Characterization and differentiation of Lactobacillus manihotivorans strains isolated from cassava sour starch.

Lactobacillus manihotivorans has been reported as one of the dominant species in cassava sour starch production process. Seven isolates that have previously been identified as belonging to this species were studied in the present work. Their molecular and phenotypic characteristics showed higher strain diversity than previously described. Differences were found in their fermentation profiles, whereas no major differences were observed in properties related to processing conditions (salt concentration, pH, temperature), or in potential functional properties (bile salt and pH 2.0 tolerance). Among the main characteristics of interest for the fermentation of cereals or cassava, blended or not with legumes, six out of seven strains were amylolytic and raffinose was fermented by all strains. Strains OND 32T and OLB 7 fermented the broadest range of carbohydrates. Most of the strains contained plasmids. Plasmid curing changed their phenotypic characteristics, particularly those of strain OND 32T, which, in addition, lost its starch and raffinose fermentation ability.

Microbial and physiological characterization of weakly amylolytic but fast-growing lactic acid bacteria: a functional role in supporting microbial diversity in pozol, a Mexican fermented maize beverage.

Pozol is an acid beverage obtained from the natural fermentation of nixtamal (heat- and alkali-treated maize) dough. The concentration of mono- and disaccharides from maize is reduced during nixtamalization, so that starch is the main carbohydrate available for lactic acid fermentation. In order to provide some basis to understand the role of amylolytic lactic acid bacteria (ALAB) in this fermented food, their diversity and physiological characteristics were determined. Forty amylolytic strains were characterized by phenotypic and molecular taxonomic methods. Four different biotypes were distinguished via ribotyping; Streptococcus bovis strains were found to be predominant. Streptococcus macedonicus, Lactococcus lactis, and Enterococcus sulfureus strains were also identified. S. bovis strain 25124 showed extremely low amylase yield relative to biomass (139 U g [cell dry weight](-1)) and specific rate of amylase production (130.7 U g [cell dry weight](-1) h(-1)). In contrast, it showed a high specific growth rate (0.94 h(-1)) and an efficient energy conversion yield to bacterial cell biomass (0.31 g of biomass g of substrate(-1)). These would confer on the strain a competitive advantage and are the possible reasons for its dominance. Transient accumulation of maltooligosaccharides during fermentation could presumably serve as energy sources for nonamylolytic species in pozol fermentation. This would explain the observed diversity and the dominance of nonamylolytic lactic acid bacteria at the end of fermentation. These results are the first step to understanding the importance of ALAB during pozol fermentation.

New efficient amylase-producing strains of Lactobacillus plantarum and L. fermentum isolated from different Nigerian traditional fermented foods.

Amylolytic lactic acid bacteria (ALAB) were isolated from Nigerian traditional fermented foods (fufu, burukutu, ogi-baba and kunu-zakki) with the aim of selecting efficient amylase-producing strains. Nine isolates were characterized on the basis of their phenotypic and taxo-molecular characteristics. Three groups could be distinguished by their fermentation profiles and this was confirmed by DNA restriction analysis. Though fermentation profiles gave good identification of strain K9 (unique representative of group III) as Lactobacillus fermentum, they could not be used to ascertain the taxonomic position of strains of groups I and II. Analysis of partial 16S rRNA sequences led to the identification of these groups as L. plantarum strains and confirmed the species of strain K9 as L. fermentum. The two distinct phenotypic groups of L. plantarum differed in their use of D-xylose, L-arabinose, melibiose and were different from the previously described amylolytic L. plantarum A6 isolated from retted cassava in Congo. L. fermentum K9 was different from L. fermentum OgiE1 and Mw2 isolated from Benin maize sourdough and it is the first amylolytic L. fermentum described from Nigerian fermented products. Enzymatic profiles showed some differences between the strains of a similar fermentation group. One of the most relevant characteristics of the isolates was a higher yield of amylase production than those reported for previously described ALAB grown under the same conditions. Furthermore, all isolates were tolerant to an exposure at pH 2 and to bile salts.

Characterization of the L. manihotivorans alpha-amylase gene.

Primers and probes were established from the sequences of the alpha-amylase genes (amyA) of L. amylovorus CIP 102989 and of L. plantarum A6 (Giraud and Cuny 1997). They were successfully used for the detection of the amyA gene in L. manihotivorans strain LMG 18010T and a 2842 bp region, containing the entire gene (2706 bp) with its putative promoter has been sequenced. More than 98% nucleotide sequence identities was found with L. amylovorus and L. plantarum amyA genes. The deduced amino acid sequence shares more than 96% amino acid sequence identities with L. amylovorus and L. plantarum alpha-amylases, and also 65% and 59% identities with the alpha-amylases of B. subtilis and S. bovis, respectively. The 3' terminal part of L. manihotivorans LMG 18010T amyA gene contained four repeated sequences (SRU). The amyA genes of the three lactobacilli species differed mainly in the number of SRU and in the size of the flanking regions of the SRU.

Effect of different cultivation conditions on Lactobacillus manihotivorans OND32T, an amylolytic lactobacillus isolated from sour starch cassava fermentation.

Study of the cassava sour starch fermentation has led to the isolation of a new homofermentative amylolytic lactic acid bacterium, Lactobacillus manihotivorans OND32T, whose nutritional requirements have been investigated in this work. The main effect of deleting one of the substrate components of the MRS-starch medium was to reduce the amylase production. When starch fermentation with nitrogen as a gas phase was compared to fermentation under aerobic conditions, both growth and amylase production were reduced whereas lactic acid formation was not affected. Addition of carbon dioxide (> or = 20% v/v) to the nitrogen gas phase restored growth and amylase production. The amylase production was high with starch, maltose or cellobiose contrary to glucose, fructose and sucrose. During mixed fermentation of glucose and maltose, a diauxic growth was observed. The maltose consumption and the amylase production started after the glucose depletion. The presence of maltose altered the carbon assimilation from glucose, whereas the energetic pathway was not affected. It is concluded that the elimination of soluble sugars by the wet extraction of starch during the processing of cassava, together with the expected in situ CO2 production, are conditions favouring the growth and the amylase synthesis. However, these are likely to be limited by the low nitrogen content in cassava.

Purification and characterization of an extracellular alpha-amylase produced by Lactobacillus manihotivorans LMG 18010(T), an amylolytic lactic acid bacterium.

This work presents the purification and characterization of an extracellular alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) produced by a new lactic acid bacterium: Lactobacillus manihotivorans able to produce L(+) lactic acid from starch. The molecular weight was found to be 135 kDa. The temperature and pH optimum were 55 degrees C and 5.5, respectively, and pI was 3.8. The alpha-amylase had good stability at pH range from 5 to 6 and the enzyme was sensitive to temperature, losing activity within 1 h of incubation at 55 degrees C. Higher thermal stability was observed when the enzyme was incubated in presence of soluble starch. K(m) value and activation energy were 3.44 mg/ml and 32.55 kJ/mol, respectively. Amylose was found to be a better substrate than soluble starch and amylopectin. Al(3+), Fe(3+), and Hg(2+) (10 mM) almost completely inhibited the alpha-amylase.

Comparative characterization of complete and truncated forms of Lactobacillus amylovorus alpha-amylase and role of the C-terminal direct repeats in raw-starch binding.

Two constructs derived from the alpha-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyADelta) forms of alpha-amylase; AmyADelta lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyADelta exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and alpha-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K(M) and V(max) values of AmyADelta were slightly higher than those of AmyA, whereas the thermal stability of AmyADelta was lower than that of AmyA. In contrast to AmyA, AmyADelta is unable to bind to beta-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyADelta cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.

Effect of pH control on lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T.

Lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T, a new amylolytic L(+) lactic acid producer, was investigated and compared with starch fermentation by Lact. plantarum A6. At non-controlled pH, growth and lactic acid production from starch by Lact. manihotivorans LMG 18010T lasted 25 h. Specific growth and lactic acid production rates continuously decreased from the onset of the fermentation, unlike Lact. plantarum A6 which was able to grow and convert starch product hydrolysis into lactic acid more rapidly and efficiently at a constant rate up to pH 4.5. In spite of complete and rapid starch hydrolysis by Lact. manihotivorans LMG 18010T during the first 6 h, only 45% of starch hydrolysis products were converted to lactic acid. When pH was maintained at 6.0, lactic acid, amylase and final biomass production by Lact. manihotivorans LMG 18010T increased markedly and the fermentation time was reduced by half. Under the same conditions, an increase only in amylase production was observed with Lact. plantarum A6. When grown on glucose or starch at pH 6.0, Lact. manihotivorans LMG 18010T had an identical maximum specific growth rate (0.35 h(-1)), whereas the maximum rate of specific lactic acid production was three times higher with glucose as substrate. Lactobacillus manihotivorans LMG 18010T did not produce amylase when grown on glucose. Based on the differences in the physiology between the two species and other amylolytic lactic acid bacteria, different applications may be expected.

Lactobacillus manihotivorans sp. nov., a new starch-hydrolysing lactic acid bacterium isolated during cassava sour starch fermentation.

Two Lactobacillus strains were isolated from sour cassava starch fermentation. The cells were Gram-positive, catalase-negative, non-spore-forming, non-motile rods. They produced only L(+)lactate and were homofermentative. Growth occurred at pH values of 5.0-7.0 and optimum growth occurred at pH 6.0. Growth was positive at 15 and 45 degrees C. The DNA G + C content was 48.4 +/- 0.2 mol%. Sequence analysis of the 16S rRNA gene revealed that strains OND 32T and YAM 1 clustered with, but were separate from Lactobacillus casei-related taxa. Protein pattern and sequence analyses of the 16S rRNA gene confirmed that the two new isolates represent a new Lactobacillus species, for which the name Lactobacillus manihotivorans is proposed; strain OND 32T is the type strain of this species.