Comparative studies of lactic acid dehydrogenases in lactic acid bacteria

The stability, pH-dependence and kinetic properties of the Mn²⁺ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM) 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiently only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn²⁺ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.Abbreviations l-LDH l-Lactic acid dehydrogenase - FDP Fructose-1,6-bisphosphate - DTE Dithioerythrol AddendumIn the case of the L. casei-LDH the shape of the NADH saturation curve is not changed by omitting the effectors FDP and Mn 2⁺. The K _M under these conditions is 3 fold higher (10.10 ^–5 M).     

Genomics of lactic acid bacteria

Lactic acid bacteria (LAB) are found to occupy a variety of ecological niches including fermented foods as well as mucosal surfaces of humans and other vertebrates. This review is based on the genomic content of LAB that is responsible for the functional and ecological diversity of these bacteria. These genomes reveal an ongoing process of reductive evolution as the LAB have specialized to different nutritionally rich environments. Species-to-species variation in the number of pseudogenes as well as genes directing nutrient uptake and metabolism reflects the adaptation of LAB to food matrices and the gastrointestinal tract. Although a general trend of genome reduction was observed, certain niche-specific genes appear to be recently acquired and appear on plasmids or adjacent to prophages. Recent work has improved our understanding of the genomic content responsible for various phenotypes that continue to be discovered, as well as those that have been exploited by man for thousands of years.     

Proteolytic systems of lactic acid bacteria

Lactic acid bacteria (LAB) have a very long history of use in the manufacturing processes of fermented foods and a great deal of effort was made to investigate and manipulate the role of LAB in these processes. Today, the diverse group of LAB includes species that are among the best-studied microorganisms and proteolysis is one of the particular physiological traits of LAB of which detailed knowledge was obtained. The proteolytic system involved in casein utilization provides cells with essential amino acids during growth in milk and is also of industrial importance due to its contribution to the development of the organoleptic properties of fermented milk products. For the most extensively studied LAB, Lactococcus lactis, a model for casein proteolysis, transport, peptidolysis, and regulation thereof is now established. In addition to nutrient processing, cellular proteolysis plays a critical role in polypeptide quality control and in many regulatory circuits by keeping basal levels of regulatory proteins low and removing them when they are no longer needed. As part of the industrial processes, LAB are challenged by various stress conditions that are likely to affect metabolic activities, including proteolysis. While environmental stress responses of LAB have received increasing interest in recent years, our current knowledge on stress-related proteolysis in LAB is almost exclusively based on studies on L. lactis. This review provides the current status in the research of proteolytic systems of LAB with industrial relevance.     

Bioconversion of ovine scotta into lactic acid with pure and mixed cultures of lactic acid bacteria

Scotta is the main by-product in the making of ricotta cheese. It is widely produced in southern Europe and particularly in Italy where it represents a serious environmental pollutant due to its high lactose content. With the aim of evaluating whether scotta bioconversion into lactic acid can be considered as an alternative to its disposal, besides providing it with an added value, here the growth, fermentative performances, and lactic acid productions of pure and mixed cultures of Lactobacillus casei, Lactobacillus helveticus, and Streptococcus thermophilus were evaluated on ovine scotta-based media, without and with the addition of nutritional supplements. The outcomes indicate that ovine scotta can be utilized for the biotechnological production of lactic acid with yields up to 92%, comparable to those obtained on cheese-whey. Indeed, the addition of nutritional supplements generally improves the fermentative performances of lactic acid bacteria leading to about 2 g l⁻¹ h⁻¹ of lactic acid. Moreover, the use of mixed cultures for scotta bioconversion reduces the need for nutritional supplements, with no detrimental effects on the productive parameters compared to pure cultures. Finally, by using L. casei and S. thermophilus in pure and mixed cultures, up to 99% optically pure l-lactic acid can be obtained.     

Isolation and characterization of a novel lactic acid bacterium for the production of lactic acid

We isolated a novel lactic acid bacterium from a Korean traditional fermented food, soybean paste. The newly isolated strain, dubbed RKY2, grew well on glucose, sucrose, galactose, and fructose, but it could not utilize xylose, starch, or glycerol. When the partially amplified 16S rDNA sequence (772 bp) of the strain RKY2 was compared with 10 reference strains, it was found to be most similar toLactobacillus pentosus JCM 1588^T, with 99.74% similarity. Therefore, the strain RKY2 was renamedLactobacillus sp. RKY2, which has been deposited in the Korean Collection for Type Cultures as KCTC 10353BP.Lactobacillus sp. RKY2 was found to be a homofermentative lactic acid bacterium, because its end-product from glucose metabolism was found to be mainly lactic acid. It could produce more than 90 g/L of lactic acid from MRS medium supplemented with 100 g/L of glucose, with 5.2 g L⁻¹ h⁻¹ of productivity and 0.95 g/g of lactic acid yield.     

Utilization of renewables for lactic acid fermentation

Originally, lactic acid was produced from pure substrates like glucose. Increasingly, however, agricultural feedstocks such as grains and green biomass are also being used as raw materials for the biotechnological production of lactic acid. A high-productivity lactic acid bacterium strain was selected, process parameters were optimized for the batch fermentation on a laboratory scale, and its performance at cultivation on a barley hydrolysate medium together with different supplements was examined. The present results for the cultivation of the Lactobacillus paracasei on complex nutrient broth are in the same range as those for another strain of the same species with pure glucose, de Man, Rogosa and Sharpe medium (MRS) minerals, peptone and yeast extract. Under these conditions, this strain was able to accumulate more than 100 g lactate/L in the MRS medium. Medium optimization experiments showed that the main part of the nitrogen-containing nutrients in the medium (peptone, yeast extract) can be replaced by protein extracts from green biomass (lucerne green juice). The green juice after pressing fresh biomass contains a series of nitrogen-containing compounds and inorganic salts, which are essential for cell growth. Thus, on laboratory scale, we have demonstrated that it is possible to substitute synthetic nutrients by renewable resources like cereals and green biomass without any loss of productivity. This high biomass concentration together with the number of living cells could increase the productivity to higher levels compared to the well-adapted synthetic nutrients of MRS.     

Preparation of concentrated lactic streptococcus starters

Single-strain cultures of Streptococcus cremoris were grown in a semisynthetic medium with automatic pH control. After centrifugation, the cells were resuspended in sterile nonfat milk (2% of the original volume). There was no significant difference in the maximum population attained when cultures were grown at pH values of 5.5, 6.0, or 6.5 with sodium hydroxide as the neutralizer. With ammonium hydroxide as the neutralizer, maximum populations obtained were increased about twofold. In most cases, the acid-producing ability of the culture concentrates was comparable to that of fresh-milk cultures. There was some variation among strains of S. cremoris with respect to the effects of different neutralizers and levels of pH control on the biological activity of the culture concentrates. The culture concentrates were stored in liquid nitrogen for as long as 231 days without significant loss in biological activity.     

Bacteriocins of lactic acid bacteria

Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow, inhibiting only those strains that are closely related to the producer organism, or wide, inhibiting a diverse group of Gram-positive microorganisms. The following review will discuss biochemical and genetic aspects of bacteriocins that have been identified and characterized from lactic acid bacteria.     

Aldolases of the lactic acid bacteria

Reciprocal qualitative and quantitative immunological experiments employing an anti-Pediococcus cerevisiae aldolase serum confirmed many of the interspecific relationships demonstrated previously among lactic acid bacteria with antisera prepared against the Streptococcus faecalis fructose diphosphate aldolase. The extent of immunological relatedness observed between the Lactobacillus and Pediococcus aldolases was markedly greater than that noted between Pediococcus and Streptococcus aldolases indicating that the pediococci share closer phylogenetic ties with the rod-shaped lactobacilli than with their spherical counterparts in the streptococci. In addition to confirming the existence of definitive, but distant, relationships between the lactic acid bacteria and certain gram positive nonsporeforming anaerobes, immunological cross-reactivity was also demonstrated between the pediococcal aldolases and those of Aerococcus viridans.This paper is dedicated with deepest appreciation to Prof. Roger Y. Stanier on the occasion of his 60th birthday in token of what his friendship and guidance have meant to me. — J. L.     

Evaluation of lactic acid bacteria autolysate for the supplementation of lactic acid bacteria fermentation

At the end of culture in a carbon-limited medium, i.e. the best conditions for subsequent autolysis, lactic acid bacteria were harvested and autolysed at 50 °C for 24 h. The resulting supernatant was then successfully tested as a substitute for industrial yeast extract for the supplementation of whey permeate and its conversion into lactic acid: for almost equivalent total nitrogen amounts of both supplements, the same growth and production rates were recorded.     

通气对乳酸发酵过程的影响

对拟干酪乳杆菌发酵产乳酸的过程进行研究,通过改变不同的通气量(不通气、0.1vvm、0.2 vvm、0.5 vvm)确定0.1vvm的通气量最有利于产生乳酸;再通过优化通气策略,在发酵0～15 h不通空气,15～50 h通0.1 vvm空气使得乳酸的产量比全程通0.1 vvm空气又提高了11.7%,同时乳酸产率也提高了16.2%。最后通过对胞内NAD~+、NADH、乳酸脱氢酶和NADH氧化酶活性、以及发酵过程氧化还原电位(Oxidation-reduction potential,ORP)变化进行分析,阐述了通气影响乳酸发酵过程的机理。     

Heteropolysaccharides from lactic acid bacteria

Microbial exopolysaccharides are biothickeners that can be added to a wide variety of food products, where they serve as viscosifying, stabilizing, emulsifying or gelling agents. Numerous exopolysaccharides with different composition, size and structure are synthesized by lactic acid bacteria. The heteropolysaccharides from both mesophilic and thermophilic lactic acid bacteria have received renewed interest recently. Structural analysis combined with rheological studies revealed that there is considerable variation among the different exopolysaccharides; some of them exhibit remarkable thickening and shear-thinning properties and display high intrinsic viscosities. Hence, several slime-producing lactic acid bacterium strains and their biopolymers have interesting functional and technological properties, which may be exploited towards different products, in particular, natural fermented milks. However, information on the biosynthesis, molecular organization and fermentation conditions is rather scarce, and the kinetics of exopolysaccharide formation are poorly described. Moreover, the production of exopolysaccharides is low and often unstable, and their downstream processing is difficult. This review particularly deals with microbiological, biochemical and technological aspects of heteropolysaccharides from, and their production by, lactic acid bacteria. The chemical composition and structure, the biosynthesis, genetics and molecular organization, the nutritional and physiological aspects, the process technology, and both food additive and in situ applications (in particular in yogurt) of heterotype exopolysaccharides from lactic acid bacteria are described. Where appropriate, suggestions are made for strain improvement, enhanced productivities and advanced modification and production processes (involving enzyme and/or fermentation technology) that may contribute to the economic soundness of applications with this promising group of biomolecules.     

Technological and economic potential of poly(lactic acid) and lactic acid derivatives

Abstract: Lactic acid has been an intermediate-volume specialty chemical (world production ∼ 40,000 tons/yr) used in a wide range of food processing and industrial applications. Lactic acid has the potential of becoming a very large volume, commodity-chemical intermediate produced from renewable carbohydrates for use as feedstocks for biodegradable polymers, oxygenated chemicals, plant growth regulators, environmentally friendly 'green' solvents, and specialty chemical intermediates. The recent announcements of new development-scale plants for producing lactic acid and polymer intermediates by major U.S. companies, such as Cargill, Ecochem (DuPont/ConAgra), and Archer Daniels Midland, attest to this potential.
In the past, efficient and economical technologies for the recovery and purification of lactic acid from crude fermentation broths and the conversion of lactic acid to the chemical or polymer intermediates had been the key technology impediments and main process cost centers. The development and deployment of novel separations technologies, such as electrodialysis (ED) with bipolar membranes, extractive distillations integrated with fermentation, and chemical conversion, can enable low-cost production with continuous processes in large-scale operations. The use of bipolar ED can virtually eliminate the salt or gypsum waste produced in the current lactic acid processes. Thus, the emerging technologies can use environmentally sound processes to produce environmentally useful products from lactic acid. The process economics of some of these processes and products can also be quite attractive. In this paper, the recent technical advances in lactic and polyactic acid processes are discussed. The economic potential and manufacturing cost estimates of several products and process options are presented. The technical accomplishments at Argonne National Laboratory (ANL) and the future directions of this program at ANL are discussed.     

Hydrolysis of chitosan in lactic acid

Effects of molecular weight and degree of acetylation on the hydrolysis of chitosan in dilute lactic acid were studied. It was demonstrated that the higher were the values of both parameters, the more rapid were the decreases in viscosity and viscosity-average molecular weight of chitosan.     

Genomic organization of lactic acid bacteria

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.     

Cancer chemopreventive effects of lactic acid bacteria

Lactic acid bacteria (LAB) provide several potential health and nutritional benefits, including improving the nutritional value of food, controlling serum cholesterol levels, and controlling some types of cancer. Numerous in vitro, in vivo, human, and epidemiological studies have provided evidence of the chemopreventive effects of LAB on colon, bladder, liver, breast, and gastric cancers. These effects act via diverse mechanisms, including alteration of the gastrointestinal microflora, enhancement of the host's immune response, and antioxidative and antiproliferative activities. This review discusses the recent progresses on the chemopreventive effects of LAB on specific cancer types and the underlying molecular mechanisms.     

Phenformin and lactic acidosis.

Thirty-eight patients who presented with diabetes and a changed state of consciousness satisfied the criteria for lactic acidosis. Sixteen patients were non-ketotic, and 15 of these were receiving phenformin on admission. In all but one of these 15 patients, however, additional renal or cardiovascular abnormalities, or both, could be identified, which supported a multifactorial aetiology for lactic acidosis. Advanced age and cardiovascular and renal disease are absolute contraindications to the use of phenformin in diabetics.     

Chromosome mapping in lactic acid bacteria

The chromosome structure of lactic acid bacteria has been investigated only recently. The development of pulsed-field gel electrophoresis (PFGE) combined with other DNA-based techniques enables whole-genome analysis of any bacterium, and has allowed rapid progress to be made in the knowledge of the lactic acid bacteria genome. Lactic acid bacteria possess one of the smallest eubacterial chromosomes. Depending on the species, the genome sizes range from 1.1 to 2.6 Mb. Combined physical and genetic maps of several species are already available or close to being achieved. Knowledge of the genomic structure of these organisms will serve as a basis for future genetic studies. Macrorestriction fingerprinting by PFGE is already one of the major tools for strain differentiation, identification of individual strains, and the detection of strain lineages. The genome data resulting from these studies will be of general application strain improvement.