Effects of High Pressure on Bacillus licheniformis Spore Germination and Inactivation

Abstract

Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at Ͻ300 MPa triggers spore germination by activating germination receptors (GRs), while pressurization at Ͼ300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHPinduced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process.IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions that maintain the sensory and nutritional qualities of the food. High-pressure (HP) processing is a nonthermal food-processing technology used to eliminate microbes from food. The application of this technology for spore eradication in the food industry requires a better understanding of how HP affects the spores of different bacterial species. The present study provides novel insights into how HP affects Bacillus licheniformis spores, a species associated with food spoilage and occasionally food poisoning. We describe the roles of different germination receptors in HPinduced germination and the effects of two different pressure levels on the germination and inactivation of spores. This study will potentially contribute to the effort to implement HP technology for spore inactivation in the food industry.KEYWORDS Bacillus licheniformis, spore germination, germination receptor, endospores, high pressure, high-pressure processing, spore inactivation, flow cytometry

Flow Cytometry Combined With Single Cell Sorting to Study Heterogeneous Germination of Bacillus Spores Under High Pressure

“…FCM has thus been used increasingly for studying spore germination in general. However, only very few studies used FCM to study spore germination under HP (Black et al, 2005;Mathys et al, 2007;Baier et al, 2011;Reineke et al, 2013a;Borch-Pedersen et al, 2017).…”

“…Previous studies on HP-treated Bacillus spores reported the usage of nucleic acid binding stains including the membrane-permeable and -impermeable stains SYTO16 and propidium iodide (PI), respectively, to distinguish between dormant, germinated and potentially inactivated spores (Black et al, 2005(Black et al, , 2007Mathys et al, 2007;Reineke et al, 2013a;Borch-Pedersen et al, 2017). The underlying assumption here is that dormant spores should not or only very poorly be stained by either SYTO16 or PI.…”

“…In a previous study investigating HP-treated Bacillus subtilis and Bacillus licheniformis spores (150 MPa, 37 • C), at least one additional sub-population with intermediate SYTO16-intensity was reported. This sub-population was found to be predominant after a 10-20 min HP-treatment, and referred to as "unknown", since its physiological state was poorly understood (Mathys et al, 2007;Reineke et al, 2013a;Borch-Pedersen et al, 2017). FCM results from previous studies were mostly verified by correlating or qualitatively comparing findings with those obtained by using classical methods such as Ca 2+ -DPA release, phase-contrast microscopy or viable plate count (Black et al, 2005;Mathys et al, 2007;Baier et al, 2011;Borch-Pedersen et al, 2017).…”

“…The cultivability of these sub-populations were proposed by comparing the FCM results with plate count data on the overall population. No previous study isolated and cultivated the single sub-populations (Mathys et al, 2007;Reineke et al, 2012;Borch-Pedersen et al, 2017). For example, some researchers found one sub-population with intermediate SYTO16 signal, and suggested that cells in this sub-population were not cultivable (Mathys et al, 2007;Reineke et al, 2012).…”

Hyperbaric Storage at Room Temperature for Fruit Juice Preservation

“…Furthermore, it is important to note that HS-RT can reduce not only the load of vegetative cells as conventional pasteurization does, but can also decrease the endospore load. Many papers in the literature have shown that relatively low pressures induce endospore germination [35][36][37][38]. Thus, during HS-RT, pressure can produce the germination of endospores, and at appropriate pressure levels, their subsequent outgrowth is not fulfilled.…”