<figure><img src="image_1.png" /></figure>

# First identification of 1-hydroxyoctan-3-one in apple purees: Use as a fruit quality segmentation marker and correlation with octane-1,3-diol concentration

Hippolyte Mouriot<sup>a,d</sup>, Lucas Suc<sup>a</sup>, Karine Gourrat<sup>b,c</sup>, Géraldine Lucchi<sup>b,c</sup>, Teddy Godet<sup>a</sup>, Valérie Nolleau<sup>a</sup>, Fabienne Remize<sup>a</sup>, Aurélie Roland<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Apple (Malus domestica)

1-hydroxyoctan-3-one

Octane-1,3-diol

Mushroom off-flavor

Penicillium spp.

Stable isotope dilution assay

Aroma

Fungal alteration

## ABSTRACT

An organoleptic defect described as musty and/or fresh mushroom can occur in cooked apple products. GC-Olfactometry was used to characterize puree spoilage associated with sensory descriptors mushroom, cellar-like, musty, dusty or metallic. This approach, coupled with pure standards analysis allowed us to identify for the first time 1-hydroxyoctan-3-one in spoiled matrices. Using a targeted approach, 1-hydroxyoctan-3-one and octane-1,3-diol, were quantified by Stable Isotope Dilution Assay LC-MS/MS. Quantification results attested that these compounds exhibited a higher concentration in spoiled purees than in control ones and could thus be relevant markers of fruit spoilage. In a second time, the origin of 1-hydroxyoctan-3-one and octane-1,3-diol was investigated by contaminating several apple purees with specific fungal species. A Penicillium digitatum strain appeared to be the highest 1-hydroxyoctan-3-one producer, while a Botrytis cinerea strain led to a decrease of octane-1,3-diol concentration in spoiled products. It opens up avenues for the quality segmentation of fruits.

## 1. Introduction

Apple (Malus domestica Borkh.) is a largely consumed fruit all over the world. In France, the most produced cultivar is Golden Delicious (GD) and represents around 30 % of apple production (Riaux et al., 2023). Food products derived from apples such as juice and fruit puree are widely produced and their quality needs to be strictly monitored. However, from the beginning of 2000s decade, a musty off-flavor was reported from long term stored apples (Seppä et al., 2013). At the same time, some off-flavors were detected in microbiologically spoiled apple juices and were described from sensory analyses as fresh mushroom, musty, moldy, earthy, cellar-like or metallic (Siegmund & Pöllinger-Zierler, 2006). Apple producers and processing companies reported that several cultivars were affected by this off-flavor, but the most affected

seems to be the cv. GD. Indeed, Hashizume and co-workers (Hashizume et al., 2007) reported a higher frequency and risk of occurrence of musty off-flavor spoilage in GD apple juice compared to other cultivars, confirming the previous empirical observations.

Volatile composition of fruit is influenced by pre and post harvest conditions, but mostly by the maturity level at harvest. The quality and quantity of released volatiles also depend on apple cultivar, harvest year and storage conditions (Rowan et al., 1999). Apples from GD cultivar are usually characterized by a sweet and floral taste, with a low acidity and a coarse texture. This profile results from different families of aroma compounds, with a dominance of esters, followed by aldehydes and alcohols in GD peel (Yang et al., 2021). In GD juice, a similar repartition was observed (Chitarrini et al., 2021).

In the case of spoiled apple products, detection of moldy-mushroom

Abbreviations: cv, apple cultivar; FID, Flame ionization detector; GC-O, Gas chromatography coupled with olfactometry; GC-MS, Gas chromatography coupled with mass spectrometry single quadrupole; GC-MS/MS, Gas chromatography coupled with tandem mass spectrometry; GD, Golden Delicious; LC-MS/MS, Liquid chromatography coupled with tandem mass spectrometry; LRI, Linear Retention Index; OA, Olfactive area; SIDA, Stable-Isotope Dilution Assay; spp, species; std, analytical standard.

E-mail address: aurelie.roland@institut-agro.fr (A. Roland).

<footnote><sup>a</sup> SPO, Univ Montpellier, INRAE, Institut Agro, 34060 Montpellier, France</footnote>

<footnote><sup>b</sup> Centre des Sciences du Goût et de l'alimentation, CNRS, INRAE, Institut Agro, Université de Bourgogne, 21000 Dijon, France</footnote>

<footnote><sup>c</sup> ChemoSens Facility, CNRS, INRAE, PROBE Research Infrastructure, 21000 Dijon, France</footnote>

<footnote><sup>d</sup> Andros SNC, 46130 Biars-sur-Cère, France</footnote>

<footnote>* Corresponding author.</footnote>
off-flavor can result from the presence of microorganisms. Indeed, in apple juices contaminated by bacteria, 15 aromatic markers have been identified to be responsible for this olfactory defect. These compounds are aromatic aldehydes and phenol derivatives, methoxypyrazine derivatives, alpha-terpineol, borneol derivatives, geosmin and C8-compounds such as 1-octen-3-ol and 3-octanone (Siegmund & Pöllinger-Zierler, 2006). Another study identified 7 volatile compounds (mainly aldehyde or ketone derivatives: pentanal, 2-methylpent-1-en-3-one, hexanal, (E)-hept-2-enal, 6-methylhept-5-en-2-one, 1-octen-3-one and (E)-oct-2-enal) responsible for metallic trigeminal sensation in cloudy apple juice, with the major contribution clearly attributed to 1-octen-3-one (Hashizume et al., 2007).

In the context of grapes and winemaking, grape musts that gave corresponding wines characterized with fresh mushroom notes showed a higher abundance of *Penicillium* spp.. The role of *Botrytis cinerea* and *Penicillium* spp. on this musty off-flavor development was reported very recently (Destanque et al., 2024). Post-harvest moldy odor of apples was previously shown to involve the same fungi (Gong et al., 2022).

C8-compounds (Table 1) could be responsible for moldy and fresh-mushroom off-flavors in grapes, pears, and apples. In grape musts, C7-C8-compounds (heptan-2-ol, 1-octen-3-one, 1-octen-3-ol, and 2-octen-1-ol) and geosmin derivatives were the main compounds responsible of fresh-mushroom off-flavor or earthy odors (La Guerche et al., 2006). More recently, it has been reported that 1-nonen-3-one (Pons et al., 2011), 1-hydroxyoctan-3-one (Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023) and octane-1,3-diol (Ployon et al., 2025) could also be specific markers of fresh-mushroom off-flavor in wine. Similarly, octane-1,3-diol, which has a chemical structure similar to those of 1-hydroxyoctan-3-one, can be found at elevated concentrations in pears (Beuerle & Schwab, 1997) but also in apple juices made from cider apple varieties up to few dozen of mg/L (Llorente et al., 2010). This pure compound is perceived with a musty note (Akedo et al., 1977) but also as green, fatty or mushroom in "Honeycrisp" apple fruit (Yan et al., 2020).

By considering the literature, C8 compounds are possibly linked to fungal colonization of fruit. Whatever the type of fruit, the origin of such C8 compounds seems to be correlated to the degradation of fatty acids by fungal alterations. Indeed, a fungal lipoxygenase (10-LOX), acts on C18 unsaturated fatty acids to form a 10-hydroperoxide of the polyunsaturated fatty acid in a first step. The action of an hydroperoxide lyase (HPL) releases specifically 1-octen-3-ol (C8) and 10-oxo-8-decanoic acid (C10) in a second step (Gong et al., 2022). Interestingly, octane-1,3-diol origin has been partly elucidated in stored apples from the enantioselective beta-oxidation of linoleic acid (Beuerle & Schwab, 1999). In this case, the metabolism seems not correlated to fruit fungal alteration and is linked with an antimicrobial activity. Even if several C8 compounds may come from the fatty acid degradation, other precursors have been also reported in literature. Indeed, some glycosidic precursors of octane-1,3-diol and 1-hydroxyoctan-3-one have been identified in apple and grape must respectively (Beuerle & Schwab, 1999; Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023). All of these data highlight the complexity of the mechanisms governing the origin of the C8 compounds responsible for the mushroom aftertaste in apple-based products.

In order to maintain the high quality of apple-based products, it is important to know precisely the origin of the compounds responsible for the 'old apple taste' defect. Consequently, the aims of this article were (i) to characterize apple products by GC-Olfactometry to identify which compounds are implicated in the off-flavor, (ii) to develop an analytical method to quantify identified spoilage markers (ii) to decipher a potential relationship between fungal development and the identified markers.

## 2. Material and methods

### 2.1. Chemicals

Dichloromethane (for GC-MS/MS analysis) (≥99.9 %), pentane (≥99 %) sodium chloride (≥99.5 %), sodium sulfate anhydrous (≥99 %) were supplied from Sigma Aldrich (St Quentin Fallavier, France). Dichloromethane solvent (≥99.9 %, distilled just before use) and absolute ethanol (≥99.2 %) were supplied from Carlo Erba Reagents (Val de Reuil, France). Ultrapure Milli-Q water, purified by Millipore Advantage A10, was purchased from Merck (Darmstadt, Germany). Helium BIP quality was supplied from Air products (Allentown, PA, USA). Analytical standards solutions of 2-heptanol (98 %), 2-octanol (≥99 %), 3-octanol (99 %), 1-octen-3-ol (98 %) and 4-nonanol (≥96.5 %) were purchased from Sigma Aldrich (St Quentin Fallavier, France). The 4-octanone (98 %) was purchased from abcr GmbH (Karlsruhe, Germany) and 1-octen-3-one (97 %) from Alfa Aesar (Haverhill, Massachusetts, USA).

The octane-1,3-diol (45 % ¹H NMR purity) was obtained by chemical reduction (NaBH₄) from 1-hydroxyoctan-3-one (39 % ¹H NMR purity) (Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023; Husain et al., 1999; Ployon et al., 2025). These compounds were both synthesized in our laboratory (UMR SPO), as well as their deuterated analogues ([8,8,8,²H]-1-hydroxyoctan-3-one 36 % ¹H NMR purity) [8,8,8,²H]-octane-1,3-diol 75 % ¹H NMR purity) (Ployon et al., 2025).

### 2.2. Biological material and fungal cultures

#### 2.2.1. Fungal strains selection

Fungi from 7 different species were selected based on the presence of a protein similar to LOX10 from Blast (Genebank) with protein sequence ABV21633.1 (cyclooxygenase-like fatty acid oxygenase) from *Aspergillus fumigatus*. Strains were obtained from:

- CIRM Champignons filamenteux, (Marseille, France) for *Talaromyces purpureogenus* (BRFM 159), *Fusarium culmorum* (BRFM 1961, *Prunus domestica* tree), *Fusarium oxysporum* (BRFM 2450), *Aspergillus terreus* (BRFM 3319, fungal culture contaminant), *Aspergillus nidulans* (BRFM 491) and *Penicillium digitatum* (BRFM 3409, orange).

- LUBEM (Brest, France) for *B. cinerea* (tomato) (UBOCC-A-117017).

- CTIFL (Prigonrieux, France) for *B. cinerea* (apple) (22-18_pomme_1).

#### 2.2.2. Spoiled sample preparation

#### 2.2.2.1. Culture medium, growth conditions and spore recovery.
Cultivation of fungi was performed on an apple juice medium. Commercial apple juice pH was adjusted to 6.0 with NaOH 1 M and the juice was mixed with agar 15 g/L, then autoclaved for 20 min at 121 °C. The medium was inoculated using the fungal stock collection, and incubated at 20 °C during a minimum of 7 days, until sufficient mycelium surface development (visual observation). Fungal spores were recovered from the mycelium by washing the medium surface with 10 mL of Tween80 solution (0.01 % in sterile water), and thereafter vortexing the suspension and filtering to remove hyphae on sterile gauze.

#### 2.2.2.2. Apple puree inoculation.
Fungal spores were counted using a particle coulter (Multisizer 4e coulter counter, Beckman Coulter, CA). Dilutions to reach the cell counting range were performed using a Microlab controller Hamilton. Spores were used to inoculate 20 g of apple puree.

A preliminary assay was performed to determine the impact of inoculation level on target compound concentration. To this end, apple
puree was inoculated with each of the eight strains at two spore populations: 10 spores/g and 10⁴ spores/g. After incubation for 14 days at 15 °C, the determination of target C8-compound concentration showed no difference according to inoculation level (Supplementary fig. 1). This experiment conducted at 15 °C would require to be repeated under real apple storage conditions, i.e. at 0–4 °C, so that any temperature related bias would be excluded.

In a second experiment, each apple puree tube was inoculated either with single strains (biological triplicate), or with a mix of two strains (biological duplicate) at the same spore concentration of 10⁴ spores/g, and then maintained at 15 °C during 14 days. One control from the same apple puree was prepared in duplicate, without inoculum.

## 2.3. Apple puree samples

Apple purees, from *Malus domestica* Borkh, from Golden Delicious, Jonagold, Gala or Pink Lady cultivars, were kindly gifted by Andros (Biars sur Cere, France). These samples were distributed as follows: control (n = 146) and spoiled (n = 11). Classification of samples (control = no retronasal defect / spoiled = fresh-mushroom notes) was made upon sensory analysis realized by the internal panel of Andros. These samples were characterized by LC-MS/MS for 1-hydroxyoctan-3-one and octane-1,3-diol (n = 146 + 11 for control and spoiled ones, respectively) and GC-O (n = 1 + 2 for control and spoiled ones, respectively).

## 2.4. Sensory characterization of apple purees by GC-O-flame ionization detector (FID) analysis

### 2.4.1. GC-O and GC-MS sample preparation

Each sample of apple puree (20.0 g) was diluted with milliQ water (20.0 g) and spiked with the internal standard (4-nonanol at 200 µg/L). The mixture was extracted with dichloromethane (3 × 10 mL) and organic fractions were combined, then dried with anhydrous Na₂SO₄ and finally, concentrated to 250 µL (adjusted volume with CH₂Cl₂) using two successive Kuderna-Danish apparatuses with a Snyder column (Merck, Darmstadt, Germany) (setup heated at 70 °C). All the corresponding extracts were stored at –20 °C before analysis both by GC-O-FID and GC-MS in Full Scan detection mode.

### 2.4.2. GC-O-FID conditions

Chromatographic separation was performed using 7890B Agilent GC (Hewlett-Packard, Palo Alto, Santa Clara, USA) equipped with a FID front detector heated at 240 °C (30 mL/min H₂, 400 mL/min airflow). Helium was used as carrier gas at a flow rate of 1.2 mL/min. Injection was done manually with a 10 µL syringe (2 µL). Separation was made on a DB-Wax column (30 m × 0.25 mm × 0.50 µm). Gradient temperature used was set as follows: 40 °C to 240 °C, 4 °C/min and 10 min final holdtime at 240 °C. At the end of the GC separation column, a plate with capillary flow technology (Agilent Technologies) was used to split the carrier gas with 50 % to an FID detector and 50 % to a sniffing port. GC-O analysis was conducted using blind coding and random order of samples. Expert panelists (n = 9) were specialized in GC-O analysis on fruit matrices and regularly sensory trained on descriptors. For descriptor selection, the panelists described the odors using their own descriptors (free choice). Each sample was analyzed once, since the number of panelists (n = 9) was enough to warrant reliability.

Enunciated descriptors were recorded with Maestro – ODP recorder (Gerstel, Mulheim an der Ruhr, Germany), and a remote - microphone system. No quantitative value has been determined by the panel.

Data was acquired using Openlab software (6850/6890 GC system, V2.3, Agilent Technologies).

### 2.4.3. GC-MS conditions

Analysis was done using a 6890 A Agilent GC (Hewlett-Packard, Palo Alto, Santa Clara, USA), equipped with a DB-Wax column (30 m × 0.25

<table><caption>Table 2
Detection parameters and analytical performances for the analysis of 1-hydroxyoctan-3-one and octane-1,3-diol by SIDA-LC-MS/MS in apple products.</caption><thead><tr><th>Compound</th><th>Internal standard</th><th>RT (min)</th><th>Quantitative transition Q1</th><th>Collision energy (V)</th><th>Qualitative transition Q2</th><th>Collision energy (V)</th><th>Linearity range (µg/L)</th><th>R<sup>2</sup></th><th>Accuracy (low spiking level)<sup>1</sup></th><th>Accuracy (medium spiking level)<sup>2</sup></th><th>Repeatability RSD</th><th>Reproducibility RSD</th><th>LOD (µg/L)</th><th>LOQ (µg/L)</th></tr></thead><tbody><tr><td>1-hydroxyoctan-3-one</td><td>[8,8,8<sup>2</sup>H]-1-hydroxyoctan-3-one</td><td>6.0</td><td>145.0<br>127.1</td><td>4</td><td>145.0<br>109.1</td><td>8</td><td>0-519</td><td>0.992</td><td>96 %</td><td>100 %</td><td>10 %</td><td>7 %</td><td>1.01</td><td>3.37</td></tr><tr><td>octane-1,3-diol</td><td>[8,8,8<sup>2</sup>H]-octane-1,3-diol</td><td>6.2</td><td>147.0<br>129.1</td><td>0</td><td>150.0<br>114.0</td><td>4</td><td>0-7130</td><td>0.999</td><td>95 %</td><td>102 %</td><td>1 %</td><td>1 %</td><td>1.41</td><td>4.68</td></tr><tr><td>[8,8,8<sup>2</sup>H]-1-hydroxyoctan-3-one</td><td>-</td><td>6.0</td><td>148.0<br>130.1</td><td>8</td><td>148.0<br>112.1</td><td>8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>[8,8,8<sup>2</sup>H]-octane-1,3-diol</td><td>-</td><td>6.2</td><td>150.0<br>132.0</td><td>0</td><td>150.0<br>114.0</td><td>4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

<footnote n="1">¹ low spiking level concentrations: [1-hydroxyoctan-3-one] = 90 µg/L; [octane-1,3-diol] = 277 µg/L.</footnote>

<footnote n="2">² medium spiking level concentrations: [1-hydroxyoctan-3-one] = 225 µg/L; [octane-1,3-diol] = 692 µg/L.</footnote>
mm × 0.5 µm) (Agilent J & W, Santa Clara, USA). Helium was used as carrier gas at a flow rate of 1.2 mL/min. The GC system was hyphenated with a mass spectrometer (Agilent 5973 N Mass Selective Detector (MSD) (Agilent J & W, Santa Clara, USA)) and the ionization was made under electron impact mode (EI) at 70 eV. Injection was made in splitless mode. Gradient temperature was linearly programmed from 40 °C to 240 °C at 4 °C/min and 10 min of final holdtime at 240 °C. MS detection was performed under Full Scan mode between m/z 29 and m/z 350. Data was acquired using ChemStation software (ver. A.03.00, Agilent J & W, Santa Clara, USA).

### 2.4.4. Linear retention index calculation

Linear retention indices (LRI) were calculated considering a mix of C7 to C30 alkanes (Sigma-Aldrich, St Quentin Fallavier, France) and using the following formula:

$$LRI = 100 \left( \frac{t_{\text{compound}} - t_{\text{n alkane}}}{t_{n+1 \text{ alkane}} - t_{\text{n alkane}}} + n \right)$$

with t = retention time of compound, n = carbon number of previous n-alkane and n + 1 = carbon number of following n-alkane (Kováts, 1958).

The volatile compounds were identified by comparing their mass spectra with mass spectra libraries (the Wiley 138, NIST, and INRAE databases achieved using standard compounds) and comparing the experimental LRI with the LRI from published data (NIST, 2022, The LRI and Odor Database).

## 2.5. Targeted analysis of 1-hydroxyoctan-3-one and octane-1,3-diol by SIDA-LC-MS/MS

### 2.5.1. Sample preparation

Apple puree samples were centrifuged during 15 min at 30000 xg. The supernatant was collected and double filtered at 0.45 µm and then 0.22 µm using syringe hydrophilic filters (33 mm diameter from Merck KGaA, Darmstadt, GermanyMillex). Filtered supernatant (500 µL) was spiked with [8,8,8,2$^H$]-1-hydroxyoctan-3-one at 93 µg/L and [8,8,8,2$^H$]-octane-1,3-diol at 1105 µg/L.

Deuterated internal standard working solutions were prepared in absolute ethanol at 2.42 mg/L for [8,8,8,2$^H$]-1-hydroxyoctan-3-one and 18.82 mg/L for [8,8,8,2$^H$]-octane-1,3-diol and stored at -20 °C. The purity and stability of stock solutions were checked by ¹H NMR as detailed in (Ployon et al., 2025).

### 2.5.2. LC-MS/MS conditions

Quantitative analyses were performed by UPLC-MS/MS (1290 Infinity II reversed phase UHPLC coupled to a 6470B Triple Quadrupole from Agilent Technologies, California, USA) as already described by Ployon et al., 2025. Briefly, compounds were separated on a Hypersil gold column (1.9 µm, 100 mm × 2.1 mm) (ThermoFisher, Waltham, Massachusetts, USA) with oven temperature set at 40 °C and a total run time of 17 min. An injection volume of 10 µL was eluted in a mobile phase composed of water (A) and methanol (B), both with formic acid

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

List of C8 pure standards injected into GC-MS and GC-O-FID and determination of their associated LRI and olfactory descriptors. The concentration of each compound injected for GC-O-FID analysis was standardized to 100 mg/L. (n = 9 panelists).

<table><thead><tr><th>LRI</th><th>Compound</th><th>Descriptors</th></tr></thead><tbody><tr><td>1250</td><td>4-octanone</td><td>Minty, aniseed</td></tr><tr><td>1318</td><td>1-octen-3-one</td><td>Mushroom, metallic</td></tr><tr><td>1333</td><td>2-heptanol</td><td>Mushroom, musty, fruity</td></tr><tr><td>1403</td><td>3-octanol</td><td>Mushroom, musty</td></tr><tr><td>1430</td><td>2-octanol</td><td>Mushroom, musty, fruity</td></tr><tr><td>1459</td><td>1-octen-3-ol</td><td>Mushroom</td></tr><tr><td>1916</td><td>hydroxyoctan-3-one</td><td>Mushroom</td></tr><tr><td>2124</td><td>octane-1,3-diol</td><td>n.d</td></tr></tbody></table>

0.1 %. The gradient went from B = 1 % to B = 99 % over 9 min, then was held at B = 99 % for 3 min and decreased to B = 1 % in 1 min, maintained at a flow rate of 0.5 mL/min for 2 min.

Source parameters have been set as follows: drying gas temperature at 260 °C and drying gas flow at 4 L/min; nebulizer set at 35 psi, sheath gas temperature at 200 °C and sheath gas flow at 10 L/min, capillary voltage at 4000 V in positive mode, nozzle voltage at 1500 V and positive Delta EMV at 400 V. Injection content was ionized using positive electrospray (ESI+) and detection was made by Multiple Reaction Monitoring (MRM), conditions defined in Table 2.

### 2.5.3. Analytical method validation

The following compounds 1-hydroxyoctan-3-one and octane-1,3-diol were quantified by SIDA-LC-MS/MS, using deuterated internal standards (1-hydroxyoctan-3-one d₃ and octane-1,3-diol d₃ respectively).

The method proposed by Ployon et al., 2025 for grapes was adapted to apple puree matrices, concerning sample preparation and concentration range. Analytical performances are given in Table 2.

For these two compounds, the analytical method was validated according to the OIV (International Organization for Vine and Wine) recommendations by considering the following parameters: linearity range, limit of detection (LOD), limit of quantification (LOQ), repeatability, reproducibility and accuracy.

The linearity range was determined by spiking a model solution at 13 concentration levels (from 0 µg/L to 519 µg/L for 1-hydroxyoctan-3-one range, and from 0 µg/L to 7130 µg/L for octane-1,3-diol range). The model solution consisted of glucose (180 g/L) and tartaric acid (4 g/L) in

<caption>**Table 4**</caption>

Olfactive areas with negative notes and putative odorant volatile compounds identified by GC-Olfactometry, using MS and LRI. The table presents the descriptors provided by the number (x) of judges out of a total of 9, with targeted initial compounds highlighted in bold. OAs were selected based on the following criteria: they must include at least one negative descriptor related to an off-flavor, present in at least one analyzed sample. For identification, “MS” indicates that the compound was detected by MS in the samples; “GC-O” means that the compound’s literature descriptors align with those detected in GC-O; “LRI” refers to the match between the literature Linear Retention Index and the compound’s LRI in the corresponding OA; “std” denotes that the pure compound was injected as an analytical standard for identification.

<table><thead><tr><th>RI</th><th>no.</th><th>compound</th><th>identification</th><th>Control<br>apple puree</th><th>Spoiled apple<br>puree</th></tr></thead><tbody><tr><td>1235</td><td>1</td><td>2-pentylfuran</td><td>MS, GC-O,<br>LRI</td><td>-</td><td>stale,<br>metallic, gas,<br>animal (4)</td></tr><tr><td>1292</td><td>2</td><td>1-octen-3-one</td><td>MS, GC-O,<br>LRI, std</td><td>mushroom,<br>metallic (9)</td><td>mushroom,<br>metallic (9)</td></tr><tr><td>1365</td><td>3</td><td>1-hexanol</td><td>MS, GC-O,<br>LRI</td><td>green,<br>metallic (9)</td><td>green,<br>metallic, sulphurous,<br>earthy, moldy (9)</td></tr><tr><td>1396</td><td>4</td><td>3-octanol</td><td>MS, GC-O,<br>LRI, std</td><td>mushroom<br>(9)</td><td>mushroom<br>(9)</td></tr><tr><td>1618</td><td>5</td><td>2-octen-1-ol</td><td>GC-O, LRI</td><td>-</td><td>roasted,<br>metallic, oil<br>(5)</td></tr><tr><td>1676</td><td>6</td><td>2-<br>furanmethanol</td><td>MS, GC-O,<br>LRI</td><td>-</td><td>cardboard,<br>dusty (2)</td></tr><tr><td>1894</td><td>7</td><td>1-<br>hydroxyoctan-<br>3-one</td><td>MS, GC-O,<br>LRI, std</td><td>floral (5)</td><td>mushroom,<br>musty, cork,<br>plastic (6)</td></tr><tr><td>1932</td><td>8</td><td>not identified</td><td>GC-O</td><td>floral, fruity<br>(4)</td><td>roasted,<br>dusty (4)</td></tr><tr><td>2070</td><td>9</td><td>octanoic acid</td><td>MS, LRI</td><td>aniseed,<br>animal (2)</td><td>aniseed,<br>fruity, dusty,<br>plastic (5)</td></tr><tr><td>2124</td><td>10</td><td>octane-1,3-<br>diol</td><td>MS, GC-O,<br>LRI, std</td><td>mushroom<br>(3)</td><td>mushroom,<br>burnt,<br>musty,<br>metallic (6)</td></tr></tbody></table>
water (1 L) with a pH adjusted to 3.5. The accuracy was measured by spiking both target compounds at two different levels (1-hydroxyoctan-3-one at 90 and 225 µg/L and octane-1,3-diol at 277 and 692 µg/L), with *n* = 3 replicates. The difference between spiked and unspiked samples allowed us to measure the corresponding recovery. Repeatability (*n* = 5) was measured at two concentration levels (125 µg/L and 179 µg/L for 1-hydroxyoctan-3-one; 376 µg/L and 537 µg/L for octane-1,3-diol) on the same day and reproducibility was measured at one concentration level (179 µg/L for 1-hydroxyoctan-3-one; 537 µg/L for octane-1,3-diol) by analyzing samples (*n* = 5) on different days (number of days *n* = 3). LOD and LOQ were calculated by determining the signal to noise (S/N) ratio equal to 3 and 10 respectively.

## 2.6. Statistical analysis

Statistical tests were performed by XLstats 2022 with Microsoft® Excel® for Microsoft 365 MSO (Version 2302 Build 16.0.16130.20894).

Wilcoxon signed ranks test was used to determine concentration differences significance between two spore concentration levels (risk α = 0.05).

ANOVA was used to determine significance between control and spoiled sample groups, and between fungal spoiled samples (Tukey test at risk α = 0.05).

To establish correlation between 1-hydroxyoctan-3-one and Octane-1,3-diol, Pearson correlation test was performed.

# 3. Results

## 3.1. GC-olfactometry profiles of control and spoiled apple purees

### 3.1.1. Implication of C8 compounds in mushroom off-flavor detection

In the light of literature, several C8 compounds (Table 1) could be responsible for the moldy, musty and mushroom notes in spoiled apple products (Siegmund & Pöllinger-Zierler, 2006; Gong et al., 2022) as well as in wines (Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023; Ployon et al., 2025). To demonstrate their implication in spoiled apple products, we decided to analyze their sensory notes by GC-O. For this purpose, we worked on pure analytical standards solutions to determine their LRI by GC-MS and then their sensory notes by GC-O-FID. (Table 3). Results showed a clear detection of the compounds 4-octanone, 1-octen-3-one, 2-heptanol, 3-octanol, 2-octanol, 1-octen-3-ol and 1-hydroxyoctan-3-one by panel members, but none of them detected the scent of octane-1,3-diol at the injected concentration (100 mg/L).

### 3.1.2. Putative identification of aroma compounds in apple products

Chemical characterization was performed on two apple products described as musty-moldy from the industry, together with a control puree. The aroma compounds were extracted by liquid-liquid extraction, concentrated by distillation and then analyzed both by GC-O and GC-MS. The gradients of temperature used both for GC-MS and GC-O-FID were the same, in order to identify the initial C8 targets or new putative compounds using LRI, MS, standard retention time and detected off-flavor (Table 4).

The olfactive areas (OA) detected in the samples by the 9 panelists were described either as positive (pleasant attributes) or negative (unpleasant attributes). As a whole, 38 OA were detected in control and spoiled apple purees. Areas described with positive descriptors (floral, fruity, caramel), were excluded from the study. Consequently, we focused on 10 OA for which negative descriptors close to our concern were used.

The OA no. 1, 3, 5, 6, 8 and 9 could match with some compounds, based on literature LRI, injected standard LRI, MS, and olfactive descriptors (Table 4). Among those, OA no. 1, 3 and 5 corresponded to descriptors associated with the metallic trigeminal sensation. Interestingly, the OA no. 1, which was specifically detected by panelists in spoiled apple puree, matched with mass spectrum, LRI and descriptors

of 2-pentylfuran. The compound 1-hexanol was identified in GC-MS chromatograms and was described as “green”. This compound could correspond to the “apple skin” descriptor in OA no. 3. Indeed, apple skin and green descriptors are often considered as synonyms. However, another unidentified co-eluted compound could be responsible for the earthy and moldy off-flavor at LRI = 1365 (OA no. 3). 2-octen-1-ol, corresponding to OA no. 5, was detected only in spoiled apple puree as roasted, metallic and oily, making it part of C8 compounds possibly linked to musty defect apparition. It is described in literature as musty-oily (Contis et al., 1998), but the lack of identification by MS suggests some coeluted compounds. Consequently, this compound was not further investigated. OA no. 6 was identified as dusty and cardboard by 2/9 panelists in spoiled apple purees. Putative compound associated with this OA was 2-furanmethanol, characterized as roasted, caramel (Yuan et al., 2023) and burnt (Zhang et al., 2022). For OA no. 8, no compound was identified, due to the lack of sensitivity on the MS signal. The OA no. 9 was detected as aniseed, animal in control puree and aniseed, fruity, dusty and plastic in spoiled puree. The only compound found in MS and corresponding to LRI in this OA was octanoic acid, described as fatty, waxy, rancid and cheesy (Zhao et al., 2024). Octanoic acid did not show suitable matching descriptors with olfactometry results. Coeluted compounds or an overtaking of perception threshold may be responsible for the perceived differences by panelists.

Considering C8 targeted compounds, 1-octen-3-one, 3-octanol, 1-hydroxyoctan-3-one and octane-1,3-diol, respectively corresponding to OA no. 2, 4, 7 and 10, were formally identified in the samples (Table 4, lines in bold) and characterized by a majority of panelists with the mushroom descriptor. Indeed, they corresponded to the mass spectrum of the pure analytical standards analyzed as preliminary results. On the other hand, 4-octanone, 2-heptanol, 2-octanol and 1-octen-3-ol were not detected.

The compounds 1-octen-3-one and 3-octanol (no. 2, 4) were detected as mushroom-like by the same number of panelists both in control and spoiled purees, making those compounds not relevant to differentiate the purees. For the first time, 1-hydroxyoctan-3-one (no. 7) was detected in apple product samples. It was described as floral in control apple puree. However, the descriptors changed to negative attributes described as mushroom, cork or and plastic notes in spoiled samples. This latter description was in accordance with GC-O detection of the pure standard. To the best of our knowledge, this compound has never been reported in apples and the sensory contribution never investigated too. Under our conditions, 1-hydroxyoctan-3-one could be discriminant to distinguish a control and spoiled apple purees but the limited number of samples did not allow us to conclude on this aspect.

Finally, octane-1,3-diol (no. 10) was detected by 3/9 panelists as mushroom in control apple puree and as mushroom, burnt, musty, metallic by 6/9 panelists in spoiled apple puree. Our results were relevant with previous works. Indeed, the octane-1,3-diol has already been described in Honeycrisp apples as an odor active compound and described as green, fatty or mushroom by GC-O (Yan et al., 2020). According to the context, octane-1,3-diol may not necessarily carry an off-flavor and could be a positive attribute in Honeycrisp apples. High concentration of this compound in other apple cultivars may not have the same impact on volatile profile, and could contribute to the off-flavor observed in apple purees.

## 3.2. Quantification of 1-hydroxyoctan-3-one and octane-1,3-diol by SIDA-LC-MS/MS

### 3.2.1. Analytical performances

The quantification of 1-hydroxyoctan-3-one and octane-1,3-diol was adapted from a recent work dedicated to grapes (Ployon et al., 2025). Briefly, these two analytes were analyzed by SIDA-LC-MS/MS using synthetic labelled and unlabelled analytical standards. The method was validated according to OIV recommendations and exhibited very satisfactory performances in terms of reliability and high analytical
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** ANOVA results for quantification of 1-hydroxyoctan-3-one and octane-1,3-diol in control (n = 146) and spoiled (n = 11) apple purees. Results were obtained by LC-MS/MS. Significance was measured using a Tukey test at risk α = 5 %.</figcaption></figure>

<caption>**Table 5**</caption>

Concentration of 1-hydroxyoctan-3-one and octane-1,3-diol (µg/kg of puree) (expressed with ± SD), in GD apple puree spoiled by filamentous fungi. Each sample was inoculated with only one fungal strain and incubated at 20 °C for 14 days. Quantification was made by SIDA LC-MS/MS. Different superscript letters indicate statistically significant differences (Tukey HSD, *p*-value<0.05).

<table><thead><tr><th>Spoilage fungal strain</th><th>[1-hydroxyoctan-3-one] µg/kg</th><th>[Octane-1,3-diol] µg/kg</th></tr></thead><tbody><tr><td>Control</td><td>14 ± 2 <sup>c</sup></td><td>2724 ± 543 <sup>a</sup></td></tr><tr><td>Penicillium digitatum</td><td>217 ± 6 <sup>a</sup></td><td>1916 ± 52 <sup>abc</sup></td></tr><tr><td>Aspergillus terreus</td><td>42 ± 26 <sup>bc</sup></td><td>2594 ± 272 <sup>a</sup></td></tr><tr><td>Aspergillus nidulans</td><td>36 ± 6 <sup>b</sup></td><td>2819 ± 329 <sup>a</sup></td></tr><tr><td>Talaromyces purpureogenus</td><td>14 ± 3 <sup>c</sup></td><td>2142 ± 571 <sup>ab</sup></td></tr><tr><td>Fusarium oxysporum</td><td>11 ± 2 <sup>c</sup></td><td>2834 ± 271 <sup>a</sup></td></tr><tr><td>Fusarium culmorum</td><td>6 ± 0 <sup>c</sup></td><td>1951 ± 53 <sup>abc</sup></td></tr><tr><td>Botrytis cinerea tomato</td><td>10 ± 1 <sup>c</sup></td><td>1369 ± 95 <sup>bc</sup></td></tr><tr><td>Botrytis cinerea apple</td><td>7 ± 2 <sup>c</sup></td><td>1164 ± 384 <sup>c</sup></td></tr></tbody></table>

throughput. Briefly, analytical performances (Table 2) demonstrated high linearity range from 0 to 519 µg/L for 1-hydroxyoctan-3-one (R² = 0.992) and from 0 to 7130 µg/L for octane-1,3-diol (R² = 0.999), allowing to use calibration on a large dynamic scale of concentration. The accuracy was evaluated at two levels of concentration (low and medium) for both compounds and recoveries ranged from 96 to 102 %. The repeatability was measured on a set of apple samples analyzed in replicates (n = 5) the same day and under the strict identical conditions. The method was highly repeatable with RSD values below to 10 % for both standards. In addition, the intermediate reproducibility (inter days measurements only) was evaluated on apple juice samples analyzed at several days. The corresponding RSD values were below 7 % for both target compounds highlighting the robustness of the method. Finally, sample preparation for the apple products was particularly simple (centrifugation and filtration), making it sufficiently sensitive (LOD above 1 µg/L for both compounds), efficient and high throughput (17 min run time) to be proposed as a routine analysis in our laboratory.

### 3.2.2. Quantification in commercial grade apple products

The analytical method was applied on a large set of apple purees collected in industry during one year of production to investigate the role of both 1-hydroxyoctan-3-one and octane-1,3-diol as analytical markers for fruit deterioration. The collected purees (n = 157) were for most of them (n = 146) of commercial grade, and n = 11 spoiled purees corresponded to apple removed batches.

The presence of 1-hydroxyoctan-3-one was scarce. Indeed, this compound was <LOD in 56 over 157 samples, <LOQ in 14 over 157 samples and quantified in 87 samples with concentration ranging from 3.4 µg/kg to 122.9 µg/kg of apple puree. Concentrations in the full sets of control (n = 146) and spoiled (n = 11) purees were 14 ± 2 µg/kg and

34 ± 7 µg/kg respectively.

On the opposite, octane-1,3-diol was quantified at very high levels in some samples with concentrations ranging from 89 µg/kg to 19,165 µg/kg in commercial grade control purees (mean value of 7715 ± 1273 µg/kg) and from 100 µg/kg to 18,427 µg/kg in the 11 spoiled products (mean value of 3770 ± 349 µg/kg). These concentrations were in coherence with previous works demonstrating levels up to 14,400 µg/kg in pears (Beuerle & Schwab, 1997) and from 2000 to 94,500 µg/L in a selection of 21 cider apple cultivars (Llorente et al., 2010). Considering these observations, octane-1,3-diol seems to be a ubiquitous compound in apple, probably impacting the aromatic profile but also possibly involved in defect depending on its level (regarding GC-O characterization in section 3.1.2).

Significantly higher concentrations were observed in spoiled samples than in control ones for both compounds (*p*-value <0.05) (Fig. 1).

In all the samples, both 1-hydroxyoctan-3-one and octane-1,3-diol levels were surprisingly well correlated with concentrations evolving in the same way (with a Pearson R² = 0.85) (Supplementary fig. 2). The same correlation has been recently shown in grape must (Ployon et al., 2025), suggesting the existence of a common biological origin. In the light of these results, we decided to go further in the knowledge of those compounds by considering that fungal alteration of fruit could be responsible for their production.

### 3.3. Targeted C8 compound production by filamentous fungi

In order to investigate the role of fungal development on apple puree and the release of both 1-hydroxyoctan-3-one and octane-1,3-diol, we contaminated apple purees with each of 8 fungal strains from the species *T. purpureogenus*, *F. culmorum*, *F. oxysporum*, *A. terreus*, *A. nidulans*, *P*.
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** 1-hydroxyoctan-3-one and octane-1,3-diol concentrations (µg/kg of puree, expressed as ± RSD, n = 2) in spoiled apple puree samples by combination of two filamentous fungi. Growth conditions were set at 15 °C during 14 days. Quantification was made by SIDA LC-MS/MS. Different superscript letters indicate statistically significant differences (Tukey HSD, p-value<0.05).</figcaption></figure>

### digitatum and B.cinerea.

The concentration of 1-hydroxyoctan-3-one was 14 ± 2 µg/kg in the control sample. A small decrease was observed in *Fusarium* spp. and *B. cinerea* spoiled samples. On the opposite, 1-hydroxyoctan-3-one concentration increased in samples spoiled by *P. digitatum*, *A. nidulans* and *A. terreus*, with a maximum concentration at 217 ± 6 µg/kg in the apple puree inoculated with *P. digitatum*.

Mono-inoculation of GD apple puree with filamentous fungi showed for most cases a stability of octane-1,3-diol concentration, determined as 2724 ± 543 µg/kg in the control sample. Surprisingly, the concentration of octane-1,3-diol decreased to 1369 ± 95 µg/kg and 1164 ± 384 µg/kg in the two *B. cinerea* contaminated samples (Table 5).

In order to observe possible synergistic or antagonistic effects resulting from fungal contaminations, co-inoculations of two species were assayed.

An heterogeneity for both 1-hydroxyoctan-3-one and octane-1,3-diol production was observed for the different samples. All samples inoculated with *P. digitatum* appeared to contain the highest concentrations of 1-hydroxyoctan-3-one, reaching 276 ± 48 µg/kg when co-inoculated with *A. terreus*. Conversely, 1-hydroxyoctan-3-one concentration did not significantly increase when other strains were used, in comparison to the control (12 ± 6 µg/kg). Those results suggest that the *P. digitatum* strain exhibited the ability to produce this compound (Fig. 2).

Regarding octane-1,3-diol, samples inoculated with *B. cinerea* strains, whatever the co-inoculated strain, had low concentration (1102 ± 190 µg/kg) compared to control samples (2478 ± 2 µg/kg).

The lowest concentration of octane-1,3-diol was observed in the sample inoculated with *B. cinerea* isolated from tomato and *F. oxysporum* (824 ± 278 µg/kg) (Fig. 2).

## 4. Discussion

Several compounds were identified by mass spectrometry and olfactometry as components of the unpleasant olfactive profile of apple purees. Octanoic acid, which was detected in our apple purees (no. 9), was quantified in higher concentration in deteriorated bayberries after cold storage (Jia-pei et al., 2024). This odor-active compound was also

detected in *Capparis spinosa* fruit (Zhang, Li, et al., 2023; Zhang, Liang, et al., 2023), with concentrations increasing after a storage period. This compound is already used as a marker of fungal infection by *Botrytis* sp. in an early detection method on strawberries (Pan et al., 2014). Octanoic acid is a lipid oxidation product and could be relevant as a marker of post-harvest storage fruit deterioration (Zhao et al., 2024).

Among others, the case of 2-pentylfuran (no.1) retained our attention because it was detected specifically in spoiled products, suggesting a new potential spoilage analytical marker. Olfactory area was described as stale, gas, animal and metallic, suggesting a potential sensory role of this compound. In literature, 2-pentylfuran has been detected in Gewurztraminer wines described with “ripe and pungent notes” and made with late harvested grapes. On the contrary, when its concentrations decreased significantly, the corresponding wines were described with floral and fruity notes (Lukić et al., 2016). More recently, a study led by Jia-pei et al., 2024 also evidenced 2-pentylfuran as a marker of fruit deterioration for *Mirica rubra* species. This compound is part of the off-flavors of oil-bearing seeds (Leonard et al., 2023). It is synthesized from linoleic acid through cyclisation in oxidative conditions, but it can be produced by bacteria and fungi (Chávez-Avilés et al., 2024). In Chardonnay grapes affected by *Penicillium expansum*, it has been reported that 2-pentylfuran was particularly overproduced without being strictly specific to this fungal species (Schueuermann et al., 2019). In addition, other studies suggest that other *Penicillium* species (Jeleń & Wasowicz, 1998), *Fusarium coeruleum* or *Phytophthora infestans* on potatoes (De Lacy Costello et al., 2001) could also produce 2-pentylfuran. Definitively, increasing concentration of 2-pentylfuran seems to be closely linked with fruit deterioration caused by fungal contamination during storage period.

In literature, 3-octanone and 1-octen-3-ol are the two most observed C8 compounds described as mushroom off-note development in apple juice. Surprisingly, we did not detect any of those in our samples by GC-O, and other C8 compounds such as 1-octen-3-one and 3-octanol were detected, but both in control and spoiled samples by the panelists. Hence, our sensory and chemical results suggested that these two compounds were not sufficient to discriminate the spoilage of apple purees. On the contrary, the accurate quantification of 1-hydroxyoctan-
3-one and octane-1,3-diol in apple products allowed us to go deeper in the understanding of apple spoilage mechanisms.

For the first time, we identified 1-hydroxyoctan-3-one in apple products and its concentration raised specifically in the case of fungal contaminated samples, making this compound a potential good spoilage analytical marker. The literature about this compound is very limited. Indeed, 1-hydroxyoctan-3-one was first identified in *Carica pubescens* fruit (Krajewski et al., 1997) and more recently in different grape must and wines affected by fresh-mushroom off-flavor in which its sensory role was formally identified (Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023; Ployon et al., 2025).

In addition to having a higher concentration of 1-hydroxyoctan-3-one, sensory descriptors obtained by GC-O analyses, both in purée and as pure standard, matched with descriptions given for spoiled purees (mushroom, musty) in the corresponding olfactory area. The perception olfactive threshold was established close to 200 µg/L in still wine and below 100 µg/L in water by Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023. Assuming a similar threshold in apple purée, the levels obtained (>200 µg/kg) in *Penicillium*-inoculated puree indicated a significant contribution to aroma profile, though matrix-specific data are lacking. Absence of negative descriptors for 1-hydroxyoctan-3-one in the olfactive area of control puree provides to this compound a real discriminative aspect for spoilage detection. The floral descriptor given by panelists for control puree could give information about an hypothetical perception of 1-hydroxyoctan-3-one at very low concentration by human nose, and below machine LOD, with another note than mushroom. However, this could be the consequence of a coelution, and determination of organoleptic detection threshold for 1-hydroxyoctan-3-one in this matrix may give more information about the mushroom off-note reveal. Currently, this compound is not commercially available as a Food Grade product and its sensory characterization in food remains problematic.

In wines, 1-hydroxyoctan-3-one production originates from a fungal development (*Crustomyces subabruptus*) notably on grape (*Delcros, Collas, et al., 2023*; *Delcros, Costis, et al., 2023*). In apple puree, we demonstrated the ability of several fungi to produce 1-hydroxyoctan-3-one, especially for *P. digitatum* strain. This observation was consistent with the clear implication of *Penicillium* spp. in the formation of musty-spoiled wines (Destanque et al., 2024; La Guerche et al., 2006).

For octane-1,3-diol, its ubiquitous occurrence was clearly observed in all the commercial grade purees. Its levels were very high (in the range of dozens of mg/kg) in comparison to 1-hydroxyoctan-3-one and completely in coherence with previous works (Beuerle & Schwab, 1997; Llorente et al., 2010). Considering GC-O experiments, octane-1,3-diol was detected in control and spoiled apple purees, with the same mushroom descriptor, suggesting that its contribution was not discriminant for both matrices. As pure compound, it was not detected in GC-O for a concentration of 100 mg/L. Previous work on Honeycrisp apples formally characterized octane-1,3-diol as mushroom by GC-O and as odor active compounds by omission tests (Yan et al., 2020). However, omission tests were performed with levels as high as 410 mg/L. Hence, the understanding of the contribution of this compound to the perceived off-flavor still requires further investigation.

Considering the LC-MS/MS quantification results, octane-1,3-diol can also be considered as discriminant for spoilage in apple purees. This information is coherent with literature, in which octane-1,3-diol has recently been proposed as a powerful analytical marker of wine affected by mushroom off-flavor together with 1-hydroxyoctan-3-one (Ployon et al., 2025). To the best of our knowledge, the olfactory detection threshold of octane-1,3-diol is not defined yet in apples and consequently, we cannot exclude its potential sensory role in apple spoilage.

Our results demonstrated that 1-hydroxyoctan-3-one and octane-1,3-diol are relevant analytical markers for characterizing the old-apple defect and may also be involved in its sensory perception. For both 1-hydroxyoctan-3-one and octane-1,3-diol, the demonstration of their

sensory contribution to the off-flavor in apple purée would deserve further investigation. The commercial unavailability of these compounds as food-grade standards is a major limitation for that purpose, that would imply spiking experiments. The AEDA approach could be the best option to gain insight into their sensory activity.

Under our conditions, we also observed a very clear correlation (R² = 0.85) between 1-hydroxyoctan-3-one and octane-1,3-diol levels in 146 commercial grade and 11 spoiled purees. This observation has been also recently demonstrated in grape must (Ployon et al., 2025) and suggests a common biogenetic origin in fruit. Surprisingly, this correlation no longer exists in fungal spoiled apple purees and highlights the necessity to continue to investigate the role of microorganisms in the production of both compounds.

An antibacterial role of 3-octanol and octan-1,3-diol was previously proposed (Akedo et al., 1977; Zhang, Li, et al., 2023; Zhang, Liang, et al., 2023). However, no correlation has been demonstrated until now between octane-1,3-diol and 1-octan-3-ol production using the lipoxygenase pathway with linoleic acid as precursor, but we can estimate that their syntheses are quite similar (Wurzenberger & Grosch, 1984). More specifically, volatile C8-compounds (3-octanol, 1-octen-3-ol, 1-octen-3-one, octane-1,3-diol and 1-hydroxyoctan-3-one) probably all originate either from linoleic acid degradation pathway or hydrolysis of glycosidic bonds (Delcros et al., 2022). Interconversions and balances between those compounds are not deciphered. Most of the previous data (Kim et al., 2019; Pennerman et al., 2022; Simonato et al., 2021) are related to the production of 1-octen-3-ol and octan-3-one. In particular, these two compounds are involved in interactions between fungi and plants and in competition mechanisms between fungi. The ability of many fungal species, in particular *Penicillium* spp., *Aspergillus* spp., *Fusarium* spp., *Talaromyces* spp. and *B. cinerea*, to produce these compounds is described. Though, the effect of *B. cinerea* contamination level on the concentration of those compounds remains largely unclear (Delcros, Collas, et al., 2023; Delcros, Costis, et al., 2023; La Guerche et al., 2006). In our study, co-inoculation of *P. culmorum* with *A. terreus*, which individually were the highest producers of 1-hydroxyoctan-3-one, resulted in an enhancement of 1-hydroxyoctan-3-one production without octane-1,3-diol degradation. Conversely, the co-inoculation of *B. cinerea* with another fungal species resulted in a decrease of octane-1,3-diol concentration compared to other co-inoculation assays. The origin of the formation of these compounds, together with other volatile C8-compounds, and the mechanisms regulating their production during fungal colonization deserve deeper investigation.

## 5. Conclusion

For the first time, a robust, high-throughput and reliable method was developed by SIDA-LC-MS/MS for the quantification of 1-hydroxyoctan-3-one and octane-1,3-diol.

Thanks to this method, 1-hydroxyoctan-3-one was detected, quantified and proposed as a new marker of fresh-mushroom off-flavor in apple transformed products. Quantification of both compounds on a larger sample set of commercial grade and spoiled purees demonstrated a clear correlation between 1-hydroxyoctan-3-one and octane-1,3-diol. As a result, we support the importance of analyzing both compounds in order to provide the most accurate diagnosis of the defect for fruit producers, particularly for apple growers.

From a metabolic point of view, employed fungal species from *P. digitatum* and *B. cinerea* presented a fairly marked difference concerning 1-hydroxyoctan-3-one and octane-1,3-diol production and consumption. In-depth research into co-metabolites from this pathway is envisaged.

Finally, this study opens avenues for putative compounds that could be implicated in this type of spoilage. Indeed, the detection of 2-pentylfuran and octanoic acid in our products is a new clue, strengthening the fungal contamination hypothesis. Further analysis of the fungal distribution on fruit during storage, in particular which species might be
responsible for 2-pentylfuran production, could provide a better understanding of fruit deterioration and assist in the adjustment of storage conditions to reduce the incidence of spoilage.

## Sensory Evaluation Ethics information

Sensory evaluation was carried out as part of this study using human volunteers. Ethical permission was not required to carry out the sensory evaluation.

## Sensory Evaluation Consent Information

We confirm that the appropriate protocols for protecting the rights and privacy of all participants were utilized during the execution of the research. All participants in the sensory study were volunteers. No formal written consent was required, as participation was entirely voluntary, anonymous, and involved no personal or sensitive data collection. Participants were informed about the nature of the study and gave their implicit consent by choosing to participate.

## CRediT authorship contribution statement

**Hippolyte Mouriot:** Writing – original draft, Visualization, Methodology, Formal analysis, Data curation. **Lucas Suc:** Methodology, Formal analysis. **Karine Gourrat:** Methodology, Formal analysis. **Géraldine Lucchi:** Supervision, Methodology. **Teddy Godet:** Methodology, Formal analysis. **Valérie Nolleau:** Supervision, Formal analysis. **Fabienne Remize:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Data curation, Conceptualization. **Aurélie Roland:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization.

## Declaration of competing interest

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

## Acknowledgement

We wanted to thank ANRT and Andros SNC for the funding of this project. This collaboration was successful thanks to resources given by Damien Chapoulart and Pierre-Alexis François (Andros SNC) and thanks to diligent work from members of CSGA, especially technical support from Noëlle Béno.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2025.146482.

## Data availability

Data will be made available on request.

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