Michael G. Gänzle

High-Pressure-Mediated Survival of Clostridium botulinum and Bacillus amyloliquefaciens Endospores at High Temperature

ABSTRACT Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure-resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120°C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110°C) accelerated the inactivation of C. botulinum spores. However, incubation at 100°C, 110°C, or 120°C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120°C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure-dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120°C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment.

Effect of ecological factors on the inhibitory spectrum and activity of bacteriocins

The effect of food components and ecological factors on the activities of nisin, sakacin P and curvacin A was evaluated. Lactobacillus curvatus, Listeria innocua, Salmonella and Escherichia coli including E. coli O157:H7 were used as target organisms. Lecithin, casein, and divalent cations were antagonists of the bacteriocins at 0.1%, 0.1% and 10 mmol l(-1), respectively. A decrease in pH as well as the presence of EDTA, propyl-parabene or NaCl at concentrations of 0-1 mmol y(-1), 0-0.16 g l(-1), and 0-6% (w/w), respectively, increased the activity of all bacteriocins. These compounds as well as a pH < 5.5 rendered the Gram-negative target organisms sensitive against bacteriocins. Of practical importance is the respective effect of NaCl at concentrations > 5% which are achieved in fermentation and ripening processes, e.g. in production of fermented sausages. A characteristic response was observed for each of the bacteriocins. It is suggested that bacteriocins of lactic acid bacteria are effective against a wide range of microorganisms including E. coli O157:H7 if applied in combination with other preservative principles prevailing in foods.

Characterization of Reutericyclin Produced by Lactobacillus reuteri LTH2584

ABSTRACT Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains ofEscherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C8 chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. Höltzel, M. G. Gänzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766–2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.

Protective effect of sucrose and sodium chloride for Lactococcus lactis during sublethal and lethal high-pressure treatments

ABSTRACT The bactericidal effect of hydrostatic pressure is reduced when bacteria are suspended in media with high osmolarity. To elucidate mechanisms responsible for the baroprotective effect of ionic and nonionic solutes, Lactococcus lactis was treated with pressures ranging from 200 to 600 MPa in a low-osmolarity buffer or with buffer containing 0.5 M sucrose or 4 M NaCl. Pressure-treated cells were characterized in order to determine viability, the transmembrane difference in pH (ΔpH), and multiple-drug-resistance (MDR) transport activity. Furthermore, pressure effects on the intracellular pH and the fluidity of the membrane were determined during pressure treatment. In the presence of external sucrose and NaCl, high intracellular levels of sucrose and lactose, respectively, were accumulated by L. lactis; 4 M NaCl and, to a lesser extent, 0.5 M sucrose provided protection against pressure-induced cell death. The transmembrane ΔpH was reversibly dissipated during pressure treatment in any buffer system. Sucrose but not NaCl prevented the irreversible inactivation of enzymes involved in pH homeostasis and MDR transport activity. In the presence 0.5 M sucrose or 4 M NaCl, the fluidity of the cytoplasmic membrane was maintained even at low temperatures and high pressure. These results indicate that disaccharides protect microorganisms against pressure-induced inactivation of vital cellular components. The protective effect of ionic solutes relies on the intracellular accumulation of compatible solutes as a response to the osmotic stress. Thus, ionic solutes provide only asymmetric protection, and baroprotection with ionic solutes requires higher concentrations of the osmolytes than of disaccharides.

Effects of high pressure on survival and metabolic activity of Lactobacillus plantarum TMW1.460

ABSTRACT The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 106cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.

Exopolysaccharide-Forming Weissella Strains as Starter Cultures for Sorghum and Wheat Sourdoughs

The addition of sourdough fermented with lactic acid bacteria synthesizing organic acids and oligo- and exopolysaccharides (EPS) from sucrose enhances texture, nutritional value, shelf life, and machinability of wheat, rye, and gluten-free bread. This study compared acetate, mannitol, and oligosaccharide formation of EPS-producing strains of Weissella and Leuconostoc spp. to the traditional sourdough starter Lactobacillus sanfranciscensis. In broth, Leuconostoc strains generally formed acetate and mannitol, whereas Weissella produced only small amounts of acetate and no mannitol in the presence of sucrose. In the presence of sucrose and maltose, Weissella and Leuconostoc strains synthesized glucooligosaccharides and EPS. Strains of Weissella were employed as starter cultures for wheat and sorghum sourdough and formed 0.8-8 g kg(-1) EPS and gluco-oligosaccharides but only low amounts of acetate and mannitol. In contrast, the formation of EPS from sucrose led to the production of high amounts of acetate and mannitol by L. sanfranciscensis LTH 2950 in wheat sourdough. This study indicates that Weissella strains are suitable starter cultures for wheat and sorghum sourdoughs and efficiently produce gluco-oligosaccharides and EPS.

Phenolic Acids and Flavonoids in Nonfermented and Fermented Red Sorghum (Sorghum bicolor (L.) Moench)

This study aimed to identify phenolic acids and flavonoids in the red sorghum variety PAN 3860 and to determine changes in their concentrations during fermentation with lactobacilli. Sorghum sourdoughs fermented with two binary strain combinations, Lactobacillus plantarum and Lactobacillus casei or Lactobacillus fermentum and Lactobacillus reuteri , were compared to chemically acidified controls. Four glycerol esters were tentatively identified, caffeoylglycerol, dicaffeoylglycerol, coumaroyl-caffeoylglycerol, and coumaroyl-feruloylglycerol, that have previously not been detected in sorghum. Chemical acidification resulted in hydrolysis of phenolic acid esters and flavonoid glucosides. During lactic fermentation, phenolic acids, phenolic acid esters, and flavonoid glucosides were metabolized. Analysis of ferulic acid, caffeic acid, and naringenin-glucoside contents in single-strain cultures of lactobacilli demonstrated that glucosidase, phenolic acid reductase, and phenolic acid decarboxylase activities contributed to polyphenol metabolism. This study demonstrates that microbial fermentation of sorghum affects the content of polyphenols and can influence the nutritional value and antimicrobial activity of sorghum.

Structure-function relationships of the antibacterial activity of phenolic acids and their metabolism by lactic acid bacteria.

Aims:  To determine structure–function relationships of antibacterial phenolic acids and their metabolites produced by lactic acid bacteria (LAB).

Comparison of pressure and heat resistance of Clostridium botulinum and other endospores in mashed carrots.

Inactivation of bacterial endospores in food requires a combination of pressure and moderate heat. Endospore resistance of seven Clostridium botulinum strains was compared with those of Bacillus spp. (B. cereus, B. subtilis, B. licheniformis, B. smithii, B. amyloliquefaciens) and Thermoanaerobacterium thermosaccharolyticum with respect to pressure (600 to 800 MPa) and temperature (80 to 116 degrees C) treatments in mashed carrots. A large variation was observed in the pressure resistance of C. botulinum spores. Their reduction after treatments with 600 MPa at 80 degrees C for 1 s ranged from more than 5.5 log units to no reduction. Spores of the proteolytic C. botulinum TMW 2.357 exhibited a greater resistance to pressure than spores from all other bacteria examined, with the exception of B. amyloliquefaciens. Heat resistance of spores did not correlate with pressure resistance, either within strains of C. botulinum or when C. botulinum spores were compared with spores of T. thermosaccharolyticum. A quantitative release of dipicolinic acid was observed from C. botulinum spores on combined pressure and temperature treatments only after inactivation of more than 99.999% of the spores. Thus, dipicolinic acid is released by a physicochemical rather than a physiological process. The resistance of spores to combined pressure and temperature treatments correlated with their ability to retain dipicolinic acid. B. amyloliquefaciens, a mesophilic organism that forms highly pressure-resistant spores is proposed as a nonpathogenic target organism for high-pressure process development.

Pressure Inactivation of Bacillus Endospores

ABSTRACT The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80°C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70°C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B. subtilis CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60°C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B. amyloliquefaciens, which form highly pressure-resistant spores.