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Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/225338571 Theabilityofblackcurrantextractstopositively modulatekeymarkersofgastrointestinal functioninrats ArticleinWorldJournalofMicrobiologyandBiotechnology·October2010 DOI:10.1007/s11274-010-0352-4 CITATIONS READS 29 36 3authors,including: Abdul-LateefMolan MarlenaCKruger UniversityofDiyala MasseyUniversity 107PUBLICATIONS1,553CITATIONS 153PUBLICATIONS2,196CITATIONS SEEPROFILE SEEPROFILE Someoftheauthorsofthispublicationarealsoworkingontheserelatedprojects: StudythepossiblelinkbetweenToxoplasmosisanddifferentkindsofcancerinIraqView project Healthbenefitsofselenium-richgreenteaViewproject Allin-textreferencesunderlinedinbluearelinkedtopublicationsonResearchGate, lettingyouaccessandreadthemimmediately. Availablefrom:Abdul-LateefMolan Retrievedon:16November2016 World J Microbiol Biotechnol (2010) 26:1735–1743 DOI 10.1007/s11274-010-0352-4 ORIGINAL PAPER The ability of blackcurrant extracts to positively modulate key markers of gastrointestinal function in rats Abdul-Lateef Molan • Zhuojian Liu Marlena Kruger • Received: 11 November 2009 / Accepted: 11 February 2010 / Published online: 27 February 2010 Ó Springer Science+Business Media B.V. 2010 Abstract An animal model was used to assess the effects of orally administered aqueous extract from two commercial health supplement ingredients; First Leaf (FL; composed of blackcurrant extract powder, lactoferrin and lutein and is developed by the Four Leaf Japan Co. Ltd, Japan) and Cassis Anthomix 30 (CAM30; blackcurrant extract powder which is developed by Just the Berries Ltd, New Zealand) on the proliferation of lactobacilli and bifidobacterial species and some undesirable bacteria in the caeca of rats. Gavaging rats with CAM30, FL or inulin three times weekly for 4 weeks resulted in a significant increase in the numbers of bifidobacteria and/or lactobacilli and a significant decrease in the numbers of bacteroides and clostridia. Moreover, rats gavaged with FL, CAM30 and inulin showed 31.5, 18 and 15% reduction in the activity of b-glucuronidase and 26, 30.4 and 18% increment in the activity of b-glucosidase when compared to the control group gavaged with water, respectively. These benefits may make these products good candidates as prebiotic agents. In conclusion, this study has shown that FL and CAM30 can positively modulate key markers of gastrointestinal function in rats. Keywords Prebiotic activity Blackcurrant products Modulation of gut microbiota Rats Abbreviations FL First Leaf product (composed of blackcurrant extract powder, lactoferrin and lutein) A.-L. Molan (&) Z. Liu M. Kruger Human Nutrition and Physiology Division, Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11 222, Palmerston North, New Zealand e-mail: [email protected] BC CAM30 FOS NOVA TAA Blackcurrant concentrates Cassis Athomix 30 (blackcurrant extract powder) Fructooligosaccharides One way analysis of variance Total antioxidant activity Introduction The human colon harbours a large and complex microbial flora and most of its members are obligate anaerobes and many of them (60–80%) have not been cultivated (Hayashi et al. 2003). However, it is now generally accepted that the composition of human intestinal microbiota has an important role in health and disease (Gionchetti et al. 2000). Many definitions have been made for the probiotics and the definition continues to be improved, and was recently made official by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO; Araya et al. 2002) as ‘‘live microorganisms which when administered in adequate amounts confer a health benefits on the host’’. Although some probiotic bacteria can survive the gastrointestinal tract transit and reach the colon safely, some studies have shown that they did not colonize and grow during short or even after prolonged feeding periods (Tannock et al. 2000). Thus, continuous consumption appears to be necessary to maintain a high population of the probiotic strain in the colon. Accordingly, there is a need for a strategy that enhances viability and promotes growth of probiotic bacteria in the colon. Some clinical studies have identified the complex interactions between gut microflora and health such as the relationship between the colonic bacteria and their 123 1736 metabolic characteristics to the risk of colon cancer (Chadwick et al. 1992). It has been shown that some species are capable of generating carcinogens, mutagens, and tumour promoting agents in the large bowel (Goldin 1990). b-glucuronidase, nitroreductase, azoreductase and nitratereductase are synthesised by some pathogenic colonic bacteria, and they are known to produce mutagens, carcinogens, and various tumour promoters (Gorbach and Goldin 1990). The importance of b-glucuronidase activity is based on its function of deconjugating with many endogenous and exogenous compounds. b-glucuronidase activity antagonises hepatic metabolism by hydrolysing the biliary conjugates, and delays its excretion. As a consequence, modification of the human intestinal microbiota has currently become an important objective in nutritional science. This goal can be achieved in three ways: either by inclusion in the diet of a significant proportion of beneficial bacteria, mainly Bifidobacterium and Lactobacillus species, with the expectation that they will be able to colonize the intestinal tract (probiotics); to give non-digestible carbohydrates (prebiotics), like fructo-oligosaccharides, which have shown an ability to promote the growth of desirable bacteria; or to give a mixture of both (synbiotics; Gopal et al. 2003). Prebiotics are generally carbohydrates that bypass the small intestine. Prebiotics are often especially intended to stimulate the growth of bifidobacteria because of the presumed beneficial role of bifidobacteria on gastrointestinal health (Grizard and Barthomeuf 1999). Presently available prebiotics (such as Fructooligosaccharides and inulin) can aid in the survival and proliferation of probiotic bacteria, but are limited by some drawbacks (Apajalahti et al. 2002). Therefore, there remains a need for alternative prebiotics with fewer or no side effects that could either be incorporated in a probiotic food matrix, or used as a stand-alone prebiotic to enhance proliferation of lactic acid bacteria naturally present in the intestine. Blackcurrant berries are rich sources of various phenolic substances with antioxidative, antibacterial, antiviral and anti-inflammatory properties including flavonoids and anthocyanidins as well as vitamin C. Consumption of fresh blackcurrant (Ribes nigrum) is limited, but is used commercially to manufacture marmalade, jelly, jam, juice, etc., including a very popular liquor in France called ‘Cassis’ (Ibarz et al. 1992). Miller and Rice-Evans (Miller and RiceEvans 1997) studied the total antioxidant activity (TAA) of orange juice, apple juice and blackcurrant drink and found that blackcurrant drink (Ribena) has the highest TAA and apple juice the lowest. The blackcurrant concentrate (BC), which had been prepared from blackcurrant juice, is reportedly known to increase resting forearm blood flow (Matsumoto et al. 2005) and to improve the peripheral circulation disorder caused by the cold water loading in 123 World J Microbiol Biotechnol (2010) 26:1735–1743 human volunteers (Takenami et al. 2004). Furthermore, BC intake effectively restored the decreased oxy-hemoglobin level during and after the typewriting workload leading to an improvement of muscle fatigue (Takenami et al. 2003). It has been reported that oral intake of BC significantly improves the peripheral circulation in human volunteers (Matsumoto et al. 2005; Takenami et al. 2004; Takenami et al. 2003). Recently, Werlein et al. (Werlein et al. 2005) studied the effect of concentrates from blackcurrant and a purified anthocyanin mix on the growth of some Grampositive and Gram-negative bacteria and yeast and found that blackcurrant concentrates inhibited the growth of Staphylococcus aureus DSM 799 and Enterococcus faecium DSM 2918, whilst Saccharomyces cerevisiae ATCC 9763 was slightly stimulated by the fruit concentrates. In the present study, the influence of FL, CAM30 and inulin on the numbers of bifidobacteria, lactobacilli, bacteroides, and certain clostridial numbers in the caecal contents of rats gavaged orally with these products was evaluated using fluorescence in situ hybridization (FISH) molecular method. In addition, the effect of these products on food intake and body weight gain and on the activity of selected metabolic indexes (b-glucuronidase and b-glucosidase enzymes) in rats was also investigated. Materials and methods Preparation of extracts The aqueous extracts from First Leaf (FL; blackcurrant powder, lactoferrin and lutein) obtained from Four Leaf Japan Co., Ltd and Cassis Anthomix 30 (CAM30; blackcurrant extract powder) obtained from Just the Berries Ltd, were prepared by dissolving the powdered extracts in sterile distilled water. Both products are commercial and are already on the market (Japan and United States). Inulin powder (Sigma, Australia) was dissolved in sterile distilled water. All the extracts were prepared on the same day of gavaging. Experimental design/animals and housing Forty Sprague–Dawley male rats aged 8 weeks that had been weaned onto a balanced semi-synthetic diet were housed individually in plastic cages in a room with a temperature of 22 ± 1°C and a 12 h light: dark cycle (light on at midnight) with free access to water and food throughout the study. The rats were obtained from the Animal Unit, Massey University, Palmerston North, New Zealand. The experimental protocol was approved by the Massey University Animal Ethics Committee (Palmerston North, New Zealand; Protocol No: 07/141; 14/12/2007). World J Microbiol Biotechnol (2010) 26:1735–1743 Administration of extracts This study involved gavaging rats with *2 ml of sterile distilled water, inulin, FL or CAM30 extracts delivered to the back of each rat’s mouth via a soft silicon rubber tube attached to a 3 ml syringe. So that the rats were comfortable with this procedure, during the 7 day acclimation period the rats were handled daily and each rat was gavaged orally with 2 ml of sterile water (at room temperature) on alternative days to match the experimental procedure. For the remainder of the study (from day 8 to the end) the rats were allocated randomly into four equal groups (n = 10). The rat groups were gavaged with 2 ml of sterile water (negative control), 2 ml of inulin extract (positive control), 2 ml of FL extract (containing 30 mg FL/kg body weight) or 2 ml of CAM30 extract (containing 13.4 mg CAM30/kg body weight), respectively, three times weekly (Monday, Wednesday and Friday) for four consecutive weeks. The same experimental diet was offered to all treatment groups during the feeding period. It was formulated to meet the nutrient requirements of growing rats. Food intake was determined three times weekly as the difference in weights of food before and after each feeding period. Similarly, the water intake was determined by weight difference. The rats were weighed at the beginning of the experiment and then thrice weekly and at the end of the trial. At the end of the experiment, rats were euthanized by CO2 inhalation according to recommendations for euthanasia of experimental animals. After dissection, the secum with contents was removed. The cecal contents were transferred to tubes and stored at -80°C until being processed. The amount of gavaging dose was based on a dose rate of 30 mg FL and 13.4 mg of CAM30/kg rat body weight, respectively as recommended by the client. For inulin, the dose rate was equivalent to 1% of food intake. Fluorescence in situ hybridization (FISH) analysis of microbiota in the cecal contents The probes used in the study were Bif164 Bac303, Chis150 and Lab158, specific for bifidobacteria, bacteroides, clostridia (perfingens/histolyticum subgroup) and lactobacilli/ enteroccocus, respectively. These were commercially synthesised and labelled with the fluorescent dye Cy3 (GeneWorks, Australia). The samples were prepared for FISH analysis as described by Molan et al. (2009). In short, caecal content samples were prepared by mixing 1 g of the digesta collected from the middle portion of the cecae with 9 ml of sterile-filtered phosphate buffer (PBS; pH 7.2). The mixture was homogenized by vortexing with a dozen glass beads for 3 min, the caecal debris was removed by 1737 centrifugation at low speed (700g), and the bacteria containing supernatant was fixed in 4% (w/v) paraformaldheyde in PBS (pH 7.2) overnight at 4°C. Fixed samples were washed in PBS and stored in a known volume of 50% (v/v) ethanol-PBS at -20°C until time of hybridization. Aliquots (5 ll) of fixed bacterial cells were applied to Teflon-coated microscopic slides (BIOLAB, New Zealand; 10 wells) and air dried. The bacterial cells were then dehydrated with a series of solutions containing 50, 80, and 99.5% ethanol (3 min for each concentration). The bacterial cells fixed on the glass slides were hybridized by addition of 8 ll of hybridization buffer (0.9 M NaCl, 0.01% sodium dodecyl sulfate, 20 mM Tris–HCl, 20% deionized formamide; pH 7.2) with 0.5 ll of Cy3-labeled oligonucleotide specific probes (50 ng/ll). The slides were hybridized at 46°C for 2 h in a plastic box containing wet sponges (soaked in hybridization buffer). After hybridization, the slides were rinsed with warm hybridization buffer at 48°C and washed in pre-warmed washing buffer (225 mM NaCl, 0.01% sodium dodecyl sulfate, 20 mM Tris–HCl; pH 7.2) for 20 min at 48°C. After washing step, the slides were rinsed with ice-cold distilled water and thoroughly dried before being observed using a fluorescence scanning microscope. The dried slides were examined with an Olympus BX51 microscope, under 4009 magnification. The images were captured using an Optronics MagnaFIRE SS99802 digital camera with MagnaFIRE frame-grabbing software on a Pentium IV computer. Fluorescent cells were counted automatically in five randomly selected fields/slide using ImageJ. Bacterial enzyme activities The samples were prepared as described by Gudiel-Urbano and Goni (2002) with some modifications. In short, cecal content samples were prepared by mixing 1 g of the digesta collected from the middle portion of the cecae with 9 ml of 0.1 M phosphate buffer (pH 7). The mixture was homogenized by vortexing and the caecal suspension (1 ml) was mixed with equal volume of 0.1 M phosphate buffer (pH 7) containing 0.2% Triton-X-100 to disrupt the bacterial cells. The mixture was centrifuged for 10 min at 5,000g at room temperature. The supernatant was assayed for the enzymatic activity of both b-glucosidase and b-glucuronidase. The enzyme activities were measured as described by Djouzzi et al. (1997) with some modifications, by the rate of release of p-nitrophenol from p-nitrophenyl-b-D-glucoside (N-7006, Sigma–Aldrich, New Zealand) as substrate for b-glucosidase and p-nitrophenyl-b-D-glucuronide (N-7009, Sigma–Aldrich, New Zealand) as substrate for b-glucuronidase. Briefly, the reaction mixture containing 123 1738 The pH of each caecal sample was measured in order to test if there is any link between the levels of b-glucuronidase, b-glucosidase, the numbers of lactobacilli and bifidobacteria and the caecal pH. The pH was measured by a digital pH-meter at room temperature (20–22°C). Data analysis Caecal concentrations of bacteria were expressed as log number of bacterial cells/g caecal content wet weight. Logarithmically-transformed data were analysed by one way analysis (ANOVA) of variance using SAS system with the level of significance set at P \ 0.05. For average comparisons, data comparisons were made using the student’s t test. Results Prebiotic and antimicrobial activities The probe technology can provide information on bacterial populations at the genus, group, and even species levels in terms of identification and enumeration. Fluorescence in situ hybridization is a powerful method for the enumeration of bacteria in complex habitats such as the gut. Most notably, it does not require cultivation of the target organisms. This method was used in this study to enumerate the good and bad bacteria in caecal contents of rats gavaged with the tested products. Gavaging rats with FL and CAM30 led to a significant increase (P \ 0.0001) in bifidobacterial numbers when compared with the rats gavaged with water (negative control; Fig. 1). In addition, bifidobacterial numbers significantly increased in rats gavaged with inulin (P = 0.0016). 123 (A) 9.5 Log number/g cecal contents pH values of caecal samples Significant increases in lactobacilli numbers were seen in rats gavaged with CAM30 (P = 0.03075) while gavaging rats with FL led to a slight but insignificant increase (P = 0.0964) in lactobacilli when compared with the control rats gavaged with water (Fig. 1b). Similarly, rats gavaged with inulin showed insignificant increase in lactobacilli in comparison with control rats. It is important to mention that the number of bifidobacteria in the caeca of rats gavaged with CAM30 was significantly higher than that in the caeca of rats gavaged with FL or inulin (Fig. 1). When compared with control rats gavaged with water, bacteroides numbers decreased to significant levels in rats gavaged with CAM30 (P = 0.0106), FL (P = 0.0115) and Bifidobacteria * *** *** 9 8.5 8 Control (B) Log number/g cecal contents 0.2 ml of a 5 mM substrate solution and 0.2 ml of caecal suspension from control and rats gavaged with inulin, FL or CAM30 was incubated at 37°C. The reaction was stopped at 30 min after the addition of 1.6 ml of 0.25 M Na2CO3 (Gudiel-Urbano and Goni 2002). The released p-nitrophenol was measured at 400 nm. The amount of p-nitrophenol released was determined by comparison with a standard nitrophenol curve. Enzyme activities were calculated as nanomoles of transformed substrate per minute per gram of cecal content wet weight (nmol/min/g).The % reduction in the activity of b-glucuronidase activity and % increase in the activity of b-glucosidase were calculated relative to the enzymatic activity in control rats gavaged with water. World J Microbiol Biotechnol (2010) 26:1735–1743 Inulin FL CAM30 ## Lactobacilli $$ 9.5 *** 9 8.5 8 Control Inulin FL CAM30 Fig. 1 Enumeration of Bifidobacterium species (a) and Lactobacillus species (b) [Log10 cells/g of wet caecal contents] from the four groups of rat caecal samples hybridized with Bifidobacterium genus-specific oligoneucleotide probes in FISH analysis. The rat groups were gavaged with water (negative control), inulin (positive control), FL or CAM30 three times weekly for four consecutive weeks. The data are expressed as means ± standard errors of the means (n = 10 rats/ group). * P B 0.05; *** P B 0.0001 by analysis of variance versus the negative control group. ## P \ 0.01 by analysis of variance versus the inulin group. $$ P \ 0.01 by analysis of variance versus the FL group World J Microbiol Biotechnol (2010) 26:1735–1743 1739 inulin (P = 0.0311; Fig. 2a). Similarly, clostridial (perfingens/histolyticum sub. grp.) numbers significantly decreased in rats gavaged with CAM30 (P = 0.0481), FL (P = 0.0346) and in inulin-treated rats (P = 0.0168) in comparison with rats gavaged with water (Fig. 2b). Concerning the activity of b-glucosidase, rats gavaged with FL, CAM30 and inulin showed 26, 30.4 and 18% increment in the activity of this enzyme when compared to the control group gavaged with water, respectively (Fig. 4). pH of caecal samples In rats gavaged with FL, CAM30 and inulin, the activity of b-glucuronidase decreased by 31.5, 18 and 15%, respectively relative to control rats gavaged with water (Fig. 3). Moreover, the FL-treated rats were characterized by a significantly lower (P \ 0.0001) activity of b-glucuronidase compared with positive control group gavaged with inulin and those gavaged with CAM30 (Fig. 3). This may explain the significant reduction in the numbers of bacteroides and clostridia in the same caecal samples. Log number/g cecal contents (A) 9.5 Bacteroides * * * 9 Figure 5 shows the pH values of the caecal samples collected from rats gavaged with water, inulin, FL and CAM30. The present study showed that the caecal pH in rats gavaged with inulin, FL and CAM30 was significantly % inhibition relative to control incubation Activity of microbial enzymes (b-glucuronidase and b-glucosidase activity) β-glucuronidase 40 *** $ 30 20 10 0 Inulin FL CAM30 Fig. 3 % reduction in the activity of b-glucuronidase in caecal contents of rats gavaged with inulin, FL or CAM30 relative to the control group gavaged with water. The data are expressed as means ± standard errors of the means (n = 10 rats/group). *** P B 0.0001 by analysis of variance versus the inulin group. $ P \ 0.05 by analysis of variance versus the CAM30 group 8.5 8 Inulin Log number/g cecal contents (B) 9.5 FL CAM30 Closteridia 9 * * * 8.5 8 Control Inulin FL CAM30 Fig. 2 Enumeration of bacteroides (a) and clostridia (b) [Log10 cells/ g of wet caecal contents] from the four groups of rat caecal samples hybridized with Bacteroides genus-specific oligoneucleotide probes in FISH analysis. The rat groups were gavaged with water (negative control), inulin (positive control), FL or CAM30 three times weekly for four consecutive weeks The data are expressed as means ± standard errors of the means (n = 10 rats/group). * P B 0.05 by analysis of variance versus the negative control group % increase in enzyme activity relative to control incubation Control β-glucosidase 40 ** 35 30 * 25 20 15 10 5 0 Inulin FL CAM30 Fig. 4 % increase in the activity of b-glucosidase in caecal contents of rats gavaged with inulin, FL or CAM30 relative to the control group gavaged with water. The data are expressed as means ± standard errors of the means (n = 10 rats/group). ** P B 0.0001 by analysis of variance versus the inulin group; * P \ 0.05 by analysis of variance versus the inulin group 123 1740 World J Microbiol Biotechnol (2010) 26:1735–1743 compared with the control group gavaged with water (Table 1). There were no significant differences detected in the body weight of rats gavaged with inulin, FL and CAM30 when compared with rats in the control group (Table 2). Overall, water intake in rats gavaged with inulin, FL or CAM30 was not affected in comparison with the rats gavaged with water only (data not shown). pH value 8 *** *** *** pH value 6 4 2 Discussion 0 Control Inulin FL CAM30 Fig. 5 Caecal pH in rats gavaged with water (control), inulin, FL and CAM30. *** P B 0.0001 by analysis of variance versus the control group gavaged with water only (negative control) lower (P \ 0.0001) than that in control rats gavaged with water. Effect on food intake, water intake and body weight gain There were no significant differences detected in the food intake of rats gavaged with inulin, FL and CAM30 when The results show for the first time that aqueous extracts from FL and CAM30 were effective at promoting the growth of lactobacilli and bifidobacteria in the caecum of rats gavaged orally with these extracts. From a health perspective, this finding is very important due to the growing interest in probiotic bacteria and the perceived benefit of increasing their numbers in the gastrointestinal tract, namely the enhancement of gut health by competitively excluding pathogens from the occupation of adhesion sites or by influencing the gut environment via secretion of simple or complex molecules. Increasing the numbers of lactic acid bacteria (lactobacilli and bifidobacteria) in the colon was found to reduce the formation of ammonia, skatole, harmful amines and other procarcino- Table 1 Food intake in Sprague–Dawley male rats fed chow pellets and gavaged orally water, FL, CAM30 or inulin three times weekly for four consecutive weeks Measurement Food intake (g/day/rat) Water group Inulin group FL group CAM30 group Week 1 30.86 ± 0.77 31.28 ± 0.44 30.48 ± 0.47 32.09 ± 0.66 Week 2 31.4 ± 0.89 30.98 ± 0.72 31.4 ± 0.54 31.74 ± 0.65 Week 3 Week 4 32.5 ± 0.96 33.86 ± 1.1 32.3 ± 0.62 33.76 ± 0.85 31.99 ± 0.58 33.25 ± 0.66 31.5 ± 0.73 33.63 ± 0.83 Mean (g/rat) 32.17 ± 0.47 32.08 ± 0.35 31.78 ± 0.29 32.24 ± 0.36 The data are expressed as means ± standard errors of the means (n = 10 rats/group) Table 2 Body weight (g) of rats gavaged with water, FL, CAM30 or inulin three times weekly for four consecutive weeks Measurement Body weight (g) Water group Week 1 Week 2 304.8 ± 7.0 348.4 ± 8.2 Inulin group 304.1 ± 4.4 348.8 ± 5.3 FL group 305.4 ± 4.4 353.5 ± 4.9 CAM30 group 308.54 ± 5.6 354.29 ± 6.1 Week 3 393.2 ± 9.7 392.3 ± 6.5 396.64 ± 5.1 395.6 ± 6.3 Week 4 431.8 ± 10.7 431.1 ± 7.5 435.24 ± 5.8 434.5 ± 6.8 369.53 ± 6.43 369.3 ± 5.7 372.7 ± 5.1 373.24 ± 5.3 Mean (g) The data are expressed as means ± standard errors of the means (n = 10 rats/group) 123 World J Microbiol Biotechnol (2010) 26:1735–1743 gens in the large intestine and the carcinogenic load on the intestine (Burns and Rowland 2000; Yamamoto et al. 1997). The production of acids by these bacteria also lowered the pH value of the colon and faeces (Yamamoto et al. 1997). Moreover, it has been found that modification of the gut microflora may interfere with the process of carcinogenesis and this opens up the possibility for dietary modification of colon cancer risk. Probiotics and prebiotics, which modify the microflora by increasing numbers of lactobacilli and/or bifidobacteria in the colon, have been a particular focus of attention in this regard (Burns and Rowland 2000). The bifidogenic activity of inulin found in the present study matches other studies performed in rats and humans. Many human intervention studies had previously found a bifidogenic effect of inulin (Bouhnik et al. 2007; Van de Wiele et al. 2004). This gives a strong indication of the validity of the results obtained in this study. The results have provided the first evidence that consumption of FL and CAM30 can beneficially affect faecal parameters related to colon cancer risk. Under the present experimental conditions, this study showed that CAM30 and FL can decrease the activity of b-glucuronidase and increase the activity of b-glucosidase, which could be perceived as potentially beneficial for the host. It has been reported that certain bacteria (such as Escherichia coli, Clostridium perfringens and Bacteroides species) have high b-glucuronidase activity (Skar et al. 1998). Moreover, this result may explain the significant increase in the numbers of bifidobacteria and lactobacilli in the same caecal samples. Results from some studies with probiotics have shown that increasing the proportion of the fecal flora represented by bifidobacteria is associated with lower activity of reductive enzymes including caecal b-glucuronidase, and inhibition of the development of Azoxymethane (AOM)-induced colon carcinogenesis (Kulkarni and Reddy 1994; Ling et al. 1994). The importance of this finding is generated from the fact that the bacterial enzyme b-glucuronidase is considered to be one of the enzymes that increases risk for colorectal cancer (Gorbach and Goldin 1990; Hambly et al. 1997). It has been suggested that b-glucuronidase is a key enzyme in the activation of the procarcinogen 1,2-dimethylhydrazine (DMH) into its toxic carcinogen (Johansson et al. 1997). Therefore, b-glucuronidase has assumed significance as colon cancer risk marker and its inhibitors have been suggested to have anticarcinogenic properties because they will increase clearance of glucuronidated carcinogens (Walaszek et al. 1986). Accordingly, lower activity of this enzyme can be considered beneficial in terms of the risk of colon cancer (Goldin 1990). Some studies have shown that certain tumours and some bacteria have high b-glucuronidase activity (Brady et al. 1741 2000), and that such tumours or bacterial infections may be treated by means of b-glucuronidase inhibitors which are toxic to the tumour cell or bacterial cell. Other studies have shown that the activities of fecal bacterial enzymes such as b-glucuronidase, azoreductase and nitroreductase increased in animals and humans consuming a high beef diet which is associated with a high risk of colon cancer (Gorbach 1982; Reddy et al. 1977). The results of the present study may suggest that CAM30 and/or FL extracts are good candidates for decreasing/inhibiting the activity of b-glucuronidase which is involved in colon cancinogenesis. The increment in the activity of b-glucosidase probably due to the stimulation of the growth of lactic acid bacteria, which have high levels of b-glucosidase activity in comparison with the other members of the gut microflora (Saito et al. 1992). Previous studies have shown that oligosaccharides have the ability to increase the activity of this enzyme and this has been attributed to the ability of oligosaccharides to stimulate the growth of lactic acid bacteria (Saito et al. 1992; Rowland and Tanaka 1993). Marteau et al. (1990) found an increase in faecal b-glucosidase activity in subjects consuming milk fermented with Lactobacillus acidophilus and Bifidobacterium bifidum. Fuller and Perdigon (Fuller and Perdigon 2000) reported that consumption of dairy products with viable L. acidophilus, which is bile resistant, can cause a significant decline in some faecal bacterial enzymes including b-glucosidase. Again, the results of the present study have provided the first evidence that consumption of FL and CAM30 decreases the caecal pH which can be considered beneficial in terms of the risk of colon cancer. An association between high colonic (or stool) pH and increased risk of colon cancer has been reported, suggesting that colonic neoplasia might be related to a reduction in the fermentation of dietary polysaccharides to organic acids in the large bowel (Pietroiusti et al. 1985). A number of potential mechanisms are suggested. Bile acids, for example, are degraded more or less efficiently depending on stool pH; the lower the pH the greater the inhibition of secondary bile acid formation, which is a key factor in development of colon cancer (Cummings and Bingham 1987). Conclusions This study has identified CAM30 and FL as good prebiotic agents that can significantly promote the growth of friendly bacteria such as bifidobacteria and lactobacilli, and lower the numbers of undesirable bacteria such as bacteroides and clostridia in the cecum of rats. This study also showed that CAM30 and FL can decrease the activity of b-glucuronidase and increase the activity of b-glucosidase, which could be perceived as potentially beneficial for the host. 123 1742 These promising results need to be confirmed in human studies involving both healthy volunteers and subjects with clinical conditions that have a supposed microbial aetiology (e.g. ulcerative colitis, bowel cancer and irritable bowel syndrome). Acknowledgments This study was supported by ‘‘Four Leaf Japan Co., Ltd.’’ and ‘‘Just the Berries Ltd’’. References Apajalahti JH, Kettunen H, Holben WE, Nurminen PH, 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 Araya JL, Morelli L, Reid G, Sanders ME, Stanton C, Pineiro M, Ben Embarek P (2002) Guidelines for the evaluation of probiotics in food. Food and Agricultural Organization and World Health Organization, London. Available at: ftp://ftp.fao.org/docrep/fao/ 009/a0512e/a0512e00.pdf Bouhnik Y, Raskine L, Champion K, Andrieux C, Penven S, Jacobs H, Simoneau G (2007) Prolonged administration of low-dose inulin stimulates the growth of bifidobacteria in humans. Nutr Res 27:187–193 Brady LJ, Gallager DD, Busta F (2000) The role of probiotic cultures in the prevention of colon cancer. J Nutr 130:410–414 Burns AJ, Rowland IR (2000) Anti-carcinogenicity of probiotics and prebiotics. Curr Issues Intest Microbiol 1:13–24 Chadwick RW, George S, Claxton LD (1992) Role of the gastrointestinal mucosa and microflora in the bioactivation of dietary and environmental mutagens and carcinogens. Drug Metab Rev 25:425–461 Cummings JH, Bingham SA (1987) Dietary fibre, fermentation and large bowel cancer. Cancer Surv 6:601–621 Djouzzi ZC, Andrieux MC, Degiury C, Szylit O (1997) The association of yoghurt starters with Lactobacillus casei DN 114.001 in fermented milk alters the composition and metabolism of intestinal microflora in germ- free rats and in human flora-associated rats. J Nutr 127:2260–2266 Fuller R, Perdigon G (2000) Probiotics 3: immunomodulation by the gut microflora and probiotics. Kiuwer Academic Publishers, Dordrecht Gionchetti P, Rizzello F, Venturi A, Campieri M (2000) Probiotics in infective diarrhoea and inflammatory bowel diseases. J Gastroenterol Hepatol 15:489–493 Goldin BR (1990) Intestinal microflora: metabolism of drugs and carcinogens. Ann Med 22:43–48 Gopal P, Prasad J, Gill H (2003) Effects of the consumption of Bifidobacterium lactis HN019 (DR10TM) and galacto-oligosaccharides on the microflora of the gastrointestinal tract in human subjects. Nutr Res 23:1313–1328 Gorbach SL (1982) The intestinalmicroflora and its colon cancer connection. Infection 10:379–384 Gorbach SL, Goldin BR (1990) The intestinal microflora and the colon cancer connection. Rev Infec Dis 12:242–261 Grizard D, Barthomeuf C (1999) Non-digestible oligosaccharides used as peebiotic agents̈mode of production and beneficial effects on animal and human health. Reproduction 39:563–588 Gudiel-Urbano M, Goni I (2002) Effect of edible sesweeds (Undaria pinnatifida and porphyra ternera) on the metabolic activities of intestinal microflora in rats. Nutr Res 22:323–331 123 World J Microbiol Biotechnol (2010) 26:1735–1743 Hambly RJ, Rummey CJ, Cunninghame M, Fletcher JM, Rowland IR (1997) Influence of diets containing high and low risk factors for colon cancer on early stages of carcinogenesis in human floraassociated (HFA) rats. Carcinogenesis 18:1535–1539 Hayashi H, Sakamoto M, Kitahara M, Benno Y (2003) Molecular analysis of fecal microbiota in elderly individuals using 16S rDNA library and T-RFLP. Microbiol Immunol 47:557–570 Ibarz A, Pagan J, Miguelsanz R (1992) Rheology of clarified fruit juices. II: blackcurrant juices. J Food Eng 15:63–73 Johansson G, Holmen A, Persson L (1997) Dietary influence of some proposed risk factors for colon cancer: fecal and urinary mutagenic activity and the activity of some intestinal bacterial enzymes. Cancer Detec Prev 21:258–266 Kulkarni N, Reddy BS (1994) Inhibitory effect of Bifidobacterium longum cultures on the azoxymethane-induced aberrant crypt foci formation and fecal bacterial beta-glucuronidase. Proc Soc Exp Biol Med 207:278–283 Ling WH, Korpela R, Mykkanen H, Salminen S, Hanninen O (1994) Lactobacillus strain GG supplementation decreases colonic hydrolytic and reductive enzyme activities in healthy female adults. J Nutr 124:18–23 Marteau P, Pochart P, Flourie B, Pellier P, Santos L, Desjeux JF, Rambaud JC (1990) Effect of chronic ingestion of a fermented dairy product containing Lactobacillus acidophilus and Bifidobacterium bifidum on metabolic activities of the colon flora in humans. Am J Clin Nutr 52:685–688 Matsumoto H, Takenami E, Iwasaki-Kurashige K, Osada T, Katsumura T, Hamaoka T (2005) Effects of black currant anthocyanins intake on peripheral muscle circulation during typing work in humans. Eur J Appl Physiol 94:36–45 Miller NJ, Rice-Evans CA (1997) Cinnamates and hydroxybenzoates in the diet: antioxidant activity assessed using the ABTS radical cation. Br Food J 99:57–61 Molan AL, Flanagan J, Wei W, Moughan PJ (2009) Seleniumcontaining green tea has higher antioxidant and prebiotic activities than regular green tea. Food Chem 114:829–835 Pietroiusti A, Caprilli R, Giuliano S, Vita S (1985) Fecal pH in colorectal cancer. Int J Gastroenterol 17:88–91 Reddy BS, Mangat S, Weisburger JH, Wynder EL (1977) Effect of high risk diets for colon carcinogenesis on intestinal mucosal and bacterial beta-glucuronidase activity in F344 rats. Cacer Res 37:3533–3536 Rowland IR, Tanaka R (1993) The effects of transgalactosylated oligosaccharides on gut flora metabolism in rats associated with a human faecal microflora. J Appl Bacteriol 74:667–674 Saito Y, Takano Y, Rowland IR (1992) Effects of soybean oligosaccharides on the human gut microflora in in vitro culture. Microb Ecol Health Dis 5:105–110 Skar V, Skar AG, Stromme JH (1998) Beta-glucuronidase activity related to bacterial growth in common bile duct bile in gallstone patients. Scand J Gastroenterol 23:83–90 Takenami EKI, Kurashige H, Matsumoto T, Nagasawa C, Ueda A, Kitamura T, Osada T, Katsumura T, Hamaoka T (2003) Effects of black currant anthocyanins intake on peripheral circulation during typing workload in humans. J Jap Coll Angiol 43:331–334 Takenami EKI, Kurashige H, Matsumoto T, Honma T, Osada M, Okubo T, Hamaoka T (2004) Improvement of cold water immersion-induced peripheral circulation impairment by black currant extract intake—the investigation on cold constitutional women. Biomed Thermol 23:194–202 Tannock GW, Munro K, Harmsen HJ, Welling GW, Smart J, Gopal PK (2000) Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20. Appl Environ Microbiol 66:2578–2588 Van de Wiele T, Boon N, Possemiers S, Jacobs H, Verstraete W (2004) Prebiotic effects of chicory inulin in the simulator of the World J Microbiol Biotechnol (2010) 26:1735–1743 human intestinal microbial ecosystem. FEMS Microbiol Ecol 51:143–154 Walaszek Z, Hanausek-Walaszek M, Minton JP, Webb TE (1986) Dietary glucarate as anti-promoter of 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis. Carcinogenesis 7:1463–1466 1743 Werlein HD, Kutemeyer C, Schatton G, Hubbermann EM, Schwarz K (2005) Influence of elderberry and blackcurrant concentrates on the growth of microorganisms. Food Control 16:729–733 Yamamoto T, Juneja LR, Chu DC, Kim M (1997) Chemistry and applications of green tea. CRC press LLC, Boca Raton 123