Abstract
Lactate racemization as a rescue pathway for supplying D-lactate to the cell wall biosynthesis machinery in Lactobacillus plantarum. Philippe Goffin;Marie Deghorain;Jean-Luc Mainardi;Isabelle Tytgat;Marie-Christine Champomier-Vergès;Michiel Kleerebezem;Pascal Hols. 2005. J Bacteriol. 187. PMID: 16166538

Lactobacillus plantarum is a lactic acid bacterium that produces d- and l-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for d-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production of d-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for d-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires l-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by l-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of l-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is d-lactate dependent. We also show that the growth defect can be partially restored by expression of the d-alanyl-d-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various d-2-hydroxy acids but not d-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in d-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in d-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for d-lactate production upon inactivation or inhibition of the LdhD enzyme.
Enantioselective regulation of lactate racemization by LarR in Lactobacillus plantarum. Benoît Desguin;Philippe Goffin;Nordine Bakouche;Aurélie Diman;Eric Viaene;Damien Dandoy;Laetitia Fontaine;Bernard Hallet;Pascal Hols. 2014. J Bacteriol. 197. PMID: 25349156

Lactobacillus plantarum is a lactic acid bacterium that produces a racemic mixture of l- and d-lactate from sugar fermentation. The interconversion of lactate isomers is performed by a lactate racemase (Lar) that is transcriptionally controlled by the l-/d-lactate ratio and maximally induced in the presence of l-lactate. We previously reported that the Lar activity depends on the expression of two divergently oriented operons: (i) the larABCDE operon encodes the nickel-dependent lactate racemase (LarA), its maturases (LarBCE), and a lactic acid channel (LarD), and (ii) the larR(MN)QO operon encodes a transcriptional regulator (LarR) and a four-component ABC-type nickel transporter [Lar(MN), in which the M and N components are fused, LarQ, and LarO]. LarR is a novel regulator of the Crp-Fnr family (PrfA group). Here, the role of LarR was further characterized in vivo and in vitro. We show that LarR is a positive regulator that is absolutely required for the expression of Lar activity. Using gel retardation experiments, we demonstrate that LarR binds to a 16-bp palindromic sequence (Lar box motif) that is present in the larR-larA intergenic region. Mutations in the Lar box strongly affect LarR binding and completely abolish transcription from the larA promoter (PlarA). Two half-Lar boxes located between the Lar box and the -35 box of PlarA promote LarR multimerization on DNA, and point mutations within one or both half-Lar boxes inhibit PlarA induction by l-lactate. Gel retardation and footprinting experiments indicate that l-lactate has a positive effect on the binding and multimerization of LarR, while d-lactate antagonizes the positive effect of l-lactate. A possible mechanism of LarR regulation by lactate enantiomers is proposed.
D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum. H Taguchi;T Ohta. 1991. J Biol Chem. 266. PMID: 1840590

The gene encoding D-lactate dehydrogenase (D-lactate: NAD+ oxidoreductase, EC 1.1.1.28) of Lactobacillus plantarum has been sequenced, and expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5'-noncoding region of the gene was replaced with the tac promoter. Comparison of the sequence of D-lactate dehydrogenase with L-lactate dehydrogenases, including the L. plantarum L-lactate dehydrogenase, showed no significant homology. In contrast, the D-lactate dehydrogenase is homologous to E. coli D-3-phosphoglycerate dehydrogenase and Lactobacillus casei D-2-hydroxyisocaproate dehydrogenase. This indicates that D-lactate dehydrogenase is a member of a new family of 2-hydroxyacid dehydrogenases recently proposed, being distinct from L-lactate dehydrogenase and L-malate dehydrogenase, and strongly suggests that the new family consists of D-isomer-stereospecific enzymes. In the reductive reaction, the enzyme showed a broad substrate specificity, although pyruvate was the most favorable of all 2-ketocarboxylic acids tested. In particular, hydroxypyruvate is effectively reduced by the enzyme, the reaction rate, and Km value being comparable to those in the case of pyruvate, indicating that the enzyme has not only D-lactate dehydrogenase activity but also D-glycerate dehydrogenase activity. The conserved residues in this family appear to be the residues involved in the substrate binding and the catalytic reaction, and thus to be targets for site-directed mutagenesis.
The effect of sodium acetate on the growth yield, the production of L- and D-lactic acid, and the activity of some enzymes of the glycolytic pathway of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T). Takao Iino;Tai Uchimura;Kazuo Komagata. 2002. J Gen Appl Microbiol. 48. PMID: 12469305

The effect of sodium acetate was studied on the change of the growth yield, the production of L- and D-lactic acid, and the activity of lactate dehydrogenases (LDHs; L-lactate dehydrogenase [EC 1.1.1.27, L-LDH] plus D-lactate dehydrogenase [EC 1.1.1.28, D-LDH]), fructose-1, 6-bisphosphate aldolase [EC 4.1.2.13, FBP-aldolase], and phosphofructokinase [EC 2.7.1.11, PFK] of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T). The growth yield of L. sakei NRIC 1071(T) was increased 1.6 times in the presence of sodium acetate compared with its absence. The activity of LDHs in L. sakei NRIC 1071(T) and L. plantarum NRIC 1067(T) was retained longer under the addition of sodium acetate in the reaction mixture. As a result, these strains produced much more lactic acid in the presence of sodium acetate compared with its absence. Furthermore, the activity of L-LDH in L. sakei NRIC 1071(T) cultivated in the presence of sodium acetate increased three times or more compared with the activity of the cells cultivated in its absence. Consequently, the type of stereoisomers of lactic acid produced by L. sakei shifted from the DL-type to the L-type because the ratio of L-lactic acid to D-lactic acid produced became larger with the addition of sodium acetate to culture media. This phenomenon was not observed in L. plantarum NRIC 1067(T). Further, the participation of lactate racemase is discussed from the viewpoint of the production of D-lactic acid by L. sakei.
Racemization of l-lactic acid in pH-swing open fermentation of kitchen refuse by selective proliferation of Lactobacillus plantarum. Kenji Sakai;Norihisa Fujii;Ekachai Chukeatirote. 2006. J Biosci Bioeng. 102. PMID: 17046538

We have shown that stable lactic acid fermentation of model kitchen refuse occurs with intermittent pH adjustment under nonsterilized conditions. Nonetheless, the optical activity of the accumulated lactic acid was low, which is disadvantageous for the production of high-quality poly-l-lactic acid. Here, we attempt to increase optical purity by introducing l-lactic acid-producing strains under nonsterilized conditions and demonstrate that the inoculation of Lactobacillus rhamnosus or Lactococcus lactis, both of which are l-lactic acid producers, is partially effective in the early fermentation stage, but does not improve the final optical purity of the accumulated lactic acid. We confirmed by fluorescence in situ hybridization using group-specific and species-specific 16S rDNA probes that this is due to the selective proliferation of naturally existing L. plantarum. L. plantarum KY-1, which is isolated from model kitchen refuse, showing lactic acid racemase activity, as well as d-lactate dehydrogenase activity, in its membrane fraction. We conclude that racemase activity associated with L. plantarum is the main cause of decreased optical purity in the accumulated lactic acid.
Production of L-lactate in Leuconostoc citreum via heterologous expression of L-lactate dehydrogenase gene. Qing Jin;Jee Yun Jung;Yu Jin Kim;Hyun-Ju Eom;So-Young Kim;Tae-Jip Kim;Nam Soo Han. 2009. J Biotechnol. 144. PMID: 19699768

D-form lactate is often found in fermented foods and excessive dietary intake of D-lactate may cause metabolic stress in both infants and patients. Leuconostoc citreum is a major lactic acid bacterium that produces D-lactate in fermented foods. The aim of this study was to change the pyruvate carbon flux in L. citreum from D-lactate into L-lactate by heterologous expression of L-lactate dehydrogenase (ldhL) gene. For this, ldhL from Lactobacillus plantarum was cloned and introduced into L. citreum using a shuttle vector pLeuCM. In the transformant, ldhL was successfully transcribed and L-lactate dehydrogenase was expressed. As a consequence of transformation, the ratio between D- and L-isomers was changed due to the increment of L-lactate and the decrement of D-lactate, but no significant differences were found in total lactate concentration between the host and transformant cells. This is the first report of metabolic engineering in Leuconostoc by modulating the central carbon flux into health-favored way.
Two-step production of D-lactate from mixed sugars by growing and resting cells of metabolically engineered Lactobacillus plantarum. Yota Tsuge;Hideo Kawaguchi;Kengo Sasaki;Tsutomu Tanaka;Akihiko Kondo. 2014. Appl Microbiol Biotechnol. 98. PMID: 24562327

To develop cost-effective systems for D-lactate production, here, the effect of high-cell density cultivation of metabolically engineered Lactobacillus plantarum on D-lactate production was evaluated. A xylose-assimilating strain of L. plantarum was anaerobically cultured with mixed sugars (glucose and xylose) as substrates. Compared to undiluted nutrient-rich de Man, Rogosa, and Sharpe (MRS) medium, D-lactate production by cultivating in 10-fold diluted MRS (0.1 MRS) medium or normal saline solution was 89.7 and 81.3 %, respectively. Notably, the xylose consumption rate was comparable in the three cultures, whereas the glucose consumption rate decreased by 18.3 and 26.1 % in 0.1 MRS medium and normal saline solution, respectively, resulting in a reduction of the D-lactate production rate. The D-lactate productivity in high-cell density cultivation was proportional to the initial cell concentrations. The use of a two-step cultivation process involving growing and resting cells in a single bioreactor revealed that the ratio of the glucose and xylose consumption rates (based on grams consumed) in resting cell conditions was 1.88, whereas that in growing conditions was 2.58. Cultivation of L. plantarum in growing conditions for 24 h produced 73.2 g/l D-lactate with the yield of 0.90 g/g, whereas cells cultivation under resting cell conditions in a saline solution for 24 h produced 68.7 g/l D-lactate with the yield of 0.93 g/g. In total, 141.9 g/l D-lactate was produced after 48 h cultivation, a value that represents the highest reported concentration of D-lactate produced from mixed sugars to date. Our findings contribute to the cost-effective, large-scale production of D-lactate.
d-lactic acid production from renewable lignocellulosic biomass via genetically modified Lactobacillus plantarum. Yixing Zhang;Amit Kumar;Philip R Hardwidge;Tsutomu Tanaka;Akihiko Kondo;Praveen V Vadlani. 2015. Biotechnol Prog. 32. PMID: 26700935

d-lactic acid is of great interest because of increasing demand for biobased poly-lactic acid (PLA). Blending poly-l-lactic acid with poly-d-lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d-lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l-lactate-deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ΔldhL1-pCU-PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d-lactic acid yield and productivity. d-lactic acid (27.3 g L(-1) ) and productivity (0.75 g L(-1) h(-1) ) was obtained from corn stover and d-lactic acid (22.0 g L(-1) ) and productivity (0.65 g L(-1) h(-1) ) was obtained from sorghum stalks using ΔldhL1-pCU-PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d-lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d-lactic acid. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271-278, 2016.
Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans. Limin Wang;Yumeng Cai;Lingfeng Zhu;Honglian Guo;Bo Yu. 2014. Appl Environ Microbiol. 80. PMID: 25217009

Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production-NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)-were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6.
Influence of polysaccharides on oxygen dependent lactate utilization by an amylolytic Lactobacillus plantarum strain. José Pintado;Maurice Raimbault;Jean-Pierre Guyot. 2004. Int J Food Microbiol. 98. PMID: 15617803

Oxygen-dependent conversion of lactate to acetate by the amylolytic strain Lactobacillus plantarum A6 was studied using MRS-grown cells, transferred to a basic medium with lactate. In the presence of oxygen, lactate was stoechiometricaly converted to acetate. When glucose, maltose or cellobiose was added to the basic medium, no utilisation of lactate was observed. However, when starch or glycogen was added, the conversion of lactate to acetate happened. To verify the possible link of this effect with sugar consumption rate, a glucose-fed batch culture was conducted with a lactic acid consuming culture grown on the basic medium with lactate. Even when glucose was fed at the same low rate as the consumption rate observed for polysaccharides, lactic acid was no more consumed. For the amylolytic strain L. plantarum A6, the transport and use of oligosaccharides resulting from polysaccharides hydrolysis might affect differently the glycolytic flux, with the putative consequence to suppress the metabolic control of glycolysis by glucose. This fact could play an important role in the fermentation of amylaceous foods.
Reduction of D-lactate content in sauerkraut using starter cultures of recombinant Leuconostoc mesenteroides expressing the ldhL gene. Qing Jin;Ling Li;Jin Seok Moon;Seung Kee Cho;Yu Jin Kim;Soo Jin Lee;Nam Soo Han. 2015. J Biosci Bioeng. 121. PMID: 26472127

The D-form of lactate, which causes metabolic stress upon excessive dietary intake, is mainly produced by Leuconostoc sp., the predominant species in sauerkraut. To shift the metabolic flux of d-lactate from pyruvate to l-lactate, we expressed the l-lactate dehydrogenase (ldhL) gene in Leuconostoc mesenteroides ATCC 8293. The ldhL gene from Lactobacillus plantarum was introduced into L. mesenteroides using the shuttle vectors pLeuCM and pLeuCM42. To elevate the expression level of ldhL in L. mesenteroides, the nucleotides for pyruvate kinase promoter were fused to ldhL and cloned into above vectors to construct pLC18pkL and pLC42pkL. As results, introduction of pLC42pkL in L. mesenteroides significantly improved both l-LDH activity and l-lactate productivity during fermentation, decreasing the d-/l-lactate ratio. When used as a starter culture for sauerkraut fermentation, recombinant L. mesenteroides harboring pLC42pkL increased l-lactate concentration and decreased d-lactate concentration compared to the wild type strain. We newly developed a recombinant L. mesenteroides which has high l-lactate dehydrogenase activity and applied this strain to minimize the harmful effect of d-lactate during the sauerkraut fermentation. To the best of our knowledge, we demonstrate for the first time the effective use of recombinant Leuconostoc sp. for quality improvement of fermented foods.
Lactobacillus plantarum ldhL gene: overexpression and deletion. T Ferain;D Garmyn;N Bernard;P Hols;J Delcour. 1994. J Bacteriol. 176. PMID: 8300514

Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing the ldhL gene without modification of its expression signals was introduced in L. plantarum. L-LDH activity was increased up to 13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)- and D-(-)-lactate. A stable chromosomal deletion in the ldhL gene was then constructed in L. plantarum by a two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activity and in exclusive production of the D isomer of lactate. However, the global concentration of lactate in the culture supernatant remained unchanged.
Knockout of the two ldh genes has a major impact on peptidoglycan precursor synthesis in Lactobacillus plantarum. T Ferain;J N Hobbs;J Richardson;N Bernard;D Garmyn;P Hols;N E Allen;J Delcour. 1996. J Bacteriol. 178. PMID: 8808932

Most bacteria synthesize muramyl-pentapeptide peptidoglycan precursors ending with a D-alanyl residue (e.g., UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala). However, it was recently demonstrated that other types of precursors, notably D-lactate-ending molecules, could be synthesized by several lactic acid bacteria. This particular feature leads to vancomycin resistance. Vancomycin is a glycopeptide antibiotic that blocks cell wall synthesis by the formation of a complex with the extremity of peptidoglycan precursors. Substitution of the terminal D-alanine by D-lactate reduces the affinity of the antibiotic for its target. Lactobacillus plantarum is a lactic acid bacterium naturally resistant to vancomycin. It converts most of the glycolytic pyruvate to L- and D-lactate by using stereospecific enzymes designated L- and D-lactate dehydrogenases, respectively. In the present study, we show that L. plantarum actually synthesizes D-lactate-ending peptidoglycan precursors. We also report the construction of a strain which is deficient for both D- and L-lactate dehydrogenase activities and which produces only trace amounts of D- and L-lactate. As a consequence, the peptidoglycan synthesis pathway is drastically affected. The wild-type precursor is still present, but a new type of D-alanine-ending precursor is also synthesized in large quantities, which results in a highly enhanced sensitivity to vancomycin.
Efficient production of optically pure D-lactic acid from raw corn starch by using a genetically modified L-lactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain. Kenji Okano;Qiao Zhang;Satoru Shinkawa;Shogo Yoshida;Tsutomu Tanaka;Hideki Fukuda;Akihiko Kondo. 2008. Appl Environ Microbiol. 75. PMID: 19011066

In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.
Production of optically pure D-lactic acid from brown rice using metabolically engineered Lactobacillus plantarum. Kenji Okano;Shinji Hama;Maki Kihara;Hideo Noda;Tsutomu Tanaka;Akihiko Kondo. 2016. Appl Microbiol Biotechnol. 101. PMID: 27832309

Simultaneous saccharification and fermentation (SSF) of D-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the ʟ-lactate dehydrogenase gene was disrupted, produced 97.7 g/L D-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8-17 h). As a result, 117.1 g/L D-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and D-lactic acid was stably produced using recycled cells (118.4-129.8 g/L). On average, D-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.
Unravelling the multiple effects of lactic acid stress on Lactobacillus plantarum by transcription profiling. Bart Pieterse;Rob J Leer;Frank H J Schuren;Mariët J van der Werf. 2005. Microbiology. 151. PMID: 16339934

The organic acid lactate is the predominant fermentation product of Lactobacillus plantarum. The undissociated form of this organic acid is a strong growth inhibitor for the organism. Different theories have been postulated to explain the inhibitory effects of lactic acid: (i) toxicity arising from the dissipation of the membrane potential, (ii) acidification of the cytosol, or (iii) intracellular anion accumulation. In general, organic acid stresses are complex to study, since their toxicity is highly dependent on their degree of dissociation and thus on the pH. In this study, transcription profiles of L. plantarum grown in steady-state cultures that varied in lactate/lactic acid concentration, pH, osmolarity and absolute and relative growth rate, were compared by microarray analysis. By doing so, the differential expression of multiple groups of genes could specifically be attributed to the different aspects of lactic acid stress. A highly coherent group of lactic acid-responsive, cell surface protein-encoding genes was identified, to which no function has previously been assigned. Moreover, a group of genes that showed increased expression in response to the combination of lactic acid and a lower growth rate is expected to be involved in the formation of the alternative fermentation end-products malate, acetate and ethanol. One of these pathways is the phosphoketolase by-pass that is typical for bifidobacteria.
Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters. S Quatravaux;F Remize;E Bryckaert;D Colavizza;J Guzzo. 2006. J Appl Microbiol. 101. PMID: 16968302

AIMS: The characterization of global aerobic metabolism of Lactobacillus plantarum LP652 under different aeration levels, in order to optimize acetate production kinetics and to suppress H2O2 toxicity. METHODS AND RESULTS: Cultures of L. plantarum were grown on different aeration conditions. After sugar exhaustion and in the presence of oxygen, lactate was converted to acetate, H2O2 and carbon dioxide with concomitant ATP production. Physiological assays were performed at selected intervals in order to assess enzyme activity and vitality of the strain during lactic acid conversion. The maximal aerated condition led to fast lactate-to-acetate conversion kinetics between 8 and 12 h, but H2O2 immediately accumulated, thus affecting cell metabolism. Pyruvate oxidase activity was highly enhanced by oxygen tension and was responsible for H2O2 production after 12 h of culture, whereas lactate oxidase and NADH-dependent lactate dehydrogenase activities were not correlated to metabolite production. Limited NADH oxidase (NOX) and NADH peroxidase (NPR) activities were probably responsible for toxic H2O2 levels in over-aerated cultures. CONCLUSION: Modulating initial airflow led to the maximal specific activity of NOX and NPR observed after 24 h of culture, thus promoting H2O2 destruction and strain vitality at the end of the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimal aeration conditions were determined to minimize H2O2 concentration level during growth on lactate.
D-lactic acid production from cellooligosaccharides and beta-glucan using L-LDH gene-deficient and endoglucanase-secreting Lactobacillus plantarum. Kenji Okano;Qiao Zhang;Shogo Yoshida;Tsutomu Tanaka;Chiaki Ogino;Hideki Fukuda;Akihiko Kondo. 2009. Appl Microbiol Biotechnol. 85. PMID: 19597813

In order to achieve direct fermentation of an optically pure D: -lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an L: -lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (ldhL1) bacterial strain. CelA expression and its degradation of beta-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27 g/l of D: -lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D: -lactic acid fermentation from barley beta-glucan was carried out with the addition of Aspergillus aculeatus beta-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D: -lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from beta-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.
Utilization of lactic acid bacterial genes in Synechocystis sp. PCC 6803 in the production of lactic acid. Ancy Joseph;Shimpei Aikawa;Kengo Sasaki;Yota Tsuge;Fumio Matsuda;Tsutomu Tanaka;Akihiko Kondo. 2013. Biosci Biotechnol Biochem. 77. PMID: 23649263

Metabolic pathway engineering of cyanobacteria for the production of industrially important chemicals from atmospheric CO2 has generated interest recently. Here, we engineered Synechocystis sp. PCC 6803 to produce lactic acid using a lactate dehydrogenase (ldh) gene from various lactic acid-producing bacteria, Lactococcus lactis (ldhB and ldhX), Lactobacillus plantarum (ldhL and ldh), and Lactobacillus rhamnosus (ldhL). The lactic acid was secreted outside the cell using a transporter (lldp) gene from L. plantarum. Expression of each ldh in Synechocystis sp. PCC6803 was ascertained by reverse-transcriptase polymerase chain reaction. Five transformants led to the production of L-lactic acid. Co-expression of lldp with ldhB from L. plantarum or ldhL from L. rhamnosus led to the secretion of lactic acid into the medium at concentration of 0.17 ± 0.02 or 0.14 ± 0.02 mM after 18 d of cultivation.
Channel-mediated lactic acid transport: a novel function for aquaglyceroporins in bacteria. Gerd P Bienert;Benoît Desguin;François Chaumont;Pascal Hols. 2013. Biochem J. 454. PMID: 23799297

MIPs (major intrinsic proteins), also known as aquaporins, are membrane proteins that channel water and/or uncharged solutes across membranes in all kingdoms of life. Considering the enormous number of different bacteria on earth, functional information on bacterial MIPs is scarce. In the present study, six MIPs [glpF1 (glycerol facilitator 1)-glpF6] were identified in the genome of the Gram-positive lactic acid bacterium Lactobacillus plantarum. Heterologous expression in Xenopus laevis oocytes revealed that GlpF2, GlpF3 and GlpF4 each facilitated the transmembrane diffusion of water, dihydroxyacetone and glycerol. As several lactic acid bacteria have GlpFs in their lactate racemization operon (GlpF1/F4 phylogenetic group), their ability to transport this organic acid was tested. Both GlpF1 and GlpF4 facilitated the diffusion of D/L-lactic acid. Deletion of glpF1 and/or glpF4 in Lb. plantarum showed that both genes were involved in the racemization of lactic acid and, in addition, the double glpF1 glpF4 mutant showed a growth delay under conditions of mild lactic acid stress. This provides further evidence that GlpFs contribute to lactic acid metabolism in this species. This lactic acid transport capacity was shown to be conserved in the GlpF1/F4 group of Lactobacillales. In conclusion, we have functionally analysed the largest set of bacterial MIPs and demonstrated that the lactic acid membrane permeability of bacteria can be regulated by aquaglyceroporins.
The effect of lactic acid bacteria on cocoa bean fermentation. Van Thi Thuy Ho;Jian Zhao;Graham Fleet. 2015. Int J Food Microbiol. 205. PMID: 25889523

Cocoa beans (Theobroma cacao L.) are the raw material for chocolate production. Fermentation of cocoa pulp by microorganisms is crucial for developing chocolate flavor precursors. Yeasts conduct an alcoholic fermentation within the bean pulp that is essential for the production of good quality beans, giving typical chocolate characters. However, the roles of bacteria such as lactic acid bacteria and acetic acid bacteria in contributing to the quality of cocoa bean and chocolate are not fully understood. Using controlled laboratory fermentations, this study investigated the contribution of lactic acid bacteria to cocoa bean fermentation. Cocoa beans were fermented under conditions where the growth of lactic acid bacteria was restricted by the use of nisin and lysozyme. The resultant microbial ecology, chemistry and chocolate quality of beans from these fermentations were compared with those of indigenous (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii, Kluyveromyces marxianus and Saccharomyces cerevisiae, the lactic acid bacteria Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in control fermentations. In fermentations with the presence of nisin and lysozyme, the same species of yeasts and acetic acid bacteria grew but the growth of lactic acid bacteria was prevented or restricted. These beans underwent characteristic alcoholic fermentation where the utilization of sugars and the production of ethanol, organic acids and volatile compounds in the bean pulp and nibs were similar for beans fermented in the presence of lactic acid bacteria. Lactic acid was produced during both fermentations but more so when lactic acid bacteria grew. Beans fermented in the presence or absence of lactic acid bacteria were fully fermented, had similar shell weights and gave acceptable chocolates with no differences in sensory rankings. It was concluded that lactic acid bacteria may not be necessary for successful cocoa fermentation.
Unexpected complexity in the lactate racemization system of lactic acid bacteria. Benoît Desguin;Patrice Soumillion;Robert P Hausinger;Pascal Hols. 2017. FEMS Microbiol Rev. 41. PMID: 28830089

Analysis of lactate racemase (Lar) in lactic acid bacteria (LAB) has been a scientific challenge for many years, as indicated by the numerous contradictory reports on this activity. Recently, genetic and biochemical studies of the Lar system of Lactobacillus plantarum have unveiled the complexity of this particular enzymatic system. Lar activity is associated with LarA and its nickel-containing cofactor, synthesized from nicotinic acid adenine dinucleotide by the three biosynthetic enzymes: LarB, LarC, and LarE. In addition to these core Lar enzymes, a nickel transporter (Lar(MN)QO), a lactic acid channel (LarD) and a transcriptional regulator (LarR) which promotes expression of the lar genes in the presence of excess L-lactate are also part of the Lar system of Lb. plantarum and of many other LAB. These proteins promote racemization of external L-lactate, in addition to carrying out intracellular racemization. This additional outcome suggests that racemization of L-lactate is not only required for cell wall biosynthesis, as reported before, but may have additional roles in lactate production and utilization in LAB. Finally, bioinformatics analyses indicate that some Lar homologs probably catalyze reactions other than lactate racemization.
Comparative studies of lactate dehydrogenases in lactic acid bacteria. Amino-acid composition of an active-site region and chemical properties of the L-lactate dehydrogenase of Lactobacillus casei, Lactobacillus curvatus, Lactobacillus plantarum, and Lactobacillus acidophilus. R Hensel;U Mayr;H Fujiki;O Kandler. 1977. Eur J Biochem. 80. PMID: 411654

The molecular weight, the amino acid composition and the N-terminal and C-terminal amino acids of two allosteric (Lactobacillus casei, L. curvatus) and two non-allosteric (L. plantarum, L. acidophilus) L-lactate dehydrogenases, purified to homogeneity by affinity chromatography, were determined. The amino acid composition of the only tryptic peptide unequivocally common to the fingerprints of the 4 enzymes is virtually identical with that of the arginine peptide, called Arg6 of the the substratebinding site of the L-lactate dehydrogenase dehydrogenase of several animals. However, the 'essential' cysteine residue 165 is replaced by threonine, as it is in the L-lactate dehydrogenase of lobster. In addition, the 4 bacterial peptides differ by one or two changes in single amino acid residues from each other as well as from those of animals. The data indicate that not only the animal L-lactate dehydrogenases, but also the allosteric and lactate dehydrogenases from bacterial sources may have evolved from a common gene.
Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake. S Kolb;H Otte;B Nagel;B Schink. 1992. Arch Microbiol. 157. PMID: 1510572

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of L-malate to L-lactate and CO2 concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C4-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.
Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria. H W Doelle. 1971. J Bacteriol. 108. PMID: 4333320

Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.
Homo-D-lactic acid production from mixed sugars using xylose-assimilating operon-integrated Lactobacillus plantarum. Shogo Yoshida;Kenji Okano;Tsutomu Tanaka;Chiaki Ogino;Akihiko Kondo. 2011. Appl Microbiol Biotechnol. 92. PMID: 21643702

In order to achieve efficient D-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (ΔldhL1-xpk1::tkt-Δxpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of ΔldhL1-xpk1::tkt-Δxpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-D-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of D-lactic acid of 99.5%. Finally, we successfully demonstrated homo-D-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-D-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.
Lactic acid production from biomass-derived sugars via co-fermentation of Lactobacillus brevis and Lactobacillus plantarum. Yixing Zhang;Praveen V Vadlani. 2015. J Biosci Bioeng. 119. PMID: 25561329

Lignocellulosic biomass is an attractive alternative resource for producing chemicals and fuels. Xylose is the dominating sugar after hydrolysis of hemicellulose in the biomass, but most microorganisms either cannot ferment xylose or have a hierarchical sugar utilization pattern in which glucose is consumed first. To overcome this barrier, Lactobacillus brevis ATCC 367 was selected to produce lactic acid. This strain possesses a relaxed carbon catabolite repression mechanism that can use glucose and xylose simultaneously; however, lactic acid yield was only 0.52 g g(-1) from a mixture of glucose and xylose, and 5.1 g L(-1) of acetic acid and 8.3 g L(-1) of ethanol were also formed during production of lactic acid. The yield was significantly increased and ethanol production was significantly reduced if L. brevis was co-cultivated with Lactobacillus plantarum ATCC 21028. L. plantarum outcompeted L. brevis in glucose consumption, meaning that L. brevis was focused on converting xylose to lactic acid and the by-product, ethanol, was reduced due to less NADH generated in the fermentation system. Sequential co-fermentation of L. brevis and L. plantarum increased lactic acid yield to 0.80 g g(-1) from poplar hydrolyzate and increased yield to 0.78 g lactic acid per g of biomass from alkali-treated corn stover with minimum by-product formation. Efficient utilization of both cellulose and hemicellulose components of the biomass will improve overall lactic acid production and enable an economical process to produce biodegradable plastics.
Microbiota-Derived Lactate Accelerates Intestinal Stem-Cell-Mediated Epithelial Development. Yong-Soo Lee;Tae-Young Kim;Yeji Kim;Su-Hyun Lee;Seungil Kim;Sung Wan Kang;Jin-Young Yang;In-Jeoung Baek;Young Hoon Sung;Yun-Yong Park;Sung Wook Hwang;Eunju O;Kwang Soon Kim;Siqing Liu;Nobuhiko Kamada;Nan Gao;Mi-Na Kweon. 2018. Cell Host Microbe. 24. PMID: 30543778

Symbionts play an indispensable role in gut homeostasis, but underlying mechanisms remain elusive. To clarify the role of lactic-acid-producing bacteria (LAB) on intestinal stem-cell (ISC)-mediated epithelial development, we fed mice with LAB-type symbionts such as Bifidobacterium and Lactobacillus spp. Here we show that administration of LAB-type symbionts significantly increased expansion of ISCs, Paneth cells, and goblet cells. Lactate stimulated ISC proliferation through Wnt/β-catenin signals of Paneth cells and intestinal stromal cells. Moreover, Lactobacillus plantarum strains lacking lactate dehydrogenase activity, which are deficient in lactate production, elicited less ISC proliferation. Pre-treatment with LAB-type symbionts or lactate protected mice in response to gut injury provoked by combined treatments with radiation and a chemotherapy drug. Impaired ISC-mediated epithelial development was found in mice deficient of the lactate G-protein-coupled receptor, Gpr81. Our results demonstrate that LAB-type symbiont-derived lactate plays a pivotal role in promoting ISC-mediated epithelial development in a Gpr81-dependent manner.
Orange peels: from by-product to resource through lactic acid fermentation. Annalisa Ricci;Ana Belen Diaz;Ildefonso Caro;Valentina Bernini;Gianni Galaverna;Camilla Lazzi;Ana Blandino. 2019. J Sci Food Agric. 99. PMID: 31353470

BACKGROUND: Considering the large amounts of by-products derived from orange processing, which are generally discarded, the present study aimed to explore the feasibility of using orange peel for lactic acid production in solid state fermentation. RESULTS: Different species of lactic acid bacteria were employed, singly and in co-culture, to evaluate their ability to ferment orange peel and produce lactic acid. Among the single cultures tested, Lactobacillus casei 2246 was the most efficient strain, reaching the highest concentration of lactic acid (209.65 g kg-1 ) and yield (0.88 g g-1 ). The use of Lactobacillus plantarum 285 and Lactobacillus paracasei 4186 in co-culture produced a comparable amount of lactic acid, showing a better performance than the same strains in single cultures. CONCLUSION: Orange peels represent a suitable raw material for solid state fermentation employing lactic acid bacteria. Lactic acid was obtained that consumed the most of sugars available, leading to high yields. Despite all the strains tested showing the same growth ability, different peculiarities in lactic acid production were revealed, dependent on the species/strains, suggesting the relevance of strain selection. © 2019 Society of Chemical Industry.
Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli. S Kochhar;H Hottinger;N Chuard;P G Taylor;T Atkinson;M D Scawen;D J Nicholls. 1992. Eur J Biochem. 208. PMID: 1396685

NAD(+)-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220-215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499-8513], a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene. Both plasmids overexpressed D-lactate dehydrogenase (greater than 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60-66]. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. alpha 2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2-oxo-acid substrates. The kinetic profiles of 2-oxo-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.
Nicotinic acid controls lactate production by K1-LDH: a Saccharomyces cerevisiae strain expressing a bacterial LDH gene. Sophie Colombié;Jean-Marie Sablayrolles. 2004. J Ind Microbiol Biotechnol. 31. PMID: 15205990

Industrial applications for lactate, such as the production of chemicals, has led to interest in producing this organic acid by metabolically engineered a yeast such as Saccharomyces cerevisiae, which is more acid tolerant than lactic acid bacteria. This paper deals with lactate production by S. cerevisiae K1-LDH, in which the Lactobacillus plantarum lactate dehydrogenase (LDH) gene is integrated into the genome of the wine yeast strain K1. We show that a vitamin, nicotinic acid (NiA), was the limiting factor for lactate production during fermentation with the K1-LDH strain. Increasing the NiA concentration in batch conditions or in the medium used to feed chemostats affected the lactate yield. Moreover, the addition of pulses of NiA or the exponential addition of NiA made it possible to control the lactate production kinetics throughout the fermentation process. The results point to the role of NiA in the regulation of metabolic pathways, but the physiological mechanisms remain poorly understood.
Influence of calcium lactate on the fate of spoilage and pathogenic microorganisms in orange juice. Jui-Yueh Yeh;Ellis Hoogetoorn;Jinru Chen. 2004. J Food Prot. 67. PMID: 15270496

Calcium lactate is used by the beverage industry as a source of calcium to fortify fruit juice. The objective of this study was to evaluate the influence of various concentrations of calcium lactate on the fate of pathogenic and spoilage microorganisms in orange juice. Commercial nonfortified orange juice was supplemented with calcium lactate at a concentration equivalent to 0, 5, 10, 15, 20, 25, or 30% dietary reference intake. The pH of each fortified juice was adjusted to 3.6 or 4.1. The prepared juice samples were inoculated separately with a three-strain mixture of salmonellae, a three-strain mixture of spoilage yeasts, and three single strains of spoilage bacteria including Alicyclobacillus acidoterrestris, Lactobacillus plantarum, and Lactobacillus sake. The contaminated juice was stored at 4 and 10 degrees C, respectively, for 6 to 7 weeks and assayed once a week for populations of salmonellae, spoilage yeasts, or spoilage bacteria. The results indicated that A. acidoterrestris was inhibited in all juice stored at 4 degrees C and low-pH juice stored at 10 degrees C. The bacterium, however, was able to grow at 10 degrees C in the high-pH juice with calcium lactate concentrations equivalent to 0 and 5% dietary reference intake. The cells of L. sake declined and eventually died off in low-pH juice stored at 4 and 10 degrees C and in high pH stored at 4 degrees C. But the organism flourished at 10 degrees C in the high-pH juice containing 0, 10, and 20% dietary reference intake of calcium lactate. The populations of L. plantarum remained approximately stable in low- as well as in high-pH juice stored at both 4 and 10 degrees C. While inhibited at 4 degrees C, the spoilage yeasts grew at 10 degrees C. Salmonellae died off in all juice stored at 4 degrees C and in low-pH juice stored at 10 degrees C. However, they persisted in the high-pH juice stored at 10 degrees C except in the samples that contained 20 to 30% dietary reference intake of calcium lactate.
Major role of NAD-dependent lactate dehydrogenases in aerobic lactate utilization in Lactobacillus plantarum during early stationary phase. Philippe Goffin;Frédérique Lorquet;Michiel Kleerebezem;Pascal Hols. 2004. J Bacteriol. 186. PMID: 15375150

NAD-independent lactate dehydrogenases are commonly thought to be responsible for lactate utilization during the stationary phase of aerobic growth in Lactobacillus plantarum. To substantiate this view, we constructed single and double knockout mutants for the corresponding genes, loxD and loxL. Lactate-to-acetate conversion was not impaired in these strains, while it was completely blocked in mutants deficient in NAD-dependent lactate dehydrogenase activities, encoded by the ldhD and ldhL genes. We conclude that NAD-dependent but not NAD-independent lactate dehydrogenases are involved in this process.
Viable, lyophilized lactobacilli do not increase iron absorption from a lactic acid-fermented meal in healthy young women, and no iron absorption occurs in the distal intestine. Stine Bering;Laila Sjøltov;Seema S Wrisberg;Anna Berggren;Jan Alenfall;Mikael Jensen;Liselotte Højgaard;Inge Tetens;Klaus Bukhave. 2007. Br J Nutr. 98. PMID: 17764597

Lactic acid-fermented foods have been shown to increase Fe absorption in human subjects, possibly by lowering pH, activation of phytases, production of organic acids, or by the viable lactic acid bacteria. In this study the effect of a heat-inactivated lactic acid-fermented oat gruel with and without added viable, lyophilized Lactobacillus plantarum 299v on non-haem Fe absorption was investigated. Furthermore, Fe absorption in the distal intestine was determined. In a randomized, double-blinded crossover trial eighteen healthy young women aged 22 (SD 3) years with low Fe status (serum ferritin < 30 microg/l) were served the two test gruels, extrinsically labelled with 59Fe and served with two enterocoated capsules (containing 55Fe(II) and 55Fe(III), respectively) designed to disintegrate in the ileum. The meals were consumed on two consecutive days, e.g. in the order AA followed by BB in a second period. Non-haem Fe absorption was determined from 59Fe whole-body retention and isotope activities in blood samples. The concentrations of Fe, lactate, phytate, and polyphenols, and the pH were similar in the heat-inactivated lactic acid-fermented oat gruels with and without added L. plantarum 299v, and no difference in Fe absorption was observed between the test gruels (1.4 and 1.3%, respectively). Furthermore, no absorption of Fe in the distal intestine was observed. In conclusion, addition of viable, lyophilized lactobacillus to a heat-inactivated lactic acid-fermented oat gruel does not affect Fe absorption, and no absorption seems to occur in the distal part of the intestine from low Fe bioavailability meals in these women.
Degradation of organic acids by dairy lactic acid bacteria. F Z Hegazi;I G Abo-Elnaga. 1980. Zentralbl Bakteriol Naturwiss. 135. PMID: 6775434

One hundred and twelve different strains of lactic acid bacteria, belonging to the genera Leuconostoc, Streptococcus, and Lactobacillus, were examined for the ability to degrade 10 organic acids by detecting gas production, using the conventional Durham tube method. All the strains did not break down succinate, glutarate, 2-oxo-glutarate, and mucate. Malate, citrate, pyruvate, fumarate, tartrate, and gluconate were variably attacked. Streptococcus cremoiris AM2, ML8, and SK11 required glucose to produce gas from citrate, whereas Leuconostoc citrovorum and Streptococcus faecalis did not. Streptococcus cremoris differed from the other streptococci in not producing gas from gluconate. From all lactic acid bacteria examined, only Lactobacillus plantarum formed gas from tartarate. Determination of acetoin and diacetyl proved to be a more reliable evidence for assessing the degradation of pyruvate, compared with detection of gas production. Homofermentative lactobacilli and Leuconostoc citrovorum produced acetoin and diacetyl from pyruvate, whereas beta-bacteria did not, a character that would be of taxonomic value. Streptobacteria degraded pyruvate in the presence of glucose with lactate as the major product together with a mean acetate of 4.1%, ethanol 7.9%, acetoin 1.7%, and diacetyl 2.6% yield on a molar basis after 60 days at 30 degrees C. L. brevis produced acetate and lactate. Formation of diacetyl from pyruvate by lactic acid bacteria may play an important role in flavour development in fermenting dairy products, especially in cheese, where lactic acid bacteria usually predominate, and pyruvate is probably excreted in the breaking down of lactose and in the oxidative deamination of alanine by the accompanying microflora.
Histidine 296 is essential for the catalysis in Lactobacillus plantarum D-lactate dehydrogenase. H Taguchi;T Ohta. 1993. J Biol Chem. 268. PMID: 8349682

Two His residues, His-205 and His-296, in Lactobacillus plantarum D-lactate dehydrogenase are highly conserved in the D-isomer-specific 2-hydroxyacid dehydrogenase family, suggesting that they are candidates for the catalytic His in the enzyme. The substitution of His-296 with Tyr by means of site-directed mutagenesis induced a drastic decrease in the reaction rate, while a His-205 to Tyr substitution induced no large change in the catalytic properties. In pyruvate reduction, the Tyr-296 enzyme showed a slightly increased Km below pH 6.0 but no significant pH dependence above pH 6.0, where the wild-type enzyme showed an increased Km value. In D-lactate oxidation, in contrast, the Tyr-296 enzyme showed a greatly increased Km value for D-lactate and strong pH dependence. An additional substitution of His-296 with Gln induced more complete loss of the catalytic activity. In contrast to the Tyr-296 enzyme, the Gln-296 enzyme showed a greatly increased Km value and a strong pH-dependent reaction rate even in pyruvate reduction. Unlike the wild-type or His-205 enzyme, both the Tyr-296 and Gln-296 enzymes showed significant resistance against diethyl pyrocarbonate. These results clearly indicate that His-296 is essential for the catalysis by D-lactate dehydrogenase, as in the case of His-195 in L-lactate dehydrogenase.
Abnormal fecal flora in a patient with short bowel syndrome. An in vitro study on effect of pH on D-lactic acid production. M I Caldarini;S Pons;D D'Agostino;J A DePaula;G Greco;G Negri;A Ascione;D Bustos. 1996. Dig Dis Sci. 41. PMID: 8769294

D-Lactic acidosis associated with encephalopathy is a clinical condition that occurs in patients with short bowel syndrome. We studied the fecal flora and the composition of fecal water of a child who developed this unusual disorder. Bacteriological studies showed that the patient's stool contained a marked predominance of gram-positive anaerobes. Two strains were identified, Lactobacillus plantarum and Lactobacillus salivarius, as the main bacteria isolated. Fecal water showed pH 4.8 and total lactic acid (sum of L- and D-lactic acids) was the principal organic anion found in the feces. We also incubated the patient's stool in a continuous culture with a view to determining the effect of the pH on the production of volatile fatty acids (VFA) and L- and D-lactic acids. The culture was maintained at pH 5.0, 5.5, 6.0, and 6.5 for four consecutive periods of four days each. We then studied the culture for a further four days at pH 5.0 once again. This study showed that with the progressive rise of the pH from 5.0 to 6.5 L- and D-lactic acids decreased and VFA production increased. D-Lactic acid formation was inhibited at pH 6.5, but when the culture was returned to pH 5.0, it increased to a high level again. These results suggest that the pH plays an important role in the ecological changes in the colonic bacteria that result in D-lactic acid production.
Lactic acid production by simultaneous saccharification and fermentation of alfalfa fiber. H K Sreenath;A B Moldes;R G Koegel;R J Straub. 2005. J Biosci Bioeng. 92. PMID: 16233139

Lactic acid was produced by simultaneous saccharification and fermentation (SSF) of liquid hot water (LHW)-pretreated and non-LHW-pretreated alfalfa fibers. The Lactobacillus plantarum and L. delbrueckii strains produced 0.464 and 0.354 g of lactic acid per g of dry matter of alfalfa fiber, respectively, by non-LHW pretreatment. L. xylosus and L. pentoaceticus produced lower yields of lactic acid from the same amount of alfalfa fiber, however, their acetic acid production was higher. These Lactobacillus strains did not require any additional nutrients during SSF of non-LHW-pretreated alfalfa fiber. After LHW pretreatment, the "raffinate" cellulosic fraction of alfalfa required additional nutrients for lactic acid production by SSF. Both L. plantarum and L. delbrueckii produced 0.606 and 0.59 g of lactic acid per g of dry matter of fiber, respectively. However, the "extract" soluble hemicellulosic fraction of alfalfa produced 0.38 to 0.62 g of lactic acid per g of dry matter extract during SSF and did not require nutrient supplementation. These results suggest that during the LHW pretreatment, alfalfa fiber nutrients are lost in cellulosic fractions but retained in hemicellulosic extract fractions.
Natural populations of lactic acid bacteria isolated from vegetable residues and silage fermentation. J Yang;Y Cao;Y Cai;F Terada. 2010. J Dairy Sci. 93. PMID: 20630231

Natural populations of lactic acid bacteria (LAB) and silage fermentation of vegetable residues were studied. Fifty-two strains of LAB isolated from cabbage, Chinese cabbage, and lettuce residues were identified and characterized. The LAB strains were gram-positive and catalase-negative bacteria, which were divided into 6 groups (A to F) according to morphological and biochemical characteristics. The strains in group A were rods that did not produce gas from glucose and formed the d and l isomers of lactate. Groups B and C were homofermentative cocci that formed l-lactic acid. Groups D, E, and F were heterofermentative cocci that formed d-lactic acid. Based on 16S rDNA gene sequence analysis, group A to F strains were identified as Lactobacillus plantarum, Lactococcus piscium, Lactococcus lactis, Leuconostoc citreum, Weissella soli and Leuconostoc gelidum, respectively. The prevalent LAB, predominantly homofermentative lactobacilli, consisted of Lactobacillus plantarum (34.6%), Weissella soli (19.2%), Leuconostoc gelidum (15.4%), Leuconostoc citreum (13.5%), Lactococcus lactis (9.6%), and Lactococcus piscium (7.7%). Lactobacillus plantarum was the dominant member of the LAB population in 3 types of vegetable residues. These vegetable residues contained a high level of crude protein (20.2 to 28.4% of dry matter). These silages prepared by using a small-scale fermentation system were well preserved, with low pH and a relatively high content of lactate. This study suggests that the vegetable residues contain abundant LAB species and nutrients, and that they could be well preserved by making silage, which is a potentially good vegetable protein source for livestock diets.
Stimulation of cadaverine production by foodborne pathogens in the presence of Lactobacillus, Lactococcus, and Streptococcus spp. Esmeray Kuley;Esra Balıkcı;Ilyas Özoğul;Saadet Gökdogan;Fatih Ozoğul. 2012. J Food Sci. 77. PMID: 22853653

The effect of Lactobacillus plantarum (FI8595), Lactococcus lactis subsp. cremoris MG 1363), Lactococcus lactis subsp. lactis (IL 1403), and Streptococcus thermophilus on cadaverine and other biogenic amine production by foodborne pathogens was investigated lysine decarboxylase broth. Both of lactic acid bacteria and foodborne pathogens used (especially Staphylococcus aureus, E. coli, Lc. lactis subsp. lactis and Lb. plantarum) had an ability to convert aminoacids into biogenic amine. The conversion of lysine into cadaverine was the highest (167.11 mg/L) by Lactobacillus spp. Gram-positive bacteria generally had a greater ability to produce cadaverine with corresponding value of 46.26, 53.76, and 154.54 mg/L for Enterococcus faecalis, S. aureus, and Listeria monocytogenes, respectively. Significant variations on biogenic amine production were observed in the presence of lactic acid bacteria strains (P < 0.05). The role of lactic acid bacteria on biogenic amine production by foodborne pathogens varied depending on strains and specific amine. Cadaverine accumulation by Enterobactericeae was increased in the presence of lactic acid bacteria strains except for St. thermophilus, which induced 2-fold lower cadaverine production by S. Paratyphi A. Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris induced 10-fold higher increases in histamine for E. coli and K. pneumoniae, respectively. Lactic acid bacteria resulted in strong increases in cadaverine production by P. aeruginosa, although remarkable decreases were observed for histamine, spermidine, dopamine, agmatine, and TMA in the presence of lactic acid bacteria in lysine decarboxylase broth . The result of the study showed that amine positive lactic acid bacteria strains in fermented food led to significant amine accumulation by contaminant bacteria and their accumulation in food product may be controlled by the use of proper starters with amine-negative activity.
In situ combination of fermentation and electrodialysis with bipolar membranes for the production of lactic acid: operational compatibility and uniformity. Xiaolin Wang;Yaoming Wang;Xu Zhang;Tongwen Xu. 2012. Bioresour Technol. 125. PMID: 23026329

An in situ combination of fermentation and electrodialysis with bipolar membranes was customized for the production of lactic acid to achieve their operational compatibility and uniformity. Primarily, fermentation experiments for lactic acid production were conducted by Lactobacillus plantarum with an average lactate productivity of 1.76 g/(Lh) and yield coefficient of 56.77%. Subsequently, an electrodialysis with bipolar membranes (EDBMs) stack was established with the fermented lactate mixtures as a feed. Effect of operating current density on the production of lactic acid and alkali liquor was investigated. Results indicated that only the current density of no less than 50 mA/cm(2) can produce enough alkali liquor to meet the need for fermentation. Ultimately, a combination of fermentation and EDBM process was carried out and the integration can achieve a lactic acid recovery ratio of 86.05% at the current density 60 mA/cm(2).
Enhanced D-lactic acid production from renewable resources using engineered Lactobacillus plantarum. Yixing Zhang;Praveen V Vadlani;Amit Kumar;Philip R Hardwidge;Revathi Govind;Tsutomu Tanaka;Akihiko Kondo. 2015. Appl Microbiol Biotechnol. 100. PMID: 26433970

D-lactic acid is used as a monomer in the production of poly-D-lactic acid (PDLA), which is used to form heat-resistant stereocomplex poly-lactic acid. To produce cost-effective D-lactic acid by using all sugars derived from biomass efficiently, xylose-assimilating genes encoding xylose isomerase and xylulokinase were cloned into an L-lactate-deficient strain, Lactobacillus plantarum. The resulting recombinant strain, namely L. plantarum NCIMB 8826 ∆ldhL1-pLEM-xylAB, was able to produce D-lactic acid (at optical purity >99 %) from xylose at a yield of 0.53 g g(-1). Simultaneous utilization of glucose and xylose to produce D-lactic acid was also achieved by this strain, and 47.2 g L(-1) of D-lactic acid was produced from 37.5 g L(-1) glucose and 19.7 g L(-1) xylose. Corn stover and soybean meal extract (SBME) were evaluated as cost-effective medium components for D-lactic acid production. Optimization of medium composition using response surface methodology resulted in 30 % reduction in enzyme loading and 70 % reduction in peptone concentration. In addition, we successfully demonstrated D-lactic acid fermentation from corn stover and SBME in a fed-batch fermentation, which yielded 61.4 g L(-1) D-lactic acid with an overall yield of 0.77 g g(-1). All these approaches are geared to attaining high D-lactic acid production from biomass sugars to produce low-cost, highly thermostable biodegradable plastics.
Low-pH production of d-lactic acid using newly isolated acid tolerant yeast Pichia kudriavzevii NG7. Hyun Joo Park;Jung-Hoon Bae;Hyeok-Jin Ko;Sun-Hee Lee;Bong Hyun Sung;Jong-In Han;Jung-Hoon Sohn. 2018. Biotechnol Bioeng. 115. PMID: 29896854

Lactic acid is a platform chemical for the sustainable production of various materials. To develop a robust yeast platform for low-pH production of d-lactic acid (LA), an acid-tolerant yeast strain was isolated from grape skins and named Pichia kudriavzevii NG7 by ribosomal RNA sequencing. This strain could grow at pH 2.0 and 50°C. For the commercial application of P. kudriavzevii NG7 as a lactic acid producer, the ethanol fermentation pathway was redirected to lactic acid by replacing the pyruvate decarboxylase 1 gene (PDC1) with the d-lactate dehydrogenase gene (d-LDH) derived from Lactobacillus plantarum. To enhance lactic acid tolerance, this engineered strain was adapted to high lactic acid concentrations, and a new transcriptional regulator, PAR1, responsible for acid tolerance, was identified by whole-genome resequencing. The final engineered strain produced 135 g/L and 154 g/L of d-LA with productivity over 3.66 g/L/hr at pH 3.6 and 4.16 g/L/hr at pH 4.7, respectively.
Antimicrobial interactions of microbial species involved in the fermentation of cassava dough into agbelima with particular reference to the inhibitory effect of lactic acid bacteria on enteric pathogens. Ebenezer Siaw Mante;Esther Sakyi-Dawson;Wisdom Kofi Amoa-Awua. 2003. Int J Food Microbiol. 89. PMID: 14580972

Lactic acid bacteria, Bacillus species and yeasts are involved in the fermentation of cassava dough into agbelima. Microbial interactions within and between these groups of microorganisms were investigated in addition to the survival of five enteric pathogens inoculated into agbelima under various conditions. Nine out of 10 cultures of lactic acid bacteria isolated at the end of agbelima fermentation showed inhibitory effect against 10 cultures of lactic acid bacteria isolated at the start of fermentation. Only 3 out of 10 isolates of Bacillus subtilis were inhibited by 10 isolates of lactic acid bacteria tested. No interactions were observed between yeasts and the lactic acid bacteria, whereas three of the Bacillus isolates showed inhibitory effects against the yeasts. Twelve isolates of Lactobacillus plantarum tested inhibited the growth of an isolate each of Lactobacillus fermentum and Lactobacillus brevis but none tested positive for bacteriocin production. The antimicrobial effect of the lactic acid bacteria was attributed to acid production. In fermenting cassava dough, enteric pathogens survived to different extents depending on pH and their sensitivity to acids. Vibrio cholerae C-230, Salmonella typhimurium 9 and Salmonella enteritidis 226 were not detectable in 10 g of sample after 4 h when inoculated into the 48-h fermented product, agbelima, whereas Shigella dysenteriae 2357T and Escherichia coli D2188 were detectable up to 8 h in the product.
Optimisation of lactic acid fermentation for improved vinegar flavour during rosy vinegar brewing. Yujian Jiang;Jianna Guo;Yudong Li;Sen Lin;Li Wang;Jianrong Li. 2010. J Sci Food Agric. 90. PMID: 20474052

BACKGROUND: Rosy vinegar is a well-known traditional Chinese product whose flavour is affected by its lactic acid content. In this study, Lactobacillus bacteria were employed to increase the content of lactic acid during the ethanol fermentation stage. RESULTS: The optimised fermentation parameters were determined as an inoculation amount of 3% (v/v), a temperature of 30 degrees C and an initial pH value of 4.0. Fermentation under these optimal conditions resulted in an alcohol degree of 6.2% (v/v), a total acidity of 49.5 g L(-1) and a lactic acid content of 4.14 g L(-1). The content of lactic acid (4.14 g L(-1)), which approached the level achieved by solid state fermentation, was 3.56-fold higher than that in vinegar fermented without lactic acid bacteria (1.16 g L(-1)). CONCLUSION: The results indicate that mixed fermentation with Lactobacillus plantarum and Saccharomyces cerevisiae strains greatly increases the lactic acid content and improves the flavour of rosy vinegar.
[Composition diversity and metabolic characters of lactic acid bacteria community SGL]. Jingjing Liu;Fuyu Yang;Xiaofen Wang;Jinhuan Liu;Xufeng Yuan;Zongjun Cui. 2016. Wei Sheng Wu Xue Bao. 55. PMID: 26915229

OBJECTIVE: We aimed to select a stable lactic acid bacteria community from switchgrass silage, that was efficient in lactic acid production. METHODS: We obtained the community by continuous restricted subcultivation in MRS broth, and analysed the composition diversity and stability of the community by 16S rRNA gene-based pyrosequencing and Denaturing Gradient Gel Electrophoresis (DGGE), respectively. In addition, we studied the effect of different nitrogen sources on growth and lactic acid production of the community, through adding different concentrations of yeast extraction, different nitrogen sources [yeast extract, peptone, urea and (NH4) 2SO4] and different proportions of (NH4)2SO4 and yeast extract leveled with elemental nitrogen 1.8 g/L. RESULTS: The microbial composition of SGL became stable from the 8th generation according to the results of DGGE. The pH value of the MRS inoculated with SGL dropped to 3.7, and the concentration of lactic acid reached 26 g/L after 24 h cultivation. The result of the pyrosequencing showed that the major composition of SGL were Lactobacillus nantensis (78.78%), Lactobacillus plantarum (7.92%), Lactobacillus pantheris (5.27%), Bacillus coagulans (4.41%) and Lactococcus lactics (3.31%). The best supplementation of yeast extraction for SGL was 20 g/L. When the elemental nitrogen ratio of (NH4) 2SO4 to yeast extract was 1:4, the growth and lactic acid production were no significant difference with 0:5 (P < 0.05). CONCLUSION: SGL had a great potential of application, as an efficient inoculant for ensilage or lactic acid production. This study would offer theoretical basis for cultivate and application of SGL in production.
Development of a new lactic acid bacterial inoculant for fresh rice straw silage. Jong Geun Kim;Jun Sang Ham;Yu Wei Li;Hyung Soo Park;Chul-Sung Huh;Byung-Chul Park. 2017. Asian-Australas J Anim Sci. 30. PMID: 28669141

OBJECTIVE: Effects of newly isolated Lactobacillus plantarum on the fermentation and chemical composition of fresh rice straw silage was evaluated in this study. METHODS: Lactic acid bacteria (LAB) from good crop silage were screened by growing them in MRS broth and a minimal medium with low carbohydrate content. Selected LAB (LAB 1821) were Gram-positive, rods, catalase negative, and were identified to be Lactobacillus plantarum based on their biochemical characteristics and a 16S rRNA analysis. Fresh rice straw was ensiled with two isolated LAB (1821 and 1841), two commercial inoculants (HM/F and P1132) and no additive as a control. RESULTS: After 2 months of storage at ambient temperature, rice straw silages treated with additives were well-preserved, the pH values and butyric and acetic acid contents were lower, and the lactic acid content and lactic/acetic acid ratio were higher than those in the control (p<0.05). Acidity (pH) was lowest, and lactic acid highest, in 1821-treated silage (p<0.05). The NH3-N content decreased significantly in inoculant-treated silage (p<0.05) and the NH3-N content in 1821-treated silage was lowest among the treatments. The dry matter (DM) content of the control silage was lower than that of fresh rice straw (p<0.05), while that of the 1841- and p1174-inoculant-treated silages was significantly higher than that of HM/F-treated silage. Microbial additives did not have any significant (p>0.05) effect on acid detergent fiber or neutral detergent fiber contents. Crude protein (CP) content and in vitro DM digestibility (IVDMD) increased after inoculation of LAB 1821 (p<0.05). CONCLUSION: LAB 1821 increased the CP, IVDMD, lactic acid content and ratio of lactic acid to acetic acid in rice straw silage and decreased the pH, acetic acid, NH3-N, and butyric acid contents. Therefore, adding LAB 1821 improved the fermentation quality and feed value of rice straw silage.
Characterization of a heat-tolerant Chlorella sp. GD mutant with enhanced photosynthetic CO2 fixation efficiency and its implication as lactic acid fermentation feedstock. Tse-Min Lee;Yu-Fei Tseng;Chieh-Lun Cheng;Yi-Chuan Chen;Chih-Sheng Lin;Hsiang-Yen Su;Te-Jin Chow;Chun-Yen Chen;Jo-Shu Chang. 2017. Biotechnol Biofuels. 10. PMID: 28919927

BACKGROUND: Fermentative production of lactic acid from algae-based carbohydrates devoid of lignin has attracted great attention for its potential as a suitable alternative substrate compared to lignocellulosic biomass. RESULTS: A Chlorella sp. GD mutant with enhanced thermo-tolerance was obtained by mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine to overcome outdoor high-temperature inhibition and it was used as a feedstock for fermentative lactic acid production. The indoor experiments showed that biomass, reducing sugar content, photosynthetic O2 evolution rate, photosystem II activity (Fv/Fm and Fv'/Fm'), and chlorophyll content increased as temperature, light intensity, and CO2 concentration increased. The mutant showed similar DIC affinity and initial slope of photosynthetic light response curve (α) as that of the wild type but had higher dissolved inorganic carbon (DIC) utilization capacity and maximum photosynthesis rate (Pmax). Moreover, the PSII activity (Fv'/Fm') in the mutant remained normal without acclimation process after being transferred to photobioreactor. This suggests that efficient utilization of incident high light and enhanced carbon fixation with its subsequent flux to carbohydrates accumulation in the mutant contributes to higher sugar and biomass productivity under enriched CO2 condition. The mutant was cultured outdoors in a photobioreactor with 6% CO2 aeration in hot summer season in southern Taiwan. The harvested biomass was subjected to separate hydrolysis and fermentation (SHF) for lactic acid production with carbohydrate concentration equivalent to 20 g/L glucose using the lactic acid-producing bacterium Lactobacillus plantarum 23. The conversion rate and yield of lactic acid were 80% and 0.43 g/g Chlorella biomass, respectively. CONCLUSIONS: These results demonstrated that the thermo-tolerant Chlorella mutant with high photosynthetic efficiency and biomass productivity under hot outdoor condition is an efficient fermentative feedstock for large-scale lactic acid production.
The occurrence and prevention of ethanol fermentation in high-dry-matter grass silage. Frank Driehuis;Piet G van Wikselaar. 2000. J Sci Food Agric. 80. PMID: 29345777

Ethanol is a common, usually minor fermentation product in ensiled forages, the major product being lactic acid. Occasionally, high levels of ethanol are found in silages. The aim of this study was to determine the incidence of high-dry-matter (DM) grass silages containing ethanol as the main fermentation product (ethanol silages), to describe the fermentation process in such silages and to determine the effect of grass maceration prior to wilting and addition of a bacterial inoculant containing Lactobacillus plantarum and Enterococcus faecium strains on fermentation. Twenty-one laboratory silages produced between 1993 and 1995, 21 farm silages produced between 1980 and 1989 and 36 farm silages produced in 1995 (all produced without additive) were examined for pH and chemical composition. Dry matter (DM) loss during ensilage was determined for the laboratory silages only. Four laboratory silages were identified as ethanol silages. Mean concentrations of ethanol, lactic acid and acetic acid were 48.1, 15.5 and 6.0 g kg-1 DM respectively. In the silages that contained lactic acid as the main fermentation product (lactic acid silages) these values were 7.7, 45.5 and 15.1 g kg-1 DM. Mean DM loss and pH were 62.8 g kg-1 DM and 5.32 respectively for ethanol silages and 24.4 g kg-1 DM and 4.69 for lactic acid silages. There was no difference between ethanol silages and lactic acid silages in the mean concentration of ammonia-N (94 g kg-1 total N), and butyric acid was not detected (<0.2 g kg-1 DM), indicating that both types of silages were well preserved. Analysis of the composition of the grass at ensiling showed a positive correlation between the concentration of soluble carbohydrates and the development into ethanol silage. Analysis of the farm silages indicated that 29% of the silages produced between 1980 and 1989 and 14% of those produced in 1995 were ethanol silages. Maceration prior to wilting and addition of silage inoculant improved lactic acid fermentation and prevented high ethanol levels. The micro-organisms responsible for ethanol fermentation as well as the implications of feeding ethanol silages to livestock remain to be resolved. © 2000 Society of Chemical Industry.
Unravelling the contribution of lactic acid bacteria and acetic acid bacteria to cocoa fermentation using inoculated organisms. Van Thi Thuy Ho;Graham H Fleet;Jian Zhao. 2018. Int J Food Microbiol. 279. PMID: 29727857

Cocoa beans (Theobroma cacao L.) are the raw material for chocolate production. Fermentation of the bean pulp by microorganisms is essential for developing the precursors of chocolate flavour. Currently, the cocoa fermentation is still conducted by an uncontrolled traditional process via a consortium of indigenous species of yeasts, lactic acid bacteria and acetic acid bacteria. Although the essential contribution of yeasts to the production of good quality beans and, typical chocolate character is generally agreed, the roles of lactic acid bacteria and acetic acid bacteria are less certain. The objective of this study was to investigate the contribution of LAB and AAB in cocoa bean fermentation by conducting small scale laboratory fermentations under aseptic conditions, inoculated with different groups of microorganisms previously isolated from spontaneous cocoa fermentations. The inoculation protocols were: (1) four yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii, Kluyveromyces marxianus and Saccharomyces cerevisiae; (2) four yeasts plus the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum; (3) four yeasts plus the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateuri and (4) four yeasts plus two lactic acid bacteria and two acetic acid bacteria. Only the inoculated species were detected in the microbiota of their respective fermentations. Beans from the inoculated fermentations showed no significant differences in colour, shell weights and concentrations of residual sugars, alcohols and esters (p>0.05), but they were slightly different in contents of lactic acid and acetic acid (p<0.05). All beans were fully brown and free of mould. Residual sugar levels were less than 2.6 mg/g while the shell contents and ethanol were in the range of 11-13.4% and 4.8-7 mg/g, respectively. Beans fermented in the presence of LAB contained higher levels of lactic acid (0.6-1.2 mg/g) whereas higher concentrations of acetic acid (1.8-2.2 mg/g) were detected in beans inoculated with AAB. Triangle and hedonic sensory evaluations of chocolates prepared from beans taken from the three fermentations showed no significant differences (p > 0.05). It was concluded that the growth of lactic acid bacteria and acetic acid bacteria may not be essential for the fermentation of cocoa beans.