Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli

A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive clone showed LDH activity and production of D(−)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH).     

Air pressure effects on biomass yield of two different Kluyveromyces strains

The use of air pressure as a way of improving oxygen transfer in aerobic bioreactors was investigated. To compare the air pressure effects with traditional air bubbled cultures, experiments using a pressure reactor and a stirred flask, with the same oxygen transfer rate, were made. Kluyveromyces marxianus is an important industrial yeast and some of it show a “Kluyver effect” for lactose: even under oxygen limited growth conditions, certain disaccharides that support aerobic, respiratory growth, are not fermented. This study deals with the effect of increased pressure on the physiological behavior of two Kluyveromyces strains: K. marxianus ATCC10022 is a lactose-fermenting strain, whereas K. marxianus CBS 7894 has a Kluyver-effect for lactose. For K. marxianus ATCC10022 an air pressure increase of 2 bar led to a 3-fold increase in biomass yield. When air pressure increased an enhancement of ethanol oxidation of cell yeasts was also observed. Batch cultures of K. marxianus CBS 7894 exhibited different growth behaviour. Its metabolism was always oxidative and ethanol was never produced. With the increase in air pressure, it was possible to increase the productivity in biomass of K. marxianus CBS 7894. As a response to high oxygen concentrations, due to the increase in oxygen partial pressure, oxidative stress in the cells was also studied. Antioxidant defences, such as superoxide dismutase, catalase, and glutathione reductase, were at high activity levels, suggesting that these yeast strains could tolerate the increased pressures applied.     

薏苡仁多菌发酵液的菌种优选及其抑制黑色素生成的作用研究

以薏苡仁作为发酵基质,确定利于提高发酵液体外活性的较优乳酸菌种,并分析优势乳酸菌种薏苡仁发酵液对斑马鱼胚体黑色素生成的抑制作用。通过比较分析乳酸乳球菌（Lactococcus lactis）、嗜热链球菌（Streptococcus thermophilus）和保加利亚乳杆菌（Lactobacillus bulgaricus）3种单一乳酸菌和三者复合乳酸菌的薏苡仁发酵液的还原糖、总酚、游离氨基酸、蛋白、总酸和乳酸含量等理化指标及体外羟自由基清除能力和酪氨酸酶活抑制率确定较优发酵菌种,采用高通量测序测定发酵过程中微生物菌群结构;利用斑马鱼模型研究发酵液对黑色素生成的抑制作用。研究结果表明,采用乳酸乳球菌、嗜热链球菌和保加利亚乳杆菌3种乳酸菌复合发酵比单一乳酸菌发酵更具优势。使用以上菌种复合发酵薏苡仁过程中,乳酸乳球菌和嗜热链球菌为发酵前期优势菌群,发酵中后期则以保加利亚乳杆菌为优势菌群。经复合乳酸菌发酵后,薏苡仁发酵液的羟自由基清除率和酪氨酸酶活抑制率分别提高了20.82%和87.26%;斑马鱼模型实验结果表明,薏苡仁发酵液可以显著减少斑马鱼体表黑色素分布,当使用含量为2.0%时,对黑色素生成抑制率达到59.45%。研究结果为利用薏苡仁多菌发酵液开发为具美白肌肤性能的功能性新原料提供了科学数据支撑,并希望进一步推进薏苡产业的升级。     

Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough

The aim of this study was to assess the interactions between Saccharomyces cerevisiae and lactic acid bacteria that either form a stable consortium in Greek wheat sourdoughs (i.e. Lactobacillus sanfranciscensis and L. brevis) or occasionally constitute the secondary microbiota (i.e. Weissella cibaria, L. paralimentarius, Pediococcus pentosaceus and Enterococcus faecium). For this purpose, wheat dough was prepared by using strains of the above mentioned species either as single starters, or in combination of the yeast with each of the lactic acid bacteria strains. The determination of the metabolic products in sourdough samples was performed by HPLC analysis. Presence of lactic acid bacteria had no effect on S. cerevisiae final cell yield but affected negatively the maximum specific growth rate. Ethanol production was primarily affected negatively while the co-culture had a variable effect on glycerol production. On the other hand, the presence of S. cerevisiae favoured mannitol and acetic acid production, had a species-dependent effect on maximum specific growth rate and had no effect on final cfu/g sourdough and lactic acid production by the lactic acid bacteria and at the same time caused the depletion of glucose, fructose and maltose.     

Resistance of Lactobacillus casei in plastic-composite-support biofilm reactors during liquid membrane extraction and optimization of the lactic acid extraction system

Lactic acid fermentations were performed with plastic-composite-support (PCS) disks in solvent-saturated media with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The PCS disks contained 50% (w/w) polypropylene, 35% (w/w) ground soybean hulls, 5% (w/w) yeast extract, 5% (w/w) soybean flour, and 5% (w/w) bovine albumin. Bioassays were performed by growing L. casei in solvent-saturated media after soaking the PCS disks. Eighteen different solvent and carrier combinations were evaluated. Overall, L. casei biofilm fermentation demonstrated the same lactic acid production in solvent-saturated medium as suspended cells in medium without solvents (control). To evaluate PCS solvent-detoxifying properties, two bioassays were developed. When solvent-saturated medium in consecutive equal volumes (10 mL then 10 mL) was exposed to PCS, both media demonstrated lactic acid fermentation equal to the control. However, when solvent-saturated medium with two consecutive unequal volumes (10 mL then 90 mL) was exposed to PCS, some degree of toxicity was observed. Furthermore, iso-octane, tributylphosphate (TBP), and Span 80 were optimized for recovery as 91%, 5%, and 4% (v/v), respectively, with a 1:1 ratio of 1.2 M Na(2)CO(3) stripping solution. Also, recovery by emulsion liquid extraction in the hollow-fiber contactor was minimal due to low recovery at pH 5.0 and incompatibility of the solvent and hollow-fiber material. These results suggest that PCS biofilm reactors can benefit lactic acid fermentation by eliminating the toxic effect from solvent leakage into the fermentation medium from liquid-liquid extractive integrated fermentations.     

Ecology of lactic acid bacteria in Italian fermented sausages: isolation, identification and molecular characterization

In this study, the ecology of the lactic acid bacteria (LAB) of three naturally fermented sausages produced in the Friuli-Venezia-Giulia region, in the North East of Italy, was investigated. A total of 465 strains isolated from three fermentations were identified by molecular methods and 12 different species of LAB were detected. Lactobacillus curvatus and Lactobacillus sakei were the most numerous (67 and 353 strains isolated, respectively) and they were subjected to RAPD-PCR. Clusters containing strains isolated from different plants were observed, underlining a coherent population distribution in three different fermentations. However, we also observed clusters formed by strains isolated from a specific fermentation, only. This could be explained considering the different technologies and recipes used for the production in three plants. Ingredient composition, fermentation and maturation parameters could play an important role in the selection of specific populations adapted in a specific environment.     

Optimization of lactic acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation was investigated. Shake flasks proved to be a better fermentation system for this purpose. Substitution of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid concentration was achieved without treatment of molasses. A Central Composite Design was employed to determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast extract, CaCO₃). A satisfactory fit of the model was realized. Lactic acid production was significantly affected both by sucrose–yeast extract and sucrose–CaCO₃ interactions, as well as by the negative quadratic effects of these variables. Sucrose and yeast extract had a linear effect on lactic acid production while the CaCO₃ had no significant linear effect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast extract and CaCO₃ of 89.93, 45.71 and 59.95 g/l, respectively.     

Study of the effects of temperature, pH, NaCl, and aw on the proteolytic and lipolytic activities of cheese-related lactic acid bacteria by quadratic response surface methodology

The individual and interactive effects of temperature, pH, NaCl, and a_w on the proteolytic and lipolytic activities of Lactobacillus delbrueckii subsp. bulgaricus B397, Lactococcus lactis subsp. lactis T12, and Lb. plantarum 2739 were studied by quadratic response surface methodology. The effects on enzyme activities depended on the interactions among the independent variables, type of activity, substrate and, especially, species. The proteinase activity of strains B397 and T12 was affected differently by pH as individual or interactive terms depending on the type of substrate _sl- or β-casein. The increase of NaCl concentration (2.5–7.5%) under cheese-like conditions had a negative effect on the proteinase activity of strain T12. The effect of NaCl was related to the corresponding decrease in a_w. Aminopeptidases N and A, iminopeptidase and endopeptidase of Lc. lactis subsp. lactis T12 were strongly inhibited by pH 5–6 and NaCl concentration higher than 3.75%. The negative effects of these independent variables was in several cases enhanced by their interaction and/or by the interaction with the lowest temperatures. In contrast, the same peptidases of Lb. plantarum 2739 retained a high activity under the very hostile environmental conditions. Iminopeptidase and especially endopeptidase activities of strain 2739 were stimulated slightly by NaCl at concentrations up to 5%. Lipase/esterase activity of Lb. delbrueckii subsp. bulgaricus B397 was markedly inhibited under cheese-like conditions.     

Evaluation of biofilm reactor solid support for mixed-culture lactic acid production

A combination of lactobacilli and biofilm-forming bacteria were evaluated in continuous fermentations for lactic acid production using various supports. Twelve different bacteria, including species of Bacillus, Pseudomonas, Streptomyces, Thermoactinomyces, and Thermomonospora were tested for biofilm-forming capabilities. Solid supports that were evaluated in either batch or continuous fermentations were pea gravels, 3M-macrolite ceramic spheres, and polypropylene mixed with 25% of various agricultural materials (e.g. corn starch, oat hulls) and extruded to form chips (pp-composite). Biofilm formation was evaluated by the extent of clumping of solid supports, weight gain and (in some instances) Gram stains of the supports after drying overnight at 70° C. The supports consistently producing the best biofilm were pp-composite chips followed by 3M-Macrolite spheres then by pea gravels. The best biofilm formation was observed with P. fragi (ATCC 4973), S. viridosporus T7A (ATCC 39115), and Thermoactinomyces vulgaris (NRRL B-5790), grown optimally at 25, 37, and 45° C, respectively, on various pp-composite chips. Lactic acid bacteria used in the fermentations were Lactobacillus amylophilus (NRRL B-4437), L. casei (ATCC 11443), and L. delbrueckii mutant DP3; these grow optimally at 25, 37 and 45° C, respectively. Lactic acid and biofilm bacteria with compatible temperature optima were inoculated into 50-ml reactors (void volume 25 ml) containing sterile pp-composite supports. Lactic acid production and glucose consumption were determined by HPLC at various flow rates from 0.06 to 1.92 ml/min. Generally, mixed-culture biofilm reactors produced higher levels of lactic acid than lactic acid bacteria alone. S. viridosporus T7A and L. casei on pp-composite chips were the best combination of those tested, and produced 13.0 g/l lactic acid in the reactors without pH control. L. casei produced 10.3 g/l lactic acid under similar conditions.Journal paper no. J-14840 of the Iowa Agriculture and Home Economics Experiment Station, Ames Iowa. Project nos. 2889 and 0178 Correspondence to: A. L. Pometto     

Kinetic Modelling of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> ssp. <Emphasis Type="Italic">rhamnosus</Emphasis> Growth and Lactic Acid Production in Batch Cultures Under Various Medium Conditions

Enrichment of medium with yeast extract and tryptone increased growth and lactic acid production in batch cultures of Lactobacillus casei ssp. rhamnosus. A reliable kinetic model that explicitly expresses the strong relationship between microbial growth, lactic acid production and medium enrichment is provided and validated using experimental data obtained with six different medium compositions. Revisions requested 2 February 2005 and 26 July 2005; Revisions received 25 July 2005 and 9 September 2005     

Lactic acid production from sugarcane bagasse hydrolysates by Lactobacillus pentosus: Integrating xylose and glucose fermentation

Lactic acid, traditionally obtained through fermentation process, presents numerous applications in different industrial segments, including production of biodegradable polylactic acid (PLA). Development of low cost substrate fermentations could improve economic viability of lactic acid production, through the use of agricultural residues as lignocellulosic biomass. Studies regarding the use of sugarcane bagasse hydrolysates for lactic acid production by Lactobacillus spp. are reported. First, five strains of Lactobacillus spp. were investigated for one that had the ability to consume xylose efficiently. Subsequently, biomass fractionation was performed by dilute acid and alkaline pretreatments, and the hemicellulose hydrolysate (HH) fermentability by the selected strain was carried out in bioreactor. Maximum lactic acid concentration and productivity achieved in HH batch were 42.5 g/L and 1.02 g/L h, respectively. Hydrolyses of partially delignified cellulignin (PDCL) by two different enzymatic cocktails were compared. Finally, fermentation of HH and PDCL hydrolysate together was carried out in bioreactor in a hybrid process: saccharification and co-fermentation with an initial enzymatic hydrolysis. The high fermentability of these process herein developed was demonstrated by the total consumption of xylose and glucose by Lactobacillus pentosus, reaching at 65.0 g/L of lactic acid, 0.93 g/g of yield, and 1.01 g/L h of productivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2718, 2019     

Growth and lactic acid production by Lactobacillus casei ssp. rhamnosus in batch and membrane bioreactor: influence of yeast extract and Tryptone enrichment

Enrichment of the medium with yeast extract (20 g.l ) and Tryptone (40 g.l ) increased the growth of Lactobacillus casei ssp. rhamnosusand its production of lactic acid in both batch and cell-recycle cultures without affecting glucose consumption and the lactic acid production rate.     

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

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

Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid’s pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.     

Enhanced production ofd(−)-lactic acid by mutants ofLactobacillus delbrueckii ATCC 9649

Summary Chemical mutagenesis with ethyl methanesulfonate (EMS) was used to develop strains ofLactobacillus delbrueckii (ATCC 9649) that tolerated increased lactic acid concentrations while continuously producing the acid. Three mutants (DP2, DP3 and DP4) were compared with wild-typeL. delbrueckii by standing fermentations with different glucose concentrations. All three mutants produced higher levels of lactic acid than the wild-type. In pH-controlled (pH 6.0) stirred-tank-batch fermentations, mutant DP3 in 12% glucose, 1% yeast extract/mineral salt/oleic acid medium produced lactic acid at a rate that was more than 2-times faster than the wild-type. Mutant DP3 also produced 77 g/l lactic acid compared with 58 g/l for the wild-type. Overall, compated with wild-type, the mutants DP2 and DP3 exhibited faster specific growth rates, shorter lag phases, greater lactic acid yields, tolerated higher lactic acid concentrations, and produced as much as 12% lactic acid in 12% glucose, 3% yeast extract/mineral salt/oleic acid medium which required an additional 9% glucose when the residual glucose concentration decreased to 3%. Mutant DP3 was stable for over 1.5 years (stored freeze dried). The strain development procedure was very successful; mutants with enhanced lactic acid-producing capacity were obtained each time the procedure was employed.Journal Paper No. J-14087 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Projects No. 2889 and 0178.     

Inhibition of yeast by lactic acid bacteria in continuous culture: nutrient depletion and/or acid toxicity?

Lactic acid was added to batch very high gravity (VHG) fermentations and to continuous VHG fermentations equilibrated to steady state with Saccharomyces cerevisiae. A 53% reduction in colony-forming units (CFU) ml^–1 of S. cerevisiae was observed in continuous fermentation at an undissociated lactic acid concentration of 3.44% w/v; and greater than 99.9% reduction was evident at 5.35% w/v lactic acid. The differences in yeast cell number in these fermentations were not due to pH, since batch fermentations over a pH range of 2.5–5.0 did not lead to changes in growth rate. Similar fermentations performed in batch showed that growth inhibition with added lactic acid was nearly identical. This indicates that the apparent high resistance of S. cerevisiae to lactic acid in continuous VHG fermentations is not a function of culture mode. Although the total amount of ethanol decreased from 48.7 g l^–1 to 14.5 g l^–1 when 4.74% w/v undissociated lactic acid was added, the specific ethanol productivity increased ca. 3.2-fold (from 7.42×10^–7 g to 24.0×10^–7 g ethanol CFU^–1 h^–1), which indicated that lactic acid stress improved the ethanol production of each surviving cell. In multistage continuous fermentations, lactic acid was not responsible for the 83% (CFU ml^–1) reduction in viable S. cerevisiae yeasts when Lactobacillus paracasei was introduced to the system at a controlled pH of 6.0. The competition for trace nutrients in those fermentations and not lactic acid produced by L. paracasei likely caused the yeast inhibition.     

On-line estimation of biological variables during pH controlled lactic acid fermentations

Indirect measurement of lactose, galactose, lactic acid, and biomass concentration from on-line sodium hydroxide weight measurements have been obtained for pure and mixed batch cultures of Streptococcus salivarius ssp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398 conducted at controlled pH and temperature. Linear correlations were established between the equivalent sodium hydroxide concentration and the lactose (substrate), galactose and lactic acid (products) concentrations while nonlinear relationships were developed between biomass and lactic acid concentrations. These nonlinear relationships took into account the inhibitory effect of lactic acid on growth and acidification. The indirect measurements of biomass concentration were introduced into a nonlinear estimator of the state variables and of the specific growth and lactic acid production rates. Good agreement was found between estimated and measured biomass concentrations (error index ranging from 10.8% to 12.6%). The results showed the feasibility of on-line estimation of biomass concentration and of the specific kinetics from NaOH addition weight measurements and its applicability for monitoring lactic acid fermentations. Using off-line measurements of L(+) and D(-) lactic acid concentrations, the evolution of the concentration of each strain in mixed cultures was obtained from the relationships proposed for the mixed cultures. (c) 1994 John Wiley & Sons, Inc.     

Lactic Acid Production in a Mixed-Culture Biofilm Reactor

Novel solid supports, consisting of polypropylene blended with various agricultural materials (pp composite), were evaluated as supports for pure- and mixed-culture continuous lactic acid fermentations in biofilm reactors. Streptomyces viridosporus T7A (ATCC 39115) was used to form a biofilm, and Lactobacillus casei subsp. rhamnosus (ATCC 11443) was used for lactic acid production. For mixed-culture fermentations, a 15-day continuous fermentation of S. viridosporus was performed initially to establish the biofilm. The culture medium was then inoculated with L. casei subsp. rhamnosus. For pure-culture fermentation, L. casei subsp. rhamnosus was inoculated directly into the reactors containing sterile pp composite chips. The biofilm reactors containing various pp composite chips were compared with a biofilm reactor containing pure polypropylene chips and with a reactor containing a suspension culture. Continuous fermentation was started, and each flow rate (0.06 to 1.92 ml/min) was held constant for 24 h; steady state was achieved after 10 h. Lactic acid production was determined throughout the 24-h period by high-performance liquid chromatography. Production rates that were two to five times faster than those of the suspension culture (control) were observed for the pure- and mixed-culture bioreactors. Both lactic acid production rates and lactic acid concentrations in the culture medium were consistently higher in mixed-culture than in pure-culture fermentations. Biofilm formation on the chips was detected at harvest by chip clumping and Gram staining.     

高糖和氮源对鼠李糖乳杆菌(Lactobacillus rhamnosus)L-乳酸发酵的影响

鼠李糖乳杆菌经实验室耐高糖高酸选育,能够在高糖浓度下高效高产L-乳酸。以酵母粉为氮源和生长因子,葡萄糖初始浓度分别为120 g/L和146 g/L,摇瓶培养120h,L-乳酸产量分别为104g/L和117.5g/L,L-乳酸得率分别为86.7%和80.5%。高葡萄糖浓度对菌的生长和乳酸发酵有一定的抑制。增加接种量,在高糖浓度发酵条件下,可以缩短发酵时间,但对增加乳酸产量效果不明显。乳酸浓度对鼠李糖乳杆菌生长和产酸有显著的影响。初始乳酸浓度到达70g/L以上时,鼠李糖乳杆菌基本不生长和产酸,葡萄糖消耗也被抑制。酵母粉是鼠李糖乳杆菌的优良氮源,使用其它被测试的氮源菌体生长和产酸都有一定程度的下降。用廉价的黄豆粉并补充微量维生素液,替代培养基中的酵母粉,可以使产酸浓度和碳源得率得以基本维持。     

Malolactic fermentation in chardonnay wine by immobilised Lactobacillus casei cells

The kinetics of malolactic fermentation in Chardonnay wine by immobilised Lactobacillus casei cells has been studied. Calcium pectate gel and chemically modified chitosan beads were used as supports for immobilisation. Repeated batch fermentations were carried out with different wine samples, some of which were treated with sulfur dioxide (free 19–25 mg/litre and total 80–88 mg/litre), in shake flask at 36, 25 and 20°C without any loss of activity. The degradation of malic acid obtained using immobilised cells was twice as high as that obtained with free cells. At an initial pH 3·2, decrease of malic acid of about 30% was observed at 25°C in one hour using L. casei cells immobilised either in pectate gel or on chitosan. Among the physico-chemical parameters studied, temperature was the main factor affecting metabolism of the organic acids as well as the rate of the malolactic fermentation. Operational stability of calcium pectate gel beads and chemically modified chitosan beads was 6 months after eight fermentations and 2 months after five fermentations, respectively, which proved the possibility of industrial application of the chosen supports in wine making.