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Engineering metabolic highways in Lactococci and other lactic acid bacteria.

Lactic acid bacteria (LAB) are widely used in industrial food fermentations and are receiving increased attention for use as cell factories for the production of food and pharmaceutical products. Glycolytic conversion of sugars into lactic acid is the main metabolic highway in these Gram-positive bacteria and Lactococcus lactis has become the model organism because of its small genome, genetic accessibility and simple metabolism. Here we discuss the metabolic engineering of L. lactis and the value of metabolic models compared with other LAB, with a particular focus on the food-grade production of metabolites involved in flavour, texture and health.

Energy Metabolism↗

Studies on gynaecological hydrophilic lactic acid preparations. Part 5: The use of Eudragit E-100 as lactic acid carrier in intravaginal tablets.

Intravaginal tablets based on hydrophilic methylcellulose and containing lactic acid complexed with Eudragit E-100 undergo deformation under standard conditions. The high flow--limit of gel originating from the tablets as well as its dynamic viscosity should enable durability of this dosage form on the vaginal mucosa. By selecting either 1 : 1 or 2 : 1 ratios of lactic acid to Eudragit E-100 it is possible to obtain tablets that disintegrate into a gelform at pH 3.8-4.4, i.e. the pH remains within the physiological range. Increasing the amount of lactic acid in the complex in relation to the polymer to a 4:1 ratio results in gels with a lower pH while possessing an acid reserve that can neutralize the excess of alkali present in severe vaginal infections.

Administration, Intravaginal↗

Inhibition of Clostridium tyrobutyricum by bacteriocin-like substances produced by lactic acid bacteria.

Lactic acid bacteria were selected for their inhibitory activity against Clostridium tyrobutyricum under conditions that eliminate the effects of lactic acid and hydrogen peroxide. Four strains were isolated belonging to the species Lactococcus lactis ssp. lactis. The sensitivity of the inhibitory substances to pronase and trypsine indicates that they are proteins or peptides different from nisin. Their resistance to phospholipase D indicates that they are also different from lactostrepcin. The inhibitory substances are produced during the exponential phase of growth. Their activity is bactericidal and directed toward some strains of Clostridium tyrobutyricum, Lactobacillus helveticus, and Streptococcus thermophilus, but strains used as dairy starters, Lactobacillus lactis, Streptococcus thermophilus, and Propionibacterium shermanii, are not all affected by the inhibition.

Animals↗

Lactate racemase. Hydroxylamine-dependent 18O exchange of the alpha-hydroxyl of lactic acid.

The lactic acid racemase (EC 5.1.2.1) derived from Clostridium butylicum catalyzes the racemization of the alpha-18O label. The proposed alpha-carbonyl intermediate for the enzyme-catalyzed reaction has been previously shown to be trapped as an enzyme-bound oxime in the presence of hydroxylamine. This report demonstrates that the formation of the inactive enzyme-bound oxime, followed by reactivation in the presence of an excess of competing free carbonyl (pyruvic acid) results in a complete loss of the alpha-18O label from an original alpha-18O-labeled lactic acid.

Clostridium↗

Determination of esterolytic and lipolytic activities of lactic acid bacteria.

Lactic acid bacteria (LAB) are considered weakly lipolytic compared with many other groups of bacteria (e.g., Pseudomonas, Bacillus, and Achromobacter). The esterolytic and lipolytic systems of dairy LAB remain poorly characterized. Esterases from lactic acid bacteria, yeasts, and Pseudomonas organisms may be involved in the development of fruity flavors in foods, and pregastric lipase and esterases are essential for the development of typical flavor in Italian cheese. Microbial lipases and esterases may improve quality or accelerate the maturation of cheeses, cured bacon, and fermented sausages. Lipases are defined as glycerol ester hydrolases (EC 3.1.1.3) that hydrolyze tri-, di-, and monoglycerides present at an oil-water interface. Esterases (EC 3.1.1.6) hydrolyze esters in solution and may also hydrolyze tri- and especially di- and monoglycerides containing short-chain fatty acids. Some probiotic strains of LAB can hydrolyze the triglycerides, releasing most short and medium chain, and essential fatty acids, which are valuable to today's health-conscious consumer. Medium chain fatty acids (C6-C14), in particular, have become accepted treatment for patients with malabsorption symptoms, a variety of metabolic disorders, cholesterol problems, and infant malnutrition. These probiotic bacteria could alleviate lipase deficiency in the digestive tract during digestion (steatorrhea). In this chapter, we describe different methods routinely used in our laboratory to determine the esterolytic and lipolytic activity of LAB. These techniques include the use of alpha- and beta-naphthyl derivatives of fatty acids (chromogenic method), the p-nitrophenyl (pNP) derivative of fatty acids (chromogenic method), and triglycerides (agar-well assay technique and titrimetric test) as substrates.

Cell Fractionation↗

Binding of mutagenic heterocyclic amines by intestinal and lactic acid bacteria.

Lactic acid bacteria have been reported to have antimutagenic/anticarcinogenic properties in vitro and in vivo. One possible mechanism for this effect involves a physical binding of the mutagenic compounds to the bacteria. The purpose of the present investigation was to study the binding capacity of eight human intestinal or lactic acid bacterial strains for mutagenic heterocyclic amines formed during cooking of protein-rich food. Binding of the mutagens Trp-P-2, PhIP, IQ and MeIQx by the bacterial strains was analyzed by HPLC. There were only minor differences in the binding capacities of the tested strains but the mutagenic compounds were bound with markedly different efficiencies. Trp-P-2 was almost completely bound and the binding tended not to be of a reversible nature. The binding of PhIP, which reached about 50%, was important as PhIP is a major mutagen in the western diet. IQ and MeIQx were slightly less well bound. pH appeared to be of importance for the binding efficacy. Binding correlated well with the reduction in mutagenicity observed after exposure of the heterocyclic amines to the bacterial strains. The results indicate that cooked food mutagenic compounds, commonly found in the western meat-rich diet, can be bound to bacteria from the normal intestinal microflora in vitro.

Amines↗

Molecular taxonomy and phylogenetic position of lactic acid bacteria.

Lactic acid bacteria, important in food technology, are Gram-positive organisms exhibiting a DNA G + C content of less than 50 mol%. Phylogenetically they are members of the Clostridium-Bacillus subdivision of Gram-positive eubacteria. Lactobacillus and streptococci together with related facultatively anaerobic taxa evolved as individual lines of descent about 1.5-2 billion years ago when the earth passed from an anaerobic to an aerobic environment. In contrast to the traditional, morphology-based classification, the genus Lactobacillus is intermixed with strains of Pediococcus and Leuconostoc. Similarly, the physiology-based clustering of lactobacilli into Thermo-, Strepto- and Betabacterium does not agree with their phylogenetic relationships. On the other hand, the phenotypically defined genus Streptococcus is not a phylogenetic coherent genus but its members fall into at least 3 moderately related genera, i.e. Streptococcus, Lactococcus and Enterococcus. The genus Bifidobacterium, frequently grouped with the lactobacilli, is the most ancient group of the second, the Actinomycetes subdivision of the Gram-positive eubacteria. In addition, propionibacteria, microbacteria and brevibacteria belong to this subdivision but the latter organisms appear as offshoots of non-lactic acid bacteria.

Bacteria↗

The interaction of glass-ionomer cements containing vinylphosphonic acid with water and aqueous lactic acid.

A glass-ionomer cement containing an acrylic acid/vinyl phosphonic acid copolymer, has been investigated for its interaction with water and with aqueous lactic acid and the results compared with those from conventional glass-ionomers based on polyacrylic acid. Cylindrical specimens (12 mm high x 6 mm diameter) were placed in 8 cm3 of aqueous lactic acid (20 mmol dm(-3); pH 2.7) for 1 week, at the end of which the pH was determined. Each specimen was then placed in a fresh 8 cm3 volume of lactic acid and the pH determined after a further week. This procedure was continued for a total of 13 weeks for each specimen. Experiments were also carried out on similar specimens exposed to 8 cm3 of water for 1 week only. Further experiments were carried out in which discs of cement were exposed to thin films (0.15 mm) of lactic acid at pH 4.5, with pH values determined at 30 s, 1, 2, 5 and 10 min. After 1 week, cements had changed the pH of the lactic acid to a mean value of 3.63 (SD 0.08) while in weeks 2-13 they changed it to a mean value of 3.31 (SD 0.11). After 1 week in aqueous lactic acid the cements had gained mass by a mean of 1.26% (SD 0.59%) compared with 1.22% (SD 0.14%) in water. After 13 weeks in lactic acid, cements had lost a mean of 2.83% (SD 0.74%) in mass. Thin films of aqueous lactic acid changed from pH 4.5-5.1 at 30 s and to a steady value of 5.6 (SD 0.3) between 2 and 10 min. These results were similar to those for conventional glass-ionomer cements based on carboxylic acid polymers. Hence, it was concluded that the presence of the vinylphosphonic acid units made no significant difference to the interaction of cements with aqueous solutions.

Glass Ionomer Cements↗

Stress responses in lactic acid bacteria.

Lactic acid bacteria (LAB) constitute a heterogeneous group of bacteria that are traditionally used to produce fermented foods. The industrialization of food bio-transformations increased the economical importance of LAB, as they play a crucial role in the development of the organoleptique and hygienic quality of fermented products. Therefore, the reliability of starter strains in terms of quality and functional properties (important for the development of aroma and texture), but also in terms of growth performance and robustness has become essential. These strains should resist to adverse conditions encountered in industrial processes, for example during starter handling and storage (freeze-drying, freezing or spray-drying). The development of new applications such as life vaccines and probiotic foods reinforces the need for robust LAB since they may have to survive in the digestive tract, resist the intestinal flora, maybe colonize the digestive or uro-genital mucosa and express specific functions under conditions that are unfavorable to growth (for example, during stationary phase or storage). Also in nature, the ability to quickly respond to stress is essential for survival and it is now well established that LAB, like other bacteria, evolved defense mechanisms against stress that allow them to withstand harsh conditions and sudden environmental changes. While genes implicated in stress responses are numerous, in LAB the levels of characterization of their actual role and regulation differ widely between species. The functional conservation of several stress proteins (for example, HS proteins, Csp, etc) and of some of their regulators (for example, HrcA, CtsR) renders even more striking the differences that exist between LAB and the classical model micro-organisms. Among the differences observed between LAB species and B. subtilis, one of the most striking is the absence of a sigma B orthologue in L. lactis ssp. lactis as well as in at least two streptococci and probably E. faecalis. The overview of LAB stress responses also reveals common aspects of stress responses. As in other bacteria, adaptive responses appear to be a usual mode of stress protection in LAB. However, the cross-protection to other stress often induced by the expression of a given adaptive response, appears to vary between species. This observation suggests that the molecular bases of adaptive responses are, at least in part, species (or even subspecies) specific. A better understanding of the mechanisms of stress resistance should allow to understand the bases of the adaptive responses and cross protection, and to rationalize their exploitation to prepare LAB to industrial processes. Moreover, the identification of crucial stress related genes will reveal targets i) for specific manipulation (to promote or limit growth), ii) to develop tools to screen for tolerant or sensitive strains and iii) to evaluate the fitness and level of adaptation of a culture. In this context, future genome and transcriptome analyses will undoubtedly complement the proteome and genetic information available today, and shed a new light on the perception of, and the response to, stress by lactic acid bacteria.

Acclimatization↗

Stability of insulin during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres.

In recent years, the acylation of peptides during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres has been described in the literature. To investigate whether insulin is prone to the covalent attachment of lactic or glycolic acid, insulin-loaded PLA and PLGA microspheres containing 5% bovine insulin were manufactured using a w/o/w multiple emulsion-solvent evaporation technique. Microspheres were characterized for their insulin encapsulation efficiency and release characteristics in phosphate-buffered saline (PBS) at pH 7.4 and 37 degrees C. Moreover, the stability of the peptide during 18 days of release was evaluated using HPLC and HPLC-MS techniques. The results showed that the insulin loading efficiencies of PLA and PLGA microspheres were 75.18% and 79.63%, respectively. The microspheres were spherical with relatively porous surfaces with an average diameter of 40 and 53 mum, respectively. Insulin release from the microspheres was characterized by an initial burst, which was attributed to the amount of protein located on or close to the microsphere surface. The total ion chromatogram (TIC) of insulin samples extracted after 18 days of erosion in phosphate buffer pH 7.4 at 37 degrees C revealed that deamidation was the major mechanism of instability. Surprisingly, no acylation products were found. Control experiments in concentrated lactic acid solutions confirmed a minimal reactivity of the peptide under these conditions.

Drug Stability↗

Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1.

In lactic acid bacteria, pentoses are metabolized via the phosphoketolase pathway, which catalyzes the cleavage of D-xylulose-5-phosphate to equimolar amounts of glyceraldehyde 3-phosphate and acetylphosphate. Hence the yield coefficient of lactate from pentose does not exceed 1.0 mol/mol, while that of Lactococcus lactis IO-1(JCM7638) at high D-xylose concentrations often exceeds the theoretical value. This suggests that, in addition to the phosphoketolase pathway, L. lactisIO-1 may possess another metabolic pathway that produces only lactic acid from xylose. In the present study, the metabolism of xylose in L. lactisIO-1 was deduced from the product formation and enzyme activities of L. lactisIO-1 in batch culture and continuous culture. During cultivation with xylose concentrations above ca. 50 g/l, the yield coefficient of L-lactate exceeded 1.0 mol/mol while those of acetate, formate and ethanol were very low. At xylose concentrations less than 5 g/l, acetate, formate and ethanol were produced with yield coefficients of about 1.0 mol/mol, while L-lactate was scarcely produced. In cells grown at high xylose concentrations, a marked decrease in the specific activities of phosphoketolase and pyruvate formate lyase (PFL), and an increase in those of transketolase and transaldolase were observed. These results indicate that in L. lactisIO-1 xylose may be catabolized by two different pathways, the phosphoketolase pathway yielding acetate, formate and ethanol, and the pentose phosphate (PP)/glycolytic pathway which converts xylose to L-lactate only. Furthermore, it was deduced that the change in the xylose concentration in the culture medium shifts xylulose 5-phosphate metabolism between the phosphoketolase pathway and the PP/glycolytic pathway in L. lactisIO-1, and pyruvate metabolism between cleavage to acetyl-CoA and formic acid by PFL and the reduction to L-lactate by lactate dehydrogenase.

Enzymes↗

Stereocomplex formation between enantiomeric poly(lactic acid)s. 12. spherulite growth of low-molecular-weight poly(lactic acid)s from the melt.

The spherulite growth of stereocomplex crystallites in the blend from low-molecular-weight poly(L-lactide) [i.e., poly(L-lactic acid) (PLLA)] and poly(D-lactide) [i.e., poly(D-lactic acid) (PDLA)] from the melt, together with that of the homocrystallites in pure PLLA and PDLA films, was investigated using polarization optical miscroscopy. The spherulite growth of stereocomplex crystallites occurred at a wider temperature range (</=190 degrees C) compared with that of homocrystallites (</=140 degrees C). At 140 degrees C, the spherulite radius growth rate (G) for the stereocomplex crystallites (136.4 microm min(-1)) was an order of magnitude higher than those for the homocrystallites of PLLA (11.8 microm min(-1)) and PDLA (15.7 microm min(-1)), whereas the induction period was shorter for the spherulties of stereocomplex crystallites (0.0 min) than for the spherulties of homocrystallites of PLLA (2.6 min) and PDLA (0.7 min). In addition to these two factors, the higher spherulite density of stereocomplex crystallites compared with those of the homocrystallites of PLLA and PDLA resulted in rapid completion of overall crystallization of stereocomplex. The front factor (G(0)) and nucleation constant (K(g)) for the stereocomplex crystallites in the temperature range of 140-190 degrees C were estimated to be 3.56 x 10(12) microm min(-1) and 8.42 x 10(5) K(2), respectively. The G(0) value for stereocomplex crystallites was 1 and 2 orders of magnitude higher than those for the homocrystallites of PLLA (9.69 x 10(11) microm min(-1)) and PDLA (8.79 x 10(10) microm min(-1)), whereas the K(g) value for stereocomplex crystallites was twice those for the homocrystallites of PLLA (4.95 x 10(5) K(2)) and PDLA (4.20 x 10(5) K(2)).

Biocompatible Materials↗

Fermentative production of DL-lactic acid from amylase-treated rice and wheat brans hydrolyzate by a novel lactic acid bacterium, Lactobacillus sp.

Rice and wheat brans, without additional nutrients and hydrolyzed by alpha-amylase and amyloglucosidase, were fermented to DL-lactic acid using a newly isolated strain of Lactobacillus sp. RKY2. In batch fermentations at 36 degrees C and pH 6, the amount of lactic acid in fermentation broth reached 129 g l(-1) by supplementation of rice bran with whole rice flour. The maximum productivity was 3.1 g lactic acid l(-1) h(-1) in rice bran medium supplemented with whole rice flour or whole wheat flour.

Glucan 1,4-alpha-Glucosidase↗

The survival and growth of acid-adapted mesophilic pathogens that contaminate meat after lactic acid decontamination.

Lactic acid decontamination (LAD) may adapt pathogens to lactic acid. Such organisms may have an increased resistance to acid and can contaminate meat after LAD. The survival and growth of acid adapted Campylobacter jejuni, Salmonella typhimurium. Escherichia coli O157:H7 and Staphylococcus aureus inoculated on skin surface of still warm pork belly cuts 2 h after LAD was examined during chilled (4 degrees C) storage and refrigeration abuse equivalent to 12.5 degrees C. Lactic acid decontamination included dipping in 1, 2 or 5% lactic acid solutions at 55 degrees C for 120 s. Lactic acid decontamination brought sharp reductions in meat surface pH, but these recovered with time after LAD at approximately 1-1.5 pH units below that of water-treated controls. A sharp decrease in the number of cfu of pathogens occurred on chilled 2-5% lactic acid treated pork belly cuts when the skin surface was less than pH 4.8-5.2. The reductions ranged from 0.1-0.3 log10 cfu cm-2 for E. coli O157:H7 to over 1.7-2.4 log10 cfu cm-2 for Camp. jejuni, respectively. Increase in storage temperature from 4 to 12.5 degrees C reduced delayed decrease in numbers of all pathogens except Camp. jejuni by a factor of two. Deaths in Camp. jejuni at 12.5 degrees C slightly exceeded those at 4 degrees C. After the initial sharp decline, the number of cfu of mesophilic pathogens decreased gradually at a rate similar to that on water-treated controls. Growth of all mesophilic pathogens except Camp. jejuni on 2-5% LAD meat occurred during storage at 12.5 degrees C when the meat surface pH exceeded 4.8-5.2, and was slower than on water-treated controls. Low temperature and acid-adapted E. coli O157:H7, Salm. typhimurium and Staph. aureus, and acid adapted Camp. jejuni that contaminate skin surface after hot 2-5% LAD, did not cause an increased health hazard, although microbiota and intrinsic parameters (lactic acid content, pH) were created that could advantage their survival and growth.

Animals↗

Acid-base balance during lactic acid infusion in the lizard Varanus salvator.

Although reptiles rely heavily on anaerobic metabolism and lactic acid production during activity, little is known concerning their ventilatory response to the attendant metabolic acidosis. We measured arterial PCO2, H+ and lactate (L)ion concentrations, and the rates of CO2 (MCO2) and O2 (MO2) exchange in Varanus salvator (n = 9) during intravenous infusions of lactic acid (HL) or sodium lactate (NaL; 250 mM) at rest. Two protocols were used: (1) 15 min infusions of 0.42 ml/min at both 25 and 35 degrees C with measurements every 5 min; (2) 4.5 min infusions of 1.73 ml/min at 35 degrees C with measurements at 4.5 min. At 35 degrees C, control pH decreased from its value at 25 degrees C with a slope of -0.007/degrees C and PaCO2 increased. The results of HL infusion were: (1) [L]a increased, (2) MCO2 increased, (3) R (MCO2/MO2) increased, (4) pHa decreased, and (5) PaCO2 remained unchanged from control at both temperatures and at both infusion rates. The only significant changes in PaCO2 observed were following the termination of fast HL infusions, when PaCO2 decreased. In NaL infusions, only small changes were observed except in [L]a. The results indicate that: (1) delta pH/delta T in V. salvator is less than in other poikilothermic vertebrates, but consistent with other varanid lizards, (2) respiratory compensation is slight in response to acute metabolic acidosis in this species, and (3) ventilation follows changes in MCO2 rather closely, accounting for precise regulation of PaCO2 despite 4-fold increases in MCO2 elicited by bicarbonate buffering and increased metabolic rate.

Acid-Base Equilibrium↗

Comparative genomics of the lactic acid bacteria.

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

Animals↗

Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology.

The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol-1. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol-1 at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h.

Analysis of Variance↗