Effects of Lactobacillus acidophilus on gut microflora metabolic biomarkers in fed and fasted rats

Clinical Nutrition 28 (2009) 318–324

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Clinical Nutrition journal homepage: http://intl.elsevierhealth.com/journals/clnu

Original Article

Effects of Lactobacillus acidophilus on gut microflora metabolic biomarkers in fed and fasted rats Konstantinos C. Mountzouris a, *, Katerina Kotzampassi b, Panagiotis Tsirtsikos a, Konstantinos Kapoutzis b, Konstantinos Fegeros a a b

Department of Nutritional Physiology and Feeding, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece Department of Surgery, Faculty of Medicine, University of Thessaloniki, Greece

a r t i c l e i n f o

s u m m a r y

Article history: Received 28 May 2008 Received in revised form 22 December 2008 Accepted 16 January 2009

Background & aims: Little is known about fasting effects on gut bacterial metabolism. As probiotics are purported to be beneficial for health, this study aimed to investigate the response of gut microbial metabolism on fasting with or without probiotic administration. Methods: Sixty male adult Wistar rats were allocated to six experimental treatments, for 6 days, arranged under three nutritional schemes namely: (a) ad libitum feeding (control), (b) fasting for 3 days and refeeding for the remainder (re-fed) and (c) fasting for 6 days combined with parenteral liquid treatment during the last 3 days (starved). Each nutritional scheme had one non-probiotic and one probiotic treatment receiving orally Lactobacillus acidophilus. Rat caecal digesta were analyzed for bacterial enzyme activities and volatile fatty acids (VFA). Results: Fasted rats had significantly lower activities of a-galactosidase, a-glucosidase and b-glucosidase and higher activities of b-galactosidase and azoreductase compared to control and re-fed rats, irrespective of probiotic administration. Results were variable regarding cholylglycine hydrolase (CGH), while there were no differences between treatments regarding b-glucuronidase and arylsulfatase activity. Fasted rats had significantly lower caecal VFA concentration and different fermentation patterns. L. acidophilus resulted in significantly reduced azoreductase activity and increased caecal acetate levels in fasted rats. Re-feeding appeared to restore most enzyme activities, fermentation intensity and to some extent fermentation patterns at control treatment levels. L. acidophilus resulted in significantly reduced CGH activity and increased butyrate levels in re-fed rats. Conclusion: The results indicate a health beneficial potential of L. acidophilus in fasted and re-fed nutritional states via reduction of harmful azoreductase and CGH activities and promotion of useful VFA components for colonic function and health. Ó 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Keywords: Probiotics Rats Fasting Microflora Enzymes Fermentation

1. Introduction Studies with germ-free and conventional animals highlight the significant role of gastrointestinal (GI) microflora on host nutrition and health.1–3 Diet is ranked among the key factors that affect the composition and metabolic activities of GI microflora in humans and animals.4–6 In clinical practice fasting is a commonly recommended prescription for examinations and perioperative procedures. Besides the obvious negative impact of fasting on host nutrition, fasting is associated with decreased gut epithelial cell proliferation,

* Corresponding author. Tel.: þ30 2105294422; fax: þ30 2105294413. E-mail address: [email protected] (K.C. Mountzouris).

increased apoptosis, mucosa atrophy7–9 and even impairment of gut barrier function.9 As a result of food restriction, fasting could have a significant effect on the quantity and quality of available substrate for fermentation by GI microflora. Gut bacteria possess a highly efficient enzymatic machinery comprising inducible and repressible hydrolytic and reductive enzymes that enable them to metabolise numerous substrates from diet and endogenous secretions. It is well known that, depending on the substrate being metabolised, gut bacterial metabolism can have protective effects as well as toxic consequences for the host.10–12 Therefore, fasting would be a critical determinant of the intensity and the direction of gut bacterial metabolism. Probiotics have been defined as live microorganisms which, when administered in adequate amounts, confer a health benefit

0261-5614/$ – see front matter Ó 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2009.01.009

K.C. Mountzouris et al. / Clinical Nutrition 28 (2009) 318–324

for the host.13 Probiotics are thought to exert their beneficial effects on the host via an array of mechanisms that: (a) improve host nutrition via enhanced digestion, energy salvage and production of vitamins (b) enhance gut barrier function via competition with pathogens for nutrients and adhesion receptors on the intestinal mucosa, anti-enterotoxic activity and stimulation of host immunity.5,14,15 In addition, probiotics stimulate gut epithelial cell proliferation,16 enhance the recovery of nutritional status and lessen gut mucosal atrophy after fasting in animal studies.17 It, therefore, seems possible that probiotics could help ameliorate the deleterious potential of fasting for host health in numerous ways. In this sense, the modulation of gut bacterial metabolism in a manner beneficial for host health could be a very interesting case. So far, a limited number of studies have looked into the effects of nutrition on gut bacterial metabolism and have examined an array of gut bacterial enzymes, some of which are considered beneficial (e.g., a-galactosidase, a-glucosidase, b-galactosidase), while others genotoxic (e.g., b-glucuronidase, azoreductase, nitroreductase) and detrimental for host nutrition and health.4,10–12,18–20 However, the effects of fasting, with or without probiotic administration, on gut bacterial metabolism have been largely unexamined so far. An understanding of gut bacterial metabolism patterns via the fingerprinting of relevant biomarkers could be deemed beneficial for devising appropriate nutritional and therapeutic schemes in clinical practice. In this context, the activity of certain gut bacterial enzymes with beneficial or harmful outcomes for host health, as well as the profile of volatile fatty acids, ranked among the major end products of bacterial fermentation, represent interesting target parameters to study. Therefore, the aim of this work was to investigate the response of gut microbial metabolism to fasting with and without probiotic administration. Lactobacillus acidophilus was considered a good candidate probiotic as it is found in many probiotic products and has a body of health supporting evidence.18,21,22 In particular, L. acidophilus DDS-1 was chosen on the basis of it’s nutritional, prophylactic and antibiotic like properties claimed by the manufacturing company (Nebraska Cultures Inc., USA). A rat animal model was used in order to approximate the fed and fasted nutritional states in clinical practice. Eight bacterial enzyme activities and the concentration of volatile fatty acids were determined in the caecum as biomarkers of gut microflora metabolic activity. 2. Materials and methods 2.1. Animals and experimental treatments Sixty male Wistar rats weighing 417  11.4 g were included in this study. The experiment was performed at the Surgical Research Laboratory of the AHEPA University Hospital. The experimental protocol was approved by the Department of Animal Care and Use Committee of the Greek Ministry of Agriculture and adhered to the European Community Guiding Principles for the Care and Use of Animals. Rats were individually caged in Plexiglas cages with wire drop bottoms and no bedding. All animals were housed in stable laboratory conditions with a 12 h dark/light cycle and free access to standard rat chow (SRC) during a 7 day pre-experimental feeding and environmental adaptation period. Water was available ad libitum throughout the experiment. At the end of the pre-experimental period, the rats were re-weighted and were randomly allocated to six experimental treatments of 10 animals (replicates) each, for 6 days, according to the three nutritional schemes summarised in Table 1. In particular, 20 rats were allocated to two experimental groups that served as controls and were fed for six consecutive days with SRC (CON) or with SRC plus L. acidophilus (CONLa). Another set of 20

319

rats were allocated to the experimental groups RC and RCLa, that were subjected to 3 days starvation followed by 3 days re-feeding SRC or to re-feeding SRC plus L. acidophilus administration during the experiment, respectively. Finally, the last set of 20 rats were allocated to experimental treatments PA and PALa, that were subjected to 6 days starvation combined with 3 days parenteral liquid treatment with Lactated Ringer’s solution or to parenteral liquid treatment plus L. acidophilus administration during the experiment, respectively. The commercial rat chow used comprised a balanced extruded natural ingredient diet (510 K, ELVIZ SA, Greece) that met and exceeded the NRC nutrient requirements for rat maintenance and growth.23 The lyophilised probiotic culture L. acidophilus DDS-1 (Nebraska Cultures Inc., USA) was used in this study. The product was determined to have a viability of 2.2  1010 CFU/g upon culture on MRS agar (Oxoid, Basingstoke, UK) in our laboratory, which was in line with the manufacturer’s claim of 1010 CFU/g. The probiotic solution was prepared fresh daily by aseptically dissolving the product in sterile normal saline (0.9% w/v) to a final concentration of 2.2  109 CFU/ml and was administered at mid-day at a volume of 1 ml per rat by gavage (intragastric). At the end of the experiment all animals were re-weighted and the body weight changes during the experiment were determined. 2.2. Tissue sampling On the morning of the 7th day the rats were anesthetised by injecting ketamine [50 mg/kg body weight] plus xylazine [10 mg/ kg body weight] and subjected to laparatomy under sterile conditions. A midline abdominal incision was performed through which the entire digestive tract from the duodenum up to peritoneal reflexion was isolated free from mesenterium and mesocolon, respectively, with care taken to minimise manipulation. The duodenum, jejunum (10 cm from the Treitz ligament), terminal ileum (10 cm from ileocecal junction), caecum and large intestine were ligated twice with 4/0 silk (Ethicon, Edinburgh, UK) and divided between ligatures. Immediately thereafter segments were snap frozen in liquid nitrogen and the animals sacrificed. Samples were subsequently stored at 80  C until all subsequent analyses. 2.2.1. Analysis of microbial metabolic biomarkers Microbial metabolic biomarkers were determined in caecal digesta. Deep frozen caeca were thawed and digesta contents were aseptically diluted 10-fold (i.e., 10% w/v) with sterile ice cold anoxic phosphate buffered saline (0.1 M PBS; pH 6.5) and subsequently homogenized for 3 min in a stomacher. Aliquots of caecal digesta homogenates were then centrifuged at 12,000 g for 10 min at 4  C. Supernatants were immediately stored at 80  C, while bacterial stock cell suspensions were made by re-dissolving the precipitated cells in PBS:glycerol (3:2) in order to protect them from the low storage temperature until subsequent analyses. 2.3. Determination of caecal microbial enzyme activities Microbial glycolytic activities of a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, and b-glucuronidase were determined in the caecal digesta homogenate supernatants through the rate of release of p-nitrophenol (pNp) from the respective p-nitrophenyl-substrates (Applichem, Darmstadt, Germany) namely a-galactoside (1 mM), b-galactoside (2 mM), a-glucoside (1 mM), b-glucoside (1 mM), and b-glucuronide (1 mM) prepared in sterile PBS (0.1 M, pH 6.5), via absorbance measurement at 405 nm as has been described by Mountzouris et al.24 All bacterial glycolytic enzyme activities were calculated

K.C. Mountzouris et al. / Clinical Nutrition 28 (2009) 318–324

Table 1 Nutritional schemes followed during the experiment. Nutrition scheme

Treatments CON CONLaa RC RCLaa PA PALaa

Ad libitum feeding during the 6 days experiment Fasting for 3 days and re-feeding ad libitum for the remaining 3 days Fasting for 6 days combined with parenteral liquid treatment with Lactated Ringer’s solution during the last three experimental days a

þ

þ

–

–

–

–

–

–

þ þ

–

–

–

–

–

þ þ

–

L. acidophilus oral administration at 9.3 log CFU/rat/day for 6 days.

using a standard curve for pNp and were expressed as mmol pNp released/min/g caecal protein. Arylsulfatase activity was determined through the rate of release of pNp from p-nitrophenyl-sulfate (Applichem, Darmstadt, Germany) according to a method adapted from McBain and MacFarlane.12 A 100 ml aliquot of cecal bacterial stock cell suspension, diluted 20-fold, was mixed with 400 ml of deoxygenized 4-nitrophenyl-sylfate (2 mM) in anoxic PBS (0.1 M, pH 6.5) and incubated for 1 h at 39  C under anaerobic conditions (N2:CO2 95%:5%). The reaction was stopped by the addition of ice cold 1 M Na2CO3. Subsequently, the samples were centrifuged (10,000 g for 10 min) to sediment the cells and the concentration of pNp released was determined in the supernatants via absorbance measurement at 405 nm. Arylsulfatase activity was determined using a pNp standard curve and was expressed as mmol pNp released per minute per g dry cell weight. The activity of cholylglycine hydrolase (CGH) was determined through the rate of cholate production from its glycine conjugate sodium glycocholate. A 100 ml aliquot of cecal digesta homogenate supernatant was added to 100 ml sodium glycocholate (Applichem Darmstadt, Germany) in PBS (1.6 mM, pH 7.0) and the reaction mix was incubated at 39  C for 5 h. Then, the reaction was stopped with the addition of 100 ml trichloroacetate (TCA) 1.2 M followed by addition of 200 ml acetonitrile (ACN). For the detection of free and glycine conjugated cholate, a reverse phase HPLC method using a 2.1  200 mm, 5 mm Hypersil ODS column (Agilent Technologies, Wilmington, US) with gradient elution using mixtures of water and ACN and fluorescence detection was used.25 The fluorogenic reagent 4-bromomethyl-7-methoxy-coumarin (Sigma–Aldrich, St. Louis, USA) was used for pre-column derivatisation of free and glycine conjugated cholate, according to the method of Abushufa et al.26 Ursodeoxycholic acid (Sigma–Aldrich, St. Louis, USA) was used as an internal standard. CGH specific activity was determined using a standard curve for sodium cholate and was expressed as mmol sodium cholate produced per minute per g caecal protein. Azoreductase activity was determined by measuring the reduction rate of amaranth (Sigma–Aldrich, St. Louis, USA) used as a chromogenic substrate, according to a method adapted from Rafii et al.27 and Russ et al.28 for amaranth. A 100 ml aliquot of caecal digesta homogenate supernatant was added to 400 ml of amaranth substrate (100 mM) in anoxic PBS (0.1 M, pH 6.5) including 5 ml FAD (20 mg/ml) and 5 ml NADPH (20 mg/ml). The reaction mix was incubated at 39  C for 8 h under anaerobic conditions (N2:CO2 95%:5%). Subsequently, the reaction was stopped by adding 1000 ml of 10% trichloroacetic acid (TCA) solution. The reduction in amaranth concentration was determined spectrophotometrically at 525 nm. Azoreductase specific activity was calculated using a standard curve for amaranth concentration and was expressed as mmol amaranth reduced per minute per g caecal protein. Protein concentration in cecal digesta was determined using the BCA assay as previously described.24

2.3.1. Volatile fatty acids (VFA) concentration Caecal digesta VFA concentrations were determined in the supernatants of homogenates by capillary gas chromatography using a PerkinElmer Autosystem XL gas chromatograph equipped with a 30 m  0.25 mm i.d. Nukol column and a flame ionization detector as described by Mountzouris et al.24 2.4. Statistical analysis Experimental data from 10 rats per treatment regarding body weight change and eight rats per treatment regarding microbial enzyme activities and VFA were analyzed by the ANOVA procedure using the SPSS for Windows statistical package program, version 8.0.0 (SPSS Inc., Chicago, IL). Statistically significant effects were further analyzed and means were compared using the Bonferroni post hoc multiple comparisons test. Statistical significance was determined at P  0.05. The results were expressed as means  SEM. 3. Results 3.1. Body weight change The changes in the body weight of rats per experimental treatment are shown in Fig. 1. The starved rats in treatments PA and PALa had a significant body weight loss (i.e., 15 and 16 g, respectively) during the experiment compared with the rats in treatments CON, CONLa, RC and RCLa that increased their body weight. There were no significant differences in body weight change between treatments CON, CONLa, RC and RCLa. 3.2. Cecal microbial enzyme activities The specific activities of the bacterial enzymes determined in the rats’ caecal digesta are shown in Figs. 2 and 3. The activity of a-galactosidase in treatments CON and CONLa was significantly higher than the respective activity in the starved rats of treatment PALa with the rest of the treatments being intermediate. Significantly higher b-galactosidase activity was found in the caecal digesta of rats in treatments PALa and PA compared to the other four treatments that did not differ between them. On the contrary, treatments PA and PALa had the lowest aglucosidase activity compared to the other four treatments. Similarly, rats in treatments PA and PALa had the lowest b-glucosidase activity compared to the other treatments, but in this case rats in

20

Body weight change (g)

320

15 10

b

b

CON

CON La

b

b

5 0 -5

RC

RCLa

PA

PALa

a

a

-10 -15 -20

Treatments Fig. 1. Rat body weight change during the experiment. Bars represent means for 10 rats per treatment  SEM. Bars with different superscripts (a, b) differ significantly (P  0.05).

K.C. Mountzouris et al. / Clinical Nutrition 28 (2009) 318–324

120

CON

CONLa

RC

RCLa

c

110

Enzyme activity (µmol pNp released/min/g caecal protein)

321

100

PA

PALa

c

90 80 70

b b ab ab

b

60

b

b

ab

50 40

b b

b b

a

30

ab

a a

20

a a

a

10 0

α-GAL

β-GAL

a

aa

α-GLU

β-GLU

β-GLN

Bacterial enzymes Fig. 2. Bacterial glycolytic enzyme activities (a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase and b-glucuronidase) in rat caecal digesta. Bars represent means for eight rats per treatment  SEM. Within each enzyme activity bars with different superscripts (a, b, c) differ significantly (P  0.05).

20 18

I ab

ab

b

ab

ab

16

a

14 12 10 8 6 4 2 0

CON

CONLa

RC

RCLa

PA

PALa

of the treatments were intermediate and not different from the treatments above. There were no statistically significant differences between treatments regarding arylsulfatase activity (Fig. 3). Regarding CGH activity, this was significantly higher in rats belonging to the re-feeding scheme of treatment RC compared to treatments RCLa, CON CONLa and PA with the CGH activity in the

Arylsulfatase activity (μmol pNp released/min/dry cell weight)

Caecal protein concentration (mg / g caecal digesta)

control treatments CON and CONLa had the highest activity compared to the rest. There were no statistically significant differences between treatments regarding b-glucuronidase activity (Fig. 2). Protein concentration in the caecal digesta of rats in treatment RC was significantly lower compared to treatment PA, while the rest

II

70 60 50 40 30 20 10 0

CON

CONLa

III

3,5 c

3,0 2,5 2,0

bc ab

ab

b

1,5 a

1,0 0,5 0,0

CON CONLa

RC

RCLa

treatments

RC

RCLa

PA

PALa

Treatments

PA

PALa

Azoreductase activity (μmol amaranth reduced/min/g caecal protein)

Cholyglycine hydrolase activity (μmol sodium cholate produced/min/g caecal protein)

Treatments

IV 180

c

160 140

b

120 100 80

ab

ab

ab a

60 40 20 0

CON

CONLa

RC

RCLa

PA

PALa

Treatments

Fig. 3. Caecal protein concentration (I) and bacterial enzyme activities in rat caecal digesta: (II) arylsulfatase, (III) cholylglycine hydrolase and (IV) azoreductase. Bars represent means for eight rats per treatment  SEM. Within each enzyme activity bars with different superscripts (a, b, c) differ significantly (P  0.05).

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latter being the lowest. CGH activity in treatment PALa was intermediate between RC and treatments RCLa, CON and CONLa. Finally, azoreductase activity was significantly higher in treatment PA compared to treatment PALa and the other four treatments. Treatments RC, CON and CONLa had intermediate azoreductase activity between treatment RCLa (i.e., lowest) and PALa (Fig. 3). 3.3. Volatile fatty acids (VFA) concentration The concentration and molar ratios of VFA in the rat caecal digesta is shown in Table 2. Total VFA concentration in the cecal digesta of rats in the control treatments CON and CONLa, as well as in the treatments with re-feeding RC and RCLa was significantly higher compared to treatments PA and PALa. Generally, for treatments belonging to similar nutritional scheme (Table 1) there were no differences in caecal VFA concentration between probiotic and non-probiotic administration. Significant differences between treatments were found for all the VFA molar ratios (MR%) determined. In particular, treatments RC and RCLa had the lowest acetate MR% compared to the other four treatments. Treatment PALa had the highest acetate MR followed by treatments PA, CON and CONLa that were not different from PALa on the contrary, propionic MR% was significantly higher in treatments RC and RCLa, followed by treatments CON and CONLa and then by treatments PA and PALa that were the lowest. The MR% of butyric acid was significantly higher in treatment RCLa compared to treatments RC, PA and PALa. Treatments CONLa and CON had intermediate butyrate MR% between treatments RC and RCLa and were significantly higher compared to treatments PA and PALa. Generally, the MR% of isobutyric, valeric, isovaleric and caproic acid as well as the collective b-VFA and o-VFA were significantly higher in treatments PA and PALa compared to the rest of treatments with several differences existing in variable patterns as shown in detail in Table 2. 4. Discussion In this work, male adult rats were arranged in six treatments in order to study the effects of fasting on elements of gut microbial metabolism. Based on feed provision mode, treatments were arranged in three nutritional schemes such that for each nutritional scheme there was one treatment with and one treatment without oral probiotic administration. L. acidophilus was chosen as it is a well known probiotic found in many probiotic products and has a significant body of supporting research on topics such as the

beneficial modulation of intestinal bacterial metabolic activity as well as on prevention of antibiotic associated diarrhoea, preservation of intestinal integrity during radiotherapy, stimulation of systemic immune response, increase in iron bioavailability, production of antimicrobial substances and reduction in bacterial vaginosis.18,21,22 It is well known that fasting results in body weight loss and as a result rats in starved groups PA and PALa significantly reduced their body weight due to feed deprivation compared to the other four treatments. In addition, re-feeding in treatments RC and RCLa resulted in body weight gain levels not significantly lower from the respective control treatments CON and CONLa. Probiotic administration had no effect in body weight change irrespective of the nutritional scheme followed. The changes in caecal microflora metabolic biomarkers among the treatments studied in this work, support the view that the gut microflora responds dynamically to dietary changes. Scientific evidence suggests that changes in the metabolic activity in the GI microflora can occur without appreciable changes in the actual numbers or types of organisms in the gut.4,24 Generally, irrespective of probiotic administration, a reduction in the specific activity of a-galactosidase, a-glucosidase and b-glucosidase could be seen in the cecal digesta of starved rats (PA and PALa) compared to the other treatments. This could be explained by the fact that fasting results in depletion of dietary carbohydrates that, in the gut, serve as substrates for the glycolytic enzymes above. For example, a-galactosidase contributes to the hydrolysis of dietary a-galactosides, such as rafinose, stachyose and other oligosaccharide components of food and feedstuffs, such as soybean meal, a-glucosidase contributes to starch fermentation19 and b-glucosidase contributes to the hydrolysis of glucose monomers from nonstarch polysaccharides (e.g., cellulose, b-glucans). While bacterial a-galactosidase and a-glucosidase could be considered as beneficial enzymes for host nutrition,19,24 things are not as clear for b-glucosidase since, depending on the nature of plant glycosides, a harmful element of b-glucosidase activity for the host could arise from the formation of toxic aglycons.29 Contrary to the three enzymes above, starved rats (PA and PALa) had significantly higher activities of b-galactosidase and azoreductase compared to the other treatments. Although b-galactosidase is also a glycolytic enzyme involved in the hydrolysis of dietary galactosides such as lactose and some prebiotics,19,24 its significantly higher activity in the starved rats is more likely to be explained by the fact that this enzyme belongs to microbial glycosidases that

Table 2 Concentration of volatile fatty acids (mmol/kg wet digesta) and their respective molar ratios (%) in the caecal digesta of fed and fasted rats. Parameter

Total VFAa Acetic (%) Propionic (%) Butyric (%) Isobutyric (%) Valeric Iso-valeric Caproic b-VFAb o-VFAc

Treatments

Statistics

CON

CONLa

RC

RCLa

PA

PALa

P

77.6B  5.17 59.3B  0.90 23.2B  0.60 11.0B,C  0.43 0.4A  0.07 2.5A  0.09 0.5A  0.03 3.2B  0.20 0.9A  0.09 5.7A,B  0.29

75.8B  4.78 59.2B  1.18 22.2B  1.06 11.2B,C  0.62 0.6A,B  0.06 2.9A  0.12 0.6A  0.05 3.2B  0.24 1.3A  0.10 6.1B  0.33

74.6B  6.75 55.5A  0.64 26.7C  0.73 10.6B  0.24 1.2A,B  0.56 2.8A  0.23 0.6A  0.03 2.7A,B  0.38 1.8A  0.54 5.5A,B  0.63

77.9B  6.12 55.5A  0.74 26.3C  0.85 12.9C  0.42 0.5A,B  0.06 2.4A  0.11 0.5A  0.05 1.9A  0.15 1.1A  0.10 4.3A  0.26

21.7A  0.63 59.7B  1.50 16.9A  0.49 5.7A  0.28 1.6B  0.15 6.9C  0.19 3.0C  0.12 6.0C  0.26 4.6B  0.25 13.0D  0.44

27.4A  1.18 62.3B,C  0.94 15.6A  0.33 7.0A  0.29 1.5B  0.14 6.1B  0.19 2.5B  0.19 4.9C  0.23 4.1B  0.32 11.0C  0.39

0.000 0.000 0.000 0.000 0.002 0.000 0.000 0.000 0.000 0.000

Data values represent treatment means  their respective standard errors. Within the same raw means with different superscripts (A,B, C, D) differ significantly (P  0.05). Treatment explanations as follows: CON: ad libitum feeding for six consecutive days; CONLa: as CON þ L. acidophilus 9.3 log CFU/day; RC: 3 days starvation followed by 3 days ad libitum re-feeding; RCLa: as RC þ L. acidophilus 9.3 log CFU/day throughout; PA: 3 days starvation followed by 3 days parenteral liquid treatment with Lactated Ringer’s solution; PALa: as PA þ L. acidophilus 9.3 log CFU/day throughout. a Total VFA ¼ acetic þ propionic þ butyric þ branched VFA þ other VFA. b Branched VFA ¼ isobutyric þ isovaleric. c Other VFA ¼ valeric þ caproic.

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participate in the degradation of oligosaccharide moieties of gut mucin glycoproteins.30 It is known that in the absence of fermentable carbohydrates, as in the case of fasting, certain gut microbes tend to direct their glycolytic activities towards the oligosaccharide side chains of gut mucins, which, in this case, play an important nutritional support role for gut bacteria.30,31 The activity of b-glucuronidase is perceived as harmful for health as it is able to release carcinogens from hepatically derived glucuronic acid conjugates and is a critical factor in the enterohepatic circulation of drugs and other foreign compounds.5 Despite the fact that probiotics have been reported to reduce b-glucuronidase activity in the gut,20,32 in this study L. acidophilus administration did not result in lowering b-glucuronidase activity, irrespective of the nutritional state of the animals. Fasting appears to have had no adverse effect on b-glucuronidase activity. Arylsulfatase is a cell bound enzyme produced by gut bacteria that is involved in the desulfation of bile acid sulfates, compounds produced by the liver in order to detoxify toxic secondary bile acids.33 Therefore, microbial desulfation could be ranked as potentially harmful for health, since it promotes the enterohepatic circulation of toxic secondary bile acids. However in this study L. acidophilus administration did not have any beneficial lowering effect of arylsulfatase activity either. Cholylglycine hydrolase (CGH) is another important gut microbial enzyme involved in bile acid metabolism. In particular, CGH catalyses the deconjugation of the glycine and taurine conjugates of primary bile acids, a prerequisite for the action of another microbial enzyme named 7a-dehydroxylase to produce the unconjugated cocarcinogenic secondary bile acids deoxycholic and lithocholic acid.34 In this study, CGH activity was not significantly different between treatments receiving probiotic (i.e., CONLa, RCLa and PALa), while it varied significantly among the three non-probiotic treatments (i.e., CON, RC and PA). L. acidophilus administration in treatment PALa resulted in higher CGH activity, compared to the respective starved non-probiotic treatment (PA). On the contrary, upon re-feeding L. acidophilus administration resulted in beneficially lowering CGH activity (RCLa vs RC). CGH activity is expressed by many gut anaerobic bacteria,34 including many species belonging to the genus Lactobacillus, such as L. acidophilus.35,36 Therefore, it could be justified that a higher CGH activity as a result of L. acidophilus administration would be more readily identified in starved animals with an empty gut compared to fed animals. In addition, it is important to note that lactobacilli and bifidobacteria do not posses the harmful 7a-dehydroxylase activity that could act synergistically with CGH to produce toxic secondary bile acids.37 It is known that certain gut anaerobic bacteria belonging to genera such as Clostridium, Bacteroides, Eubacterium and Butyrivibrio express harmful azoreductase activity that catalyses the reduction of azo dye compounds to carcinogenic aromatic amines.27 In this study, it is not clear why fasted rats displayed higher azoreductase activity compared to fed and to starved re-fed treatments, since in treatments PA and PALa there was no case of dietary intake of azo dye compounds. Perhaps, it could be speculated that this enzymatic activity could be associated with an overall increased nitrogen metabolism (e.g., proteolysis) evidenced by lower VFA concentration and the higher molar ratio of branched VFA in the caecum of fasted rats. Lactobacilli are purported to act in a manner beneficial for health through their contribution for the reduction of azoreductase activity in the gut.20,21 This was also confirmed in this study, since L. acidophilus administration in starved rats did significantly reduce harmful azoreductase activity (PALa vs PA). In most cases re-feeding after 3 days of starvation restored microbial enzyme activities to the respective control levels. As expected, fasting severely affected the fermentation intensity in the caecum due to depletion of available fermentable dietary

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substrates. In addition, since VFA concentration in the gut is the outcome of production and absorption processes,10 the significantly lower VFA concentration in the caecal digesta of the starved rats compared to the control and re-fed ones could also be due to a higher host absorption rate in the latter in order to meet increasing energy requirements. The depletion of fermentable carbohydrates was also evidenced by the increased molar ratios of isobutyric, isovaleric and collectively branched VFA determined in the starved rats, as it is known that their production is concomitant with an increase in proteolysis.10 The lower molar ratios for propionic and butyrate determined in starved rats compared to the other treatments could be explained by their higher uptake by colonocytes in order to meet their energy needs. Butyrate, in particular, is known to serve as the preferable fuel source for the colonic epithelium.10,38 Treatment PALa had the higher acetate molar ratio compared to the other treatments, which could be due to the fact that the metabolic stimulation of L. acidophilus could be more readily identified in starved animals with an empty gut compared to fed animals. Starved rats in treatments PALa and PA also had significantly higher molar ratios of valeric, caproic and the collective o-VFA compared to the other treatments, indicating that fasting results in different fermentation profiles as a result of the higher contribution that endogenous substrates such as gut mucin glycoproteins make to the gut fermentation process.30 In conclusion, fasting results in significant changes in the activities of caecal bacterial a-galactosidase, a-glucosidase, bglucosidase, b-galactosidase, cholylglycine hydrolase and azoreductase. In particular, enzyme activities appeared to adapt according to substrate availability in the caecum. Due to depletion of fermentable dietary substrates, starved rats had significantly lower caecal VFA concentration compared to the control and re-fed ones. Re-feeding appeared to restore enzyme activities, fermentation intensity and to some extent fermentation patterns at control treatment levels. Generally, L. acidophilus was beneficial in terms of significantly reducing harmful azoreductase activity in fasted rats and cholylglycine hydrolase activity in re-fed rats. In addition, it appears that L. acidophilus contributed to beneficial changes in the caecal ecosystem that directed the fermentation towards VFA, such as acetate (i.e., PALa) and butyrate (i.e., RCLa) that are highly important for colonic epithelium function and health. Clearly, the beneficial potential for host health of L. acidophilus in fasted and refed nutritional states in clinical practice will have to be further assessed by examining additional parameters related to host health, such as intestinal function and physiology. Conflict of interest The authors have no conflict of interests. Acknowledgements The authors would like to thank Dr. M. Shahani from Nebraska Cultures, Inc., USA for the kind provision of L. acidophilus DDS-1 culture and Dr. E. Zoidis for his assistance in the preparation of caecal homogenates. Authors: KCM participated in the conception, design, sample analysis, data analysis and drafted the manuscript, KKot and KKap participated in the conception, design, execution of the animal experiment and revision of the study, PT participated in sample analysis and data analysis and KF participated in design and revision of the study. References 1. Berg RD. The indigenous gastrointestinal microflora. Trends Microbiol 1996;4:430–5.

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2. Falk PG, Hooper LV, Midtvedt T, Gordon JI. Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology. Microbiol Mol Biol Rev 1998;62:1157–70. 3. O’Hara AM, Shanahan F. The gut flora as a forgotten organ. Eur Mol Biol Org 2006;7:688–93. 4. Goldin B, Gorbach SL. Alterations in fecal microflora enzymes related to diet, age, lactobacillus supplements, and dimethylhydrazine. Cancer 1977;40:2421–6. 5. Salminen S, Bouley C, Boutron-Ruault MC, et al. Functional food science and gastrointestinal physiology and function. Br J Nutr 1998;80:S147–71. 6. Snel J, Harmsen HJM, Van der Wielen PWJJ, Williams BA. Dietary strategies to influence the gastrointestinal microflora of young animals and its potential to improve intestinal health. In: Blok MC, Vahl HA, De Lange L, Van de Braak AE, Hemke G, Hessing M, editors. Nutrition and health of the gastrointestinal tract. The Netherlands: Wageningen Academic Publishers; 2002. p. 37–69. 7. Iwakiri R, Gotoh Y, Noda T, et al. Programmed cell death in rat intestine: effect of feeding and fasting. Scand J Gastroenterol 2001;36:39–47. 8. Chappell VL, Thompson MD, Jeschke MG, Chung DH, Thompson JC, Wolf SE. Effects of incremental starvation on gut mucosa. Dig Dis Sci 2003;48:765–9. 9. Ulusoy H, Usul H, Aydin S, et al. Effects of immunonutrition on intestinal mucosal apoptosis, mucosal atrophy, and bacterial translocation in head injured rats. J Clin Neurosci 2003;10:596–601. 10. Cummings JH, Macfarlane GT. The control and consequences of bacterial fermentation in the human colon. J Appl Bacteriol 1991;70:443–59. 11. Hughes R, Rowland IR. Metabolic activities of the gut microflora in relation to cancer. Microb Ecol Health Dis 2000;12:179–85. Suppl. 2. 12. McBain AJ, MacFarlane GT. Modulation of genotoxic enzyme activities by nondigestible oligosaccharide metabolism in in-vitro human gut bacterial ecosystems. J Med Microbiol 2001;50:833–42. 13. FAO/WHO guidelines for the evaluation of probiotics in food. Joint working group report on drafting. London, Ontario, 2002: 1–11. 14. Rastall RA, Gibson GR, Gill HS, et al. Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications. FEMS Microbiol Ecol 2005;52:145–52. 15. Mountzouris KC. Nutritional strategies targeting the beneficial modulation of the intestinal microflora with relevance to food safety. In: McElhatton A, Marshall RJ, editors. Food safety. A practical and case study approach. USA: Springer; 2007. p. 128–47. 16. Ichikawa H, Kuroiwa T, Inagaki A, et al. Probiotic bacteria stimulate gut epithelial cell proliferation in rat. Dig Dis Sci 1999;44:2119–23. 17. Dock DB, Latorraca MQ, Aguilar-Nacimento JE, Gomes-da-Silva MHG. Probiotics enhance recovery from malnutrition and lessen colonic mucosal atrophy after short-term fasting in rats. Nutrition 2004;20:473–6. 18. Goldin BR, Gorbach SL. The effect of milk and Lactobacillus feeding on human intestinal bacterial enzyme activity. Am J Clin Nutr 1984;39:756–61. 19. Djouzi Z, Andrieux C. Compared effects of three oligosaccharides on metabolism of intestinal microflora in rats inoculated with a human faecal flora. Br J Nutr 1997;78:313–24. 20. Haberer P, du Toit M, Dicks LMT, Ahrens F, Holzapfel WH. Effect of potentially probiotic lactobacilli on faecal enzyme activity in minipigs on a high-fat,

21. 22. 23. 24.

25.

26. 27.

28. 29.

30.

31.

32.

33.

34.

35. 36.

37. 38.

high-cholesterol dietda preliminary in vivo trial. Int J Food Microbiol 2003;87:287–91. Lee YK, Nomoto K, Salminen S, Gorbach SL. Handbook of probiotics. USA: John Wiley & Sons, Inc.; 1999. Reid G. Testing the efficacy of probiotics. In: Tannock GW, editor. Probiotics a critical review. Norfolk: Horizon Scientific Press; 1999. p. 129–40. National Research Council. Nutrient requirements of laboratory animals. 4th ed. Washington D.C: The National Academies Press; 1995. p. 11–79. Mountzouris KC, Balaskas C, Fava F, Tuohy KM, Gibson GR, Fegeros K. Profiling of composition and metabolic activities of the colonic microflora of growing pigs fed diets supplemented with prebiotic oligosaccharides. Anaerobe 2006;12:178–85. Gatti R, Cavrini V, Roveri P. 2-Bromoacetyl-6-methoxynaphthalene: a useful fluorescent labeling reagent for HPLC analysis of carboxylic acids. Chromatographia 1992;33:13–8. Abushufa R, Reed P, Weinkove C. Fatty acids in erythrocytes measured by isocratic HPLC. Clin Chem 1994;40:1707–12. Rafii F, Franklin W, Cerniglia CE. Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora. Appl Environ Microbiol 1990;56:2146–51. Russ R, Rau J, Stolz A. The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria. Appl Environ Microbiol 2000;66:1429–34. Pool-Zobel B, Van Loo J, Rowland I, Roberfroid MB. Experimental evidences on the potential of prebiotic fructans to reduce the risk of colon cancer. Br J Nutr 2002;87:S273–81. Hoskins LC, Agustines M, McKee WB, Boulding ET, Kriaris M, Niedermeyer G. Mucin degradation in human colon ecosystems. Isolation and properties of fecal strains that degrade ABH blood group antigens and oligosaccharides from mucin glycoproteins. J Clin Invest 1985;75:944–53. Miller RS, Hoskins LC. Mucin degradation in human colon ecosystems. Fecal population densities of mucin-degrading bacteria estimated by a ‘‘most probable number’’ method. Gastroenterology 1981;81:759–65. Ling WH, Korpela R, Mykkanen H, Salminen S, Hanninen O. Lactobacillus strain GG supplementation decreases colonic hydrolytic and reductive enzymes activities in healthy female adults. J Nutr 1994;124:18–23. Huijghebaert SM, Mertens JA, Eyssen HJ. Isolation of a bile salt sulfataseproducing Clostridium strain from rat intestinal microflora. Appl Environ Microbiol 1982;43:185–92. Thomas LA, Veysey MJ, French G, Hylemon PB, Murphy GM, Dowling RH. Bile acid metabolism by fresh human colonic contents: a comparison of caecal versus faecal samples. Gut 2001;49:835–42. Corzo G, Gilliland SE. Bile salt hydrolase activity of three strains of Lactobacillus acidophilus. J Dairy Sci 1999;82:472–80. Liong MT, Shah NP. Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains. Int Dairy J 2005;15:391–8. Takahashi T, Morotomi M. Absence of cholic acid 7a-dehydroxylase activity in the strains of Lactobacillus and Bifidobacterium. J Dairy Sci 1994;77:3275–86. Scheppach W. Effects of short chain fatty acids on gut morphology and function. Gut 1994;S1:S35–8.