Lactic acid bacteria of the sour cassava starch fermentation

In Brazil and Colombia, 'sour starch' is traditionally obtained by a submerged lactic fermentation of crude cassava starch followed by sun drying. It is used by local bakers to prepare breadlike products which display the same expanded crumb texture as in wheat bread.
In this process, suspended starch is settled down and left aside for a few weeks under anaerobic conditions where natural lactic populations develop.
Three collections of clones isolated from local fermentations have been identified using the API procedure and further characterized. Most of them belong to different species of Lactobacillus . Many display a ropy phenotype, typical for exopolysaccharide (EPS) excretion. A possible role of these EPS in the special properties of sour starch is discussed.     

解淀粉乳酸细菌在L-乳酸发酵生产中的应用

对解淀粉乳酸细菌及其产生的淀粉酶和发酵工艺等方面的国内外研究现状进行了综述。解淀粉乳酸细菌具有分泌淀粉酶的能力,可免去原料水解处理工序直接发酵淀粉质原料生产乳酸,可以简化生产工艺,并可节约设备投资,进而降低生产成本。解淀粉乳酸细菌主要分离自传统发酵食品,也可从有机废弃物和厨余垃圾中分离得到。介绍了解淀粉乳酸细菌直接利用淀粉质原料的机理,比较了解淀粉乳酸菌发酵生产L-乳酸的工艺。提出通过诱变育种和基因工程育种等方法获得更加高效的解淀粉乳酸细菌,并结合先进的发酵、分离技术来提高乳酸生产效率。     

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.     

Batch fermentations on synthetic mixed sugar and starch medium with amylolytic lactic acid bacteria

The green crop drying industry in Denmark uses Italian rye grass, clover, and alfalfa as raw materials for the production of green pellets. The green crop drying industry solves its energy economical problems by heating and pressing of the green crop before drying. The produced sidestream is called brown juice. Brown juice was shown to be an excellent medium for lactic acid fermentation. The aim of this study was to investigate the utilisation of brown juice in the production of polylactic acid, where wheat starch would be added to increase the lactic acid yield and, thus, the feasibility of the process. A number of amylolytic lactic acid bacteria have been identified, and in this work, six different strains were tested for their ability to produce α-amylase and to utilise all sugars with high lactic acid yield in a medium with a complex composition of free sugars (brown juice) and starch. Lactobacillus plantarum A6 was the only strain that showed both a good lactic acid production and utilisation of starch in this medium. The growth rate of this strain was approximately 0.4 h⁻¹ and the lactic acid yield was 0.7.     

Design and evaluation of a Lactobacillus manihotivorans species-specific rRNA-targeted hybridization probe and its application to the study of sour cassava fermentation

Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.     

Parakari, an indigenous fermented beverage using amylolytic Rhizopus in Guyana

The alcoholic beverage parakari is a product of cassava (Manihot esculenta Crantz) fermentation by Amerindians of Guyana. While fermented beverage production is nearly universal among indigenous Amazonians, parakari is unique among New World beverages because it involves the use of an amylolytic mold (Rhizopus sp., Mucoraceae, Zygomycota) followed by a solid substratum ethanol fermentation. The mycological significance of this dual fermentation process previously was unrecognized. A detailed study of parakari fermentation was made in the Wapisiana Amerindian village of Aishalton, South Rupununi, Guyana. Thirty steps were involved in parakari manufacture, and these exhibited a high degree of sophistication, including the use of specific cassava varieties, control of culture temperature and boosting of Rhizopus inoculum potential with purified starch additives. During the fermentation process, changes in glucose content, pH, flavor, odor and culture characteristics were concomitant with a desirable finished product. Parakari is the only known example of an indigenous New World fermentation that uses an amylolytic mold, likely resulting from domestication of a wild Rhizopus species in the distant past. Parakari production is remarkably similar to dual fermentations of Asia, yet it was independently derived.     

Use of inexpensive nitrogen sources and starch for L(+) lactic acid production in anaerobic submerged fermentation

L(+) Lactic acid fermentation was studied by Lactobacillus amylophilus GV6 under the influence of inexpensive nitrogen sources (red lentil-RL, and Baker's yeast cells-YC) and starch by response surface methodology (RSM). Central composite rotatable design (CCRD) was employed to determine maximum lactic acid production at optimum values for process variables RL, YC and incubation period (IP) and a satisfactory fit model was realized. Lactic acid production was significantly affected by RL and IP interactions as well as by independent variables RL and YC. Maximum lactic acid production of 13.5 g/15.2g starch was obtained with RL 0.8%, YC 1% and IP of 48 h, with 92% lactic acid yield efficiency (g lactic acid produced/g substrate utilized) and 40% increase (from 50 g to 92 g/100 g starch utilized) in lactic acid production. This is the first report on response optimization in direct fermentation of starch to lactic acid using inexpensive nitrogen sources substituting peptone and yeast extract in anaerobic submerged fermentation by amylolytic lactic acid bacteria (LAB).     

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.     

Molecular diversity of lactic acid bacteria from cassava sour starch (Colombia)

Lactic acid bacteria and more particularly lactobacilli and Leuconostoc, are widely found in a wide variety of traditional fermented foods of tropical countries, made with cereals, tubers, meat or fish. These products represent a source of bacterial diversity that cannot be accurately analysed using classical phenotypic and biochemical tests. In the present work, the identification and the molecular diversity of lactic acid bacteria isolated from cassava sour starch fermentation were assessed by using a combination of complementary molecular methods: Randomly Amplified Polymorphic DNA fingerprinting (RAPD), plasmid profiling, hybridization using rRNA phylogenetic probes and partial 16S rDNA sequencing. The results revealed a large diversity of bacterial species (Lb. manihotivorans, Lb. plantarum, Lb. casei, Lb. hilgardii, Lb. buchneri, Lb. fermentum, Ln. mesenteroides and Pediococcus sp.). However, the most frequently isolated species were Lb. plantarum and Lb. manihotivorans. The RAPD analysis revealed a large molecular diversity between Lb. manihotivorans or Lb. plantarum strains. These results, observed on a rather limited number of samples, reveal that significant bacterial diversity is generated in traditional cassava sour starch fermentations. We propose that the presence of the amylolytic Lb. manihotivorans strains could have a role in sour starch processing.     

Design and Evaluation of a Lactobacillus manihotivorans Species-Specific rRNA-Targeted Hybridization Probe and Its Application to the Study of Sour Cassava Fermentation

Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.     

Breeding and growth of Rhizopus in raw cassava by solid state fermentation

Nineteen Rhizopus strains were selected and tested for their growth capacity on raw cassava starch and their ability to produce amylase when grown on solid-state fermentations. Only three strains grew significantly on this natural substrate. Glucoamylase production was higher on raw cassava than on cooked cassava. After 48 h of fermentation, the protein content of cassava was increased from 1.75% to 11.3%. The byproducts of fermentation were fumaric acid, lactid acid and ethanol.     

New amylolytic yeast strains for starch and dextrin fermentation

Yeast strains capable of fermenting starch and dextrin to ethanol were isolated from samples collected from Brazilian factories in which cassava flour is produced. Considerable alcohol production was observed for all the strains selected. One strain (DI-10) fermented starch rapidly and secreted 5 times as much amylolytic enzyme than that observed for Schwanniomyces alluvius UCD 54-83. This strain and three other similar isolates were classified as Saccharomyces cerevisiae var. diastaticus by morphological and physiological characteristics and molecular taxonomy.     

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.     

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and Alpha‐amylase to produce ethanol

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α‐amylase from Aspergillus oryzae as well as α‐amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:338–347, 2014     

Ethanol production from native cassava starch by a mixed culture of Endomycopsis fibuligera and Zymomonas mobilis

The conversion of starch from unhydrolyzed cassava flour to ethanol by a pure culture of Endomycopsis fibuligera and by a co-culture of this amylolytic yeast and the bacterium Zymomonas mobilis was studied. The best overall results were obtained using the mixed culture. After 96 h of fermentation of a medium containing 150 g/l initial cassava starch, an ethanol concentration of 31.4 g/l, a productivity of 0.33 g ethanol/l × h and a yield of 0.21 g ethanol/g initial starch were reached. The highest yield (0.37 g/g) was obtained after 48 h when using a medium containing 50 g/l initial starch.     

Amylolytic bacterial lactic acid fermentation - a review

Lactic acid, an enigmatic chemical has wide applications in food, pharmaceutical, leather, textile industries and as chemical feed stock. Novel applications in synthesis of biodegradable plastics have increased the demand for lactic acid. Microbial fermentations are preferred over chemical synthesis of lactic acid due to various factors. Refined sugars, though costly, are the choice substrates for lactic acid production using Lactobacillus sps. Complex natural starchy raw materials used for production of lactic acid involve pretreatment by gelatinization and liquefaction followed by enzymatic saccharification to glucose and subsequent conversion of glucose to lactic acid by Lactobacillus fermentation. Direct conversion of starchy biomass to lactic acid by bacteria possessing both amylolytic and lactic acid producing character will eliminate the two step process to make it economical. Very few amylolytic lactic acid bacteria with high potential to produce lactic acid at high substrate concentrations are reported till date. In this view, a search has been made for various amylolytic LAB involved in production of lactic acid and utilization of cheaply available renewable agricultural starchy biomass. Lactobacillus amylophilus GV6 is an efficient and widely studied amylolytic lactic acid producing bacteria capable of utilizing inexpensive carbon and nitrogen substrates with high lactic acid production efficiency. This is the first review on amylolytic bacterial lactic acid fermentations till date.     

Fermentation of starch by Klebsiella oxytoca p2, containing plasmids with alpha-amylase and pullulanase genes.

Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 degrees C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12-24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower alpha-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.     

An economic approach for L-(+) lactic acid fermentation by Lactobacillus amylophilus GV6 using inexpensive carbon and nitrogen sources

AIMS: Development of cost-effective production medium by applying statistical designs for single-step fermentation of starch (corn flour - CF) to L-(+) lactic acid, using inexpensive nitrogen sources as substitutes for peptone and yeast extract in MRS medium by amylolytic Lactobacillus amylophilus GV6. METHODS AND RESULTS: A two-level Plackett-Burman design was employed for screening various available crude starches (flours) for L-(+) lactic acid production by Lact. amylophilus GV6 using red lentil flour (RL) and bakers yeast cells (YC) as substitutes for commercial peptone and yeast extract in MRS medium in anaerobic submerged fermentation. Of all the tested flours, CF was found to be the most significant. Central composite rotatable design was employed to determine maximum production of L-(+) lactic acid at optimum values of process variables, CF, RL, YC, CaCO(3) and incubation period (IP). minitab analyses showed that lactic acid production was significantly affected by the linear terms CF, RL, CaCO(3) and IP. The interactions of CF-RL, CF-YC, CF-CaCO(3), RL-YC and RL-CaCO(3) and the square terms CF and IP were significant. The maximum lactic acid production of 29 g/37 g of starch present in 50 g of CF was obtained at optimized concentrations of CF 5%, RL 0.7%, YC 0.8%, CaCO(3) 0.8% and IP 2.9 days. CONCLUSIONS: Successful application of Plackett-Burman design helped in identifying CF as the best carbon source among the tested flours for L-(+) lactic acid production using inexpensive nitrogen sources. Further optimization of the process variables by response surface methods (RSMs) led to maximum production of lactic acid (29 g lactic acid from 37 g of starch present in 50 g of flour). SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus amylophilus GV6 showed 78.4% lactic acid production efficiency (g lactic acid produced/g starch taken) and 96% lactic acid yield efficiency (g lactic acid produced/g starch utilized). Information from the present studies provides a better understanding on production of L-(+) lactic acid on fermentation of CF using inexpensive nitrogen sources and on changes in the production as a response from interaction of factors. Use of inexpensive nitrogen sources and starch as substrate in MRS medium for single-step fermentation of lactic acid can become an efficient, economic and viable process. This report is on optimization of inexpensive nitrogen sources completely replacing peptone and yeast extract in single-step submerged fermentation of starch (present in CF) to lactic acid with high production efficiency.     

Direct conversion of starch to L(+) lactic acid by amylase producing Lactobacillus amylophilus GV6

Lactobacillus amylophilus strain GV6, isolated from corn starch processing industrial wastes, was amylolytic and produced 0.96?g L(+) lactic acid per gram of soluble starch. The optimum temperature and pH for growth and L(+) lactic acid production were 37?°C and 6.5, respectively. At low substrate concentrations, the lactic acid production on corn starch was almost similar to soluble starch. The strain is fermenting various naturally available starches directly to lactic acid. The total amylase activity of the strain is 0.59?U/ml/min. The strain produced 49 and 76.2?g/l L(+) lactic acid from 60?g/l corn starch and 90?g/l soluble starch, respectively. This is the highest L(+) lactic acid among the wild strains of L. amylophilus reported so far.     

Isolation and characterization of new amylolytic strains of Lactobacillus fermentum from fermented maize doughs (mawè and ogi) from Benin

Twelve amylolytic heterofermentative lactic acid bacteria were isolated in Benin from the fermentation processes of maize sour dough, namely ogi and mawè. Discrimination of strains was performed by DNA restriction patterns and compared with carbohydrate fermentation profiles. This allowed two new amylolytic strains, Ogi E1 and Mw2, belonging to the species Lactobacillus fermentum , to be distinguished. Strains Ogi E1 and Mw2 presented different amylolytic activities; amylase from strain Mw2 was more acidophilic and mesophilic than amylase produced by strain Ogi E1.