Paola Labaky

Lidwine Grosmaire

Julien Ricci

Christelle Wisniewski

Nicolas Louka

Layal Dahdouh
email: layaldahdouha@cirad.fr

Innovative non-destructive sorting technique for juicy stone fruits: textural properties of fresh mangos and purees

Keywords: Mango, purees, texture, rheology, maturity, sorting tool

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mangifera indica L. known as mango, is a tropical fruit originated from the Indo-Burmese region. Mango is one of the most produced (mainly in India, China, Thailand, Indonesia and Pakistan) and consumed fruits worldwide after banana [START_REF] Chantalak | Handbook of Mango Fruit[END_REF][START_REF] Masud Parvez | Pharmacological Activities of Mango (Mangifera Indica): A Review[END_REF]. There is a large variety of mangos that differ in size, color, texture, and nutritional properties. The most consumed mangos varieties are Kent, Keitt, Haden, Tommy Atkins, Cogshall, Alphonso, Amelie and Valencia pride [START_REF] Djioua | Amélioration de la conservation des mangues 4 ème gamme par application de traitements thermiques et utilisation d ' une co[END_REF].

According to the FAO statistics, mango production worldwide increased by 26 million metric tons over a period of 17 years to reach 50 million metric tons in 2017 (FAO, 2019). In developing countries, post-harvest fruit losses due to the abundant mango production can be estimated up to 40% [START_REF] Boateng | Analysis of Post Harvest Losses in the Mango Marketing Channel in Southern Ghana 99[END_REF][START_REF] Memon | Estimation of mango post harvest losses in sindh[END_REF]. Hence, processing and transforming mangos into purees, juices, jams, canned products and dried slices [START_REF] Evans | Mango Production, Global Trade, Consumption Trends, and Postharvest Processing and Nutrition[END_REF] is an alternative not only to minimize post-harvest losses but also to provide local incomes. Nevertheless, the quality of processed products (physical, physico-chemical and organoleptic characteristics) depends on the maturity stage of raw mangos [START_REF] Ellong | Physicochemical, Nutritional, Organoleptic Characteristics and Food Applications of Four Mango (<i>Mangifera indica</i>) Varieties[END_REF]. Indeed, the heterogeneity of the maturity stages encountered in a same batch of mangos for processing constitute a barrier for controlling the process and the quality of the finished product [START_REF] Rivier | Le séchage des mangues[END_REF] In the light of the above, fruit sorting prior to mango processing is generally performed in processing units to reduce batch heterogeneity in relation with maturity stages. To date, mango sorting is mainly manual, time consuming and highly labor-dependent (visual and tactile know-how) leading in some cases to inaccurate fruits sorting and disparate quality of processed products. Several tools (e.g. textural, spectral, fluorescence and biochemical measurements) have been evaluated to predict the maturity stage or the quality of mango fruits [START_REF] Pronprasit | Prediction of Mango Fruit Quality from NIR Spectroscopy using an Ensemble Classification[END_REF][START_REF] Valente | Instrumental and sensory characterization of mango fruit texture[END_REF][START_REF] Zakaria | Improved maturity and ripeness classifications of Magnifera Indica cv. harumanis mangoes through sensor fusion of an electronic nose and acoustic sensor[END_REF]. However, as far as the authors are aware, in these studies, none of the proposed tools were conceived as to predict the quality of processed products.

Nowadays, the lack of instrumental tools suitable to sort mango fruits according to their ability to be processed into products with specific quality is a main setback for up-grading mango fruits. In this context, the aim of this work was to develop new methods and tools, mainly non-destructive, allowing to sort mangos according not only to their maturity stage but also to the specific properties of their processed products (puree). To this end, an experimental strategy combining mechanical (rheology and texture), physical and physicochemical (particles size measurement, color, pH, titratable acidity, °Bx, dry matter) analyses was proposed to characterize fresh mangos and mango purees. Firstly, textural (penetrometry and compression), rheological (oscillatory), physical (weight, density) and color analyses were performed on fresh mangos at different stages of maturity. In a second place, fresh mangos were processed into purees that were characterized (particles size, rheology and physico-chemistry). Finally, statistical analyses were used to (i) identify pertinent instrumental indicators that describe the maturity stages of mangos, (ii) evaluate the impact of mangos maturity stage on purees characteristics and finally to (iii) propose new tools for sorting fresh mangos according to their ability to be processed into purees.

Materials and methods

Fruits

Fifty green mangos (Mangifera indica L., cv. Kent, Peru) previously stored at 10°C during 10 days (to slow down maturation) were purchased from a local warehouse (Georges Helfer SA, Plan d'Orgon, France). All mangos were stored in controlled conditions (18°C, 80% RH) between one and twenty-five days. Daily, one or two fruits were characterized. Each mango was transformed separately into puree and all measurements were carried out on the same fruit and its puree. Data of twenty-eight mangos were selected for this study.

Fresh mango characterization

Before the analyses, mango fruits, were soaked in chlorinated water (200 ppm sodium hypochlorite, Chem-Lab, Zedelgem, Belgium) and wiped with 70% ethanol (Honeywell, Riedel-de Haën, absolute, ≥ 99.9, Charlotte, North Carolina, USA) in order to remove the latex layer that covers the fruits [START_REF] Palafox-Carlos | Effect of ripeness stage of mango fruit (Mangifera indica L., cv. Ataulfo) on physiological parameters and antioxidant activity[END_REF][START_REF] Penchaiya | Measurement of mango firmness by non-destructive limited compression technique[END_REF].

Fruit density

Fruit density was calculated using Archimedes' principle by measuring the fresh fruits mass in air and in water (each mango was placed in a basket hanging from the balance and fully immersed in water) according to the method described by [START_REF] Joas | Postharvest Biology and Technology Comparison of postharvest changes in mango ( cv Cogshall ) using a Ripening class index ( Rci ) for different carbon supplies and harvest dates[END_REF][START_REF] Joas | Postharvest Biology and Technology Comparison of postharvest changes in mango ( cv Cogshall ) using a Ripening class index ( Rci ) for different carbon supplies and harvest dates[END_REF].

Texture analysis

Firmness of mangos was measured using a texture analyzer (TA-XT2, Stable micro Systems, London, UK) equipped with a 5 kg load cell and an Exponent software (version 5.1.1.0) to record data. Two different methods were evaluated: a non-destructive compression test and a destructive penetrometry test.

Non-destructive compression test

The compression test in this study was proposed to simulate the tactile perception of the operator during sorting mango fruits according to their firmness. Compression tests were performed on both larger sides of mango since water potential of the two sides may differ due to the position of the fruit on the tree and to its exposition to the sun. Measurements were performed on three different positions on each side of the fruit. The test was carried out to reach a maximum compression force of 10 N with a 2 cm spherical probe and probe speed of 1 mm.s -1 . The maximum force was carefully chosen in order to avoid damaging the fruit structure and subsequently proposing a non-destructive test. The obtained force-distance curves were recorded and the distances of compression (D5N or D10N) at 5 or 10 N were identified as the most consistent parameters to describe mango firmness. Similar results of D5N and D10N values were recorded regardless the side of mango (R 2 > 0.99) for linear correlations of D5N data and D10N data for side 1 and side 2 (data not shown). Considering this observation, results concerning compression tests will be presented only for D5N and for one side in the following.

Destructive penetrometry test

Penetrometry test was performed on slices (h=15 mm, using an electric ham slicer generally used for processed meat) from each side of mango using a cylindrical stainless steel probe of 5 mm diameter. The test was carried out on the inner flesh tissue to reach a maximum distance of 5 mm with a probe speed of 1 mm.s -1 . The aim of this additional test was to evaluate the firmness of the mango flesh in a destructive way as it is conventionally done. The firmness of mangos was evaluated by the average of the maximum value of the peak force (Fmax) of the resulting force-distance curves. Measurements were realized on three different positions of the slice.

Rheological measurements

Rheological measurements were performed using a Haake Mars 60 rheometer (Thermofisher, Waltham, Massachusetts, USA) equipped with a 35 mm serrated parallel plates geometry and a "RheoWin" software (version 4.82.0002) to record rheological data. A strain amplitude sweep test was performed in low strain amplitude range (from 0.01 to 1%) at a constant frequency of 1 Hz and a controlled normal force of 4 N. All experiments were conducted at temperature of 25°C ± 0.1 controlled by a Peltier system. The storage modulus values G'(Pa) at 0.1% of strain were selected as rheological indicator reflecting the solid-like behavior of mango slices. Measurements were performed in quadruplicate.

Flesh color

Color was determined on grinded flesh using a Minolta CR-300 colorimeter (Konica Minolta Sensing, Inc, Ramsey, New Jersey, USA) according to the CIELAB color system (L*, a*, b*).

L* represents the lightness varying from 0 to 100, (0 representing black and 100 representing white), a* represents the variation from green (-) to red (+) and the b* reflects the variation from blue (-) to yellow (+).

Characterization of mango puree

Puree preparation

Each mango was manually peeled, the seed was removed and the flesh was cut into small pieces (around 1 cm 3 ). The flesh was grinded at 25°C with a Thermomix (Vorwerk, Typ 31-1, Wuppertal, Germany) at a speed of 1000 rpm for 1 min, 7000 rpm for 1 min and 10000 rpm for 2 min. After grinding, the puree (approximately 180g) was stored at 4°C before the analyses.

Rheological measurements

Rotational measurements

Rotational measurements of mango purees were performed using a Physica MCR301 rheometer (Anton Paar Gmbh, Graz, Austria) equipped with "Start Rheoplus" software (version RHEOPLUS/32 V3.40) to record the rheological data. A six blades vane geometry (ST22-6V-16, radius of 22 mm) was used with stationary cup with a radius of 27 mm giving a gap of 2.5 mm. The shear rate varied from 0.1 to 500 s -1 and temperature was held at 25°C ± 0.1 using a Peltier system. The evolution of dynamic viscosity µ (Pa.s), of each mango puree, as function of the shear rate (s -1 ) was recorded.

Oscillatory measurements

Strain amplitude sweep tests were conducted using the Haake Mars 60 rheometer equipped with a 35 mm serrated parallel plates geometry. Frequency was held constant at 1 Hz, while strain amplitude varied between 0.01 and 100%. All experiments were conducted at a temperature of 25°C ± 0.1 controlled by a Peltier system. For each puree, the storage and loss moduli G' and G" were recorded as function of the strain (%). The values of the storage G' (Pa) and loss G" (Pa) moduli (at 1% of strain in the linear viscoelastic domain) were selected as rheological indicators reflecting the solid-like behavior and liquid-like behavior of mango purees, respectively.

Particles size measurements of suspended insoluble solids

Particles size distribution was determined by LASER diffraction using a Malvern Mastersizer (Mastersizer 3000, Malvern Instruments Limited, Worcestershire, UK). This particles size analyzer can provide theoretically particles size distribution from 10 nm to 3500 μm.

Measurements were carried out in a wet-mode using distilled water as the suspension medium. The values 1.73 and 1.33 were used for the refractive indices of cloud particles and dispersion phase (water), respectively, and 0.1 was used for the absorption index of cloud particles [START_REF] Dahdouh | Rheological study of orange juices for a better knowledge of their suspended solids interactions at low and high concentration[END_REF]. Samples were introduced into the volume presentation unit, which already contained deionized water (obscuration of 20%). In this unit, the diluted sample was stirred at 1500 rpm and pumped through the optical cell. The initial particles size distribution of puree was verified to be not modified in such conditions of stirring and pumping. For each measurement, size distribution (volume density against particles size) was provided and statistical volume diameters, D10, D50 and D90 were given (Dx indicates a particles size for which x% of the particles are below that size). The surface area average diameter D [3;2] (Sauter mean diameter) and the volume mean diameter D [4;3] (Brouckere mean diameter) were also provided [START_REF] Dahdouh | Identification of relevant physicochemical characteristics for predicting fruit juices filterability[END_REF].

The D [3;2] indicates the diameter of a sphere that has the same volume/surface ratio as the set of particles Eq. ( 1):

D [3;2] = ∑ ∑ (1)
The D [4;3] indicates the diameter of a sphere whose volume is equal to the average volumes of all the particles in the sample Eq. ( 2):

D [4;3] = ∑ ∑ (2)
With ni the number of particles of diameter di.

Particles size measurements performed separately on mango purees and on their isolated suspended insoluble solids (centrifugation 18000 g /30 min) provided identical particles size distributions, smaller compounds being not detectable in the Mastersizer operating conditions.

Hence, results presented in this work concern mainly the particles size distribution of suspended insoluble solids (> 1µm) of purees.

Physico-chemical analyses

Mango is a climacteric fruit for which biochemical and nutritional changes occurring during tree-ripening can continue after harvesting. Titratable acidity (TA), total soluble solids (TSS) and dry matter (DM) were performed on purees according to protocols and methods used for fruit suspensions [START_REF] Dahdouh | Rheological study of orange juices for a better knowledge of their suspended solids interactions at low and high concentration[END_REF]. pH and titratable acidity were measured using an automatic Titroline apparatus (Schott Schweiz AG, St. Gallen, Switzerland). Titratable acidity was assessed by titration with 0.025 N NaOH until a pH 8.2. Titratable acidity (TA) was expressed in g of citric acid /100 g of puree since it is well known that citric acid is one of the major organic acids present in mango contributing to fruit acidity and flavor. Total soluble solids (TSS, expressed in °Bx) were measured with an Abbe refractometer (Atago, Japan).

Dry matter (DM, expressed in g/100 g of puree) was determined by drying 3 g of puree at 70°C under vacuum for 24 h. For all physico-chemical analyses, measurements were performed at 25°C in triplicate and the average values were used.

Statistical analyses

Multivariate analyses were carried out using XLSTAT (version 16.0.4744 Addinsoft, Paris, France). Principal component analysis (PCA), a multivariate projection method designed to reduce the dimensionality and to describe the variation of the data [START_REF] Azira | Use of principal component analysis for differentiation of gelatine sources based on polypeptide molecular weights[END_REF] TA, TSS and DM) were set as variables. The score plot was extracted from the first two principal components, PC 1 and PC 2, as they presented the maximum variability of the data.

Results and discussion

3.1 Fresh mangos 3.1.1 Texture characterization Results regarding compression test are presented in Fig. 1(a). The distance achieved by the probe when applying 5 N (D5N) on the raw mango increased significantly with maturation highlighting a significant loss of mango firmness. The loss of firmness of fruits occurring during the maturation period could be due to biochemical changes such as the degradation of pectins, cellulose and hemicellulose [START_REF] Lawson | Characterization of Southeast Asia mangoes (Mangifera indica L) according to their physicochemical attributes[END_REF][START_REF] Padda | Methods to analyze physico-chemical changes during mango ripening: A multivariate approach[END_REF][START_REF] Yashoda | Mango ripening: Changes in cell wall constituents in relation to textural softening[END_REF]. Fig. 1(b) shows that the force (Fmax) needed to puncture 15 mm of mango slice decreased significantly during maturation confirming the loss of firmness. However, penetrometry test seems to be less discriminating regarding mango firmness than compression test after 15 days of maturation. Indeed, a significant decrease of Fmax (92%) was observed before 15 days of maturation whereas beyond this maturation stage the Fmax was almost steady. After 15 days, the difference of the flesh firmness was not high enough to be detectable by the penetrometry test. Contrariwise, in the case of compression test, the firmness of the whole fruit (peel and flesh) was measured, leading to better results.

Rheological characterization

Since texture tests showed that the firmness of mango flesh decreased during maturation, rheological measurements were performed to assess the evolution of the solid-like behavior of mango flesh in relation with firmness loss during maturation. To this end, the storage modulus G' (Pa) reflecting the solid-like behavior of a material was evaluated through oscillatory test in small amplitude range (0.1 to 1%) within the linear viscoelastic range [START_REF] Lee | Erratum to: Objective Measurements of Textural and Rheological Properties of Cheese[END_REF]. Indeed, the storage modulus of mango slices at the end of maturation was 10 times lower than the one recorded for green mango at early maturation stages. This observation emphasizes that biochemical and physico-chemical phenomena (starch, cell wall, cellulose and hemicellulose degradation, etc.,) [START_REF] Nambi | Texture and rheological changes of Indian mango cultivars during ripening[END_REF] occurring during maturation led to a modification of the viscoelastic properties of mango flesh and subsequently to its loss of firmness.

Moreover Fig. 2 (b) and (c), point up interesting correlations between the solid-like behavior (G') of mango flesh and the firmness of mango flesh (Fmax) and the firmness of the whole fruit (D5N). It is important to notice that the D5N is a better indicator than Fmax regarding the solidlike behavior of mango flesh at stages of maturation above 10 days (when Fmax < 5 N), Fig. 2 (b) and (c). This result confirms that it is possible to trace back the solid-like behavior of To sum up, both penetrometry (destructive) and compression (non-destructive) tests allowed to measure significant loss of mango firmness during maturation. Compression test provided better information about mango firmness after 15 days of maturation without damaging the fruits. Moreover, this simple and non-destructive compression test makes possible to trace back the solid-like behavior (G') of mango flesh, since D5N, is a good indicator of the solidlike behavior of mango flesh even at stages of maturation above 15 days.

Other physical characterization: flesh color, fruit weight and density

Fruit weight, density and flesh color, were measured during maturation in order to assess the effect of maturation on these physical and physico-chemical characteristics of mangos [START_REF] Gentile | Food quality and nutraceutical value of nine cultivars of mango (Mangifera indica L.) fruits grown in Mediterranean subtropical environment[END_REF][START_REF] Lawson | Characterization of Southeast Asia mangoes (Mangifera indica L) according to their physicochemical attributes[END_REF].

Fruit density decreased during maturation as presented in Fig. 3(a). A weight loss of mango fruits during maturation is mentioned by [START_REF] Lawson | Characterization of Southeast Asia mangoes (Mangifera indica L) according to their physicochemical attributes[END_REF] and explained by the loss of water through the stomata and pores [START_REF] Lawson | Characterization of Southeast Asia mangoes (Mangifera indica L) according to their physicochemical attributes[END_REF].

Concerning color, the variation of three parameters L*, a* and b* is generally used to monitor the variation of flesh color. The decrease of the lightness L* point up a darkening of fleshes during maturation due to the activity of the polyphenol oxidases forming brown pigments [START_REF] Liu | Physico-chemical and antioxidant properties of four mango (Mangifera indica L.) cultivars in China[END_REF]. The increase of a* and b* reveal biochemical changes (formation of carotenoids, chlorophyll degradation, etc.) during maturation [START_REF] Lawson | Characterization of Southeast Asia mangoes (Mangifera indica L) according to their physicochemical attributes[END_REF][START_REF] Liu | Physico-chemical and antioxidant properties of four mango (Mangifera indica L.) cultivars in China[END_REF][START_REF] Rumainum | Fruit quality and antioxidant capacity of six Thai mango cultivars[END_REF]. In this study, the obtained results, Fig. 3 (b), (c), were similar to those previously reported by many authors regarding L* (L* was 1.2 times lower at the end of the maturation) and a* (a* was 7.4 times higher at the end of the maturation), but no significant evolution of b* was observed (Data not shown). 3.2 Mango puree characterization and relations with fresh mangos characterization

Particles size of suspended insoluble solids

Fruit suspensions consist in two phases: a serum phase containing soluble compounds such as sugars, acids and soluble pectins and insoluble phase containing insoluble suspended solids such as pectins, fibers and cell walls fragments [START_REF] Lopez-Sanchez | Shear Elastic Deformation and Particle Packing in Plant Cell Dispersions[END_REF]. For fruits-based suspensions, it is well known that the concentration and the size of insoluble suspended solids have a major role in the structural and rheological characteristics of these products [START_REF] Dahdouh | Rheological study of orange juices for a better knowledge of their suspended solids interactions at low and high concentration[END_REF]. Therefore, particles size measurements were performed on mango purees to assess the size distribution of their suspended insoluble solids in relation with mango maturation stage and puree rheological properties. Fig. 4 presents examples of particles size distributions for mango purees at 5 different days of maturation. These distributions are typical of polydisperse suspensions with a monomodal size distribution as reported in the literature for many fruits suspensions such as apple purees and fruit juices [START_REF] Dahdouh | Rheological study of orange juices for a better knowledge of their suspended solids interactions at low and high concentration[END_REF][START_REF] Leverrier | Influence of Particle Size and Concentration on Rheological Behaviour of Reconstituted Apple Purees[END_REF]. It can be noticed that purees obtained from the greenest stage were characterized by the higher volume density for particles around 1000 µm. The volume density of these particles larger than 1000 µm decreased considerably as the stage of maturation increased to disappear in late stage of maturation. For purees obtained from mango beyond 10 days of maturation, the highest volume density for particles shifted from 1000 µm to 200 µm. Changes in particles size in purees in relation with mango maturation were also highlighted by the evolution of the statistical diameters as they all decreased significantly with maturation, Fig. 5 show some examples of the evolution of D10, D50 and D [3;2] during maturation. Results concerning D90 and D [4;3] were not depicted in this section as they provided redundant information, they will be presented only in the statistical analyses. This observation could be explained by the hydrolysis and degradation, during maturation, of large insoluble suspended solids (starch, pectins and cell walls) into smaller compounds leading to a decrease of the size of suspended insoluble particles [START_REF] Venkatesan | Effect of Ethrel on the Starch Sugar Changes of Off-Season Fruits of Mango (Mangifera indica l. Var. Neelum) during Ripening[END_REF]. These results show that the loss of mango firmness and the hydrolysis of specific compounds (e.g. cell walls) during the maturation could lead to mango purees with smaller particles size. Indeed, Fig. 6 (a), (b) point up significant correlations between the average surface diameter D [3;2] of the purees and the firmness of mango flesh (Fmax) and the whole fruit (D5N). These observations emphasize the impact of mango firmness on the particles size distribution of the puree. Moreover, the same trend is observed between the average surface diameter D [3;2] and the storage modulus G' of mango flesh on Fig. 6 (c), showing that particles size distribution of mango puree is closely dependent on the solid-like behavior of mango flesh. In fact, purees grinded from the greenest mangos with a higher storage modulus G' of the flesh presented purees with larger particles. Since it is well known that grinding conditions have an important impact on the particles size distribution of purees [START_REF] Espinosa | Effect of processing on rheological, structural and sensory properties of apple puree[END_REF], standardized conditions of grinding were carefully used in this work, for all stages of maturation, allowing to show the specific role of mango firmness and solid-like behavior on the particles size of the purees. Hence, this work proposes for the first time a simple and fast tool to predict the particles size of mango purees (obtained in specific conditions) based on a non-destructive compression test (D5N), since the same trends were observed when destructive texture and rheological analyses were performed.

Rheological characterization

Rotational measurements

The effect of maturation on the flow behavior of mango purees was investigated throughout the measurements of the viscosity of different mango purees. Several published studies, reported that fruits purees are characterized by shear-thinning behavior as their viscosity decreases with the increase of the shear rate [START_REF] Espinosa-Muñoz | Structural parameters that determine the rheological properties of apple puree[END_REF][START_REF] Gundurao | Effect of soluble solids concentration and temperature on thermo-physical and rheological properties of mango puree[END_REF][START_REF] Phaokuntha | Rheological properties of mango puree and process development of mango sheet[END_REF]. Since all mango purees had also shear-thinning behavior in this study, a power Law (specific of this behavior) was used to determine the consistency index (K, Pa.s n ) and the flow behavior index (n) of each puree (with significant determination coefficient R 2 , for α = 5%, degree of freedom = 27), Eq. ( 3):

Power Law: = (3)
Where, , the modeled viscosity (Pa.s), K, the consistency index (Pa.s n ), n, the flow behavior index and the shear rate ( s -1 ). clearly as the maturation stage of mango increased whereas the consistency indexes (K) and the dynamic viscosity (µ) noticeably decreased only when comparing purees at very early and other stages of maturation. Indeed, consistency indexes were higher when considering purees at maturity stage between 0 and 5 days and slightly decreased for the other maturation stages.

All these results highlight the effect of the fresh mango maturation on its puree's viscosity. This is in line with the hydrolysis of protopectins, a binding substance between cell walls and the degradation of starch, etc. [START_REF] Sánchez-Gimeno | White guava fruit and purees : textural and rheological properties and effect on the temperature[END_REF] occurring during maturation and inducing a decrease of the size of suspended insoluble particles, thus, leading to a decrease of the viscosity of mango puree [START_REF] Espinosa | Effect of processing on rheological, structural and sensory properties of apple puree[END_REF]. *For all data the given are the average of three trials and all standard deviation values are inferior to 5% of the average value.

Table 1. The ranges of variation of K, the consistency index (Pa.s n ), n, the flow behavior index and (Pa.s) the experimental dynamic viscosity at 50 s -1 of mango purees.

Fig. 7 point up a decreasing trend between the experimental dynamic viscosity at 50 s -1 (µ) of mango puree and the firmness of the whole fruit (D5N) mainly when comparing early maturation stages [0-5 days] and [6-10 days] with the other stages. This observation emphasizes the impact of mango firmness at early stages of maturation not only on particles size of purees but also on their viscosity even if the correlation between µ and D5N is not as good as for D [3;2].

Oscillatory Strain Sweep

The viscoelastic behavior of different mango purees was evaluated through an oscillatory strain sweep test. For all purees the storage modulus G' and the loss modulus G" were constant in the domain of low strain amplitude (< 1%) and characterized a linear viscoelastic region (LVE-R). In this region, the storage modulus was higher than the loss modulus, showing a viscoelastic solid-like behavior. This behavior was independent of the maturity stage since all purees showed similar trends. G' and G" values declined and when a specific strain was reached, G" exceeded G' indicating a transition from a solid-like (G' > G") to a viscous-like (G" > G') behavior and a dependency of the rheological properties of purees on the strain. These results showed that, all purees can be considered as viscoelastic solids in the domain of low strain amplitude (< 1%) and viscoelastic liquids for higher strain amplitude.

Althought, all purees showed a viscoelastic solid-like behavior, a decrease in the storage modulus G' within the linear viscoelastic region (LVE-R) with maturity was observed indicating a significant decrease of the solid-like behavior of mango purees during maturation (Fig. 8 (a)). Indeed, the storage modulus of mango purees decreased significantly by 70% These results show a decrease of the solid-like behavior of mango purees during maturation which is consistent with the loss of mango firmness (D5N, Fmax) and the loss of flesh solid-like behavior (G'). This observation highlights that the loss of mango firmness has an impact on the viscoelastic properties of the mango puree.

Physico-chemical analyses

Several physico-chemical characteristics of mango purees were assessed during maturation. In this study, the titratable acidity (TA, g citric acid/100g of puree) of mango purees decreased overall during maturation. Indeed, TA values were significantly higher for mangos in stages [0-5 days] and [6-10 days] than for the other stages as presented in Fig. 9. This decrease is in accordance with literature and could be explained by metabolic reactions including respiration in which organic acids are used as substrates [START_REF] Gill | Physico-chemical changes during progressive ripening of mango (Mangifera indica L.) cv. Dashehari under different temperature regimes[END_REF][START_REF] Lawson | Characterization of Southeast Asia mangoes (Mangifera indica L) according to their physicochemical attributes[END_REF]. After 15 days of maturation, no significant decrease of titratable acidity was observed showing that no more significant degradation of organic acids occurred after this maturation stage. Concerning TSS and DM of purees, no significant changes regarding these parameters were noticed before 20 days of maturation as presented in Fig. 9 (b). This observation is not in accordance with literature since several studies showed an increase in sugar content (hydrolysis of starch)

and dry matter (loss in water) during maturation [START_REF] Dea | Optimal ripeness stage for processing "Kent" mangoes into fresh-cut slices[END_REF][START_REF] Elbandy | Aloe vera Gel as a Functional Ingredient and Natural Preservative in Mango Nectar[END_REF][START_REF] Maldonado-Celis | Chemical Composition of Mango (Mangifera indica L.) Fruit: Nutritional and Phytochemical Compounds[END_REF][START_REF] Palafox-Carlos | Effect of ripeness stage of mango fruit (Mangifera indica L., cv. Ataulfo) on physiological parameters and antioxidant activity[END_REF]. This result could be explained by the fact that, in this study, TSS and DM of mango purees reached their highest values even at green stages [0-5 days], showing that these two parameters have evolved during storage of mango before purchasing (mango have been stored at 10°C before purchasing). Indeed, for this mango variety (Mangifera indica cv. Kent), several works reported that the highest values of TSS are around 20° Bx [START_REF] Jha | Modeling of color values for nondestructive evaluation of maturity of mango[END_REF]. After 20 days of maturation, DM and TSS decreased significantly, highlighting the degradation of sugars. These observations could be explained by the fermentation phenomenon occurring at very late stages of maturation (after 20 days) during which the sugars are consumed and volatile compounds are generated.

Moreover, the evaporation of volatile compounds (ethanol) during fermentation could explain the DM decrease. To sum up, the studied variables for fresh mangos and mango purees allowed discriminating in both cases fresh mangos and mango purees into 3 main groups according to their maturation stages: early, intermediate and late maturation stage. In this study, mangos at early stages of maturation could be distinguished by their high firmness, solid-like behavior, viscosity and particles size whereas mangos at very late maturation stage are mainly characterized by low values of total soluble solids and dry matter. Furthermore, PCA results allowed identifying relevant indicators to (i) characterize the heterogeneity of fresh mangos and mango purees and (ii) discriminate these products according to their stage of maturation.

Finally, results of PCA confirmed the main trends obtained previously when considering the evolution of each variable separately as function of maturation.

Conclusion

The aim of this study consisted in developing innovative tools for sorting mangos according to their maturity stages and to the specific properties of their purees. Instrumental characterization of fresh mangos and mango purees were carried out to identify relevant indicators of mangos heterogeneity and purees variability. Results showed that mango firmness is a great indicator of mango maturity and has an important impact on the properties of mango purees. In this work, a non-destructive compression test was proposed to measure accurately the loss of mango firmness during maturation without damaging the fruits. This fast and easy measurement allowed also to trace-back the solid-like behavior of mango measured by destructive rheological analysis. In addition, results indicated that the firmness of fresh mangos governed the particles size distributions and the rheological properties of This work provided new knowledge in mango field and an innovative and simple tool to sort mango according to firmness in relation with their maturity stage. This tool could be also of great interest to anticipate the characteristics of mango puree according to mango firmness.

As the proposed compression test is fast and easy to perform, this sorting strategy could be easily applied not only for mango fruits but also for many other juicy stone fruits in transformation units to reduce post-harvest losses.

Formatting of funding sources

Funding: this work was carried out as part of "Interfaces" flagship project, publicly funded throught ANR (the French National Research Agency) under the "Investissements d'avenir"

program with the reference ANR-10-LABX-001-01 Labex Agro and coordinated by Agropolis Fondation under the reference ID 1603-001.

  , was performed to analyze the total variability between samples and to identify groups with similar characteristics among fresh mangos and puree. Eight indicators for fresh mango (Fmax, D5N, G', L*, a*, b*, weight, density) and twelve indicators for purees (particles size: D [3;2], D [4;3], D10, D50, D90; rheological behavior: G', G", K, n and physico-chemical characteristics:

Fig. 1 .

 1 Fig. 1. Evolution of (a) D5N (mm) and (b) Fmax (N) during maturation.

Fig. 2

 2 Fig. 2 (a) depicts the evolution of the storage modulus G' (Pa) as function of maturation daysshowing that the solid-like behavior of mango flesh decreased significantly during maturation.

Fig. 2 .

 2 Fig. 2. Evolution of the storage modulus G' (Pa) of mango slices as function of (a) maturation (days), (b) Fmax (N) and (c) D5N (mm).

Fig. 3 .

 3 Fig. 3. Evolution of (a) density, (b) and (c) L*and a* (color parameters) during maturation (days).

Fig. 4 .

 4 Fig. 4. Particles size distributions of mango purees at 5 different days of maturation.

Fig. 6 .

 6 Fig. 6. Variation of the average surface diameter D [3;2] (µm) with (a) Fmax (N), (b) D5N (mm) and (c) G' of mango flesh (Pa).

Fig. 7 .

 7 Fig. 7. Variation of the experimental dynamic viscosity µ at 50 s -1 (Pa. s) as function of the D5N (mm).

  of maturation whereas beyond this maturation stage, G' was almost steady.

Fig. 8 .

 8 Fig. 8. Evolution of the storage modulus G' (Pa) as function of (a) the maturation (days) and (b) the D5N (mm).

Fig. 9 .

 9 Fig. 9. Variation of (a) the titratable acidity, TA (g citric acid / 100g puree) and (b) the total soluble solids, TSS (°Bx) and dry matter, DM (g / 100g puree) during maturation.

Fig. 10 .

 10 Fig. 10. Principal component analysis (PCA) on instrumental indicators of (a) mangos and (b) purees at different maturity stages. a*: red to green transition , b*: blue to yellow transition, DM: dry matter (g / 100 g puree), D5N: distance of compression at 5N (mm), D [3;2]: Sauter mean diameter (µm), D [4,3]: Brouckere mean diameter (µm), D10, D50 and D90: particle size statistical diameters (µm), Fmax: maximum value of the peak force (N) , G': storage modulus (Pa), G": loss modulus (Pa), K: consistency index (Pa.s n ), L*: lightness (black to white transition), n: flow behavior index, TA: titratable acidity (g citric acid/ 100 g puree), TSS: total soluble solids (°Bx).

  mango purees. According to these observations, it was possible to predict the particles size of the mango purees based on the firmness of the fresh mangos. Concerning, purees rheological properties (viscosity and solid-like behavior), compression test enabled discriminating mainly mango purees at early stages of maturation [0-10 days] from the other stages [10-25 days].PCA results confirmed that the investigated variables in this work (textural, rheological, physical and physico-chemical) seem to be good indicators to characterize the heterogeneity of fresh mangos and mango purees. It was possible to discriminate mango and puree into 3 main groups (early maturation stages, intermediate stages and a late maturation stage) based mainly on total soluble solids, dry matter, firmness, particles size and rheological properties.

Table 1

 1 summarize the ranges of variation of K, n and the experimental viscosity at 50 s -1 of mango purees at different stages of maturity. To ensure the clarity of results concerning rheological properties of purees, authors presented results according to 5 stages of maturity, a

priori ([0-5 days], [6-10 days], [11-15 days], [16-20 days], [21-25 days]). As expected, shearthinning behavior of all purees was confirmed by the flow behavior indexes as they presented all values below 1 (n < 1). It can be noticed that the flow behavior indexes (n) decreased


Vincent Visconti

Karim Rigalma

Emmanuel Coton

Philippe Dantigny
email: philippe.dantigny@univ-brest.fr

Impact of intraspecific variability and physiological state on Penicillium commune inactivation by 70% ethanol

Keywords: mold, conidia, fungicide, disinfectant, predictive mycology

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Fungi are a major source of contamination in agri-food industrial environments due to their ability to grow on a large number of food matrices such as dairy products, bakery products, cereals, vegetables, meats, processed products, high-sugar products and oilseed [START_REF] Dagnas | Predicting and preventing mold spoilage of food products[END_REF][START_REF] Filtenborg | Moulds in food spoilage[END_REF]. Development of spoilage molds on these products impacts their organoleptic properties, and causes food waste and economic losses. Although it is difficult to get the exact figures, it is estimated that 5 to 10% of global food losses and waste are attributed to spoilage fungi [START_REF] Pitt | Introduction, in: Fungi and food spoilage[END_REF]. Among those, molds are able to easily disseminate in processing plant through the production of conidia by asexual reproduction [START_REF] Salustiano | Microbiological air quality of processing areas in a dairy plant as evaluated by the sedimentation technique and a one-stage air sampler[END_REF]. Airborne conidia are produced in large quantities, facilitating mold dissemination in the processing plant and on equipment, thus leading to food contamination [START_REF] Heldman | Factors influencing air-borne contamination of foods. A review[END_REF].

To prevent the risk of contamination, manufacturers use a wide variety of chemical surface disinfectants. Liquid disinfectants that contain ethanol as the fungicide molecule are sprayed on machine surfaces that can be in contact with food products. In Europe, the fungicidal effect of disinfectants is defined by the European standard EN 13697 (2001). While this standard is widely used, three major limits can be highlighted.

Firstly, Aspergillus brasiliensis (ATCC 16404) and Candida albicans (ATCC 10231) are the reference species for testing disinfectants for their fungicidal activity. However, depending on the considered type of food production, these species may be not representative of the encountered contaminants. For example, species from the Penicillium genus would be more representative of molds responsible for cheese spoilage [START_REF] Ledenbach | Microbiological Spoilage of Dairy Products[END_REF]. Among them, Penicillium commune is often isolated from spoiled dairy products [START_REF] Filtenborg | Moulds in food spoilage[END_REF][START_REF] Garnier | Diversity of spoilage fungi associated with various French dairy products[END_REF][START_REF] Hocking | Fungi causing thread mould spoilage of vacuum packaged Cheddar cheese during maturation[END_REF]. The choice of the fungal spoiler representative of contamination is important because the efficacy of a disinfectant depends greatly on the considered species. For example 0.15% peracetic acid decreased the population of A. brasiliensis, C. albicans and P. commune (CCT 7683) by 0.67, 1.3 and 2.3 log10, respectively [START_REF] Bernardi | Efficacy of commercial sanitizers against fungi of concern in the food industry[END_REF]. These authors showed that the difference between the species was even more pronounced at 1.5% peracetic acid as these species exhibited decrease of 3 log for A. brasiliensis and more than 5 log for C. albicans and

P. commune.
Secondly, the use of only one strain of the selected species can raise questions concerning its representativeness of the species behavior towards the studied factor (i.e. biocide resistance or sensitivity). Several studies have shown intraspecific differences on inactivation for a given biocide [START_REF] Bundgaard-Nielsen | Fungicidal effect of 15 disinfectants against 25 fungal contaminants commonly found in bread and cheese manufacturing[END_REF][START_REF] Korukluoglu | The fungicidal efficacy of various commercial disinfectants used in the food industry[END_REF][START_REF] Salo | Disinfectant efficacy on foodborne spoilage yeast strains[END_REF]. For example, the application of 1.5% benzalkonium chloride for 10 min at 20 °C inactivated 1.2 and 4 log10 Aspergillus versicolor conidia depending on the 2 studied strains [START_REF] Bundgaard-Nielsen | Fungicidal effect of 15 disinfectants against 25 fungal contaminants commonly found in bread and cheese manufacturing[END_REF].

Finally, in these standards, conidia are hydrated to obtain conidial suspension. However, this condition may not represent the physiological state of airborne conidia present in an industrial environment. Moreover, it is unlikely that conidia are produced under optimal conditions in dairies. It is suggested that molds may have rather encountered various abiotic stresses before and during the production of conidia. It has been shown previously that inactivation of conidia depended greatly on the strain, the physiological state, and the medium used [START_REF] Dantigny | Relevant issues in predictive mycology[END_REF]. Water activity (aw) during the production of conidia is an essential factor that impacts the physiological state of conidia. It was shown to play an important role in many biological responses in fungi such as germination, mycotoxin production, and resistance to heat and chemical stresses [START_REF] Dantigny | Relevant issues in predictive mycology[END_REF][START_REF] Dantigny | Significance of the physiological state of fungal spores[END_REF]. [START_REF] Dao | Preparation of fungal conidia impacts their susceptibility to inactivation by ethanol vapours[END_REF] showed that a reduced aw during conidial production was associated with a greater resistance of Penicillium chrysogenum, Penicillium digitatum and Penicillium italicum conidia to ethanol vapors (0.67 kPa).

In this context, this study aimed at assessing the impact of the intraspecific variability and the physiological state on the efficacy of 70% ethanol solution to inactivate four Penicillium commune strains. For each tested strain, 4 types of conidia, corresponding each to a different physiological state, were obtained by modifying the water activity during conidial production (0.950 aw and 0.995 aw) and harvesting conditions (hydrated and non-hydrated).

Materials and methods

Molds

The four studied P. commune strains were provided by the University de Bretagne

Occidentale Culture Collection (UBOCC). They were all isolated from contaminated cheeses, their source is listed in Table 1. The molds were maintained on Potato Dextrose Agar (PDA) medium at 4 °C.

Conidial production

Conidia were produced from mycelium grown on PDA at 2 aw levels (0.995 and 0.950) for 7 days at 25 °C. aw was adjusted by substituting a part of the water with an equal weight of glycerol. The relative amount of glycerol was 20.6% (w/w) to obtain PDA media at aw 0.950.

The aw value of the culture media was controlled after autoclaving and solidification at 20 °C using a Tunable Diode Laser aw-meter (TDL Aqualab, Decagon Devices, Pullman, WA, USA) with an accuracy of 0.005 aw unit.

Conidia harvesting protocols

Dry-harvested conidia were obtained mechanically without any contact with water according to [START_REF] Dao | Preparation of fungal conidia impacts their susceptibility to inactivation by ethanol vapours[END_REF]. Ten sterile glass beads (diameter 3 mm) were deposited on the agar surface and the Petri dish was shaken gently to detach the conidia.

Then, the plate was turned upside-down and the conidia were harvested on the lid by gently tapping 100 times on the bottom of the Petri dish.

Hydrated conidia were obtained by flooding the lid with 5 ml of sterile saline solution (NaCl, 9 g/l of water) containing Tween 80 (0.015% v/v). For this condition, conidia were hydrated for 30 minutes before applying the disinfectant testing procedure.

Disinfectant testing procedures

Seventy percent ethanol solution was prepared from anhydrous ethanol diluted in distilled water containing Tween 80 (0.015%, v/v). Treatment of dry harvested conidia began when 10 ml of a 70% ethanol solution was applied to the dry-conidia on the lid. Before the first sampling time, the suspension (ethanol containing conidia) was transferred to a Falcon tube and homogenized by vortexing for 10 s. Treatment of hydrated conidia began when 1 ml of conidial suspension was transferred in 9 ml of a 78% ethanol solution. After homogenization by vortex (10 s), the final ethanol concentration of the mixture was 70%. Disinfection tests were performed at ambient temperature, 20 ± 1 °C.

After 1, 2 and 5 minutes of exposure, ethanol was neutralized by diluting ten-fold the suspension in a saline solution (NaCl, 9 g/l of water) containing Tween 80 (0.015% v/v). The initial amount of dry-harvested conidia (N0) was evaluated by counting on a Malassez cell according to [START_REF] Dao | Inactivation of conidia of Penicillium chrysogenum, P. digitatum and P. italicum by ethanol solutions and vapours[END_REF]. The method was based on the principle that the application of ethanol did not affect the morphology of the conidia during the time of the experiment so it is possible to count under the microscope the initial conidia treated even after the experiment was started.

Viability assessment

After neutralization, the treated conidia were grown on PDA at 25 °C for 3 days by spreading 100 µl of sample and 4 subsequent decimal dilutions. If no visible mycelium was observed after 3 days, the dishes were incubated for 4 additional days. Only counts (Nt) in the range 10 to 100 colonies per plate were considered. Conidial inactivation was expressed as the logarithmic reduction factor log10 (Nt/N0).

Results

Log10 inactivation values of conidia of the 4 studied P. commune strains exposed to 70% ethanol for 1, 2 and 5 minutes are shown in Table 2. The obtained data showed that after five minutes exposure to 70% ethanol, total inactivation of conidia was achieved, regardless of the considered strain, aw for conidial production and harvesting conditions. For all strains, after 1 minute exposure, only dry-harvested conidia produced at aw 0.950 exhibited survivors. Under these production and harvesting conditions, P. commune UBOCC-A-116003 was the most resistant with an inactivation value of 2.35 log10. The three other P. commune strains had inactivation values in the range 4.62 log10 to 5.40 log10. In addition, P. commune UBOCC-A-116003 was the only one to survive at 2 minutes exposure with an inactivation value of 3.89 log10 (Table 2) and even at 3 min (inactivation value of 4.78 log10) (data not shown).

Two different groups can be distinguished according to the inactivation data obtained for 1 min treatment. A "weak" physiological state group (i.e. hydrated conidia, regardless of the aw used for conidia production, and non-hydrated conidia produced at aw 0.990), for which all conidia were inactivated, and a "strong" physiological state group (i.e. non-hydrated conidia produced at aw 0.950) for which some conidia remained viable. All conidia from the "weak" physiological state were inactivated, inactivation was greater than 3.86, 3.85, and 4.02 log10

for UBOCC-A-119012, UBOCC-A-112050 and UBOCC-A-112059, respectively. The number of conidia obtained for the conditions of the "weak" physiological state group was lower than that the one produced for the "strong" physiological state with the exception of UBOCC-A-112059. Because more conidia were obtained for the "strong" physiological state, inactivation seemed greater than for the "weak" one. In fact, the effect of the physiological state can be accurately determined for the strain UBOCC-A-116003, as all conidia from the "weak" physiological state group were inactivated. Inactivation was greater than 3.89 log10 for this group, whereas the "strong" group exhibited only a 2.35 log10 inactivation value. In this case, the difference between the two groups was greater than 1.54 log10.

The impact of intraspecific variability in P. commune could be quantified for the "strong" physiological state only. A group of "sensitive" strains, namely UBOCC-A-112050, UBOCC-A-112059 and UBOCC-A-119012 could be distinguished from the "resistant" UBOCC-A-116003strain. For 1 min treatment, the difference between the inactivation value of the UBOCC-A-116003 and the mean of the 3 "sensitive" strains was 2.61 log10.

Discussion

In this study, 5 minutes exposure allowed total inactivation (inactivation values greater than 4 log10) of conidia of P. commune regardless of the tested strain, and the production or harvesting conditions. Despite little information in the literature on the susceptibility of P.

commune to ethanol, our results are in agreement with those of [START_REF] Bundgaard-Nielsen | Fungicidal effect of 15 disinfectants against 25 fungal contaminants commonly found in bread and cheese manufacturing[END_REF] who observed the absence of growth of three P. commune strains within 14 days after an exposition to 70% ethanol solution for 10 minutes.

The choice of a species representative of the actual contamination of a given food sector, food product and even plant is an important question. Indeed, according to standards, the fungicidal activity of a disinfectant must be evaluated on the two reference strains belonging each to the A. brasiliensis and C. albicans species. However, these species might not be the most representative contaminants of a studied food sector. In this context, [START_REF] Bernardi | Efficacy of commercial sanitizers against fungi of concern in the food industry[END_REF] showed that these 2 strains behaved differently than 4 actual food contaminant strains (belonging to 4 different species, including P. commune) when exposed to 5 disinfectants (benzalkonium chloride, biguanide, peracetic acid, quaternary ammonium and sodium hypochlorite). Moreover, sensitivity variability was observed between the 4 contaminant species (Cladosporium cladosporioides, P. commune, Penicillium polonicum and Penicillium roqueforti) representative strains. These observations underline the importance of the initial choice of the species to be tested that might allow optimizing disinfectant procedure and potentially reduce their concentrations, the use of water for rinsing and therefore the associated cost, while guarantying safety.

Concerning the representativeness of the tested strains, in this study, the time necessary to inactivate 10 4 P. commune conidia with 70% ethanol varied according to the considered strain. It was less than 1 min for the UBOCC-A-119012, UBOCC-A-112050 and UBOCC-A-112059 strains, and greater than 2 min (and even 3 min -data not shown-) for the UBOCC-A-116003 strain. The effect of the fungal strain on the susceptibility to a disinfectant was previously reported [START_REF] Bernardi | Antifungal activity of commercial sanitizers against strains of Penicillium roqueforti, Penicillium paneum, Hyphopichia burtonii, and Aspergillus pseudoglaucus: Bakery spoilage fungi[END_REF][START_REF] Korukluoglu | The fungicidal efficacy of various commercial disinfectants used in the food industry[END_REF]. [START_REF] Korukluoglu | The fungicidal efficacy of various commercial disinfectants used in the food industry[END_REF] described disinfectant susceptibility of 5 strains of Aspergillus niger and Penicillium roqueforti to 8 disinfectants and different concentrations. Depending on the strain, the time to inactivate 10 6 A. niger conidia using isopropyl alcohol ranged from 10 to 25 min, while that time varied between 5 and 16 min to inactivate 10 6 P. roqueforti conidia using 0.3% peracetic acid. The observed intraspecific susceptibility was dependent on the disinfectant and the concentration used. [START_REF] Bernardi | Antifungal activity of commercial sanitizers against strains of Penicillium roqueforti, Penicillium paneum, Hyphopichia burtonii, and Aspergillus pseudoglaucus: Bakery spoilage fungi[END_REF] also showed evidence of strain-dependent inactivation. When applying 3% peracetic acid for 15 min on 3 P. roqueforti strains, the inactivation values were 3.69, 3.70 and 5.66 log10 with 1.97 log10 difference observed between the sensitive strain and the 2 other resistant strains.

Concerning the impact of the physiological state on inactivation, our results clearly showed that production of conidia and harvesting conditions have a strong impact on the survival to the disinfectant treatment. In the tested conditions, dry-harvested conidia produced at aw 0.950 survived after 1 minute exposure. These conditions could be considered as more representative of the processing plant than the standard conditions. First, in the dairy industry, fungal contamination of food products is due to airborne conidia that are disseminated in the air [START_REF] Burgain | Inoculation of airborne conidia of Penicillium chrysogenum on the surface of a solid medium[END_REF][START_REF] Salustiano | Microbiological air quality of processing areas in a dairy plant as evaluated by the sedimentation technique and a one-stage air sampler[END_REF]. Secondly, it is very unlikely that conidia are produced in dairy plants under optimum aw conditions. As suggested by previous studies, as compared to aw 0.995, a reduced aw induced major changes in the physiological state and in the resistance of conidia to ethanol [START_REF] Dao | Preparation of fungal conidia impacts their susceptibility to inactivation by ethanol vapours[END_REF][START_REF] Nesci | Osmotic and matric potential effects on growth, sugar alcohol and sugar accumulation by Aspergillus section Flavi strains from Argentina[END_REF].

In our study, it was not possible to determine whether aw during production of conidia was more important than the harvesting conditions on inactivation. In fact, compared to hydrated conidia produced at aw 0.995, the effect of modifying one condition only, either the harvesting condition or the aw during conidial production, could not be quantified as these 3 conidia types were inactivated. The impact of the physiological state was also tested on 3 Penicillium species exposed to ethanol vapors [START_REF] Dao | Preparation of fungal conidia impacts their susceptibility to inactivation by ethanol vapours[END_REF]. As compared to dry-harvested conidia that remained unaffected by ethanol treatments, hydrated conidia exhibited inactivation in the range 1 to 3.5 log10 depending on species and treatment conditions. The effect of aw during production of conidia on inactivation appeared even greater, after 48h treatments with 0.67 kPa ethanol vapors, the difference between conidia produced at 0.99 and 0.95 aw was about 4 log10 whatever the species.

Conclusion

Identification of the species and strains responsible for fungal spoilage in dairy industries is important to improve food quality, to control fungal spoilage, but also to optimize disinfection procedures according to the reality of the threat. Moreover, the strain is another important factor to take into account to assess the efficacy of disinfectants, as disinfectant sensitivity variability was observed suggesting that at least few strains should be studied for each considered species.

By combining a reduced aw for the production of conidia and a dry harvesting protocol, a "strong" physiological state, which can be considered as more representative of dairy plant conditions, was obtained. This physiological state can be compared to the "weak" physiological state that results from the preparation of conidial suspensions as recommended by the international standards to assess the efficacy of disinfectants against molds. The impact of the physiological state on the inactivation of conidia could be greater than 1.5 log10, thus suggesting that disinfectants may not be as efficient as expected against conidia that are produced in a dairy environment.

Table 1 . List of Penicillium commune strains tested and their origin. The strains were provided by the University de Bretagne Occidentale Culture Collection (UBOCC).

 1

	Strain	Area	Source
	UBOCC-A-112050	French dairy plant	Contaminated blue veined cheese
	UBOCC-A-112059	French dairy plant	Contaminated blue veined cheese
	UBOCC-A-116003	French dairy plant	Contaminated hard cheese
	UBOCC-A-119012	Consumer fridge	Contaminated hard cheese

Table 2 . Log10 inactivation values (± standard variation) of Penicillium commune conidia exposed to 70% ethanol for 1, 2 and 5 minutes.

 2 White cells: total inactivation of conidia. Grey cells: presence of visible mycelium after 3 and 7 days at 25 °C.

	Inactivation value log10

Acknowledgements

This work was funded by the French Dairy Interbranch Organization (CNIEL) (Moisibio Project, 2019_00621) and the French Association for Research and Technology (ANRT) (CIFRE 2018/0036).