# Heat resistance of five spoilage microorganisms in a carbonated broth

Fabien Saubade, Noëmie Cossec, Luc Giguelay Gesret, Christelle Kouamé, Mariem Ellouze, Cédric Gérard, Olivier Couvert, Noémie Desriac

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Fabien Saubade, Noëmie Cossec, Luc Giguelay Gesret, Christelle Kouamé, Mariem Ellouze, et al. Heat resistance of five spoilage microorganisms in a carbonated broth. Food Microbiology, 2024, 122, pp.104545. 10.1016/j.fm.2024.104545. hal-05386712

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# Heat resistance of five spoilage microorganisms in a carbonated broth

Fabien Saubade<sup>a, *</sup>, Noëmie Cossec<sup>a</sup>, Luc Giguelay Gesret<sup>a</sup>, Christelle Kouamé<sup>b</sup>, Mariem Ellouze<sup>c</sup>, Cédric Gérard<sup>c</sup>, Olivier Couvert<sup>a</sup>, Noémie Desriac<sup>a</sup>

<figure><img src="image_2.png" /></figure>

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## ARTICLE INFO

### Keywords:

Beverage

Carbonation

Modified atmosphere

Thermal treatment

Spoilage

Predictive microbiology

## ABSTRACT

Despite their acidic pH, carbonated beverages can be contaminated by spoilage microorganisms. Thermal treatments, before and/or after carbonation, are usually applied to prevent the growth of these microorganisms. However, the impact of CO₂ on the heat resistance of spoilage microorganisms has never been studied. A better understanding of the combined impact of CO₂ and pH on the heat resistance of spoilage microorganisms commonly found in carbonated beverages might allow to optimize thermal treatment.

Five microorganisms were selected for this study: *Alicyclobacillus acidoterrestris* (spores), *Aspergillus niger* (spores), *Byssochlamys fulva* (spores), *Saccharomyces cerevisiae* (vegetative cells), and *Zygosaccharomyces parabailii* (vegetative cells). A method was developed to assess the impact of heat treatments in carbonated media on microbial resistance.

The heat resistances of the five studied species are coherent with the literature, when data were available. However, neither the dissolved CO₂ concentration (from 0 to 7 g/L), nor the pH (from 2.8 to 4.1) have an impact on the heat resistance of the selected microorganisms, except for *As. niger*, for which the presence of dissolved CO₂ reduced the heat resistance. This study improved our knowledge about the heat resistance of some spoilage microorganisms in presence of CO₂.

## 1. Introduction

Carbonated beverages include several types of drinks, such as carbonated soft drinks and waters, beers, and sparkling wines (Abu-R-eidah, 2020; Ashurst, 2016; Azeredo et al., 2016). These beverages are characterized by the presence of carbon dioxide (CO₂; between 3 and 7g of dissolved CO₂/L), and usually an acidic pH (comprised between 2 and 4). Thanks to these drastic conditions, most of the pathogens and spoilage microorganisms are not able to grow (Azeredo et al., 2016). However, numerous carbonated beverages contain sugars, which can be used by some spoilage microorganisms able to grow in acidic conditions (Shankar et al., 2021). For example, yeasts can contaminate carbonated beverages, since they are commonly found in raw materials, in the packaging and even in the processing environment (Shankar et al., 2021). In addition, they can grow at pH values as low as 1.5. Among yeasts, some genera are commonly implicated in spoilage of carbonated

beverages, such as *Saccharomyces* and *Zygosaccharomyces* (Azeredo et al., 2016). Some bacteria, such as *Alicyclobacillus acidoterrestris*, are also able to spoil carbonated beverages at pH values as low as 2.5 (Huang et al., 2021; Smit et al., 2011; Steyn et al., 2011). Moulds involved in spoilage of carbonated beverages have been less studied, while spoilage due to moulds, such as *Aspergillus* and *Byssochlamys* species, has been recorded in carbonated beverages (Silva and Evelyn, 2020).

One solution to prevent the growth of these microorganisms, and consequently the spoilage of the beverage, consists in heating the product in order to inactivate the microorganisms, but the most resistant moulds and sporulated bacteria can survive. Thermal treatments are the most used process to prevent microbial spoilage (Rawson et al., 2011). However, heat treatments can have negative impacts on the nutritional and organoleptic properties of fruit and vegetables juices and beverages, as reviewed by Petruzzi et al. (2017). According to this review, it is

<footnote><sup>a</sup> Univ Brest, INRAE, Laboratoire Universitaire de Biodiversité et Écologie Microbienne, F-29000, Quimper, France</footnote>

<footnote><sup>b</sup> NPTC Nestlé Waters, 1020 avenue Georges Clémenceau, F-88800, Vittel, France</footnote>

<footnote><sup>c</sup> Nestlé Research Center, Lausanne, Switzerland</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: fabien.saubade@univ-brest.fr (F. Saubade), noemie.cossec07@outlook.fr (N. Cossec), luc.giguelay.gesret@gmail.com (L. Giguelay Gesret), christelle.kouame@waters.nestle.com (C. Kouamé), mariem.ellouze@rdls.nestle.com (M. Ellouze), cedric.gerard@rdls.nestle.com (C. Gérard), olivier.couvert@univ-brest.fr (O. Couvert), noemie.desriac@univ-brest.fr (N. Desriac).</footnote>
important to optimize the thermal treatment for each juice or beverage, in order to optimize the microbiological quality, nutritional and organoleptic quality.

In the case of carbonated drinks and beverages, a flash pasteurisation (85–95 °C, for up to 20 s) is usually performed before carbonation. According to the data available in the literature, most of microorganisms are eliminated thanks to this thermal treatment. Indeed, the heat resistance of microorganisms can be quantified thanks to different methods, such as the capillary tubes method (Zottola and Jezeski, 1969). From that, heat treatment parameters are usually calculated: the *D* values obtained at different temperatures to assess the *D*ref and *z*<sub>T</sub> values. A *D*-value is defined as the time needed at a given temperature to reduce the population of the studied microorganism by one log (a loss of 90% of the microbial population). A *z*<sub>T</sub> value is the variation of temperature needed to obtain a tenfold variation of *D* value. According to the LEMGO *D*- and *z*-value database, numerous vegetative cells of spoilage microorganisms are not able to survive high temperature thermal treatments (Althoff and Schwarzer, 2023). For example, *S. cerevisiae* *D*<sub>60</sub> value is usually lower than 10 min in fruit juices, even for the ascospores, and its *z*<sub>T</sub> value is usually comprised between 4 and 7 °C (Shearer et al., 2002; Splittstoesser et al., 1986). *Aspergillus niger* conidia also seem to have a low heat resistance, since *D*<sub>60</sub> values lower than 1 min, and *z*<sub>T</sub> values lower 4 °C were observed in citrate buffer (Shearer et al., 2002).

However, in some fruit juices and carbonated beverages, some spoilage microorganisms can significantly be more resistant to thermal treatments. For example, spores of *A. acidoterrestris* have a *D* value at 90 °C that is usually higher than 5 min and can be as high as 37.5 min, and *z*<sub>T</sub> values usually comprised between 6 and 13 °C (Leguerinel et al., 2020). This means that some *A. acidoterrestris* spores might not be affected at 95 °C for 20 s, and might even be activated to form new vegetative cells (Bevilacqua and Corbo, 2011). Spores of moulds can also be very resistant. Another example, *D*<sub>90</sub> values comprised between 1 and 20 min have been recorded for *Byssochlamys fulva* ascospores (Bayne and Michener, 1979).

In some cases, a second pasteurisation (pasteurisation tunnel) can be performed after carbonation, to control potential cross-contaminations (Ashurst, 2016; Azeredo et al., 2016). This pasteurisation can also be used as a replacement of the flash pasteurisation. The temperature of the pasteurisation tunnel rarely exceeds 70 °C, for about 20 min, even if different time/temperature couples can be found in the literature (Rachon et al., 2018; Wray, 2015). However, the impact of the thermal treatment in carbonated drinks and beverages on the potential spoilage microorganisms has rarely been studied.

Research on the impact of CO₂ on microorganisms has mainly been focused on the use of high pressure CO₂ to inactivate both the vegetative cells and spores of bacteria, yeasts, and moulds, as summarized by Zhang et al. (2006). The inactivation of few microorganisms, such as Fusarium oxysporum, Lactobacillus fructivorans, and S. cerevisiae by low pressure CO₂ microbubbles has been investigated (Kobayashi et al., 2012, 2013, 2014). Shimoda et al. (2001), developed a continuous flow system to study the impact of dissolved CO₂ and pressure on the thermal resistance of microorganisms. Dissolved CO₂ (dCO₂) can be defined as the CO₂ gas dissolved in the liquid medium, and the carbonate ions (H₂CO₃, HCO₃⁻, and CO₃²⁻). Their studies showed that dCO₂ can have a significant impact on the thermal inactivation of vegetative cells. However, as far as the authors are aware, the impact of dCO₂ on the thermal inactivation of spores of spoilage microorganisms has never been studied.

The goal of this study was to better understand the combined effects of low pH, carbonation, and heat treatments on the inactivation of spoilage microorganisms. For this purpose, five spoilage microorganisms commonly found in acidic drinks (carbonated fruit juices) were selected: *Alicyclobacillus acidoterrestris*, *Aspergillus niger*, *Byssochlamys fulva*, *Saccharomyces cerevisiae*, and *Zygosaccharomyces parabailii*. A newly developed method was used to perform thermal treatments with small volumes of carbonated media.

## 2. Material and methods

### 2.1. Microorganisms

Five spoilage microorganisms commonly found in carbonated beverages and fruit juices were selected. *A. acidoterrestris* DSM 3922, *As. niger* M750, and *B. fulva* Equasa 362 (origin: apple juice), *S. cerevisiae* UBOCC-A-217014, and *Z. parabailii* MUCL 51627 (ATCC MYA-4549). Thermal treatments were performed on the bacterium (*A. acidoterrestris*) spores, the moulds (*As. niger* and *B. fulva*) spores, and the yeasts (*S. cerevisiae*, and *Z. parabailii*) vegetative cells.

### 2.2. Preparation of moulds spores

Spore stocks of both mould strains were produced at EQUASA ESIAB, following the same protocol. Each mould was inoculated into ten Roux culture bottles containing Malt extract agar (Sigma Aldrich M9802). To ensure constant aeration of the moulds, the bottles were only partially closed during the incubation at 25 °C for *As. niger*, and 30 °C for *B. fulva*, for two weeks. The presence of spores all over the surface was checked on the bottles. In order to collect the spores, few millilitres of a sterile mix of distilled water and tween were poured into each bottle. A spreader was then used to scrap the surface. This step was performed gently in order to avoid collecting hyphae along with the spores. For each mould, the suspensions were pooled in a stomacher bag for homogenisation and filtration of eventual agar and hyphae pieces. Both mould suspensions were then centrifuged at 8000 rpm for 10 min (room temperature), and the supernatants were discarded. Each pellet was suspended in sterile glycerol 30 % for conservation at -80 °C.

The concentrations of the spore suspensions were 2.3 × 10⁸ conidia/mL and 2.2 × 10⁶ asci/mL for *As. niger* and *B. fulva*, respectively. While glycerol has a preservative effect for conservation of microorganisms at low temperatures, it might also increase the apparent heat resistance of mould spores during thermal treatments. Therefore, spore suspensions were tenfold diluted in peptone dextrose broth (PDB) before each experiment.

### 2.3. Preparation of bacterial spores

*A. acidoterrestris* was refreshed on YSG (Yeast extract, Sigma aldrich Y1625; Starch, Sigma aldrich S9765; Glucose, Sigma aldrich 47249) agar plates at pH 3.8. The plates were incubated at 45 °C for 72 h. Then one colony was used to inoculate 10 mL of BAT broth (Döhler, 204.717.020) at pH 4. This suspension was incubated overnight at 45 °C with agitation (100 rpm). A new suspension was prepared in an Erlenmeyer flask, by inoculating 1 mL of the overnight suspension into 80 mL of BAT at pH 4. This new suspension was incubated at 45 °C with agitation (100 rpm) for 8 h. The whole suspension was then used to inoculate BAT agar plates at pH 4. For each Petri dish, 1 mL of suspension was deposited and spread all over the surface. The remaining liquid was collected and discarded. The Petri dishes were then incubated at 45 °C and regular samples were collected to observe the microbial population under microscope. After 5 days of incubation, more than 99.9 % of spores were observable, and the spores were collected. In this purpose, 1 mL of sterile distilled water was poured onto each plate, before scraping gently the surface. The spore suspensions were collected and the last step was repeated in order to collect as many spores as possible. The suspensions were pooled before applying three successive centrifugations at 6000 g for 10 min.

The pellet was suspended into 100 mL sterile distilled water and the concentration was determined. For that, *A. acidoterrestris* spores were heat treated at 80 °C for 10 min. The spore stock concentration was 2.3 × 10⁸ spores/mL. This stock was stored at 4 °C until use.
## 2.4. Preparation of yeasts vegetative cells

Both yeast strains were stored in cryotubes at -80 °C. Once a month, the content of a cryotube, for each strain, was used to inoculate a Petri dish containing YPA (Sigma Aldrich Y1500). The plates were incubated at 30 °C for at least two days, to obtain isolated colonies. These plates were kept at 4 °C for up to one month.

Before each experiment, a colony was collected and inoculated in 10 mL of YPD (Sigma Aldrich Y1375). The suspensions were incubated at 30 °C with agitation (100 rpm) until they reached an OD<sub>600 nm</sub> of 1.00 ± 0.05. New suspensions were prepared by inoculating 1 mL or 100 µL into 50 mL of YPD at pH = 3.2 for *S. cerevisiae* and *Z. parabailii*, respectively. A new incubation was performed at 30 °C with agitation (100 rpm) for 26 h and 22 h for *S. cerevisiae* and *Z. parabailii*, respectively. The suspensions were ready to use once they reached an OD<sub>600 nm</sub> of 1.40 ± 0.07. This correspond to a concentration of 2.4 × 10<sup>6</sup> CFU/mL and 2.1 × 10<sup>7</sup> CFU/mL for *S. cerevisiae* and *Z. parabailii*, respectively.

## 2.5. Thermal treatments in non carbonated media using capillary tubes

Thermal treatments in non-carbonated media were performed in capillary tubes (HIRSCHMANN ringcaps 960.02.22). Each strain was studied at four selected temperatures to obtain *D* values comprised between 50 and 0.3 min. Each condition was performed at least in duplicates. The capillary method used in this publication has previously been described in the literature (Leguerinel et al., 2005; Zottola and Jezeski, 1969). Briefly, sterile capillary tubes were filled with 100 µL of the selected suspension, before sealing the extremities thanks to a blow-torch. Yeast suspensions were used directly, as they were prepared in YPD. However, before filling the tubes, the spore suspensions were tenfold diluted in the appropriate medium (PDB at pH = 3.2 for the moulds, and PDB at pH = 3.8 for *A. acidoterrestris*). The tubes were immersed in either a water or oil (for *A. acidoterrestris*) bath (Julabo TW20 Water Bath), for a determined time and immediately cooled in a mix of water and ice, in order to avoid any further damages or germination.

The content of each capillary tube was then enumerated. When necessary, serial tenfold dilutions where performed before spreading 0.1 mL of the suspension on the appropriate solid medium. For *A. acidoterrestris* and for both moulds, the suspensions were spread on PDB supplemented with agar (Sigma Aldrich M9802; 15 g/L) at pH 3.8 and 3.2, respectively. Yeast were spread on YPD supplemented with

agar. The plates were then incubated at 45 °C, 30 °C, and 25 °C, for *A. acidoterrestris*, yeasts, and moulds, respectively. The colonies were enumerated after 24–48 h.

## 2.6. Thermal treatment in carbonated media using stainless steel tubes

Thermal treatments in carbonated media were performed in “tailor-made” gas-tight stainless steel tubes.

For each organisms and each experiment, 15g of an acidified liquid medium (usually lower than pH 2) was poured in a stainless-steel tube. The medium was YPD (Sigma Aldrich Y1375) for both yeasts, while PDB (Sigma-Aldrich P6685) was used for both moulds and *A. acidoterrestris*. Six tubes corresponding to six sampling times were used for each experiment, as shown in Tables 2–6. Indeed, the analysis requires the opening of the tubes. Therefore each tube could only be used for a sampling time.

To generate the targeted CO<sub>2</sub> level, a predetermined amount of sodium bicarbonate powder (NaHCO<sub>3</sub>; Sigma Aldrich reference S6297) was added in the tubes using hypromellose capsules (LGA reference A5003) previously exposed to UV for 1 h. Hypromellose capsules dissolve in few minutes in acidic media, releasing their content.

They were used to delay CO<sub>2</sub> gas formation from the NaHCO<sub>3</sub> powder, in order to prevent any CO<sub>2</sub> loss before closing the tubes. Indeed, in an acidic medium, sodium bicarbonate forms carbonate ions (H<sub>2</sub>CO<sub>3</sub>, HCO<sub>3</sub><sup>-</sup>, and CO<sub>3</sub><sup>2-</sup>), as well as CO<sub>2</sub> in both dissolved and gas forms (Couvert et al., 2017).

The CO<sub>2</sub> formation requires H<sup>+</sup> protons, which consequently increases the pH to the targeted value. One tube was dedicated to check the final pH and the pressure, which indirectly gives the amount of CO<sub>2</sub> formed, as described by Couvert et al. (2017), after the equilibrium has been reached.

The five other tubes were used to perform thermal treatments with five pre-defined sampling times.

For that, inoculum of the studied strain was poured into empty hypromellose capsules before adding them to the five thermal treatment tubes. Here, the hypromellose capsules were used to ensure that the microorganisms would be diluted in the medium only after the increase of pH.

Similarly to the capillary tubes, all stainless-steel tubes were immersed in either a water or oil (for *A. acidoterrestris*) bath for a determined time. They were then cooled down in iced-water before opening for enumeration, as described for capillary tubes. Each

<caption>**Table 1**</caption>

D and z<sub>T</sub> values for Alicyclobacillus acidoterrestris 3922, Aspergillus niger M750, Byssochlamys fulva, Saccharomyces cerevisiae, and Zygosaccharomyces parabailii. For each D value, the coefficient of determination of the curve used to calculate this D value is indicated in brackets. For each z<sub>T</sub> value, the confidence interval (95%) calculated with MATLAB is indicated in brackets.

<table><thead><tr><th>Microorganism</th><th>Cell type</th><th>Medium</th><th>pH</th><th>Temperature (°C)</th><th>D value (min)</th><th>R<sup>2</sup></th><th>z<sub>T</sub> value (°C)</th></tr></thead><tbody><tr><td rowspan="4">A. acidoterrestris</td><td rowspan="4">spores</td><td rowspan="4">PDB</td><td rowspan="4">3.8</td><td>89.8</td><td>20.3</td><td>0.980</td><td rowspan="4">7.64 [6.99–8.30]</td></tr><tr><td>91.8</td><td>10.0</td><td>0.982</td></tr><tr><td>96.9</td><td>2.75</td><td>0.968</td></tr><tr><td>100</td><td>0.84</td><td>0.976</td></tr><tr><td rowspan="4">As. niger</td><td rowspan="4">Spores</td><td rowspan="4">PDB</td><td rowspan="4">3.2</td><td>53.9</td><td>23.3</td><td>0.953</td><td rowspan="4">3.80 [3.46–4.14]</td></tr><tr><td>55.9</td><td>5.25</td><td>0.978</td></tr><tr><td>59</td><td>0.82</td><td>0.926</td></tr><tr><td>61</td><td>0.31</td><td>0.841</td></tr><tr><td rowspan="4">B. fulva</td><td rowspan="4">Spores</td><td rowspan="4">PDB</td><td rowspan="4">3.2</td><td>81.5</td><td>17.3</td><td>0.939</td><td rowspan="4">6.53 [5.26–7.81]</td></tr><tr><td>83.6</td><td>10.4</td><td>0.857</td></tr><tr><td>86.7</td><td>3.58</td><td>0.854</td></tr><tr><td>89.8</td><td>0.97</td><td>0.823</td></tr><tr><td rowspan="4">S. cerevisiae</td><td rowspan="4">Vegetative cells</td><td rowspan="4">YPD</td><td rowspan="4">3.2</td><td>45.6</td><td>7.29</td><td>0.985</td><td rowspan="4">5.82 [2.72–8.91]</td></tr><tr><td>48.7</td><td>2.81</td><td>0.701</td></tr><tr><td>50.8</td><td>1.43</td><td>0.420</td></tr><tr><td>52.8</td><td>0.50</td><td>0.517</td></tr><tr><td rowspan="4">Z. parabailii</td><td rowspan="4">Vegetative cells</td><td rowspan="4">YPD</td><td rowspan="4">3.2</td><td>45.6</td><td>40.8</td><td>0.839</td><td rowspan="4">3.66 [3.41–3.90]</td></tr><tr><td>48.7</td><td>4.99</td><td>0.934</td></tr><tr><td>50.8</td><td>1.40</td><td>0.922</td></tr><tr><td>52.8</td><td>0.37</td><td>0.873</td></tr></tbody></table>
<caption>**Table 2**</caption>

δ and z values for A. acidoterrestris. For each δ, z, and p value, the 95% CI was given in brackets, and the precise pH and dCO₂ concentrations (g/L) were also indicated. Only one p value was calculated for the totality of the curves. ND: not determined. NS: not significant (the confidence interval values were very high).

<table><caption>**Table 3**</caption><thead><tr><th></th><th>Temperature</th><th>pH</th><th>[dCO<sub>2</sub>] (g/L)</th><th>δ value (min)</th><th>z value</th><th>p value</th></tr></thead><tbody><tr><td rowspan="10">Temperature variable; pH and dCO<sub>2</sub> constant</td><td>91.8</td><td>3.06</td><td>4.610</td><td>3.92 [3.16; 4.68]</td><td rowspan="10">z<sub>T</sub> = 6.51 [5.51; 7.51]<br>°C</td><td rowspan="10">1.24 [1.11; 1.37]</td></tr><tr><td>89.8</td><td>3.37</td><td>4.562</td><td>3.91 [3.24; 4.58]</td></tr><tr><td>87.7</td><td>3.00</td><td>4.572</td><td>7.54 [6.20; 8.89]</td></tr><tr><td>85.7</td><td>3.23</td><td>4.456</td><td>6.95 [5.74; 8.17]</td></tr><tr><td>81.5</td><td>3.39</td><td>4.440</td><td>15.26 [12.68; 17.85]</td></tr><tr><td>85.7</td><td>3.58</td><td>4.505</td><td>13.85 [11.52; 16.17]</td></tr><tr><td>81.5</td><td>3.24</td><td>4.504</td><td>28.53 [23.75; 33.32]</td></tr><tr><td>85.7</td><td>3.74</td><td>4.697</td><td>36.53 [30.01; 43.05]</td></tr><tr><td>85.7</td><td>3.27</td><td>4.537</td><td>130.79 [107.31; 154.28]</td></tr><tr><td>85.7</td><td>3.43</td><td>4.638</td><td>117.09 [96.95; 137.23]</td></tr><tr><td rowspan="10">dCO<sub>2</sub>variable; pH and temperature constant</td><td>85.7</td><td>3.23</td><td>0.000</td><td>32.23 [26.32; 38.13]</td><td rowspan="10">z<sub>CO<sub>2</sub></sub> = -38.95 [-97.74; 19.84] g/L</td><td rowspan="10"></td></tr><tr><td>85.7</td><td>3.84</td><td>0.000</td><td>39.72 [31.73; 47.72]</td></tr><tr><td>85.7</td><td>3.40</td><td>4.013</td><td>19.12 [15.57; 22.68]</td></tr><tr><td>85.7</td><td>3.54</td><td>4.074</td><td>20.85 [17.36; 24.35]</td></tr><tr><td>85.7</td><td>3.25</td><td>5.161</td><td>32.23 [25.04; 39.42]</td></tr><tr><td>85.7</td><td>3.37</td><td>5.066</td><td>49.21 [40.10; 58.32]</td></tr><tr><td>85.7</td><td>3.02</td><td>5.906</td><td>75.79 [58.87; 92.70]</td></tr><tr><td>85.7</td><td>3.22</td><td>6.007</td><td>66.33 [53.08; 79.59]</td></tr><tr><td>85.7</td><td>2.88</td><td>7.174</td><td>42.92 [34.79; 51.05]</td></tr><tr><td>85.7</td><td>2.56</td><td>6.975</td><td>44.65 [36.77; 52.53]</td></tr><tr><td rowspan="10">pH variable; temperature and dCO<sub>2</sub> constant</td><td>85.7</td><td>2.94</td><td>ND</td><td>31.84 [26.49; 37.20]</td><td rowspan="10">z<sub>pH</sub> = -113.91 [NS]</td><td rowspan="10"></td></tr><tr><td>85.7</td><td>2.89</td><td>4.461</td><td>28.55 [23.60; 33.49]</td></tr><tr><td>85.7</td><td>3.67</td><td>4.560</td><td>29.66 [24.49; 34.82]</td></tr><tr><td>85.7</td><td>3.91</td><td>4.478</td><td>32.43 [26.98; 37.88]</td></tr><tr><td>85.7</td><td>4.23</td><td>4.427</td><td>31.77 [26.36; 37.18]</td></tr><tr><td>85.7</td><td>4.21</td><td>ND</td><td>47.17 [38.16; 56.18]</td></tr></tbody></table>

δ and z values for As. niger. For each δ, z, and p value, the 95% CI was given in brackets, and the precise pH and dCO₂ concentrations (g/L) were also indicated. Only one p value was calculated for the totality of the curves.

<table><thead><tr><th></th><th>Temperature</th><th>pH</th><th>[dCO<sub>2</sub>] (g/L)</th><th>δ value (min)</th><th>z value</th><th>p value</th></tr></thead><tbody><tr><td rowspan="10">Temperature variable; pH and dCO<sub>2</sub> constant</td><td>58</td><td>3.09</td><td>4.482</td><td>1.36 [0.99; 1.74]</td><td rowspan="10">z<sub>T</sub> = 4.25 [3.94; 4.57]<br>°C</td><td rowspan="10">1.03 [0.91; 1.15]</td></tr><tr><td>55.9</td><td>3.42</td><td>4.464</td><td>1.31 [0.94; 1.68]</td></tr><tr><td>53.9</td><td>3.34</td><td>4.632</td><td>3.99 [3.05; 4.93]</td></tr><tr><td>51.8</td><td>3.24</td><td>4.478</td><td>3.42 [2.63; 4.20]</td></tr><tr><td>49.7</td><td>3.16</td><td>4.460</td><td>8.57 [6.53; 10.61]</td></tr><tr><td>49.7</td><td>3.38</td><td>4.518</td><td>8.73 [6.91; 10.55]</td></tr><tr><td>49.7</td><td>3.17</td><td>4.491</td><td>26.80 [16.00; 37.59]</td></tr><tr><td>49.7</td><td>3.00</td><td>4.614</td><td>22.07 [17.38; 26.75]</td></tr><tr><td>49.7</td><td>3.56</td><td>4.621</td><td>122.42 [97.11; 147.72]</td></tr><tr><td>49.7</td><td>3.26</td><td>4.509</td><td>103.46 [63.77; 143.16]</td></tr><tr><td rowspan="10">dCO<sub>2</sub>variable;<br>pH and temperature constant</td><td>51.8</td><td>3.29</td><td>0.000</td><td>132.40 [105.16; 159.65]</td><td rowspan="10">z<sub>CO<sub>2</sub></sub> = 7.14 [5.94; 8.33] g/L</td><td rowspan="10"></td></tr><tr><td>51.8</td><td>3.22</td><td>0.000</td><td>229.31 [173.76; 284.86]</td></tr><tr><td>51.8</td><td>3.61</td><td>4.001</td><td>35.25 [27.98; 42.52]</td></tr><tr><td>51.8</td><td>3.45</td><td>4.024</td><td>47.31 [35.73; 58.89]</td></tr><tr><td>51.8</td><td>3.31</td><td>4.945</td><td>30.75 [24.03; 37.48]</td></tr><tr><td>51.8</td><td>3.10</td><td>5.122</td><td>30.60 [24.28; 36.92]</td></tr><tr><td>51.8</td><td>3.17</td><td>5.943</td><td>28.98 [22.96; 35.00]</td></tr><tr><td>51.8</td><td>3.04</td><td>5.906</td><td>24.85 [19.58; 30.13]</td></tr><tr><td>51.8</td><td>3.30</td><td>7.050</td><td>16.73 [13.02; 20.44]</td></tr><tr><td>51.8</td><td>3.28</td><td>7.453</td><td>16.28 [12.92; 19.63]</td></tr><tr><td rowspan="10">pH variable; temperature and dCO<sub>2</sub> constant</td><td>51.8</td><td>2.67</td><td>4.467</td><td>33.33 [22.25; 44.40]</td><td rowspan="10">z<sub>pH</sub> = 31.94 [-75.71; 139.6]</td><td rowspan="10"></td></tr><tr><td>51.8</td><td>2.75</td><td>4.467</td><td>27.50 [21.84; 33.16]</td></tr><tr><td>51.8</td><td>3.69</td><td>4.563</td><td>30.84 [24.09; 37.60]</td></tr><tr><td>51.8</td><td>4.07</td><td>4.500</td><td>34.28 [27.05; 41.51]</td></tr><tr><td>51.8</td><td>4.09</td><td>4.624</td><td>30.82 [24.44; 37.20]</td></tr><tr><td>51.8</td><td>4.01</td><td>4.382</td><td>40.64 [31.56; 49.73]</td></tr></tbody></table>

experiment was performed in duplicate.

For each strain, five temperatures (dCO₂ = 4.5 g/L, pH = 3.2) were selected in order to obtain δ values comprised between 200 and 1 min. Then a temperature giving a δ-value comprised between 10 and 30 min was selected among these five temperatures. At this temperature, five additional dCO₂ concentrations (0, 4, 5, 6, and 7 g/L) and three additional pH (2.8, 3.8, and 4.1) were studied. The conditions tested for the five microorganisms are given in Tables 2–6.

## 2.7. Models

Two models were fitted on the heat destruction kinetics depending on their shape. For the capillary tubes, the linear model was used to obtain *D* values (eq. (1)).

For the stainless steel tubes, the Weibull model has been used instead (Couvert et al., 2005). This model (eq. (2)), which gives *δ* and *p* values, is used when the curve is or might be non-linear. Similarly to *D* values in linear inactivations, a *δ*-value corresponds to the time needed at a given temperature to achieve the first decimal reduction (the first loss of 90%
<caption>**Table 4**</caption>

δ and z values for B. fulva. For each δ, z, and p value, the 95% CI was given in brackets, and the precise pH and dCO₂ concentrations (g/L) were also indicated. Only one p value was calculated for the totality of the curves.

<table><caption>**Table 5**</caption><thead><tr><th></th><th>Temperature</th><th>pH</th><th>[dCO<sub>2</sub>] (g/L)</th><th>δ value (min)</th><th>z value</th><th>p value</th></tr></thead><tbody><tr><td rowspan="10">Temperature variable; pH and dCO<sub>2</sub> constant</td><td><strong>86.7</strong></td><td>3.70</td><td>4.439</td><td>2.47 [1.96; 2.98]</td><td rowspan="10">z<sub>T</sub> = 5.28 [4.72; 5.83]<br>°C</td><td rowspan="10">1.43 [1.19; 1.66]</td></tr><tr><td>3.67</td><td>4.514</td><td>2.40 [1.92; 2.88]</td></tr><tr><td><strong>83.6</strong></td><td>3.33</td><td>4.400</td><td>8.40 [6.67; 10.12]</td></tr><tr><td>3.68</td><td>4.486</td><td>8.17 [6.57; 9.76]</td></tr><tr><td><strong>81.5</strong></td><td>3.51</td><td>4.420</td><td>23.24 [18.56; 27.93]</td></tr><tr><td>3.41</td><td>4.441</td><td>23.17 [18.51; 27.84]</td></tr><tr><td><strong>79.5</strong></td><td>3.08</td><td>4.585</td><td>66.35 [46.95; 85.74]</td></tr><tr><td>3.27</td><td>4.443</td><td>63.27 [50.39; 76.15]</td></tr><tr><td><strong>77.4</strong></td><td>3.07</td><td>4.512</td><td>102.51 [76.48; 128.53]</td></tr><tr><td>3.13</td><td>4.439</td><td>73.70 [58.97; 88.44]</td></tr><tr><td rowspan="10">dCO<sub>2</sub>variable; pH and temperature constant</td><td>81.5</td><td>3.28</td><td><strong>0.000</strong></td><td>27.17 [21.08; 33.26]</td><td rowspan="10">z<sub>CO<sub>2</sub></sub> = 21.69 [9.72; 33.65] g/L</td><td rowspan="10"></td></tr><tr><td>3.32</td><td><strong>0.000</strong></td><td>31.35 [25.28; 37.42]</td></tr><tr><td>81.5</td><td>ND</td><td><strong>4.119</strong></td><td>27.12 [21.05; 33.19]</td></tr><tr><td>3.35</td><td><strong>4.058</strong></td><td>21.30 [17.15; 25.46]</td></tr><tr><td>3.66</td><td><strong>5.028</strong></td><td>25.49 [20.05; 30.92]</td></tr><tr><td>3.20</td><td><strong>5.045</strong></td><td>26.57 [20.72; 32.42]</td></tr><tr><td>81.5</td><td>3.19</td><td><strong>6.006</strong></td><td>14.05 [11.25; 16.85]</td></tr><tr><td>3.44</td><td><strong>5.970</strong></td><td>13.77 [11.22; 16.32]</td></tr><tr><td>3.16</td><td><strong>6.988</strong></td><td>14.31 [11.31; 17.31]</td></tr><tr><td>4.47</td><td><strong>7.130</strong></td><td>21.53 [17.32; 25.75]</td></tr><tr><td rowspan="10">pH variable; temperature and dCO<sub>2</sub> constant</td><td>81.5</td><td>2.74</td><td>4.453</td><td>14.17 [10.94; 17.40]</td><td rowspan="10">z<sub>pH</sub> = 5.04 [2.34; 7.74]</td><td rowspan="10"></td></tr><tr><td>2.77</td><td>4.480</td><td>13.16 [10.33; 16.00]</td></tr><tr><td>81.5</td><td>3.51</td><td>4.420</td><td>21.99 [17.66; 26.32]</td></tr><tr><td>3.84</td><td>4.387</td><td>29.47 [22.34; 36.59]</td></tr><tr><td>81.5</td><td>3.91</td><td>4.525</td><td>26.80 [20.86; 32.75]</td></tr><tr><td>4.14</td><td>4.579</td><td>34.20 [22.85; 45.55]</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

δ and z values for S. cerevisiae. For each δ, z, and p value, the 95% CI was given in brackets, and the precise pH and dCO₂ concentrations (g/L) were also indicated. Only one p value was calculated for the totality of the curves.

<table><thead><tr><th></th><th>Temperature</th><th>pH</th><th>[dCO<sub>2</sub>] (g/L)</th><th>δ value (min)</th><th>z value</th><th>p value</th></tr></thead><tbody><tr><td rowspan="10">Temperature variable; pH and dCO<sub>2</sub> constant</td><td><strong>50.8</strong></td><td>3.61</td><td>4.395</td><td>9.32 [6.54; 12.10]</td><td rowspan="10">z<sub>T</sub> = 6.50 [5.03; 7.98]<br>°C</td><td rowspan="10">1.20 [0.98; 1.42]</td></tr><tr><td>3.21</td><td>4.388</td><td>4.01 [2.51; 5.51]</td></tr><tr><td>3.13</td><td>4.398</td><td>5.03 [3.50; 6.57]</td></tr><tr><td>3.22</td><td>4.445</td><td>12.45 [8.41; 16.49]</td></tr><tr><td><strong>46.7</strong></td><td>ND</td><td>ND</td><td>33.78 [23.48; 44.07]</td></tr><tr><td>3.28</td><td>4.67</td><td>34.59 [23.49; 45.68]</td></tr><tr><td>3.41</td><td>4.444</td><td>70.86 [47.59; 94.14]</td></tr><tr><td>3.35</td><td>4.362</td><td>68.69 [48.00; 89.38]</td></tr><tr><td><strong>40.5</strong></td><td>3.34</td><td>4.467</td><td>131.52 [90.92; 172.12]</td></tr><tr><td>3.45</td><td>4.39</td><td>249.80 [158.91; 340.69]</td></tr><tr><td rowspan="10">dCO<sub>2</sub>variable; pH and temperature constant</td><td>46.7</td><td>3.25</td><td><strong>0.000</strong></td><td>18.89 [12.83; 24.95]</td><td rowspan="10">z<sub>CO<sub>2</sub></sub> = -15.29 [-27.64;-2.94] g/L</td><td rowspan="10"></td></tr><tr><td>3.22</td><td><strong>0.000</strong></td><td>11.00 [6.12; 15.88]</td></tr><tr><td>3.22</td><td><strong>4.071</strong></td><td>15.19 [10.35; 20.03]</td></tr><tr><td>3.66</td><td><strong>4.205</strong></td><td>13.37 [9.42; 17.31]</td></tr><tr><td>3.43</td><td><strong>5.023</strong></td><td>25.98 [18.79; 33.16]</td></tr><tr><td>3.36</td><td><strong>5.156</strong></td><td>51.36 [27.77; 74.94]</td></tr><tr><td>3.38</td><td><strong>6.005</strong></td><td>41.67 [29.15; 54.20]</td></tr><tr><td>3.39</td><td><strong>5.745</strong></td><td>49.21 [32.62; 65.80]</td></tr><tr><td>3.16</td><td><strong>6.988</strong></td><td>33.59 [24.30; 42.88]</td></tr><tr><td>4.47</td><td><strong>7.130</strong></td><td>38.50 [28.02; 48.97]</td></tr><tr><td rowspan="10">pH variable; temperature and dCO<sub>2</sub> constant</td><td>46.7</td><td>3.02</td><td>4.469</td><td>17.93 [12.64; 23.22]</td><td rowspan="10">z<sub>pH</sub> = 12.30 [-19.59; 44.19]</td><td rowspan="10"></td></tr><tr><td>2.86</td><td>4.429</td><td>11.85 [8.15; 15.55]</td></tr><tr><td>46.7</td><td>3.88</td><td>4.499</td><td>27.37 [19.45; 35.29]</td></tr><tr><td>46.7</td><td>4.25</td><td>4.489</td><td>16.08 [11.35; 20.81]</td></tr><tr><td>4.01</td><td>4.459</td><td>19.11 [13.82; 24.40]</td></tr><tr><td>4.01</td><td>4.459</td><td>37.73 [22.83; 52.62]</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

of the microbial population). The p-values indicates the inclination of the curve. The Weibull model equation is given in equation (2).

$$ \log N = \log N_0 - \frac{t}{D} \qquad (1) $$

$$ \log N = \log N_0 - \left(\frac{t}{\delta}\right)^p \qquad (2) $$

In these equations, N and N₀ are the number of microorganisms at a given time (t), and at the beginning of the experiment (t₀), respectively.

Secondary models were fitted to quantify the effect of temperature (eq. (3)); from Couvert et al. (2005)), pH (eq. (4)); from Couvert et al. (2005)) and dissolved carbon dioxide (eq. (5)); adapted from the inhibitory model in Coroller et al. (2015)) on the heat resistance parameter (D or δ).

$$ \log \delta = \log \delta^* - \frac{T - T^*}{z_T} \qquad (3) $$

Where δ (or D) is the heat resistance at the temperature T, δ* (or D*) is the heat resistance at the reference temperature T*, and z_T is the increase
<caption>**Table 6**</caption>

δ and z values for Z. parabailii. For each δ, z, and p value, the 95% CI was given in brackets, and the precise pH and dCO₂ concentrations (g/L) were also indicated. Only one p value was calculated for the totality of the curves.

<table><thead><tr><th></th><th>Temperature</th><th>pH</th><th>[dCO<sub>2</sub>] (g/L)</th><th>δ value (min)</th><th>z value</th><th>p value</th></tr></thead><tbody><tr><td rowspan="10">Temperature variable; pH and dCO<sub>2</sub> constant</td><td><strong>50.8</strong></td><td>3.32</td><td>4.393</td><td>0.49 [0.34; 0.63]</td><td rowspan="10">z<sub>T</sub> = 3.93 [3.23; 4.64]<br>°C</td><td rowspan="10">0.57 [0.51; 0.62]</td></tr><tr><td rowspan="2"><strong>48.7</strong></td><td>3.22</td><td>4.605</td><td>0.38 [0.26; 0.49]</td></tr><tr><td>ND</td><td>ND</td><td>1.19 [0.84; 1.54]</td></tr><tr><td rowspan="2"><strong>46.7</strong></td><td>3.23</td><td>4.534</td><td>2.46 [1.65; 3.28]</td></tr><tr><td>3.25</td><td>4.613</td><td>6.78 [4.78; 8.79]</td></tr><tr><td rowspan="2"><strong>45.6</strong></td><td>3.29</td><td>4.451</td><td>6.88 [4.71; 9.06]</td></tr><tr><td>3.26</td><td>4.731</td><td>12.14 [8.38; 15.90]</td></tr><tr><td rowspan="2"><strong>43.6</strong></td><td>3.27</td><td>4.345</td><td>12.94 [9.08; 16.79]</td></tr><tr><td>3.29</td><td>4.929</td><td>33.55 [23.65; 43.46]</td></tr><tr><td rowspan="2">dCO<sub>2</sub>variable; pH and temperature constant</td><td rowspan="2">45.6</td><td>3.30</td><td>4.429</td><td>39.20 [27.55; 50.85]</td></tr><tr><td>3.17</td><td><strong>0.000</strong></td><td>9.18 [6.35; 12.00]</td></tr><tr><td rowspan="10">pH variable; temperature and dCO<sub>2</sub> constant</td><td rowspan="2">45.6</td><td>3.23</td><td><strong>0.000</strong></td><td>7.13 [4.85; 9.41]</td><td rowspan="10">z<sub>CO<sub>2</sub></sub> = 34.44 [-27.11; 95.99] g/L</td><td rowspan="10"></td></tr><tr><td>3.50</td><td><strong>3.990</strong></td><td>9.37 [6.61; 12.14]</td></tr><tr><td rowspan="2">45.6</td><td>3.30</td><td><strong>3.989</strong></td><td>9.54 [6.72; 12.35]</td></tr><tr><td>3.52</td><td><strong>5.051</strong></td><td>17.30 [11.30; 23.30]</td></tr><tr><td rowspan="2">45.6</td><td>3.20</td><td><strong>5.051</strong></td><td>14.13 [9.89; 18.38]</td></tr><tr><td>3.41</td><td><strong>5.911</strong></td><td>6.03 [4.18; 7.87]</td></tr><tr><td rowspan="2">45.6</td><td>3.48</td><td><strong>5.884</strong></td><td>5.65 [3.93; 7.38]</td></tr><tr><td>3.36</td><td><strong>6.773</strong></td><td>3.52 [2.30; 4.75]</td></tr><tr><td rowspan="2">45.6</td><td>3.41</td><td><strong>7.013</strong></td><td>3.71 [2.52; 4.90]</td></tr><tr><td>45.6</td><td><strong>2.96</strong></td><td>4.435</td><td>12.73 [8.94; 16.52]</td><td rowspan="5">z<sub>pH</sub> = -22.82 [-150.74; 105.10]</td></tr><tr><td rowspan="2">45.6</td><td>3.12</td><td>4.313</td><td>12.08 [8.50; 15.66]</td></tr><tr><td>4.11</td><td>4.483</td><td>7.40 [5.11; 9.70]</td></tr><tr><td rowspan="2">45.6</td><td>3.80</td><td>4.292</td><td>5.81 [4.02; 7.59]</td></tr><tr><td>4.10</td><td>4.352</td><td>16.11 [11.18; 21.05]</td></tr><tr><td rowspan="2">45.6</td><td>4.32</td><td>4.495</td><td>9.30 [6.55; 12.04]</td></tr></tbody></table>

of temperature leading a ten-fold reduction of δ.

$$ \log \delta = \log \delta^* + \frac{pH - pH^*}{z_{pH}} \quad (4) $$

Where δ (or D) is the heat resistance at the pH, δ* (or D*) is the heat resistance at the reference pH*, and z<sub>pH</sub> is the variation of pH leading a ten-fold variation of δ.

$$ \log \delta = \log \delta^* - \frac{dCO_2 - dCO_2^*}{z_{CO_2}} \quad (5) $$

Where δ (or D) is the heat resistance at the dissolved CO₂ concentration dCO₂, δ* (or D*) is the heat resistance at the reference dCO₂ concentration (4.5 g/L), and z<sub>CO₂</sub> is the variation of dCO₂ leading a ten-fold variation of δ (or D).

The fitting of all the models were performed using function nlinfit (Statistical Toolbox, MATLAB R2020a, The Mathworks, Natick, USA).

## 3. Results and discussion

### 3.1. Heat resistance in capillary tubes without CO₂

The thermal resistance of the five selected microorganisms were firstly determined in non-carbonated media, thanks to the capillary tube method. For each microorganism, one medium and one pH was selected. The logarithmic reductions of spores (bacterium and moulds) and vegetative cells (yeasts) suspensions were studied at four temperatures in duplicate. The D and z<sub>T</sub> values were calculated for each microorganism, in a given pH and medium, for four temperature values and are given in Table 1. The results obtained for As. niger are presented in Figs. 1 and 2, as an illustration. All the other figures are given in supplementary data (Supplementary Figs. 1–8).

According to the results, A. acidoterrestris 3922 was the most resistant microorganism of this study, with D-value of 2.75 min at 96.9 °C and a z<sub>T</sub> value of 7.64 °C. This is in agreement with previous studies. Indeed, z<sub>T</sub> values comprised between 6.1 and 10.8 °C have been recorded for several strains of A. acidoterrestris when thermal treatments have been performed in fruit juices with pH values close to 3.8 (López et al., 2011;

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Changes in the concentrations (log(CFU/mL) of spores of As. niger M750 in PDB, pH = 3.2 when exposed to different static temperatures: 53.9 °C (lozenges), 55.9 °C (squares), 96.9 °C (triangles), and 100 °C (stars). For each condition, replicates are indicated by black and empty symbols. At 55.9 °C, a third replicate has been performed (blue squares).</figcaption></figure>

Molva and Baysal, 2015; Splittstoesser et al., 1994). Furthermore, Leguerinel et al. (2020) calculated the D<sub>90</sub> (D-value at 90 °C) of different strains of A. acidoterrestris, including the A. acidoterrestris 3922 strain, from the data of different publications. The D<sub>90</sub> obtained for this strain in different media is comprised between 6.5 and 12.6 min. The D<sub>91.8</sub> found in this study (10.0 min) is therefore in agreement with the literature.

For B. fulva Equasa 362 asci, the D values recorded in this study are lower than those recorded in the literature for some other strains. Indeed, our strain had a D<sub>86.7</sub> and a D<sub>89.8</sub> of 3.58 min and 0.97 min, respectively. Bayne and Michener (1979) studied the thermal resistance of ascospores from 25 B. fulva strains in tartrate buffer at pH values comprised between 3.6 and 5.0. They found D<sub>90</sub> values comprised
<figure><img src="image_4.png" /><figcaption>Fig. 2. logarithmic reductions of the D values of As. niger.</figcaption></figure>

between 1.3 and 17 min. This means that our strain is up to 17 times less resistant. However, they also excluded nine strains that did not survive 20 min at 87.8 °C, which would have been the case of our strain.

As. niger M750 spores were the less resistant spores of this study. However, the D (D₆₁ = 0.31 min) and zT (3.8 °C) values obtained in this study are coherent with values obtained in citrate medium in acidic conditions (pH close to 3.0–4.0). Indeed, another As. niger strain exhibited a D₆₀ value comprised between 0.376 and 0.451 min and a zT value comprised between 3.3 and 3.7 °C (Shearer et al., 2002). The As. niger spores tested in our study are nevertheless significantly more resistant than the two yeasts vegetative cells studied in this study.

The vegetative cells of the two yeast strains of this study have been submitted to thermal treatments over the same range of temperature (45–53 °C). They exhibited similar resistance for medium temperature. For example, the D₅₀.₈ values are 1.40 ± 0.26 and 1.43 ± 0.27 min for Z. parabailii and S. cerevisiae, respectively. However, Z. parabailii exhibited a higher resistance at low temperature. Indeed, the D₄₅.₆ (40.8 min) of this strain was about 5 times higher than for S. cerevisiae UBOCC-A-217014 (D₄₅.₆ = 7.29 min). On the contrary, the D₅₂.₈ (0.50 min) value of S. cerevisiae was times 1.35 higher than for Z. parabailii (D₅₂.₈ = 0.37 min), even though both values are not significantly different (p > 0.05). S. cerevisiae is therefore less impacted by the increase of temperature than Z. parabailii, which can be quantified by the zT values (5.82 and 3.66 °C for S. cerevisiae and Z. parabailii, respectively). The values obtained for Z. parabailii MUCL 51627 can hardly be compared with the literature, since no other Z. parabailii strains have been studied. Shearer et al. (2002) found D₆₀ values of 0.039 and 0.008 min, and zT values of 3.3 and 2.1 °C, for a Z. rouxii strain in citrate buffer at pH 3.5 and 4.0, respectively. Our Z. parabailii strain could therefore be considered as ten times more resistant than this Z. rouxii. However more investigations are necessary since the conditions used are different and only one strain was studied in each study. Vegetative cells of S. cerevisiae UBOCC-A-217014 exhibited a low heat resistance compared to other S. cerevisiae strains. Indeed, Shearer et al. (2002) estimated the D₆₀ values of vegetative cells of a S. cerevisiae strain in citrate buffer, and obtained values comprised between 1.3 and 2.8 min depending on the pH, as well as zT values comprised between 3.5 and 4.0 °C. Similarly, Put and Jong (1982) reported D₆₀ values of 0.11 and 0.32 min in citrate-phosphate buffer for 18 S. cerevisiae strains, which is about four times and ten times higher than the estimated D₆₀ value (2.90 × 10⁻² min) of our strain. In fruit juices, Shearer et al. (2002) also obtained D₅₇ values comprised between 9 and 32 min, as well as zT values comprised between 4.0 and 6.1 °C in the same conditions. The D₅₇ value of our strain can be estimated at 8.93 × 10⁻² min. Also in a fruit juice, reported a D₅₅ value of 0.9 min for

vegetative cells of S. cerevisiae, while the D₅₅ value of our strain can be estimated at 0.19 min. More research on the intraspecific variability is needed to better understand the heat resistance of S. cerevisiae.

## 3.2. Heat resistance in carbonated broth (in stainless steel tubes)

In order to assess the combined impact of pH and dCO₂ on the thermal resistance of the five selected microorganisms, thermal treatments were performed in stainless steel tubes. Firstly, thermal treatments at different temperatures (from 49.7 to 91.8 for spores and from 40.5 to 50.8 for vegetative cells) were performed at pH 3.2, with 4.5g of dCO₂/L, for each strain, allowing the determination of zT values. Then a temperature (central point) giving a δ value comprised between 10 and 40 min was selected for each microorganism. Thermal treatments were performed at this temperature, at pH 3.2, and at dCO₂ concentrations comprised between 0 and 7 g/L, allowing the determination of zCO₂ values. A last set of experiment was performed at the same temperature, with 4.5g of dCO₂/L, and at pH comprised between 2.6 and 4.3 for determining zpH values. The precise temperature, pH, dCO₂ concentrations, δ, p and z values collected are summarized in Tables 2–6 for A. acidoterrestris, As. niger, B. fulva, S. saccharomyces, and Z. parabailii, respectively. The results obtained for As. niger are presented in Figs. 3 and 4, as an illustration. All the other figures are given in supplementary data (Supplementary Figs. 9–16).

For A. acidoterrestris, the δ-values were estimated at five different temperatures between 81.5 and 91.8 °C giving a zT value of 6.51 [5.51; 7.51] °C.

Thermal treatments performed with dCO₂ concentration from 0 to 7 g/L showed that dCO₂ concentration does not have an impact on heat resistance. Consequently, the zCO₂ value is estimated at -38.95 [-97.74; 19.84]g/L.

Likewise the heat resistance is not impacted by the pH in a range from pH 2.94 and 4.21, given a zpH value of -113.9.

For B. fulva, δ-values were estimated at temperatures comprised between 77.4 and 86.7 °C, which gave a zT value of 5.28 [4.72; 5.83] °C. In addition, the results obtained at different dCO₂ concentrations show that CO₂ (up to 7 g/L) does not have an impact on the thermal resistance of this microorganism (zCO₂ = 21.69 [9.72; 33.65]g/L). pH also have a no effect on its thermal resistance, since the zpH obtained was 5.04 [2.34; 7.74]. However, more data would be needed to really draw a conclusion.

For As. niger, a zT value of 4.25 [3.94; 4.57] °C was obtained from δ-values estimated at temperatures comprised between 49.7 and 58.0 °C. The thermal resistance of this microorganism was impacted by dCO₂ concentration over the range 0–7 g/L. Indeed, a zCO₂ value of 7.14 [5.94; 8.33]g/L has been obtained. This means that the δ-value (17 min) at 7 g/L is approximately ten times lower than the δ-value (181 min) at 0 g/L. On the contrary, pH does not have an impact on the thermal resistance of As. niger, since its zpH value is estimated at 31.94 [-75.71; 139.6].

The zT value obtained for S. cerevisiae vegetative cells was 6.50 [5.03; 7.98] °C, based on the δ-values estimated at temperatures comprised between 40.5 and 50.8 °C. For this microorganism, the thermal resistance is not affected by both pH and dCO₂ concentration, since the zpH and zCO₂ obtained were 12.30 [-19.59; 44.19] and -15.29 [-27.64; -2.94]g/L, respectively. This last result would be in disagreement with the literature. Indeed, Shimoda et al. (2001) found that D values were significantly decreased when the dCO₂ concentration was increased. However, they did not precise the dCO₂ concentration unit. After that, two other studies also showed the impact of dCO₂ on the thermal resistance of S. cerevisiae (Kobayashi et al., 2010, 2014). The confidence intervals obtained in our study for both zpH and zCO₂ are very wide. Therefore, in order to really be able to conclude, wider ranges of pH and dCO₂ concentration should be studied.

With Z. parabailii vegetative cells, temperatures between 43.6 and 50.8 °C were selected to estimate δ-values. A zT value of 3.93 [3.23; 4.64] °C was then obtained. Based on the zCO₂ value obtained (34.44 [-27.11; 95.99]g/L), the dCO₂ concentration does not have an impact
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Changes in the concentration of As. niger spores in PDB at different conditions of pH, dCO₂, and temperatures (central point: pH = 3.2, dCO₂ = 4.5 g/L, and T = 51.8 °C). For each condition, dots and circles represent replicates.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** logarithmic reductions of the delta values depending on temperature, pH or dCO₂ for As. niger.</figcaption></figure>

on the heat resistance of *Z. parabailii*. pH has no impact on the thermal resistance of this microorganism, since a zₚH value of -22.82 [-150.7; 105.1] was obtained. However, the confidence interval is too wide to be able to conclude, and a wider range of pH should be tested to conclude.

### 3.3. Limitations of the study

While these results enhance our knowledge on spoilage microorganisms' heat resistance, some limitations need to be highlighted. Firstly, only five microorganisms, and one strain per species, has been
studied. In addition, the selected strains have been isolated from fruit juices, but not from carbonated beverages. As. niger strains frequently exposed to CO2, might exhibit higher heat resistance in carbonated beverages. More strains from different sources should be studied in order to have enough data for predictive microbiology. The possible presence of persister/viable but not culturable (VBNC) cells has not been taken into account. Indeed, even if the formation of such cells usually takes weeks or month, in some conditions it might occur in less than an hour (Liu et al., 2023; Pazos-Rojas et al., 2024; Xu et al., 2020). Persister/VBNC cells can not be quantified on agar plates, which was the only enumeration method used in this study. This limitation should be considered in further studies.

The heat resistance measurement method is also different from heat treatments in carbonated beverage industries. Indeed, for a quick and repeatable heat transfer inside the stainless steel tubes, we had to agitate the tubes at the beginning of the heat treatments. In industries, cans or bottles would not be agitated. In addition, to measure the dCO₂ concentrations, we recorded the pressure in tubes at 25 °C. During heat treatments, the temperature will lead to a changing in the equilibrium, and therefore the dCO₂ concentration will not be the same inside the tube depending on the heat treatment temperature. The ratio headspace/liquid volume also impacts this equilibrium. This means that results obtained in stainless steel tubes are not directly comparable to those obtained in cans or bottles with different headspace/liquid volume ratio. Verification should therefore be performed in the final recipient with the right volumes, before to really conclude. Finally, the experiment were performed in broth media (PDB, and YPD), in order to insure microorganisms' viability. Experiments performed in carbonated fruit juices might lead to different results and should be further investigated.

This study considered only the heat resistance of spoilage microorganisms in carbonated or not acidic beverages. As we saw earlier, some of the selected microorganisms (spores) might not be fully eliminated by a pasteurisation after carbonation. However, to truly assess the potential spoilage by these microorganisms, their ability to grow in carbonated beverages should also be taken into consideration. This means not only to study their ability to grow in an acidic medium with CO₂ concentrations up to 7 g/L, but also to assess their recovery abilities in carbonated beverages after a thermal treatment.

## 4. Conclusion

A. acidoterrestris and B. fulva spores were the most heat resistant cells of this study, even though B. fulva spores were significantly less resistant than A. acidoterrestris spores. Indeed, B. fulva spores had a D₈₈°C value of only 1.6 min, against 20.3 min for A. acidoterrestris. In addition, A. acidoterrestris spores are also the less impacted by temperature variations, with a zT value of 7.49 °C. Spores of both these microorganisms might therefore be able to survive a flash pasteurisation at 95 °C for 20 s, depending on the initial spore concentration (only 0.08 and 1.90 decimal reductions for A. acidoterrestris and B. fulva, respectively).

As. niger spores are significantly more resistant than the yeast vegetative cells, with a D₅₃°C value of 23.3 ± 4.2 min, while the D₅₂°C values for both yeasts are 0.50 ± 0.35 and 0.37 ± 0.10 min for S. cerevisiae and Z. parabailii, respectively. B. fulva asci and S. cerevisiae vegetative cells exhibit similar sensitivity to heat variation, with zT values of 6.36 and 6.15 °C, respectively. As. niger spores and Z. parabailii vegetative cells are the most sensitive cells to heat variations, with zT values of 3.72 and 3.43 °C, respectively. None of these three microorganisms could survive a flash pasteurisation at 95 °C for 20 s.

The five microorganisms were exposed to heat treatments in stainless steel tubes with 4.5g of dCO₂/L at different temperatures, or with different dCO₂ concentrations, but one temperature. According to the results, only spores of As. niger exhibited clearly lower δ-values when the dCO₂ concentration was increased. The thermal resistance of S. cerevisiae vegetative cells might be increased while increasing the dCO₂ concentration. However, a wider range of dCO₂ should be studied

to obtain a more precise zCO₂ value. Therefore, based on these results, dCO₂ addition may not be used in order to reduce time and temperatures of thermal treatments of carbonated food products.

The impact of pH on the thermal resistance of the selected microorganisms in a carbonated medium (4.5 g/L) has also been assessed. According to the results, pH seems to have an impact on the thermal resistance of B. fulva only in the conditions tested. However, the range of pH studied (2.8-4.1) is likely too narrow to be able to draw a conclusion. Therefore, neither pH alone, nor in combination with CO₂, seem to be potential ways to reduce time and temperatures of thermal treatments of carbonated food products. Further investigations on the impact of pH on a wider range would allow to draw a more precise conclusion.

Data obtained in this study still can be used, as it improves our knowledge on thermal resistance of the five tested spoilage microorganisms. Based on the microorganisms studied here, the addition of CO₂ will reduce neither the duration nor the pasteurisation temperature. Indeed, since dCO₂ has no impact on the thermal resistance of the most resistant microorganisms (spores of A. acidoterrestris and B. fulva), a high temperature remains needed, even for carbonated beverages.

## Funding

This study was financed by NESTLE WATERS®

## Data availability statement

The data, tools, and material (or their source) that support the protocol are available from the corresponding author upon reasonable request.

## CRediT authorship contribution statement

Fabien Saubade: Writing – original draft, Supervision, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Noémie Cossec: Methodology, Investigation. Luc Giguelay Gesret: Methodology, Investigation. Christelle Kouamé: Writing – review & editing, Validation. Mariem Ellouze: Writing – review & editing, Validation. Cédric Gérard: Writing – review & editing, Validation. Olivier Couvert: Writing – review & editing, Visualization, Validation, Project administration, Methodology, Funding acquisition, Conceptualization. Noémie Desriac: Writing – review & editing, Validation, 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.

none

## Acknowledgment

This study was supported by the UMT ACTIA 24.02 TRANSISPORE (Joint Technological Unit - French Ministry responsible for Food).

The authors thank the EQuASA technicians who contributed to the mould spore suspensions.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fm.2024.104545.
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