The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of carbohydrates

Interactions betweenLactobacillus brevis subsp.lindneri CB1,L. plantarum DC400,Saccharomyces cerevisiae 141 andS.exiguus M14 from sourdoughs were studied in a co-culture model system using a synthetic medium. The lack of competition for maltose whenS.exiguus M14 was present in co-culture with each of the lactic acid bacteria (LAB) enhanced the bacterial cell yield and lactic and acetic acid production.L.brevis subsp.lindneri CB1 resting cells hydrolysed maltose and accumulated glucose in the medium, allowing the growth of maltose negative yeast.S.cerevisiae 141 competed greatly with each of the LAB for glucose and only withL.plantarum DC400 for fructose, causing a decrease in the bacterial cell number and in acid production. As a result of the glucose and fructose availability after the invertase activity of both yeasts,L.plantarum DC400 grew optimally in the presence of sucrose as a carbon source. All of the interactions indicated were confirmed by studying the behaviour of the co-cultures in wheat flour hydrolysate.     

Taxonomic Structure and Monitoring of the Dominant Population of Lactic Acid Bacteria during Wheat Flour Sourdough Type I Propagation Using Lactobacillus sanfranciscensis Starters

The structure and stability of the dominant lactic acid bacterium population were assessed during wheat flour sourdough type I propagation by using singly nine strains of Lactobacillus sanfranciscensis. Under back-slopping propagation with wheat flour type 0 F114, cell numbers of presumptive lactic acid bacteria varied slightly between and within starters. As determined by randomly amplified polymorphic DNA-PCR and restriction endonuclease analysis-pulsed-field gel electrophoresis analyses, only three (LS8, LS14, and LS44) starters dominated throughout 10 days of propagation. The others progressively decreased to less than 3 log CFU g⁻¹. Partial sequence analysis of the 16S rRNA and recA genes and PCR-denaturating gradient gel electrophoresis analysis using the rpoB gene allowed identification of Weissella confusa, Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rossiae, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Pediococcus pentosaceus, and Lactobacillus spp. as the dominant species of the raw wheat flour. At the end of propagation, one autochthonous strain of L. sanfranciscensis was found in all the sourdoughs. Except for L. brevis, strains of the above species were variously found in the mature sourdoughs. Persistent starters were found in association with other biotypes of L. sanfranciscensis and with W. confusa or L. plantarum. Sourdoughs were characterized for acidification, quotient of fermentation, free amino acids, and community-level catabolic profiles by USING Biolog 96-well Eco microplates. In particular, catabolic profiles of sourdoughs containing persistent starters behaved similarly and were clearly differentiated from the others. The three persistent starters were further used for the production of sourdoughs and propagated by using another wheat flour whose lactic acid bacterium population in part differed from the previous one. Also, in this case all three starter strains persisted during propagation.     

Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy)

A total of 41 strains of lactic acid bacteria (LAB) isolated from durum wheat sourdoughs used to produce Cornetto di Matera bread, were identified by SDS-PAGE of whole cell proteins (WCP) and screened for acid production ability, antimicrobial activity and exopolysaccharide (EPS) production. The isolates were identified as Lactobacillus plantarum (49%), Leuconostoc mesenteroides (17%), Lactobacillus curvatus (15%), Lactobacillus paraplantarum (12%), Weissella cibaria (5%) and Lactobacillus pentosus (2%). Several strains of Lb. plantarum and Leuc. mesenteroides showed a high acid production ability. The antagonistic activity was tested using an agar-spot deferred antagonism assay against a set of five indicators. The species had different profiles of inhibition. Lb. plantarum had the largest spectrum of inhibition, while no isolates of W. cibaria and Leuc. mesenteroides showed antimicrobial activity. No strains had antimicrobial activity against Bacillus cereus. The inhibitory activity of five strains was confirmed to be sensitive to proteolytic enzymes and thus potentially due to bacteriocin production. All Leuc. mesenteroides and W. cibaria strains produced EPS from sucrose. Some Lb. plantarum and Lb. paraplantarum strains produced EPS from different sugars in solid media. EPS production in liquid media was different within the species, with the highest production in liquid media containing glucose and maltose. A defined strain starter culture (W. cibaria DBPZ1006, Lb. plantarum DBPZ1015 and S. cerevisiae MTG10) was selected on the basis of technological properties and tested in model sourdough fermentations.     

DNA Fingerprinting of Lactic Acid Bacteria in Sauerkraut Fermentations

Previous studies using traditional biochemical identification methods to study the ecology of commercial sauerkraut fermentations revealed that four species of lactic acid bacteria, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis, were the primary microorganisms in these fermentations. In this study, 686 isolates were collected from four commercial fermentations and analyzed by DNA fingerprinting. The results indicate that the species of lactic acid bacteria present in sauerkraut fermentations are more diverse than previously reported and include Leuconostoc citreum, Leuconostoc argentinum, Lactobacillus paraplantarum, Lactobacillus coryniformis, and Weissella sp. The newly identified species Leuconostoc fallax was also found. Unexpectedly, only two isolates of P. pentosaceus and 15 isolates of L. brevis were recovered during this study. A better understanding of the microbiota may aid in the development of low-salt fermentations, which may have altered microflora and altered sensory characteristics.     

Characterization of Lactose Utilization and β-Galactosidase in Lactobacillus brevis KB290, the Hetero-Fermentative Lactic Acid Bacterium

Unlike dairy lactic acid bacteria, Lactobacillus brevis cannot ferment milk. We characterized the lactose utilization by L. brevis KB290. In a carbohydrate fermentation assay using API 50 CHL, we showed during 7?days L. brevis did not ferment lactose. L. brevis grew to the stationary phase in 2?weeks in MRS broth containing lactose as the carbon source. L. brevis slowly consumed the lactose in the medium. L. brevis hydrolyzed lactose and a lactose analog, o-nitrophenyl-β-d-galactopyranoside (ONPGal). This β-galactosidase activity for ONPGal was not repressed by glucose, galactose, fructose, xylose, or maltose showing the microorganism may not have carbon catabolite repression. We purified the L. brevis β-galactosidase using ammonium sulfate precipitation and several chromatographies. The enzyme’s molecular weight is estimated at 72 and 37?kDa using SDS-PAGE analysis. The N-terminal amino acid sequence of the larger protein was 90?% similar to the sequence of the putative β-galactosidase (YP_796339) and the smaller protein was identical to the sequence of the putative β-galactosidase (YP_796338) in L. brevis ATCC367. This suggests the enzyme is a heterodimeric β-galactosidase. The specific activity of the purified enzyme for lactose is 55?U/mg. We speculate inhibition of lactose transport delays the lactose utilization in L. brevis KB290.     

Maltose-fructose co-fermentation by Lactobacillus brevis subsp. lindneri CB1 fructose-negative strain

The Lactobacillus brevis subsp. lindneri CB1 fructose-negative strain utilized fructose in co-fermentation with maltose or glucose. Compared to the maltose (17 g/l) fermentation, the simultaneous fermentation of maltose (10 g/l) and fructose (7 g/l) increased cell yield (A ₆₂₀from 2.6 to 3.3) and the concentrations of lactic acid and especially of acetic acid (from 2.45 g/l to 3.90 g/l), produced mannitol (1.95 g/l) and caused a decrease in the amount of ethanol (from 0.46 g/l to 0.08 g/l). The utilization of fructose depended on the continuous presence of maltose in the growth medium and the two carbohydrates were consumed in a molar ratio of about 2:1. The presence of tagatose (a fructose stereoisomer) partially inhibited fructose consumption and consequently caused a decrease of the end products of the co-metabolism. Since maltose was naturally present during sourdough fermentation, the addition of only 6 g fructose/kg wheat dough enabled the co-fermentation of maltose and fructose by L. brevis subsp. lindneri CB1. A higher titratable acidity and acetic acid concentration, and a reduced quotient of fermentation (2.7) were obtained by co-fermentation compared with normal sourdough fermentation. Some interpretations of the maltose-fructose co-fermentation are given.     

Identification and characterization of lactic acid bacteria in ragi tape

One hundred and eighteen lactic acid bacteria (LAB) were isolated from five different types of ragi tape, a traditional dry-starter of Balinese rice wine. The isolates could be classified into three groups based on the cell shape and capability to produce gas from glucose. Group I contained 66 homofermentative cocci, group II contained seven homofermentative rods, and group III contained 45 heterofermentative rods. Among these 118 isolates, 21 isolates representing these groups were selected and were first identified using phenotypic characters. The identification performed phenotypically was confirmed by sequencing of variable region 8 (V8) of the 16S rDNA. The comparative studies led to the identification of Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Weissella confusa, and W. paramesenteroides from the ragi tape examined.     

Production of lactic acid from date juice extract with free cells of single and mixed cultures of Lactobacillus casei and Lactococcus lactis

The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3 g l⁻¹) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46 g l⁻¹, respectively. In the case of single Lactobacillus casei or Lactococcus lactis, the total percentage of glucose and fructose utilized were 82.2; 94.4% and 93.8; 60.3%, respectively, whereas in the case of mixed culture, the total percentage of glucose and fructose were 96 and 100%, respectively. These results showed that the mixed culture system gave better results than single cultures regarding lactic acid concentration, and sugar consumption.     

Antimicrobial susceptibility of microflora from ovine cheese

Strains identified in ovine cheese and bryndza by matrix-assisted laser desorption/ionization time-of-flight analysis belonged to ten species of non-enterococcal lactic acid bacteria and included Lactobacillus casei/Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Lactococcus lactis, Pediococcus pentosaceus and Pediococcus acidilactici. The susceptibility toward antibiotics was determined in lactobacilli, lactococci and pediococci and also in Escherichia coli for comparison. Analysis of L. fermentum and pediococci revealed the presence of non-wild-type epidemiological cut-offs in streptomycin, clindamycin or gentamicin. E. coli were resistant to ampicillin, tetracycline, enrofloxacin and florfenicol. No extended spectrum β-lactamases were detected.     

Preliminary characterization of wine lactobacilli able to degrade arginine

Lactobacillus strains able to degrade arginine were isolated and characterized from a typical red wine. All the strains were gram-positive, catalase-negative and produced both D- and L-lactate from glucose. Strains L2, L3, L4, and L6 were able to produce CO₂ from glucose; however, production of CO₂ from glucose was not observed in strains L1 and L5, suggesting that they belong to the homofermentative wine lactic acid bacteria (LAB) group. All of the lactobacilli were tested for their ability to ferment 49 carbohydrates. The sugar fermentation profile of strain L1 was unique, suggesting that this strain belonged to Lactococcus lactis ssp. cremoris, a non-typical wine LAB. Furthermore, a preliminary typing was performed by using a random amplified polymorphic DNA analysis (RAPD-PCR analysis).     

Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products

Thirty samples of Italian durum wheat semolina and whole durum wheat semolina, generally used for the production of Southern Italy's traditional breads, were subjected to microbiological analysis in order to explore their lactic acid bacteria (LAB) diversity and to find strains with antifungal activity. A total of 125 presumptive LAB isolates (Gram-positive and catalase-negative) were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and sequence analysis of the 16S rRNA gene, leading to the identification of the following species: Weissella confusa, Weissella cibaria, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus rossiae and Lactobacillus plantarum. The REP-PCR results delineated 17 different patterns whose cluster analysis clearly differentiated W. cibaria from W. confusa isolates. Seventeen strains, each characterized by a different REP-PCR pattern, were screened for their antifungal properties. They were grown in a flour-based medium, comparable to a real food system, and the resulting fermentation products (FPs) were tested against fungal species generally contaminating bakery products, Aspergillus niger, Penicillium roqueforti and Endomyces fibuliger. The results of the study indicated a strong inhibitory activity – comparable to that obtained with the common preservative calcium propionate (0.3% w/v) – of ten LAB strains against the most widespread contaminant of bakery products, P. roqueforti. The screening also highlighted the unexplored antifungal activity of L. citreum, L. rossiae and W. cibaria (1 strain), which inhibited all fungal strains to the same or a higher extent compared with calcium propionate. The fermentation products of these three strains were characterized by low pH values, and a high content of lactic and acetic acids.     

Rapid investigation of French sourdough microbiota by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region

The aim of the present research is to identify rapidly the lactic acid bacteria (LAB) microflora of four natural French sourdoughs (GO, BF, VB and RF), applying a biphasic (restriction length polymorphism (RFLP) and sequencing) approach for bacterial identification. For this purpose, a database with the RFLP patterns of 30 lactobacilli type strains was created. So-developed ISR-RFLP algorithm was further applied for the differentiation and identification of 134 sourdough isolates. The 16S-23S rDNA intergenic spacer region was amplified by primers t^Ala and 23S/10, and then digested by HindIII, HinfI and α-TaqI enzymes. Nucleotide sequences of the cloned 16S-23S intergenic spacer region (ISR) were determined by the dideoxynucleotide chain termination method. The T7Prom and M13rev primers flanking the multiple cloning site of pCR2.1 DNA were used to sequence both DNA strands. The RFLP profile obtained upon digestion with HindIII, HinfI and α-TaqI enzymes can be used to discriminate Lactobacillus sanfranciscensis (66%), Lactobacillus panis (17%), Lactobacillus nantensis (11%) and Lactobacillus hammesii(6%) in sourdough GO, Lactobacillus sanfranciscensis (80%), Lactobacillus spicheri (14%) and Lactobacillus pontis(6%) in sourdoughs BF. In sourdoughs VB, which differed in the process temperature, we can differentiate Lactobacillus sanfranciscensis (89%) and Leuconostoc mesenteroidessubsp. mesenteroides (11%). Lactobacillus frumenti(47%), Lactobacillus hammesii (8%), and Lactobacillus paralimentarius (45%) were differentiated in sourdough RF.     

Isolation and characterization of a novel lactic acid bacterium for the production of lactic acid

We isolated a novel lactic acid bacterium from a Korean traditional fermented food, soybean paste. The newly isolated strain, dubbed RKY2, grew well on glucose, sucrose, galactose, and fructose, but it could not utilize xylose, starch, or glycerol. When the partially amplified 16S rDNA sequence (772 bp) of the strain RKY2 was compared with 10 reference strains, it was found to be most similar toLactobacillus pentosus JCM 1588^T, with 99.74% similarity. Therefore, the strain RKY2 was renamedLactobacillus sp. RKY2, which has been deposited in the Korean Collection for Type Cultures as KCTC 10353BP.Lactobacillus sp. RKY2 was found to be a homofermentative lactic acid bacterium, because its end-product from glucose metabolism was found to be mainly lactic acid. It could produce more than 90 g/L of lactic acid from MRS medium supplemented with 100 g/L of glucose, with 5.2 g L⁻¹ h⁻¹ of productivity and 0.95 g/g of lactic acid yield.     

Characterisation of IS153, an IS3-family Insertion Sequence Isolated from Lactobacillus sanfranciscensis and its use for Strain Differentiation

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451^T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by –1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family.In L. sanfranciscensis type strain DSM 20451^T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS 153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS 153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Functional Compounds in Fermented Buckwheat Sprouts

Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.     

Molecular Structure of the Lactobacillus plantarum Sucrose Utilization Locus: Comparison with Pediococcus pentosaceus

Structure of the sucrose utilization locus in a Lactobacillus plantarum type strain was studied using PCR and Southern hybridization. Restriction map analysis revealed its high similarity to the sequenced sucrose utilization locus of Pediococcus pentosaceus pSRQ1. The L. plantarum locus proved to contain oppositely oriented scrA and the scrBRagl operon, but not agaS. The L. plantarum sucrase gene (scrB) was partly sequenced. A higher (98.6%) homology was revealed between scrB than between the 16S rRNA genes of L. plantarum and P. pentosaceus, suggesting horizontal transfer of the sucrose utilization locus between the genera of lactic acid bacteria. Amino acid sequence analysis showed that the ScrB proteins of the two species belong to a subfamily of glycosyl hydrolase family GH32 which includes various -fructosidases.     

Use of starter cultures containingStreptococcus diacetilactis,Lactobacillus brevis andSaccharomyces cerevisiae for fermenting milk for production of Nigeriannono

Starter cultures consisting ofStreptococcus diacetilactis, Strept. cremoris, Lactobacillus brevis andSaccharomyces cerevisiae were tested singly and in mixtures for ability to ferment milk to producenono with organoleptically acceptable qualities. Only mixed cultures containing eitherStrept. diacetilactis orStrept. cremoris andL. brevis were suitable. Presence of yeast adversely affected either acid formation or diacetyl production.Nono containingStrept. diacetilactis was acceptable, even in the presence ofSacch. cerevisiae, because of the high diacetyl production. A mixed starter containing two of these organisms,Strept. diacetilactis orStrept. cremoris andL. brevis, is recommended fornono production.Sacch. cerevisiae is not essential.R. N. Okagbue and M. O. Bankole were formerly with the Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria.     

The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation

Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's yeast. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the species Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus paralimentarius, and Weissella cibaria. This consortium seems to be unique for the Greek traditional wheat sourdoughs studied. Strains of the species W. cibaria have not been isolated from sourdoughs previously. No Lactobacillus pontis or Lactobacillus panis strains were found. An L. brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species. In addition, fermentation capabilities associated with the LAB detected have been studied. During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol. Mannitol was produced from fructose that served as an additional electron acceptor. In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested except L. sanfranciscensis. Two of the L. paralimentarius isolates tested did not ferment maltose; all strains were homofermentative. In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly. No maltose phosphorylase activity was found either. These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.