# Oil-in-water emulsion and acid concentrations impact Bacillus weihenstephanensis KBAB4 growth and culturability

Agathe Dutoit, Clément Trunet, Nicolas Decourcelle, Anne-Gabrielle Mathot, Louis Coroller

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## To cite this version:

Agathe Dutoit, Clément Trunet, Nicolas Decourcelle, Anne-Gabrielle Mathot, Louis Coroller. Oil-in-water emulsion and acid concentrations impact Bacillus weihenstephanensis KBAB4 growth and culturability. International Journal of Food Microbiology, 2025, 434, pp.111132. 10.1016/j.ijfoodmicro.2025.111132. hal-05386727

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Submitted on 28 Nov 2025

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# Oil-in-water emulsion and acid concentrations impact *Bacillus weihenstephanensis* KBAB4 growth and culturability

Agathe Dutoit, Clément Trunet, Nicolas Decourcelle, Anne-Gabrielle Mathot, Louis Coroller*

Univ Brest, INRAE, Laboratoire Universitaire de Biodiversité et Écologie Microbienne, F-29000 Quimper, France

## ARTICLE INFO

**Keywords:**

Growth inhibition

Acids

Non-culturability

Flow cytometry

Viability

VBNC

*Bacillus cereus* group

## ABSTRACT

Organic acids have long been used to limit microbial growth and preserve food products from spoilage and pathogens. However, the food complexity can affect the efficiency of preservatives in inhibiting bacterial growth. The inhibitory activity of acetic, lactic, caprylic and lauric acid in nutrient broth and in a model food system, i.e. an oil-in-water (O/W) emulsion at two pH levels, were compared using the plate count method.

The fate of *B. weihenstephanensis* KBAB4 cells incubated in O/W emulsions revealed a loss of culturability on Brain Heart Infusion agar (BHI agar) medium, occurring at different times according to the tested conditions. Cell viability in O/W emulsion was assessed through flow cytometry measurements which confirmed that a large majority of cells possessed an intact membrane, despite only 3 % being able to recover on BHI agar. Decreasing the pH of the emulsions delayed the time at which cells lost their culturability as well as the time at which it was restored. Using acetic, caprylic and lauric acid in the emulsion impacted strain recovery on BHI agar whereas using lactic acid enhanced the cell recovery from the emulsion exposure, on BHI agar surface.

The culturability of *B. weihenstephanensis* KBAB4 cells incubated in an O/W emulsion without acid has been restored using other recovery media, highlighting ROS involvement and limited strain catalase activity in the recovery ability. This study put in evidence the susceptibility of this strain to oxidative stress and challenges the use of given media for enumerating spore-forming bacteria in food and other complex matrices.

## 1. Introduction

Organic acids including fatty acids are commonly employed as antimicrobials to guarantee the safety of food items (Davidson et al., 2021; Theron and Lues, 2011). Naturally occurring or naturally produced compounds in food have gained popularity in the food industry, as they meet consumer expectations for more transparency and naturalness in their daily food (Coban, 2020; Lucera et al., 2012). While the inhibitory effects of organic acids against various foodborne pathogens have been well-established in liquid laboratory media (Ababouch et al., 1994; Batovska et al., 2009; Coroller et al., 2005; Hsiao and Siebert, 1999), there is a growing demand to optimize their utilization in food and to assess their inhibitory potential in these complex environments.

Foods are complex systems composed of different microenvironments with distinct physicochemical properties, such as oil-in-water emulsion (O/W emulsion). The chemical composition, structure, and storage conditions of food play a significant role in microbial growth and survival (Wilson et al., 2002). The impact of the composition and structure of an O/W emulsion on bacterial growth has long been studied

(Brocklehurst et al., 1995; Parker et al., 1995b; Robins and Wilson, 1994) and it is recognized that microbial growth occurs in the aqueous phase of an O/W emulsion in planktonic form (Wilson et al., 2002). The intrinsic environment of an O/W emulsion is therefore important for a microorganism because it allows bacterial mobility but also transport of nutrients to the cell and diffusion of metabolites into the medium, which can all influence bacterial growth (Alisherovich et al., 2023). Indeed, the physicochemical properties of inhibiting acids, and their interactions with components of the food matrix, can modify their distribution and thus their ability to inhibit microbial development in an oil-in-water emulsion (Brocklehurst and Wilson, 2000; Weiss et al., 2015). Increasing concentrations of the oil phase inhibiting bacterial germination and outgrowth (Léonard-Akkari et al., 2018). This decline is attributed to the lipophilic character of hexanoic acid. On the other hand, the inhibitory efficiency of caprylic acid in oil-in-water emulsion is mainly led by the concentration of surfactant rather than the oil droplet size (El Kadri et al., 2017).

As few studies have been performed on the activity of organic acids in O/W emulsion against spore-forming bacteria such as *Bacillus cereus*

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: louis.coroller@univ-brest.fr (L. Coroller).</footnote>
group species, it is necessary to better understand the impact of the physicochemical properties of acid on their antimicrobial activity in a complex matrix. The main objective of this study was to evaluate the inhibitory activity of acetic, lactic, caprylic, and lauric acids on *B. weihenstephanensis* KBAB4 in nutrient broth and a complex system, i.e. O/W emulsion, chosen to represent a model food matrix. The four chosen acids are found in numerous food such as dairy products where they act as preservatives. They were selected because they are characterized by different physicochemical properties: chemical structure, dissociation constant, and partition coefficient (log P). The strain is considered to be representative of the various independently isolated, moderately psychrotolerant *B. weihenstephanensis* strains (Guinebretière et al., 2008; Lapidus et al., 2008; Lechner et al., 1998). This strain was well documented with more than twenty papers of different research group describing its physiology and its metabolism from 2007 to 2024, including in food-based medium (Guérin et al., 2019). The complete genome sequence of strain KBAB4 is available. The O/W emulsion is composed of nutrient broth, as aqueous phase and hexadecane, as oil phase. Although hexadecane is never used in food, its use as the fatty phase in the model emulsion avoids any traces of saturated or polyunsaturated fatty acids in the medium, which could affect the behaviour and growth of the microorganisms (Mvou Lekogo et al., 2010). Furthermore, pure hexadecane is regularly used in the scientific literature to formulate emulsions for cosmetic or pharmaceutical applications but also in the food industry to obtain stable emulsions and to visualise the growth of microorganisms (Brocklehurst et al., 1995; Kim et al., 2004; Parker et al., 1995a). In these studies, it was shown that bacterial growth in hexadecane emulsions was similar to that observed in a control aqueous nutrient medium.

## 2. Materials and methods

### 2.1. Bacterial strain and spore production

*B. weihenstephanensis* KBAB4, reclassified as *B. mycoides* (Liu et al., 2018), was kindly provided by INRAE (Avignon, France). The strain was stored at -80 °C, as 1 mL aliquots of vegetative cell suspension in Brain Heart Infusion (BHI) broth (Condalab, Madrid, Spain) diluted in 20 % glycerol (v/v). A 1 mL aliquot was inoculated in 100 mL BHI broth contained in 250 mL flasks and incubated for 8 h at 30 °C, the optimal growth temperature of the strain. Flasks were shaken at 100 rpm. Then, 1 mL of this culture was diluted in 100 mL BHI broth and was incubated overnight. The final cell concentration of the second culture was approximately $1 \times 10^8$ CFU/mL.

Sporulation of *B. weihenstephanensis* KBAB4 was performed in a two-step process. First, 100 mL of BHI broth was inoculated with 0.1 mL of the overnight preculture of *B. weihenstephanensis* KBAB4 and incubated at 30 °C. After 6 h, cells were harvested by centrifugation at 6000 g for 15 min at 12 °C and then resuspended in 100 mL of sporulation mineral

buffer, SMB (Baril et al., 2011). Spores were collected in 5 mL of sterile distilled water and stored at 4 °C. The final concentration of the spore suspension was approximately $1 \times 10^8$ spores/mL and corresponded to the number of cells recovering on BHI agar at 30 °C after a 10-min heat treatment at 80 °C, the concentration of cells was not significant. These spores were used to inoculate nutrient broth or emulsion media.

### 2.2. Tested acids and solutions preparation

Two weak acids and two fatty acids were studied as antimicrobial compounds: acetic acid (99 % pure), lactic acid (90 % pure), caprylic acid (98 % pure), and lauric acid (98 % pure, all the acids were purchased from Sigma-Aldrich, St. Quentin Fallavier, France). The tested acids were chosen for their physicochemical properties: their hydrophobicity (logP), their ability to dissociate (pKa), and their carbon chain length (molar mass), as shown in Table 1. Stock solutions of 1 M acetic and lactic acids were prepared in distilled water. Caprylic acid was prepared in ethanol 70 % to a final concentration of 1 M. The final ethanol concentration in the medium had no impact on bacterial growth. Lauric acid was mixed at 55 °C in Tween 80 (0.1 % p/v), then added to the nutrient broth to reach a final concentration of 0.075 mM.

### 2.3. Emulsion preparation

The aqueous phase of the emulsion consisted of nutrient broth, allowing bacterial growth, (Biokar Diagnostics, Beauvais, France) supplemented with 3 % (w/v) Tween 80. Appropriate amounts of stock solutions of acetic, lactic, and caprylic acid, were added to the aqueous phase to obtain a final concentration of 3.0 mM, 1.8 mM, and 0.020 mM of undissociated acetic, lactic, and caprylic acid. The final concentration of lauric acid was equal to 0.075 mM of total acid. These concentrations of undissociated and total acids were selected in accordance with a previous study (Dutoit et al., 2024), because they are lower than the MIC value against *B. weihenstephanensis* KBAB4, so growth can be visualized as the growth inhibition caused in the nutrient broth is approximately 40 %. Aqueous phases were then adjusted to pH 5.5 or 6.0 using HCl or NaOH 1 M. The oil phase of the emulsion consisted of hexadecane (99 % pure) supplemented with 3 % (w/v) Span 80. The oil and aqueous phases were filtered through a 0.22 µm filter (Stericup, Millipore). Then, 50 mL of the aqueous phase was mixed with 50 mL of the oil phase using a 19 mm diameter sonication probe for 1 min at 60 % amplitude with a Branson 450 W-20 kHz digital sonifier. A temperature probe and an ice bath were added to control temperature variation in the medium during sonication. The temperature was recorded below 40 °C for each sample.

### 2.4. Particle size determination and stability

The emulsion particle size distributions were measured by laser granulometry with a Malvern MS3000, in 0.1 % SDS dispersant at 2000

<table><caption>**Table 1**

Physicochemical properties of acetic, lactic, caprylic and lauric acid and their concentrations in the medium.</caption><thead><tr><th rowspan="2">Name (CAS)</th><th rowspan="2">Chemical formula</th><th rowspan="2">Molecular structure</th><th rowspan="2">logP</th><th rowspan="2">pKa</th><th rowspan="2">Molar mass (g/mol)</th><th colspan="3">Medium condition</th></tr><tr><th>pH</th><th>Total form (mM)</th><th>Undissociated form (mM)</th></tr></thead><tbody><tr><td rowspan="2">Acetic acid (64-19-7)</td><td rowspan="2">C<sub>2</sub>H<sub>4</sub>O<sub>2</sub></td><td rowspan="2"></td><td rowspan="2">-0.21</td><td rowspan="2">4.76</td><td rowspan="2">60.05</td><td>5.5</td><td>20</td><td>3.0</td></tr><tr><td>6.0</td><td>60</td><td>3.0</td></tr><tr><td rowspan="2">Lactic acid (79-33-4)</td><td rowspan="2">C<sub>3</sub>H<sub>6</sub>O<sub>3</sub></td><td rowspan="2"></td><td rowspan="2">-0.72</td><td rowspan="2">3.86</td><td rowspan="2">90.08</td><td>5.5</td><td>80</td><td>1.8</td></tr><tr><td>6.0</td><td>250</td><td>1.8</td></tr><tr><td rowspan="2">Caprylic acid (124-07-2)</td><td rowspan="2">C<sub>8</sub>H<sub>16</sub>O<sub>2</sub></td><td rowspan="2"></td><td rowspan="2">3.05</td><td rowspan="2">4.89</td><td rowspan="2">144.21</td><td>5.5</td><td>0.090</td><td>0.020</td></tr><tr><td>6.0</td><td>0.250</td><td>0.020</td></tr><tr><td rowspan="2">Lauric acid (143-07-7)</td><td rowspan="2">C<sub>12</sub>H<sub>24</sub>O<sub>2</sub></td><td rowspan="2"></td><td rowspan="2">4.20</td><td rowspan="2">5.30</td><td rowspan="2">200.32</td><td>5.5</td><td>0.075</td><td>0.030</td></tr><tr><td>6.0</td><td>0.075</td><td>0.012</td></tr></tbody></table>
rpm. The mean volume diameters and the particle size dispersions were monitored for 24 h after emulsion preparation with storage at 30 °C which was the temperature used for the microbial assay. The optical parameters selected were: oil globule absorbance of 0.01, dispersed phase refractive index of 1.53, dispersant liquid refractive index of 1.33 and laser obscuration between 5 % and 15 %.

The emulsion stability (flocculation, creaming, clarification) was monitored by multiple light scattering with a Turbiscan (Formulaction, France). The destabilization phenomena were monitored for 10 days at 30 °C, without bacteria. Particle size and stability measurements were performed in triplicate for the oil-in-water emulsion without inhibitors at pH 6.0 and 5.5 and oil-in-water emulsions with acids at pH 6.0.

## 2.5. Enumeration of culturable cells in nutrient broth and emulsion medium with plate counting

To study the growth of *B. weihenstephanensis* KBAB4 in O/W model emulsions and their corresponding nutrient broth at pH 6.0 and 5.5, media were inoculated with a heat-treated (10 min/80 °C) suspension of spores to reach a final concentration of $10^5$ CFU/mL in 100 mL nutrient broth or emulsion. The same concentrations of acids were present in the oil-in-water emulsions and the nutrient broth (Table 1). Erlens were incubated at 30 °C with 110 rpm stirring for a maximum of 35 h. Cells were enumerated at regular intervals by collecting 150 µL of nutrient broth or 300 µL of emulsion (ratio 50/50) in 1350 µL of 9 g/L saline water and then serially diluted and plated using a spiral plater (easy-Spiral Dilute, Interscience, France) on BHI agar plates (Condalab, Madrid, Spain). Colony forming units were enumerated after 24 h of incubation at 30 °C and log(CFU/mL) values were calculated. Experiments were performed in duplicate for each condition.

## 2.6. Flow cytometry measurements

To study the membrane permeability of vegetative cells of *B. weihenstephanensis* KBAB4 grown in oil-in-water emulsion, flow cytometry analysis was carried out using the Live/Dead BacLight Bac-terial Viability Kit (Invitrogen, Thermo Fisher Scientific, USA). SYTO9 and Propidium Iodide (PI) are nucleic acid dyes, SYTO9 stains all cells green and PI penetrates the non-intact cell membrane and stains them red. Double staining was used to identify and quantify the percentage of the different subpopulations within the sampled cell population: in green, cells presenting an intact membrane, in red, the permeable membrane cells and a transient state with green and red characteristics, assumed to be “damaged cells”. To prepare flow cytometry samples, 200 µL of oil-in-water emulsion were added to 900 µL of demineralized water (sterilized by filtration on 0.22 µm, Stericup, Millipore) and immediately stained with 1.500 µM of SYTO9 and 20.00 µM of PI. Measurements were then performed using a flow cytometer (Cyflow space, Sysmex Europe Gmbh, Norderstedt, Germany) equipped with a 488 nm excita-tion laser and four detectors: FSC, SSC (detecting light emission at 488 nm) and two fluorescence light detectors. Green fluorescence is detected by a 536/40 nm filter (FL1) and red fluorescence by a 610 nm high-pass filter (FL2). The trigger was set for the SSC and events were absolutely enumerated in 0.2 mL as the instrument allows. Compensation was applied in order to correct fluorescence spillover, based on the median fluorescence value of the negative population. For this, dead cells were prepared by heat inactivation at 80 °C for 10 min, as a negative control.

Experiments were repeated three times and analysed using Flow Max software (Cyflow Space, Sysmex Europe Gmbh, Norderstedt, Germany). The subpopulations of cells with intact, damaged or permeable membranes were identified and gated on dot plots of FL1 versus FL2 and quantified by counting the number of events inside the corresponding gate (Fig. 1).

<figure><img src="image_5.png" /><figcaption>Fig. 1. Flow cytometry dot plots of a control oil-in-water emulsion previously inoculated with dead and viable cells of *B. weihenstephanensis* KBAB4. Populations with a permeable membrane appeared in quadrant A and intact membrane cells appeared in quadrant C, damaged double-stained cells appeared in quadrant B.</figcaption></figure>

## 2.7. Effect of the recovery medium on culturability of *B. weihenstephanensis* KBAB4 incubated in emulsion

Three media were used to investigate the recovery ability of *B. weihenstephanensis* KBAB4 following its exposure to a model emulsion without acid. First, an agar medium was developed from the liquid O/W emulsion. This medium consisted of mixing 80 mL of sterile oil-in-water model emulsion pre-heated to 55 °C with 20 mL of autoclaved agar solution to obtain a final concentration of 15 g/L. Then, 1 mL of 0.05 % filter sterilized solution of 2,3,5-triphenyl tetrazolium chloride (TTC) was plated on Petri dishes before adding the agar-emulsion mixture and carefully mixing. TTC was used to facilitate the visualization of colonies by dyeing them red. Then, two modified BHI agars were used to study the recovery. These media were supplemented by ROS scavengers, either with catalase or dimethylthiourea (DMTU). Solutions of catalase and DMTU (Sigma-Aldrich, St. Quentin Fallavier, France) were sterilized by filtration and overlayed (100 µL) onto BHI agar plates, to reach a final concentration of 2000 units/mL and 25 mM respectively. The fate of *B. weihenstephanensis* KBAB4 in O/W model emulsion without acid was determined in triplicate using these three media.

## 2.8. Modelling

The growth of vegetative cells ($Y_{tot}$) in nutrient broth or in oil-in-water emulsion recovered in supplemented media (see results section) was described by Rosso's model (Rosso et al., 1995) (Eq. 1).

$$Y_{tot} = \ln[N(t_i)] = \begin{cases} \ln(N_0), & t_i < \lambda \\ \ln\left(\frac{N_{max}}{1 + \left(\frac{N_{max}}{N_0}\right) * e^{-\mu_{max}(t_i - \lambda)}}\right), & t_i \ge \lambda \end{cases} \quad (1)$$

with $N(t_i)$ the bacterial concentration at time $t_i$ (CFU/mL), $N_0$ the initial cell concentration (CFU/mL), $N_{max}$ the maximum cell concentration (CFU/mL), $\lambda$ the lag period (h), and $\mu_{max}$ the maximum specific growth rate (h⁻¹).
A model was developed to describe the loss of culturability on BHI agar during the growth in oil-in-water emulsions. To assess the total vegetative cell concentration ($Y_{tot}$), it was considered as the sum of two subpopulations: culturable cells ($Y_{culturable}$) and non-culturable cells on BHI agar ($Y_{non-culturable}$):

$$Y_{tot} = Y_{culturable} + Y_{non-culturable} \qquad (2)$$

In emulsions, the total vegetative cells population ($Y_{tot}$) is assumed to grow (following the model 1). At a given time, a portion of the population loses the ability to grow on BHI agar (represented by the proportion $P_{max}$). The time at which individual cells lose culturability varies among the population and is assumed to follow a normal distribution, characterized by a mean time ($t_{non-culturable}$) and a standard deviation ($\sigma$) (Fig. 2). A lower standard deviation indicates a faster or more synchronized loss of culturability across the population. At the end of the kinetic, the cells regain culturability on BHI agar after a specific recovery time ($t_{rec}$), with no scattering applied to $t_{rec}$. Following these hypotheses, the colonies which were enumerated on BHI agar can be computed as the culturable fraction of the population:

$$Y_{culturable} = Y_{tot} - Y_{non-culturable} \qquad (3)$$

Where the total population ($Y_{tot}$) is computed by the growth model of Rosso (see above), and the non-culturable cells on BHI agar ($Y_{non-culturable}$) are as follows:

$$Y_{non-culturable} = (\%Y_{loosing culturability} - \%Y_{recovering culturability}) Y_{tot} \qquad (4)$$

Where the percentage of cells losing their culturability ($\%Y_{loosing culturability}$) is derived from the maximal proportion of cells losing the culturability ($P_{max}$) and the normal cumulative distribution function of the time at which the cells lose their culturability within the total population, with the mean time $t_{non-culturable}$ and the standard deviation $\sigma$:

$$\%Y_{loosing culturability} = P_{max} \cdot \left[ \frac{1}{\sigma\sqrt{2\pi}} \int_{-\infty}^{t} \exp\left(-\frac{(t - t_{non-culturable})^2}{2\sigma^2}\right) dt \right] \qquad (5)$$

And the percentage of cells recovering their culturability ($\%Y_{recovering culturability}$) is:

$$\%Y_{recovering culturability} = \begin{cases} 0 & \text{if } t \le t_{rec} \\ P_{max} & \text{if } t > t_{rec} \end{cases} \qquad (6)$$

## 2.9. Statistical analysis

The growth parameters, i.e. the lag phase ($\lambda$) and the maximum specific growth rate ($\mu_{max}$), were estimated using Rosso's model by minimizing the sum of squared errors (SSE; lsqcurvefit function, MatlabR2020a, MathWorks, Natick, MA, USA). The 95 % confidence intervals were assessed using the nlparci function (nlparci function, Matlab2020a, MathWorks, Natick, MA, USA). Finally, the goodness of the fit of the model was evaluated by the root mean square error (RMSE), determined using Eq. 7.

<figure><img src="image_6.png" /><figcaption>Fig. 2. Theoretical modelling diagram showing the impact of culturability loss on BHI agar used for bacterial enumeration after exposure to O/W emulsion where growth occurs. The black dotted line represents the kinetic of the total cells ($Y_{tot}$) in O/W emulsion, characterized by the growth model of Rosso. The red line is probability density of time when the cells lose their ability to grow on BHI agar used for enumeration, characterized by maximum proportion of cells losing their ability to grow on BHI agar ($P_{max}$), the mean time ($t_{non-culturable}$) and the dispersion within the population ($\sigma$). The time $t_{rec}$, in green, corresponds to the time at which the proportion of non-culturable cells can fully recover on BHI agar. The black continuous line represents the evolution of the population of culturable cells (log(CFU/mL)) enumerated on BHI agar in O/W emulsion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
$$RMSE = \sqrt{\frac{ESS}{n-p}} \qquad (7)$$

with ESS the sum of squared errors, n the number of experimental data and p the number of parameters of the model. The smaller the RMSE was, the better the model was fitted on the data.

One-way analysis of variance (ANOVA) was performed with the F-statistic test to compare particle size dispersion or bacterial concentration (log(CFU/mL)) obtained on the different recovery media over time and also to compare estimated growth parameters (μmax and λ) of B. weihenstephanensis KBAB4 on those media. ANOVA was performed using Matlab software (anova1 function, Matlab2020a, MathWorks, Natick, MA, USA) with a p-value equal to 0.05.

The fate of the bacterial population in emulsion was simulated by the model describing the loss of culturability on BHI agar. Knowing growth parameters (meaning N₀, Nmax, λ and μmax) from growth kinetics in closely related conditions and the experimental setup (see Section 3.5 “Simulating the temporary loss of culturability” for details); it was possible to compute the total vegetative cells population (Ytot) using the Rosso's model (Eq. 1). Then, the culturable population kinetics (log (CFU/ml) over incubation time) were used to estimate Pmax, tnon-culturable, σ and trec by minimizing the sum of squared errors (SSE; fminsearch function, MatlabR2020a, MathWorks, Natick, MA, USA).

# 3. Results

## 3.1. Particle size determination and stability

The model O/W emulsion at pH 6.0 was characterized in terms of oil droplet size by laser granulometry. Measurements showed that the O/W emulsion without an inhibitor, or with acetic, caprylic and lauric acid added at pH 6.0, presented a monomodal distribution of oil droplets with a diameter equal to 0.717 ± 0.070 µm, 0.626 ± 0.013 µm, 0.727 ± 0.038 µm and 0.634 ± 0.010 µm respectively. However, the oil droplet diameter of the O/W emulsion with lactic acid is significantly different

from the other four and equal to 1.194 ± 0.238 µm, reflecting a different emulsion structure. The emulsion stability measurements conducted with a Turbiscan confirmed this observation. The Turbiscan Stability Index (TSI) was used to compare O/W emulsions after 24 h of storage at 30 °C. The emulsions (without acid or with acetic, caprylic and lauric acid) did not exhibit any creaming or sedimentation after 24 h (TSI < 0.5) except for the emulsion containing lactic acid. The TSI value of this latter emulsion was found to be 0.53 ± 0.40, indicating the occurrence of a destabilization phenomenon.

## 3.2. Growth kinetics of B. weihenstephanensis KBAB4 in nutrient broth and in O/W emulsion, with organic acids

To investigate the inhibition of B. weihenstephanensis KBAB4 by organic acids in an O/W emulsion, the experimental cell fates in O/W emulsion at pH 6.0 and pH 5.5 were compared to the cell fates in nutrient broth in the same conditions (Fig. 3). The conditions tested are without weak or fatty acid (Fig. 3A), and in presence of a specific concentration of undissociated acetic acid (HAce = 3.0 mM — Fig. 3B), undissociated lactic acid (HLac = 1.8 mM — Fig. 3C), undissociated caprylic acid (HCap = 0.020 mM — Fig. 3D) and total lauric acid (Lau = 0.075 mM — Fig. 3E). The strain growth parameters were estimated in nutrient broth for each condition (Table 2). Estimated lag phase (λ) includes spores germination and outgrowth and vegetative cells latency. RMSE values indicated a good fitting property of the model to the experimental data. The addition of organic acids to the nutrient broth significantly decreases the μmax at both pH levels. μmax estimated in nutrient broth with undissociated acetic, undissociated caprylic and total lauric acid are similar at both pH values. Lactic acid appeared to be more effective at pH 5.5 than at pH 6.0 in nutrient broth, as the μmax of B. weihenstephanensis KBAB4 with 1.8 mM of lactic acid was equal to 0.46 h⁻¹ and 0.74 h⁻¹ respectively. Moreover, decreasing the pH increased the strain lag time (λ) for each tested condition.

However, the kinetics in O/W emulsion shown a drop in log(CFU/mL) over time, followed by a sudden return to higher concentration, close to 8 log(CFU/mL) at the end of the kinetics (Fig. 3A). This pattern

<figure><img src="image_7.png" /><figcaption>Fig. 3. Experimental kinetics of B. weihenstephanensis KBAB4, monitored by enumeration on BHI agar during their exposure to oil-in-water emulsion (circle), and in nutrient broth (triangle) without acid (A) or in presence of acetic (B), lactic (C), caprylic (D) or lauric (E) acid at pH 6.0 (black) or pH 5.5 (grey). Symbols with error bars represent the data average value and respective standard deviation due to replicates. The red arrows point to the drop in cell concentration in optimal conditions (without acid). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<table><caption>**Table 2**
Growth parameters of *B. weihenstephanensis* KBAB4 estimated in nutrient broth for each tested condition and their standard deviations.</caption><thead><tr><th>pH</th><th>Growth parameters</th><th>Without acid</th><th>[HAce] = 3.0 mM</th><th>[HLac] = 1.8 mM</th><th>[HCap] = 0.02 mM</th><th>[Lau] = 0.075 mM</th></tr></thead><tbody><tr><td rowspan="3">6.0</td><td>μ<sub>max</sub> (h<sup>-1</sup>)</td><td>1.59 ± 0.29</td><td>0.68 ± 0.07</td><td>0.74 ± 0.10</td><td>0.69 ± 0.07</td><td>0.57 ± 0.04</td></tr><tr><td>λ (h)</td><td>3.89 ± 1.04</td><td>11.27 ± 0.52</td><td>6.49 ± 0.59</td><td>4.28 ± 0.56</td><td>9.61 ± 0.43</td></tr><tr><td>RMSE</td><td>0.35</td><td>0.41</td><td>0.36</td><td>0.43</td><td>0.43</td></tr><tr><td rowspan="3">5.5</td><td>μ<sub>max</sub> (h<sup>-1</sup>)</td><td>1.32 ± 0.14</td><td>0.60 ± 0.06</td><td>0.46 ± 0.05</td><td>0.63 ± 0.05</td><td>0.57 ± 0.09</td></tr><tr><td>λ (h)</td><td>8.75 ± 0.34</td><td>19.04 ± 0.55</td><td>12.87 ± 0.98</td><td>9.15 ± 0.48</td><td>18.81 ± 0.67</td></tr><tr><td>RMSE</td><td>0.37</td><td>0.41</td><td>0.34</td><td>0.60</td><td>0.38</td></tr></tbody></table>

was observed even after prolonged 72 h incubation of petri dishes at 30 °C and made it difficult to estimate reliable parameters. This phenomenon was observed during the exposure to O/W emulsions and with the use of BHI agar as enumeration media, at both pH levels but at different times during the kinetics and different intensity, according to the acid used. For example, the decrease appeared at 6 h in the O/W emulsion without an inhibitor at pH 6.0 but it appeared at 10 h at pH 5.5. Only the emulsion with added lactic acid did not show any drop in concentration in log(CFU/mL) over time on BHI agar (Fig. 3C). Thus, the O/W emulsions studied here have an impact on the fate of *B. weihenstephanensis* KBAB4, resulting in its inactivation over time on BHI agar medium.

### 3.3. Membrane permeability assessment of *B. weihenstephanensis* KBAB4 cells during growth by flow cytometry

To further assess the physiological state of *B. weihenstephanensis* KBAB4 cells, membrane permeability was measured by flow cytometry during exposure to an O/W emulsion without an inhibitor at pH 6.0. SYTO9/PI were used for the staining procedure (see Materials and methods section). Fig. 4A shows the evolution of the cell population in O/W emulsion without acid at pH 6.0 quantified by flow cytometry and also enumerated on BHI agar. The appearance of a population of vegetative cells was shown by flow cytometry (FSC/FL1) to occur from 5 h of incubation in the emulsion and increased steadily up to 10 h. Before 5 h, the concentration of vegetative cells in the emulsion was difficult to detect because of the oil droplet background, preventing cell population detection below 4 log. The concentration of cells quantified by flow cytometry is 1.5 log, 1.8 log and 0.9 log higher than the concentration enumerated on BHI agar at 7 h, 8 h and 9 h respectively, indicating that about 3 %, 1.4 % and 13 % of the total cells present in the emulsion are able to recover on BHI agar. Fig. 4B represents the percentage of intact,

damaged and permeable membrane cells (FL1/FL2) among the population of *B. weihenstephanensis* KBAB4 cells between 5 h and 24 h in O/W emulsion without acid at pH 6.0. A large majority of the population possessed an intact membrane during the decrease in log(CFU/mL) on BHI agar (between 7 h and 9 h). The percentage of cells with a damaged or permeable membrane increased after 10 h of growth in O/W emulsion. Complementary microscopic observations shown motile cells in the water phase of the O/W emulsion (data not show). These results confirmed that cells of *B. weihenstephanensis* KBAB4 were able to grow in O/W emulsion at pH 6.0 (intact membrane cells and increasing concentration), thus the decrease observed in log(CFU/mL) on BHI agar plates is a decrease in cell culturability on BHI agar.

### 3.4. Effect of the recovery medium on culturability of *B. weihenstephanensis* KBAB4 cells in emulsion

Cells exposed to O/W emulsion at pH 6.0 without acid were plated on a medium with an identical composition to the O/W emulsion but supplemented with 15 g/L of agar and TTC (30 mg/mL). Cells were also plated onto BHI agar containing catalase or DMTU. Fig. 5 shows the culturable cell concentration in log(CFU/mL) during their exposure to O/W emulsion at pH 6.0 on BHI agar, BHI agar supplemented with catalase or DMTU and O/W emulsion agar medium. It was observed that the culturability of *B. weihenstephanensis* KBAB4 during exposure to O/W emulsion is enhanced on the modified media compared to BHI agar. Indeed, log(CFU/mL) between 7 h and 9 h of incubation in emulsion is significantly lower when cells are cultivated on BHI agar (between 1.22 and 1.45 log lower). Thus, the use of a solid O/W emulsion as a recovery medium as well as the use of H₂O₂-degrading agents on BHI agar enabled the strain to fully regain its ability to recover.

The growth parameters of *B. weihenstephanensis* KBAB4 in O/W emulsion at pH 6.0 free from organic acid were estimated from the

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Cell population evolutions of *B. weihenstephanensis* KBAB4 in oil-in-water emulsion (A), quantified by flow cytometry (●) and enumerated on BHI agar (◆). (B) Proportion of intact ( ), damaged (■), and permeable (■) membrane cells in oil-in-water emulsion between 5 and 10 h of incubation at 30 °C.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Recovery of *B. weihenstephanensis* KBAB4 during their incubation in oil-in-water emulsion. Colonies were enumerated on BHI agar (black), BHI agar supplemented with catalase (light grey), BHI agar supplemented with thiourea (dark grey) and O/W emulsion agar medium (hatched grey). Error bars correspond to standard deviations between replicates. Asterisks represents a significant difference between the concentrations (log(CFU/mL)) (p value = 0.05), NS means non-significant difference. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

kinetics of log(CFU/mL) over time obtained on each recovery medium. The $\mu_{max}$ and $\lambda$ estimated for each recovery media (BHI agar + catalase, BHI agar + DMTU and O/W emulsion agar) were not significantly different (data not shown). The growth rate of *B. weihenstephanensis* KBAB4 in O/W emulsion at pH 6.0, without inhibitor, was 1.46 ± 0.11 $h^{-1}$ and the lag time was equal to 3.85 ± 0.57 h. No difference was observed between growth parameters in nutrient broth and in O/W emulsion at pH 6.0 without acid.

### 3.5. Simulating the temporary loss of culturability of *B. weihenstephanensis* KBAB4 on BHI agar

As previously shown, *B. weihenstephanensis* KBAB4 cells grown in O/

W emulsion without acid at pH 6.0 and growth parameters were estimated for this condition. The evolution of culturable cells on BHI agar was then described by a model derived from the logistic Rosso model, in order to compare cell non-culturability parameters of *B. weihenstephanensis* KBAB4 for all tested conditions in O/W emulsions (Fig. 6). Four parameters ($P_{max}$, $t_{non-culturable}$, $\sigma$ and $t_{rec}$) were used to describe and compare the evolution of the culturable cell population in O/W emulsion with or without acid and enumerated on BHI agar. To simulate the evolution of the culturable cell population ($Y_{culturable}$), it was necessary to estimate the growth of the total vegetative cell population ($Y_{tot}$) previously and to define growth parameters ($N_0$, $N_{max}$, $\mu_{max}$ and $\lambda$) (see Eq. 1). To do so, we tested several hypotheses and then assumed that i) growth parameters ($\mu_{max}$ and $\lambda$) of the strain are similar

<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Monitoring the culturable cells on BHI agar (circles) of *B. weihenstephanensis* KBAB4 exposed to O/W emulsion at pH 6.0 (A) or pH 5.5 (B) with 5 different conditions. The evolution over time of the theoretical culturable cell population on BHI agar is fitted with a black line and the simulated growth kinetics in O/W emulsion are represented with a dotted line. Symbols with error bars represent the data average value and respective standard deviation due to replicates. The parameters correspond to the parameters described in Fig. 2.</figcaption></figure>
in nutrient broth and in O/W emulsion without acid, as shown previ-
ously; ii) growth kinetics in O/W emulsions containing weak acids
(acetic and lactic acid) follow the same kinetics (same parameters) as in
nutrient broth with the same acid concentrations and the same pH
levels, and iii) growth kinetics in O/W emulsion containing fatty acids
(caprylic and lauric acid) follow the same kinetics (same parameters) as
in O/W emulsion without acid.

As shown in Fig. 6, the evolution of culturable cells on BHI agar can
be described considering the assumption on the growth parameters,
obtained in nutrient broth, except for emulsions with lactic acid. In this
condition, cells were able to recover on the medium so there is no
appearance of non-culturable cells (Pmax = 0) and the simulation of the
culturable cell population over time is therefore not reliable. Fig. 6 also
shows that lowering pH did not seem to have an impact on the pro-
portion of non-culturable cells, except if the emulsion is enriched with
acetic acid (Pmax = 0.93 at pH 6.0 and Pmax = 0.82 at pH 5.5). Moreover,
the presence of lauric acid seemed to decrease this proportion at both pH
levels, compared to other conditions (Pmax = 0.67 and 0.62). However,
decreasing pH delayed the time at which non-culturability occurred
(tnon-culturable) and the time at which cells regained their recovery ca-
cacity (trec) for all tested conditions.

4. Discussion

The main objective of this study was to evaluate and compare the
inhibitory activity of two weak acids (acetic and lactic acid) and two
fatty acids (caprylic and lauric acid) against *B. weihenstephanensis*
KBAB4 in nutrient broth and an O/W emulsion. The results showed that
adding 3.0 mM of undissociated acetic acid, 1.8 mM of undissociated
lactic acid, 0.020 mM of undissociated caprylic acid and 0.075 mM of
total lauric acid to nutrient broth caused growth inhibition, by
decreasing the growth rate and increasing the lag time of the strain
compared to the conditions without acid at both pH levels (Table 2), as
expected. It is known that weak or short chain fatty acids (C4–C8) in
their undissociated form can easily diffuse across bacterial cell mem-
branes, causing acidification of the cytoplasm and cell death (Stratford
and Eklund, 2003). On the other hand, long chain fatty acids have a
lipophilic character, resulting in a greater affinity for bacterial cell
membranes and leading to cell damage such as disruption or disorga-
nization (Casillas-Vargas et al., 2021; Desbois and Smith, 2010).

However, comparing the acid inhibition in nutrient broth and a more
complex system, i.e. an O/W emulsion, was not possible in this study due
to the sudden decrease in cell concentration enumerated on BHI agar
over time (Fig. 3). The fate of *B. weihenstephanensis* KBAB4 was thus
impacted by the complex system and the presence of oil droplets,
leading to a loss of culturability of cells on BHI agar for all tested con-
ditions except in O/W emulsions with lactic acid where no drop in cell
concentration was observed on BHI agar. A larger oil droplet size was
the only difference observed concomitantly and could perhaps play a
role in this phenomenon.

This plate count method has therefore shown its limits for measuring
bacterial growth and assessing bacterial inhibition in O/W emulsion.
Flow cytometry offers an alternative method for determining the phys-
iology of individual cells by providing data on cell survival and mem-
brane permeability, directly within a complex matrix (Berney et al.,
2007; Raymond and Champagne, 2015; Wilkinson, 2018). In this study,
membrane permeability was assessed by using nucleic acid dyes: when
the permeability barrier of the cell is lost, the presence of the PI dye in
the cell is correlated with cell damage. The results showed that cell
multiplication did occur in the O/W emulsion without acid over time,
confirming the loss of culturability on BHI agar. Moreover, the results
also showed that cell membrane integrity was intact during the non-
culturability phenomenon observed on BHI agar (Fig. 4). The cells of
*B. weihenstephanensis* KBAB4 have therefore entered a Viable But Non
Culturable (VBNC) state on BHI agar following their exposure to the O/
W emulsion. Bacteria in the VBNC state are no longer able to grow on a

routine culture medium, but are still alive, maintaining their metabolic
activities, gene expression capacities and integrated cytoplasmic mem-
branes (Oliver, 2005). This phenomenon is adopted by a microorganism
as a survival strategy in response to a stressful environment, but resus-
citation can occur with appropriate environmental conditions (Oliver,
2010; Pan and Ren, 2023). Stressful environments inducing the VBNC
state are varied and include nutrient starvation, incubation outside the
growth temperature range, inappropriate osmotic concentrations, oxy-
gen concentrations, etc. (Oliver, 2010). Very few studies have been re-
ported on the VBNC state of *Bacillus cereus* species (Karmakar and
Roychoudhury, 2017; Morawska and Kuipers, 2022a; Rowan, 2004).
The presence of a subpopulation of metabolically viable cells in
matrices, but with no ability to be cultured on media, represents a threat
to public health and food safety (Ayrapetyan and Oliver, 2016). The
determination of the conditions required to regain full strain cultur-
ability is thus valuable.

Catalase and DMTU have long been studied in bacteria media for the
recovery of stressed cells because they are effective and specific scav-
engers of H₂O₂ (Kong et al., 2004; Martin et al., 1976; O'Donoghue et al.,
2016). Cells of *B. weihenstephanensis* KBAB4 totally regain their ability to
recover on BHI agar supplemented with catalase and DMTU, after being
exposed to O/W emulsion, underlining the potential impact of hydrogen
peroxide on the culturability of stressed cells. Similar results were ob-
tained for other microorganisms (Hong et al., 2019; Morawska and
Kuipers, 2022b; Wai et al., 2000). Moreover, using a recovery medium
with an identical composition to the O/W emulsion with added agar and
free of ROS, also enabled the strain to fully recover, showing that cell
culturability depends on their ability to neutralize ROS concentrations
generated on BHI agar. This pattern should be verified by testing further
culture media, used as a control to guarantee the health risk posed by the
*B. cereus* group.

Indeed, transferring VBNC cells directly to nutrient media will likely
lead to their demise given that dormant cells attempting to resuscitate
poorly withstand the oxidative stress imposed by nutrient media
(Ayrapetyan et al., 2018). The exposure to hydrogen peroxide leads to
the induction of oxidative stress associated genes and mechanisms (Mols
and Abee, 2011). Genes encoding catalases, thioredoxin reductases and
peroxidases, constituting the antioxidant defense system of *B. cereus*
(Duport et al., 2016), are induced to remove hydrogen peroxide from the
cells or extracellular environment. Otherwise, accumulated ROS results
in proteins and DNA damage (Ceragioli et al., 2010). However, studies
have indicated that despite VBNC cells continuing to maintain their
membrane integrity, the cells exhibit a lower metabolic activity. Mor-
awska and Kuipers (2022a), showed a decreased induction of H₂O₂
detoxifying enzymes in VBNC cells of *B. subtilis* compared to culturable
cells. Similarly, Kong et al. (2004) demonstrated that Vibrio vulnificus
cells in the VBNC state are nonculturable on laboratory media largely
due to reduced catalase activity. Therefore, the accumulation of
hydrogen peroxide attributed to low catalase activity has been identified
as the main factor contributing to the reduced colony-forming ability of
cells expose to stress (Juven and Pierson, 1996). This pattern prevents
replication of these cells on media containing ROS.

In our study, the culturability of *B. weihenstephanensis* KBAB4 cells incubated in O/W emulsion was affected differently by the different organic acids. Notably, we found that the addition of lactic acid to the emulsion did not lead to a loss of culturability. Lactic acid, an antimicrobial compound with multiples technological purposes in food systems, also exhibits antioxidant properties that may protect cells from oxidative stress induced by the nutritive medium (Silva and Lidon, 2016). Under other conditions, a loss of culturability occurred during the early growth phase, specifically during the assumed lag time, and before the exponential phase for the majority of the conditions (between 4 and 16 h). An exception was observed for the O/W emulsion at pH 6.0 without acid, where the cell concentration on BHI agar started to increase at 5 h then suddenly decrease during the exponential growth between 7 h and 9 h. Cells may encounter difficulties adapting to the
nutritive agar media during exponential phase and thus enter in a dormancy state to survive. However, cell capacity to recover on BHI agar was instantaneous and occurred during the stationary phase between 8 h and 28 h depending on the tested condition. It is recognized that the expression of catalase increases as cells of *B. cereus* species enter the stationary growth phase due to the establishment of a stress adaptive response induced by the lack of nutrients (Bol and Yasbin, 1994; den Besten et al., 2013). Thus, it may induce a higher resistance to oxidative stress and restore cell culturability on BHI agar. Moreover, it was found that VBNC cells resuscitate in response to a quorum sensing signal produced by cells when permissive conditions are reached (Ayrapetyan et al., 2014), thus cells incubated in the emulsions may be more sensitive to the quorum sensing signal when the maximal cell concentration is reached.

## 5. Conclusion

This work underlined the limits of comparing growth inhibition by acids in nutrient broth and in an emulsified system using a plate count method and a routine laboratory medium. An unexpected decrease in cell concentrations of *B. weihenstephanensis* KBAB4 followed by a sudden rise were observed when cells were incubated in emulsion and recovered on BHI agar. The cytometric method allows the quantification of non-permeable membrane cells and confirmed the membrane integrity of the cells incubated in the emulsion. Therefore, the complex matrix, i.e. an oil-in-water emulsion, allowed bacterial growth, did not impact cell survival and cell membrane permeability, but induced a physiological state of the cells leading to their non-culturability on a routine medium. The addition of anti-ROS agent on the medium enabled the strain to regain its ability to recover. Determination of catalase activity or proteomic analyses on cells incubated in the oil-in-water emulsion could provide valuable insights into the cause of their non-culturability on BHI agar medium. This work may be relevant for studying the occurrence of non-culturable cells following their exposure to complex matrices, especially spore-forming bacteria. The underestimation of pathogenic bacteria cells of the *B. cereus* group is a significant issue in food safety controls. Exposure to emulsion might lead to false negative results in microbiological quality indicators and compromising public health.

## CRediT authorship contribution statement

**Agathe Dutoit:** Writing – original draft, Project administration, Methodology.
**Clément Trunet:** Writing – review & editing, Methodology.
**Nicolas Decourcelle:** Writing – review & editing, Supervision, Project administration, Methodology.
**Anne-Gabrielle Mathot:** Writing – review & editing, Supervision, Project administration, Methodology, Formal analysis.
**Louis Coroller:** Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This work is a part of Agathe Dutoit's PhD project, funded by the "Conseil Régional de Bretagne" and "Quimper Bretagne Occidentale". The authors wish to thank our partners which also financially support the IGAB project, the "Centre National Interprofessionnel de l'Economie Laitière" (CNIEL, France). This work was supported by the UMT ACTIA ALTER'iX (Joint Technological Unit - French Ministry responsible for Food). The authors gratefully acknowledge Noémie Cossec for her technical support, and Fabienne Courand (ADRIA Food Expertise) for

her help with the granulometry experimentations.

## Data availability

Data will be made available on request.

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