# Use of Penicillium roqueforti in plant-based veined-blue cheese: a source of mycotoxin hazards?

Oriane Gauthier, Stéphane Bernillon, Rayan Khireddine, Capucine Saupique, Nathalie Gallegos, Cécile Callon, Christophe Chassard, F. Richard-Forget

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Oriane Gauthier, Stéphane Bernillon, Rayan Khireddine, Capucine Saupique, Nathalie Gallegos, et al.. Use of Penicillium roqueforti in plant-based veined-blue cheese: a source of mycotoxin hazards?. Food Control, 2025, 171, pp.111130. 10.1016/j.foodcont.2024.111130. hal-04872724

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# Use of *Penicillium roqueforti* in plant-based veined-blue cheese: A source of mycotoxin hazards?

Oriane Gauthier<sup>a, ID</sup>, Stéphane Bernillon<sup>a, ID</sup>, Rayan Khireddine<sup>a, ID</sup>, Capucine Saupique<sup>a</sup>, Nathalie Gallegos<sup>a</sup>, Cécile Callon<sup>b, ID</sup>, Christophe Chassard<sup>b</sup>, Florence Richard-Forget<sup>a, *</sup>

## ARTICLE INFO

**Keywords:**

Cheese analogue

Fermented food model

Food safety

Roquefortine C

Andrastin A

## ABSTRACT

While the consumer demand for plant-based cheese is increasingly growing, there is insufficient knowledge to allow anticipating potential hazards related to mycotoxins in these new products and notably in plant-based blue veined cheese inoculated with *Penicillium roqueforti*. Actually plant-based matrices, characterized by a higher C/N ratio than dairy milk, are likely to promote the yield of mycotoxins by *P. roqueforti*. This study aims to provide first scientific insights to fulfill this gap of knowledge. As a first step, the capacity of 15 *P. roqueforti* strains belonging to the Roquefort and Non-Roquefort populations to yield mycotoxins was assessed. The medium used allowed to evidence a significant higher production of roquefortine C and a lower yield of andrastin A by strains of the Non-Roquefort group compared to those of the Roquefort one. In a second step a microbiological challenge testing approach was implemented using five matrices of plant (almond, cashew and soy) and animal (cow and sheep) origin. Results suggested that animal matrices were more prone to contamination with andrastin A while cashew and soy matrices promoted the yield of roquefortine C and mycophenolic acid, and in a less extent of fumigaclavin A. Besides, while warning on the mycotoxin risk associated with some plant matrices, our results also evidenced that the risk was strongly dependent on choice of the combination *P. roqueforti* strain/matrix.

## 1. Introduction

Plant-based cheese (PBC) is a non-dairy alternative to traditional cheese, designed to mimic the texture and sensory attributes of animal-based cheese (Grossmann & McClements, 2021). Using plant ingredients including soy, almond or cashew milk, PBC are popular among the growing vegetarian and vegan population and their market is constantly expanding. The sensory and textural characteristics of PBC are mainly conditioned by the nature and the processing of the plant ingredient or the blend of plant ingredients and of some key processing steps such as fermentation or ripening (Ben-Harb et al., 2020). In order to mimic blue-veined cheese, inoculation with fungal starter cultures (most frequently *Penicillium roqueforti*) is commonly used in PBC production processes. Since many decades, *P. roqueforti* has been used as a ripening culture in the making of dairy blue-veined cheeses such as Roquefort, Gorgonzola, Stilton, and others. Its technological interest mainly relies on its proteolytic and lipolytic activities responsible for the production of various volatile and non-volatile metabolites that determine the

aroma and flavor of blue-veined cheeses (Caron et al., 2021; Cerning et al., 1987). As described by Coton et al. (2020) and recently reviewed by Crequer et al. (2023), the *P. roqueforti* genus gathers five genetically different clusters also called populations, two of them characterized by strains from environmental origin (lumber or silage). The three others are associated with cheese-making: the Roquefort and Non-Roquefort populations that are used in industrial and traditional cheese-making and a newly identified one, specific to French Termignon cheese which is produced with spontaneous blue mould colonization. In a recent report (Caron et al., 2021), it has been clearly demonstrated that according to the population it belongs to, a *P. roqueforti* strain will differentially impact the sensory attributes of blue veined cheese. Strains from the Roquefort and Non-Roquefort clusters have the capacity to produce a wide range of mycotoxins including the roquefortine C, PR-toxin, mycophenolic acid, andrastin A, isofumigaclavine A and eremofortins (Chávez et al., 2023); one main phenotypic difference between these two clusters being related to the production of mycophenolic acid since only the Roquefort strains own the genetic

<footnote><sup>a</sup> INRAE, UR 1264 Mycology and Food safety, 33882, Villenave D'Ornon, France</footnote>

<footnote><sup>b</sup> INRAE, UMR 545 Fromage, VetAgro Sup, Université Clermont Auvergne, 15000, Aurillac, France</footnote>

<footnote>* Corresponding author.
E-mail address: florence.forget@inrae.fr (F. Richard-Forget).</footnote>
capacity to yield this metabolite (Coton et al., 2020). Eremofortins are biosynthetic intermediates of the PR-toxin which is the *P. roqueforti* mycotoxin with the greatest toxicological concern for animal and human (Dubey et al., 2018). Numerous cellular processes including transcription and protein biosynthesis have been shown to be affected by PR-toxin which, in mice, exhibits a LD50 of 5.8 mg/kg when administered intraperitoneally (Chen et al., 1982). In animal-based cheeses, the PR-toxin is rarely detected due to its high instability and degradation into the less toxic PR-amine and PR-imide (Chang et al., 1993; Dubey et al., 2018). Compared to PR-toxin, roquefortine C characterized by a LD50 close to 180 mg/kg in mice after intraperitoneal administration (Arnold et al., 1978) could be considered a less threatening contaminant. But, unlike the PR-toxin, roquefortine C is commonly found in blue veined cheese, with amounts ranging from 10 to 14000 µg/kg (Finoli et al., 2001; Kokkonen et al., 2005; Fontaine et al., 2015). The presence of mycophenolic acid has also been evidenced in blue-veined cheese, quantified concentrations being comprised between 15 and 6000 µg/kg (Usleber et al., 2008). Due to its very low toxicity to mammals (LD50 of 450 mg/kg in mice), the classification of mycophenolic acid as a mycotoxin remains still a subject of debate (Bentley, 2000). Main information related to its toxicity in human includes gastrointestinal disorders and is related to adverse side effects of mycophenolic acid-based treatments, which are prescribed after organ transplantation for preventing graft rejection. Indeed, due to its capacity to inhibit the inosine 5'-monophosphate dehydrogenase (IMPDH) enzyme, mycophenolic acid is a powerful inhibitor of lymphocyte proliferation (Davies et al., 2007). Regarding andrastin A, fumigaclavin A and also eremofortins, in-depth studies are still required to precisely clarify their toxic effects. Whatever, while blue cheeses can be contaminated with mycotoxins produced by *P. roqueforti*, it is largely acknowledged that their levels are too low to pose significant health risk to consumers (Fontaine, 2015). Such a statement is reinforced by the long history of safe use of *P. roqueforti* as cheese starter culture without any case of human intoxication being reported. However, the new use of *P. roqueforti* to ferment plant-based matrices to produce PBC could raise new safety issues related to mycotoxins. Knowing the significant effect of abiotic conditions including nutrient factors on the yield of various mycotoxins (Wang et al., 2022), it is more than likely that the amounts and stability of mycotoxins produced by *P. roqueforti* will significantly differ between PBC and dairy cheese. In comparison with other toxigenic fungi (*Aspergillus* spp., *Fusarium* spp.), information on the impact of abiotic factors on the biosynthesis of mycotoxins produced by *P. roqueforti* remains however incomplete. One can mention the study by Fontaine et al. (2015) that underlined the impact of strains and genetic diversity on mycotoxin production and showed the negative effect of decreasing the C/N ratio, the O₂ content and temperature (from 25 to 12 °C) and of increasing NaCl concentration on the biosynthesis of roquefortine C and mycophenolic acid but using only one strain from the Non-Roquefort population. Information related to the genetic regulation of the biosynthesis of mycotoxins by *P. roqueforti* is also very scarce. As recently reviewed by Chávez et al. (2023), the effect of key global regulators such as CreA and AreA involved in the regulation by nutrient sources, the pH-regulator PacC, or the oxidative stress-responsive transcription factors AP1 and Skn7 have not been investigated so far.

As aforementioned, public health issues related to mycotoxins produced by *P. roqueforti* in blue-veined PBC cannot be excluded. The currently available knowledge is however totally insufficient to determine whether such a safety risk might exist. In this context, the present study aims to provide first key scientific insights to fulfill these gaps of knowledge. Using a set of Roquefort and Non-Roquefort strains and fermented food models, the potential of various plant-based milks to promote or restrain mycotoxin production by *P. roqueforti* will be investigated.

# 2. Materials and methods

## 2.1. Biological material

The 15 *P. roqueforti* strains used in the present study are part of the UMRF INRAE culture collection of technological fungi. All the strains are food-grade and have been isolated from dairy blue-veined cheeses. The strains were identified as *Penicillium roqueforti* species thanks to their β-tubulin sequence. Seven strains belong to the Roquefort genetic group (R1, R2, R3, R4, R5, R6 and R7) and eight to the Non-Roquefort genetic group (NR1, NR2, NR3, NR4, NR5, NR6, NR7 and NR8). Non-Roquefort (NR) strains display two genetic markers, Wallaby and CheesyTer, that Roquefort (R) strains do not (Gillot, Jany, Dominguez-Santos, et al., 2017). All strains were sequenced and their belonging to each group determined based on the presence of the previous markers. The strains were supplied in a milk +8 % glycerol solution and stored at -80 °C. Before use, the solutions were transferred to a PDA (Potato Dextrose Agar) medium in inclined tubes for 7 days at 25 °C, then stored at 4 °C.

## 2.2. Chemicals

Chemicals used for the experiments were: ethyl acetate, acetonitrile, hexane (HPLC grade) and water (LC-MS grade) from VWR BDH Chemicals (Leuven, Belgium); acetonitrile (LC-MS grade) from Sigma Aldrich (Saint Louis, MO, USA) and formic acid (LC-MS grade) from Honeywell Fluka (Charlotte, NC, USA). Besides, the following commercial mycotoxins were used as analytical standards: isofumigaclavine A, eremofortin A, eremofortin B, roquefortine C (BioViotica, Dransfeld, Germany); mycophenolic acid (BioWORLD, Visalia, USA); patulin (Cayman Chemicals, Ann Arbor, USA); andrastin A, cyclopiazonic acid (Sigma Aldrich, Saint Louis, USA); citrinin (Adipogen Life Science, Fuellinsdorf, Switzerland); marcfortine A, penicillic acid, orsellinic acid, roquefortine E, penitrem A (VWR International, Pennsylvania, USA).

## 2.3. In vitro growth conditions and mycotoxin extraction

Three mL of sterile water were added to each PDA tube containing the *P. roqueforti* strains. After 30 s of gentle mixing, the spore solution was filtered through 40 µm filters (Fisher Scientific, Waltham, MA, USA). Spore counting was done on a Malassez cell; spore concentrations close to $10^7$ spores/mL were obtained for each of the 15 strains. The solution was diluted with sterile water to achieve a concentration of $10^6$ spores/mL.

The CDNY growth medium (CDNY for Czapek Dox No Yeast extract) was used throughout this study. Lack of yeast extract supplementation allowed controlling carbon and nitrogen concentrations. CDNY was adapted from the Czapek Dox medium and composed with the following ingredients: glucose (40 g/L), $MgSO_4 \cdot 7H_2O$ (0.5 g/L), $KH_2PO_4$ (1 g/L), KCl (0.25 g/L), NaCl (0.25 g/L), $NaNO_3$ (1 g/L), $NH_4Cl$ (0.25 g/L), $ZnSO_4 \cdot 7H_2O$ (0.01 g/L), $FeSO_4 \cdot 7H_2O$ (0.01 g/L). 200 µL of spore solution were inoculated in each Petri dish filled with 15 mL of medium (5 replicates/per condition), further incubated at 25 °C for 14 days.

Following incubation, mycelia and culture media were separated by centrifugation (4 °C, 4816 rcf, 15 min). Four mL of culture media were extracted with 6 mL of ethyl acetate. After agitation (30 min) using a Multi Reax shaker (Heidolph Instruments, Schwabach, Germany) and centrifugation for 10 min (4 °C, 4816 rcf), 4 mL of supernatant were collected and evaporated to dryness with a Speedvac (Thermo Fisher Scientific, Waltham, USA) vacuum concentrator (45 °C, 2 h). The remaining pellet was solubilized in 1 mL of methanol (LC-MS grade) and filtered (0.2 µm, Acrodisc PTFE, VWR International, Pennsylvania, USA) into a HPLC vial. Extracts were stored at -20 °C until LC-HRMS analysis.

## 2.4. Fermented food model production and mycotoxin extraction

Thirty fermented food models were produced using six *P. roqueforti*
strains (R1, R2, R3 and NR1, NR2, NR3) and five types of milks: two animal-based (cow and sheep) and three plant-based (almond, soy and cashew, all sugar free). The applied protocol was fixed to mimic production processes employed by industrials. Each milk was mixed with refined sunflower oil, agar agar (Biokar Diagnostics, Allone, France), glucono-delta-lactone (GDL, Merck, Fontenay sous Bois, France) and salt, according to the proportions reported in Table 1.

The formulation was defined to minimize differences in total fat content, pH and density, so that all matrices have the same properties which was a required condition to specifically assess the effect of the type of milk (plant and animal milk). Oil was added to each plant-based mix to match the fat levels found in the animal-based milks. Agar agar was used to replace rennet. Agar proportions were adjusted in each mixture to achieve a final dry extract of 50 % (±1 %). Dry extract was measured after 24 h in an oven at 100 °C. GDL was used to replace lactic starter as done previously by Ben-Hard et al. (2020). GDL was added to adjust the pH to a value close to 4.9 after 24h as observed in dairy blue cheeses. After homogenization, mixes were heated for 10 min at 84 °C, then rapidly poured into aluminium moulds placed under a laminar flow hood and cooled down to reach 43 °C. They were then inoculated with 0.1 mL of *P. roqueforti* spore solution (10⁶ spores/mL) with a sterile cone and gently homogenized. After solidification, they were dried for 24 h at ambient temperature. The curds were removed from the mould, pricked on each side with sterile cones and ripened for 15 days at 9 °C in sterile boxes (relative humidity of 95%), these conditions being the refining conditions commonly applied for plant-based alternatives to cheese in industrial processes. The 30 obtained fermented-food models were stored at -20 °C until analysis. Fermented food models were lyophilized for 5 days using a Cryotech (Saint-Gély-Du-Fesc, France) freeze dryer and ground with a mortar. Mycotoxin analysis was done using 1 g of powder of each fermented food product, using a protocol adapted from Hammerl et al. (2019). Six mL of acetonitrile (HPLC grade) and then 6 mL of hexane were added to the powder. After 30 min, the mix was homogenized with an Ultra Turrax - T25 dispersion device (IKA, Staußen, Germany) for 3 min at 20000 rcf. After centrifugation for 20 min (4 °C, 4816 rcf), 1 mL of the acetonitrile phase was collected, evaporated to dryness and the remaining pellet was solubilized in 1 mL of methanol (LC-MS grade), and filtered with 0.2 µm syringe filters (Pall Corp, Port Washington, USA) before LC-HRMS analysis.

## 2.5. Mycotoxin analysis

Mycotoxins of *P. roqueforti* were analyzed according to a LC-HRMS method adapted from Hammerl et al. (2019). Analysis was performed using a Q-Exactive Focus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization (HESI) source and a Vanquish UHPLC System (Thermo Fisher Scientific, Bremen, Germany). Separation was achieved on a Kinetex Phenyl-Hexyl column (2.1 × 100 mm, 1.7 mm, x 100 Å) (Phenomenex Inc., Torrance, CA, USA) maintained at 30 °C. The mobile phase consisted of H₂O and 1 % (v:v) formic acid (solvent A) and acetonitrile and 1 % (v:v) formic acid (solvent B). The elution gradient was as follows: 100 % solvent A from 0 to 5min, 60 % solvent A from 5 to 9.5 min, 0 % solvent A from 9.5 to 11.5 min and 100 % solvent 1 from 11.5 to 19.5 min. The flow was kept at 0.4 mL/min and injection volume set at 2 µL.

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

Composition of the different mixes used to produce fermented food models.</caption><thead><tr><th>Source</th><th>Milk (mL)</th><th>Oil (mL)</th><th>Agar agar (g)</th><th>GDL* (g)</th><th>Salt (g)</th></tr></thead><tbody><tr><td>Cow</td><td>100</td><td>0</td><td>3.1</td><td>1.0</td><td>2</td></tr><tr><td>Sheep</td><td>100</td><td>0</td><td>1.5</td><td>1.3</td><td>2</td></tr><tr><td>Soy</td><td>100</td><td>4</td><td>1.5</td><td>0.5</td><td>2</td></tr><tr><td>Almond</td><td>100</td><td>4</td><td>3</td><td>1.2</td><td>2</td></tr><tr><td>Cashew</td><td>100</td><td>4</td><td>2.5</td><td>0.25</td><td>2</td></tr></tbody></table>

GDL*: Glucono Delta Lactone.

The Xcalibur software (version 4.4.16.14 ThermoFisher Scientific) was used to control the LC-MS system, acquire, and process the data. The mass spectrometer was operated in positive mode with 70000 @ m/z 200 resolving power. Full scans were acquired in the 120–1000 m/z range. Nitrogen was used as auxiliary gas. The following parameters were used: Spray voltage: 3.5 kV; Sheath gas: 50 a.u.; aux gas: 13; sweep gas: 3 a.u; capillary temperature: 263 °C; auxiliary temperature: 425 °C; S-lens RF: 50 %. m/z calibration was performed each week. Quantification was performed by external calibration using HRMS EIC with standard solutions of mycotoxins ranging from 0.01 to 20 mg/mL.

## 2.6. Statistical analysis

Principal component analysis (PCA) was conducted to explore the relationship between mycotoxin profiles and the various factors (*P. roqueforti* strain, type of milk). Variables were UV-scaled before PCA. *p* value of Barlett's sphericity test was <0.001. Two principal components were kept, explaining more than 70 % of variance. Pearson correlation coefficient was calculated to evaluate correlation between the variables. To compare mycotoxins production between *P. roqueforti* strain or type of milk, one-way ANOVA analyses were used, with a Tukey or Dunnett *post-hoc* test. A Shapiro-Wilk test was done to check that data were normally distributed. Reported values are mean ± standard deviation including five biological replications, except in Fig. 2 where median ± standard deviation is displayed. Those statistical analyses were conducted with XLstat (Addinsoft, France, version 23.3.1) and statistical significance was considered when *p* < 0.05. To compare mycotoxin yields in CDNY and fermented food models, a correlation analysis was performed (Supplementary Table 1). It was conducted with RStudio (Posit Software, PBC, USA, version 2024.04.2 + 764) using cor.test function to calculate Pearson's correlation coefficient.

# 3. Results

## 3.1. *P. roqueforti* strains: common features and specificity

### 3.1.1. Mycotoxins production profile varies between Roquefort (R) and Non-Roquefort (NR) strains and between strains within the same population

The results of the mycotoxin analysis for the 15 *P. roqueforti* strains grown for 14 days in CDNY medium are gathered in Table 2. The procedure used within this study allowed to quantify six mycotoxins including roquefortine C, isofumigaclavine A, mycophenolic acid, eremofortins A and B and andrastin A among all those tested. The highest amounts were obtained for eremofortin B with values close to 13 mg/mL for the NR7 strain and the lowest levels measured at 0.02 mg/mL for both eremofortin B for the R2 strain and isofumigaclavine A for the R5 strain. Differences in the ability to produce mycotoxins were evidenced between R and NR strains and between strains of the same population. As expected, no mycophenolic acid was detected in media inoculated with NR strains. According to our data, R strains appeared to produce higher amounts of andrastin A (quantities ranging from 0.39 to 3.31 mg/mL) compared to NR strains (quantities ranging from 0.03 to 0.20 mg/mL). An opposite observation was made for roquefortine C, with a higher amount of the toxin quantified for NR strains (quantities ranging from 0.19 to 1.09 mg/mL) than for R strains (quantities ranging from 0.03 to 0.45 mg/mL). Regarding the frequency of yield within each population, some mycotoxins seemed to be produced by a greater number of strains than others. For R strains, isofumigaclavine A and eremofortin A were quantified in very few inoculated media (Isofumigaclavine was detected only in R1 broths and eremofortin A in 3 of the 6 cultures inoculated with R strains) whereas andrastin A and mycophenolic acid were detected for each strain considered. For the NR strains, isofumigaclavine A was never quantified and eremofortin A was quantified only for the NR1 and NR7 strains whereas roquefortine C and andrastin A were produced by almost all strains.
<caption>**Table 2**</caption>

Concentration (mg/mL) of roquefortine C, isofumigaclavine A, mycophenolic acid, andrastin A, eremofortin A and B in 14-day-old broths inoculated with R and NR strains. *Limit of quantification: 0.003 mg/mL; nd: not detected.*

<table><thead><tr><th>Strains</th><th>Isofumigaclavine A (mg/mL)</th><th>Roquefortine C (mg/mL)</th><th>Mycophenolic acid (mg/mL)</th><th>Andrastin A (mg/mL)</th><th>Eremofortin A (mg/mL)</th><th>Eremofortin B (mg/mL)</th></tr></thead><tbody><tr><td>R1</td><td>nd</td><td>nd</td><td>3.35 ± 0.18</td><td>0.26 ± 0.02</td><td>0.40 ± 0.03</td><td>5.43 ± 0.28</td></tr><tr><td>R2</td><td>0.05 ± 0.01</td><td>nd</td><td>0.03 ± 0.01</td><td>3.31 ± 0.65</td><td>nd</td><td>0.01 ± 0.01</td></tr><tr><td>R3</td><td>nd</td><td>0.03 ± 0.05</td><td>1.07 ± 0.16</td><td>0.39 ± 0.06</td><td>nd</td><td>0.96 ± 0.20</td></tr><tr><td>R4</td><td>nd</td><td>0.21 ± 0.02</td><td>0.01 ± 0.00</td><td>1.31 ± 0.07</td><td>nd</td><td>nd</td></tr><tr><td>R5</td><td>0.01 ± 0.00</td><td>0.45 ± 0.06</td><td>0.58 ± 0.19</td><td>2.80 ± 0.32</td><td>nd</td><td>0.09 ± 0.01</td></tr><tr><td>R6</td><td>nd</td><td>0.24 ± 0.01</td><td>0.02 ± 0.01</td><td>1.38 ± 0.18</td><td>nd</td><td>0.02 ± 0.01</td></tr><tr><td>R7</td><td>0.02 ± 0.00</td><td>0.18 ± 0.01</td><td>0.12 ± 0.03</td><td>0.64 ± 0.04</td><td>nd</td><td>0.16 ± 0.07</td></tr><tr><td>NR1</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>0.08 ± 0.07</td><td>3.32 ± 0.94</td></tr><tr><td>NR2</td><td>nd</td><td>0.97 ± 0.04</td><td>nd</td><td>0.20 ± 0.04</td><td>nd</td><td>nd</td></tr><tr><td>NR3</td><td>nd</td><td>0.52 ± 0.08</td><td>nd</td><td>0.19 ± 0.02</td><td>nd</td><td>nd</td></tr><tr><td>NR4</td><td>nd</td><td>0.05 ± 0.01</td><td>nd</td><td>0.13 ± 0.03</td><td>nd</td><td>nd</td></tr><tr><td>NR5</td><td>nd</td><td>0.87 ± 0.07</td><td>nd</td><td>0.06 ± 0.01</td><td>nd</td><td>nd</td></tr><tr><td>NR6</td><td>nd</td><td>1.09 ± 0.06</td><td>nd</td><td>0.56 ± 0.22</td><td>nd</td><td>0.14 ± 0.02</td></tr><tr><td>NR7</td><td>nd</td><td>0.18 ± 0.01</td><td>nd</td><td>0.03 ± 0.00</td><td>0.86 ± 0.03</td><td>13.33 ± 0.23</td></tr><tr><td>NR8</td><td>nd</td><td>0.60 ± 0.15</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td></tr></tbody></table>

### 3.1.2. Mycotoxins production differentiates strains from the R and NR populations

In order to visualize how the production of mycotoxins in CDNY medium could discriminate R and NR strains, a PCA analysis was performed using the six variable values quantified for each of the 15 strains (Fig. 1). The two principal components accounted for 72.68% (F1 + F2) of the variance in our data. Eremofortins A and B exhibited the greatest positive contribution to F1 while F2 was strongly and positively

correlated with roquefortine C and negatively correlated with andrastin A and isofumigaclavine A. A clear clustering of R and NR strains according to their mycotoxin profiles was evidenced such as the occurrence of few outgroup strains. One can mention R1 and R2 for the Roquefort group and NR 7 for the Non-Roquefort one. The outgrouping of these three strains seemed to be related to their different ability to produce eremofortins. Fig. 1 also suggests some positive correlations between the production of some mycotoxins. The highest Pearson's

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Principal component analysis (PCA) biplot depicting the relationships between the estimated mycotoxin quantities and *P. roqueforti* strains from the Roquefort population/R and the Non-Roquefort one/NR (15 observations, 6 variables). F1 on the x-axis accounted for 41,26% of the total variability, while F2 explained 31.42% of total variability. The contribution of traits to F1 and F2 is indicated by the direction and length of the lines.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Differences in mycotoxin production in 14-day-old cultures between R and NR strains. A: Boxplots of isofumigaclavine A. B: Boxplots of roquefortine C. C: Boxplots of mycophenolic acid. D: Boxplots of andrastin A. E: Boxplots of eremofortin A. F: boxplots of eremofortin B. Dark gray boxplots correspond to R strains and light gray boxplots to NR strains. Statistical significance ⚫ (p < 0.05) and ns (non-significant).</figcaption></figure>

coefficient values were obtained for eremofortins A and B (r = 0.985) and for andrastin A and isofumigaclavine A (r = 0.772).

To assess the contribution of mycotoxins to the discrimination between R and NR *P. roqueforti* strains, non-parametric univariate statistical analyses were applied. The boxplots reported in Fig. 2 clearly demonstrated the statistically higher capacity of R strains to yield andrastin A (Fig. 2D) and in a lesser extent, isofumigaclavine A (Fig. 2A) in CDNY medium, compared to NR strains. An opposite trend was observed for roquefortine C (Fig. 2B), the quantified amounts of which

were statistically greater in the supernatants of R cultures compared to NR ones: the median's value of the R group was close to 0.2 mg/mL while it reached 0.6 mg/mL for the NR group. Regarding eremofortins A and B, no difference in the production capacities of the two groups of *P. roqueforti* strains was visualized (Fig. 2E and F).

<caption>**Table 3**</caption>

Minimum, maximum and mean values of mycotoxin contents detected in fermented food models using 5 types of milk and 6 different *P. roqueforti* strains. Min and max values are means of 5 replicates for one strain (indicated in brackets). Mean is the mean of all samples. LOQ: 0.006 µg/g of dry fermented food.

<table><thead><tr><th>Milk/Juice</th><th>Value</th><th>Isofumigaclavine A (µg/g dry fermented food)</th><th>Eremofortin B (µg/g dry fermented food)</th><th>Roquefortine C (µg/g dry fermented food)</th><th>Andrastin A (µg/g dry fermented food)</th><th>Mycophenolic acid (µg/g dry fermented food)</th></tr></thead><tbody><tr><td rowspan="2">Cow</td><td>Min</td><td>0.06 ± 0.01 (NR3)</td><td>nd</td><td>0.06 ± 0.01 (NR3)</td><td>0.35 ± 0.07 (NR3)</td><td>nd</td></tr><tr><td>Mean</td><td>0.30 ± 0.24</td><td>0.02 ± 0.08</td><td>0.25 ± 0.13</td><td>2.16 ± 1.30</td><td>0.02 ± 0.01</td></tr><tr><td rowspan="2">Sheep</td><td>Max</td><td>0.71 ± 0.11 (NR1)</td><td>0.13 ± 0.05 (NR1)</td><td>0.47 ± 0.12 (R3)</td><td>4.20 ± 0.78 (NR1)</td><td>0.04 ± 0.02 (R2)</td></tr><tr><td>Min</td><td>0.10 ± 0.02 (NR3)</td><td>nd</td><td>0.03 ± 0.00 (NR3)</td><td>0.70 ± 0.13 (NR3)</td><td>nd</td></tr><tr><td rowspan="2">Almond</td><td>Mean</td><td>0.30 ± 0.14</td><td>0.02 ± 0.02</td><td>0.10 ± 0.06</td><td>1.72 ± 1.01</td><td>0.05 ± 0.04</td></tr><tr><td>Max</td><td>0.41 ± 0.12 (R2)</td><td>0.07 ± 0.04 (NR1)</td><td>0.17 ± 0.04 (R1)</td><td>3.58 ± 0.73 (NR1)</td><td>0.14 ± 0.03 (R1)</td></tr><tr><td rowspan="2">Soy</td><td>Min</td><td>0.03 ± 0.02 (NR3)</td><td>nd</td><td>0.06 ± 0.02 (NR3)</td><td>0.18 ± 0.09 (NR3)</td><td>nd</td></tr><tr><td>Mean</td><td>0.27 ± 0.22</td><td>0.44 ± 0.46</td><td>0.16 ± 0.07</td><td>0.87 ± 0.97</td><td>nd</td></tr><tr><td rowspan="2">Cashew</td><td>Max</td><td>0.54 ± 0.23 (NR1)</td><td>1.34 ± 0.74 (NR1)</td><td>0.27 ± 0.08 (R1)</td><td>2.82 ± 1.17 (R3)</td><td>nd</td></tr><tr><td>Min</td><td>0.15 ± 0.12 (NR2)</td><td>nd</td><td>0.26 ± 0.06 (R3)</td><td>0.24 ± 0.04 (NR2)</td><td>nd</td></tr><tr><td rowspan="2"></td><td>Mean</td><td>0.35 ± 0.23</td><td>0.00 ± 0.01</td><td>0.74 ± 0.46</td><td>0.68 ± 0.67</td><td>0.12 ± 0.03</td></tr><tr><td>Max</td><td>0.78 ± 0.12 (R2)</td><td>0.03 ± 0.02 (R1)</td><td>1.35 ± 0.17 (R1)</td><td>1.99 ± 0.49 (R2)</td><td>0.37 ± 0.03 (R2)</td></tr><tr><td rowspan="2"></td><td>Min</td><td>0.05 ± 0.04 (NR3)</td><td>nd</td><td>0.25 ± 0.13 (NR1)</td><td>nd</td><td>nd</td></tr><tr><td>Mean</td><td>0.55 ± 0.42</td><td>0.01 ± 0.00</td><td>0.78 ± 0.58</td><td>0.71 ± 0.37</td><td>0.25 ± 0.30</td></tr><tr><td rowspan="2"></td><td>Max</td><td>1.17 ± 0.04 (R1)</td><td>0.03 ± 0.03 (NR1)</td><td>1.66 ± 0.05 (R1)</td><td>2.89 ± 0.09 (R1)</td><td>0.79 ± 0.12 (R1)</td></tr></tbody></table>
## 3.2. Fermented food models: mycotoxins and food matrices

### 3.2.1. Milk origin influences the type and levels of mycotoxins produced by *P. roqueforti* in fermented food matrix

Fermented food models were processed using five types of milk, each being inoculated with six different *P. roqueforti* strains (R1, R2, R3 and NR1, NR2, NR3). The analytical procedure used throughout this study led to the quantification of five mycotoxins, their minimum, mean and maximal amounts being reported in Table 3. Unlike CDNY cultures, no eremofortin A was evidenced whatever the type of fermented model.

Regarding fermented food models using cow milk, andrastin A was by far the most produced mycotoxin with a mean value slightly higher than 2 µg/g of dry matter, followed by roquefortine C and iso-fumigaclavine A (0.25 and 0.30 µg/g of dry matter) while very low levels of mycophenolic acid and eremofortin B were quantified (0.02 µg/ g of dry matter). The same trend was observed with sheep milk inocu-lated by *P. roqueforti* where the andrastin A was the predominant mycotoxin (1.72 µg/g of dry matter) and eremofortin B and mycophe-nolic acid showed the lowest concentrations (0.02 and 0.05 µg/g of dry matter). In almond-based fermented models, andrastin A was once again found at highest levels (0.87 µg/g of dry matter) but unlike to what was observed in cow and sheep matrices, eremofortin B was produced in significant amounts (0.44 µg/g of dry matter). In this last model, no mycophenolic acid was produced, whatever the inoculated *P. roqueforti* strain. Lastly, when considering fermented models using soy and cashew, the highest amounts were quantified for roquefortine C and

andrastin A which were produced at very close concentrations (mean of 0.72 µg/g of dry matter). In these two matrices, iso-fumigaclavine A (0.55 µg/g of dry matter) followed by mycophenolic acid (0.12 µg/g of dry matter) were also produced in significant concentrations while only traces of eremofortin B were quantified. Overall, according to our data, the highest concentrations of roquefortine C and mycophenolic acid were obtained using soy and cashew matrices, the highest amounts of andrastin A was observed with cow and sheep matrices, and the highest levels of eremofortin B was quantified in almond-based fermented models. For andrastin A, the five types of milk were shown to allow its production with mean concentrations ranging between 0.27 and 0.55 µg of dry matter.

### 3.2.2. Distribution of fermented food models according to their mycotoxin content

A PCA analysis was performed using the concentrations of the 5 mycotoxins quantified in the 30 fermented food models (5 types of milk, 6 inoculated *P. roqueforti* strains). Results reported on Fig. 3 indicate the clustering of the almond and sheep matrices (blue ellipse) and of the cashew and soy matrices (red ellipse) whereas the food models using cow's milk appeared to be separated from the others. When looking at the variables that could explain this distribution, roquefortine C, andrastin A and eremofortin B were the main contributors to the F2 axis whereas the F1 axis was correlated with iso-fumigaclavine A and mycophenolic acid. Most of cashew and soy samples were characterized by negative F2 coordinates, which could be associated with their higher

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Principal component analysis (PCA) biplot depicting the relationships between the estimated mycotoxin quantities and fermented food models inoculated with strains from the Roquefort population/R and the Non-Roquefort one/NR (30 observations, 5 variables). F1 on the x-axis accounted for 49,53 % of the total variability, while F2 explained 21,57% of total variability. The contribution of traits to F1 and F2 is indicated by the direction and length of the lines.</figcaption></figure>
concentrations in roquefortine C, compared to the others. Besides, the PCA biplots also suggest that some measured mycotoxins could be correlated (according to Pearson correlation coefficient), such as mycophenolic acid with both roquefortine C (r = 0.708) and isofumigaclavine A (r = 0.735). A medium correlation was also suggested between andrastin A and isofumigaclavine A (r = 0.526) while eremofortin B seems to not be correlated with any of the other mycotoxins.

### 3.2.3. Mycotoxins production in fermented food models is influenced by both the inoculated *P. roqueforti* strain and the type of milk

As suggested by the distribution of samples in Fig. 3, and notably the occurrence of outgroup samples such as R1-Cashew or NR1-Almond and negative coordinates on the F1 axis for most of the samples associated with NR strains, the nature of the inoculated *P. roqueforti* strain impacts the profile of mycotoxins quantified in the fermented food models. This impact can be clearly visualized on Fig. 4A, 4B, 4C and 4D which focus on roquefortine C, mycophenolic acid, andrastin A and isofumigaclavine A respectively. Regarding roquefortine C (Fig. 4A), for most of the six strains, the highest production was by far obtained with soy and cashew matrices but for two strains, R3 and NR1, the levels of roquefortine C quantified in fermented models using cow's milk were not statistically different from those associated with the two previously mentioned vegetable milks. As shown in Fig. 4B, mycophenolic acid was only quantified in matrices inoculated with R strains, corroborating the lack of genetic capacity of NR strains to produce this mycotoxin (Coton et al., 2020). Besides, whatever the considered R strain, mycophenolic acid was never detected in almond-based fermented models. For R1 and R2, the highest concentrations were quantified in soy and cashew matrices whereas for the R3 strain, no production occurred in cashew models and the amounts quantified in sheep and soy-based models were not markedly different. Results are less clear-cut when considering andrastin A (Fig. 4C), particularly in models inoculated with R strains. For NR strains, the highest production of andrastin A was observed in animal matrices, i.e. cow and sheep matrices. For R strains, additional matrices than animal ones seemed to promote the production of andrastin A: cashew for R1, soy for R2 and almond for R3. Finally, while isofumigaclavine A production by R strains (Fig. 4D) appeared to be favored by vegetal milks and particularly by cashew milk for R1, no such clear tendency was observed with NR strains.

## 4. Discussion

To our knowledge, the present study is the first addressing the issue of *P. roqueforti* mycotoxins when this fungal species is used as a starter culture to produce plant alternatives to blue-veined cheese. Innovatively, a microbiological challenge testing approach was implemented using *P. roqueforti* strains from the Roquefort and Non-Roquefort populations and five matrices of plant (almond, cashew and soy) and animal (cow and sheep) origin. As a first step, the capacity of *P. roqueforti* strains to produce mycotoxins was assessed in CDNY, a synthetic medium that deliberately does not contain yeast extract. This choice was driven by the opened possibility to further investigate the impact of nutrient and carbon source on the yield of mycotoxins by *P. roqueforti*. Nevertheless, even if CDNY medium was less favouring the production of *P. roqueforti* mycotoxins compared to the commonly used YES medium (Crequer et al., 2023; Fontaine, 2015), the quantified amounts allowed characterising the metabolic diversity of the set of considered strains. While corroborating the lack of mycophenolic acid production by NR strains, which has been ascribed to a deletion in the key MpaC polyketide synthase enzyme responsible for its biosynthesis (Crequer et al., 2023), our data also indicated that R and NR strains could be differentiated according to their production of roquefortine C and andrastin A. Indeed, in CDNY medium, NR strains seemed to produce higher levels of roquefortine C compared to R ones while an inverse trend was observed for andrastin A. Such differences require however to be confirmed using a larger set of strains. Compared to previously published data (Cakmakci

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Concentration of mycotoxins produced by Roquefort (R1, R2 and R3) and Non-Roquefort (NR1, NR2 and NR3) strains in fermented food models using five different milks, cow milk in red, sheep milk in yellow, almond milk in dark green, soy milk in blue and cashew milk in light green. A: roquefortine C. B: mycophenolic acid. C: andrastin A. D: isofumigaclavine A.</figcaption></figure>

Stars: ⭐ (p < 0.05), ⭐STER (p < 0.01), ⭐STER (p < 0.001) and ns (non-significant) indicate significant differences with control group, i.e. cow milk (Dunnett test). Values are expressed in mg/g of dry fermented food.
et al., 2015; Finoli et al., 2001) our data corroborate the widespread capacity of *P. roqueforti* strains to produce roquefortine C and andrastin A. However, to our knowledge, such a contrasting production of roquefortine C and andrastin A between R and NR strains has never been reported before. Indeed, in the study of Gillot, Jany, Poirier, et al. (2017), no specific distribution among 55 strains from cheese populations according to their production of roquefortine C and andrastin A was evidenced. More recently (Crequer et al., 2023), *P. roqueforti* NR strains were reported to produce the lowest quantities of mycotoxins, including roquefortine C and andrastin A, compared to strains belonging to the four other populations; this phenotype was ascribed to the intense domestication of NR strains. Our data (i. e. a higher yield of roquefortine C by NR strains) did not support this statement. One rational for these contradictory results could be related to differences in the culture media since the aforementioned studies have used YES agar medium while in the present work, a liquid CDNY medium was employed.

When *P. roqueforti* strains were inoculated in fermented models using plant and animal milks, the profiles of mycotoxins were varied according to the matrix. Overall, our data suggested that animal matrices were more prone to contamination with andrastin A while cashew and soy matrices promoted the yield of roquefortine C and mycophenolic acid, and to a lesser extent of isofumigaclavine A. Regarding almond-based fermented models, they exhibited the highest amounts of eremofortins but appeared to inhibit the yield of mycophenolic acid. However, for some *P. roqueforti* strains, the results differed from the previously described tendency. One can mention the high production of andrastin A by R1 when inoculated in Almond matrix or the equivalent yields of roquefortine C by NR1 and R3 in cow, cashew and soy fermented models. Therefore, while warning on the mycotoxin risk associated with some plant matrices, our results also evidenced that the choice of the combination *P. roqueforti* strain/type of milk required specific consideration. In our study, the highest concentrations of roquefortine C and mycophenolic acid were obtained with R1 and cashew milk, of andrastin A with R2 and cow milk, of eremofortins with NR1 and Almond milk and of isofumigaclavine A with R2 and cashew milk. According to our data, use of some strains as starters to produce PBC, such as R1 and R2, could be considered at a risky choice. When comparing the mycotoxin production of the *P. roqueforti* strains in fermented models and in CDNY medium, with the exception of the lack of mycophenolic production by NR strains in any of the matrices, no clear correlation was obtained. Indeed, Pearson's coefficient values higher than 0.5 were only obtained for roquefortine C production in CDNY and in sheep and almond fermented models, and for andrastin A and isofumigaclavine A yields in CDNY and in soy model (see Supplementary Table 1). This lack of correlation clearly indicates that mycotoxin production in CDNY medium cannot be used as an indicator to predict the yield of mycotoxins in plant and animal matrices. This lack of correlation, that illustrates how the nature of the substrate may redirect metabolic pathways associated with mycotoxin yield, corroborates previously published data (Fontaine, 2015). The regulation of the production of mycotoxin by abiotic factors including, pH, water activity, substrate composition, carbon and nitrogen sources and oxidative and salt stress, has been intensively investigated for mycotoxins legislated by the EU regulation (Commission Regulation (EU) 2023/915 of April 2023 on Maximum Levels for Certain Contaminants in Food and Repealing Regulation (EC) No 1882/2006, 2024) and mainly for aflatoxin, ochratoxin, deoxynivalenol, zearalenone, and fumonisin (Cary & Calvo, 2008; Ferrara et al., 2022; Li et al., 2022; Merhej et al., 2011). As recently reviewed by Wang et al. (2022) the regulation of the biosynthesis of mycotoxins under environmental factors is a complex and orchestrated process involving pathway-specific regulators, global regulators and epigenetic modification. Regarding the influence of carbon and nitrogen sources, which may be one reason to explain the different accumulation of *P. roqueforti* mycotoxins in animal and plant-based fermented foods, the transcription factor CreA, a major transcriptional repressor of carbon catabolite metabolism in fungi, and AreA, a core transcription factor of nitrogen

metabolism repression, have been the subject of intense research (Wang et al., 2022). For instance, a role of CreA and AreA in aflatoxin biosynthesis has been evidenced through the use of ΔcreA or ΔareA deleted strains (Fasoyin et al., 2018). A similar reverse genetic approach was employed by Tannous et al. (2017) to highlight the role of CreA in patulin biosynthesis and by Kim and Woloshuk (2007) for studying the influence of AreA in fumonisin biosynthesis. In contrast, only limited data are available on the environmental factors underlying the production of mycotoxins by *P. roqueforti*. One can mention the study of Fontaine (2015) who showed the positive impact of decreasing NaCl concentration and increasing C/N ratio on the production of secondary metabolites by a strain of *P. roqueforti* and of Hammerl et al. (2019) who demonstrated significant alterations in the exometabolome of one *P. roqueforti* strain induced by tryptophan and tyrosine. While the gene clusters and enzymatic pathways involved in the biosynthesis of roquefortine C, mycophenolic acid, andrastin A and isofumigaclavine A have been elucidated (Coton et al., 2020; Metin, 2023), the current knowledge related to their regulation remains scarce (Chávez et al., 2023). So far, no specific transcription factor has been identified for any of the *P. roqueforti* mycotoxins and only four global regulators - pga1, pcz1, sfk1 and prLaeA - have been investigated for their role in regulating the yield of roquefortine C, andrastin A and mycophenolic acid (Chávez et al., 2023; Marcano et al., 2023). The scientific knowledge is therefore too restricted to understand while no eremofortin A was quantified in any of the fermented food models while a production of this mycotoxin was evidenced for the R1 and NR1 strains in CDNY. Since eremofortin A is an intermediate in the PR-toxin biosynthetic pathway, resulting from the epoxidation of eremofortin B catalysed by an enzyme encoded by the *prx8* gene and a further acetylation step (Chávez et al., 2023), we could hypothesize that the expression of the *prx8* gene is repressed in the fermented food model conditions. This requires however to be demonstrated. Moreover, the current state of scientific evidence does not allow explaining the mechanisms that govern the differential accumulation of *P. roqueforti* mycotoxins according to the culture substrate or the fermented food model and the reasons why cashew and soy matrices seem more prone to roquefortine C and mycophenolic acid contamination.

## 5. Conclusion

Overall, our data provide first scientific evidence underscoring the need to assess the risk of mycotoxins produced by *P. roqueforti* when strains of this species are used as culture starters to produce new plant-based alternatives to dairy products. These data were obtained using *in vitro* or model studies and a restricted set of *P. roqueforti* strains and will need to be subsequently validated in industrial conditions. In our study, soy and cashew matrices were demonstrated as the most prone to contamination with roquefortine C, the risk being however dependent of the inoculated strain. There is currently no European legislation setting maximum permissible values for mycotoxins in plant-based alternatives to dairy cheese. Outcomes of the present study could feed discussions on the best way to consider *P. roqueforti* mycotoxins in plant-based alternatives to blue cheese. In fact, even if they need to be further validated, these first data indicating a lack of associations between the type of *P. roqueforti* strain and the type of plant milk support a legislation targeting the total amount of *P. roqueforti* mycotoxins rather than focusing on specific ones. The present study also highlights the need to deepen the knowledge on the factors and molecular mechanisms that govern the yield of *P. roqueforti* mycotoxins. Indeed, the current understanding is by far too limited to allow anticipating the occurrence of roquefortine C, mycophenolic acid but also PR-toxin when new matrices are used to produce fermented foods. Lastly, it should not be overcome that the vegetable raw material used to produce plant-based milk alternatives can also be contaminated by mycotoxins. In the survey recently published by Pavlenko et al. (2024), the occurrence of enniatins, deoxynivalenol, T~2~ and HT~2~ and aflatoxins was reported in various plant-based
milks including oat, nut, rice and pea drinks.

### CRediT authorship contribution statement

**Oriane Gauthier:** Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Formal analysis, Data curation. **Stéphane Bernillon:** Writing – review & editing, Supervision, Project administration, Formal analysis, Data curation.

**Rayan Khireddine:** Investigation, Formal analysis, Data curation. **Capucine Saupique:** Investigation, Formal analysis, Data curation.

**Nathalie Gallegos:** Investigation, Formal analysis, Data curation. **Cécile Callon:** Writing – review & editing, Investigation, Formal analysis. **Christophe Chassard:** Writing – review & editing, Project administration, Funding acquisition. **Florence Richard-Forget:** Writing – review & editing, Validation, Supervision, Funding acquisition, Conceptualization.

### Data availability

Data supporting these findings are available upon request from the corresponding author.

### 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 research has benefited from an INRAE MICA department funding (ROQUEFORTI project, 2022–2023).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodcont.2024.111130.

### Data availability

Data will be made available on request.

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