Bacillus Spores in the Food Industry: A Review on Resistance and Response to Novel Inactivation Technologies Aswathi Soni, Indrawati Oey, Pat Silcock, and Phil Bremer Abstract: The importance and challenges presented by Bacillus spores in the food industry are briefly outlined with a focus on Bacillus cereus. The structure and the mechanism of resistance exhibited by Bacillus spores are described, and the steps involved in their germination are included. Novel technologies, using no or only mild heat treatments, to inactivate Bacillus spores are covered, including ultraviolet radiation, pulsed electric field, and high-pressure processing, both as stand-alone techniques or techniques as part of a hurdle approach. Keywords: Bacillus spores, ultraviolet, pulsed electric field, Hurdle technology, resistance, high-pressure processing Introduction Bacterial spores are of concern to the food industry due to their ability to survive processing, the various steps designed to kill the vegetative cells, and their potential to subsequently germinate and grow in food, thereby decreasing its safety and shelf-life (Daelman and others 2013). Spores are tough, metabolically inert structures produced by some vegetative bacterial cells as a survival strategy in response to adverse environmental conditions (pH, nutrient limi- tations, desiccation, and temperature). A few Gram-positive (phy- lum Firmicutes, classes Bacilli and Clostridia) and Gram-negative (Sporamusa ovata, phylum Firmicutes, class Negativicutes) bacterial cells can produce spores (Poehlein and others 2013). Spores can be produced by anaerobic, aerobic, or facultative aerobic bacteria, for example, by Clostridium botulinum, Thermoactinomyces vulgaris, and Bacillus cereus, respectively. Spore-forming bacteria such as C. bo- tulinum and B. cereus cause food poisoning, whereas others like Alicyclobacillus acidoterrestris, C. tyrobutyricum, and Geobacillus stearothermophilus produce enzymes which break down food com- ponents. Hence, in foods they decrease nutritional value, func- tionality, and sensory quality (Sakaguchi 1982; Hauschild 1990; Lindb¨ ack and Granum 2006). Some spore-forming bacteria cause no apparent harm to food or consumers, but their occurrence in foods in high numbers reduces the value of a food product, for example, Anoxybacillus flavithermus or G. stearothermophilus in milk powder (Bremer and others 2009; Burgess and others 2010). Spore-forming organisms have traditionally been controlled by the application of heat (121 ºC) for a duration depending on the organ- ism targeted for sterilization. However, the increasing demand for foods with a fresh-like quality (texture, color, and aroma) and the MS 20161000 Submitted 22/6/2016, Accepted 29/8/2016. Authors are with Authors Soni, Oey, Silcock, and Bremer are with Dept. of Food Science, Univ. of Otago, PO Box 56, Dunedin 9054, New Zealand. Direct inquiries to author: Bremer (E-mail: phil.bremer@otago.ac.nz). desire to produce foods with functional ingredients has increased interest in the development of low-heat operations to either inac- tivate spores or to prevent their germination and growth. B. cereus is an aerobic or facultative anaerobic Gram-positive, spore-forming bacterium commonly present in soil, grains, veg- etables, raw and cooked rice, egg white, milk, and meat (Helgason and others 1998; Magnusson and others 2007; Arnesen and others 2008; Bottone 2010). B. cereus has the ability to grow over a wide range of temperatures (4 to 50º C) and is resistant to heat (wet and dry), chemical treatments, and radiation (ultraviolet [UV], X- rays, protons, and high-energy charged ions) (Borge and others 2001; Setlow 2006; Moeller and others 2014). B. cereus is of par- ticular concern to food producers as its vegetative cells produce 2 types of toxins, an emetic toxin (heat-stable cerulide) and diar- rheal enterotoxins (nonhemolytic enterotoxin [Nhe], cytotoxin K [CytK], hemolysin BL [Hbl]), which all are known to cause food poisoning (Granum and others 1993, 1999; Ehling-Schulz and others 2005). This review focuses on the importance of B. cereus to the food industry, on the conventional methods for its control, including the mechanism of resistance to various treatments, and on the potential of emerging technologies such as UV irradiation, high-pressure processing (HPP), and pulsed electric field (PEF) to control Bacillus spores in food. Other nonthermal techniques in- volving microfiltration and ultrasound have been investigated for the removal of Bacillus spores (Guerra and others 1997; Raso and others 1998; G´ esan-Guiziou 2010) but are outside the scope of this paper. Bacillus Spores Members of the species Bacillus are rod shaped, and most species are nonmotile with the exception of B. subtilis that shows swarm- ing motility (Kearns and Losick 2003). Bacillus species produce en- dospores that have the ability to resist heat, radiation, and chemical treatments. The external layer of the spores produced by Bacillus species, such as B. megaterium, B. anthrax, and B. cereus, is called an C  2016 Institute of Food Technologists ® doi: 10.1111/1541-4337.12231 Vol. 15, 2016  Comprehensive Reviews in Food Science and Food Safety 1139