Inactivation mechanisms of lactic acid starter cultures preserved by drying processes

The preservation of lactic acid starter cultures by drying are of increased interest. A further improvement of cell viability is, however, still needed, and the insight into inactivation mechanisms of the cells is a prerequisite. In this present work, we review the inactivation mechanisms of lactic acid starter cultures during drying which are not yet completely understood. Inactivation is not only induced by dehydration inactivation but also by thermal- and cryo-injuries depending on the drying processes employed. The cell membrane has been reported as a major site of damage during drying or rehydration where transitions of membrane phases occur. Some drying processes, such as freeze drying or spray drying, involve subzero or very high temperatures. These physical conditions pose additional stresses to cells during the drying processes. Injuries of cells subjected to freezing temperatures may be due to the high electrolyte concentration (solution effect) or intracellular ice formation, depending on the cooling rate. High temperatures affect most essential cellular components. It is difficult to identify a critical component, although ribosomal functionality is speculated as the primary reason. The activation during storage is mainly due to membrane lipid oxidation, while the storage conditions such as temperature moisture content of the dried starter cultures are important factors.     

Changes in synaptic transmission produced by hydrogen peroxide

The effect of hydrogen peroxide (H₂O₂) on excitatory and inhibitory synaptic transmission was studied at the lobster neuromuscular junction. H₂O₂ produced a dose dependent decrease in the amplitude of the junction potential (V_ejp). This decrease was due to changes in both presynaptic transmitter release and the postsynaptic response to the neurotransmitter. Observed presynaptic changes due to exposure to H₂O₂ were a decrease in the amount of transmitter released, that is, quantal content, as well as a decrease in the fast facilitation, that is, the amplitude increase of successive excitatory junction potentials at a rate of 3 Hz. To discern postsynaptic changes, glutamate, the putative excitatory neurotransmitter for this preparation was applied directly to the bathing medium in order to bypass the presynaptic release process. H₂O₂ produced a decreased response of the glutamate receptor/ ionophore. The action of H₂O₂ was not selective to excitatory (glutamate-mediated) transmission because inhibitory (GABA-mediated) transmission was also depressed by H₂O₂. This effect was primarily presynaptic since H₂O₂ produced no change in the postsynaptic response to applied GABA.     

The oxidation produced by hydrogen peroxide on Ca-ATP-G-actin

We report here that in vitro exposure of monomeric actin to hydrogen peroxide leads to a conversion of 6 of the 16 methionine residues to methionine sulfoxide residues. Although the initial effect of H2O2 on actin is the oxidation of Cys374, we have found that Met44, Met47, Met176, Met190, Met269, and Met355 are the other sites of the oxidative modification. Met44 and Met47 are the methionyl sites first oxidized. The methionine residues that are oxidized are not simply related to their accessibility to the external medium and are found in all four subdomains of actin. The conformations of subdomain 1, a region critical for the functional binding of different actin-binding proteins, and subdomain 2, which plays important roles in the polymerization process and stabilization of the actin filament, are changed upon oxidation. The conformational changes are deduced from the increased exposure of hydrophobic residues, which correlates with methionine sulfoxide formation, from the perturbations in tryptophan fluorescence, and from the decreased susceptibility to limited proteolysis of oxidized actin.     

A kinetic method for calculating the viability of lactic starter cultures

Summary A simple method of assessing the viability of a lactic starter culture is presented. The method is based on the kinetics of growth of a culture during the lag and early exponential phases. The microorganisms used throughout this work were Lactobacillus spp. The method was tested with inocula samples of different ages and with samples taken during chemostat runs, at different dilution rates. The results obtained are similar to ones describes in the literature for similar situations. The method is very easy to operate once the intrinsic parameters have been established (_max and _max). It needs only standard laboratory equipment and is very economical. Offprint requests to: M. T. O. Barreto     

Detection of hydrogen peroxide by lactoperoxidase-mediated dityrosine formation

The aim of this work was to study the dityrosine-forming activity of lactoperoxidase (LPO) and its potential application for measuring hydrogen peroxide (H₂O₂). It was observed that LPO was able to form dityrosine at low H₂O₂ concentrations. Since dityrosine concentration could be measured in a simple fluorimetric reaction, this activity of the enzyme was utilized for the measurement of H₂O₂ production in different systems. These experiments successfully measured the activity of NADPH oxidase 4 (Nox4) by this method. It was concluded that LPO-mediated dityrosine formation offers a simple way for H₂O₂ measurement.     

Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yogurts

Folate is a B-group vitamin that cannot be synthesized by humans and must be obtained exogenously. Although some species of lactic acid bacteria (LAB) can produce folates, little is known about the production of this vitamin by yogurt starter cultures. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were isolated from artisanal Argentinean yogurts and were grown in folate-free culture medium (FACM) and nonfat milk after which intracellular and extracellular folate production were evaluated. From the initial 92 isolated LAB strains, 4 L. delbrueckii subsp. bulgaricus and 32 S. thermophilus were able to grow in the absence of folate. Lactobacillus delbrueckii subsp. bulgaricus CRL 863 and S.?thermophilus CRL 415 and CRL 803 produced the highest extracellular folate levels (from 22.3 to 135?μg/L) in FACM. In nonfat milk, these strains were able to increase the initial folate concentrations by almost 190%. This is the first report where native strains of L. delbrueckii subsp. bulgaricus were shown to produce natural folate. The LAB strains identified in this study could be used in developing novel fermented products bio-enriched in natural folates that could in turn be used as an alternative to fortification with the controversial synthetic chemical folic acid.     

Survival kinetics of lactic acid starter cultures during and after freeze drying

Survival kinetics of lactic acid starter cultures were modeled considering the microorganism and external medium interfacial area as the critical factors determining the resistance of the microorganisms to freeze-drying. Surviving fraction of the microorganisms increased with the increasing biomass concentration during freeze-drying, and this is attributed to the mutual shielding effect of the microorganisms against the severe conditions of the external medium. Survival of the microorganisms over the storage period after freeze drying was enhanced by the presence of dead microorganisms which reduce the interfacial area between the live cells and the external medium. Streptococcus thermophilus was found to be more resistant to freeze-drying conditions than Lactobacillus bulgaricus. Storage under vacuum or nitrogen was superior to storage under air. Poor survival rates under air was attributed to oxygen diffusion into the dry cells through the interfacial area.     

Alternative drying processes for the industrial preservation of lactic acid starter cultures

The preservation of lactic acid starter cultures by alternative drying processes has attracted increasing attention due to the high costs and energy consumption of freezing and freeze drying. This review thus aims to provide a survey regarding the state of knowledge of starter culture production at high levels of viability. The results from numerous studies on various drying processes and lactic acid bacteria are summarized. The alternative drying processes considered, such as spray drying, fluidized bed drying, and vacuum drying, are mainly of industrial interest. The features, advantages, and disadvantages of these drying processes are described. In conclusion, the important factors that need to be considered, standardized, or optimized to achieve high levels of viability include intrinsic tolerance of cultures, growth media and conditions, stress induction, cell harvesting conditions, protective agents, rehydration conditions, enumeration of cells, and storage conditions.     

Evaluation of the lipolytic activity of starter cultures for meat fermentation purposes

A modified pork fat based agar method was specially developed to determine the lipolytic activity of starter strains used for meat fermentations. The lipolytic ability of strains of lactobacilli, pediococci, staphylococci and micrococci, was examined on agar plates using different substrates and in a lipid broth model system. The screening results indicate that lipase activity was confined to strains of Staphylococcus and Micrococcus . None of the lactic acid bacteria tested showed lipolytic activity. A strain of Staphylococcus xylosus , which displayed high lipolytic activity during the screening process was examined to find its optimum conditions for lipase activity regarding pH, temperature and NaCl concentration. The lipolytic activity of seven bacterial strains was determined under optimum conditions (0% salt, pH 7 and 30 °C) and sausage conditions (3·5% salt, pH 5 and 20 °C) using fat buffer model systems. High lipolytic activity was determined for all seven strains under optimal conditions, whereas no activity was detected under fermented sausage conditions.     

Evaluation of lactic acid Streptococci for the preparation of frozen concentrated starter cultures

Eight strains of Streptococcus diacetilactis and Streptococcus lactis were examined for viability, growth rate, lactic acid and diacetyl production in milk and proteolytic activity before and after freezing at --30 degrees C. Concentrations of yeast autolysate, peptone, lactose and citrate as well as the usefulness of milk and whey culture media for active biomass production were investigated. After freezing and storage at --30 degrees C, with the use of non-fat milk as a cryoprotective agent, high survival and endocellular proteolytic activity of the frozen concentrate was achieved. S. diacetilactis sp. and S. lactis 115 were shown to be more biologically active than other strains. Their physiological properties remained unaffected by freezing.     

Robust oxygen supply by controlled addition of hydrogen peroxide to microbial cultures

The supply of oxygen can be improved by the direct addition of hydrogen peroxide to cultures of aerobic microbes expressing sufficient amounts of catalase. This is of special interest if normal aeration has to be kept low, for instance, in order to minimize evaporation of volatile compounds (either substrates or products) or to minimize foaming. Also, if the mechanical power input to the bioreactor is or has to be limited, addition of hydrogen peroxide may be useful. The appropriate dosage of hydrogen peroxide can be simply determined by a controller of the oxygen partial pressure or of the oxygen content in the exhaust gas using various control algorithms. The added hydrogen peroxide can be either a stabilized concentrate, e.g. 30%, or any dilute form of this. In high density cultures, Pseudomonas cells tolerated even harsh controller disturbances. This approach proved to be very robust and reliable.     

Decolourization of paprika dye effluent with hydrogen peroxide produced by glucose oxidase

Abstract

Hydrogen peroxide was produced from bran by a two-step process using cellulase/xylanase and glucose oxidase, sequentially. The decolourization efficiency of the produced reagent was tested using paprika oil dye (effluent from industrial source) and high levels of colour removal (96%) were achieved after saponification pre-treatment and hydrogen peroxide application. The method is economically and environmentally advantageous since lower energy and chemical input are needed and wastewater pollution is considerably reduced. At the same time, the utilization of waste materials was successfully achieved.     

Molecular imaging of hydrogen peroxide produced for cell signaling

Hydrogen peroxide (H2O2) is emerging as a newly recognized messenger in cellular signal transduction. However, a substantial challenge in elucidating its diverse roles in complex biological environments is the lack of methods for probing this reactive oxygen metabolite in living systems with molecular specificity. Here we report the synthesis and application of Peroxy Green 1 (PG1) and Peroxy Crimson 1 (PC1), two new fluorescent probes that show high selectivity for H2O2 and are capable of visualizing endogenous H2O2 produced in living cells by growth factor stimulation, including the first direct imaging of peroxide produced for brain cell signaling. The combined features of reactive oxygen species selectivity, sensitivity to signaling levels of H2O2, and live-cell compatibility presage many new opportunities for PG1, PC1 and related synthetic reagents for exploring the physiological roles of H2O2 in living systems with molecular imaging.     

Detection of hydrogen peroxide with chemiluminescent micelles

The overproduction of hydrogen peroxide is implicated in the progress of numerous life-threatening diseases and there is a great need for the development of contrast agents that can detect hydrogen peroxide in vivo. In this communication, we present a new contrast agent for hydrogen peroxide, termed peroxalate micelles, which detect hydrogen peroxide through chemiluminescence, and have the physical/chemical properties needed for in vivo imaging applications. The peroxalate micelles are composed of amphiphilic peroxalate based copolymers and the fluorescent dye rubrene, they have a ‘stealth’ polyethylene glycol (PEG) corona to evade macrophage phagocytosis, and a diameter of 33 nm to enhance extravasation into permeable tissues. The peroxalate micelles can detect nanomolar concentrations of hydrogen peroxide (>50 nM) and thus have the sensitivity needed to detect physiological concentrations of hydrogen peroxide. We anticipate numerous applications of the peroxalate micelles for in vivo imaging of hydrogen peroxide, given their high sensitivity, small size, and biocompatible PEG corona.     

Site-specific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide

Oxidative DNA damage has been implicated in diverse biological processes including mutagenesis, carcinogenesis, aging, radiation effects, and chemotherapy. We examined the in vitro effect of low concentrations of Cu(II) or H2O2 alone and in combination on supercoiled plasmid DNA. As much as 10(-2) M Cu(II) or 10(-2) M H2O2 alone did not break the DNA. However, a mixture of 10(-6) M Cu(II) plus 10(-5) M H2O2 produced strand breaks and inactivated transforming ability. Strand breakage was proportional to incubation time, temperature, and Cu(II) and H2O2 concentrations. Abasic sites were not detected. Strand breakage was inhibited by metal chelators, catalase, and by high levels of free radical scavengers implying that Cu(II), Cu(I), H2O2, and .OH were involved in the reaction. The extent of DNA strand breakage was not affected by superoxide dismutase indicating that superoxide was not a major contributor to the DNA damage. DNA sequence analysis demonstrated that hot piperidine-sensitive DNA lesions were produced preferentially at sites of 2 or more adjacent guanosine residues. This sequence specificity was observed with Cu(II) plus H2O2 but not with Cu(I) alone. Polyguanosine sequence specificity for DNA damage induction appears to be unique among simple chemical systems. This reaction may be important in mechanisms of oxidative damage in vivo.     

Characteristics of some lactic Acid bacteria used as starter cultures in dry sausage production

The fermentation characteristics of two commercial (Duploferment 66 and Saga II) and five Norwegian lactic acid bacteria used in dry sausage production were compared with those of Lactobacillus plantarum ATCC 8014. The Norwegian strains lacked the ability to ferment mannitol, sorbitol, lactose, and d-(+)-raffinose and grew at 8 but not at 42 degrees C, in contrast to the ATCC culture and the two commercial strains. The lactate dehydrogenase activity of the Norwegian strains was not stimulated by pyruvate. All strains were capable of peroxide destruction when grown in the presence of myoglobin.     

Changes in the plasmid profile of Lactobacillus plantarum obtained from commercial meat starter cultures

The stability of plasmids in Lactobacillus plantarum used commercially as a starter culture in the production of dry sausage was studied. Individual isolates of one strain taken from the starter culture produced over a period of 10 years were examined, and certain changes in the plasmid profiles identified. From molecular sizing and restriction enzyme analysis it appeared that two of the six plasmids became fused over this period of time to give one new, larger plasmid. The other plasmids remained unchanged.