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

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

Electrotransformation of Lactobacillus manihotivorans LMG 18010T and LMG 18011

An electroporation procedure was developed for the genetic transformation of intact cells of Lactobacillus manihotivorans , a new Lactobacillus species isolated from cassava sour starch fermentation in Colombia. Transformation efficiency of Lact. manihotivorans strains LMG 18010^T and LMG 18011 was measured and compared with electroporability of Lact. plantarum strains NCIMB 8299 and LMG 9211, by investigating the effect of changes in various parameters. For Lact. manihotivorans strain LMG 18010^T, the composition of the culture medium, such as the type of peptone and the presence of Tween-80, was found to be the most critical parameter, as well as the aeration conditions of the culture; better electroporation was obtained without air. The presence of MgCl₂ in the recovery medium was favourable to regeneration of electroporated cells. Plasmid-curing of the cells did not improve their electroporability. Transformants were obtained with Lact. manihotivorans strain LMG 18010^T and the plasmids pLZ12 and pGK13, whereas Lact. manihotivorans strain LMG 18011 was transformable with plasmids pLP825 and pLZ12, with different electroporation conditions.     

Direct production of L+-lactic acid from starch and food wastes using Lactobacillus manihotivorans LMG18011

This study describes several essential factors for direct and effective lactic acid production from food wastes by Lactobacillus manihotivorans LMG18011, and optimum conditions for simultaneous saccharification and fermentation using soluble starch and food wastes as substrates. The productivity was found to be affected by three factors: (1) initial pH, which influenced amylase production for saccharification of starch, (2) culture pH control which influenced selective production of L(+)-lactic acid, and (3) manganese concentration in medium which improved in production rate and yield of lactic acid. The optimum initial pH was 5.0-5.5, and the fermentation pH for the direct and effective fermentation from starchy substrate was 5.0 based on the yield of L(+)-lactic acid. Under these conditions, 19.5 g L(+)-lactic acid was produced from 200 g food wastes by L. manihotivorans LMG18011. Furthermore, the addition of manganese stimulated the direct fermentation significantly, and enabled complete bioconversion within 100 h.     

Direct fermentation of starch to lactic acid byLactobacillus amylovorus

Summary Fermentation production of lactic acid directly from starch was studied in a batch fermentor usingLactobacillus amylovorus. At an initial concentration of 120 g/L starch, 96.2 g/L of lactic acid was produced from liquefied starch in 20 hours while 92.5 g/L of lactate was produced from the raw starch in 39 hours. High initial glucose levels (100 g/L) in the medium inhibited the organism, unless it had been adapted by growing it in a low-glucose medium. The direct production of lactic acid from starch could reduce overall production costs significantly.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

Effect of low pH start-up on continuous mixed-culture lactic acid fermentation of dairy effluent

Mixed-culture fermentation that does not require an energy-intensive sterilization process is a viable approach for the economically feasible production of lactic acid (LA) due to the potential use of organic waste as feedstock. This study investigated mixed-culture LA fermentation of whey, a high-strength organic wastewater, in continuous mode. Variations in the hydraulic retention time (HRT) from 120 to 8 h under different pH regimes in two thermophilic reactors (55 °C) were compared for their fermentation performance. One reactor was maintained at a low pH (pH 3.0) during operation at HRTs of 120 to 24 h and then adjusted to pH 5.5 in the later phases of fermentation at HRTs of 24 to 8 h (R1), while the second reactor was maintained at pH 5.5 throughout the experiment (R2). Although the LA production in R1 was negligible at low pH, it increased dramatically after the pH was raised to 5.5 and exceeded that in R2 when stabilized at HRTs of 8 and 12 h. The maximum yield (0.62 g LA/g substrate fed as the chemical oxygen demand (COD) equivalent), the production rate (11.5 g/L day), and the selectivity (95 %) of LA were all determined at a 12-h HRT in R1. Additionally, molecular and statistical analyses revealed that changes in the HRT and the pH significantly affected the bacterial community structure and thus the fermentation characteristics of the experimental reactors. Bacillus coagulans was likely the predominant LA producer in both reactors. The overall results suggest that low pH start-up has a positive effect on yield and selectivity in mixed-culture LA fermentation.     

Production of lactic acid by direct fermentation of starchy wastes by an amylase-producing Lactobacillus

Lactobacillus cellobiosus, isolated from city wastes, produced an extracellular alpha-amylase and produced lactic acid by direct fermentation of waste potato mash. Using a 5% (w/v) potato mash with 3% (w/v) CaCO to neutralise the lactic acid produced, 50% conversion of starch to lactic acid occurred in 48 h without any other media supplement.     

Semi-continuous lactic fermentation of whey by Lactobacillus bulgaricus

Semi-continuous tests of lactic fermentation of whey by Lactobacillus buigaricus were carried out using a mixture of hydrolysed milk and vitamins. The volume of the Inoculum varied from 20% to 50% of the reactor working volume. A Monod-like equation correlates the lactic acid productivity and the volume fraction of inoculum.The authors are with the Centro de Desenvolvimento Biotecnológico de Santa Catarina (Biotechnological Development Centre of Santa Catarina), R. Princesa isabel, 114, 89200, Joinville, SC, Brazil. CDEB. L.A. Kulay was an undergraduate student, in 1989, of the Escola de Engenharia Mauá (Mauá Engineering School), São Caetano do Sul, SP, Brazil. W. Borzani is the corresponding author.     

Simultaneous enzymatic wheat starch saccharification and fermentation to lactic acid by Lactococcus lactis

Simultaneous saccharification of starch from whole-wheat flour and fermentation to lactic acid (SSF) was investigated. For saccharification the commercial enzyme mixture SAN Super 240 L, having α-amylase, amyloglucosidase and protease activity, was used, and Lactococcus lactis ssp. lactis ATCC 19435 was used for the fermentation. SSF was studied at flour concentrations corresponding to starch concentrations of 90 g/l and 180 g/l and SAN Super concentrations between 3 μl/g and 8 μl/g starch. Kinetic models, developed for the saccharification and fermentation, respectively, were used for simulation and data from SSF experiments were used for model verification. The model simulated SSF when sufficient amounts of nutrients were available during fermentation. This was achieved with high wheat flour concentrations or with addition of yeast extract or amino acids. Nutrient release was dependent on the level of enzyme activity. Received: 26 January 1999 / Accepted: 20 February 1999     

Evaluation of plastic-composite supports in repeated fed-batch biofilm lactic acid fermentation by Lactobacillus casei

A customized stirred-tank biofilm reactor was designed for plastic-composite supports (PCS). In repeated-batch studies, the PCS-biofilm reactors outperformed the suspended-cell reactors by demonstrating higher lactic acid productivities (2.45 g l(-1) h(-1) vs 1.75 g l(-1) h(-1)) and greater glucose consumption rates (3.27 g l(-1) h(-1) vs 2.09 g l(-1) h(-1)). In the repeated fed-batch studies, reactors were spiked periodically with concentrated glucose (75%) to maintain a concentration of approximately 80 g of glucose l(-1) in the bioreactor. In suspended-cell fermentations with 10 g of yeast extract (YE) l(-1) and zero, one, two, and three glucose spikes, the lactic acid productivities were 2.64, 1.58, 0.80, and 0.62 g l(-1) h(-1), respectively. In comparison, biofilm reactors with 7 g of YE l(-1) and zero, one, two, and three glucose spikes achieved lactic acid productivities of 4.20, 2.78, 0.66, and 0.94 g l(-1) h(-1), respectively. The use of nystatin (30 U ml(-1)) subdued the contaminating yeast population with no effect on the lactic acid productivity of the biofilm reactors, but it did affect productivity in the suspended-cell bioreactor. Overall, in repeated fed-batch fermentations, the biofilm reactors consistently outperformed the suspended-cell bioreactors, required less YE, and produced up to 146 g of lactic acid l(-1) with 7 g of YE l(-1), whereas the suspended-cell reactor produced 132 g l(-1) with 10 g of YE l(-1).     

Production of mannitol and lactic acid by fermentation with Lactobacillus intermedius NRRL B-3693

Lactobacillus intermedius B-3693 was selected as a good producer of mannitol from fructose after screening 72 bacterial strains. The bacterium produced mannitol, lactic acid, and acetic acid from fructose in pH-controlled batch fermentation. Typical yields of mannitol, lactic acid, and acetic acid from 250 g/L fructose were 0.70, 0.16, and 0.12 g, respectively per g of fructose. The fermentation time was greatly dependent on fructose concentration but the product yields were not dependent on fructose level. Fed-batch fermentation decreased the time of maximum mannitol production from fructose (300 g/L) from 136 to 92 h. One-third of fructose could be replaced with glucose, maltose, galactose, mannose, raffinose, or starch with glucoamylase (simultaneous saccharification and fermentation), and two-thirds of fructose could be replaced with sucrose. L. intermedius B-3693 did not co-utilize lactose, cellobiose, glycerol, or xylose with fructose. It produced lactic acid and ethanol but no acetic acid from glucose. The bacterium produced 21.3 +/- 0.6 g lactic acid, 10.5 +/- 0.3 g acetic acid, and 4.7 +/- 0.0 g ethanol per L of fermentation broth from dilute acid (15% solids, 0.5% H(2)SO(4), 121 degrees C, 1 h) pretreated enzyme (cellulase, beta-glucosidase) saccharified corn fiber hydrolyzate.     

Effect of environmental pH on fermentation balance of Lactobacillus bulgaricus.

When Lactobacillus bulgaricus NLS-4 was grown anaerobically in continuous culture with limiting glucose, a shift in the pH of the medium from the acidic to the alkaline range caused this normally homofermentative bacterium to catabolize glucose in a heterofermentative fashion. The change in the nature of the fermentation was accompanied by a decrease in lactate dehydrogenase biosynthesis in alkaline conditions. The lactate dehydrogenase from this organism did not require fructose 1,6-diphosphate or manganese ions (Mn2+) for catalytic activity. Involvement of the phosphoroclastic split in the pyruvate conversion in an alkaline environment was also confirmed. The high lactate dehydrogenase synthesis in acidic medium together with the participation of the phosphoroclastic split under alkaline conditions may explain the shift from homolactic to heterolactic fermentation of L. bulgaricus NLS-4 with the change of environmental pH.     

Effect of environmental pH on the fermentation balance of Lactobacillus reuteri

The influence of environmental pH on the regulation of glucose catabolism by Lactobacillus reuteri was examined in anaerobic batch cultures. Under acidic conditions both glucose consumption and end-products formation were low. Maximum biomass was reached at pH 5·0, with a specific growth rate of µ= 0·78 h^-1. The shift in pH values from 4.3 to 6.5 reflected an increase in glucose uptake as well as in the yield ( Y _p/x) of acetate, lactate and ethanol after 12 h of incubation. Ethanol was the major metabolite produced at all pH values assayed.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Direct fermentation of starch to L-(+)-lactic acid using Lactobacillus amylophilus

Summary Fermentation of L-(+)-lactic acid from soluble starch by Lactobacillus amylophilus was studied. The bacterium produced about 30 g of L-(+)-lactic acid from 50 g of soluble starch when the pH of the culture was ranging from pH 5 to pH 6.8 at 28°C. 53.4 g of L-(+)-lactic acid was produced when 100 g of starch was added in the medium. The fermentation procedures will reduce the cost of complete hydrolysis of starch to glucose prior to fermentation.     

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.     

Behaviour of different Lactobacillus species in the production of lactic acid by the fermentation of whey

The behaviour of different Lactobacillus casei and Lactobacillus plantarum species in the fermentation of Manchego whey was experimentally studied and the results were statistically analyzed using a hypothesis contrast method. The steadiness of the velocity of the production of lactic acid during the fermentation process allowed the use of this variable to compare the different microorganisms. From this comparison it was inferred that the individuals of the same population behave alike and that the L. casei population produces lactic acid at a higher rate than the L. plantarum population. A competitive effect among the members of the L. casei population was also observed.     

Optimization of the conditions of the fermentation of beet molasses to lactic acid by Lactobacillus delbrueckii

Optimization studies were carried out for the production of L-lactic acid from the fermentation of beet molasses by Lactobacillus delbrueckii. A Central Composite Design was used to determine the optimum values of the process variables (temperature, pH, inoculum concentration, and initial sucrose concentration) for obtaining the maximum yield and the maximum volumetric productivity of lactic acid. Among the variables selected for study, it was found that all of them apart from the temperature significantly affected the responses (yield and volumetric productivity of lactic acid). The Central Composite Design also permitted formulating two second-order polynomial empirical models relating to the responses and the significant variables. From these models it was possible to determine the value of the variables giving the maximum yield of lactic acid production (87.8%) and the maximum volumetric rate of lactic acid biosynthesis (2.7 g/l · h). Finally, the dependence of the lactic acid yield and productivity on the model variables was investigated. All conclusions are restricted to the experimental range studied.     

Uptake of lactose and continuous lactic acid fermentation by entrapped non-growing Lactobacillus helveticus in whey permeate

 Continuous production of lactic acid from lactose has been carried out in a stirred-tank reactor with non-growing Lactobacillus helveticus entrapped in calcium alginate beads. A considerably longer operation half-life was obtained in a continuously operated reactor than in a batch-operated reactor. It is possible to simulate the action of entrapped non-growing cells on the basis of information from diffusion and kinetic experiments with suspended free cells. The simulation fit the experimental data over a broad range of substrate concentrations if the specific lactic acid production rate, q _P, was used as a variable parameter in the model. The dynamic mathematical model used is divided into three parts: the reactor model, which describes the mass balance in a continuously operated stirred-tank reactor with immobilized biomass, the mass-transfer model including both external diffusion and internal mass transfer, and the kinetic model for uptake of substrate on the basis of a Michaelis-Menten-type mechanism. From kinetic data obtained for free biomass experiments it was found, with the use of non-linear parameter estimation techniques, that the conversion rate of lactose by L. helveticus followed a Michaelis-Menten-type mechanism with K _S at half-saturation=0.22±0.01 g/l. The maximum specific lactose uptake rate for growing cells, q _S,max, varied between 4.32±0.02 g lactose g cells^-1 h^-1 and 4.89 ±0.02 g lactose g cells^-1 h^-1. The initial specific lactose uptake rate for non-growing cells, q _S,0, was found to be approximately 40% of the maximum specific lactose uptake rate for growing cells. Received: 4 October 1995/Received last revision: 23 April 1996/Accepted: 29 April 1996