Production, recovery and purification of bacteriocins from lactic acid bacteria

Bacteriocins produced by lactic acid bacteria are a heterogeneous group of peptide inhibitors which include lantibiotics (class I, e.g. nisin), small heat-stable peptides (class II, e.g. pediocin AcH/PA1) and large heat-labile proteins (class III, e.g. helveticin J). Many bacteriocins belonging to the first two groups can be successfully used to inhibit undesirable microorganisms in foods, but only nisin is produced industrially and is licensed for use as a food preservative in a partially purified form. This review focuses on the production and purification of class I and class II bacteriocins from lactic acid bacteria. Bacteriocin production is growth associated but the yield of bacteriocin per unit biomass is affected by several factors, including the producing strain, media (carbohydrate and nitrogen sources, cations, etc.) and fermentation conditions (pH, temperature, agitation, aeration and dilution rate in continuous fermentations). Continuous fermentation processes with cell recycle or immobilized cells can result in a dramatic improvement in productivity over batch fermentations. Several simple recovery processes, based on adsorbing bacteriocin on resins or silica compounds, have been developed and can be used to build integrated production processes. Received: 29 December 1998 / Received revision: 23 April 1999 / Accepted: 23 April 1999     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Escherichia coli and Lactobacillus plantarum responses to osmotic stress

Escherichia coli and Lactobacillus plantarum were subjected to final water potentials of −5.6 MPa and −11.5 MPa with three solutes: glycerol, sorbitol and NaCl. The water potential decrease was realized either rapidly (osmotic shock) or slowly (20 min) and a difference in cell viability between these conditions was only observed when the solute was NaCl. The cell mortality during osmotic shocks induced by NaCl cannot be explained by a critical volume decrease or by the intensity of the water flow across the cell membrane. When the osmotic stress is realized with NaCl as the solute, in a medium in which osmoregulation cannot take place, the application of a slow decrease in water potential resulted in the significant maintenance of cell viability (about 70–90%) with regard to the corresponding viability observed after a sudden step change to same final water potential (14–40%). This viability difference can be explained by the existence of a critical internal free Na⁺ concentration. Received: 20 May 1998 / Received revision: 31 July 1998 / Accepted: 31 July 1998     

Sublethal injury makes Gram-negative and resistant Gram-positive bacteria sensitive to the bacteriocins, pediocin AcH and nisin

Gram-negative and some Gram-positive bacteria that are resistant to bacteriocins of lactic acid bacteria (LAB) were subjected to sublethal stresses and treated with nisin and pediocin AcH. Both bacteriocins reduced the viability of cells surviving sublethal stresses. The results explain the possible mechanisms by which bacteriocins of LAB enter through the walls (or outer membranes) to destabilize the cytoplasmic (or inner) membranes and kill cells of sensitive Gram-positive and resistant, but injured, Gram-negative and Gram-positive bacteria.     

Improving nisin production by increasing nisin immunity/resistance genes in the producer organism Lactococcus lactis

The effect on nisin production of increasing nisin immunity/resistance genes in Lactococcus lactis subsp. lactis MG1363 was investigated. The 60-kb nisin immunity/resistance plasmid pND300, which was isolated from a non-nisin-producing strain, encodes five genes involved in nisin immunity/resistance, which are very similar to those of the immunity/resistance system encoded by the nisin-production transposon. The introduction of pND300 into MG1363(TnNip) resulted in the construct being able to produce significantly more nisin than the parent MG1363(TnNip). The introduction of pND314, which contains the nisin immunity/resistance genes subcloned into pSA3, into MG1363(TnNip) allowed the strain to grow more rapidly than the parent MG1363(TnNip) with a concomitant increase in the rate of nisin production. This work illustrates that introduction of pND300 and a derivative containing the nisin immunity/resistance system of pND300 into MG1363 (TnNip) can result in significant alterations to the kinetics of nisin production. These observations indicate approaches that may be used successfully to improve the economics of nisin production. Received: 11 February 1998 / Received revision: 25 June 1998 / Accepted: 27 June 1998     

细菌素的合成与作用机制

细菌素是由细菌产生的抗菌蛋白,可以杀死与产生菌相近的细菌。很多乳酸菌产生不同多样性的细菌素,虽然这些细菌素都是由发酵或非发酵食品中发现的乳酸菌产生的,但是迄今只有乳酸链球菌素(Nisin)作为食品防腐剂被广泛应用。和抗生素不同的是,细菌素由核糖体合成,需经翻译后修饰活化并且通过特定转运系统输到胞外才能发挥其功能,它一般通过作用于靶细胞膜来抑制靶细胞的生长,同时本身合成细菌素的细胞对其产物具有免疫性。细菌素能安全有效地抑制病原体生长,在食品行业中具有广阔的应用前景。     

Microbial production of bacteriocins: Latest research development and applications

Bacteriocins are low molecular weight peptides secreted by the predator bacterial cells to kill sensitive cells present in the same ecosystem competing for food and other nutrients. Exceptionally few bacteriocins along with their native antibacterial property also exhibit additional anti-viral and anti-fungal properties. Bacteriocins are generally produced by Gm+, Gm– and archaea bacteria. Bacteriocins from Gm?+?bacteria especially from lactic acid bacteria (LAB) have been thoroughly investigated considering their great biosafety and broad industrial applications. LAB expressing bacteriocins were isolated from fermented milk and milk products, rumen of animals and soil using deferred antagonism assay. Nisin is the only bacteriocin that has got FDA approval for application as a food preservative, which is produced by Lactococcus lactis subsp. Lactis. Its crystal structure explains that its antimicrobial properties are due to the binding of NH₂ terminal to lipid II molecule inhibiting the peptidoglycan synthesis and carboxy terminal forming pores in bacterial cell membrane leading to cell lysis. The hinge region connecting NH₂ and carboxy terminus has been mutated to generate mutant variants with higher antimicrobial activity. In a 50 ton fermentation of the mutant strain 3807 derived from L. lactis subsp. lactis ATCC 11454, 9,960?IU/mL of nisin was produced. Currently, high purity of nisin (>99%) is very expensive and hardly commercially available. Development of more advanced tools for cost-effective separation and purification of nisin would be commercially attractive. Chemical synthesis and heterologous expression of bacteriocins ended in low yields of pure proteins. At present, bacteriocins are almost solely applied in food industries, but they have a great potential to be used in other fields such as feeds, organic fertilizers, environmental protection and personal care products. The future of bacteriocins is largely dependent on getting FDA approval for use of other bacteriocins in addition to nisin to promote the research and applications.     

EPR study and structural aspects of ferredoxins obtained from Thiobacillus ferrooxidans

A comparison of iron-sulfur proteins obtained from Thiobacillus ferrooxidans was carried out. The microorganisms were grown on iron(II)- or sulfur-containing nutrients. In both cases different, broad elctron paramagnetic resonance (EPR) lines, originating from an iron(III) compound, were detected. Additional EPR lines of tetrahedral iron(III) and free radicals were observed. The UV spectra of these compounds also differ. Received: 15 July 1998 / Received revision: 8 October 1998 / Accepted: 16 October 1998     

Dialysis cultures

Dialysis techniques are discussed as a means for effective removal of low-molecular-mass components from fermentation broth to reach high cell density. Reactor systems and process strategies, the relevant properties of membranes and examples for high-density fermentation with dialysis, and problems related to scale-up are addressed. The dialysis technique has turned out to be very efficient and reliable for obtaining high cell densities. As in dialysis processes the membranes are not perfused, membrane clogging is not a problem as it is for micro- and ultrafiltration. By applying a “nutrient-split” feeding strategy, the loss of nutrients can be avoided and the medium is used very efficiently. The potential of dialysis cultures is demonstrated on the laboratory scale in a membrane dialysis reactor with an integrated membrane and in reactor systems with an external dialysis loop. In dialysis cultures with different microorganisms (Staphylococci, Escherichia coli, extremophilic microorganisms, Lactobacilli) the cell densities achieved were up to 30 times higher than those of other fermentation methods. The technique enables high cell densities to be attained without time-consuming medium optimization. For animal cell cultures the concept of a fixed bed coupled with dialysis proved to be very effective. Received: 24 March 1998 / Received revision: 18 June 1998 / Accepted: 19 June 1998     

Biotransformation of citronellol by the basidiomycete Cystoderma carcharias in an aerated-membrane bioreactor

The basidiomycete Cystoderma carcharias transformed citronellol into 3,7-dimethyl-1,6,7-octanetriol as the main product. 3,7-Dimethyl-6,7-epoxy-1-octanol was identified as important intermediary product of the biotransformation, and the allylic diols 2,6-dimethyl-2-octene-1,8-diol, 3,7-dimethyl-5-octene-1,7-diol and 3,7-dimethyl-7-octene-1,6-diol were found to be minor products. Microbial formation of rose oxide, a flavour-impact component, was observed for the first time. The formation of the main products was inhibited by 70% after addition of 0.1 mmol l⁻¹ cytochrome monooxygenase inhibitors. Formation of 3,7-dimethyl-1,6,7-octanetriol was effective in a bioreactor with aeration over a coil of a hydrophobic microporous polypropene capillary membrane. Production rates of up to 150 mg l⁻¹ day⁻¹ were reached and led to a product concentration of 866 mg l⁻¹ (conversion rate: 52%). The total loss of the added volatile substrate via the exhaust air was 4.5% when this aeration method was used. Received: 30 July 1998 / Received revision: 2 November 1998 / Accepted: 7 November 1998     

Production of sophorolipids from whey: development of a two-stage process with Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214 using deproteinized whey concentrates as substrates

In order to produce sophorolipids from whey, thereby lowering the lactose content and biological oxygen demand, a two-step batch cultivation process was developed including medium sterilization by filtration. In the first step, whey was sterilized by a combination of crossflow and sterile filtration. Because the sophorolipid-producing yeast Candida bombicola ATCC 22214 was not able to use lactose as a carbon source directly, the oleaginous yeast Cryptococcus curvatus ATCC 20509 was grown on deproteinized whey concentrates (DWC). With 1: 1 diluted DWC-20, lactose was consumed as the carbon source and biomass (24 g/l dry weight content) as well as single-cell oil (SCO, 10 g/l) were produced. The cultivation broth was disrupted with a glass bead mill and it served as medium for growth (29 g cell dry mass/l) and sophorolipid production (12 g/l) of the yeast C. bombicola. Received: 29 July 1998 / Received revision: 5 October 1998 / Accepted: 11 October 1998     

Isolation and characterization of two bacteriocins of Lactobacillus acidophilus LF221

Lactobacillus acidophilus LF221 produced bacteriocin-like activity against different bacteria including some pathogenic and food-spoilage species. Besides some lactic acid bacteria, the following species were inhibited: Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus D. L. acidophilus LF221 produced at least two bacteriocins, acidocin LF221 A and acidocin LF221 B, which were purified by ammonium sulphate precipitation, ion-exchange chromatography, hydrophobic interaction and reverse-phase FPLC. The antibacterial substances were heat-stable, sensitive to proteolytic enzymes (trypsin, pepsin, pronase, proteinase K) and migrated as 3500- to 5000-Da proteins on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The sequences of 46 amino-terminal amino acid residues of peptide A and 35 of peptide B were determined. Among the residues identified, no modified amino acids were found. No significant homology was found between the amino acid sequences of acidocin LF221 A and other bacteriocins of lactic acid bacteria and 26% homology was found between acidocin LF221 B and brevicin 27. L. acidophilus LF221 may be of interest as a probiotic strain because of its human origin and inhibition of pathogenic bacteria, especially Clostridium difficile. Received: 2 October 1997 / Received revision: 12 January 1998 / Accepted: 13 January 1998     

Antibacterial metabolites of lactic acid bacteria: their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

Antibacterial metabolites of lactic acid bacteria: Their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

Reversal of the inhibitory effect of surfactants upon germination and growth of a consortium of two strains of Bacillus

Anionic, cationic, amphoteric and non-ionic surfactants inhibited spore germination and subsequent growth of a mixture of two Bacillus strains at surfactant concentrations ranging from 1 ppm to 50 ppm. Germination appeared to be more affected than cell growth by the presence of surfactants, the inhibitory thresholds being largely increased when media were inoculated with vegetative cells. The bacterial species forming the consortium were incapable of growing on liquid and agar-solidified media prepared with non-diluted domestic wastewater. Addition of hydrolases (protease, cellulase, α-amylase and lipase) to the wastewater medium allowed the germination of spores and their vegetative growth. Received: 9 July 1998 / Received revision: 26 October 1998 / Accepted: 30 October 1998     

Rhamnose lipids – biosynthesis, microbial production and application potential

Biosurfactants containing rhamnose and β-hydroxydecanoic acid and called rhamnolipids are reviewed with respect to microbial producers, their physiological role, biosynthesis and genetics, and especially their microbial overproduction, physicochemical properties and potential applications. With Pseudomonas species, more than 100 g l⁻¹ rhamnolipids were produced from 160 g l⁻¹ soybean oil at a volumetric productivity of 0.4 g l⁻¹h⁻¹. The individual rhamnolipids are able to lower the surface tension of water from 72 mN m⁻¹ to 25–30 mN m⁻¹ at concentrations of 10–200 mg l⁻¹. After initial testing, rhamnolipids seem to have potential applications in combating marine oil pollution, removing oil from sand and in combating zoosporic phytopathogens. Rhamnolipids are also a source of l-rhamnose, which is already used for the industrial production of high-quality flavor components. Received: 1 July 1998 / Received revision: 11 September 1998 / Accepted: 13 September 1998     

PsbY, a novel manganese-binding, low-molecular-mass protein associated with photosystem II

We describe two related manganese-binding polypeptides with L-arginine metabolizing enzyme activity that can be detected as distinct components (designated PsbY-A1 and PsbY-A2, previously called L-AME) in membranes containing Photosystem II (PS II) from spinach. The polypeptides are bitopic and appear to exist in a heterodimeric form, but only in the chlorophyll a/b lineage of plants. Both proteins are encoded in the nucleus. In spinach and in Arabidopsis thaliana they are both derived from a single-copy gene (psbY) that is translated into a precursor polyprotein of approximately 20 kDa. The processing of the polyprotein is complex and includes at least four cleavage steps. Both polypeptides are exposed N-terminally to the lumenal and C-terminally to the stromal face of the thylakoid membrane. Received: 9 June 1998 / Accepted: 9 July 1998     

Subharmonic resonance and chaos in forced excitable systems

 Forced excitable systems arise in a number of biological and physiological applications and have been studied analytically and computationally by numerous authors. Existence and stability of harmonic and subharmonic solutions of a forced piecewise-linear Fitzhugh-Nagumo-like system were studied in Othmer ad Watanabe (1994) and in Xie et al. (1996). The results of those papers were for small and moderate amplitude forcing. In this paper we study the existence of subharmonic solutions of this system under large-amplitude forcing. As in the case of intermediate-amplitude forcing, bistability between 1 : 1 and 2 : 1 solutions is possible for some parameters. In the case of large-amplitude forcing, bistability between 2 : 2 and 2 : 1 solutions, which does not occur in the case of intermediate-amplitude forcing, is also possible for some parameters. We identify several new canonical return maps for a singular system, and we show that chaotic dynamics can occur in some regions of parameter space. We also prove that there is a direct transition from 2 : 2 phase-locking to chaos after the first period-doubling bifurcation, rather than via the infinite sequence of period doublings seen in a smooth quadratic interval map. Coexistence of chaotic dynamics and stable phase-locking can also occur. Received: 6 July 1998 / Revised version: 2 October 1998     

The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms

Currently available microbiological techniques are not designed to deal with very slowly growing microorganisms. The enrichment and study of such organisms demands a novel experimental approach. In the present investigation, the sequencing batch reactor (SBR) was applied and optimized for the enrichment and quantitative study of a very slowly growing microbial community which oxidizes ammonium anaerobically. The SBR was shown to be a powerful experimental set-up with the following strong points: (1) efficient biomass retention, (2) a homogeneous distribution of substrates, products and biomass aggregates over the reactor, (3) reliable operation for more than 1 year, and (4) stable conditions under substrate-limiting conditions. Together, these points made possible for the first time the determination of several important physiological parameters such as the biomass yield (0.066 ± 0.01 C-mol/mol ammonium), the maximum specific ammonium consumption rate (45 ± 5 nmol/mg protein/min) and the maximum specific growth rate (0.0027 · h⁻¹, doubling time 11 days). In addition, the persisting stable and strongly selective conditions of the SBR led to a high degree of enrichment (74% of the desired microorganism). This study has demonstrated that the SBR is a powerful tool compared to other techniques used in the past. We suggest that the SBR could be used for the enrichment and quantitative study of a large number of slowly growing microorganisms that are currently out of reach for microbiological research. Received: 14 May 1998 / Received last revision: 30 July 1998 / Accepted: 31 July 1998     

Bacterial magnetosomes: microbiology, biomineralization and biotechnological applications

Magnetotactic bacteria orient and migrate along geomagnetic field lines. This ability is based on intracellular magnetic structures, the magnetosomes, which comprise nanometer-sized, membrane-bound crystals of the magnetic iron minerals magnetite (Fe₃O₄) or greigite (Fe₃S₄). Magnetosome formation is achieved by a mineralization process with biological control over the accumulation of iron and the deposition of the mineral particle with specific size and orientation within a membrane vesicle at specific locations in the cell. This review focuses on the current knowledge about magnetotactic bacteria and will outline aspects of the physiology and molecular biology of the biomineralization process. Potential biotechnological applications of magnetotactic bacteria and their magnetosomes as well as perspectives for further research are discussed. Received: 2 December 1998 / Received revision: 2 March 1999 / Accepted: 5 March 1999