Effect of Temperature, pH, and aw on Cereulide Synthesis and Regulator Genes Transcription with Respect to Bacillus cereus Growth and Cereulide Production

Bacillus cereus is a food-borne pathogen that can produce cereulide in the growth period, which causes food poisoning symptoms. Due to its resistance to heat, extreme pH, and proteolytic enzymes, cereulide poses a serious threat to food safety. Temperature, pH, and aw can influence cereulide production, but there is still a lack of research with multi-environmental impacts. In this study, the effects of temperature (15~45 °C), pH (5~8), and aw (0.945~0.996) on the emetic reference strain B. cereus F4810/72 growth, cereulide production, relevant ces genes (cesA, cesB, cesP), and transcription regulators genes (codY and abrB) expression at transcription level were studied. B. cereus survived for 4~53 h or grew to 6.85~8.15 log10 CFU/mL in environmental combinations. Cereulide accumulation was higher in mid-temperature, acidic, or high aw environments. Increased temperature resulted in a lower cereulide concentration at pH 8 or aw of 0.970. The lowest cereulide concentration was found at pH 6.5 with an increased aw from 0.970 to 0.996. Water activity had a strong effect on transcriptional regulator genes as well as the cesB gene, and temperature was the main effect factor of cesP gene expression. Moreover, environmental factors also impact cereulide synthesis at transcriptional levels thereby altering the cereulide concentrations. The interaction of environmental factors may result in the survival of B. cereus without growth for a period. Gene expression is affected by environmental factors, and temperature and pH may be the main factors influencing the correlation between B. cereus growth and cereulide formation. This study contributed to an initial understanding of the intrinsic link between the impact of environmental factors and cereulide formation and provided valuable information for clarifying the mechanism of cereulide synthesis in combined environmental conditions.


Introduction
Bacillus cereus is a Gram-positive and facultative anaerobic foodborne pathogen that can pose a serious threat to food safety [1,2].Approximately 1.4~12% of foodborne outbreaks worldwide are attributed to B. cereus [3].In Europe, 413 foodborne outbreaks caused by B. cereus were reported between 2007 and 2014 [4].Most outbreaks of foodborne illness Toxins 2024, 16, 32 2 of 14 caused by B. cereus have usually been associated with cell concentrations above 10 5 CFU/g or CFU/mL in foods because B. cereus may produce cereulide (emetic toxin) when bacterial concentrations reach this level [4].Cereulide is extremely stable in thermal, so food heating processing might inactivate the microorganism but would not destroy cereulide that is pre-formed in foods [5].Cereulide can resist proteolytic enzymes in the gastrointestinal environment, be absorbed, and distributed throughout the body, causing severe multi-organ failure or even death after it is taken into the body [6,7].Serious food poisoning outbreaks usually occur worldwide due to the high toxicity and stability of cereulide [8][9][10][11].
Cereulide ([D-O-Leu-D-Ala-L-O-Val-L-Val]3) is a cyclic dodecadepsipeptide (1.2 kDa) encoded by the ces cluster and produced by the activated non-ribosomal peptide synthetase (NRPS) [1].The cereulide gene cluster comprises cesPTABCD operon and cesH.The ce-sPTABCD is transcribed as a polycistronic transcript from the main ces promoter located upstream of cesP and cesH is transcribed from its own promoter, which controls cereulide formation on a transcriptional level [12,13].CesP encodes phosphotransferase to activate NRPS; the structural genes of cesA and cesB can integrate amino acid monomers into polypeptide chains which play an important role in the structure formation of cereulide [1,14].Furthermore, cereulide-NRPS is the result of a complex and multi-layered process and is tightly regulated by the metabolite-responsive transcriptional regulators of AbrB, Spo0A, and CodY [13,15].These regulators may promote or inhibit ces gene expression at the post-transcriptional level, thereby affecting the process of cereulide synthesis [16].Environmental factors, such as temperature, pH value, and water activity (a w ), are usually involved in food processing [17][18][19].Currently, the temperature has been proven to have an impact on cereulide production and the expression of the cereulide synthetase gene [20,21].Nevertheless, there is a notable scarcity of comprehensive investigations covering the (interactions) impact of pH and a w on cereulide production [22][23][24].These limitations hinder the ability to closely simulate real food production scenarios.Therefore, the aim of the paper was to monitor the B. cereus growth in combinations of temperature, pH, and a w with cereulide production.Furthermore, an RT-qPCR assay was included to examine the influence of temperature, pH, and a w on the expression of cereulide synthesis-related genes at a transcriptional level.

Effect of Environments on Emetic B. cereus Behavior and Cereulide Production 2.1.1. B. cereus Survival
Under certain conditions, the growth of B. cereus was not observed, and the number of B. cereus counts gradually decreased to the detected limit (<50 CFU/mL) with an initial inoculation of 2~3 log 10 CFU/mL (Table 1, Group I).Notably, B. cereus had the longest survival time (183 h) at the condition of 45 • C-pH 6.5-a w 0.945, whereas the bacteria survived for the shortest time (approximately 4 h) at 45 • C-pH 5-a w 0.970 (Figure 1).Similarly, the time that B. cereus was maintained without growth in BHI at pH of 5 and a w of 0.970 (53 h) was less than that at pH 6.5-a w 0.945 (125 h) at 15 • C (Figure 1).These findings suggest that the combination of pH 5-a w 0.970 is unfavorable for B. cereus survival and that elevated temperatures may further reduce B. cereus counts in a shorter time.

B. cereus Growth with or without Cereulide Production
Figure 2 shows the growth curves of B. cereus growth under different conditions by fitting the Gompertz model.The estimated parameters of the maximum population (Y max ) obtained from it are summarized in Table 1 (Group II and Group III).The adj.R 2 was in the range of 0.946~0.995 in each condition, showing the goodness-of-fit of this model.The Y max was in the range of 6.85~8.15log 10 CFU/mL at the combination effects of temperature, pH, and a w .The highest Y max was obtained at 30 • C, pH of 8, and a w of 0.996, while the lowest Y max was obtained at 15 • C, pH of 8, and a w of 0.970.It was suggested that a decrease in temperature and a w would result in a decrease in Y max and would not favor B. cereus growth.The Y max was around 7 log 10 CFU/mL for other combinations with a slight difference.Meanwhile, as depicted in Figure 2a, it is evident that cereulide is usually produced in the mid to late exponential phase and gradually increases as B. cereus grows.The rate of cereulide production will gradually slow down or even tend to stabilize when B. cereus enters the growth stationary phase.However, no cereulide (<0.1 ng/g) was detected at the conditions combined with 45 • C, even though B. cereus grew to 7.05~7.16log 10 CFU/mL (Figure 2b).
The concentrations of cereulide in the stationary phase of B. cereus growth under each condition are summarized in Table 1 (Group II).The maximum cereulide concentrations were 367 ng/g, observed at the condition of T:30 • C-pH 8-a w 0.996, followed by 125 ng/g at the condition of T:30 • C-pH 5-a w 0.996, and the minimum cereulide concentrations observed at the condition of T:15 • C-pH 8-aw 0.970 were just 3 ng/g.Cereulide concentrations were 119.16 ng/g at 15 • C, while no toxin was detected at 45 • C, indicating that higher temperatures were not conducive to cereulide production.Cereulide concentrations at pH 5 (78.05 ng/g) were higher than those at pH 8, suggesting that acidic conditions favored cereulide production.Similarly, cereulide concentrations at a w 0.996 (130.95ng/g) were higher than those at a w 0.945, indicating that reduced a w was disadvantageous for cereulide production.peratures coupled with increased water activity resulted in higher cereulide concentrations.Furthermore, cereulide concentrations at pH 8-aw 0.996 were 132.49ng/g higher than at pH 6.5-aw 0.970, and concentrations at pH 5-aw 0.996 were 210.54 ng/g higher than at pH 6.5-aw 0.970.It was indicated that the least favorable conditions for cereulide production were at pH 6.5 when aw increased from 0.970 to 0.996, and that increased acidity led to higher cereulide concentrations.In addition, both effects of factors can also be analyzed from Table 1.The condition of 15 • C-pH 6.5 reduced the cereulide concentration by 101.57ng/g compared to the condition of 30 • C-pH 5 at a w of 0.996, while the condition of 15 • C-pH 8 reduced the concentration by 7.04 ng/g compared to the condition of 30 • C-pH 6.5 at a w of 0.970.It was indicated that cereulide concentration increased when the temperature increased with the pH decreased in the a w range of 0.970~0.996.And decreased pH had a greater impact on cereulide concentration changes in acidic conditions.Cereulide concentrations increased by 139.53 ng/g when a w increased from 0.970 to 0.996, while concentrations increased by 8.58 ng/g with a w decreased from 0.970 to 0.945 when the temperature increased from 15 to 30 • C.And the lowest concentration was found at a w 0.970.It was suggested that higher temperatures coupled with increased water activity resulted in higher cereulide concentrations.Furthermore, cereulide concentrations at pH 8-a w 0.996 were 132.49ng/g higher than at pH 6.5-a w 0.970, and concentrations at pH 5-a w 0.996 were 210.54 ng/g higher than at pH 6.5-a w 0.970.It was indicated that the least favorable conditions for cereulide production were at pH 6.5 when a w increased from 0.970 to 0.996, and that increased acidity led to higher cereulide concentrations.

Effect of a Single Factor
Compared to 15 • C, the mRNA levels of cesA and cesB expression increased 0.16-fold and 0.6-fold at 30 • C, respectively, while they were down-regulated 0.21-fold and 0.39-fold at 45 • C, respectively (Figure 3a).This suggests a temperature-dependent modulation of cesA and cesB expression, initially enhanced and subsequently suppressed with increasing temperature.Conversely, the expression of the cesP gene showed a 0.32-fold reduction at 30 • C and a significant 0.78-fold reduction at 45 • C (Figure 3a), indicating suppression of gene expression with elevated temperatures.The transcriptional regulator genes, codY and abrB, were significantly down-regulated 0.72-fold and 0.83-fold at 30 • C, respectively.At 45 • C, codY was up-regulated 0.75-fold, while abrB was significantly down-regulated 0.73-fold.Both codY and abrB exhibited suppression when the temperature increased from 15 • C to 30 • C, with abrB showing a greater inhibition of gene expression.While the expression of the codY gene increased from 30 • C to 45 • C, the abrB gene was less affected but still up-regulated (Figure 3a).This indicates that the mRNA expression of both genes decreased and then increased with the rising temperature from 15 to 45 • C, reaching the lowest expression at 30 • C.
Compared to the pH of 6.5, all tested genes exhibited significant up-regulation, ranging from 3.28-to 15.23-fold at pH 5. Notably, the relative mRNA levels expressed by cesA, cesB, and cesP (6.32-15.23-fold)surpassed those of codY and abrB (3.28~4.71-fold)(Figure 3b), and a lower pH environment was more conducive to the expression of ces genes.Among these, cesP demonstrated the most significant up-regulation (14.23 folds) within the ces gene cluster (Figure 3b).CesA, cesB, codY, and abrB genes experienced a 0.65-0.87-folddown-regulation, while cesP was significantly up-regulated (3.37-fold) at pH 8 (Figure 3b).This suggests that the pH range from 6.5 to 8 had a more substantial effect on cesP gene transcription, but other toxin synthesis-related genes were less affected.Furthermore, the mRNA levels expressed by cesP fluctuated with changes in pH, being lowest at a pH of 6.5, indicating that the acidic environment favored cesP gene expression.
Compared to a w of 0.996, cesA, cesB, cesP, codY, and abrB were significantly upregulated: 1.64-, 5.25-, 3.07-, 1.19-, and 14.92-fold at a w 0.970, respectively, with the abrB exhibiting the highest mRNA level.At a w of 0.945, all genes were significantly up-regulated 1.60-, 5.12-, 1.61-, 29.76-, and 14.21-fold, respectively, with the highest mRNA level observed for codY (Figure 3c).The mRNA expression levels of cesA, cesB, and abrB increased and then stabilized as a w gradually decreased.The mRNA expression levels of cesP followed a pattern of increase followed by a decrease, peaking at a w of 0.970.The a w of 0.945 had the most pronounced effect on codY expression.

Effects of Two Factors
The cesA and cesB up-regulated 0.17 and 0.60-fold at 30 • C, respectively (Figure 3a).At pH 5, the cesA and cesB significantly up-regulated 5.32 and 9.67-fold, respectively (Figure 3b).Simultaneous exposure to temperature and pH conditions led to the upregulation of cesA and cesB: 1.55 and 2.71-fold, respectively, at 30 • C-pH 5 compared to 15 • C-pH 6.5 (Figure 4a).The combined impact of temperature and pH on the mRNA expression levels of the cesA and cesB genes was intermediate between the individual effects, suggesting a greater combined effect of temperature and pH on these genes compared to either factor alone.The relative mRNA levels of the cesP, codY, and abrB (0.62~1.75-fold up-regulated) were higher at 30 • C-pH 5 than at 15 • C-pH 6.5 (Figure 4a).In addition, these genes were significantly up-regulated at pH 5 alone while down-regulated at 30 • C alone (Figure 3a,b), indicating that the enhancement of their expression due to reduced pH outweighed the inhibition caused by increased temperature.exhibiting the highest mRNA level.At aw of 0.945, all genes were significantly up-regulated 1.60-, 5.12-, 1.61-, 29.76-, and 14.21-fold, respectively, with the highest mRNA level observed for codY (Figure 3c).The mRNA expression levels of cesA, cesB, and abrB increased and then stabilized as aw gradually decreased.The mRNA expression levels of cesP followed a pattern of increase followed by a decrease, peaking at aw of 0.970.The aw of 0.945 had the most pronounced effect on codY expression.

Effects of Two Factors
The cesA and cesB up-regulated 0.17 and 0.60-fold at 30 °C, respectively (Figure 3a).At pH 5, the cesA and cesB significantly up-regulated 5.32 and 9.67-fold, respectively (Figure 3b).Simultaneous exposure to temperature and pH conditions led to the up-regulation of cesA and cesB: 1.55 and 2.71-fold, respectively, at 30 °C-pH 5 compared to 15 °C-pH 6.5 (Figure 4a).The combined impact of temperature and pH on the mRNA expression levels of the cesA and cesB genes was intermediate between the individual effects, suggesting a greater combined effect of temperature and pH on these genes compared to either factor alone.The relative mRNA levels of the cesP, codY, and abrB (0.62~1.75-fold up-regulated) were higher at 30 °C-pH 5 than at 15 °C-pH 6.5 (Figure 4a).In addition, these genes When water activity and temperature acted simultaneously on cesA and cesB expression (Figure 4b), both genes up-regulated 1.75 and 7.15-fold at 30 • C-a w 0.945, respectively.Notably, the combination of water activity and temperature resulted in a higher mRNA expression level for cesB compared to each factor alone, suggesting a synergistic promotion of gene expression.At 30 • C-a w 0.945, cesP, codY, and abrB significantly up-regulated 1.52, 10.90, and 7.78-fold, respectively (Figure 4b).Comparatively, the expression of these genes increased when influenced by both water activity and temperature, indicating that the reduction in water activity had a more substantial impact on gene expression than the effect of increased temperature alone.
At pH 6.5-a w 0.945, cesB, codY, and abrB significantly up-regulated 1.26, 5.47, and 4.59-fold, whereas the mRNA levels of the cesA and cesP genes were less changed compared to pH 5-a w 0.996 (Figure 4c). Figure 3b illustrates that the pH effect alone significantly decreased transcription of all genes in the increased pH from 6.5 to 5. Conversely, a decrease in water activity (from 0.996 to 0.945) alone resulted in a significant up-regulation of all genes (Figure 3c).When both factors acted on the cesA and cesP genes, the inhibitory effect from the increase in pH may have neutralized the enhancement effect from the decrease in water activity, resulting in no significant change in the expression of both.For cesB, codY, and abrB expression, the impact of water activity may be greater than the effect of pH, resulting in the up-regulation of these genes.When water activity and temperature acted simultaneously on cesA and cesB expression (Figure 4b), both genes up-regulated 1.75 and 7.15-fold at 30 °C-aw 0.945, respectively.Notably, the combination of water activity and temperature resulted in a higher mRNA expression level for cesB compared to each factor alone, suggesting a synergistic promotion of gene expression.At 30° C-aw 0.945, cesP, codY, and abrB significantly up-regulated 1.52, 10.90, and 7.78-fold, respectively (Figure 4b).Comparatively, the expression of these genes increased when influenced by both water activity and temperature, indicating that the reduction in water activity had a more substantial impact on gene expression than the effect of increased temperature alone.
At pH 6.5-aw 0.945, cesB, codY, and abrB significantly up-regulated 1.26, 5.47, and 4.59fold, whereas the mRNA levels of the cesA and cesP genes were less changed compared to pH 5-aw 0.996 (Figure 4c). Figure 3b illustrates that the pH effect alone significantly  The emetic B. cereus F4810/72 strain exhibits growth across a temperature range of 15~45 • C, pH levels of 5~8, and a w of 0.945~0.996(NaCl concentrations of 0.5~8%) [25,26].However, diverse environmental factors within this range may hinder B. cereus growth, allowing for substrate survival.Martínez et al. [27] demonstrated that B. cereus can endure without growth for 50 days in nutrient broth at 12-40 • C with a salt concentration exceeding 1% and a pH of 4.5.Similarly, in our result (Figure 1), B. cereus survived for 4~53 h at 15~45 • C with the pH of 5~6.5 and a w of 0.945~0.970(corresponding salt concentration of 4.25~8%).Increased salt concentration may impede B. cereus survival due to reduced water activity, limiting water availability for bacterial metabolism and consequently reducing survival time [28].Survival of B. cereus may have the potential for further growth [29] and even result in cereulide production if the counts are over 5 log 10 CFU/mL (Figure 2).Therefore, minimizing B. cereus survival time under appropriate conditions is crucial to reduce the risk of growth and subsequent cereulide production.
In most conditions, the growth of B. cereus accompanied by cereulide production was observed across various temperature, pH, and a w combinations (Figure 2a).The optimal condition for cereulide production was found at 30 • C, pH 5, and a w 0.996 (Table 1), which is within the typical pH and water activity range of most foods.Therefore, prioritizing temperature control is essential to manage cereulide production.At 45 • C, temperature becomes dominant, potentially rendering cereulide undetectable (Figure 2b), aligning with previous studies [20,21].Elevating the temperature to 45 • C was suggested to reduce the risk of cereulide production by B. cereus.Regarding the effect of pH on cereulide formation, our results indicated a higher cereulide production at pH 5 compared to pH 8 (Table 1).A similar phenomenon has been observed in acidic foods (pH 5.8) with higher concentrations of cereulide than alkaline foods (pH 7.9) [22].However, this trend may not be universal for all cereulide-producing B. cereus.Guérin et al. [30] found that, when the pH decreased from 7 to 5.4, the cereulide concentration produced by B. weihenstephanensis (belonging to the B. cereus phylogenetic group) decreased.This may be due to the individual differences between different B. cereus.groups.In addition, our results indicated that cereulide concentrations decreased by 130.95 ng/g when the a w was reduced from 0.996 to 0.945 (Table 1).Dommel et al. [24] also showed that the cereulide concentration decreased when the NaCl concentration increased.The increase in NaCl concentration led to a decrease in the available water in the environment and also adversely affected the B. cereus growth (Table 1).Therefore, cereulide produced in the growth phase may be inhibited in low a w environments resulting in a reduction in cereulide concentration.Above all, mid-temperatures, acidic, or high a w environments may result in higher cereulide concentrations.It is worth noting that temperature may act as a major factor influencing cereulide production at higher temperatures and the risk of cereulide formation may be lower at 45 • C.
In addition, we also explored the multifactorial situations that reduced cereulide concentration.The increased temperature with an increase in pH decreased cereulide concentration.Furthermore, the lowest concentration was found at an a w of 0.970 at increased temperatures.When a w increased from 0.970 to 0.996, a pH of 6.5 was the least favorable condition for cereulide production.These provided the reference for the environmental prevention and control of cereulide.

The Effect of Temperature, pH, and a w on Cereulide-Related Gene Expression
The ces operon (cesPTABCD) controls cereulide formation and is expressed during B. cereus growth [13,30].Environmental factors have been shown to influence gene expression at the transcriptional level.The cesA and cesB are structural genes, encoding the cereulide NRPS, which are responsible for the D-Ala-D-O-Leu fragment and L-Val-L-O-Val fragment, respectively [12].The cesA and cesB were often significantly up-regulated by the decreased pH (from 6.5~5) or a w (0.996 to 0.945) (Figure 3b,c), indicating that these conditions were favorable for the formation of D-Ala-D-O-Leu and L-Val-L-O-Val fragments.Dommel et al. [24] found that the relative expression of the cesA decreased with decreasing a w during the exponential growth phase of B. cereus, whereas the present study found that the relative expression of this gene increased with decreasing in a w in the stationary growth phase, suggesting that the expression of cesA may not be consistently suppressed by increasing salt concentration during the B. cereus growth cycle.In addition, we found that the maximum relative expression of cesA and cesB occurred at 30 • C, which might be the optimal temperature for the formation of cereulide structures, which was consistent with the trend of results from Kranzler et al. [21].The cesP gene encodes a phosphopantetheinyl transferase to activate NRPS [1].And the cesP 1 promoter is located upstream of cesP and drives the transcription of the cesPTABCD operon [22].Currently, changes in the expression of this gene as influenced by environmental factors are unknown.Our study found that the increased temperature from 15 • C to 45 • C or pH of 6.5 was detrimental to cesP expression (Figure 3a,b), thereby inhibiting the synthesis of the phosphotransferase and affecting the transcription of ces operon.While a w of 0.970 may have a positive effect on the cereulide biosynthesis process due to the transcription level of cesP being enhanced.Therefore, reduced pH or a w favored the formation of the cereulide structure, and the most optimal temperature was 30 • C. pH and a w had opposite trends on cesP expression, and increased temperature was detrimental to NRPS activation.
Transcriptional regulators are also involved in the process of ces expression [12].CodY, a global transcriptional regulator, is linked to the metabolic state and virulence expression of the bacterial cell [31].In B. cereus, CodY acts as a repressor of the ces operon, thereby diminishing the cereulide formation [32].Another transcriptional regulator, AbrB, negatively influences cereulide synthesis by binding to the promoter of the cesP gene and inhibiting its expression [15].The mRNA levels of codY and abrB were at their lowest at 30 • C (Figure 3a), thereby reducing their inhibitory impact on cereulide biosynthesis at the transcriptional level.In contrast, the expression of codY and abrB was significantly up-regulated in environments with pH 5 or a w 0.945 (Figure 3b,c), suggesting that these conditions are conducive to the synthesis of CodY and AbrB.Consequently, their heightened presence enhances the negative regulation of the cereulide formation process.Therefore, the negative effect of transcription factors on cereulide synthesis was minimal at 30 • C. While the negative regulation increased progressively with decreasing pH or a w , thus unfavorably affecting cereulide formation.
Furthermore, the impact of temperature and pH, or temperature and a w , on the expression of cesA and cesB does not appear to follow a single factor.There may be interactive effects between these factors, giving rise to either antagonistic or synergistic effects on gene expression.Consequently, the mRNA expression levels may deviate from a mere summation of individual effects.Notably, the influence of water activity on cesB expression surpassed that of pH, whereas both factors exhibited similar effects on the expression of cesA and cesP.Temperature emerged as a pivotal factor governing cesP expression.Interestingly, water activity had the greatest effect on the expression of transcriptional regulator genes of codY and abrB, followed by pH, with temperature exerting the least effect.

Relationship between B. cereus Growth and Cereulide Production
The observed lack of strict correlation between cereulide formation and B. cereus growth may be attributed to extrinsic environmental factors playing a direct role in the expression of cereulide synthetase genes, which agreed with the findings by Kranzler et al. [21] and Dommel et al. [30].In our study, the Y max of B. cereus could reach 7 log 10 CFU/mL at 45 • C, which was not much different from that at 15 • C. Despite this, cereulide was not detected, possibly due to the down-regulation of cesA, cesB, and cesP genes at 45 • C, as illustrated in Figure 3a.This down-regulation inhibits cereulide synthesis by affecting both the formation of the cereulide structure and the activation of cereulide synthase.Meanwhile, the substantial up-regulation of the codY may increase the inhibitory effect of the transcription factor CodY on the process of cereulide synthesis.In addition, the effect of the negative regulation of abrB on cereulide production may be less than that of other genes because no toxins were detected at 45 • C.Besides temperature, pH also contributed to the differences in B. cereus growth and cereulide formation.Table 1 shows that a pH value of 5 was unfavorable for B. cereus growth but favorable for cereulide production.The promotion of cereulide synthesis, as evidenced by higher relative mRNA levels of cesA, cesB, and cesP compared to codY and abrB, likely contributed to the increase in final cereulide concentration under the former condition.Above all, temperature and pH are important factors that may affect the correlation between B. cereus growth and cereulide formation.
To the best of our knowledge, this study represents the inaugural investigation into cereulide production under the influence of diverse environmental factors and its consequential impact on B. cereus gene expression at the transcriptional level.Gaining insight into the influence of extracellular factors on cereulide synthesis is crucial for delineating inhibitory conditions, thereby facilitating the formulation of appropriate measures for preventing and controlling cereulide.Additionally, this paper analyzed the pattern of gene expression associated with cereulide synthesis in response to environmental factors, providing an initial understanding of the mechanism governing cereulide synthesis in combined environmental conditions.Nonetheless, the pathways through which B. cereus cells perceive environmental signals and the mechanisms orchestrating the integration of these signals into physiological metabolism to activate the cereulide biosynthesis process remain unclear.Further elucidation of these aspects would contribute to a deeper understanding of the cereulide biosynthesis pathway in B. cereus under environmental influences.

Conclusions
B. cereus exhibits the capability to endure without active growth in the culture medium over a specific duration, contingent upon the interplay of temperature, pH, and water activity (a w ).The association between B. cereus growth and cereulide production is noteworthy; however, a precise correlation may not always exist.This divergence may be attributed to the direct influence of environmental conditions on the transcriptional activity of B. cereus cereulide-related genes, potentially resulting in variations in cereulide concentration.It is imperative to recognize that diverse environmental factors exert distinct impacts on gene expression, and the degree of influence varies among different genes.Notably, temperature emerges as a key determinant governing cesP gene expression.Furthermore, water activity significantly influences the expression of transcriptional regulator genes during cereulide synthesis, with temperature exhibiting a comparatively lesser impact.The interaction of these environmental factors and their potential effects on gene expression adds complexity to our understanding of the regulatory mechanisms underlying B. cereus behavior in specific conditions.

Preparation of Stock Culture
The standard emetic strain of B. cereus F4810/72 was used in this study (purchased from DSMZ, Braunschweig, Germany).The strain stored in 50% glycerin at −80 • C was inoculated in 10 mL Brain Heart Infusion (BHI) broth (Hopebiol, Qingdao, China) and incubated for 20 h at 30 • C.Then, the culture was enumerated on Tryptone Soya Agar (TSA) plates (Hopebiol, Qingdao, China) and incubated for 20 h at 30 • C. One isolated colony was inoculated into BHI broth and incubated for 18 h at 30 • C to have a working culture.To prove the absence of spores, the culture was heated at 80 • C for 10 min and spread on TSA plates.After overnight incubation, there was no colony growth, and no spores were observed in the culture by a microscope, indicating that there were no spores in the initial bacterial culture [33].

Experimental Design
The experimental ranges were determined according to the single-factor growth range of emetic B. cereus strain F4810/72, three common factors in food processing, temperature of 15~45 • C, pH of 5~8, and a w of 0.996~0.945(conversed from 0.5~8% NaCl) were selected to explore the growth of B. cereus under multiple factors [24,25], and the experimental groups were designed by Box-Behnken design [34].Additional conditions of 30 • C-pH 6.5-a w 0.996 and 30 • C-pH 6.5-a w 0.945 were supplemented in gene transcription results.

Samples Inoculation and Growth Quantification
A conical flask (200 mL volume) containing 120 mL of BHI broth was adjusted to the desired a w and pH with NaCl and HCl/NaOH, respectively.The conversion relationship between NaCl concentration and a w was referenced in the study of Carlin et al. [22].BHI broth (200 mL volume) was supplemented with the desired NaCl so that the NaCl concentration of the final solution was 0.5%, 4.2%, and 8% (w/v).The a w of 0.945, 0.970, and 0.996 correspond to NaCl concentrations of 8%, 4.2%, and 0.5% (w/v), respectively.Then, it was inoculated with an overnight culture of B. cereus F4810/72 to reach an initial target concentration of about 10 2 ~10 3 CFU/mL vegetative cells.The inoculum was incubated in tested conditions.Samples were taken at intervals depending on the preliminary experiments to ensure at least 1 to 2 observations in each logarithmic level of the bacterial population during the growth of B. cereus.Test samples were serially diluted 10-fold in saline and 60 µL were transferred for drop and spread plating three times on TSA plates and then incubated for colony counting.Three countable drops were used to calculate the average of the total number of colonies [35].Three biologically independent replicates were performed for each condition.

Growth Modelling
The growth curves of B. cereus under different conditions were fitted by the Gompertz model using the GraphPad Prism version 8.3 (La Jolla, CA, USA, 2019), presented in Equation ( 1) [36].The maximum population (Y max ) was obtained from the bacterial growth curves to compare the ability to grow in different environmental conditions.If the increase in B. cereus counts did not exceed 2 log 10 CFU/mL, the growth was considered not significant, and no fit was applied [20].The adjusted coefficient of determination (Adj.R 2 ) was used to evaluate the goodness-of-fit of the model [37].
where Y(t) is the bacterial population at t in ln CFU/mL, Y 0 is the initial population in ln CFU/mL, µ max is the maximum specific growth rate (1/h), Y max is the maximum population in ln CFU/mL, and λ is the lag time in h.

Cereulide Extraction
The experimental conditions for cereulide extraction were in accordance with bacterial growth conditions.Interval extraction of the cereulide during the B. cereus exponential growth phase in each condition.The B. cereus cell pellets were obtained from the 120 mL bacterial culture after centrifugation at 8600× g for 10 min.The 13 C 6 -cereulide (Chiralix, Nijmegen, The Netherlands) was added to the pellets as internal standard and then equilibrated for 30 min at 25 • C. Each sample was extracted by shaking with acetonitrile on a rocking table for 18 h at 20 • C. The extract was centrifuged at 8600× g for 15 min, the supernatant obtained was centrifuged at 11,000× g for 8 min, then membrane filtered (0.2 µm, PTFE membrane) to remove cell debris [38,39].

Cereulide Quantification
The analysis was performed on a Shimadzu Nexera X2 LC-30AD UPLC system coupled with a Shimadzu LCMS-8060 mass spectrometer (Kyoto, Japan).The samples from cell extractions were injected into the 1.7 µm 50 × 2.1 mm column (Waters, Milford, MA, USA) at 40 • C. The isocratic elution was made with 90% acetonitrile-0.1% (v/v) formic acid (solvent A) and 10% 10 mM ammonium formate-0.1% (v/v) formic acid solution (solvent B) and the flow rate was 0.4 mL/min [18].The MS-MS analysis was carried out using a Shimadzu LCMS-8060 triple quadrupole with an electrospray ionization (ESI) interface.The instrument was operated in positive mode in the multiple reaction monitoring (MRM) mode.The collision energy for the cereulide was set to 55 eV.The transitions (m/z) used for obtaining the daughter fragments of cereulide were 1170.7 → 314.4,1170.7 → 499.4.The collision energy for the 13 C 6 -cereulide was set to 90 and 74 eV, and the transitions (m/z) used for obtaining the daughter fragments of 13 C 6 -cereulide were 1176.7 → 172.2, 1176.7 → 315.4 [39].
The limit of quantification (LoQ) was defined based on a S/N ratio > 10.The LoQ in this study was determined at 0.1 ng/g in the tested bacterial cell pellets.The recovery rates were 103%, 97%, and 92% in the cereulide concentrations were 0.02, 0.15, and 5 ng/mL, respectively (Table S1).The standard calibration curve was established by cereulide (Chi-

Figure 1 .Figure 1 .
Figure 1.The survival of B. cereus in the condition of temperature, pH, and aw.2.1.2.B. cereus Growth with or without Cereulide Production Figure 2 shows the growth curves of B. cereus growth under different conditions by fitting the Gompertz model.The estimated parameters of the maximum population (Ymax)

Figure 3 .
Figure 3.Effect of single factor on the transcription of cesA, cesB, cesP, codY, and abrB genes.(a) The temperature effect; (b) The pH effect; (c) The aw effect.The fold change value for the control group was 1. 0 < 2 −ΔΔCT < 0.5 or 2 −ΔΔCT > 2, representing significant (p < 0.05) down-or up-regulation of genes, represented as *.Data represented as the mean ± standard deviations of three biological replicates.

Figure 3 .
Figure 3.Effect of single factor on the transcription of cesA, cesB, cesP, codY, and abrB genes.(a) The temperature effect; (b) The pH effect; (c) The a w effect.The fold change value for the control group was 1. 0 < 2 −∆∆CT < 0.5 or 2 −∆∆CT > 2, representing significant (p < 0.05) down-or up-regulation of genes, represented as *.Data represented as the mean ± standard deviations of three biological replicates.

Toxins 2024 ,
16, 32 7 of 15were significantly up-regulated at pH 5 alone while down-regulated at 30 °C alone (Figure3a,b), indicating that the enhancement of their expression due to reduced pH outweighed the inhibition caused by increased temperature.

Figure 4 .
Figure 4. Effect of two factors on the transcription of cesA, cesB, cesP, codY, and abrB genes.(a) The temperature and pH effects; (b) The temperature and aw effects (c) The pH and aw effects.The fold change value for the control group was 1. 0 < 2 −ΔΔCT < 0.5 or 2 −ΔΔCT > 2, representing significant (p < 0.05) down-or up-regulation of genes, represented as *.Data represented as the mean ± standard deviations of three biological replicates.

Figure 4 .
Figure 4. Effect of two factors on the transcription of cesA, cesB, cesP, codY, and abrB genes.(a) The temperature and pH effects; (b) The temperature and aw effects (c) The pH and a w effects.The fold change value for the control group was 1. 0 < 2 −∆∆CT < 0.5 or 2 −∆∆CT > 2, representing significant (p < 0.05) down-or up-regulation of genes, represented as *.Data represented as the mean ± standard deviations of three biological replicates.

1 .
The Effect of Temperature, pH, and a w on B. cereus Behavior and Cereulide Production

Table 1 .
The maximum population and cereulide concentration of B. cereus at combinations of temperature, pH, and a w .