Elsevier

Food Microbiology

Volume 72, June 2018, Pages 112-127
Food Microbiology

Effects of lowering water activity by various humectants on germination of spores of Bacillus species with different germinants

https://doi.org/10.1016/j.fm.2017.11.012Get rights and content

Highlights

  • Low water activity (aw) inhibited germination with all nutrient and non-nutrient germinants, including high pressure.

  • Low aw most strongly inhibited the commitment step in germination, with smaller effects exerted on subsequent steps in germination.

Abstract

The effect of water activity (aw), as lowered by different dietary humectants, on the germination of Bacillus subtilis, Bacillus megaterium and Bacillus cereus spores with germinants that act by different mechanisms has been investigated and compared. Germination of spores of these species by all of the germinants investigated was inhibited as aw decreased, with the general order of efficacy for these non-ionic humectants being sucrose > trehalose > glycerol. The effect of lowering aw on germination by germinant receptor (GR)-dependent germinants was not appreciably altered by varying germinant concentrations, was generally not much more effective with spores lacking coats or an outer membrane, and was less pronounced with heat-activated spores. Analysis of the effect of aw on spore germination via different mechanisms showed that GR-dependent germination was least sensitive to aw, while germination via activation of spore cortex peptidoglycan hydrolysis or dipicolinic acid release was more sensitive. However, germination by high hydrostatic pressure was less sensitive to inhibition by low aw, than was germination by other germinants. Examination of the GR-dependent germination of individual spores indicated that aw acted most strongly in inhibiting the commitment step of germination, while exerting smaller effects on dipicolinic acid release or cortex peptidoglycan hydrolysis.

Introduction

Water activity (aw) and pH are important parameters in food preservation, stabilization, and processing for preventing or limiting the growth of microorganisms, including molds, fungi and bacteria, as well as growth from bacterial spores, which can be infectious or have deleterious effects on food quality and safety (Fontana et al., 2008, Troller and Christian, 1978, Feeherry et al., 2003, Taub et al., 2003, Gulati et al., 2015). As a physical measure of the free water available to microorganisms and for chemical reactions in a food system, aw is defined as the ratio of the water vapor pressure of a substance to the vapor pressure of pure water at the same temperature. Examples of aw levels that control microorganisms in foods include: aw = 0.950 controls Pseudomonas, Shigella, Bacillus, and Clostridium perfringens in highly perishable foods – canned and fresh fruits; aw = 0.910 controls Salmonella, Vibrio parahaemolyticus, Lactobacillus and Clostridium botulinum in cured ham, or Cheddar, Swiss, Muenster, or Provolone cheeses; and aw = 0.870 controls many yeasts in fermented sausage, dry cheeses, sponge cakes; and aw = 0.800 controls most molds in fruits juice concentrates, chocolate and maple syrups, country style ham, and high-sugar cakes (adapted from Beuchat, 1981). According to international and national standards, foods with aw ≤ 0.85 and pH ≤ 4.6 are rendered non-potentially hazardous and do not require refrigeration to maintain safety from the rapid and progressive growth of infectious or toxigenic microorganisms. Foods with any component(s) having aw > 0.85 and pH > 4.6 require a microbial challenge test to validate their safety for consumption (IFT/FDA, 2003). Food products with physical properties of {aw > 0.93 and pH > 5.0} or {aw > 0.93 and pH > 5.5} require challenge tests to ensure the products do not support the rapid and progressive growth specifically of the pathogenic endospore-formers Bacillus cereus or Clostridium perfringens, respectively.

Water activity (aw) and pH are important parameters in food preservation, stabilization, and processing for preventing or limiting the growth of microorganisms, including molds, fungi and bacteria, as well as growth from bacterial spores, which can be infectious or have deleterious effects on food quality and safety (Fontana et al., 2008, Troller and Christian, 1978, Feeherry et al., 2003, Taub et al., 2003, Gulati et al., 2015). As a physical measure of the free water available to microorganisms and for chemical reactions in a food system, aw is defined as the ratio of the water vapor pressure of a substance to the vapor pressure of pure water at the same temperature. Examples of aw levels that control microorganisms in foods include: aw = 0.950 controls Pseudomonas, Shigella, Bacillus, and Clostridium perfringens in highly perishable foods – canned and fresh fruits; aw = 0.910 controls Salmonella, Vibrio parahaemolyticus, Lactobacillus and Clostridium botulinum in cured ham, or Cheddar, Swiss, Muenster, or Provolone cheeses; and aw = 0.870 controls many yeasts in fermented sausage, dry cheeses, sponge cakes; and aw = 0.800 controls most molds in fruits juice concentrates, chocolate and maple syrups, country style ham, and high-sugar cakes (adapted from Beuchat, 1981). According to international and national standards, foods with aw ≤ 0.85 and pH ≤ 4.6 are rendered non-potentially hazardous and do not require refrigeration to maintain safety from the rapid and progressive growth of infectious or toxigenic microorganisms. Foods with any component(s) having aw > 0.85 and pH > 4.6 require a microbial challenge test to validate their safety for consumption (IFT/FDA, 2003). Food products with physical properties of {aw > 0.93 and pH > 5.0} or {aw > 0.93 and pH > 5.5} require challenge tests to ensure the products do not support the rapid and progressive growth specifically of the pathogenic endospore-formers Bacillus cereus or Clostridium perfringens, respectively.

Low-water activity foods (aw < 0.6) can occur naturally or be dried deliberately, and include cereals, chocolate, cocoa powder, dried fruits and vegetables, egg powder, fermented dry sausage, flour, meal and grits, herbs, spices and condiments, honey, hydrolyzed vegetable protein powder, meat powders, dried meat, milk powder, pasta, peanut butter, peanuts and tree nuts, powdered infant formula, grains, and seeds (Beuchat et al., 2013). Recent recalls and foodborne illnesses associated with low aw foods (Salmonella in spices, dry nuts, chocolate, and peanut butter; Cronobacter in powdered infant formula; Clostridium botulinum in honey; and Bacillus cereus in rice cereal) have increased public concern for the safety of these foods, such that viable pathogenic microorganisms may not grow but may survive and persist for extended periods (Syamaladevi et al., 2016). These target pathogens may require additional processing and validation steps with low aw foods, to ensure food safety, with particular consideration of the influence of low aw on the thermal resistance of these and other microorganisms (Syamaladevi et al., 2016). With spores, for instance, lowering aw from 0.9 to 0.2 is known to increase the thermal resistance of Geobacillus stearothermophilus and C. botulinum spores at temperature T = 110 °C (Murrell and Scott, 1966). Similarly, lowering aw in the region of 1.0–0.9 with NaCl or sucrose increases the resistance of B. amyloliquefaciens spores to inactivation by heat (T = 105 and 115 °C) or to high pressure (HP) processing treatments that combine HP (P = 600 MPa (MPa)) with elevated heat (T = 105 and 115 °C), presumptively by retarding dipicolinic acid (DPA) release through interactions of the humectant molecules with the spore inner membrane (Sevenich et al., 2015).

Bacillus cereus is a gram-positive, facultative anaerobic rod-shaped endospore-forming bacterium found ubiquitously in soil and in many raw and processed foods, such as rice, vegetables, milk and dairy products, and spices. Food poisoning with B. cereus has been associated with rice, meats, sauces, desserts, rice, cereal grains and related products (pasta, focaccia); fats, oils and salad dressings; and milk and milk products (Beuchat et al., 2013). B. cereus infections occur as two types of gastrointestinal disorders: the emetic syndrome, which is characterized by vomiting and caused by ingestion of heat-stable toxin, which is usually pre-formed in starchy foods (cakes, pasta, cooked rice). The diarrhoeal syndrome is caused by a diarrheagenic toxin that can be formed in the food or in the small intestine. There are concerns of B. cereus contamination in pasteurized, refrigerated foods that may contain viable spores that can germinate and outgrow during storage, even at low temperatures (de Vries et al., 2004, Guinebretiere et al., 2003, Choma et al., 2000, Carlin et al., 2000). B cereus spores can also survive in dry foods such as rice cereal and in dry food processing environments for long periods of time and can germinate and grow in reconstituted products that are not properly processed or stored. Wet processing of dry foods (cereals) can also introduce conditions for growth and production of heat-stable toxins. While infection with B. cereus usually produces mild symptoms, a B. cereus-associated food poisoning outbreak from the consumption of pasta salad demonstrates the potential severity of the emetic syndrome and importance of determining factors for controlling B. cereus in foods for public health (Dierick et al., 2005).

The nonthermal technology of HP at high temperatures has also been used at P ≤ 600 MPa, and temperatures of ≤60 °C for 30 min in the study of the germination and inactivation of B. cereus spores (Van Opstal et al., 2004, Wei et al., 2009, Ju et al., 2008). If germination and outgrowth are not adequately controlled during food storage, HP treatments will have to eliminate B. cereus spores from foods, to ensure safety. The HP inactivation kinetics of B. cereus have also been studied under HP – high temperature conditions (H Luu-Thi et al., 2014, Daryaei et al., 2013). An important aspect to consider is that spores of Bacillus cereus are known to become more resistant to germination and inactivation by HP as aw decreases (Al-Holy et al., 2007). Specifically, lowering aw from 0.99 to 0.92 with sucrose inhibits germination of B. cereus spores by HP (250 MPa, 25 °C, 15 min) for spores made at temperatures of 37, 30, or 20 °C (there was no inactivation at these HPP conditions). Similarly, lowering aw to 0.92 reduces germination of B. cereus spores by 3–5 logs and prevents 1–3 logs of inactivation that occurred at aw ∼0.99 with HP conditions of 690 MPa, 40 °C for 2 min (Raso et al., 1998). Together, these factors show the importance of determining mechanisms of spore resistance and germination, particularly as they relate to processing foods with low aw.

Spore germination in Bacillus species is normally initiated by a variety of nutrient germinants; these include specific amino acids, sugars and purine nucleosides, presumably molecules that indicate the environment is favorable for growth of a particular organism (Setlow, 2013). The different nutrient germinants trigger spore germination by activating one or more germinant receptors (GRs) located in spores’ inner membrane (IM) (Griffiths et al., 2011, Hudson et al., 2001, Paidhungat and Setlow, 2001). In Bacillus subtilis, spore germination is triggered by l-alanine or l-valine activating the GerA GR, or a mixture of l-asparagine, d-glucose, d-fructose, and KCl (AGFK) simultaneously activating both the GerB and GerK GRs (Setlow, 2013). Normally, l-asparagine alone does not trigger B. subtilis spore germination. However, a mutant form of GerB, termed GerB*, can be activated by l-asparagine alone (Atluri et al., 2006, Paidhungat and Setlow, 1999). Bacillus megaterium and Bacillus cereus spores also have multiple IM GRs, and their GR-dependent germinants include d-glucose and KBr for B. megaterium spores, and l-alanine and inosine for B. cereus spores (Abee et al., 2011, Gupta et al., 2013).

A number of events occur in a defined sequence during Bacillus spore germination. First, after mixing spores with germinants, the germinants must pass through spores' outer layers including the outer membrane (OM) to reach the IM where GRs are located. This germinant permeation is likely facilitated by GerP proteins located in spores' outer layers (Behravan et al., 2000, Butzin et al., 2012). Following germinant-GR interaction, spores can become committed to germinate, such that germination proceeds even if the germinants are removed or further GR-germinant interaction is blocked (Setlow, 2013, Yi and Setlow, 2010). Subsequent events in germination take place in two stages. In Stage I the germination signal leading to commitment is transduced and amplified in some manner (Setlow, 2013, Setlow, 2014), first leading to release of H+, Na+, and K+ and then the spores' large depot of pyridine-2,6-dicarboxylic acid (dipicolinic acid, abbreviated DPA), all present in the spore core. DPA is present as a 1:1 chelate with divalent cations, primarily Ca2+ (CaDPA), and CaDPA comprises ∼ 25% of the spore core dry wt (Setlow, 2013, Setlow, 2014). CaDPA is released via specific channels in spores IM composed largely or perhaps completely of multiple SpoVA proteins (Setlow, 2013, Setlow, 2014). Replacement of the released CaDPA by water raises spore core water content somewhat, and completes Stage I of germination. Stage II of germination can be triggered by completion of Stage I, as CaDPA release leads to the activation of cortex lytic enzymes (CLEs) that initiate the degradation of spores’ large peptidoglycan (PG) cortex that likely is important in maintaining the low water content in the dormant spore core. Bacillus spores have two redundant CLEs, CwlJ and SleB. CwlJ is likely activated by CaDPA released from the spore core and by exogenous CaDPA, while changes in cortex structure caused by CaDPA release in Stage I may allow SleB to now act on the cortex PG (Setlow, 2013, Setlow, 2014). Either or both of these two CLEs then degrade the cortex PG, allowing core expansion and water uptake to levels found in growing cells and completion of germination.

In addition to nutrient germinants that act via IM GRs, Bacillus spore germination can also be initiated by several non-nutrient agents, including: (i) dodecylamine, that activates a specific SpoVA protein in the CaDPA channel and triggers CaDPA release; this release then triggers Stage II of germination as described above (Setlow, 2013, Setlow, 2014, Velásquez et al., 2014); (ii) exogenous CaDPA, that activates the CLE CwlJ triggering cortex PG degradation, which then triggers CaDPA release from the spore core through the IM CaDPA channels (Paidhungat et al., 2001); and (iii) HP treatment, that activates GRs at 100–200 MPa or opens the SpoVA protein CaDPA channel at ≥ 400 MPa (Reineke et al., 2013). Notably, HP is increasingly used in the food industry to generate pasteurized, refrigerated foods, although has not yet been implemented commercially for the production of shelf stable foods (Knorr et al., 2011, Balasubramaniam et al., 2015). HP is also a chemical-free method to inactivate spores of the bio-terrorism agent Bacillus anthracis (Cléry-Barraud et al., 2004), which is closely related to B. cereus.

As mentioned above, a variety of reports have indicated that lowering aw prevents the growth of bacteria from spores presumably by inhibiting spore germination and outgrowth. Generally, a lower aw is required for inhibition of spore germination than for inhibition of the outgrowth of germinated spores (Baird-Parker and Freame, 1967, Gould, 1964, Hagen et al., 1967, Jakobsen et al., 1972, Smoot and Pierson, 1982), but how low aw inhibits spore germination is not clear. Since germination is an early and essential event in the return to life of bacterial spores, determining how aw inhibits spore germination by various stimuli (e.g., nutrients, HP, etc) could provide information that might be helpful in designing processing regimens to ensure food safety and improve food quality. Given the multiple events occurring during spore germination and the different ways in which germination can be triggered, the obvious question is what specific event(s) in germination are inhibited by lowering aw? In this paper, we report the results of experiments to determine the effects of aw on germination of spores of three Bacillus species, and by both GR-dependent and GR-independent germinants, including HP. The effects of aw on some individual events in GR-dependent germination have also been determined.

Section snippets

Bacterial strains and spore preparation and purification

The B. subtilis strains used are isogenic derivatives of a laboratory 168 strain and are: (i) PS533 (wild-type), carrying plasmid pUB110 encoding resistance to kanamycin (Kmr, 10 μg/ml) (Setlow and Setlow, 1996) – this antibiotic resistant strain was used as the wild-type to allow its easy discrimination on plates in a lab with large numbers of spores of different B. subtilis strains; (ii) FB10 (gerB*), with a mutation in the gerB operon, termed gerB*, such that the GerB* GR triggers spore

Effects of aw on GR-dependent spore germination with low mol wt germinants

Previous work has found that lowering the aw of germination incubations can inhibit bacterial spore germination, with different solutes used to adjust aw exhibiting different inhibition efficiencies (Anagnostopoulos and Sidhu, 1981, Jakobsen et al., 1972). With B. subtilis wild-type spores, germination with l-valine was strongly inhibited by increasing concentrations of different humectants used to decrease the aw of germination incubations (Fig. 1A–C). To precisely examine the effect of aw on

Discussion

As has been reported by others, decreasing aw decreases rates of bacterial spore germination, and this was found in the current work, and that different humectants have different efficiencies in reducing spore germination rates. This was seen for GR-dependent germination via different GRs from the same Bacillus species, and with different Bacillus species. In general, the effect of aw due to different humectants was sucrose > trehalose > glycerol, as found previously for sucrose and glycerol (

Acknowledgements

L. Rao was supported by a fellowship from the China Scholarship Council (CSC) (201506350152). The efforts of P. Setlow were supported by a grant from the U.S. Department of Defense | United States Army | RDECOM | Army Research Office (ARO) (W911NF-16-1-0024).

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