Braised sauce beef is a traditional pickled ready-to-eat meat product in China and South-East Asia. It is generally produced by boiling and braising beef in a sauce that is seasoned and circularly used. Owing to its unique taste and flavor, braised sauce beef is widely popular among the consumers. However, the rich nutrition and high-water activity also provide a favorable environment for microbial growth, which results in the quality deterioration of braised sauce beef. Therefore, braised sauce beef treated by sterilization or pasteurization is typically packaged in vacuum or modified atmosphere to prolong the shelf-life in the industrial process (Li et al., 2019), which makes Clostridium botulinum the target bacteria for developing a thermal processing for the products.

Clostridium sporogenes is a spore-forming, nontoxigenic surrogate for modeling thermal inactivation of neurotoxin-producing C. botulinum due to its high heat resistance, ease of monitoring its presence through off-odor and gas formation (Butler et al., 2017; Soni et al., 2021). A thermal process that insufficiently kills C. botulinum spores allows them to survive and subsequently produce toxin. In industrial sterilization, it is common practice to achieve a 12 log reduction of proteolytic C. botulinum to allow the safe storage of foods at ambient temperature, while in heat-treated chilled food it is aimed a 6 log reduction of nonproteolytic C. botulinum for a severe pasteurization to allow a product shelf-life up to 6 weeks at 5 °C (Peng et al., 2017). Therefore, it is of great importance to understand the heat resistance of C. sporogenes spores and its mechanisms in establishing thermal processes applicable to inactivate C. botulinum.

The heat resistance of C. sporogenes spores is closely related with strain type, sporulation condition and thermal environment (Dong, 2011; Lekogo et al., 2010; Parker et al., 2015; Valero et al., 2020). The inherent difference in heat resistance of C. sporogenes spores is partially explained by the acquisition of a second spoVA operon, which is responsible for transportation of dipicolinic acid into the spore core during sporulation and therefore increase spore heat tolerance. For food thermal process, plenty of studies have demonstrated that external environment including temperature, pH and antimicrobial agents also plays an important role in heat resistance of Clostridium spores (Boix et al., 2022; Boix et al., 2021; Hofstetter et al., 2013; Liang et al., 2021; Paredes-Sabja et al., 2007; Ros-Chumillas et al., 2015). Some studies investigated the relationship between different food media and the heat resistance of C. sporogenes spores. For instance, Cameron et al. (1980) found that the heat resistance of C. sporogenes spores in phosphate buffer significantly differed from that in pea puree. The study conducted by Liato et al. (2015) also pointed out that the heat resistance of C. sporogenes spores varied from different model solution and vegetable puree. For braised meat products, some studies investigated the effects of external environment factors on the growth and heat resistance of Clostridium spp., including thermal temperature and cooling after cooking (Li et al., 2019; Wang et al., 2020). However, there has been a lack of published data on the relationship between the food matrix and heat resistance of C. sporogenes spores in braised beef and products thereof, which is the key for development of thermal processed braised sauce beef.

Lipid is an important component of food and may impact the heat resistance of spores in foods. Our preliminary experiments found that during thermal processing of braised sauce beef, lipid content in sauce increased evidently due to the lipid migration from beef to sauce (Table S1). However, limited information is available on the influence of lipid on the heat resistance of C. sporogenes spores, particularly in real foods. It has been reported that bacterial cells or spores were more heat resistant in oil than that in aqueous system, but most of previous works were conducted on Bacillus species and focused on their microbial survivor rate in oil-water system (Ababouch and Busta, 1987; Nakagawa et al., 1998). One research conducted by Oliveira et al. (2018) found that C. sporogenes spores were less sensitive to heat in the full-fat “requeijão cremoso” formulation than that in the low-fat one, without further exploring the underlying mechanism. The possible causes of higher heat resistance of microorganisms in oil may be the low water activity in oil, or the increased hydrophobic interaction between bacterial surfaces and oil (Nakagawa et al., 1998; Shigemoto et al., 2010). However, the above hypothesis was proposed for Bacillus spores in oil-water system, without further exploration in the real foods. It lacks of investigations of the protective effect of oil on spores of Clostridium species, particularly in real food system, which is essential for developing thermal process to control these spores in food matrices with lipid.

Therefore, the objectives of this study are as following: (1) characterize the heat resistance of C. sporogenes spores in braised sauce beef; (2) evaluate the effect of oil addition on the heat resistance of C. sporogenes spores; (3) assess the changes of spore surface characteristics and microstructure with oil addition under heating to obtain their relations with the spore heat resistance.