# Functional activities of French raw-milk cheeses on aging, mobility, muscle integrity, and chondrocyte protection using in vivo and in vitro models

Anna Diet, Christophe Poix, Muriel Bonnet, Christian Coelho, Isabelle Ripoche, Yohann Wittrant, Caroline Decombat, Julien Priam, Etienne Saunier, Pierre Chalard, et al.

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Anna Diet, Christophe Poix, Muriel Bonnet, Christian Coelho, Isabelle Ripoche, et al. Functional activities of French raw-milk cheeses on aging, mobility, muscle integrity, and chondrocyte protection using in vivo and in vitro models. Journal of Functional Foods, 2025, 129, pp.106899. 10.1016/j.jff.2025.106899. hal-05261325

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# Functional activities of French raw-milk cheeses on aging, mobility, muscle integrity, and chondrocyte protection using *in vivo* and *in vitro* models

Anna Diet<sup>a, *</sup>, Christophe Poix<sup>a</sup>, Muriel Bonnet<sup>a</sup>, Christian Coelho<sup>a</sup>, Isabelle Ripoche<sup>b</sup>, Yohann Wittrant<sup>c</sup>, Caroline Decombat<sup>c</sup>, Julien Priam<sup>d</sup>, Etienne Saunier<sup>d</sup>, Pierre Chalard<sup>b</sup>, Florence Caldefie-Chézet<sup>c</sup>, Stéphanie Bornes<sup>a</sup>, Laurent Rios<sup>a</sup>

## ARTICLE INFO

### Keywords:

Raw-milk cheeses

Aging

Mobility

Muscle

Caenorhabditis elegans

Chondrocytes

## ABSTRACT

Aging is associated with various musculoskeletal disorders (muscle and/or cartilage disorders) that can decrease both mobility and overall health span. This study investigated the biological activity of seven french raw-milk cheeses on longevity, mobility, muscle integrity during aging, and chondrocyte protection over time using both *in vivo* (*Caenorhabditis elegans*) and *in vitro* (human articular chondrocytes) models. All cheeses enhanced both longevity and mobility during aging in *C. elegans*. They all protected the muscle integrity of *C. elegans* over time. The investigations *in vitro* on an osteoarthritic model of chondrocytes were conducted with fractions (one polar and one apolar) from two cheeses. Among the four tested fractions, we observed a reduction of NO, PGE₂ and MMP-13 productions in the chondrocytes. These findings highlight the biological activity of apolar fraction (containing the cheese lipid components) and suggest, re-evaluating cheese as a functional food with potential benefits for mobility and aging.

## 1. Introduction

One of humanity's key aspirations is to live longer while maintaining good health (Organization, 2002). Although medical advancements extend life expectancy, aging does not always equate to good health. Indeed, aging leads to various physiological changes, significantly increasing the prevalence of age-related diseases (Kwon et al., 2023). Aging is therefore related to various health issues (osteoarticular affections (Greene & Loeser, 2015), neurodegenerative disorders (Hou et al., 2019; Jo et al., 2023), cardiovascular diseases (Costantino et al., 2016), cancers (Campisi, 2013), etc.) and the locomotory system is often affected. Such is the case with the prevalence of muscular disorders (Frontera, 2017) or joint affections (Loeser et al., 2016; Seo et al., 2023; Steinmetz et al., 2023), negatively affecting functional mobility, thereby impacting the elderly quality of life.

Skeletal muscles, essential for movement, degrade with age, leading to loss of mass and function, mobility restriction, and joint instability (McCormick & Vasilaki, 2018; Tieland et al., 2018). In addition to enabling movement, the function of periarticular muscles is to provide articulation stability by distributing loads evenly, which supports overall joint health. Preserving muscles represents an effective approach to managing articulation diseases such as osteoarthritis (OA), a major joint affection leading to a loss of articular cartilage (Hunter & Bierma-Zeinstra, 2019). Chondrocytes, the articular cartilage cells, are responsible for maintaining the extracellular matrix by secreting its components as well as factors that promote matrix degradation (Chevalier & Richette, 2005). In OA, damages to chondrocytes, such as oxidative stress (Zahan et al., 2020) and inflammation (Goldring & Otero, 2011), result in their deregulation and promote the degradation of the articular cartilage.

E-mail addresses: anna.diet@vetagro-sup.fr (A. Diet), christophe.poix@vetagro-sup.fr (C. Poix), muriel.bonnet@uca.fr (M. Bonnet), christian.coelho@vetagro-sup.fr (C. Coelho), isa-belle.ripoche@sigma-clermont.fr (I. Ripoche), yohann.wittrant@uca.fr (Y. Wittrant), caroline.decombat@uca.fr (C. Decombat), j.priam@domespharma.com (J. Priam), e.saunier@domespharma.com (E. Saunier), pierre.chalard@sigma-clermont.fr (P. Chalard), florence.caldefie-chezet@uca.fr (F. Caldefie-Chézet), stephanie.bornes@uca.fr (S. Bornes), laurent.rios@vetagro-sup.fr (L. Rios).

https://doi.org/10.1016/j.jff.2025.106899

Received 20 January 2025; Received in revised form 12 May 2025; Accepted 15 May 2025

Available online 19 May 2025

1756-4646/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

<footnote><sup>a</sup> Université Clermont Auvergne, INRAE, VetAgro Sup, UMRF, F-15000 Aurillac, France</footnote>

<footnote><sup>b</sup> Université Clermont Auvergne, CNRS, Clermont Auvergne INP, ICCF, F-63000 Clermont-Ferrand, France</footnote>

<footnote><sup>c</sup> Université Clermont Auvergne, INRAE, UNH, Unité de Nutrition Humaine, CRNH-Auvergne, F-63000 Clermont-Ferrand, France</footnote>

<footnote><sup>d</sup> Dômes Pharma, ZAC de Champ Lamet, 3 Rue Andrée Citröen, F-63284 Pont-du-Château, France</footnote>

<footnote>* Corresponding authors.</footnote>
Strategies aimed at preserving muscle integrity and protecting articular chondrocytes from oxidative stress and inflammation may help prevent or mitigate disease onset, thereby maintaining mobility with advancing age and extending the span of active living. Among the identified approaches to promoting healthy aging, dietary strategies and the consumption of foods with potential health benefits have garnered significant attention (Chandra et al., 2022; Duan et al., 2022; Longo & Anderson, 2022). Over time, fermented foods have attracted growing interest in the fields of food science, nutrition, and health. This interest arises from the natural fermentation process during which compounds can exert positive biological effects upon consumption (Marco et al., 2017; Şanlier et al., 2019). Among fermented foods, cheese is a rich source of nutrients widely consumed in the Western diet (Temme et al., 2015). During fermentation, various metabolites are generated in cheeses, some of which may exert beneficial effects on health. Notably, bioactive peptides derived from milk, whey proteins, and casein have been particularly studied for their health-promoting properties, including antioxidant, antimicrobial, and antihypertensive effects (Kaur et al., 2022; Khan et al., 2019; Y. W. Park & Nam, 2015; Silva & Malcata, 2005). The presence and concentration of these metabolites depend on the cheese's chemical composition and the microbiota of the cheese, which are influenced by the wide variety of cheese-making processes, milk types, or ripening conditions (Díaz-Castro et al., 2012; Gupta et al., 2009; Kumar et al., 2006; Nam et al., 2021; Santiago-López et al., 2018). In a previous study, we showed that a goat raw-milk cheese had beneficial effects on longevity and against oxidative processes in the *in vivo* biological model *Caenorhabditis elegans* (*C. elegans*) (Cardin et al., 2021). We expanded these studies by investigating a broader range of raw milk cheeses (from different animal origins and/or various manufacturing processes) and explored their antioxidant potential using human leukocytes and *C. elegans* (Diet et al., 2024). We demonstrated their ability to extend *C. elegans* survival rate under oxidative conditions by reducing reactive oxygen species (ROS) accumulation *in vivo*. They reduced ROS production in human leukocytes and enhanced antioxidant gene expression in *C. elegans*. In the current study, we pursued our investigations about the effects of seven french raw-milk cheeses on aging and associated mobility disorders.

*Caenorhabditis elegans* is a well-established *in vivo* model for aging research (Finch & Ruvkun, 2001; Shen et al., 2018) due to its short life cycle (about 20 days). During aging, *C. elegans* is concerned with physiological conserved processes such as reduced movement (Marck et al., 2017) and a progressive loss of muscle function comparable to human muscle aging-deterioration (Herndon et al., 2002). In addition, its body transparency allowed for the use of fluorescent mutants.

Using *C. elegans*, we assessed the effect of the seven french raw-milk cheeses on longevity. In addition, we evaluated their effects on the mobility of the nematodes measuring their movement over time. We pursued our investigation by using a GFP-coupled mutant to further examine the impact of these cheeses on muscle maintenance and structure by exploring the muscle mitochondrial network during aging. We then selected two cheeses to explore their effects *in vitro* on articular cartilage. We used two fractions from various polarities to study both apolar and polar fractions for each cheese and evaluated their impacts on the secretion of cartilage-degrading factors using an IL-1β-stimulated primary human articular chondrocytes model.

# 2. Material & Method

## 2.1. Reagents, kits, and solvents

Cyclohexane was purchased from Carlo Erba (Val de Reuil, France). Amphotericin B, agarose, cholesterol, NaCl, MgSO₄, CaCl₂, NaHCO₃, Na₂HPO₄, NH₄Cl, KH₂PO₄, EDTA, gentamycin, type IV-S hyaluronidase (750–3000 units/mg), trypsin (≥9000 BAEE units/mg), type II collagenase (125 units/mg), Cell Proliferation Kit II (XTT), sodium azide, and NaOH were obtained from Sigma Aldrich (Saint-Louis, USA). Lysogeny

Broth (LB, Miller's Modification), peptone, and agar were purchased from Conda (Madrid, Spain). Yeast fraction, 5-fluoro-2'-deoxyuridine (FUDR), Dulbecco's Modified Eagle Medium (DMEM) were acquired from ThermoFisher Scientific (Waltham, MA, USA). Nitrate/Nitrite colorimetric assay and prostaglandin E2 Enzyme Immunoassay (EIA) kit were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Hank's Balanced Sodium Salt (HBSS) and penicillin/streptomycin were obtained from Life Technologies (Villebon-Sur-Yvette, France). The Fetal calf serum (FCS) was provided by Pan-Biotech (Aidenbach, Germany). The human recombinant IL-1 β was purchased from Millipore Corporation (Molsheim, France). The human ELISA kit for MMP-13 detection was acquired from Abcam® (Paris, France).

## 2.2. Flowchart of the study

The detailed steps of the research conducted are graphically summarized in a flowchart, presented in Fig. 1, providing an overview of the experimental design and the analyses performed.

## 2.3. Cheese selection

Seven french raw-milk cheeses were selected based on theirs characteristics as in the previous study (Diet et al., 2024). To guarantee the reliability and reproducibility of our research, we chose cheeses recognized for their high production consistency and year-round availability. The seven cheeses were selected at their most popular and more consumed stage of ripening (see Table 1).

## 2.4. Obtention of the cheese fractions (FDC, W70 and ApF)

Each cheese and its rind was cut into small slices. They were freeze-dried and the slices were crushed with mortar and pestle as previously described (Cardin et al., 2020). The powder obtained was named freeze-dried cheese (FDC) and conserved in a waterproof container at -20 °C until use.

In addition to FDC, two fractions were made for Goat cheese and Saint-Nectaire as previously described (Diet et al., 2024). Each of these two FDCs was mixed with distilled cyclohexane (ratio 1/10 (w/v)), stirred for 4 h and filtered with Büchner. The resulting filtrate was concentrated under vacuum and a dry fraction was obtained and conserved. These steps were repeated twice but stirred for 2 h, and then 1 h. The three dry fractions were pooled and named « cheese apolar fraction » (ApF).

The resulting solid obtained after cyclohexane extraction was extracted with HPLC-grade water (ratio 1/10 (w/v)). The mixture was mechanically stirred for 1 h at 70 °C and centrifuged (15 min, 8000 rpm; Avanti J26S XPI, Beckman Coulter, Brea, CA, USA). The supernatant recovered was evaporated under a vacuum to obtain a dry fraction. The cheese matrix was exhausted by repeating this procedure three times. All the dry fractions obtained were combined, ground using a mortar and pestle, and designated as « W70 ».

Each apolar (ApF) and polar (W70) fractions of Goat cheese and Saint-Nectaire were kept in a waterproof container at -20 °C until use.

## 2.5. *In vivo* study on *Caenorhabditis elegans*

### 2.5.1. Microbial strain, growth conditions and heat-killed preparation

The *Escherichia coli* strain OP50 (*E. coli* OP50) was purchased at the *Caenorhabditis* Genetics Center (Minneapolis, MN, USA), and cultivated in a lysogeny broth medium (overnight, 37 °C) as previously described (Poupet et al., 2019). The next day, the microbial suspension was centrifuged (4000 rpm, 15 min; Rotofix 32 A, Hettich Zentrifugen, Tuttlingen, Germany) and the pellet obtained was washed three times with M9 buffer (per L: 3 g of KH₂PO₄; 5 g of NaCl; 6 g of Na₂HPO₄; 1 mL of 1 M MgSO₄). The live *E. coli* OP50 suspension was adjusted to 100 mg/mL final concentration.
<figure><img src="image_3.png" /><figcaption>Fig. 1. Flowchart of the study.</figcaption></figure>

<table><caption>**Table 1**
The selected cheeses and their characteristics (Diet et al., 2024).</caption><thead><tr><td>Cheese</td><td>Milk origin</td><td>Paste</td><td>Rind</td><td>Cheese-making process</td><td>Ripening</td></tr></thead><tbody><tr><td>Goat cheese</td><td>goat</td><td>-</td><td>-</td><td>fresh cheese</td><td>3 weeks</td></tr><tr><td>Saint-Nectaire</td><td>cow</td><td>soft cheese</td><td>surface molds</td><td>uncooked pressed cheeses</td><td>6 weeks</td></tr><tr><td>Cantal</td><td>cow</td><td>hard cheese</td><td>washed rind</td><td>uncooked cheeses</td><td>12 to 17 weeks</td></tr><tr><td>Bleu d'Auvergne</td><td>cow</td><td>soft cheese internal molds</td><td>-</td><td>uncooked unpressed cheeses</td><td>8 weeks</td></tr><tr><td>Roquefort</td><td>ewe</td><td>soft cheese internal molds</td><td>-</td><td>uncooked unpressed cheeses</td><td>8 weeks</td></tr><tr><td>Comté</td><td>cow</td><td>hard cheese</td><td>smear rind</td><td>cooked cheeses</td><td>17 weeks</td></tr><tr><td>Epoisses</td><td>cow</td><td>soft cheese</td><td>washed rind</td><td>uncooked unpressed cheeses</td><td>4 weeks</td></tr></tbody></table>

The heat-killed (HK) *E. coli* OP50 was obtained from live *E. coli* OP50 (100 mg/mL) held for 1 h at 80 °C.

These two solutions were kept at 4 °C until use.

### 2.5.2. *Caenorhabditis elegans* maintenance

The *Caenorhabditis elegans* N2 wild-type strain and the CB5600 mutant strain (ccls4251 I; him-8(e1489) IV, expressing green fluorescent protein (GFP) in nuclei and mitochondria of body wall muscles) were obtained from the *Caenorhabditis* Genetics Center (Minneapolis, MN, USA).

The maintenance of strains was made on nematode growth medium (NGM) plates (per L: 3 g of NaCl; 17 g of agar; 2.5 g of peptone; 1 mM of CaCl₂; 5 mg of cholesterol; 1 mM of MgSO₄; 25 mL of 1 M potassium phosphate buffer at pH 6), supplemented with yeast extract (4 g/L) (NGMY) as previously described (Poupet et al., 2019). The plates were seeded with live *E. coli* OP50 as a source of food. The nematodes were kept on plates at 20 °C.

### 2.5.3. Synchronization of *Caenorhabditis elegans* strains

All experiments were performed using a synchronized population of nematodes as previously described (Veisseire et al., 2020). The gravid worms and eggs were gathered from NGMY plates, washed off, and centrifuged (1500 rpm, 2 min). After removing as much supernatant as possible, 5 mL of worm bleach (700 µL of bleach at 12.5 % of sodium hypochlorite; 1 mL of sodium hydroxide 5 M; 3.3 mL of M9 buffer) were added to the pellet. The suspension was vigorously shaken until the adult worm bodies were disrupted and 40 mL of M9 buffer were added to stop the effect of the worm bleach. The suspension was centrifuged (1500 rpm, 2 min) and the pellet was collected and washed twice with M9 buffer. The isolated eggs hatched in 20 mL of M9 buffer under slown agitation (24 h, 25 °C).

The resulting L1 larvae were then transferred onto NGMY plates with live *E. coli* OP50 as a food source (48 h, 20 °C) until they reached the L4 stage corresponding to the young adult stage.

### 2.5.4. Preparation of the 24-well plates for the assay

An agar medium (per L: 3 g of NaCl and 6 g of agarose) was prepared,
split into individual aliquots supplemented with amphotericin B (1.6 µg/
mL), FUDR (0.12 mM), and stored at 40 °C. Each one was supplemented
with 0,5 % (for the longevity assay) or 1 % of freeze-dried cheese (w/v),
except for the control group. Three wells per plate were poured (500 µL
per well) and the plates were immediately moved onto ice to densify the
agar. The plates were kept at 4 °C until use.

2.5.5. *Caenorhabditis elegans longevity assay*

The impact of freeze-dried cheeses (FDCs) on the life expectancy of
worms was evaluated by performing a longevity assay on the *C. elegans*
N2 strain as previously described (Cardin et al., 2021).

About fifteen L4-synchronized worms were placed per well of a 24-
well plate supplemented with 0,5 % of FDC (w/v). To avoid starva-
tion, 30 µL of HK E. coli OP50 (100 mg/mL) were added, every 3 days as
a source of food. The number of living nematodes was scored daily. The
assay was performed as four independent experiments with three wells
per condition each time.

2.5.6. *Caenorhabditis elegans mobility assay*

The impact of cheeses on the mobility of N2 wild-type *C. elegans*
during aging was measured by counting the body movement (body
bends) in *C. elegans* while swimming in a drop of buffer, as described in
previous studies (Hughes et al., 2022; Wu et al., 2022). Their body bends
were recorded at the beginning of the experiment (day 0) and after
spending 5, 7, 10, 12, and 15 days on agar plates as described previously
with slight modifications (Wu et al., 2022).

For that purpose, five 24-well plates supplemented with 1 % of FDC
were made in parallel, corresponding to the five times of incubation.
About thirty L4-synchronized worms per well were deposited on each
plate. Every 3 days, 30 µL of HK E. coli OP50 (100 mg/mL) per well were
added to avoid starvation.

After 0, 5, 7, 10, 12 or 15 days, about 10 worms were placed in a droplet of M9 buffer and allowed to swim. The droplet was placed under a microscope and worm videos were recorded (FLEXACAM C1, LEICA S9D) for 30 s (H. Wang et al., 2019). The videos were exported on ImageJ software and the worm mobility behavior (body bends per second) was analyzed using wrMTrck (wrMTrck multiple object tracker - phage.dk). The assay was performed in at least 3 independent experiments for each condition.

2.5.7. *Caenorhabditis elegans muscle structure assay*

The impact of the FDCs on the *C. elegans* muscle structure during
aging was evaluated. The CB5600 mutant strain, labeled with GFP, was
used to visualize and qualitatively assess the structure of the mito-
chondrial network of body wall muscle cells, as previously described
(Giron et al., 2022). The structure of the mitochondrial network is a
precursor to the loss of muscle fiber integrity (Gaffney, Pollard, Barratt,
Constantin-Teodosiu, Greenhaff & Szewczyk, 2018), thus its structure
reflected the fiber quality and muscle structure. The mitochondrial
network structure was observed at the beginning of the experiment (day
0) and after spending 5, 7, 10, 12, or 15 days of adulthood on different
conditions.

For the experiment, five 24-well plates supplemented with 1 % of
FDC were made in parallel, corresponding to the five times of incuba-
tion. About 30 worms per well were deposited on each plate. They were
fed every 3 days with HK E. coli OP50 (30 µL of 10 mg/mL) to avoid
starvation.

After 0, 5, 7, 10, 12 or 15 days, about 20 worms were collected on a
slide, immobilized using sodium azide (0.5 M), and observed under a
fluorescence microscope. Pictures of the mitochondrial network of
worms were made at 100× magnification with a GFP filter (EVOS XL
Core Imaging System, ThermoFisher, Hampton, USA). The assay was
performed in at least 3 independent experiments for each condition.

The 2253 photos obtained were anonymized and sent to four quali-
fied persons for qualitative notation of the structure. The mitochondrial
network structure of each worm was categorized as: (1) "linear" when

the network was mostly long and great interconnected; (2) “intermedi-
ate” when the network was interconnected but occurred in tandem with
smaller fragmented mitochondria; or (3) “fragmented” when it was no
longer a network but only sparse, small, and round mitochondria visible.

For each population on the various condition, we obtained the per-
centage of the population with either a linear, intermediate, or frag-
mented mitochondrial network structure.

2.6. *In vitro* study using chondrocytes

2.6.1. Chondrocytes culture

Human articular chondrocytes (HACs) were obtained from osteoar-
thritic patients following a knee replacement surgery. Only intact
cartilage areas from the tibial plateau and femoral condyles were used
for chondrocyte isolation following the previously described process
with slight modification (Wauquier et al., 2019). Cartilage was sliced
and the chips obtained were successively digested with HBSS supple-
mented with 0.05 % type IV-S hyaluronidase (750–3000 units/mg) for
10 min at 37 °C, then with 0.2 % trypsin (≥9000 BAEE units/mg) for 15
min at 37 °C and with 0.2 % type II collagenase (125 units/mg) for 30
min at 37 °C. They were transferred in control medium (DMEM com-
plemented with 10 % (v/v) fetal cal serum (FCS) and 1 % (v/v) peni-
cillin/streptomycin (P/S)) supplemented with 0.03 % type II collagenase
overnight at 37 °C.

Chondrocytes were harvested in control medium (10 % FCS, 1 % P/
S) at a density of 100,000 cells/cm² in F225 flasks at passage 1 and
maintained at 37 °C in a humidified atmosphere of 5 % CO₂.

At confluency, cells were subcultured in 96-well plates for experi-
ments. Chondrocytes were preincubated in control medium (10 % FCS,
1 % P/S) supplemented with cheese fractions (0, 50, 100, or 200 µg/mL)
for 24 h at 37 °C. *C. media* was replaced and cells were then incubated for
an additional 24 h with human recombinant IL-1 β at 0.5 ng/mL and
cheese fractions (0, 50, 100, or 200 µg/mL).

2.6.2. *Chondrocyte proliferation*

The chondrocyte proliferation was determined using a Cell Prolif-
eration Kit II, based on an XTT method, in accordance with the manu-
facturer's instructions. Optical density was measured at 450 nm.

2.6.3. NO, PGE₂, and MMP-13 quantification in chondrocytes

The NO, PGE₂, and MMP-13 quantification were measured on su-
pernatants using the Nitrate/Nitrite colorimetric assay kit, the prosta-
glandin E₂ Enzyme Immunoassay (EIA) kit, and the human ELISA kit for
MMP-13 detection, respectively, following the supplier's instructions.

2.7. Statistical analysis

The data were expressed as mean ± standard error of the mean
(SEM). The ternary plot were made on ternaryplot.com (TernaryPlot.
Com, n.d.).

The *C. elegans* longevity assay was examined using the script (Grol-
lemond, n.d.) and the Kaplan–Meier method. The differences were
determined with the log-rank test with R software version 3.5.0 (R
Project, n.d.) survival (GitHub, n.d.) and survminer (Survminer, n.d.) R
packages. Each experiment was performed in four independent experi-
ments with three wells per group. The null hypothesis H0 is that of the
equality of the survival functions between two groups. A *p*-value <0.05
was considered as significant.

Statistical significance between two groups was performed with
GraphPad Prism version 8.2.1 for Windows (GraphPad Software, La
Jolla, CA, USA). Groups were compared to the control using a two-tailed
Student's t-test. Differences were considered statistically significant at a
*p*-value <0.05.
# 3. Results

## 3.1. Influence of freeze-dried cheeses on *C. elegans* longevity

We investigated the impact of freeze-dried cheese (FDC) of the seven raw-milk cheeses on the longevity of *C. elegans*. Worms were placed on agar medium supplemented with FDC or not (CTRL population) and fed every 3 days to avoid starvation. All the survival curves, except Comté, were significantly different (*p* < 0.001) from the CTRL condition and showed a significant positive impact on the longevity of *C. elegans* (Fig. 2).

To delve deeper, we extracted median survival and maximum longevity from the survival curves and presented them in a table for easier access (Table 2). Moreover, we focused on day 22 (the death of the CTRL) for each population.

The median survival (13 days for the CTRL), increased by two days (+15 %) for Cantal, Bleu d'Auvergne, and Epoisses, reaching 15 days and up to seven days (+54 %), reaching 20 days for Roquefort.

The FDC-supplemented group increased the maximum longevity (of 22 days for the CTRL) to 29 days for Comté allowing it to gain 7 days (+32 %), and up to 39 days for the two FDCs of Goat cheese and Bleu d'Auvergne, providing it with 17 extra days (+77 %). Interestingly, on day 22 (death of the CTRL), at least 7 % of the population was still alive on Comté and up to 42 % on Saint-Nectaire.

In conclusion, all cheeses except Comté positively impacted the worm population's longevity compared to the CTRL. Among them, Goat cheese, Saint-Nectaire, and Bleu d'Auvergne emerged as the most effective, based on the three parameters analyzed.

## 3.2. Impact of the freeze-dried cheeses on the mobility of *C. elegans* during aging

After assessing lifespan, we evaluated the impact of the seven FDCs on *C. elegans* health span by measuring mobility over time. Based on CTRL longevity, worm movement was monitored on days 0, 5, 7, 10, 12, and 15 (Fig. 3 a). Mobility, measured as body bends per second (bds/s) in liquid, started at 4.58 bds/s on day 0.

On day 5, in comparison with the CTRL (3.5 bds/s), all seven FDCs of cheese preserved the mobility of *C. elegans* ranging from 3.97 bds/s for Cantal (+13 %; *p* < 0.01) and up to 4.9 bds/s for Goat cheese (+40 %; *p* < 0.001).

CTRL mobility declined steadily, reaching 2.9 bds/s on day 10. In

<table><caption>**Table 2**

Survival of N2 strain.</caption><thead><tr><th></th><th>Mean lifespan (in days)</th><th>Maximum longevity (in days)</th><th>Population remaining alive after 22 days (in %)</th></tr></thead><tbody><tr><td>CTRL</td><td>13</td><td>22</td><td>0</td></tr><tr><td>Goat cheese</td><td>18</td><td>39</td><td>33</td></tr><tr><td>Saint-Nectaire</td><td>18</td><td>35</td><td>42</td></tr><tr><td>Cantal</td><td>15</td><td>35</td><td>25</td></tr><tr><td>Bleu d'Auvergne</td><td>15</td><td>39</td><td>29</td></tr><tr><td>Roquefort</td><td>20</td><td>35</td><td>15</td></tr><tr><td>Comté</td><td>13</td><td>29</td><td>7</td></tr><tr><td>Epoisses</td><td>15</td><td>33</td><td>18</td></tr></tbody></table>

L4-synchronized worms were incubated on a standard culture medium (CTRL) or supplemented with FDC (1 %) and fed every 3 days with HK OP50 *E. coli*. Mean lifespan, maximum longevity, the percentage of the population still alive after 22 days (maximum longevity of the CTRL) were obtained from the longevity curves (Fig. 1).

contrast, mobility increased with all FDCs except Bleu d'Auvergne, ranging from 3.8 bds/s for Goat cheese (+39 %; *p* < 0.0001) to 4.6 bds/s for Cantal (+48 %; *p* < 0.0001).

At day 15, comparing with the CTRL (2.02 bds/s), the FDCs of Saint-Nectaire, Bleu d'Auvergne, Comté, and Epoisses significantly protected worm mobility, ranging from 3.02 bds/s for Epoisses (+33 %; *p* < 0.05) and up to 4.26 bds/s for Comté (+41 %; *p* < 0.0001).

Finally, out of the 7 cheeses, the two FDCs of Saint-Nectaire and Comté significantly improved (*p* < 0.05) mobility compared to the CTRL at all measured times, making them the most consistent and uniform FDCs in significantly preserving the mobility of *C. elegans* during its aging process.

In addition to these results, mobility was also measured at days 7, and 12 (Supplementary Data 1). All together, these results allowed us to make a line graph based on these results to examine the mobility evolution of *C. elegans* over the overall 15-day duration rather than at specific points (Fig. 3 b). From this representation, we calculate the average of the curve to obtain a smoothed average of a worm's activity (bds/s) over the 15 days, without considering the daily fluctuations or variations in worm activity. As expected, the CTRL group had the lowest mobility, averaging 3.16 bds/s over 15 days, followed by Cantal (3.82 bds/s, +21 %), Bleu d'Auvergne (4.00 bds/s, +27 %),

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Influence of the FDC on *C. elegans* wild-type strain (N2) longevity.</figcaption></figure>

L4 synchronized worms were incubated on a standard culture medium supplemented with FDC (1 %) or not (CTRL condition) and fed every 3 days with HK OP50 *E. coli*. The "N" value represented the number of nematodes obtained for each condition from four independent experiments. Longevity curves were the average of the individual longevity curves, and were compared with the CTRL condition; *** = *p* < 0.001.
a.

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Influence of the FDC on *C. elegans* wild-type strain (N2) mobility. L4-synchronized worms were incubated on a standard culture medium supplemented with FDC (1%) or not (CTRL condition), and fed every 3 days with HK OP50 *E. coli*. Mobility was measured (body bends/s) on days 5, 7, 10, 12, and 15. Results are presented as mobility assays at days 5, 10, and 15 (a) and as a line graph over 15 days (b). Data are shown as the mean ± SEM. For each day (a), results were compared with the CTRL. \* = *p* < 0.05; \*\* = *p* < 0.01; \*\*\* = *p* < 0.001; \*\*\*\* = *p* < 0.0001.</figcaption></figure>

Saint-Nectaire (4.05 bds/s, +28 %), Roquefort (4.12 bds/s, +30 %), and
Goat cheese (4.21 bds/s, +33 %). The highest average activity was seen
with Comté and Epoisses (4.24 and 4.25 bds/s, +34 %).

Overall, all the cheeses induced a highest mobility in comparison
with the CTRL. Among all of them, Goat cheese, Comté, and Epoisses
provided the highest mobility, suggesting these FDCs most effectively
maintained nematode activity over time.

3.3. Impact of the freeze-dried cheeses on *C. elegans* muscle structure during aging

In *C. elegans*, mobility depends on body-wall muscles, which decline with age. Markers of muscle aging included an age-dependent increase in mitochondrial fragmentation (*Christian & Benian, 2020*; *Gaffney, Pollard, Barratt, Constantin-Teodosiu, Greenhaff, & Szewczyk, 2018*). Using a mutant strain with GFP-labeled mitochondria in muscle cells, we studied mitochondria as an output of muscle function and structure over time. We described the mitochondrial network as linear, intermediate or fragmented (Fig. 4).

To assess the effect of FDC supplementation on muscle structure
during aging, we analyzed mitochondrial networks at days 0, 5, 7, 10,
12, and 15. For each population, we measured the percentage of in-
dividuals with linear, intermediate, or fragmented networks (see **Sup-plementary Data 2**). Since the percentages sum to 100 %, ternary plots
were used to represent the network distribution over time (Fig. 4 **b-e**).

In the ternary plot, data points for each day were connected to
illustrate the temporal evolution of each population. A trajectory mov-
ing toward the top of the triangle reflects an increasing proportion of
population with fragmented networks, and a decline in those with linear
networks. In the CTRL group (Fig. 4 b), we observed a shift toward
fragmented networks starting by day 5, which dominated over half the
population by day 10, with almost no linear networks remaining.

In contrast to CTRL, a FDC supplementation significantly slowed
mitochondrial network deterioration during aging. Among all the
cheeses, this representation revealed three distinct patterns of evolution
(Fig. 4c-e).

Goat cheese, Bleu d'Auvergne, Roquefort, Comté, and Epoisses exhibited a similar worm population trend (Fig. 4 c). Their distributions
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Influence of the FDCs on age-dependent mitochondrial changes in body-wall muscle cells.</figcaption></figure>

(a) Representative images of the mitochondrial changes observed in body-wall muscle cells. The transgenic *C. elegans* strain (CB5600) expressing GFP in the mitochondria of the body-wall muscle cells has been used. Images were acquired using a fluorescence microscope with a GFP filter at 100× magnification. Scale bar: 10 µm.

(b-e) L4-synchronized transgenic *C. elegans* strain CB5600 was incubated on a standard culture medium (CTRL) or supplemented with FDC (1%) and fed every 3 days with HK OP50 *E. coli*. Mitochondrial network structure were assessed in populations at days 0, 5, 7, 10, 12, and 15. Ternary plots were used to visualize the distribution of mitochondrial network (linear, intermediate, fragmented) over time. Each vertex of the triangle corresponds to 100% of one of the three modalities. A point positioned inside the triangle allows for a quick visualization of the distribution for the three parameters (their sum being 100%). The distribution at days 0, 5, 7, 10, 12, and 15 were linked together for all populations to observe the evolution of each distribution (b) The CTRL condition showed a progressive shift in network structure across 15 days. Comparing the cheeses revealed three main profiles: (c) an average profile where the population distributions were similar between them; (d) a less favorable profile showing a faster shift toward fragmented network, and (e) the most favorable profile, indicating strong preservation of mitochondrial network structure.

were composed of varying representations of all possible network types, effectively limiting the appearance of fragmented networks while also preserving the percentage of linear networks up to day 15 compared to the CTRL.

Among all cheeses supplementation, Cantal cheese provided the least effective protection among all cheeses (Fig. 4 d). The deterioration of the mitochondrial network of its population was the fastest of the seven FDCs, with no longer population having linear network by day 15. Cantal appeared to provide the least protection to the mitochondrial

network over time with the highest percentage of the population with fragmented mitochondrial networks, although it did not exceed 50% of the population.

Finally, the Saint-Nectaire cheese presented a distinct evolution compared to the others (Fig. 4 e). At all time points studied, Saint-Nectaire was more effective in preserving the mitochondrial network by significantly limiting the percentage of its population with a fragmented network from day 0 to day 15.

In conclusion, all FDCs preserved mitochondrial network structure
over time compared to the CTRL. Among them, Saint-Nectaire, Goat cheese, and Comté provided the strongest protection, as indicated by the lowest proportion of individuals with fragmented networks. This suggests a better preservation of muscle structure during aging.

### 3.4. Influence of saint-Nectaire goat cheeses on NO, PGE₂, and MMP-13 productions in human chondrocytes

Based on the results for longevity, mobility, and mitochondrial network architecture during aging, we aimed to identify the two cheeses with the most promising overall effects. To this end, we created a summary table (Table 3) comparing all cheeses from each study. From this comparison, Saint-Nectaire, Comté, and Goat cheeses appeared to be the three most effective. As Comté cheese did not significantly improve C. elegans longevity, we further explore Saint-Nectaire and Goat cheeses effects on cartilage degradation.

Primary human chondrocytes (HACs) stimulated with IL-1β were used to mimic an osteoarthritic profile and enhance catabolic compound production. This model allowed us to assess the impact of Goat cheese and Saint-Nectaire on matrix degradation and inflammatory factors overexpressed with osteoarthritis (NO, PGE₂, MMP-13; Fig. 5). For this study, direct use of the FDC was not feasible, we utilized two fractions (W70 for polar, and ApF for apolar fractions) to represent the cheeses.

As expected, IL-1β stimulation increased CTRL-NO production (+132%; *p* < 0.001) compared to the Negative CTRL (Fig. 5a). Neither Goat cheese fraction significantly reduced NO production. Only Saint-Nectaire W70 fraction reduced NO in a dose-dependent reaction (*p* < 0.05): -25% at 50 µg/mL, -27% at 100 µg/mL, and -33% at 200 µg/mL.

IL-1β stimulation increased PGE₂ production in the CTRL (+224%; *p* < 0.01) compared to the negative CTRL (Fig. 5b). Both polar and apolar fractions of the two cheeses reduced PGE₂ production. Goat cheese polar fraction (W70) reduced PGE₂ by -69% at 50 µg/mL (*p* < 0.05) to -72% at 200 µg/mL (*p* < 0.01). Its apolar fraction (ApF) reduced it by -39% at 200 µg/mL (*p* < 0.05). Saint-Nectaire W70 fraction reduced PGE₂ by -50% at 50 µg/mL (*p* < 0.05) and -48% at 100 µg/mL (*p* < 0.05), while its ApF fraction reduced it by -54% at 50 µg/mL (*p* < 0.05) and -43% at 200 µg/mL (*p* < 0.05).

IL-1β stimulation increased MMP-13 production in the CTRL by +832% (*p* < 0.0001) compared to the Negative CTRL (Fig. 5c). Both Goat cheese fractions significantly reduced MMP-13 production. The W70 fraction reduced it by -92% at 100 µg/mL (*p* < 0.0001) and -90% at 200 µg/mL (*p* < 0.0001). The apolar fraction reduced it by -16% at 50 µg/mL (*p* < 0.05) to -32% at 100 µg/mL (*p* < 0.01). For Saint-Nectaire, only its W70 fraction significantly reduced MMP-13 by -20% at 50 µg/mL (*p* < 0.05) to -41% at 100 µg/mL (*p* < 0.001).

In conclusion, polar and/or apolar fractions of Saint-Nectaire and Goat cheeses reduced the production of biomarkers involved in inflammation and matrix degradation in human chondrocytes. Only the Saint-Nectaire cheese fractions reduced the three studied biomarkers.

## 4. Discussion

During aging, a range of ailments can influence the musculoskeletal system, impacting muscles and/or joints (Frontera, 2017; Larsson et al., 2019; Sacitharan, 2019). These conditions often compromise health

span, leading to mobility loss. Many processes, such as oxidative stress, could be implicated in deteriorating muscles and/or joints. We previously demonstrated the antioxidant potential of raw-milk cheeses on oxidative processes (Diet et al., 2024), and the current study aimed to pursue the investigations by exploring the impact of seven raw-milk cheeses - Goat cheese, Saint-Nectaire, Cantal, Bleu d'Auvergne, Roquefort, Comté and Epoisses - on various health aspects of the aging.

We used the previously developed methodology (Cardin et al., 2020) to study cheese on aging-associated biological functions by using a whole fraction, the freeze-dried cheese (FDC). With the model organism C. elegans, we found that six of the seven raw-milk cheeses tested showed a beneficial effect on the C. elegans longevity by enhancing not only the maximum longevity but also the median survival of the population. A key pathway influencing longevity is the insulin/insulin-like growth factor-1 signaling (IIS) pathway (Finch & Ruvkun, 2001; Henderson & Johnson, 2001; Kenyon et al., 1993; Murphy & Hu, 2013). This pathway includes DAF-2, a protein closely related to vertebrate insulin and insulin-like growth factor-1 receptors, and DAF-16, a transcription factor belonging to the FOXO family. In a previous study, we observed that raw-milk cheeses increased the expression of the gene *daf-16*, coding for the transcription factor DAF-16, in response to oxidative stress (Diet et al., 2024). The mechanism by which raw-milk cheeses contributes to longevity may involve the activation of the IIS pathway and the upregulation of *daf-16* during aging. As a perspective, to clarify the mechanisms underlying the role of raw-milk cheeses in promoting longevity in C. elegans, it would be interesting to investigate the effects using mutant strains, such as *daf-2* (−/−) or *daf-16* (−/−).

In addition to enhancing the life expectancy of C. elegans, all the raw-milk cheeses had a beneficial effect on the mobility over time. Locomotory impairment during C. elegans aging could result from a decline in muscle function (Herndon et al., 2002), thus we investigated if the cheeses could be implicated in muscle integrity maintenance with age. Aging in C. elegans muscle is characterized by altered structure and reduced function (Gaffney, Pollard, Barratt, Constantin-Teodosiu, Greenhaff & Szewczyk, 2018). One of the hallmarks of aging is mitochondrial dysfunction (Alway et al., 2017; Cai et al., 2023; Leduc-Gaudet et al., 2021) which precedes the loss of muscle fiber structure in aging (Gaffney et al. 2018) and plays a key role in age-related decline in muscle integrity and function (Wang et al., 2019). With GFP-labeled nematodes, it is possible to visualize mitochondrial structure and network in the muscle tissue via fluorescence microscopy (Giron et al., 2022; Hewitt et al., 2018) reflecting the muscle integrity over time. Using one of them, we found that all the cheeses maintained the mitochondrial network over time revealing their ability to preserve muscle integrity and structure compared to the CTRL.

All the cheeses showed beneficial effects on at least one tested aspect of aging in C. elegans (longevity, mobility, muscle integrity), with varying efficacy. Comté did not significantly extend lifespan but was among the most effective at improving mobility, indicating some protection against aging. Cantal enhanced longevity but showed moderate improvement on mobility and muscle structure preservation. Saint-Nectaire stood out as one of the top cheeses for extending lifespan and maintaining mobility, likely due to its great ability to preserve the mitochondrial network and muscle structure. Goat cheese, previously studied (Cardin et al., 2020, 2021), was among the top three for both lifespan extension and average activity over 15 days, due to its great

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

Summary of the three best cheeses for each of the different parameters measured across the various studies.

<table><thead><tr><th>Study</th><th>Goat cheese</th><th>Saint-Nectaire</th><th>Cantal</th><th>Bleu d'Auvergne</th><th>Roquefort</th><th>Comté</th><th>Epoisses</th></tr></thead><tbody><tr><td>Longevity</td><td>✓</td><td>✓</td><td></td><td>✓</td><td></td><td></td><td></td></tr><tr><td>Mobility</td><td>✓</td><td></td><td></td><td></td><td></td><td>✓</td><td>✓</td></tr><tr><td>Mitochondrial network architecture</td><td>✓</td><td>✓</td><td></td><td></td><td></td><td>✓</td><td></td></tr></tbody></table>

The top three cheeses for both the longevity and mobility studies were selected based on all the parameters analyzed. For the study on mitochondrial network architecture, the top three cheeses were selected by identifying those with the lowest percentages of populations exhibiting a fragmented network over time.
<figure><img src="image_8.png" /><figcaption>Saint-Nectaire</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>b. Goat cheese</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Saint-Nectaire</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>c. Goat cheese</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>Saint-Nectaire</figcaption></figure>

<figure><img src="image_13.png" /></figure>

**Fig. 5.** Effect of Goat cheese and Saint-Nectaire (W70 and ApE extracts) on NO, PGE₂, and MMP-13 production in primary human articular chondrocytes. Human articular chondrocytes (HAC) were isolated from tibial plateau and femoral condyles following knee replacement surgery. These HAC were pre-incubated for 24 h with polar (W70) and apolar (ApE) extracts of Goat cheese and Saint-Nectaire at several concentrations (0, 50, 10, and 200 µg/mL). After that, they were incubated for an additional 24 h with the extracts and stimulated with IL-1 β (0.5 ng/mL). NO (a), PGE₂ (b), and MMP-13 (c) release were measured in the culture media and compared with the IL-1β-stimulated control (CTRL). Results are presented as mean ± SD; \* = *p* < 0.05; \*\* = *p* < 0.01; \*\*\* = *p* < 0.001; \*\*\*\* = *p* < 0.0001.

protection of the mitochondrial network. The other cheeses showed similar positive effects, making it difficult to distinguish between them. Overall, this study highlights the positive impact of raw-milk cheeses on aging in *C. elegans* and opens new perspectives for exploring both cheese as a whole and their mechanisms of action.

Cheese is an interesting source of protein and calcium (Rozenberg et al., 2016) that can contribute to musculoskeletal health. Previous studies demonstrated the beneficial effect of cheese consumption on muscle protection, contributing to its strength maintenance, recovery after physical activity, and therefore to muscle health (de Hart et al.,
2021; Hermans et al., 2022). In the elderly, consumption of cheese helped to preserve skeletal muscle mass over time (Radavelli-Bagatini et al., 2013, 2014) and mitigate aging-associated muscle strength loss in people with sarcopenia (age-related loss of muscle mass, strength, and function) (Chen et al., 2024) and non-sarcopenic elderly subjects (Alemán-Mateo et al., 2014). These studies showed that cheese consumption has a positive impact on muscle integrity over time. Our results support these studies and go further showing that the cheese's beneficial effects on preserving skeletal muscle structure and function during aging could be, at least in part, due to the protection of mitochondrial network integrity in muscle. Further studies are needed to understand the mechanisms of action involved in human muscle cells during the aging process.

In this study, we showed the beneficial effects of seven raw-milk cheeses on muscle health. Preserving muscle structure and function during aging could help in preventing and/or limiting diseases such as sarcopenia (Frontera, 2017) or osteoarthritis (Carvalho et al., 2022; Krishnasamy et al., 2018). Those improvements could promote social life, psychological well-being, and quality of life. From an economic perspective, preventing/limiting mobility disorders in the elderly could largely reduce the costs (Steinmetz et al., 2023).

We pursued our study by investigating of the effects of Saint-Nectaire and Goat cheeses on synovial joints. In OA, chondrocytes, the cells of the synovial joint, are damaged by processes including oxidative stress and inflammation. As cheeses showed beneficial effects against oxidative processes (Diet et al., 2024), they might also alleviate chondrocyte damages observed in OA. We examined the effects of two cheeses (Goat cheese and Saint-Nectaire) *in vitro* on IL-1β-stimulated primary human articular chondrocytes using for each cheese polar (W70) and apolar (ApF) fractions.

In the cartilage, pro-inflammatory cytokines like IL-1β activate chondrocytes to express iNOS and COX-2, leading to the production of NO and PGE₂, which promote chondrocyte apoptosis, inhibit extracellular matrix synthesis, and cause joint swelling and pain (Goldring, 2000; Rahmati et al., 2017). NO is a free radical and a tissue-damaging pro-inflammatory mediator when expressed in excessive amounts (Ahmad et al., 2021). Among the fractions from Goat cheese and Saint-Nectaire, only the W70 fraction from Saint-Nectaire significantly reduced NO production in chondrocytes starting at 50 µg/mL. These results suggested that Saint-Nectaire had bioactive polar metabolites able to reduce NO levels. PGE₂ is a key mediator of inflammation and pain (J. Y. Park et al., 2006). We showed here that all the 4 fractions were able to significantly reduce the PGE₂ production in IL-1β-stimulated chondrocytes. Thus, Goat cheese and Saint-Nectaire appeared to contain both polar and apolar molecules with bioactivity to reduce PGE₂ production in chondrocytes. Apart from anti-inflammatory activities, the present study also investigated the chondroprotective activity of Saint-Nectaire and Goat cheeses through the inhibition of MMP-13 production. MMP-13 is a distinctive metalloproteinase expressed in chondrocytes. In inflammatory conditions, MMP-13 is upregulated and therefore plays an important role in the early stages of OA. MMP-13 is responsible for the cleaving of the type-II collagen, a major component of the extracellular matrix in human articular cartilage (Rahmati et al., 2017). Interestingly, the two fractions of Goat cheese were efficient in reducing MMP-13 production in IL-1β-stimulated chondrocytes suggesting a bioactivity of both polar and apolar molecules of Goat cheese. For Saint-Nectaire, only the polar fraction reduced the production of MMP-13 starting from 50 µg/mL. Together, these results showed that Goat cheese and Saint-Nectaire have interesting metabolites, both polar and apolar, to alleviate the inflammation on IL-1β-stimulated chondrocytes through the reduction of NO, PGE₂, and MMP-13.

Recent studies have explored the link between human diet and health, particularly its role in OA development. A study (Lu et al., 2014) found that high cheese consumption (over 7 servings per week) increased knee osteoarthritis pathophysiology by reducing joint space width, whereas milk had a positive impact against OA. However, little

information was provided about the type(s) of cheese considered, and the serving size was not specified. Contradictory findings emerged from more recent studies. In a previous study (Xie & Qin, 2023), the authors found that cheese consumption reduced OA risk, particularly in the knee and spine. Another study (Lv et al., 2024) also showed a negative correlation between cheese consumption and knee OA risk. A study (Denissen et al., 2019) noted that full-fat dairy and Dutch cheeses (e.g., Gouda) were inversely related to knee OA. Another study (Zhu et al., 2024) found that certain dietary habits, such as cheese consumption, were linked to fewer osteophytes in knee OA patients. These studies suggest that cheese may help prevent OA. Our research supported these findings, showing that raw-milk cheeses positively affected aging processes and muscle function *in vivo*, and reduced catabolic factors in an IL-1β-stimulated chondrocyte model *in vitro*. The benefits observed in muscle and cartilage suggest cheese may offer a promising intervention for OA. Further research is needed to explore the mechanisms behind these effects, potentially using arthritic models as a perspective to see their impact on both muscle and cartilage *in vivo* such as in rats (Silva et al., 2018) or larger animals (McCoy, 2015).

Although the risks of foodborne pathogens being present is not zero rin raw-milk cheeses (Brooks et al., 2012), our study shows that raw-milk cheeses contain bioactive metabolites that may have beneficial effects on health. It may be of interest for the dairy industry to preserve these beneficial effects across different cheese varieties.

Cheese has high saturated fat content. However, it is often recommended to limit the consumption of saturated fat due to its proposed impact on serum total cholesterol levels, which can be linked to the development of cardiovascular disease (CVD) (Mensink et al., 2003; Siritarino et al., 2010). In addition, saturated fatty acid consumption may be associated with other pathologies, such as the pathogenesis of osteoarthritis (Felson et al., 2003; Y. Wang et al., 2009) or the increased mortality among people with diabetes (Trichopoulou et al., 2006). Therefore, the recommendations generally advise limiting cheese consumption due to its fat content (WHO, 2018). However, an increasing number of studies indicate that saturated fat consumption is not always associated with an increase in risk factors for developing pathologies (Chowdhury et al., 2014; Dehghan et al., 2017) and is highly dependent on the type of food consumed (De Oliveira Otto et al., 2012). In accordance, our findings on *C. elegans* suggested that cheese consumption may have beneficial effects on the aging process and the maintenance of mobility over time through the preservation of muscle integrity. Furthermore, we have demonstrated bioactivity from the FDC (corresponding to the whole cheese) as well as specific bioactivity from apolar extract (ApE), containing the lipid component from the cheese. The ApE extracts (of Goat cheese and Saint-Nectaire) exhibited anti-inflammatory activity *in vitro*, using IL-1β-stimulated chondrocytes. Additionally, we previously showed that these same apolar extracts from all the tested cheeses possessed antioxidant potential by protecting *C. elegans* with the ability to both limiting ROS accumulation *in vivo* and reducing ROS production *in vitro* in human leukocytes (Diet et al., 2024). Collectively, these results suggest that the apolar fraction (containing the lipid component of the cheese) has biological activity and may be as important as the remaining matrix. In addition, previous studies (Monfoulet et al., 2015; Philippe et al., 2017; Wauquier et al., 2013) showed that a fatty acid-enriched diet had a positive impact on osteoarticular affections by limiting bone loss through the stimulation of the free fatty acid receptor, G-coupled protein receptor 40 (GPR40). Together, these findings suggest that some fatty acids may also play a role in the nutritional prevention of osteoarticular diseases.

For this study, we selected seven cheeses produced from the milk of different animal species (cow, ewe, and goat). We showed that the apolar cheese fraction presented a bioactivity as well as the polar one. It is known that the lipidic fraction can change in quantity and quality among cheeses made with milks from different animal species (Paszczyk & Łuczyńska, 2020) and among cheese-making processes (fermentation, ripening). In our study, this quantity of apolar fraction by freeze-drying
was standardized for each test. The observed differences in bioactivity among cheese fractions could arise from metabolite diversity.

Earlier studies have linked cheese consumption to the French paradox through its potential to reduce systemic inflammation (Petyaev & Bashmakov, 2012) and metabolic syndrome (Biong et al., 2004; Kawakami, 2023) contributing to better cardiovascular health, despite its high saturated fat content. Aging (Paneni et al., 2017) and sedentary behavior (Yang et al., 2023) are risk factors for developing cardiovascular diseases. In addition to showing that the apolar fractions of cheeses (containing the lipid component of cheeses) had antioxidant (Diet et al., 2024) and anti-inflammatory properties, we demonstrated that cheeses could have a protective effect on aging and could improve the individual mobility, by preserving movement, thus, limiting sedentary behavior. These results reinforced the hypothesis that cheese might be a significant contributor to the French paradox.

## 5. Conclusion

To the best of our knowledge, this study is the first to investigate the biological effects of supplementation with various raw-milk cheeses on biological models, in order to assess their impact on aging and age-related impairments. We showed that supplementation with these cheeses extended lifespan and improved mobility in C. elegans, while preserving muscle integrity and function over time. Their polar and apolar fractions (including lipids) protected cartilage from degradation in primary human articular chondrocytes. This study allowed us to observe that cheeses may contain bioactive metabolites of interest, which may offer health and aging-related benefits, suggesting that consuming raw-milk cheeses as part of a balanced diet could help maintain mobility with age by protecting both muscles and cartilage. This challenges current recommendations regarding the health implications of raw-milk cheeses, raises questions about their role in the French paradox, and opens up new perspectives for exploring their effects on other health processes.

### CRediT authorship contribution statement

**Anna Diet:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Christophe Poix:** Writing – review & editing, Validation, Software, Formal analysis, Data curation.
**Muriel Bonnet:** Writing – review & editing, Methodology, Investigation.
**Christian Coelho:** Writing – review & editing, Data curation.
**Isabelle Ripoche:** Writing – review & editing.
**Yohann Wittrant:** Writing – review & editing, Methodology.
**Caroline Decombat:** Writing – review & editing, Methodology.
**Julien Priam:** Writing – review & editing, Supervision, Funding acquisition.
**Etienne Saunier:** Writing – review & editing, Supervision, Resources, Funding acquisition.
**Pierre Chalard:** Writing – review & editing, Validation, Supervision, Resources, Methodology, Funding acquisition.
**Florence Caldefie-Chézet:** Writing – review & editing, Validation, Supervision, Resources, Methodology, Funding acquisition, Conceptualization.
**Stéphanie Bornes:** Writing – review & editing, Validation, Supervision, Resources, Methodology.
**Laurent Rios:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.

### Ethics statement

This work does not involve humans or vertebrate animals. Therefore, it is not necessary to provide an ethical statement.

### Funding

This research was financed by La Région Auvergne-Rhône-Alpes (Pack Ambition Recherche 2020 - FRIMOB project; P0890002).

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Authors J.P. and E.S. were employed by the company Dômes Pharma. The company was a partner in the research project funded by La Région Auvergne-Rhône-Alpes, in which this study was conducted. J.P. and E.P. participated in the funding acquisition and in the supervision of this study. The remaining authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

### Acknowledgements

We thank Fabien WAUQUIER for his assistance and Angélique BEVIA for her technical support.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jff.2025.106899.

### Data availability

Data will be made available on request.

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