Effects of Cocoa Husk Feeding on the Composition of Swine

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Effects of cocoa husks feeding on the composition of swine intestinal microbiota Damiano Magistrelli, Raffaella Zanchi, Luca Malagutti, Gianluca Galassi, Enrica Canzi, and Fabia Rosi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05732 • Publication Date (Web): 13 Feb 2016 Downloaded from http://pubs.acs.org on February 18, 2016

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Journal of Agricultural and Food Chemistry

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Effects of cocoa husks feeding on the composition of swine intestinal microbiota

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Damiano Magistrelli#, Raffaella Zanchi§, Luca Malagutti#, Gianluca Galassi#, Enrica Canzi§ and

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Fabia Rosi*#,

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#

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20133 Milan, Italy

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§

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2 – 20133 Milan, Italy

Department of Agricultural and Environmental Sciences, University of Milan, via G. Celoria, 2 –

Department of Food, Environmental and Nutritional Sciences, University of Milan, via G. Celoria,

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Corresponding author * Phone: +39 02 50316443. Fax: +39 02 50316434. e-mail: [email protected]

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Funding

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There was no specific funding for this work, so all the authors declare no competing financial

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interest.

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ACS Paragon Plus Environment

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ABSTRACT

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A two-diets/two-periods change over experiment was performed in order to investigate the effects

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of cocoa husks, as source of dietary fibre and polyphenols, on pig intestinal microbial composition.

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Six pigs were fed a conventional cereal-based diet or a diet obtained by substitution of 7.5% of the

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conventional diet with cocoa husks, for 3 weeks. Experimental diets were isoproteic and

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isoenergetic. At the end of each 3-week testing period, samples of fresh feces were collected and

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analyzed for microbial composition by fluorescence in situ hybridization. Cocoa husks did not

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affect feed intake, weight gain and feed efficiency. Analysis of fecal microbial populations, grouped

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by phyla, showed a decrease of Firmicutes and an increase of Bacteroidetes in cocoa husks-fed

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pigs. Particularly, cocoa husks reduced fecal populations of the Lactobacillus-Enterococcus group

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and Clostridium histolyticum and increased the Bacteroides-Prevotella group and Faecalibacterium

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prausnitzii, suggesting a potential for cocoa husks in the improvement of intestinal microbial

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balance.

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Keywords: cocoa husks, polyphenols, pigs, intestinal microbiota.

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INTRODUCTION

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The intestinal microbial ecosystem is made by a wide array of bacterial species, which develop

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important metabolic and immune functions, with a marked effect on health status of the host.1 Some

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dietary components, such as fibre (in particular oligosaccharides) have been shown to influence the

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balance among intestinal microbial populations.2 Beside fibre, other chemicals, such as polyphenols,

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can influence the composition of intestinal microbiota.1,3 Some dietary polyphenols resist to gastric

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digestion, are not absorbed by the small intestine and accumulate in the large intestine, where they

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can modify microbiota composition and/or its activity.4,5 Moreover, some dietary polyphenols can

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be converted by the colonic microbiota to bioactive compounds that may exert physiological effects,

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including also anti-inflammatory activity.4,6 The effects of tea polyphenols on intestinal microbes

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has been proven both in vivo and in vitro.6,7 In particular, the growth of pathogenic bacteria, such as

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Clostridium perfringens and Clostridium difficile is significantly repressed by tea polyphenols and

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their derivatives, while commensal anaerobes and potential probiotics, such as Bifidobacterium spp.

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and Lactobacillus spp., are less severely affected.6 Recently, some wastes from crops and agro-

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industry have been used as feed additives, because of their polyphenolic content. It is the case of

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cocoa husks. Cocoa husks are the integuments of the beans of Theobroma cacao L., a plant tree

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originated in the rain forests of America, whose culture has extended to equatorial areas of Africa

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and Asia.8 Cocoa beans are used worldwide for chocolate production. After the harvest, cocoa

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beans are fermented, dried, roasted, packaged and sent to industrialized countries where the seeds

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are shelled and husks are removed.9 The shelled seeds are used for the production of chocolate,

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while the husks constitute a by-product to discard, despite they contain 57-72 g of polyphenols per

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kg of dry matter.8,10 Cocoa husks have been used in pig nutrition with good effects on some indices

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of health and welfare and no negative effects on productive performances;10,11 however, the

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influence of cocoa husks feeding on the composition of intestinal microbiota has not been studied,

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although cocoa husks are a rich source of dietary fibre and polyphenols, which have both been

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observed to affect intestinal microbial composition.12 According to this, the present study aimed at ACS Paragon Plus Environment

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analyzing the in vivo effects of cocoa husks feeding on the composition of intestinal microbial

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ecosystem of finishing pigs. Pigs share many more anatomical and physiological similarities with

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humans than other small or large domestic animals do. Consequently, pig models mimic the human

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situation in various ways more accurately than other species.13 Moreover, among farm animals,

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fecal microbiota of pigs exhibit the least difference to that of humans14,15 and the pig may serve as

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attractive non-primate animal model to study the effect of cocoa polyphenols on human microbial

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ecosystem.

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MATERIALS AND METHODS

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Animals and diets

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Six castrated male Landrace x Large White pigs (Sus scrofa domesticus) homogeneous for weight

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(135 ± 1.47 kg; mean ± SE), age (9 months) and genetics (same father) were studied for 7 weeks.

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Animals were allocated into individual cages with slatted floor, in order to remove feces and urines

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and keep the animals clean. Cages were in the same room, under identical natural conditions for

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light (14.5 ± 0.75 h/d), temperature (25.6 ± 3.63 °C) and humidity (58.6 ± 6.99%). Pigs were

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initially fed a conventional commercially available meal based on cereals (Consorzio Agrario di

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Milano e Lodi, Milan, Italy). After one week of adaptation to the environmental conditions, pigs

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were randomly divided into two groups and fed two different diets for two consecutive periods of 3

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weeks, in a change-over designed experiment. The control diet (CTRL) consisted in the

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conventional cereal-based diet, while the test diet (COCOA) was obtained by substitution of 7.5%

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of the conventional diet with cocoa husk meal (Icam S.p.a., Lecco, Italy). Such percentage of cocoa

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husks was considered to have no detrimental effects on dietary palatability and feed intake. Cocoa

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husk meal was accurately mixed with the feedstuff in a screw mixer, so that animals weren’t

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allowed to select the ingredients. The CTRL diet consisted of maize, barley, wheat, soy, wheat bran,

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fat and vitamin/mineral supplements, choline chloride, L-Lys HCl and DL-Met. The CTRL diet was

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formulated using standard nutrient requirements of INRA.16 Pigs were fed twice a day (8:30 am and ACS Paragon Plus Environment

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5:30 pm). Leftovers were removed from the trough and weighed before the administration of the

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diets. Feeding and fresh water were ad libitum. The trial was carried out during July and August, in

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the North of Italy. All the animals used were reared in accordance with European Union guidelines

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(Directive 86/609/CEE) and Italian legislation (DL 116, 1992 and DL 473, 1993) and considered

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clinically healthy by the local sanitary authority, over the entire study period.

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Measures and samples

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During the study period, samples of the experimental diets and cocoa husks were collected for the

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analysis of chemical composition. Moreover, cocoa husks may constitute a potential source of

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pathogens, due to poor hygiene conditions during bean harvesting, fermenting and drying in the

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Countries of origin.17 According to this, the experimental diets and cocoa husks were also subjected

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to microbiological analysis.

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During the study period, individual DM intake and body weight of pigs were recorded daily and

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weekly, respectively, in order to calculate weight gain and feed efficiency (G:F).

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After the one-week adaptation period, at the time of allotment into the experimental groups (t0) and

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at the end of each 3-week testing period (t1 and t2), samples of fresh feces of about 50 g were

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collected directly from the anus of each animal, during the first defecation of the day, and

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introduced in sterile plastic bags. On the same days, rectal temperatures were recorded.

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Analysis of chemical composition of the experimental diets and cocoa husks

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Collected samples of diets and cocoa husks were analyzed for DM by drying at 105 °C until

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constant weight in a forced air oven (AOAC method 945.15)18 and then re-ground through a 1 mm

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screen (Fritsch GmbH - Idar-Oberstein, Germany). After grinding, samples were analyzed for crude

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protein by the macro-Kjeldhal technique (AOAC method 984.13),18 using a 2300 Kjeltec Analyzer

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Unit (FOSS, Hillerød, Denmark). Minerals were determined by incineration at 550 °C for 3 h in a

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muffle furnace (AOAC method 942.05),18 ether extract by extraction with petroleum ether in a ACS Paragon Plus Environment

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Tecator Soxtec 2050 apparatus (FOSS, Hillerød, Denmark) (AOAC method 920.29).18 NDF was

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determined by the method of Mertens,19 with addition of sodium sulphite and α-amylase to the

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neutral detergent solution; ADF and ADL were determined by the method of Van Soest et al.,20

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using the Ankom 200 fibre apparatus (ANKOM Technology Corp., Fairport, NY). Fibrous fractions

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are reported on a mineral-free basis. Samples were analyzed for the content of total polyphenols

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using the Prussian Blue method21 and for gross energy using an IKA 4000 adiabatic bomb

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calorimeter (IKA Werke GmbH K.G., Staufen, Germany).

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Microbiological analysis of the experimental diets and cocoa husks

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To check an eventual microbial contamination of the experimental diets and cocoa husks, viable

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counts of total aerobic bacteria, fecal coliforms and Eumycetes were evaluated, after 3 days of

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aerobic incubation on Difco (Becton, Dickinson and Company, Franklin Lakes, NJ) Plate Count

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agar at 28 °C, on Difco Mac Conkey agar at 37 °C, and on Difco Malt agar at 28 °C, respectively.

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The counts were done in duplicate.

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Microbiological analysis of fecal microbiota

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Within 30 minutes after collection, feces in plastic bags were manually homogenized, then 1 g of

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homogenized feces was transferred in a sterile tube added with 9 mL of filtered PBS (2 mM

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NaH2PO4, 8 mM Na2HPO4, 130 mM NaCl, pH 7.4) and 12 glass balls (3 mm diameter), and

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vortexed for 3 min at 40 Hz. Moreover, the tubes were immerged for 10 s in a 45 kHz ultrasonic

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bath at 37 °C, in order to facilitate separation of microbial cells from the fecal particles. Fecal

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suspensions were centrifuged at 400 x g for 5 min at 4 °C, in order to remove large particles. After

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that, 1 mL of each supernatant was transferred in a 2 mL Eppendorf® sterile tube and centrifuged at

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13000 x g for 5 min at 4 °C. After discarding the supernatant, the pellets of cells were suspended in

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1.8 mL of particle free PBS pH 7.4 containing 3% (w/v) of paraformaldehyde, mixed up completely,

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and then stored at 4 °C for 4 h in horizontal position, in order to favour the distribution of the cells ACS Paragon Plus Environment

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on a thin layer, thereby facilitating cells fixation. The fixed samples were finally washed twice with

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PBS, and stored in PBS/ethanol (1/1, w/w) at −20 °C until further processing. Defrosted samples

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were washed twice with PBS. Two replicate aliquots of each cell suspension were opportunely

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diluted with PBS, and bacteria were collected by filtration onto 0.2 µm pore-size white

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polycarbonate filters (ISOPORE GTTP02500, diameter 25 mm; Merck Millipore, Darmstadt,

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Germany) mounted in a glass holder (3 cm2 filtration area) applying a vacuum of 30 kPa. The filters

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were rinsed with 3 mL of filtered PBS, air dried, dehydrated by dipping them in 50, 80 and 96%

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aqueous ethanol subsequently, air dried, and stored in dark at room temperature. For the in situ

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hybridization, 4 small sections were cut out of the whole polycarbonate filters. Each filter section

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was aligned on silicon coated slides, covered with 96 µL of hybridization buffer (900 mM NaCl, 20

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mM Tris-HCl pH 8.0, 0.01% SDS) set to different formamide concentration according to different

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probes (Table 1) and 4 mL of the labelled probe (50 ng/µL) (MWG-Biotech, Ebersberg, Germany).

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Each slide was placed in a 50 mL plastic tube, which was humidified with the surplus of

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hybridization buffer, as a moisture chamber. Hybridization was performed at 46 °C for 4 h. After

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hybridization, each filter was transferred into 2 mL of warmed washing buffer (20 mM Tris-HCl pH

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8.0, 5 mM EDTA, 0.01 % SDS, pH 8.0) with different NaCl concentration to achieve appropriate

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washing stringency (Table 1), for 10 min at 48 °C and was then washed into 2 mL refrigerated

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Milli-Q water for few seconds. Finally, filters were dried at 65 °C for 2 min, and stored at -20 °C,

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until fluorescence microscopy counting. The filters were mounted on a microscope slide with anti-

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fading oil (Citifluor Ltd, London, United Kingdom) to prevent a fast bleaching of probe signals, and

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examined at epifluorescence microscope Axioskope (Zeiss, Oberkochen, Germany) equipped with a

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50 W type HBO high-pressure mercury lamp (Osram, Munich, Germany) and the Zeiss 15 filter set.

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An eyepiece with a calibrated reticule was used for bacterial counting. At least 30 randomly

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selected microscopic fields with 10 x 10 square units of the reticule, with objectives 100× (Plan-

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Neofluar, Zeiss, Oberkochen, Germany) were inspected for each filter. Three replicates were used,


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and at least 300 cells were counted for each sample. When the probes had a very low cell densities,

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a total of 60 microscopic fields were inspected, which corresponded to 0.5% of the sample filter.

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Statistical analysis

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Analysis of the experimental diets was purely descriptive and no statistical comparison has been

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done. Data on intestinal microbial composition, obtained on t0, were analyzed by one-way ANOVA

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(PROC GLM). All the other results were analyzed by a mixed procedure for repeated measures

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(PROC MIXED RM) using SAS software version 8.01 (SAS Institute Inc., Cary, NC), considering

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dietary treatment as fixed effect and animal as randomized effect. The effect of period was not

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significant, so it was removed from the statistical model. Data on microbial cells count were Log10-

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transformed, before the analysis. The level of significance was set at P ≤ 0.05.

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RESULTS

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As shown in Table 2, inclusion of cocoa husks increased fibrous fractions of the diet, in particular,

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redoubled the percentage of ADL, without affecting dietary protein and energy content. Substitution

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of 7.5% by weight of the control diet with cocoa husks increased by 50% dietary content of

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polyphenols (Table 2).

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Microbiological analysis showed that feedstuffs were in good hygiene conditions (Table 3), since

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all the investigated parameters were even below the limits allowed by law for ready-to-use foods

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intended for human consumption (EU Commission Regulation No. 2073/2005).

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As reported in Table 4, dietary treatment did not affect feed intake, weight gain and feed efficiency.

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Along the entire study period, rectal temperature ranged between 38.1 ± 0.64 °C (recorded on t2)

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and 38.9 ± 0.23 °C (recorded on t1) and never differed between the experimental groups.

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In adult pigs, the investigated bacterial communities account for more than 95% of total intestinal

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bacteria.32 Analysis of intestinal microbial composition of pigs at the time of allotment into the

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experimental groups (t0) did not show any difference for the investigated microbial groups (Table 5 ACS Paragon Plus Environment

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and 6). By contrast, analysis of data recorded on t1 and t2 showed that cocoa husks feeding for 3

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weeks reduced intestinal populations of the Lactobacillus-Enterococcus group and Clostridium

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histolyticum and

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Faecalibacterium prausnitzii, whereas no difference has been observed for Bifidobacteria, the

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Clostridium Coccoides-Eubacterium Rectale group, Escherichia Coli and Ruminococci (Table 7).

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Analysis of the intestinal microbial populations grouped by phyla, showed a tendency toward a

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decrease of Firmicutes and a significant increase of Bacteroidetes, after consumption of cocoa

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husks for 3 weeks (Table 8). Consequently, cocoa husks feeding largely increased Bacteroidetes to

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Firmicutes ratio (Table 8).

increased

the

populations

of the

Bacteroides-Prevotella

group

and

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Discussion

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One limitation of the present study lies in the small number of experimental subjects. Anyway, this

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limit is counterbalanced by the homogeneity in the composition of intestinal microbiota of the

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selected pigs, and the lack of difference, at the time of allotment into the experimental groups (t0),

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testifies for the homogeneity among subjects.

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Cocoa husks feeding for 3 weeks reduced intestinal population of bacteria belonging to the group

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Lactobacillus-Enterococcus, which belongs to the phylum Firmicutes. Microorganisms of the genus

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Lactobacillus are considered beneficial for the host’s health, as most probiotic bacterial strains are

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ascribable to this genus.33,34 Lactobacilli survive the transport through the gastro-intestinal tract and

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colonise the large intestine, where they produce lactic acid by fermentation of sugars, reducing

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intestinal pH and thus making the environment unfavourable to the growth of pathogens. Moreover,

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Lactobacilli produce peptides (bacteriocins) having antimicrobial activity against several Gram-

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negative and Gram-positive harmful bacteria.35 Lactobacilli also produce antioxidants, which

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provide a beneficial effect in scavenging reactive oxygen species, such as free radicals, and perform

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potential anti-inflammatory and anti-carcinogenic actions: Lactobacilli have been observed to

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reduce the risk of a wide range of intestinal inflammatory diseases and could be effective in binding ACS Paragon Plus Environment

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mutagens (ingested or produced from foods) and secreting anti-mutagenic substances, thus being

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useful in preventing carcinogenesis.35,36

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Enterococci are common components of the intestinal microbiota of pigs. As Lactobacilli, some

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species of Enterococcus may provide defence against pathogenic bacteria, thanks to the production

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of bacteriocins.37 Enterococcus faecium also competes for adhesion to porcine intestinal mucosa

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with pathogens, such as enterotoxigenic Escherichia coli K88.38 On the other hand, Enterococcus

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villorum and Enterococcus porcinus are associated with enteric disorders in pigs.39,40 In humans,

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Enterococcus faecalis is an opportunistic pathogen that can trigger inflammatory bowel diseases

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(IBD), which affect several million patients in the world; moreover, Enterococcus faecalis has the

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ability to acquire virulence factors and antibiotic resistance genes, hindering treatment of the

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infections it causes.41 Enterococci represent a sub-dominant group of pig intestinal microbial

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community, whereas the genus Lactobacillus is one of the most represented.32

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The COCOA diet decreased the population of Clostridium histolyticum. It is a motile, Gram-

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positive, facultative anaerobe belonging to the predominant groups of the pig intestine.42 It is a

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potential pathogen: when the integrity of the intestinal mucous barrier is compromised (by stress,

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illness or malnutrition), Clostridium histolyticum can adhere to the intestinal epithelium and cause

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inflammation by producing a mixture of proteolytic enzymes and a necrotizing toxin.43,44

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Cocoa husks feeding for 3 weeks increased intestinal populations of Bacteroides-Prevotella and

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Faecalibacterium prausnitzii. Microorganisms of the genera Bacteroides and Prevotella, which are

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taxonomically and functionally similar,45 are among the dominant populations in the distal intestine

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of mammals.32,46 This group of Gram-negative anaerobes, belonging to the phylum Bacteroidetes,

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can degrade and ferment a wide variety of polysaccharides with production of SCFA, in particular

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acetate.46 The three principal end-products of fermentation may have different functions.1 Butyrate

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is metabolized by the colonic epithelium and it’s generally regarded as a healthy metabolite, since it

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positively influences growth and differentiation of enterocytes, and exerts anti-inflammatory

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effects: a wide range of intestinal inflammatory diseases is associated to a reduction of ACS Paragon Plus Environment

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Bacteroides.36 Acetate stimulates goblet cell differentiation, but, if produced in excess, it may cause

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overexpression of mucus-related genes.47 Acetate and propionate can access the portal circulation

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and oppositely impact lipid metabolism: acetate seems to contribute to lipid and cholesterol

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synthesis in the liver, propionate inhibits the effects of acetate.1 Moreover, SCFA, in particular

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acetate, are efficient growth inhibitors of many common pathogens.48

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According to Massot-Cladera et al., who have recently observed that a cocoa-enriched diet

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significantly decreases microbial fecal population of Bacteroides in rats,49 the increase of the

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Bacteroides-Prevotella group in our cocoa husks-fed pigs should not be ascribable to cocoa

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polyphenols. Our result can be rather attributed to the higher level of fibre in the cocoa diet, as

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observed by Ivarsson, who has reported that dietary fibre increases the abundance of Bacteroides-

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Prevotella in ileal digesta of growing pigs.50 In the last cited work, feeding a high-fibre diet, in

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addition to increasing Bacteroides-Prevotella, which are the major dietary fibre degrading genera in

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the pig gastrointestinal tract, also decreased the counts of Lactobacilli, as here reported.50

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With regard to Faecalibacterium prausnitzii, it is an anaerobic commensal bacterium, belonging to

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the phylum Firmicutes, which colonises the large intestine of both humans and pigs.51 This

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bacterium may be crucial to gut homeostasis and is considered as a protective factor for the

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intestinal mucosa.52 Faecalibacterium prausnitzii is the principal butyrate-producing bacterial

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species of mammalian intestine and many studies confirm its protective properties against a wide

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range of intestinal inflammatory diseases.36,52,53 Moreover, the abundance of Faecalibacterium

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prausnitzii is decreased in obese and diabetic humans.54 The increase in the population of

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Faecalibacterium prausnitzii may have been favoured by the increase of the Bacteroides-Prevotella

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group. Several species of Bacteroides, in fact, produce acetate as end product of polysaccharides

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fermentation, which has a positive effect on growth of Faecalibacterium prausnitzii.47 It has been

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clearly demonstrated in gnotobiotic rats that Bacteroides thetaiotaomicron, a major acetate producer,

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and Faecalibacterium prausnitzii, a major acetate consumer and butyrate producer, are

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metabolically complementary47. When Faecalibacterium prausnitzii is associated with Bacteroides ACS Paragon Plus Environment

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thetaiotaomicron, it may help intestinal epithelium to maintain the efficiency of the mucus layer

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and appropriate proportions of different cell types of the secretory lineage, by limiting the effects of

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acetate on goblet cells.47

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These latter data suggest potential positive effects of cocoa husks intake on intestinal microbial

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ecosystem. Moreover, striking information comes from the analysis of the intestinal microbial

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populations grouped by phyla, which showed an increase of the Bacteroidetes to Firmicutes ratio, in

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cocoa husks-fed pigs. The Bacteroidetes to Firmicutes ratio is regarded to be of significant

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relevance

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Bacteroidetes/Firmicutes has been linked to the risk of metabolic disorders. In obese subjects, the

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microbiota shows an elevated proportion of Firmicutes and a reduced population of Bacteroides.

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Conversely, a greater proportion of Bacteroidetes than Firmicutes has been shown to be useful in

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the regulation of energy balance and reduction of adiposity.55 These findings indicate that obesity

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has a microbial component, which may be regulated by diet, and cocoa husks may contribute to

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improve intestinal microbial balance.

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In conclusion, our results suggest that the use of cocoa husks in pig nutrition may have a positive

280

effect on the balance of intestinal microbial ecosystem. Despite a reduction of Lactobacilli, cocoa

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husks feeding for 3 weeks increased microbial populations of the Bacteroides-Prevotella group and

282

Faecalibacterium prausnitzii, which are considered beneficial for the host’s health because of the

283

production of SCFA, in particular butyrate, which is regarded as a healthy metabolite, since it

284

positively influences growth and differentiation of enterocytes, and exerts anti-inflammatory effects,

285

thereby reducing the incidence of a wide range of intestinal inflammatory diseases. Moreover,

286

cocoa husks feeding improved the proportion between the main phyla of the intestinal ecosystem,

287

which may help reducing the risk of excessive fattening, that is considered to be detrimental for the

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quality of the end products.56 Further studies will clarify if the effect of cocoa husks on intestinal

289

microbial populations can be ascribed to cocoa polyphenols, to the content of dietary fibre, or to a

in

gut

microbiota

composition,

in

both

humans


and

rodents.

Moreover,

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possible synergistic activity of these two dietary components or to the decrease of other dietary

291

ingredient.

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ABBREVIATIONS USED

294

PBS, phosphate-saline buffer; SDS, sodium dodecyl sulphate; FISH, fluorescent in situ

295

hybridization; AOAC, association of official analytical chemists; SCFA, short chain fatty acids.

296 297

Acknowledgments

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There was no specific funding for this work and the research was made possible thanks to the

299

invaluable help of Davide De Angeli, Paolo Roveda, Marco Misitano, Irene Bergamaschi and

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Emanuele Silva. Special thanks to Icam S.p.a. for the supply of cocoa husks.

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REFERENCES 1. Laparra, J. M.; Sanz, Y. Interactions of gut microbiota with functional food components and Nutraceuticals. Pharmacol. Res. 2010, 61, 219–225.

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2. Gibson, G. R.; Probert, H. M.; Van Loo, J.; Rastall, R. A.; Roberfroid, M. B. Dietary

306

modulation of the human colonic microbiota: updating the concept of prebiotics. Nutr. Res. Rev.

307

2004, 17, 259–275.

308

3. Etxeberria, U.; Fernández-Quintela, A.; Milagro, F. I.; Aguirre, L.; Martínez, J. A.; Portillo,

309

M. P. Impact of polyphenols and polyphenol-rich dietary sources on gut microbiota composition. J.

310

Agric. Food Chem. 2013, 61, 9517–9533.

311

4. Larrosa, M.; Luceri, C.; Vivoli, E.; Pagliuca, C.; Lodovici, M.; Moneti, G.; Dolara, P.

312

Polyphenol metabolites from colonic microbiota exert anti-inflammatory activity on different

313

inflammation models. Mol. Nutr. Food Res. 2009, 53, 1044–1054.


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Page 14 of 28

314

5. Kemperman, R. A.; Bolca, S.; Roger, L. C.; Vaughan, E. E. Novel approaches for analysing

315

gut microbes and dietary polyphenols: challenges and opportunities. Microbiology 2010, 156,

316

3224–3231.

317 318 319 320

6. Lee, H. C.; Jenner, A. M.; Lowa, C. S.; Lee, Y. K. Effect of tea phenolics and their aromatic fecal bacterial metabolites on intestinal microbiota. Res. Microbiol. 2006, 157, 876–884. 7. Zanchi, R.; Canzi, E.; Molteni, L.; Scozzoli, M. Effect of Camellia sinensis L. whole plant extract on piglet intestinal ecosystem. Ann. Microbiol. 2008, 58, 147–152.

321

8. Lecumberri, E.; Mateos, R.; Izquierdo-Pulido, M.; Rupérez, P.; Goya, L.; Bravo, L. Dietary

322

fibre composition, antioxidant capacity and physico-chemical properties of a fibre-rich product

323

from cocoa (Theobroma Cacao L.). Food Chem. 2007, 104, 948–954.

324 325 326 327 328 329

9. Beckett, S. T. Traditional chocolate making. In Industrial chocolate manufacture and use, 4th ed.; Beckett, S. T., Ed.; John Wiley & Sons Ltd: Chichester, U.K., 2009, pp. 1–9. 10. Magistrelli, D.; Malagutti, L.; Rosi, F. Cocoa husks feeding improves some indices of health and welfare of pigs. Indian J. Res. 2014, 3(7), 210–213. 11. Magistrelli, D.; Malagutti, L.; Galassi, G.; Rosi, F. Cocoa husks in diets of Italian heavy pigs. J. Anim. Sci. 2012, 90 (Suppl. 4), 230–232.

330

12. Tuohy, K. M.; Conterno, L.; Gasperotti, M.; Viola, R. Up-regulating the human intestinal

331

microbiome using whole plant foods, polyphenols, and/or fiber. J. Agric. Food Chem. 2012, 60,

332

8776–8782.

333 334

13. Bähr, A.; Wolf, E. Domestic animal models for biomedical research. Reprod. Dom. Anim. 2012, 47 (Suppl. 4), 59–71.

335

14. Furet, J. P.; Firmesse, O.; Gourmelon, M.; Bridonneau, C.; Tap, J.; Mondot, S.; Doré, J.;

336

Corthier, G. Comparative assessment of human and farm animal fecal microbiota using real-time

337

quantitative PCR. FEMS Microbiol. Ecol. 2009, 68, 351–362.

338 339

15. Lee, J. E.; Lee, S.; Sung, J.; Ko, G. P. Analysis of human and animal fecal microbiota for microbial source tracking. ISME J. 2011, 5, 362–365. ACS Paragon Plus Environment

14

Page 15 of 28

340 341


16. Ferrando, R. ; Blum, J. C. L’alimentation des animaux monogastriques: porc, lapin, volaille, 2nd ed., INRA: Paris, France, 1989.

342

17. Burndred, F. Food safety in chocolate manufacture and processing. In Industrial chocolate

343

manufacture and use, 4th ed.; Beckett, S. T., Ed.; John Wiley & Sons Ltd: Chichester, U.K., 2009,

344

pp. 530–550.

345 346 347 348 349 350 351 352 353 354

18. AOAC Official Methods of Analysis, 15th ed.; Association of Official Analytical Chemists International: Gaithersburg, MD, 1995. 19. Mertens, D. R. Gravimetric determination of amylase-treated neutral detergent fiber in feeds using refluxing in beakers or crucibles: collaborative study. J. AOAC 2002, 85, 1217–1240. 20. Van Soest, P. J.; Robertson, J. B.; Lewis, B. A. Methods of dietary fiber, neutral detergent fiber and non-polysaccharides in relation to animal nutrition. J. Dairy Sci. 1991, 74, 3583–3597. 21. Budini, R.; Tonelli, D.; Girotti, S. Analysis of total phenols using the Prussian Blue method. J. Agric. Food Chem. 1980, 28, 1236–1238. 22. Amann, R. I.; Ludwig, W.; Schleifer, K. H. Phylogenetic identification and in-situ detection of individual microbial-cells without cultivation. Microbiol. Rev. 1995, 59, 143–169.

355

23. Daims, H.; Bruhl, A.; Amann, R.; Schleifer, K. H.; Wagner, M. The domain-specific

356

probe EUB338 is insufficient for the detection of all Bacteria: Development and evaluation of a

357

more comprehensive probe set. Syst. Appl. Microbiol. 1999, 22, 434–444.

358

24. Wallner, G.; Amann, R.; Beisker, W. Optimizing fluorescent in situ hybridization with

359

ribosomal-RNA-targeted

oligonucleotide

probes

360

microorganisms. Cytometry 1993, 14, 136–143.

for

flow

cytometric

identification

of

361

25. Harmsen, H. J. M.; Elfferich, P.; Schut, F.; Welling, G. W. A 16S rRNA-targeted probe

362

for detection of lactobacilli and enterococci in fecal samples by fluorescent in situ hybridisation.

363

Microbiol. Ecol. Health. Dis. 1999, 11, 3–12.


15


Page 16 of 28

364

26. Manz, W.; Amann, R.; Ludwig, W. Application of a suite of 16S rRNA-specific

365

oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-

366

bacteroides in the natural environment. Microbiology 2006, 142, 1097–1106.

367

27. Langendijk, P. S.; Schut, F.; Jansen, G. J.; Raangs, G. C.; Kamphuis, G. R.; Wilkinson, M.

368

H.; Welling, G. W. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with

369

genus-specific 16S rRNA-targeted probes and its application in fecal samples. Appl. Environ.

370

Microbiol. 1995, 61, 3069–3075.

371

28. Franks, A. H.; Harmsen, H. J.; Raangs, G. C.; Jansen, G. J.; Schut, F.; Welling, G. W.

372

Variations of bacterial populations in human feces measured by fluorescent in situ hybridization

373

with group-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 1998, 64,

374

3336–3345.

375 376

29. Kempf, V. A.; Trebesius, K.; Autenrieth, I. B. Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures. J. Clin. Microbiol. 2000, 38, 830–838.

377

30. Poulsen, L. K.; Lan, F.; Kristensen, C. S.; Hobolth, P.; Molin, S.; Krogfelt, K. A. Spatial

378

distribution of Escherichia coli in the mouse large intestine inferred from rRNA in situ

379

hybridization. Infect. Immun. 1994, 62, 5191–5194.

380

31. Suau, A.; Rochet, V.; Sghir, A.; Gramet, G.; Brewaeys, S.; Sutren, M.; Rigottier-Gois, L.;

381

Doré, J. Fusobacterium prausnitzii and related species represent a dominant group within the human

382

fecal flora. Syst. Appl. Microbiol. 2001, 24, 139–145.

383

32. Kim, H. B.; Borewicz, K.; White, B. A.; Singer, R. S.; Sreevatsan, S.; Tu, Z. J.; Isaacson

384

R. E. Longitudinal investigation of the age-related bacterial diversity in the feces of commercial

385

pigs. Vet. Microbiol. 2011, 153, 124–133.

386

33. Guarner, F.; Schaafsma, G. J. Probiotics. Int. J. Food Microbiol. 1998, 39, 237–238.

387

34. Collins, M. D.; Gibson, G. R. Probiotics, prebiotics, and synbiotics: approaches for

388

modulating the microbial ecology of the gut. Am. J. Clin. Nutr. 1999, 69 (Suppl.), 1052S–1057S.


16

Page 17 of 28

389 390 391 392 393 394


35. Ljungh, A.; Wadström, T. Lactic acid bacteria as probiotics. Curr. Issues Intest. Microbiol. 2006, 7, 73–90. 36. Sasaki, M.; Klapproth, J. M. A. The role of bacteria in the pathogenesis of ulcerative colitis. J. Signal Transduct. 2012, Article ID 704953, 6 pages. DOI: 10.1155/2012/704953. 37. Foulquié Moreno, M. R.; Sarantinopoulos, P.; Tsakalidou, E.; De Vuyst, L. The role and application of enterococci in food and health. Int. J. Food Microbiol. 2006, 106, 1–24.

395

38. Jin, L. Z.; Marquardt, R. R.; Zhao, X. A Strain of Enterococcus faecium (18C23) inhibits

396

adhesion of enterotoxigenic Escherichia coli K88 to porcine small intestine mucus. Appl. Environ.

397

Microbiol. 2000, 66, 4200–4204.

398

39. Teixeira, L. M.; Carvalho, M. d G. S.; Espinola, M. M. B.; Steigerwalt, A. G.; Douglas, M.

399

P.; Brenner, D. J.; Facklam, R. R. Enterococcus porcinus sp. nov. and Enterococcus ratti sp. nov.,

400

associated with enteric disorders in animals. Int. J. Syst. Evol. Microbiol. 2001, 51, 1737–1743.

401

40. Vancanneyt, M.; Snauwaert, C.; Cleenwerck, I.; Baele, M.; Descheemaeker, P.; Goossens,

402

H.; Pot, B.; Vandamme, P.; Swings, J.; Haesebrouck, F.; Devriese, L. A. Enterococcus villorum sp.

403

nov., an enteroadherent bacterium associated with diarrhoea in piglets. Int. J. Syst. Evol. Microbiol.

404

2001, 51, 393–400.

405 406

41. Balish, E.; Warner, T. Enterococcus faecalis induces inflammatory bowel disease in interleukin-10 knockout mice. Am. J. Pathol. 2002, 160, 2253–2257.

407

42. Collado, M. C.; Sanz, Y. Characterization of the gastrointestinal mucosa–associated

408

microbiota of pigs and chickens using culture-based and molecular methodologies. J. Food Prot.

409

2007, 12, 2708–2934.

410

43. Hatheway, C. L. Toxigenic Clostridia. Clin. Microbiol. Rev. 1990, 3, 66–98.

411

44. Swidsinski, A.; Loening-Baucke, V.; Herber, A. Mucosal flora in Crohn’s disease and

412 413 414

ulcerative colitis - an overview. J. Physiol. Pharmacol. 2009, 60 (Suppl. 6), 61–71. 45. Wu, G. D.; Bushmanc, F. D.; Lewis, J. D. Diet, the human gut microbiota, and IBD. Anaerobe 2013, 24, 117–120. ACS Paragon Plus Environment

17


415 416

Page 18 of 28

46. Hooper, L. V.; Midtvedt, T.; Gordon, J. I. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Ann. Rev. Nutr. 2002, 22, 283–307.

417

47. Wrzosek, L.; Miquel, S.; Noordine, M. L.; Bouet, S.; Chevalier-Curt, M. J.; Robert, V.;

418

Philippe, C.; Bridonneau, C.; Cherbuy, C.; Robbe-Masselot, C.; Langella, P.; Thomas, M.

419

Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii influence the production of mucus

420

glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent.

421

BMC Biol. 2013, 11, 61–73.

422

48. van Limpt, C.; Crienen, A.; Vriesema, A.; Knol, J. Effect of colonic short chain fatty acids

423

and pH on the growth of pathogens and gut commensals. Book of Abstracts of the 24th

424

International Congress of Pediatrics, Cancun, Mexico, 2004, p. 487.

425

49. Massot-Cladera, M.; Pérez-Berezo, T.; Franch, A.; Castell, M.; Pérez-Cano, F. J. Cocoa

426

modulatory effect on rat faecal microbiota and colonic crosstalk. Arch. Biochem. Biophys. 2012,

427

527, 105–112.

428

50. Ivarsson, E. Chicory (Cichorium intybus L) as fibre source in pig diets: effects on

429

digestibility, gut microbiota and performance. Acta Universitatis Agriculturae Sueciae: Doctoral

430

Thesis n° 19, 2012. URL (http://pub.epsilon.slu.se/8693/1/ivarsson_emma_120416.pdf).

431

51. Leser, T. D.; Amenuvor, J. Z.; Jensen, T. K.; Lindecrona, R. H.; Boye, M.; Møller, K.

432

Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited. Appl.

433

Environ. Microbiol. 2002, 68, 673–690.

434

52. Sokol, H.; Seksik, P.; Furet, J. P.; Firmesse, O.; Nion-Larmurier, I.; Beaugerie, L.; Cosnes,

435

J.; Corthier, G.; Marteau, P.; Doré, J. Low counts of Faecalibacterium prausnitzii in colitis

436

microbiota. Inflamm. Bowel Dis. 2009, 15, 1183–1189.

437 438 439 440

53. Pryde, S. E.; Duncan, S. H.; Hold, G. L.; Stewart, C. S.; Flint, H. J. The microbiology of butyrate formation in the human colon. FEMS Microbiol. Lett. 2002, 217, 133–139. 54. Karlsson, F.; Tremaroli, V.; Nielsen, J.; Bäckhed, F. Assessing the human gut microbiota in metabolic diseases. Diabetes 2013, 62, 3341–3349. ACS Paragon Plus Environment

18

Page 19 of 28

441 442


55. Ley, R. E.; Turnbaugh, P. J.; Klein, S.; Gordon, J. I. Microbial ecology: Human gut microbes associated with obesity. Nature 2009, 444, 1022–1023.

443

56. Magistrelli, D.; Galassi, G.; Crovetto, G. M.; Rosi, F. Influence of high levels of beet pulp

444

in the diet on endocrine/metabolic traits, slaughter dressing percentage, and ham quality in Italian

445

heavy pigs. It. J. Anim. Sci. 2009, 8, 37–49.

446


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447

Table 1 Probes used for target bacterial groups in FISH analysis performed on pig fecal samples,

448

concentration of formamide in the hybridization buffer and concentration of NaCl in the washing

449

solution. Superscript numbers in probes’ acronyms indicate the reference literature. Probe EUB33822 EUB338II23 EUB338III23 NONEUB24 LAB15825 Bac30326 Bif16427 Chis15028 Erec48229

Sequence (5’-3’) GCTGCCTCCCGTAGGAGT GCAGCCACCCGTAGGTGT GCTGCCACCCGT AGGTGT ACTCCTACGGGAGGCAGC GGT ATT AGC AYC TGT TTC CA CCA ATG TGG GGG ACC TT CAT CCG GCA TTA CCA CCC TTA TGC GGT ATT AAT CTY CCT TT GCT TCT TAG TCA RGT ACC G

Target organisms

FA*

NaCl#

Bacteria Planctomycetales Verrucomicrobiales Negative control LactobacillusEnterococcus Bacteroides-Prevotella Bifidobacterium Clostridium hystolyticum Clostridium coccoidesEubacterium rectale

30 30 30 30

102 102 102 102

0

900

0 0

900 900

0

900

0

900

CAC CGT AGT GCC TCG TCA E. coli 35 TCA CCT CTG CAC TAC TCA AGA Faecalibacterium Fprau64531 30 AAA AC prausnitzii Rbro73029 TAA AGC CCA GYA GGC CGC Ruminococcus 30 * Concentration of formamide in the hybridization buffer expressed as percentage (%). Ec151330

450 451

#

70 102 102

Concentration of NaCl in the washing solution expressed as mM.


20

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452

Table 2 Chemical analysis of the experimental diets and cocoa husks. CTRL = control diet;

453

COCOA = 7.5% cocoa husks diet; DM = dry matter; NDF = neutral detergent fibre; ADF = acid

454

detergent fibre; ADL = acid detergent lignin. Item Dry Matter Organic Matter Crude Proteins Crude Fats NDF ADF ADL Minerals Gross Energy Total Polyphenols

Unit % % DM % DM % DM % DM % DM % DM % DM kJ/g DM mg/g DM

Diet CTRL 89.2 93.9 19.6 4.84 18.4 8.84 1.85 6.14 19.0 8.20

COCOA 89.5 93.8 19.4 4.75 20.7 11.6 3.75 6.25 19.0 12.3

Cocoa husks 92.6 92.4 16.7 3.69 49.3 48.9 27.1 7.62 18.5 61.5

455 456


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457

Table 3 Microbiological analysis of the experimental diets and cocoa husks. CTRL = control diet;

458

COCOA= 7.5% cocoa husks diet; cfu=colony forming unit; DM = dry matter. Item

459

Aerobic colony count Salmonella Listeria monocytogenes Escherichia coli Eumycetes * ND = not detected

Unit cfu/g DM cfu/g DM cfu/g DM cfu/g DM cfu/g DM

Diet CTRL 2.60 x 103 Absent Absent Absent Absent

COCOA 1.83 x 105 Absent Absent Absent ND*

Cocoa husks 2.41 x 106 Absent Absent Absent 102

460


22

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461

Table 4 Feed intake, weight gain and feed efficiency for pigs fed a negative control diet (CTRL)

462

and a 7.5% cocoa husks diet (COCOA). Data are given as means per treatment. CTRL = control diet;

463

COCOA = 7.5% cocoa husks diet; G:F = gain to feed ratio; SE = Standard Error. Item

Unit

Feed intake Weight gain G:F

g/d g/d g/g

CTRL 2369 601 0.26

Group COCOA 2321 601 0.26

SE

P-value

209 80.2 0.03

0.83 0.66 0.99

464


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465

Table 5 Intestinal microbial populations of pigs at the time of allotment into the experimental

466

groups (t0). Data are expressed as (Log10 n)/g dry matter. CTRL = control diet; COCOA = 7.5%

467

cocoa husks diet; SE = Standard Error. Domain/Genus/Species Eubacteria Lactobacillus-Enterococcus Bacteroides-Prevotella Bifidobacterium Clostridium histolyticum Clostridium coccoidesEubacterium rectale Escherichia coli Faecalibacterium prausnitzii Ruminococcus

MEAN 9.84 8.86 8.92 7.49 7.99

Group CTRL COCOA 8.97 9.21 8.75 8.97 9.20 8.64 7.84 7.14 7.87 8.11

SE

P-value

0.30 0.22 0.80 0.48 0.67

0.95 0.40 0.53 0.27 0.78

7.81

8.11

7.51

0.30

0.22

6.93 6.57 7.32

6.90 6.77 7.22

6.95 6.36 7.42

0.39 0.44 0.30

0.89 0.41 0.57

468 469


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470

Table 6 Intestinal microbial populations (grouped by phyla) of pigs at the time of allotment into the

471

experimental groups (t0). Data are expressed as (Log10 n)/g dry matter, with the exception of the

472

Bacteroidetes to Firmicutes ratio. CTRL = control diet; COCOA = 7.5% cocoa husks diet; SE =

473

Standard Error. Phylum Firmicutes Bacteroidetes Actinobacteria Proteobacteria Bacteroidetes/Firmicutes

MEAN 9.09 8.92 7.49 6.93 1.68

CTRL 8.97 9.20 7.84 6.90 1.72

Group COCOA 9.21 8.63 7.14 6.96 1.64

SE

P-value

0.22 0.80 0.48 0.39 0.36

0.46 0.53 0.27 0.89 0.86

474


25


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475

Table 7 Analysis of the effect of dietary treatment on intestinal microbial composition. Data are

476

given as means per treatment and expressed as (Log10 n)/g dry matter. CTRL = control diet; COCOA

477

= 7.5% cocoa husks diet; SE = Standard Error. Domain/Genus/Species Eubacteria Lactobacillus-Enterococcus Bacteroides-Prevotella Bifidobacterium Clostridium histolyticum Clostridium coccoidesEubacterium rectale Escherichia coli Faecalibacterium prausnitzii Ruminococcus

Group CTRL 9.88 9.14 8.20 8.21 8.68

COCOA 9.91 8.70 9.39 7.71 7.93

SE

P-value

0.03 0.15 0.31 0.45 0.26

0.38 0.04 0.01 0.31 0.05

7.82

7.87

0.28

0.87

7.21 6.47 7.53

7.72 7.42 7.64

0.28 0.35 0.21

0.13 0.04 0.62

478 479


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480

Table 8 Analysis of the effect of dietary treatment on intestinal microbial populations, grouped by

481

phyla. Data are given as means per treatment and expressed as (Log10 n)/g dry matter, with the

482

exception of the Bacteroidetes to Firmicutes ratio. CTRL = control diet; COCOA = 7.5% cocoa

483

husks diet; SE = Standard Error. Phylum Firmicutes Bacteroidetes Actinobacteria Proteobacteria Bacteroidetes/Firmicutes

Group CTRL 9.40 8.20 8.21 7.21 0.27

COCOA 8.98 9.39 7.71 7.72 3.40

SE

P-value

0.15 0.31 0.45 0.28 0.74

0.07 0.01 0.31 0.13 0.01

484 485


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486 487

TOC graph


28

Effects of Cocoa Husk Feeding on the Composition of Swine

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