Isolation and identification of microbial species found in cocoa fermentation as microbial starter culture candidates for cocoa bean fermentation in Colombia.
Microbial diversity in cocoa fermentation in Colombia
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Dec 31, 2020
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Microbial activity involved in the cocoa beans fermentation process is essential to maintain and improve the organoleptic and nutritional qualities of chocolate; therefore, the aim of this investigation was to search and select microbial isolates with the potential to improve the quality of cocoa beans. Fermentation experimentswere conducted on farms located in Maceo (Antioquia), San Vicente de Chucurí (Santander), and Rivera and Algeciras (Huila), Colombia. Yeast, lactic acid bacteria (LAB), acetic acid bacteria (AAB), and mesophilic aerobic microorganisms were obtained from different fermentation batches. The growth of these microorganismswas tested in six treatments as follows: 50% cocoa pulp agar (CPA), high concentrations of glucose (10%), ethanol (5%), and acetic acid (7%), an acidic pH of 3.0, and a high temperature of 50oC for 24 h. The isolates with the highest growth were identified by 18S and 16S rRNA gene analysis, revealing a high diversity ofspecies associated with cocoa fermentation, including eight species of yeasts (Debaryomyces hansenii, Meyerozyma guillermondii, Wickerhanomyces anomalus, Pichia guillermondii, Pichia kudriavzevii, Trichosporon asahii, Candida parapsilosis, and Pichia manshurica), six species of LAB (Pediococcus acidilactici, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus farraginis, Lactobacillus rhamnosus, and Leuconostoc mesenteroides), four species of AAB (Gluconobacter japonicus, Acetobacter tropicalis, Acetobacter pasteurianus, and Acetobacter malorum/tropicalis), and three species of Bacillus spp. (Bacillusaryabhattai /megaterium, Bacillus subtilis, and Bacillus coagulans). In general, microbial populations increased in cocoa batches after 12 h of fermentation and decreased after 84-96 h. All the yeast isolates grew in 10% glucose and CPA, 85.7% in 5% ethanol, and 95% at a pH of 3.0. All the yeast isolates were affectedby 7% acetic acid and incubation at 50oC for 24 h. Eighty-five percent of the LAB grew in 10% glucose, 100% in 5% ethanol, 42.8% in CPA, 64% at a pH of 3.0, and 35.7% grew after being exposed to 50oC for 24 h; all were affected by 7% acetic acid. As for the AAB, 100% grew in 10% glucose, 71% in 7% ethanol, 100% grew in CPA, in 7% acetic acid, and at a pH of 3.0, while 100% were affected by incubation at 50oC. Three yeast isolates, W. anomalus, D. hansenii and M. guillermondii, three LAB isolates, P. acidilactici, L. brevis, and L. plantarum, and three AAB isolates, A. tropicalis, A. pasteurianus and G. japonicus, were selected as promising strains to be used in a microbial starter culture for cocoa bean fermentation to improve the organoleptic quality of cocoa.
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Lozano Tovar, M., Tibasosa, G., González, C., Ballestas Alvarez, K., Lopez Hernandez, M., & Rodríguez Villamizar, F. (2020). Isolation and identification of microbial species found in cocoa fermentation as microbial starter culture candidates for cocoa bean fermentation in Colombia. Pelita Perkebunan (a Coffee and Cocoa Research Journal), 36(3), 236-248. https://doi.org/10.22302/iccri.jur.pelitaperkebunan.v36i3.443
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References
Ardhana, M.M. & G.H. Fleet (2003). The microbial ecology of cocoa bean fermentations in Indonesia. Int. J. Food Microbiol, 86, 87-99.
Barros, R.R.; M.D. Carvalho; J.M. Peralta; R.R. Facklam & L.M. Teixeira (2001). Phenotypic and genotypic characterisation of Pediococcus strains isolated from human clinical sources. JCM, 39, 1241-1246.
Boekhout, T. & J. Phaff (2003). Yeast biodiversity. In: T. Boekhout & V. Robert (Eds.) .Yeasts in food, beneficial and detrimental aspects, p. 1 – 29. CRC Press, Florida.
Breuer, U. & H. Harms (2006). Debaryomyces hansenii—an extremophilic yeast with biotechnological potential. Yeast, 23, 415-437
Camu, N.; T. De Winter; S.K. Addo; J.S. Takrama; H. Bernaert & L. De Vuyst (2008). Fermentation of cocoa beans: influence of microbial activities and polyphenol concentrations on the flavour of chocolate. J Sci Food Agr, 88, 2288-2297.
Caporaso, N.; M. Whitworth; M. Fowler & I. Fisk (2018). Hyperspectral imaging for non-destructive prediction of fermentation index, polyphenol content and antioxidant activity in single cocoa beans. Food Chem. 258, 343-351.
Coda, R.; A. Cassone; C.G. Rizzello; L. Nionelli; G. Cardinali & M. Gobbetti ( 2011). Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: identification of novel compounds and long-term effect during storage of wheat bread. Appl. Environ. Microbiol, 77, 3484–3492.
De Melo, G.V.; K.T. Magalhaes; E.G. de Almeida; I. Da Silva & R.F. Schwan (2013). Spontaneus cocoa bean fermentation carried out in a novel-desing stainless Steel tank: influence on the dynamics of microbial populations and physical-chemical properties. Int. J. Food Microbiol, 161, 121-133.
De Vuyst, L.; T. Lefeber; Z. Papalexandratou & N. Camu (2010). The functional role of lactic acid bacteria in cocoa bean fermentation. In: F. Mozzi; R.R Raya & G.M. Vignolo (Eds.) Biotechnology of lactic acid bacteria: novel applications, p 301–25. Wiley-Blackwell, Oxford.
De Vuyst, L. & S. Weckx (2016). The cocoa bean fermentation process: from ecosystem analysis to starter culture development. J. Appl. Microbiol, 121, 5–17.
Della Riccia, D.N.; F. Bizzini; M. Perilli; A. Polimeni; V. Trinchieri; G. Amicosante & M. Cifone (2007). Anti-inflammatory effects of Lactobacillus brevis (CD2) on periodontal disease. Oral Dis. 13, 376–385.
Duarte, W.F.; D.R. Dias; J.M. Oliveira; J.A. Teixeira; J.B. Almeida & R.F. Schwan (2010). Characterizarion of different fruit wines made from cacao, cupuassu, gabiroba, jaboticaba and umbu. LWT-food Sci Technol, 43, 1564-1572.
Freitas, S. (1998). Cocoa Fermentations Conducted with a Defined Microbial Cocktail Inoculum. Appl. Environ. Microbiol, 64, 1477-1483.
García, J.M.; S.M. Castro; R. Casquete; J. Silva; R. Queirós; J.A. Saraiva & P. Teixeira (2017). Enhancement of bacteriocin production and antimicrobial activity of Pediococcus acidilactici HA-6111-2. Acta Aliment Hung, 46, 92-99.
Guerber, J.C.; B. Liu & J.C. Correll (2003). Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intro RFLPs, and mating compatibility. Mycologia, 95, 872-895.
Hansen, C.E; M. Del Olmo & C. Burri (1998). Enzyme activities in cocoa beans during fermentation. J. Sci. Food Agri, 77, 273-281.
Ho, V.T.; J. Zhao & G. Fleet (2014). Yeast are essential for cocoa bean fermentation. Int. J. Food Microbiol, 174, 72-82.
Lavermicocca, P.; F. Valerio; A. Evidente; S. Lazzaroni; A. Corsetti & M. Gobbetti (2000). Purification and characterization of novel antifungal compounds from the Sourdough Lactobacillus plantarum Strain 21 B. Appl. Environ. Microbiol, 66, 4084-4090.
Lefeber, T.; W. Gobert; G. Vrancken; N. Camu & L.D. Vuyst (2011). Dynamics and species diversity of communities of lactic acid bacteria and acetic acid bacteria during spontaneous cocoa bean fermentation in vessels. Food Microbiol, 28, 457-464.
Lefeber, T.; Z. Papalexandratou; W. Gobert; N. Camu & L. De Vuyst (2012). On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof. Food Microbiol, 30, 379-392
Lima, L.J.R.; M.H. Almeida; M.J. Rob Nout & M.H. Zwietering (2011). Theobroma cacao L., “the food of the gods”: Quality determinants of commercial cocoa beans, with particular reference to the impact of fermentation. Crit Rev Food Sci Nutr, 51, 731-761.
López, M.; J. Criollo; M. Hernández; M.D. Lozano-Tovar (2019). Physicochemical and microbiological dynamics of the fermentation of the CCN51 cocoa material in three maturity stages. Rev.Bras. Frutic, 41, 1-13.
Mancini, A. & F. Fava (2016). Probiotic potential of a high GABA producing strain, Lactobacillus brevis FEM 1874, isolated from traditional “wild” Alpine cheese. J. Clin. Gastroenterol, 50, 220–221.
Meersman, E.; J. Steensels; T. Paulus; N. Struyf; V. Saels; M. Mathawan & K.J. Verstrepen (2015). Breeding strategy to generate robust yeast starter cultures for cocoa pulp fermentations. Appl. Environ. Microbiol, 81, 6166-6176.
Meersman, E.; J. Steensels; N. Struyt; T. Paulus; V. Saels; M. Mathawan; L. Allegaert; G. Vrancken & K. Verstrepen (2015a). Tuning chocolate flavor through development of thermotolerant Saccharomyces cerevisiae starter cultures with increased acetate ester production. Appl. Environ. Microbiol, 82, 732-746.
Merrifield, D.L. (2013). Probiotic Pediococcus acidilactici modulates both localised intestinal-and peripheral-immunity in tilapia (Oreochromis niloticus). Fish Shellfish Immun. 35, 1097–1104.
Moens, F.; T. Lefeber & L. De Vuyst (2014). Oxidation of metabolites highlights the microbial interactions and role of Acetobacter pasteurianus during cocoa bean fermentation. Appl. Environ. Microbiol, 80, 1848–1857
Moreira, I.M.; M.G. Miguel; W.F. Duarte; D.R. Dias & R.F. Schwan (2013). Microbial succession and the dynamics of metabolites and sugars during the fermentation of three different cocoa (Theobroma cacao L.) hydrids. Food Res Int, 54, 9-17.
Nakayan, P; A. Hameed; S. Singh; L. Young; M. Hung & C. Youn (2013). Phosphate-solubilizing soil yeast Meyerozyma guillermondii CC1 improves maize (Zea mays L.) productivity and minimizes requisite chemical fertilization. Plant Soil, 373, 1-2.
Navarro, D.; E. Mateo; M. Torija & A. Mas (2013). Acetic acid bacteria in grape must. Acetic Acid Bacteria, 2, 19-23.
Nguyen, T.D.; J.H. Kang & M.S. Lee (2007). Characterization of Lactobacillus plantarum PHO4, a potential probiotic bacterium with cholesterol-lowering effects. Int J Food Microbiol, 113, 358-361.
Nielsen, D.S.; O.D. Teniola; L. Ban-Koffi; M. Owusu; T.S. Andersson & W.H. Holzapfel (2007). The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods. Int J Food Microbiol, 114, 168–186.
Ohmori, S.; T. Uozumi & T. Beppu (1982). Loss of acetic acid resistence and etanol oxidizing ability in an Acetobacter strain. Agric. Biol. Chem, 46, 381-389.
Pelaez, P.; S. Guerra & D. Contreras (2016). Changes in physical and chemical characteristics of fermented cocoa (Theobroma cacao) beans with manual and simi-mechanized transfer, between fermentation boxes. Scientia Agropecuaria, 7, 111-119.
Rodríguez, S.Y. (2008). Identificación molecular y establecimiento del código de barras, Bar Code, de levaduras nativas. Pontificia Universidad Javeriana - Puj - Sede Bogotá
Ronka, E.; E. Malinen; M. Saarela; M. Rinta-Koski; J. Aarnikunnas & A. Palva (2003). Probiotic and milk technological properties of Lactobacillus brevis. Int J Food Microbiol, 83, 63-74.
Schultz, M.; C. Vektkamp; L. Dieleman; W. Grenther; P. Wyrick; S. Tonkonogy & S. Sartor (2002). Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice. Inflamm Bowel Dis, 8, 71-80.
Schwan, R. & A. Wheals (2004). The microbiology of cocoa fermentation and its role in chocolate quality. Crit Rev Food Sci Nutr, 44, 205-221.o
Ström, K.; J. Sjorgen; A. Broberg & J. Schnurer (2002). Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo (L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid. Applied Environ. Microbiol, 68, 4322-4327.
Sunoj, S.; C. Igathinathane & R. Visvanathan (2016). Nondestructive determination of cocoa bean quality using FT-NIR spectroscopy. Compu Electron Agr, 124, 234-242.
Syukur, S.; B. Bisping; Z. Noli & E. Purwati (2013). Antimicrobial properties and lactase activities from selected probiotic Lactobacillus brevis associated with green cacao fermentation in west Sumatra, Indonesia. J Prob Health, 1, 4.
Thompson, S.S.; K.B. Miller; A. Lopez & N. Camu (2013). Cocoa and coffee. In: M.P. Doyle & R. L. Beuchat (Eds.) Food. Microbiology: Fundamentals and Frontiers, 4th edition.p 881-889. ASM Press, Washington DC.
Tortoló, C.K. & G.A. Bell (2015). Producción de proteínas recombinantes en Bacillus megaterium: estado del arte ICIDCA. Sobre los Derivados de la Caña de Azúcar. 49, 22-26.
Wullt, M.; M.L. Hagslatt & I. Odenhoit (2003). Lactobacillus plantarum 299v for the treatment of recurrent Clostridium difficile-associated diarrhoea: a double-blind, placebo-, controlled trial. Scand J. Infect Dis. 35, 365-367.
Barros, R.R.; M.D. Carvalho; J.M. Peralta; R.R. Facklam & L.M. Teixeira (2001). Phenotypic and genotypic characterisation of Pediococcus strains isolated from human clinical sources. JCM, 39, 1241-1246.
Boekhout, T. & J. Phaff (2003). Yeast biodiversity. In: T. Boekhout & V. Robert (Eds.) .Yeasts in food, beneficial and detrimental aspects, p. 1 – 29. CRC Press, Florida.
Breuer, U. & H. Harms (2006). Debaryomyces hansenii—an extremophilic yeast with biotechnological potential. Yeast, 23, 415-437
Camu, N.; T. De Winter; S.K. Addo; J.S. Takrama; H. Bernaert & L. De Vuyst (2008). Fermentation of cocoa beans: influence of microbial activities and polyphenol concentrations on the flavour of chocolate. J Sci Food Agr, 88, 2288-2297.
Caporaso, N.; M. Whitworth; M. Fowler & I. Fisk (2018). Hyperspectral imaging for non-destructive prediction of fermentation index, polyphenol content and antioxidant activity in single cocoa beans. Food Chem. 258, 343-351.
Coda, R.; A. Cassone; C.G. Rizzello; L. Nionelli; G. Cardinali & M. Gobbetti ( 2011). Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: identification of novel compounds and long-term effect during storage of wheat bread. Appl. Environ. Microbiol, 77, 3484–3492.
De Melo, G.V.; K.T. Magalhaes; E.G. de Almeida; I. Da Silva & R.F. Schwan (2013). Spontaneus cocoa bean fermentation carried out in a novel-desing stainless Steel tank: influence on the dynamics of microbial populations and physical-chemical properties. Int. J. Food Microbiol, 161, 121-133.
De Vuyst, L.; T. Lefeber; Z. Papalexandratou & N. Camu (2010). The functional role of lactic acid bacteria in cocoa bean fermentation. In: F. Mozzi; R.R Raya & G.M. Vignolo (Eds.) Biotechnology of lactic acid bacteria: novel applications, p 301–25. Wiley-Blackwell, Oxford.
De Vuyst, L. & S. Weckx (2016). The cocoa bean fermentation process: from ecosystem analysis to starter culture development. J. Appl. Microbiol, 121, 5–17.
Della Riccia, D.N.; F. Bizzini; M. Perilli; A. Polimeni; V. Trinchieri; G. Amicosante & M. Cifone (2007). Anti-inflammatory effects of Lactobacillus brevis (CD2) on periodontal disease. Oral Dis. 13, 376–385.
Duarte, W.F.; D.R. Dias; J.M. Oliveira; J.A. Teixeira; J.B. Almeida & R.F. Schwan (2010). Characterizarion of different fruit wines made from cacao, cupuassu, gabiroba, jaboticaba and umbu. LWT-food Sci Technol, 43, 1564-1572.
Freitas, S. (1998). Cocoa Fermentations Conducted with a Defined Microbial Cocktail Inoculum. Appl. Environ. Microbiol, 64, 1477-1483.
García, J.M.; S.M. Castro; R. Casquete; J. Silva; R. Queirós; J.A. Saraiva & P. Teixeira (2017). Enhancement of bacteriocin production and antimicrobial activity of Pediococcus acidilactici HA-6111-2. Acta Aliment Hung, 46, 92-99.
Guerber, J.C.; B. Liu & J.C. Correll (2003). Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intro RFLPs, and mating compatibility. Mycologia, 95, 872-895.
Hansen, C.E; M. Del Olmo & C. Burri (1998). Enzyme activities in cocoa beans during fermentation. J. Sci. Food Agri, 77, 273-281.
Ho, V.T.; J. Zhao & G. Fleet (2014). Yeast are essential for cocoa bean fermentation. Int. J. Food Microbiol, 174, 72-82.
Lavermicocca, P.; F. Valerio; A. Evidente; S. Lazzaroni; A. Corsetti & M. Gobbetti (2000). Purification and characterization of novel antifungal compounds from the Sourdough Lactobacillus plantarum Strain 21 B. Appl. Environ. Microbiol, 66, 4084-4090.
Lefeber, T.; W. Gobert; G. Vrancken; N. Camu & L.D. Vuyst (2011). Dynamics and species diversity of communities of lactic acid bacteria and acetic acid bacteria during spontaneous cocoa bean fermentation in vessels. Food Microbiol, 28, 457-464.
Lefeber, T.; Z. Papalexandratou; W. Gobert; N. Camu & L. De Vuyst (2012). On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof. Food Microbiol, 30, 379-392
Lima, L.J.R.; M.H. Almeida; M.J. Rob Nout & M.H. Zwietering (2011). Theobroma cacao L., “the food of the gods”: Quality determinants of commercial cocoa beans, with particular reference to the impact of fermentation. Crit Rev Food Sci Nutr, 51, 731-761.
López, M.; J. Criollo; M. Hernández; M.D. Lozano-Tovar (2019). Physicochemical and microbiological dynamics of the fermentation of the CCN51 cocoa material in three maturity stages. Rev.Bras. Frutic, 41, 1-13.
Mancini, A. & F. Fava (2016). Probiotic potential of a high GABA producing strain, Lactobacillus brevis FEM 1874, isolated from traditional “wild” Alpine cheese. J. Clin. Gastroenterol, 50, 220–221.
Meersman, E.; J. Steensels; T. Paulus; N. Struyf; V. Saels; M. Mathawan & K.J. Verstrepen (2015). Breeding strategy to generate robust yeast starter cultures for cocoa pulp fermentations. Appl. Environ. Microbiol, 81, 6166-6176.
Meersman, E.; J. Steensels; N. Struyt; T. Paulus; V. Saels; M. Mathawan; L. Allegaert; G. Vrancken & K. Verstrepen (2015a). Tuning chocolate flavor through development of thermotolerant Saccharomyces cerevisiae starter cultures with increased acetate ester production. Appl. Environ. Microbiol, 82, 732-746.
Merrifield, D.L. (2013). Probiotic Pediococcus acidilactici modulates both localised intestinal-and peripheral-immunity in tilapia (Oreochromis niloticus). Fish Shellfish Immun. 35, 1097–1104.
Moens, F.; T. Lefeber & L. De Vuyst (2014). Oxidation of metabolites highlights the microbial interactions and role of Acetobacter pasteurianus during cocoa bean fermentation. Appl. Environ. Microbiol, 80, 1848–1857
Moreira, I.M.; M.G. Miguel; W.F. Duarte; D.R. Dias & R.F. Schwan (2013). Microbial succession and the dynamics of metabolites and sugars during the fermentation of three different cocoa (Theobroma cacao L.) hydrids. Food Res Int, 54, 9-17.
Nakayan, P; A. Hameed; S. Singh; L. Young; M. Hung & C. Youn (2013). Phosphate-solubilizing soil yeast Meyerozyma guillermondii CC1 improves maize (Zea mays L.) productivity and minimizes requisite chemical fertilization. Plant Soil, 373, 1-2.
Navarro, D.; E. Mateo; M. Torija & A. Mas (2013). Acetic acid bacteria in grape must. Acetic Acid Bacteria, 2, 19-23.
Nguyen, T.D.; J.H. Kang & M.S. Lee (2007). Characterization of Lactobacillus plantarum PHO4, a potential probiotic bacterium with cholesterol-lowering effects. Int J Food Microbiol, 113, 358-361.
Nielsen, D.S.; O.D. Teniola; L. Ban-Koffi; M. Owusu; T.S. Andersson & W.H. Holzapfel (2007). The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods. Int J Food Microbiol, 114, 168–186.
Ohmori, S.; T. Uozumi & T. Beppu (1982). Loss of acetic acid resistence and etanol oxidizing ability in an Acetobacter strain. Agric. Biol. Chem, 46, 381-389.
Pelaez, P.; S. Guerra & D. Contreras (2016). Changes in physical and chemical characteristics of fermented cocoa (Theobroma cacao) beans with manual and simi-mechanized transfer, between fermentation boxes. Scientia Agropecuaria, 7, 111-119.
Rodríguez, S.Y. (2008). Identificación molecular y establecimiento del código de barras, Bar Code, de levaduras nativas. Pontificia Universidad Javeriana - Puj - Sede Bogotá
Ronka, E.; E. Malinen; M. Saarela; M. Rinta-Koski; J. Aarnikunnas & A. Palva (2003). Probiotic and milk technological properties of Lactobacillus brevis. Int J Food Microbiol, 83, 63-74.
Schultz, M.; C. Vektkamp; L. Dieleman; W. Grenther; P. Wyrick; S. Tonkonogy & S. Sartor (2002). Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice. Inflamm Bowel Dis, 8, 71-80.
Schwan, R. & A. Wheals (2004). The microbiology of cocoa fermentation and its role in chocolate quality. Crit Rev Food Sci Nutr, 44, 205-221.o
Ström, K.; J. Sjorgen; A. Broberg & J. Schnurer (2002). Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo (L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid. Applied Environ. Microbiol, 68, 4322-4327.
Sunoj, S.; C. Igathinathane & R. Visvanathan (2016). Nondestructive determination of cocoa bean quality using FT-NIR spectroscopy. Compu Electron Agr, 124, 234-242.
Syukur, S.; B. Bisping; Z. Noli & E. Purwati (2013). Antimicrobial properties and lactase activities from selected probiotic Lactobacillus brevis associated with green cacao fermentation in west Sumatra, Indonesia. J Prob Health, 1, 4.
Thompson, S.S.; K.B. Miller; A. Lopez & N. Camu (2013). Cocoa and coffee. In: M.P. Doyle & R. L. Beuchat (Eds.) Food. Microbiology: Fundamentals and Frontiers, 4th edition.p 881-889. ASM Press, Washington DC.
Tortoló, C.K. & G.A. Bell (2015). Producción de proteínas recombinantes en Bacillus megaterium: estado del arte ICIDCA. Sobre los Derivados de la Caña de Azúcar. 49, 22-26.
Wullt, M.; M.L. Hagslatt & I. Odenhoit (2003). Lactobacillus plantarum 299v for the treatment of recurrent Clostridium difficile-associated diarrhoea: a double-blind, placebo-, controlled trial. Scand J. Infect Dis. 35, 365-367.
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