Skip to main content

Reducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines

  • C. Poelaert (a1) (a2), G. Nollevaux (a3), C. Boudry (a1), B. Taminiau (a4), C. Nezer (a5), G. Daube (a4), Y.-J. Schneider (a3), D. Portetelle (a2), A. Théwis (a1) and J. Bindelle (a1)...

Over the past decade, in vitro methods have been developed to study intestinal fermentation in pigs and its influence on the digestive physiology and health. In these methods, ingredients are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na2S or cysteine-HCl generates the required anaerobic environment by releasing metabolites similar to those produced when protein is fermented, possibly inducing a dysbiosis. An experiment was conducted to study the impact of two reducing agents on results yielded by such in vitro fermentation models. Protein (soybean proteins, casein) and carbohydrate (potato starch, cellulose) ingredients were fermented in vitro by bacteria isolated from fresh feces obtained from three sows in three carbonate-based incubation media differing in reducing agent: (i) Na2S, (ii) cysteine-HCl and (iii) control with a mere saturation with CO2 and devoid of reducing agent. The gas production during fermentation was recorded over 72 h. Short-chain fatty acids (SCFA) production after 24 and 72 h and microbial composition of the fermentation broth after 24 h were compared between ingredients and between reducing agents. The fermentation residues after 24 h were also evaluated in terms of cytotoxicity using Caco-2 cell monolayers. Results showed that the effect of the ingredient induced higher differences than the reducing agent. Among the latter, cysteine-HCl induced the strongest differences compared with the control, whereas Na2S was similar to the control for most parameters. For all ingredients, final gas produced per g of substrate was similar (P>0.10) for the three reducing agents whereas the maximum rate of gas production (R max) was reduced (P<0.05) when carbohydrate ingredients were fermented with cysteine-HCl in comparison to Na2S and the control. For all ingredients, total SCFA production was similar (P>0.10) after 24 h of fermentation with Na2S and in the control without reducing agent. Molar ratios of branched chain-fatty acids were higher (P<0.05) for protein (36.5% and 9.7% for casein and soybean proteins, respectively) than for carbohydrate (<4%) ingredients. Only fermentation residues of casein showed a possible cytotoxic effect regardless of the reducing agent (P<0.05). Concerning the microbial composition of the fermentation broth, most significant differences in phyla and in genera ascribable to the reducing agent were found with potato starch and casein. In conclusion, saturating the incubation media with CO2 seems sufficient to generate a suitable anaerobic environment for intestinal microbes and the use of a reducing agent can be omitted.

  • View HTML
    • Send article to Kindle

      To send this article to your Kindle, first ensure is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

      Note you can select to send to either the or variations. ‘’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

      Find out more about the Kindle Personal Document Service.

      Reducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines
      Available formats
      Send article to Dropbox

      To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

      Reducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines
      Available formats
      Send article to Google Drive

      To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

      Reducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines
      Available formats
Corresponding author
Hide All
Altschul, SF, Gish, W, Miller, W, Myers, EW and Lipman, DJ 1990. Basic local alignment search tool. Journal of Molecular Biology 215, 403410.
Andriamihaja, M, Davila, AM, Eklou-Lawson, M, Petit, N, Delpal, S, Allek, F, Blais, A, Delteil, C, Tomé, D and Blachier, F 2010. Colon luminal content and epithelial cell morphology are markedly modified in rats fed with a high-protein diet. American Journal of Physiology - Gastrointestinal and Liver Physiology 299, G1030G1037.
Artursson, P, Palm, K and Luthman, K 2012. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Advanced Drug Delivery Reviews 64, 280289.
Attene-Ramos, MS, Wagner, ED, Plewa, MJ and Gaskins, HR 2006. Evidence that hydrogen sulfide is a genotoxic agent. Molecular Cancer Research 4, 914.
Awano, N, Wada, M, Mori, H, Nakamori, S and Takagi, H 2005. Identification and functional analysis of Escherichia coli cysteine desulfhydrases. Applied and Environmental Microbiology 71, 41494152.
Bindelle, J, Buldgen, A, Wavreille, J, Agneessens, R, Destain, JP, Wathelet, B and Leterme, P 2007. The source of fermentable carbohydrates influences the in vitro protein synthesis by colonic bacteria isolated from pigs. Animal 1, 11261133.
Bindelle, J, Pieper, R, Montoya, CA, Van Kessel, AG and Leterme, P 2011. Nonstarch polysaccharide-degrading enzymes alter the microbial community and the fermentation patterns of barley cultivars and wheat products in an in vitro model of the porcine gastrointestinal tract. FEMS Microbiology Ecology 76, 553563.
Cardarelli, HR, Martinez, RCR, Albrecht, S, Schols, H, BDGM, Franco, SMI, Saad and Smidt, H 2016. In vitro fermentation of prebiotic carbohydrates by intestinal microbiota in the presence of Lactobacillus amylovorus DSM 16998. Beneficial Microbes 7, 119133.
Christl, SU, Murgatroyd, PR, Gibson, GR and Cummings, JH 1992. Production, metabolism, and excretion of hydrogen in the large intestine. Gastroenterology 102, 12691277.
Cremin, JD, Fitch, MD and Fleming, SE 2003. Glucose alleviates ammonia-induced inhibition of short-chain fatty acid metabolism in rat colonic epithelial cells. American Journal of Physiology - Gastrointestinal and Liver Physiology 285, G105G114.
Fukamachi, H, Nakano, Y, Yoshimura, M and Koga, T 2002. Cloning and characterization of the l-cysteine desulfhydrase gene of Fusobacterium nucleatum . FEMS Microbiology Letters 215, 7580.
Fukushima, RS, Weimer, PJ and Kunz, DA 2003. Use of photocatalytic reduction to hasten preparation of culture media for saccharolytic Clostridium species. Brazilian Journal of Microbiology 34, 2226.
Gomez-Alarcon, RA, O’Dowd, C, Leedle, JA and Bryant, MP 1982. 1,4Naphthoquinone and other nutrient requirements of Succinivibrio dextrinosolvens . Applied and Environmental Microbiology 44, 346350.
Groot, JCJ, Cone, JW, Williams, BA, Debersaques, FMA and Lantinga, EA 1996. Multiphasic analysis of gas production kinetics for in vitro fermentation of ruminant feeds. Animal Feed Science and Technology 64, 7789.
Guo, X, Xia, X, Tang, R, Zhou, J, Zhao, H and Wang, K 2008. Development of a real-time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs. Letters in Applied Microbiology 47, 367373.
Hughes, R, Magee, EA and Bingham, S 2000. Protein degradation in the large intestine: relevance to colorectal cancer. Current Issues in Intestinal Microbiology 1, 5158.
Jha, R, Woyengo, TA, Li, J, Bedford, MR, Vasanthan, T and Zijlstra, RT 2015. Enzymes enhance degradation of the fiber–starch–protein matrix of distillers dried grains with solubles as revealed by a porcine in vitro fermentation model and microscopy. Journal of Animal Science 93, 10391051.
Kim, HB, Borewicz, K, White, BA, Singer, RS, Sreevatsan, S, Tu, ZJ and Isaacson, RE 2011. Longitudinal investigation of the age-related bacterial diversity in the feces of commercial pigs. Veterinary Microbiology 153, 124133.
Koecher, KJ, Noack, JA, Timm, DA, Klosterbuer, AS, Thomas, W and Slavin, JL 2014. Estimation and interpretation of fermentation in the gut: coupling results from a 24 h batch in vitro system with fecal measurements from a human intervention feeding study using fructo-oligosaccharides, inulin, gum acacia, and pea fiber. Journal of Agricultural and Food Chemistry 62, 13321337.
Kumagai, H, Sejima, S, Choi, Y, Tanaka, H and Yamada, H 1975. Crystallization and properties of cysteine desulfhydrase from Aerobacter aerogenes. FEBS Letters 52, 304307.
Leser, TD, Lindecrona, RH, Jensen, TK, Jensen, BB and Møller, K 2000. Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae. Applied and Environmental Microbiology 66, 32903296.
Medani, M, Collins, D, Docherty, NG, Baird, AW, O’Connell, PR and Winter, DC 2011. Emerging role of hydrogen sulfide in colonic physiology and pathophysiology. Inflammatory Bowel Diseases 17, 16201625.
Menke, KH and Steingass, H 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development 28, 755.
Morgan, R, Kliem, KE and Mould, FL 2004. The use of a nitrogen free medium for in vitro fermentation studies. Paper presented at the British Society of Animal Science Annual Meeting, 5–7 April, York, UK, p. 232.
O’Herrin, SM and Kenealy, WR 1993. Glucose and carbon dioxide metabolism by Succinivibrio dextrinosolvens . Applied and Environmental Microbiology 59, 748755.
Payne, AN, Zihler, A, Chassard, C and Lacroix, C 2012. Advances and perspectives in in vitro human gut fermentation modeling. Trends in Biotechnology 30, 1725.
Pieper, R, Bindelle, J, Rossnagel, B, Van Kessel, A and Leterme, P 2009a. Effect of carbohydrate composition in barley and oat cultivars on microbial ecophysiology and proliferation of Salmonella enterica in an in vitro model of the porcine gastrointestinal tract. Applied and Environmental Microbiology 75, 70067016.
Pieper, R, Janczyk, P, Urubschurov, V, Korn, U, Pieper, B and Souffrant, WB 2009b. Effect of a single oral administration of Lactobacillus plantarum DSMZ 8862/8866 before and at the time point of weaning on intestinal microbial communities in piglets. International Journal of Food Microbiology 130, 227232.
Pruesse, E, Quast, C, Knittel, K, Fuchs, BM, Ludwig, W, Peplies, J and Glöckner, FO 2007. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Research 35, 71887196.
Rakoff-Nahoum, S, Coyne, MJ and Comstock, LE 2014. An ecological network of polysaccharide utilization among human intestinal symbionts. Current Biology 24, 4049.
Rodriguez, C, Taminiau, B, Korsak, N, Avesani, V, Van Broeck, J, Brach, P, Delmée, M and Daube, G 2016. Longitudinal survey of Clostridium difficile presence and gut microbiota composition in a Belgian nursing home. BMC Microbiology 16, 229.
Roediger, WE, Moore, J and Babidge, W 1997. Colonic sulfide in pathogenesis and treatment of ulcerative colitis. Digestive Diseases and Sciences 42, 15711579.
Sappok, M, Pellikaan, WF, Verstegen, MW and Sundrum, A 2009. Assessing fibre-rich feedstuffs in pig nutrition: comparison of methods and their potential implications. Journal of the Science of Food and Agriculture 89, 25412550.
Schloss, PD, Westcott, SL, Ryabin, T, Hall, JR, Hartmann, M, Hollister, EB, Lesniewski, RA, Oakley, BB, Parks, DH, Robinson, CJ, Sahl, JW, Stres, B, Thallinger, GG, Van Horn, DJ and Weber, CF 2009. Introducing mothur: open-source, platformindependent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology 75, 75377541.
Su, Y, Bian, G, Zhu, Z, Smidt, H and Zhu, W 2014. Early methanogenic colonisation in the faeces of Meishan and Yorkshire piglets as determined by pyrosequencing analysis. Archaea 2014, 547908.
Theodorou, MK, Williams, BA, Dhanoa, MS, McAllan, AB and France, J 1994. A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Animal Feed Science and Technology 48, 185197.
Tilley, JMA and Terry, RA 1963. A two-stage technique for the in vitro digestion of forage crops. Grass and Forage Science 18, 104111.
Tran, THT, Boudry, C, Everaert, N, Théwis, A, Portetelle, D, Daube, G, Nezer, C, Taminiau, B and Bindelle, J 2015. Adding mucins to an in vitro batch fermentation model of the large intestine induces changes in microbial population isolated from porcine feces depending on the substrate. FEMS Microbial Ecology (,, Published online by Oxford University Press 20 December 2015.
Watanabe, Y, Suzuki, R, Koike, S, Nagashima, K, Mochizuki, M, Forster, RJ and Kobayashi, Y 2010. In vitro evaluation of cashew nut shell liquid as a methane-inhibiting and propionate-enhancing agent for ruminants. Journal of Dairy Science 93, 52585267.
Weiss, E, Rist, V, Rink, F and Mosenthin, R 2015. Discrepancies in porcine microbiota composition between in vivo measurements and in vitro fermentation with the modified Hohenheim Gas Test. Livestock Science 176, 141145.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 1751-7311
  • EISSN: 1751-732X
  • URL: /core/journals/animal
Please enter your name
Please enter a valid email address
Who would you like to send this to? *


Type Description Title
Supplementary materials

Poelaert et al. supplementary material
Table S1 and Figure S1

 Word (29 KB)
29 KB


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed