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Chapter 7 : Effects of Pressure on Lactic Acid Bacteria
Category: Applied and Industrial Microbiology
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This chapter focuses on the high hydrostatic pressure (HHP)-mediated stress responses of selected strains of Lactic acid bacteria (LAB). The selected strains included Lactococcus lactis subsp. cremoris MG1363, Lactobacillus plantarum TMW1460, and Lactobacillus sanfranciscensis DSM20451. The chapter discusses the cellular basis of a sublethal injury demonstrated to reside in the impaired or rerouted metabolism, membrane integrity and functionality, and effects on ribosomal biosynthesis. The study of cross tolerances provides some insight into the sensors, pathways, and effectors of HHP stress. Studies of the stress response, not only on the proteome level, often seem to give contradictory results at first glance even if they are done with the same bacterium. This is because two types of stress responses must be distinguished with respect to the experimental setup. First, bacteria can be exposed to a shock for minutes, which they would not survive for long, and are subsequently held under optimum conditions in a recovery phase. Second, bacteria can be grown at suboptimal growth conditions, typically at 10% of their maximal growth rate, for several hours. The current understanding of HHP-induced microbial inactivation of vegetative cells enables establishment of HHP food processes from a microbial food safety point of view. The challenge to use HHP in microbial studies has, however, changed its focus, looking at HHP as a tool to study macromolecular interaction in cellular systems or models thereof.
Time course of HHP inactivation of Lactobacillus plantarum demonstrating areas of sublethal injury.
Range of sublethal pressure stress in Lactobacillus sanfranciscensis. (a) Metabolic activity as determined by maltose consumption (bars) and survival (lines) upon 20 min of HHP treatment of two different strains (black and grey). (b) Filamentation upon growth at 45 MPa (10% of maximal growth velocity).
Effect of pressure treatment at 20°C on metabolic activity (▼), membrane integrity (○), and viability (●) of Lactococcus lactis MG1363.
Different effects of solutes on the metabolic activity and membrane integrity of Lactococcus lactis MG1363 after treatment with 300 MPa at 20°C. Metabolic activity and membrane integrity were determined as described by Ulmer et al. ( 75 ).
Cross-protection in Lactobacillus sanfranciscensis as determined by survival of prestressed cells to a second stress of the same or another type.
Changes in the intracellular pH of Lactococcus lactis subsp. cremoris. Samples were treated at 300 MPa and 20°C in milk buffer. The compression rate was 200 MPa min-1; the ramp-up time was 90 s (shaded area). The measurement of the intracellular pH was performed as described by Molina-Gutierrez et al. ( 55 ).
Two-dimensional electrophoretic analysis of cytoplasmic protein extracts of Lactobacillus sanfranciscensis left untreated and after incubation for 1 h at 80 MPa. Proteins were silver stained. High-pressure-affected proteins are indicated by arrows. Isoelectric points and molecular weights are indicated.
HHP-induced generation of genetic diversity. (a) Induction of transcription of IS elements in L. sanfranciscensis by high pressure. L. san-franciscensis was subjected to 45 MPa (black bars) or 80 MPa (grey bars) for 30 min, and transcription ratios (high pressure to atmospheric pressure) of several transposable elements were determined. Data were normalized against xpk transcription; shown are the means of three independent experiments. (b) High-pressure-induced changes of hybridization patterns of ISLsf6 of L. sanfranciscensis. EcoRV-digested DNA was hybridized with a probe for orfB of ISLsf6. Lane 1 shows genomic DNA of the ancestor. Lanes 2 through 5 show genomic DNA after 1 growth cycle at 0.1 MPa (lane 2), 25 growth cycles at 0.1 MPa (lane 3), 1 growth cycle at 50 MPa (lane 4), and 25 growth cycles at 50 MPa (lane 5). Lane 6 shows DNA markers. Hybridization with a probe for orfA of ISLsf6 resulted in the same pattern. The arrow indicates the loss of a fragment in high-pressure-treated cells.
Summary of HHP-sensitive cellular targets identified and studied in LAB. Indicated are methods cited in the text, which were used to study these cellular functions. PI, propidium iodide; PI*DNA, fluorescent complex of PI with DNA; TOTO, 1,1’(4,4,8,8-tetramethyl-4,8-diazaundecamethylene) bis[4-(3-methyl-2,3-dihydrobenzo-1,3-thiazolyl-2-methylidene)quinolinium] tetraiodide; TOTO*DNA, fluorescent complex of TOTO with DNA; DISC3( 5 ), dipropylthiadicarbocyanin iodide; cFDASE, carboxyfluorescein diacetate lyase; INT, 2-(-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride; EB, ethidium bromide; EB*DNA, fluorescent complex of EB with DNA. Laurdan was used for measurements of membrane fluidity.
Cellular processes along increasing pressure.
Genes responsive to high pressure (45 MPa for 30 min) as determined by array hybridization and/or real-time PCR a
Susceptibilities of wild-type Lactobacillus sanfranciscensis and the pressure-adapted mutant to various antibiotics acting on the ribosomes or on translational processes