Impact of the spore production strategy of Penicillium camemberti in a solid medium on its germination abilities

Abstract

The production process of Penicillium camemberti, a filamentous fungus of dairy interest, involves transition from a solid to a liquid medium, allowing acquisition of a sufficient quantity of spores for transfer to a bioreactor. This step is hardly referenced whereas its impact on growth can be substantial. The aim of this study was to define the best condition for spore production on solid medium that maximizes the quality of produced spores for the transition to liquid medium. A relationship between pre-culture time on a solid medium and future ability to germinate in a liquid medium was demonstrated. Spores tend to lose their ability to germinate with time. This behavior was less notable on a new solid medium. Washing the spores has not been shown to be beneficial for maximizing germination. Finally, a population effect was also observed with a total inhibition of germination at high spore concentration in liquid medium.

Introduction

Penicillium camemberti is traditionally used for the production of white mould cheese. The genus Penicillium develops asexual spores, more commonly known as conidiospores for reproduction and dissemination. Growth and sporulation in submerged batch culture were first studied by the pharmaceutical industry for production of antibiotics (Foster et al., 1945) then by cheese industry (Bockelmann et al., 1999) in order to obtain large quantities of P. camemberti conidiospores.

Morton (1961), has extensively studied the induction of Penicillium sporulation during submerged culture. Production of P. camemberti spores in instrumented bioreactors is now the most cost-effective and widespread method (Canteri, 2014). Pre-culturing on a solid medium enables the production of a sufficient quantity of spores necessary for transitioning to a bioreactor (Bockelmann et al., 1999). Traditionally, P. camemberti spores are generated through surface cultivation on agar medium in small units known as Roux trays. Protocols vary from one author to another but typically the spores are harvested between 1 week (Boualem et al., 2008) and 1 month (Siegbert, 1988) and then used to inoculate bioreactor. There is a strong interest in understanding this step of the bioproduction process.

Growth of microorganisms is very sensitive to environmental parameters such as temperature (Gervais et al., 1988b; Suutari et al., 1990) or water activity (Gervais and Molin, 2003; Scott, 1957). It has been shown for example that Trichoderma rossicum hyphae are significantly thinner in nutrient-poor media (Bernier et al., 2024).

In the case of Fungi, the lag phase represents a period during which spores adapt to a new environment. Adaptation includes formation of enzymes and intermediates to support resumption of growth. The length of this phase is dependent not only on the physiological state of the fungus, but also on the morphology and level of inoculum (Gillot et al., 2016). Then spore inoculum requires a germination period (Smith and Calam, 1980). Germination is characterized by the absorption of water (Martín and Nicolás, 1970) and the biosynthesis of various cell components due to the formation of enzymes and intermediates, which begins in the lag phase. Germination triggers early synthesis of RNA, proteins, and ATP. Spores contain lipid and protein reserves that are mobilized for development. Nuclear division and nuclear migration are observed, involving cytoskeletal components (Giovannetti, 2000). Morphologically, the spore swells (Mandels and Darby, 1953) and then forms a germ tube to eventually develop into a mature thallus (Martín and Nicolás, 1970). Spore germination relies on various factors, including the presence of essential nutrients, favorable environmental conditions for metabolic activity, and inherent spore regulatory mechanisms (Gareth Jones, 1994). It has been demonstrated that the conidia of Penicillium notatum can only germinate effectively when provided with both carbon and nitrogen sources (Martín and Nicolás, 1970). Dormancy can also be disrupted by activation processes such as heat, osmotic and pH shocks or chemical treatment (Canteri and Ghoul, 2015). Chemicals like cupric sulfate inhibit spore germination in water, but this inhibitory effect can be reversed by adding agar (Ko et al., 1975). Environmental parameters such as moisture, but also temperature and the substrate therefore directly influence the germination of fungal spores (Hassouni et al., 2007). For example, regarding the germination of Aspergillus niger spores in solid-state fermentation, it has been observed that a decrease in initial moisture leads to a reduction in germination rates (Hassouni et al., 2007). As well optimal germination of Penicillium roqueforti has been observed at water activity levels between 0.98 and 1, whereas spores exhibit difficulties in germinating at water activity levels below 0.94 (Gervais et al., 1988a).

Concentration of fungal spores would have an impact on growth morphology in submerged culture (Metz and Kossen, 1977). A relationship between Aspergillus niger spore concentration and the number of spores per pellet was found (Metz and Kossen, 1977). This type of relationship is now well known in Fungi. However, it has been shown more recently that the number of spores could impact their own germination in a liquid medium (Gillot et al., 2016). Indeed, microbial cells have the ability to detect the density of their surrounding population through extracellular signals, a phenomenon observed in various microorganisms, including Fungi (Fuqua et al., 1994). It has been demonstrated that the germination of P. camemberti spores is self-regulated through a mechanism known as "quorum sensing" otherwise called "crowding effect" (Gillot et al., 2016). This phenomenon was observed under conditions of high spore density. Indeed P. camemberti displays self-inhibitory effect. The volatile nature of the compounds involved was first demonstrated in liquid medium and 1-octanol was found to be the main volatile compound produced at high spore density (10⁸ spores/ml) (Gillot et al., 2016). There could also be a correlation between the pre-culture duration on a solid medium and its subsequent ability to germinate in a liquid medium. While there is no specific reference for P. camemberti, it is known that the age of the inoculum is of prime importance in determining the morphology of Fungi (Papaggiani, 2004). In Neurospora crassa, a clear correlation is observed between spore age and a decrease in their ability to germinate (Kawanabe, 1986). If germinability generally decreases with age, this effect is species and strain dependent (Dantigny and Nanguy, 2009). This inability of fungal spores to germinate is maintained by several factors (Sephton-Clark and Voelz, 2018): The resistant cell wall, sometimes characterized by the presence of melanin, chitosan, or hydrophobic rodlets; internal compartmentalization separating energy substrates and enzymes; low water content and reduced metabolic activity; dormancy factors such as the transcription factor Afta. During dormancy release in Aspergillus spp., a significant transcriptional reprogramming is observed as early as the first two hours, notably involving the roles of the RAS/MAPK and cAMP/PKA pathways in germination (Dantigny and Nanguy, 2009). In Fusarium spp., specific gene expressions such as GEA1 or FgATG15 are involved. Metabolic reprogramming linked to ergosterol is also observed (Dantigny and Nanguy, 2009).

Washing phases of the inoculum spores are found in many protocols. Indeed, inhibitory compounds can be introduced with the inoculum (Prosser, 1994) and criticism has been raised about using unwashed spores to assess spore germination in distilled water (Sheridan and Sheehan, 1980).

The complexity of germination arises from the multiplicity of signaling pathways involved, which respond to various stimuli, and from the lack of clear molecular signals allowing the isolation of specific events (Osherov and May, 2001). Rigorous standardization of spore harvesting conditions is essential to ensure comparability between studies (Dantigny and Nanguy, 2009). In this context, the aims of this study are first to investigate the correlation between the pre-culture duration of P. camemberti on a solid medium and its subsequent ability to germinate in a liquid medium. Additionally, the study aimed to show the potential interest of washing the spores and to examine the crowding effect. Thereby this study provides a comprehensive reference for this bioprocess step with the intention of highlighting the critical nature of the pre-culture stage on biotechnological production of P. camemberti, in submerged conditions for its use in the dairy industry. This study presents experimental results specific to the strain under investigation, which is of interest to the dairy industry, and lets to broaden scientific discussion on the importance of this stage that is scarcely documented for P. camemberti. Furthermore, the discussions are intended to open debate on the potentially involved mechanisms.

Materials and methods

Strains, culture conditions and spore suspension preparations

A P. camemberti strain isolated from mould-ripened soft cheese was used for this study. This strain was chosen for its organoleptic qualities in the context of the dairy industry and then isolated and stored in Toulouse Biotechnology Institute (Toulouse, France). Spore suspensions of the strain were preserved at − 70 °C in 10% (v/v) glycerol. To produce spores on solid medium a spore suspension was inoculated on slant Potato Dextrose Agar (PDA, Biokar, Netherlands: 4 g/L potato extract, 20 g/L glucose, 15 g/L agar) and tubes were incubated for 7 days with controlled temperature and humidity of respectively 23,5 °C and 85% (Memmert, Germany). Then spores were harvested adding 8 mL of sterile distilled water supplemented with 0.1% (v/v) Tween 80 using a sterile cell scraper. Spore suspension was collected using 10 mL sterile pipette and used to inoculate Roux-flask containing 100 mL PDA. They are incubated under the same conditions for a duration between 13 and 27 days. To harvest spores, same protocol has been completed with 25 mL of sterile distilled water supplemented with 0.1% (v/v) Tween 80. The Penicillium spore suspension is then cleared of any potential filaments recovered during the previous step, using a 70 µm cutoff threshold sterile cell sieve (Sarstedt, Germany). The resulting spore suspension was transferred into a tube and centrifuged for 5 min at 4000g at 4 °C (Eppendorf, Germany). Depending on the experiments, spores were resuspended either in 10 mL sterile distilled water supplemented with 0.1% (v/v) Tween 80 (spore washing condition) or in 10 mL of supernatant solution (unwashed spores condition), with the aim of having a similar treatment of the spores for both conditions.

Finally, suspensions of 1 × 10⁶ and 5 × 10⁷ spores/mL were prepared.

Spore counts were all carried out by the same operator using a Thoma counting chamber, where 10 large squares of the grid were counted. The cell concentration is obtained by Eq. 1.

$$\frac{Quantityofsporescountedsquares\times dilutionfactor\times volumeconversion\left({\text{mm}}^{3}to\text{mL}\right)}{Surfaceareaofasquare\left({\text{cm}}^2\right)\times Depthoftheslide\left(\text{cm}\right)\times numberofcountedsquares}$$ 1

Effects of spore age on the ability to germinate in solid medium

The ability of spores to germinate on solid medium was determined for durations of Roux flasks PDA medium cultures from 14 to 27 days. One pre-culture on solid medium was carried out for each condition between 14 and 27 days. The spore suspensions collected after PDA medium Roux flasks cultivation were prepared in series of one-tenth dilutions, from 3 to 5, and then 0.1 mL of the dilution was spread on a rich solid medium in duplicate, Plant Count Agar (PCA, Biomérieux, France: 2.5 g/L yeast extract, 5 g/L tryptone, 1 g/L glucose, 15 g/L agar). The agar plates are incubated at 23.5 °C and 85% of humidity, and the colonies are counted after 48–72 h. Each count was assessed in duplicate.

The germination percentage on solid medium is calculated by dividing the average values of plates counts by the suspension total spore count determined by Thoma chamber counting.

Effects of spore age, spore washing and spore concentration on the ability to germinate in liquid medium

The ability of P. camemberti spores to germinate in liquid medium under the studied conditions was assessed in sterile tubes with a useful volume of 2 mL: 50% spore solution and 50% synthetic medium inspired by Canteri (2014) (10 g of D( +)glucose, 2.05 g of CaCI₂ × 2H₂0, 9.3 g of sodium acetate x 3H₂0, 5 g sodium citrate tribasic x 2H₂0, 4.6 g ammonium sulfate, 0.15 g MgS0₄ × 7H₂0, 0.13 g KH₂PO₄, 0.05 g KCl, 0.0002 g FeS0₄ × 7H₂0 and 1 mL stock solution of trace elements, 5 mg/L CuS0₄ × 5H₂0, 3.4 mg/L MnS0₄ × 1H₂0, 2 mg/L ZnS0₄ × 7H₂0, were dissolved in 1 L distilled water). The tubes were left for 20 h at 25 °C with a rotary shake of 150 rpm (Minitron, Infors, Switzerland) to allow the microorganism to complete its lag and germination phases. The germinated spore amount was determined on Thoma chamber counting. A minimum of 100 conidia were counted. A spore is considered germinated if its germ tube or tubes are of a length equal to the diameter of the swollen spore. The germination percentage was calculated for each condition by dividing these amounts by the suspension total spore count. For example, if the operator observes 120 spores and considers 90 of them to have germinated, according to the morphological criteria defined above, the germination percentage in liquid medium will be 75%. To minimize potential operator-related bias, the same person performed all Thoma chamber counts throughout the study. Each count was assessed in duplicate. In addition, biological duplicates were performed for the conditions with 14 days of pre-culture.

Effects of spore age and concentration on germination kinetics in liquid medium

Germination kinetics were carried out on cultures made in 50 mL Erlenmeyer flasks with 10 mL of synthetic medium incubated at 25 °C with a rotary shake of 150 rpm. The germinated spores’ amount was determined on Thoma chamber counting at 15, 20, 26, 32, 39 and 48 h by the same operator. Each count was assessed in duplicate. Two biological replicates were performed at 39 h.

Statistical analysis

All counts were performed in duplicate, whether on PCA solid medium to assess the ability of spores to germinate on solid medium or using a Thoma chamber to evaluate their ability to germinate in a synthetic liquid medium. The results presented therefore represent the values obtained from both counts. In addition, biological duplicates were performed for the 14 days pre-culture condition as part of the measurement of germination percentage in liquid medium (Fig. 2). Two biological replicates were performed at 39 h as part of the study on the effects of spore age and concentration on germination kinetics in liquid medium. The standard deviation from the mean was then calculated and represented (Fig. 4).

Fig. 2.

Effect of spore washing on the ability to germinate in liquid medium at low concentration (1 × 10⁶ sp/mL), with washed (•) and unwashed (□) spores

Fig. 4.

Effect of pre-culture time, 13 days (•) and 27 days (□), on germination kinetics in liquid medium (washed spores and low concentration: 1 × 10⁶ sp/mL)

To characterize the overall trend of germination rates in solid and liquid medium while minimizing the influence of isolated extreme values, we calculated the variation ratio between rolling 5-day extremes values. These extreme values correspond to the mean germination percentages between 14 and 18 days pre-culture time, as well as between 29 and 33 days pre-culture time.

Results and discussion

Effects of spore age on the ability to germinate in solid medium

First, in the context of spore pre-cultures on solid media, it is important to distinguish the age of the spores and the duration of the pre-culture period, as the spores produced at any given pre-culture time are heterogeneous in age. However, the longer the pre-culture period, the higher the average age of the spore population under study is.

The quality of spores is interesting to analyze and optimize within the framework of the bioprocess for producing this filamentous fungus of dairy interest. As initial indicator of spore quality, the ability of P. camemberti to germinate on a solid medium was studied over time. This refers to the ability of spores obtained from preculture on PDA medium at different durations (age of spores) to form colonies on a solid medium. Regarding this indicator, a slight decrease in the ability to germinate on solid medium is observed when the pre-culture duration of P. camemberti is extended (Fig. 1).

Fig. 1.

Effect of pre-culture time on the ability to germinate in solid medium (washed spores)

The variation ratio calculated between rolling 5-day extreme values, that is, between spores obtained from pre-cultures considered as young (14–18 days) and old (29–33 days), is − 41% (Table 1). This approach captures general directional changes over time and suggests a potential loss of quality in the spores of P. camemberti between 14 and 33 pre-culture days.

Table 1.

Evolution of spore germination percentage in solid medium (washed spores) and liquid medium (washed spores and low concentration: 1 × 10⁶ sp/mL) for different pre-culture time between 14 and 33 days

Effects of spore age, spore washing and spore concentration on the ability to germinate in liquid medium

The quality of the spores was studied based on their ability to germinate in synthetic liquid medium. This indicator is the most relevant in the context of scaling up this microorganism to submerged culture in bioreactors. Spores from PDA medium preculture were used to inoculate liquid synthetic medium tubes and the germination percentages in liquid medium were calculated after precisely 20 h of growth. This analysis time is relevant because it allows the microorganism enough time to complete its lag phase and then begin germination, but without observing complex filamentous structures microscopically.

Effect of spore age

The ability of spores to germinate in liquid medium was studied as a function of pre-culture duration. The ability of spores to germinate in liquid medium decreases sharply with spore age (Fig. 2). The results show a very high majority of germinated spores from 14-day pre-cultures (> 80%) to less than 10% germination after about thirty days of pre-culture. For a 33-day pre-culture, the germination rate in liquid medium is nearly zero (3%). It is therefore worth noting that the age of the spores has a more pronounced impact on submerged culture. Since this method is now used for the bio-production of P. camemberti, it represents a finding of interest for better understanding and could help improve the production strategy. Moreover, these results contradict the bibliographic references on this topic where pre-culture times are frequently described vaguely around a month (Siegbert, 1988). A correlation between spore age and a decrease in their ability to germinate has been also demonstrated in Neurospora crassa (Kawanabe, 1986). Therefore, the results obtained for our strain are not entirely surprising. Germinability generally decreases with age (Dantigny and Nanguy, 2009). That said, no previous study has shown this phenomenon in P. camemberti, and this study highlights the sensitivity of spore age in a strain of this microorganism, which is widely used in the cheese industry and currently produced in liquid media by suppliers. While it is true that strain-dependence may play a role in the observed phenomenon, it is very important not to overlook this pre-culture step, as it has a strong impact on the microorganism’s growth (Gillot et al., 2016). Rigorous standardization of spore harvesting conditions is essential to ensure comparability between studies (Dantigny and Nanguy, 2009) but also appears necessary for the development of industrial scale bioproduction protocols. However, it is also possible to consider other steps upstream of the bioreactor and downstream of the solid medium pre-culture (Canteri, 2014) but this protocol has not proven to be suitable for our strain.

In the context of our results, it is important to note that the loss of spore germination capacity as a function of pre-culture age on solid medium, is clearly more obvious in relation to the transfer to liquid medium (Figs. 1, 2). The variation ratio calculated between rolling 5-day extreme values (Table 1), that is, between spores obtained from pre-cultures corresponding to roughly 2 to 4 weeks, is − 41% in a fresh solid medium, whereas the loss of germination ability is enhanced in a fresh liquid medium (− 94%). This observation raises another scientific question and could help guide reflections on the cause of this loss of spore germination capacity in liquid medium. As such, it may also steer the search for potentially associated cellular mechanisms. Given the near-complete loss of germination capacity in liquid medium, it is worth considering the potential role of water in the experimental results observed for P. camemberti. It was highlighted that the hydration state of fungal spores could significantly influence their physiological condition (Dantigny and Nanguy, 2009). Indeed, several studies have reported that fungal spores may lose endogenous reserves when exposed to environments with water potential higher than that within their cells (Kelly, 1991; Pascual et al., 2002). It is therefore reasonable to question whether spores transfer to a liquid medium could have an impact, due to the higher water potential of the medium compared to that within the spores themselves. This potential sensitivity appears to be more pronounced in spores originating from older pre-cultures. It would nevertheless be of interest to elucidate the cellular mechanisms linking spore age to the loss of germination capacity observed in liquid medium. Our study therefore also highlights the importance of developing and tailoring protocols to the specificities of each process. Indeed, the complexity of spore germination lies in the multitude of signaling pathways involved, each responsive to different stimuli (Osherov and May, 2001). In our case, it was necessary to consider not only the duration of the pre-culture but also the impact of transferring spores from a solid to a liquid medium.

Given that our germination rates are measured at precisely 20 h of culture, a significant question arises: how can we be sure that this is a loss of the spores' ability to germinate rather than a slowdown in germination? To address this, several germination kinetics were subsequently performed on our strain at different pre-culture times where germination in liquid medium is significantly different: 14 days or 27 days pre-culture time. These results are analyzed later (Fig. 4).

Effect of spore washing

Washing phases of the inoculum spores are found in protocols (Gillot et al., 2016) but it is not easy to find studies that prove their usefulness. The washing effect on spores was studied on spores from pre-cultures ranging from 14 to 33 days (Fig. 2). The results show that for washed or unwashed spores from pre-cultures, their ability to germinate in liquid medium is not affected. For example, for 22-day pre-cultures, the germination rate of spores in liquid medium is close to 50%, whether or not they are washed. Similarly, the germination rate in liquid medium at 33 days is close to 10%, with or without spore washing. A hypothesis was put forward that inhibitory compounds might be introduced into the medium by preculture (Prosser, 1994) but, in our study, our unwashed spore solution is then diluted in a liquid medium where its ability to germinate is studied and then we can’t verify this hypothesis. This could also explain why spore washing has no relevance in the case studied. That said, the effect of spore age on their ability to germinate in liquid medium is observed regardless of whether the spores are washed or not. This suggests that the washing step of P. camemberti spores may have limited importance when it comes to controlling and optimizing the germination of spores in liquid medium, particularly those obtained from solid pre-cultures. This supports the idea that the impact of pre-culture duration on spore germination in liquid medium is unlikely to be explained solely by the introduction of inhibitory compounds from the pre-culture (Prosser, 1994), suggesting the involvement of an additional mechanism.

Effect of spore concentration

The next effect we aimed to demonstrate is the population effect. This inhibitory effect on germination has been described in the literature for P. camemberti at high population density (1 × 10⁸ sp/mL) (Gillot et al., 2016). In our study, we compare the germination rates of a high spore concentration (5 × 10⁷ sp/mL) with a population 50 times less dense (1 × 10⁶ sp/mL). The population effect was directly identified under these conditions (Fig. 3). Indeed, for spores from 14-day pre-cultures on solid medium, their ability to germinate in liquid medium is high (86%) at low population density (1 × 10⁶ sp/mL), whereas the proportion of germinated spores at high density (5 × 10⁷ sp/mL) is quite low (7%). This population effect is significant, and it is therefore crucial to take it into account when moving to a bioreactor. It will be important to consider the concentration of P. camemberti spore inoculum in bioreactors, not only by optimizing this ratio for microorganism growth and sporulation but also for its ability to germinate in submerged culture. For spores from 33-day pre-cultures, the population effect is no longer visible because the effect of spore age is predominant, as explained earlier. In P. camemberti, the volatile nature of the compounds involved was first demonstrated (Gillot et al., 2016), and 1-octanol was identified as the main volatile compound produced at high spore density (10⁸ spores/mL). It is quite likely that similar volatile compounds are produced by our strain under high cell density conditions, even though the density in our system is approximately twofold lower. The production of these inhibitory compounds would occur in liquid medium under high cell density conditions, which is consistent with the observation that washing the spores during inoculum preparation does not affect this phenomenon.

Fig. 3.

Effect of spore concentration on the ability to germinate in liquid medium for washed spores, at low concentration (•): 1 × 10⁶ sp/mL and high concentration (□): 5 × 10⁷ sp/mL

Effect of spore age and concentration on germination kinetics in liquid medium

The spore germination kinetics were performed over 48 h using spores from 13-day and 27-day pre-cultures (Fig. 4). Focusing first on the germination ratio at exactly 20 h, we observe that the germination ratio from spores of a short pre-culture is good at 67%, while it is only 3% for a 27-day pre-culture. This confirms the effect of spore age on their ability to germinate at 20 h as previously shown. The germination ratio in the context of a short pre-culture was even 76% after 15 h of culture in liquid medium. It then gradually decreased to 0% after 48 h. This can be easily explained by the observed morphological changes. Indeed, the spores undergo a latency phase in the early hours of culture in liquid medium, then primarily germinate in isolation from one another, leading to the maximum germination rate around 15–20 h. Subsequently, the germinated spores will undergo vegetative growth resulting in the formation of complex filamentous structures. Therefore, there will be fewer and fewer isolated spores, which are mostly non-germinated, as they actually correspond to the spores with lowest initial quality. The second very interesting aspect of Fig. 4 is that spores from 27-day pre-cultures hardly germinate during the 48-h observation period. Therefore, these spores are not able to exit their latency phase, unlike younger spores. This further demonstrates the effect of spore age on their future quality in liquid medium.

In the same way, the behavior of high spore populations (5 × 10⁷ sp/mL) was studied in terms of germination kinetics over time (Fig. 5). As with the age effect, it results in an inhibition of the spores' ability to germinate if they are exposed to too high population density. Under these conditions, the spores, like all microorganisms, sense their environment and are not in favorable conditions to initiate their growth.

Fig. 5.

Effect of spore concentration, 1 × 10⁶ sp/mL (•) and 5 × 10⁷ sp/mL (□), on germination rates in liquid medium (13 days pre-culture time and washed spores)

The aim of this study was to define the conditions for spore production on a solid medium that maximize the quality of the produced spores for the transition to liquid medium. A correlation between the pre-culture time on a solid medium and its future ability to germinate in a liquid medium was demonstrated. The impact of spore age on their germination capacity was particularly more pronounced in liquid medium than on solid medium. A population effect was also observed: too high spore population concentration in a liquid medium leads to total inhibition of germination. The effects of age and spore population are such that germination is inhibited rather than delayed. It would be interesting to identify potential molecules responsible for this inhibition, in order to possibly uncover the underlying cellular mechanisms as it was discovered regarding the population effect (Gillot et al., 2016). Several studies have reported that fungal spores may lose endogenous reserves when exposed to environments with water potential higher than that within their cells (Kelly, 1991; Pascual et al., 2002). This more pronounced impact in liquid medium could possibly be explained by a combined effect of this mechanism and the duration of the pre-culture.

It is therefore crucial to consider these two parameters, age and spore population, when transitioning from solid to liquid medium in the bioproduction process of P. camemberti. In our specific case, for the strain studied, 2 weeks of pre-culture and a concentration of 1 × 10⁶ sp/mL are favorable for good submerged germination. Our study therefore also highlights the critical nature of the pre-culture stage on biotechnological production of P. camemberti, in submerged conditions, for its use in the dairy industry. This study presents experimental results specific to the strain under investigation. It also underlines the importance of developing and tailoring protocols to the specificities of each strain and process, especially considering transitions between solid and liquid media, given the sensitivity of P. camemberti.

Declarations

Conflict of interest

The corresponding author states that there is no conflict of interest.