and soybean-oligosaccharides (SOS; Calpis, Tokyo, Japan).
2.2. Diets and animals Nine diets were prepared for the study: a basal diet and different diets composed by a basal diet supplemented with one of the eight prebiotics studied (FOS, INU, LAC, XOS, GOS, TOS, IMO and SOS). The test substances were added to the basal diet at a level of 1% (w/w). The basal diet was prepared in accordance to the American Institute of Nutrition 93 (AIN-93) (Reeves et al., 1993). Five-month-old female Swiss mice were used after a 1month quarantine period. The diet feedings were started when the animals were 6 months old. Sixteen females were employed in each dietary group. Animals were housed four per cage, and cages were changed twice a week. Water was available continuously from bottles. All animals were housed in a building that was specially equipped for animal experiments. The animals were maintained under controlled environmental conditions. Animal caretakers, technicians, or anyone entering the animal holding area had to dress in a special suit, including cap, face mask, and shoe covers, to minimize the possibility of contamination. Body weights and food intakes were recorded twice weekly. For examination of organs and sampling, mice were killed by carbon dioxide anaesthesia followed by cervical dislocation. Samples were taken 6 months after the start of the diet treatment. The intestine was immediately removed from each animal and cut to separate small and large intestine. Homogenates were prepared from lengths (5 cm) of small intestine and large intestine. These tissues were homogenized in brain–heart infusion broth (BHI) by using a Teflon homogenizer. The resulting homogenates were maintained in a reduced condition in GasPak jars (BBL Microbiology Systems, Cockeysville, Maryland) and then, they were diluted (10 fold to 1010) in prerreduced BHI.
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2.3. Enumeration of gastrointestinal microbes Microbial counts were obtained by culturing homogenates of gastrointestinal organs on selective media as previously reported (Roach and Tannock, 1979; Tannock, 1979). Viable counts were obtained from BHI agar that was incubated aerobically and anaerobically at 37 1C for 3 days (aerobic and anaerobic counts, respectively). Gram-positive bacterial counts were obtained from phenylethanol agar (Difco, Detroit, Michigan) that was incubated aerobically and anaerobically at 37 1C for 3 days. From Azide Blood Agar (ABA, Oxoid Ltd, Basingstoke, UK) that was incubated aerobically and anaerobically at 37 1C for 3 days, mainly streptococcal counts. From Kanamycin Esculin Azide Agar (KAA, Oxoid) agar that was incubated aerobically at 37 1C for 2 days, Enterococcus counts. From SPS agar (Oxoid) that was incubated anaerobically at 37 1C for 3 days, sulphite-reducing clostridial counts. From Eosin Methylene blue (EMB, Oxoid) agar that was incubated aerobically at 37 1C for 1 day (Enterobacteriaceae counts), and from BHI agar containing a Gram-negative anaerobic supplement (Oxoid) that was incubated anaerobically at 37 1C for 3 days (Gram-negative anaerobic bacterial counts). Anaerobic incubations were performed in Gaspak jars. For bifidobacteria, Neomycin–Paromomycin–Nalidixic acid–Lithium chloride agar (NPNL) was used according to the instructions by the authors (Teraguchi et al., 1978; Hartemink and Rombouts, 1999). LAMVAB medium was selected for the determination of lactobacilli. The medium was prepared according to the instructions by the authors (Hartemink et al., 1997). Kanamycin–vancomycin blood agar (Merck, Darmstadt, Germany) was used in anaerobic conditions to determine Bacteroides/Fusobacteria counts. 2.4. Persistence of the prebiotic effects Nine groups of eight animals, fed with the same diets as described above, were studied. At the end of the experimental period, 6 months, prebiotic diets were suspended and a basal diet was administered. Daily, one animal of each group was killed and the number of microbial counts determined. Experiences were done by triplicate. 3. Results and discussion The aim of the present study was to investigate the changes in the microbial ecology of the bowel of mice due to the effects of a wide number of substances with reported prebiotic properties. These dietary supplements were administered with a basal diet, which conformed to the nutritional composition of the standard AIN-93 diet (Reeves et al., 1993). Only a small number of studies have addressed the effects of the prebiotics on the microbial population during its continued long-term administration (Kleessen et al., 1997). The data in this study suggest that the composition
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of the bowel microflora is influenced by the type of prebiotic in the diet (Fig. 1). Furthermore, the feeding of a diet containing a prebiotic resulted in the stimulation of various bacterial groups, in particular acidogenic bacteria such as bifidobacteria and lactobacilli. Other bacteria tested (Enterococcus spp.) remained more or less unchanged. These variations in the microbial populations were observed with no significant alterations in body weight and the amount of food eaten, with all animals eating approximately 7 g per day. The mean body weight gain of the mice during the experiences ranged from 6.670.3 g (basal diet) to 7.170.2 g (mean of the prebiotic diets) and there were no growth differences at any interval throughout the study. Total aerobes in the large bowel changed little due to the ingestion of oligosaccharides compared with the basal diet. Furthermore, the small bowel of mice consuming XOScontaining diets showed a reduction in the aerobic microflora concomitantly with an increased number of anaerobes. Total anaerobes were higher for XOS-fed mice compared with all other treatments. In general, total anaerobes were also higher for mice fed with prebiotics compared with the basal diet. The effect of the prebiotics on the enterobacteria counts was variable as showed in Fig. 1, although this bacterial population, in general terms, was reduced. Enterococcus counts and aerobic and anaerobic counts in Azide Blood Agar were slightly affected or unaffected by the different diets. The number of Bacteroides/Fusobacteria was higher in the large bowel compared with small bowel and the most important changes were observed in the XOS containing diet. In this microbial group, counts were variable or unaffected in the small bowel (Fig. 1). Despite the evidence that the gut microflora is affected by the administration of prebiotics, it is difficult to identify the specific micro-organisms responsible for these changes. It seems likely that a number of prebiotic-degrading bacteria, which are present in the gastrointestinal tract are complementary, or antagonic, in their metabolic activities. Although prebiotics offer one rational approach to the probiotic concept, the health consequences have not yet been defined. In theory, a number of potential benefits may arise (Hussein et al., 1999; Loo et al., 1999). However, it may be that improved resistance to pathogens offers the most feasibility. The lactic acid bacteria of the gastrointestinal tract are thought to play a significant role in improved colonization resistance. Increased bifidobacterial numbers in the gut may be one factor that contributes towards improved competitive exclusion of pathogens seen in prebiotic-treated animals. Moreover, it is noteworthy that the feeding of prebiotic-containing diets resulted in a significant decrease of bacteria belonging to the clostridial group. A high proportion of these micro-organisms may be pathogenic through their proteolytic capabilities and toxin production. Our results showed that mice fed diets containing prebiotics significantly reduced large bowel concentrations of sulphite-reducing clostridia and XOS induced the strongest reduction.
Lactobacilli concentrations were greatest in small and large intestine as a result of ingestion of prebiotics (Fig. 1). In the large bowel, the most important effect was due to the ingestion of XOS and INU-diets, but all of the tested prebiotics increased lactobacilli. Bifidobacteria counts were statistically greater in LAC, IMO and XOS-diets but also in other prebiotics-supplemented dietary groups. In these groups, the greater bifidobacteria count was evident in absolute concentration and in the percentage of bifidobacteria among total anaerobes (Fig. 1). The increase in lactobacilli and bifidobacteria, especially in XOS-diet, may contribute to the stabilization of the microflora and to the health of the host. Many lactobacilli and bifidobacteria are able to produce natural antibiotics, which can have a broad spectrum of activity against various intestinal pathogens (Gibson and Wang, 1994; Shiba et al., 2003). Lactobacillus and Bifidobacterium are non-pathogenic bacteria of the bowel tract. These micro-organisms may increase resistance to disease by reducing the growth of pathogenic and putrefactive bacteria by producing inhibitory substances, competing directly for substrates and mucosal attachment sites (Coconier et al., 1998; Finlay and Falkow, 1989; Jacobsen et al., 1999). Although it is generally accepted that prebiotics are not metabolised by rat digestive enzymes in the small intestine and thus reach the caecum and colon, a partial bacterial hydrolysis and fermentation of these NDO in the distal part of the small intestine cannot be excluded. This view is supported by the small intestine numbers of microorganisms, such as bifidobacteria (Fig. 1), which tended to be higher in mice fed with TOS (6.5 106 UFC g 1) than in control mice (5.1 106 UFC g 1). Any carbohydrate that reaches the cecum is a potential substrate for fermentation by the microbiota, and much evidence supports the belief that the currently identified prebiotics are fermented. Many different prebiotics support bacterial growth and produce various fermentation-derived end products (Wang and Gibson, 1993; Gibson, 1999). It has been demonstrated that the nature of the prebiotic determines its fermentability (Loo et al., 1999). Fructans are resistant to clostridial breakdown; however, XOS, lactulose and fructans are extensively fermented by other enteric bacteria including strains of Bifidobacterium, Lactobacillus and Bacteroides. The hydrolysis of prebiotics by these micro-organisms may lead to the accumulation of intermediates, which may be available for cross feeding of non-degrading-prebiotic species (Macfarlane and Englyst, 1986). It has been observed that in pure culture experiments different strains of bifidobacteria grew well, as did Clostridium perfringens and Escherichia coli (Wang and Gibson, 1993). However, it has been shown, in competition experiments between B. infantis, E. coli and C. perfringens, with oligofructose as sole source of carbon and energy, that the bifidobacteria grew well and showed an inhibitory effect on the growth of the other two (Gibson and Wang, 1994; Rycroft et al., 2001; Sghir et al., 1998; Loo et al., 1999).
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Fig. 1. Microbial counts in small and large bowel after a six-month treatment with various prebiotics. Black columns: small bowel. White columns: large bowel. (A) Aerobic bacteria; (B) anaerobic bacteria; (C) Gram-positive aerobic bacteria; (D) Gram-positive anaerobic bacteria; (E) Gram-negative anaerobic bacteria; (F) aerobic bacteria on Azide Blood Agar; (G) anaerobic bacteria on Azide Blood Agar; (H) lactobacilli; (I) bifidobacteria; (J) Enterobacteriaceae; (K) Enterococcus spp.; (L) sulphite-reducing bacteria; (M) Bacteroides/Fusobacteria.
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Fig. 2. Time course evolution of different microbial populations of the large bowel. The effect of the basal diet on the microbial populations of previously prebiotic-treated mice was studied. (A): (’), Aerobic bacteria; (K), anaerobic bacteria; (m), Gram-positive aerobic bacteria; (.), Gram-positive anaerobic bacteria; (~), Gram-negative anaerobic bacteria; (&), aerobic bacteria on Azide Blood Agar; (J), anaerobic bacteria on Azide Blood Agar. (B): (’), Lactobacilli; (K), bifidobacteria; (m), Enterobacteriaceae; (.), Enterococcus spp.; (~), sulphite-reducing bacteria; (&), Bacteroides/ Fusobacteria.
To our knowledge, this is the first long-term study that shows that prebiotic-containing diets changed some microbial populations of the small and large bowel of mice. In the present work, it was shown that the beneficial microbiological status induced by prebiotics disappeared by the suppression of prebiotic-enriched diets (Fig. 2). The effect of the basal diet in the previously prebiotic-treated mice was studied and time effects on the different bacterial counts in the large bowel were observed in the 6-monthprebiotic-treated animals. The observed prebiotic effects (increased anaerobes, lactobacilli and bifidobacteria counts, etc.) were gradually reverted by basal diet (Fig. 2). Furthermore, in the first week without prebiotic diets, microbial counts were similar to that obtained in control animals with a 6-month-basal diet (Fig. 1). In summary, the use of a basal diet as a replacement for prebiotics in diets caused a marked decrease in the number of total anaerobes, lactobacilli and bifidobacteria, whereas total aerobes, enterobacteria and clostridia all increased. The study suggests that the benefits of a prebiotic-enriched diet disappeared quickly and so, a continuous prebiotic diet must be followed to maintain the reported benefits in the host. References Apajalahti, J.H.A., Kettunen, H., Kettunen, A., Holben, W.E., Nurminen, P.H., Rautonen, N., Mutanen, M., 2002. Culture-independent microbial community analysis reveals that inulin in the diet primarily affects previously unknown bacteria in the mouse cecum. Appl. Environ. Microbiol. 68, 4986–4995. Coconier, M.-H., Lievin, V., Hemery, E., Servin, A.L., 1998. Antagonistic activity against Helicobacter infection in vitro and in vivo by the human Lactobacillus acidophilus strain LB. Appl. Environ. Microbiol. 64, 4573–4580. Cummings, J.H., Macfarlane, G.T., 1997. Role of intestinal bacteria in nutrient metabolism. Clin. Nutr. 16, 3–11. Finlay, B.B., Falkow, S., 1989. Common themes in microbial pathogenicity. Microbiol. Rev. 53, 210–230.
Gibson, G.R., 1999. Dietary modulation of the human gut microflora using the prebiotics oligofructose and inulin. J. Nutr. 129, 1438–1441. Gibson, G.R., Wang, X., 1994. Inhibitory effects of bifidobacteria on other colonic bacteria. J. Appl. Bacteriol. 77, 412–420. Gismondo, M.R., Drado, L., Lombardi, A., 1999. Review of probiotics available to modify gastrointestinal flora. Int. J. Antimicrob. Agents 12, 287–292. Greene, J.D., Klaenhammer, T.R., 1994. Factors involved in adherence of lactobacilli to human Caco-2 cells. Appl. Environ. Microbiol. 60, 4487–4494. Hartemink, R., Rombouts, F.M., 1999. Comparison of media for the detection of bifidobacteria, lactobacilli and total anarobes from fecal samples. J. Microbiol. Methods 36, 181–192. Hartemink, R., Kok, B.J., Weenk, G.H., Rombouts, F.M., 1997. LAMVAB—a new selective medium for the isolation of lactobacilli from faeces. J. Microbiol. Methods 29, 77–84. Hussein, S., Flickinger, E.A., Fahey, G.C., 1999. Petfood applications of inulin and oligofructose. J. Nutr. 129, 1454–1456. Jacobsen, C.N., Nielsen, V.R., Hayford, A.E., Moller, P.L., Michaelsen, K.F., Paerregaard, A., Sandstro¨m, B., Tvede, M., Jakobsen, M., 1999. 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. 65, 4949–4956. Kleessen, B., Stoof, G., Proll, J., Schmiedl, D., Noak, J., Blaut, M., 1997. Feeding resistant starch affects fecal and cecal microflora and shortchain fatty acids in rats. J. Animal Sci. 75, 2453–2462. Kleessen, B., Hartmann, L., Blaut, M., 2001. Oligofructose and long-chain inulin: influence on the gut microbial ecology of rats associated with a human faecal flora. Br. J. Nutr. 86, 291–300. Loo, J.V., Cummings, J., Delzenne, N., Englyst, H., Frank, A., Hopkings, M., Kok, N., Macfarlane, G., Newton, D., Quigley, M., Roberfroid, M., van Vliet, T., van den Heuvel, E., 1999. Functional food properties of non-digestible oligosaccharides report from the ENDO project (DGXII AIRII-CT94-1095). Br. J. Nutr. 81, 121–132. Macfarlane, G.T., Englyst, H.N., 1986. Starch utilization by the human large intestinal microflora. J. Appl. Bacteriol. 60, 195–201. Marquina, D., Santos, A., Corpas, I., Mun˜oz, J., Zazo, J., Peinado, J.M., 2002. Dietary influence of kefir on microbial activities in the mouse bowel. Lett. Appl. Microbiol. 35, 136–140. Pool-Zobel, B.L., Bertram, B., Knoll, M., Lambertz, R., Neudecker, C., Schillinger, U., Schmezer, P., Holzapfel, W.H., 1993. Antigenotoxic properties of lactic acid bacteria in vivo in the gastrointestinal tract of rats. Nutr. Cancer 20, 271–282.
ARTICLE IN PRESS A. Santos et al. / Food Microbiology 23 (2006) 498–503 Reeves, P.G., Nielson, F.H., Fahey, G.C., 1993. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951. Roach, S., Tannock, G.W., 1979. Indigenous bacteria influence the number of Salmonella typhimurium in the ileum of gnotobiotic mice. Can. J. Microbiol. 25, 1352–1358. Rycroft, C.E., Jones, M.R., Gibson, G.R., Rastall, R.A., 2001. A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. J. Appl. Microbiol. 91, 878–887. Santos, A., Corpas, I., Martinez, J., Marquina, D., 2002. The potential therapeutical applications of probiotics. Recent Res. Dev. Microbiol. 6, 1–10. Sghir, A., Chow, J.M., Mackie, R.J., 1998. Continuous culture selection of bifidobacteria and lactobacilli from human faecal samples using fructooligosaccharide as selective substrate. J. Appl. Microbiol. 85, 769–777.
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Shiba, T., Aiba, Y., Ishikawa, H., Ushiyama, A., Takagi, A., Mine, T., Koga, Y., 2003. The suppressive effect of bifidobacteria on Bacteroides vulgatus, a putative pathogenic microbe in inflammatory bowel disease. Microbiol. Immunol. 47, 371–378. Simmering, R., Blaut, M., 2001. Pro- and prebiotics—the tasty guardian angels? Appl. Microbiol. Biotechnol. 55, 19–28. Tannock, G.W., 1979. Coliforms and enterococci isolated from the intestinal tract of conventional mice. Microbiol. Ecol. 5, 27–34. Tannock, G.W., Crichton, C., Weelling, G.W., Koopman, J.P., Midtvet, T., 1988. Reconstitution of the gastrointestinal microflora of Lactobacillus-free mice. Appl. Environ. Microbiol. 54, 2971–2975. Teraguchi, S., Uehara, M., Ogasa, K., Mitsuoka, T., 1978. Enumeration of bifidobacteria in dairy products. Jpn. J. Bacteriol. 33, 753–761. Wang, X., Gibson, G.R., 1993. Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. J. Appl. Bacteriol. 75, 373–380.
Short communication
Prebiotics and their long-term influence on the microbial populations of the mouse bowel A. Santos, M. San Mauro, D. Marquina Dı´ az Department of Microbiology III, Biology Faculty, Complutense University of Madrid, Jose´ Antonio Nova´is, 2, 28040 Madrid, Spain Received 1 September 2004; received in revised form 1 April 2005; accepted 1 July 2005 Available online 10 October 2005
Abstract Various prebiotics were orally administered to mice and the evolution of different microbial populations was studied. The administration of prebiotics significantly increased lactobacilli and bifidobacteria in the large bowel content. Ingestion of prebiotics specifically lowered microbial populations of sulphite-reducing clostridia. Xylo-oligosaccharides (XOS) increased lactobacilli by 10 fold and produced the highest counts of bifidobacteria. In XOS-treated mice, levels of sulphite-reducing clostridia decreased significantly. Prebiotics slightly reduced the amount of aerobic bacteria and significantly increased the number of anaerobes in both the small and the large bowel. These effects of prebiotics were reverted by the basal diet. r 2005 Elsevier Ltd. All rights reserved. Keywords: Prebiotics; Mice; Microbial populations; Small bowel; Large bowel
1. Introduction The functional effect of prebiotics and probiotics is a growing area of research. The gastrointestinal tract contains a large variety of bacteria, which are either beneficial (Lactobacillus, Bifidobacterium, etc.) or detrimental (Clostridium, Shigella, etc.) to the host’s health. Species of the lactic acid bacteria Bifidobacterium and Lactobacillus are the most widely studied probiotic strains and have been shown to exert a wide number of health benefits (Gismondo et al., 1999; Simmering and Blaut, 2001). It is known that Lactobacillus and Bifidobacterium have low activities of enzymes involved in the formation of mutagens and carcinogens (b-glucuronidase, b-glucosidase, nitrate reductase, urease, azoreductase) compared with other anaerobes of the gastrointestinal tract (Pool-Zobel et al., 1993). Lactobacillus and Bifidobacterium strains have been shown to possess inhibitory activity towards the growth of pathogenic bacteria, the ability to adhere to the intestinal epithelial cells and other positive effects on the host health (Greene and Klaenhammer, 1994). Increased Corresponding author. Tel./fax: +34 91 394 49 64.
E-mail address: [email protected] (D.M. Dı´ az). 0740-0020/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2005.07.004
counts of these lactic acid bacteria in the bowel may be achieved by consumption of substances that are known to stimulate probiotic growth (Marquina et al., 2002; Santos et al., 2002). These substances are named as prebiotics. The premise is based on the fact that the large gut contains bacteria that are beneficial or detrimental to health. It has become clear over the last years that the group of nondigestible oligosaccharides (NDO) is likely to play an important nutritional role (Cummings and Macfarlane, 1997). Currently, food components that are seen to exert the best prebiotic effects are inulin-type fructans (Apajalahti et al., 2002; Kleessen et al., 2001). Although differing in their chemical characteristics, all the NDO resist digestion in the small intestine and they are potential substrates for bacteria of the large intestine. Simple stimulation of a particular micro-organism is not sufficient to demonstrate a true prebiotic effect. In vivo, the micro-organisms that survive in the mice bowel have been studied over the recent decades (Marquina et al., 2002; Tannock, 1979; Tannock et al., 1988). The mouse therefore provides an excellent model to determine the effect of a diet on the microbial populations of the gastrointestinal tract. The aims of the present work were to study the influence of the administration of prebiotics on the microbial ecology
ARTICLE IN PRESS A. Santos et al. / Food Microbiology 23 (2006) 498–503
of the gastrointestinal tract of mice and to evaluate the changes on these microbial populations. Furthermore, the persistence of the observed effects was studied.
2. Materials and methods 2.1. Prebiotics The following commercial preparations of prebiotic oligosaccharides were used: fructo-oligosaccharides (FOS, Raftilose P95, Orafti, Tienen, Belgium), long chain inulin (INU; Raftiline HP, Orafti), lactulose (LAC; Sigma Chemical Co., St. Louis, Missouri), xylo-oligosaccharides (XOS; Suntory, Osaka, Japan), galacto-oligosaccharides (GOS; Oligomate55, Yakult Institute, Tokyo, Japan), transgalactosylated-oligosaccharides (TOS; Yakult Institute), isomalto-oligosaccharides (IMO; Isomalt900, Sweetener Industry) and soybean-oligosaccharides (SOS; Calpis, Tokyo, Japan).
2.2. Diets and animals Nine diets were prepared for the study: a basal diet and different diets composed by a basal diet supplemented with one of the eight prebiotics studied (FOS, INU, LAC, XOS, GOS, TOS, IMO and SOS). The test substances were added to the basal diet at a level of 1% (w/w). The basal diet was prepared in accordance to the American Institute of Nutrition 93 (AIN-93) (Reeves et al., 1993). Five-month-old female Swiss mice were used after a 1month quarantine period. The diet feedings were started when the animals were 6 months old. Sixteen females were employed in each dietary group. Animals were housed four per cage, and cages were changed twice a week. Water was available continuously from bottles. All animals were housed in a building that was specially equipped for animal experiments. The animals were maintained under controlled environmental conditions. Animal caretakers, technicians, or anyone entering the animal holding area had to dress in a special suit, including cap, face mask, and shoe covers, to minimize the possibility of contamination. Body weights and food intakes were recorded twice weekly. For examination of organs and sampling, mice were killed by carbon dioxide anaesthesia followed by cervical dislocation. Samples were taken 6 months after the start of the diet treatment. The intestine was immediately removed from each animal and cut to separate small and large intestine. Homogenates were prepared from lengths (5 cm) of small intestine and large intestine. These tissues were homogenized in brain–heart infusion broth (BHI) by using a Teflon homogenizer. The resulting homogenates were maintained in a reduced condition in GasPak jars (BBL Microbiology Systems, Cockeysville, Maryland) and then, they were diluted (10 fold to 1010) in prerreduced BHI.
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2.3. Enumeration of gastrointestinal microbes Microbial counts were obtained by culturing homogenates of gastrointestinal organs on selective media as previously reported (Roach and Tannock, 1979; Tannock, 1979). Viable counts were obtained from BHI agar that was incubated aerobically and anaerobically at 37 1C for 3 days (aerobic and anaerobic counts, respectively). Gram-positive bacterial counts were obtained from phenylethanol agar (Difco, Detroit, Michigan) that was incubated aerobically and anaerobically at 37 1C for 3 days. From Azide Blood Agar (ABA, Oxoid Ltd, Basingstoke, UK) that was incubated aerobically and anaerobically at 37 1C for 3 days, mainly streptococcal counts. From Kanamycin Esculin Azide Agar (KAA, Oxoid) agar that was incubated aerobically at 37 1C for 2 days, Enterococcus counts. From SPS agar (Oxoid) that was incubated anaerobically at 37 1C for 3 days, sulphite-reducing clostridial counts. From Eosin Methylene blue (EMB, Oxoid) agar that was incubated aerobically at 37 1C for 1 day (Enterobacteriaceae counts), and from BHI agar containing a Gram-negative anaerobic supplement (Oxoid) that was incubated anaerobically at 37 1C for 3 days (Gram-negative anaerobic bacterial counts). Anaerobic incubations were performed in Gaspak jars. For bifidobacteria, Neomycin–Paromomycin–Nalidixic acid–Lithium chloride agar (NPNL) was used according to the instructions by the authors (Teraguchi et al., 1978; Hartemink and Rombouts, 1999). LAMVAB medium was selected for the determination of lactobacilli. The medium was prepared according to the instructions by the authors (Hartemink et al., 1997). Kanamycin–vancomycin blood agar (Merck, Darmstadt, Germany) was used in anaerobic conditions to determine Bacteroides/Fusobacteria counts. 2.4. Persistence of the prebiotic effects Nine groups of eight animals, fed with the same diets as described above, were studied. At the end of the experimental period, 6 months, prebiotic diets were suspended and a basal diet was administered. Daily, one animal of each group was killed and the number of microbial counts determined. Experiences were done by triplicate. 3. Results and discussion The aim of the present study was to investigate the changes in the microbial ecology of the bowel of mice due to the effects of a wide number of substances with reported prebiotic properties. These dietary supplements were administered with a basal diet, which conformed to the nutritional composition of the standard AIN-93 diet (Reeves et al., 1993). Only a small number of studies have addressed the effects of the prebiotics on the microbial population during its continued long-term administration (Kleessen et al., 1997). The data in this study suggest that the composition
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of the bowel microflora is influenced by the type of prebiotic in the diet (Fig. 1). Furthermore, the feeding of a diet containing a prebiotic resulted in the stimulation of various bacterial groups, in particular acidogenic bacteria such as bifidobacteria and lactobacilli. Other bacteria tested (Enterococcus spp.) remained more or less unchanged. These variations in the microbial populations were observed with no significant alterations in body weight and the amount of food eaten, with all animals eating approximately 7 g per day. The mean body weight gain of the mice during the experiences ranged from 6.670.3 g (basal diet) to 7.170.2 g (mean of the prebiotic diets) and there were no growth differences at any interval throughout the study. Total aerobes in the large bowel changed little due to the ingestion of oligosaccharides compared with the basal diet. Furthermore, the small bowel of mice consuming XOScontaining diets showed a reduction in the aerobic microflora concomitantly with an increased number of anaerobes. Total anaerobes were higher for XOS-fed mice compared with all other treatments. In general, total anaerobes were also higher for mice fed with prebiotics compared with the basal diet. The effect of the prebiotics on the enterobacteria counts was variable as showed in Fig. 1, although this bacterial population, in general terms, was reduced. Enterococcus counts and aerobic and anaerobic counts in Azide Blood Agar were slightly affected or unaffected by the different diets. The number of Bacteroides/Fusobacteria was higher in the large bowel compared with small bowel and the most important changes were observed in the XOS containing diet. In this microbial group, counts were variable or unaffected in the small bowel (Fig. 1). Despite the evidence that the gut microflora is affected by the administration of prebiotics, it is difficult to identify the specific micro-organisms responsible for these changes. It seems likely that a number of prebiotic-degrading bacteria, which are present in the gastrointestinal tract are complementary, or antagonic, in their metabolic activities. Although prebiotics offer one rational approach to the probiotic concept, the health consequences have not yet been defined. In theory, a number of potential benefits may arise (Hussein et al., 1999; Loo et al., 1999). However, it may be that improved resistance to pathogens offers the most feasibility. The lactic acid bacteria of the gastrointestinal tract are thought to play a significant role in improved colonization resistance. Increased bifidobacterial numbers in the gut may be one factor that contributes towards improved competitive exclusion of pathogens seen in prebiotic-treated animals. Moreover, it is noteworthy that the feeding of prebiotic-containing diets resulted in a significant decrease of bacteria belonging to the clostridial group. A high proportion of these micro-organisms may be pathogenic through their proteolytic capabilities and toxin production. Our results showed that mice fed diets containing prebiotics significantly reduced large bowel concentrations of sulphite-reducing clostridia and XOS induced the strongest reduction.
Lactobacilli concentrations were greatest in small and large intestine as a result of ingestion of prebiotics (Fig. 1). In the large bowel, the most important effect was due to the ingestion of XOS and INU-diets, but all of the tested prebiotics increased lactobacilli. Bifidobacteria counts were statistically greater in LAC, IMO and XOS-diets but also in other prebiotics-supplemented dietary groups. In these groups, the greater bifidobacteria count was evident in absolute concentration and in the percentage of bifidobacteria among total anaerobes (Fig. 1). The increase in lactobacilli and bifidobacteria, especially in XOS-diet, may contribute to the stabilization of the microflora and to the health of the host. Many lactobacilli and bifidobacteria are able to produce natural antibiotics, which can have a broad spectrum of activity against various intestinal pathogens (Gibson and Wang, 1994; Shiba et al., 2003). Lactobacillus and Bifidobacterium are non-pathogenic bacteria of the bowel tract. These micro-organisms may increase resistance to disease by reducing the growth of pathogenic and putrefactive bacteria by producing inhibitory substances, competing directly for substrates and mucosal attachment sites (Coconier et al., 1998; Finlay and Falkow, 1989; Jacobsen et al., 1999). Although it is generally accepted that prebiotics are not metabolised by rat digestive enzymes in the small intestine and thus reach the caecum and colon, a partial bacterial hydrolysis and fermentation of these NDO in the distal part of the small intestine cannot be excluded. This view is supported by the small intestine numbers of microorganisms, such as bifidobacteria (Fig. 1), which tended to be higher in mice fed with TOS (6.5 106 UFC g 1) than in control mice (5.1 106 UFC g 1). Any carbohydrate that reaches the cecum is a potential substrate for fermentation by the microbiota, and much evidence supports the belief that the currently identified prebiotics are fermented. Many different prebiotics support bacterial growth and produce various fermentation-derived end products (Wang and Gibson, 1993; Gibson, 1999). It has been demonstrated that the nature of the prebiotic determines its fermentability (Loo et al., 1999). Fructans are resistant to clostridial breakdown; however, XOS, lactulose and fructans are extensively fermented by other enteric bacteria including strains of Bifidobacterium, Lactobacillus and Bacteroides. The hydrolysis of prebiotics by these micro-organisms may lead to the accumulation of intermediates, which may be available for cross feeding of non-degrading-prebiotic species (Macfarlane and Englyst, 1986). It has been observed that in pure culture experiments different strains of bifidobacteria grew well, as did Clostridium perfringens and Escherichia coli (Wang and Gibson, 1993). However, it has been shown, in competition experiments between B. infantis, E. coli and C. perfringens, with oligofructose as sole source of carbon and energy, that the bifidobacteria grew well and showed an inhibitory effect on the growth of the other two (Gibson and Wang, 1994; Rycroft et al., 2001; Sghir et al., 1998; Loo et al., 1999).
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Fig. 1. Microbial counts in small and large bowel after a six-month treatment with various prebiotics. Black columns: small bowel. White columns: large bowel. (A) Aerobic bacteria; (B) anaerobic bacteria; (C) Gram-positive aerobic bacteria; (D) Gram-positive anaerobic bacteria; (E) Gram-negative anaerobic bacteria; (F) aerobic bacteria on Azide Blood Agar; (G) anaerobic bacteria on Azide Blood Agar; (H) lactobacilli; (I) bifidobacteria; (J) Enterobacteriaceae; (K) Enterococcus spp.; (L) sulphite-reducing bacteria; (M) Bacteroides/Fusobacteria.
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Fig. 2. Time course evolution of different microbial populations of the large bowel. The effect of the basal diet on the microbial populations of previously prebiotic-treated mice was studied. (A): (’), Aerobic bacteria; (K), anaerobic bacteria; (m), Gram-positive aerobic bacteria; (.), Gram-positive anaerobic bacteria; (~), Gram-negative anaerobic bacteria; (&), aerobic bacteria on Azide Blood Agar; (J), anaerobic bacteria on Azide Blood Agar. (B): (’), Lactobacilli; (K), bifidobacteria; (m), Enterobacteriaceae; (.), Enterococcus spp.; (~), sulphite-reducing bacteria; (&), Bacteroides/ Fusobacteria.
To our knowledge, this is the first long-term study that shows that prebiotic-containing diets changed some microbial populations of the small and large bowel of mice. In the present work, it was shown that the beneficial microbiological status induced by prebiotics disappeared by the suppression of prebiotic-enriched diets (Fig. 2). The effect of the basal diet in the previously prebiotic-treated mice was studied and time effects on the different bacterial counts in the large bowel were observed in the 6-monthprebiotic-treated animals. The observed prebiotic effects (increased anaerobes, lactobacilli and bifidobacteria counts, etc.) were gradually reverted by basal diet (Fig. 2). Furthermore, in the first week without prebiotic diets, microbial counts were similar to that obtained in control animals with a 6-month-basal diet (Fig. 1). In summary, the use of a basal diet as a replacement for prebiotics in diets caused a marked decrease in the number of total anaerobes, lactobacilli and bifidobacteria, whereas total aerobes, enterobacteria and clostridia all increased. The study suggests that the benefits of a prebiotic-enriched diet disappeared quickly and so, a continuous prebiotic diet must be followed to maintain the reported benefits in the host. References Apajalahti, J.H.A., Kettunen, H., Kettunen, A., Holben, W.E., Nurminen, P.H., Rautonen, N., Mutanen, M., 2002. Culture-independent microbial community analysis reveals that inulin in the diet primarily affects previously unknown bacteria in the mouse cecum. Appl. Environ. Microbiol. 68, 4986–4995. Coconier, M.-H., Lievin, V., Hemery, E., Servin, A.L., 1998. Antagonistic activity against Helicobacter infection in vitro and in vivo by the human Lactobacillus acidophilus strain LB. Appl. Environ. Microbiol. 64, 4573–4580. Cummings, J.H., Macfarlane, G.T., 1997. Role of intestinal bacteria in nutrient metabolism. Clin. Nutr. 16, 3–11. Finlay, B.B., Falkow, S., 1989. Common themes in microbial pathogenicity. Microbiol. Rev. 53, 210–230.
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