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1 Laboratoire de Microbiologie Alimentaire et Industrielle (LMAI), Unité de Recherche QM2A, ENITIAA, rue de la Géraudière, BP 82225, 44322 Nantes Cedex 3, France
2 Department of Microbiology, Faculty of Biology, University Sofia, 8 Dragan Tzankov Street, 1162 Sofia, Bulgaria
3 Lehrstuhl für Technische Mikrobiologie, Technische Universität München, Weihenstephaner Steig 16, 85350 Freising, Germany
Correspondence
Maher Korakli
maher.korakli{at}wzw.tum.de
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Published online ahead of print on 4 November 2005 as DOI 10.1099/ijs.0.63619-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LP33T is AY690834.
RAPD and AFLP patterns of Lactobacillus nantensis LP33T and selected reference sourdough lactobacilli are shown in supplementary figures available in IJSEM Online.
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). Because of the superior sensory quality and the prolonged shelf life of the resulting baked goods, sourdough processes have retained their importance in modern baking technology (Stolz et al., 1996; Ottogalli et al., 1996). Yeasts contribute predominantly to dough leavening, whereas LAB are responsible for acidification, aroma formation and sensorial as well as nutritional improvement of the fermented product (Vogel et al., 1999; Böcker et al., 1990). Knowledge regarding LAB diversity is thus essential in sourdough investigations.
Recent studies of the LAB microbiota of sourdoughs have employed traditional cultivation methods in combination with phenotypic (physiological and biochemical) and/or genotypic [e.g. randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) analyses] identification methods (Galli et al., 1988; Mäntynen et al., 1999; Rocha & Malcata, 1999; Corsetti et al., 2001; Bervas, 1991; Onno & Roussel, 1994; Gabriel et al., 1999; Valcheva et al., 2005). The majority of LAB isolated from sourdoughs belong to the genus Lactobacillus, and more than 50 Lactobacillus species have been isolated in relevant cell counts from sourdough. Sourdoughs prepared by traditional procedures, type I sourdough, are characterized by the association between homo- and heterofermentative lactobacilli (Vogel et al., 1999). Lactobacillus sanfranciscensis is most frequently isolated and was thus designated as a key organism in type I sourdough. The homofermentative species Lactobacillus plantarum and Lactobacillus alimentarius are also usually isolated (Vogel et al., 1999; Corsetti et al., 2001; De Vuyst et al., 2002; Valcheva et al., 2005). Industrial fermentations are carried out at elevated temperature and over prolonged periods. In these fermentations, producing type II sourdough, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus acidophilus and Lactobacillus amylovorus are often isolated (Vogel et al., 1999; Hammes & Gänzle, 1998; Meroth et al., 2003). Recent aims to characterize sourdough microflora by using a polyphasic taxonomic approach have resulted in the description of several novel Lactobacillus species, namely Lactobacillus frumenti (Müller et al., 2000), L. mindensis (Ehrmann et al., 2003), L. spicheri (Meroth et al., 2004), L. hammesii (Valcheva et al., 2005), L. zymae and L. acidifarinae (Vancanneyt et al., 2005) and L. rossii (Corsetti et al., 2005).
In the present study, the LAB of a traditional French wheat sourdough were investigated. A preliminary screening based on observed phenotypic characteristics led to the isolation of 30 bacterial strains. Additional information regarding the taxonomic status at species and strain level was obtained by amplified-fragment length polymorphism (AFLP) and RAPD analyses. These methods allowed the identification to species level of almost all LAB involved in this sourdough fermentation. However, a consistent group of 14 strains could be clearly discriminated from recognized Lactobacillus species. On the basis of the phenotypic and genotypic results obtained from a representative strain of this group, LP33T, a novel Lactobacillus species is described.
The traditional French wheat sourdough used for this study was a firm sourdough originating from initially spontaneous fermentation of flour and water (dough yield 160 kg dough per 100 kg flour) at 18–20 °C. It was maintained by back slopping (repeated cycles of re-inoculation of flour, water and 30 % inoculum from the previous fermentation) under the same conditions. Sourdough samples used for bread preparation were taken aseptically, stored at 4 °C and analysed immediately. The LAB population was enumerated as described by Valcheva et al. (2005). After 48–72 h incubation at 30 °C under anaerobic conditions, the number of colonies with similar morphology and the total number of LAB (as c.f.u. g–1) were estimated. The distribution of various colony forms was recorded and the relative distribution of the various isolates was determined. Thirty colonies representing these different morphological forms were picked and purified on the same medium by successive subculturing at 30 °C. Pure isolates were stored in 20 % (w/v) glycerol stock cultures at –80 °C.
Lactobacillus reference strains used were L. alimentarius LMG 9187T (=DSM 20249T), L. alimentarius LMG 9188t2, Lactobacillus brevis DSM 20054T, L. farciminis LMG 9200T (=DSM 20184T), L. hammesii DSM 16381T, Lactobacillus kimchii DSM 13961T, L. kimchii LMG 19822T, L. mindensis DSM 14500T (=LMG 21932T), L. paralimentarius DSM 13238T (=LMG 19152T), L. sanfranciscensis DSM 20451T, L. spicheri DSM 15429T and L. versmoldensis LMG 21929T (=DSM 14857T). They were grown anaerobically on mMRS4 medium at the appropriate temperature (Stolz et al., 1996). All tests were performed at 30 °C unless otherwise stated. Colony morphology, Gram staining and cellular morphology were determined on cells grown anaerobically on mMRS4 incubated for 2 days. All tests for biochemical characterizations were carried out at least in duplicate, and as described by Valcheva et al. (2005).
DNA was isolated according to the method of Marmur (1961) with the modification described by Ehrmann et al. (2003). The isolated DNA was used for 16S rRNA gene sequence amplification and for RAPD analysis. The complete 16S rRNA gene was amplified using primers fD1 and rD1 as described by Weisburg et al. (1991). A clone library of the 16S rRNA gene amplified with primers fD1 and rD1 of strain LP33T was constructed in Escherichia coli JM109 using the pDrive cloning kit (Qiagen) and the insert of positive clones was sequenced. Primers T7-pro and SP6 flanking the multiple cloning site of pDrive DNA were used for sequencing. Internal primers 609R, 612R, 607R, 606R and 607V were additionally used for sequence verification (Ehrmann et al., 2003). The complete 16S rRNA gene sequence of LP33T was included in the phylogenetic analysis. A phylogenetic tree was constructed according to the neighbour-joining method using BioNumerics software (Applied Maths). RAPD-PCR was carried out as described by Ehrmann et al. (2003). The AFLP patterns of the sourdough isolates were compared with those of the reference strains. AFLP analysis was performed according to a modification of the procedure described by Gevers et al. (2001). DNA–DNA relatedness analysis was carried out as described by De Ley et al. (1970), with the modifications described by Escara & Hutton (1980) and Huß et al. (1983), and was performed using the fluorometric method as described by Ezaki et al. (1989). AFLP and DNA–DNA hybridization experiments were carried out in duplicate by BCCMTM/LMG. Cell wall composition and DNA G+C content were determined by the DSMZ according to the methods of Schleifer & Kandler (1972) and Mesbah & Whitman (1989), respectively.
The LAB microflora of the sourdough described above was determined qualitatively and quantitatively. This sourdough had a pH of 3·65 and total titratable acidity (TTA) of 26·6 ml (10 g)–1. The LAB population was 1·7x109 c.f.u. g–1. Thirty morphologically different colonies were isolated and were further characterized morphologically and physiologically. On this basis, the bacterial population was divided into three groups of Gram-positive, catalase-negative rods: (i) heterofermentative with growth at 15 °C but not at 45 °C, (ii) homofermentative with growth at 15 and 45 °C and (iii) homofermentative with growth at 15 °C but not at 45 °C. Additionally, these isolates were clustered based on RFLP analysis of 16S–23S rRNA intergenic spacer regions (data not shown). According to RFLP and phenotypic analysis, the Lactobacillus microflora of the sourdough was characterized as a consortium of 37 % L. hammesii (11 isolates), 17 % L. plantarum (five isolates) and 46 % unknown (14 isolates). One representative strain of each cluster was subsequently identified based on 16S rRNA gene sequencing, RAPD and AFLP analyses. The results confirmed the initially presumed phylogenetic status of the first two groups of isolates as representing L. hammesii and L. plantarum. RAPD and carbohydrate fermentation patterns of five selected strains of the third group were identical, indicating that at least five of the 14 isolates represent the same species. Isolate LP33T, representative of the third group, was not assignable to any recognized species and was therefore included in a polyphasic approach using RAPD and AFLP genotyping methods. Strategies combining different typing methods, e.g. SDS-PAGE of cellular proteins, RAPD-PCR and AFLP, have increasingly been used to identify closely related species and strains (Valcheva et al., 2005; Gancheva et al., 1999). RAPD-PCR of isolate LP33T and 11 reference Lactobacillus species usually found in sourdough was performed. The dendrogram thus constructed clearly indicated the separate taxonomic position of strain LP33T. Similarly, strain LP33T was included in AFLP analysis. The AFLP dendrogram constructed placed strain LP33T in a cluster together with L. kimchii LMG 19822T, L. paralimentarius LMG 19152T and seven isolates of L. paralimentarius. The RAPD and AFLP patterns are available as Supplementary Figs S1 and S2 in IJSEM Online.
The complete sequence (1561 bp) of the 16S rRNA gene sequence of LP33T was determined. In a neighbour-joining dendrogram based on the sequence of LP33T from this study and sequences from the GenBank database, the phylogenetic position of LP33T was determined. LP33T was placed within the L. plantarum group and was phylogenetically most closely related to L. farciminis, L. alimentarius, L. paralimentarius, L. mindensis and L. versmoldensis (Fig. 1). 16S rRNA gene sequence similarity between LP33T and L. farciminis DSM 20184T, L. mindensis DSM 14500T and L. paralimentarius DSM 13238T was 97·5, 97·4 and 96·7 %, respectively.
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), indicating that strain LP33T represents a separate genomic species. The DNA G+C content of LP33T was 38·6 mol%, which is within the range 32–55 mol% reported for Lactobacillus species (Kandler & Weiss, 1986). Analysis of the cell wall composition of strain LP33T revealed the presence of lysine and aspartic acid, indicating A4
L-lys–D-Asp peptidoglycan type.
Cells of strain LP33T were Gram-positive, non-motile, lacked endospores and had irregular internal granulation as revealed by methylene blue staining. After incubation on MRS agar for 3 days, colonies were white, circular to slightly irregular, convex, with a smooth to rough surface and diameter of 0·8–1·5 mm. Strain LP33T exhibited no oxidase or catalase activities. In mMRS4 at pH 6·5 and 30 °C, the specific growth rate of strain LP33T was 0·48±0·01 h–1. The optimal temperature for growth was in the range 30–35 °C; the specific growth rate was only 46 and 44 % (100 % at 30 °C) at 20 and 40 °C, respectively. LP33T grew well at initial pH values of 4·4–7·7; the specific growth rate was only 4 and 22 % (100 % at pH 6·5) at pH 3·0 and 9·2, respectively. LP33T grew well at an NaCl content of up to 2 %; the specific growth rate was 73 and 56 % (100 % without NaCl) at 3 and 5 % NaCl, respectively. The carbohydrate fermentation patterns of strain LP33T are given in Table 1.
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Description of Lactobacillus nantensis sp. nov.
Lactobacillus nantensis (nan.ten'sis. M.L. masc. adj. nantensis pertaining to Nantes, from where the first strain of this species was isolated).
Cells are Gram-positive, 2–5 µm long and 1·0 µm wide, non-motile, non-spore-forming rods with irregular internal granulation. They occur singly, in pairs or in chains. Colonies are small (1·5 mm), circular to slightly irregular, convex, with a smooth to rough surface and white when grown on MRS agar. Cells grow well in liquid and solid MRS under anaerobic conditions. The optimum growth temperature is 30 °C and the optimal initial pH is 4·4–7·7. Strain LP33T is able to grow at up to 8 % NaCl. Cells are catalase- and oxidase-negative. Glucose is metabolized homofermentatively and lactate is the sole final product. Strain LP33T produces L(+)- and D(–)-lactic acid. Acid is produced from glucose, fructose, mannose, galactose, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, 
-gentiobiose, tagatose, mannitol, sorbitol, methyl -D-mannoside, N-acetylglucosamine, amygdalin, arbutin, aesculin and salicin. Ribose is weakly fermented. Ammonia is not produced from arginine. Citrate is utilized weakly in the absence of glucose.
The type strain is LP33T (=DSM 16982T=CIP 108546T=TMW 1.1265T).
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ACKNOWLEDGEMENTS |
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