# Comparison of the biological activity of different probiotic strains under aerobic and anaerobic conditions

Justine Guilbaud, Khaled Fadhlaoui, Sandrine Chalancon, Eric Beyssac, Emmanuelle Laine, Ghislain Garrait

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Justine Guilbaud, Khaled Fadhlaoui, Sandrine Chalancon, Eric Beyssac, Emmanuelle Laine, et al. Comparison of the biological activity of different probiotic strains under aerobic and anaerobic conditions. BMC Microbiology, 2025, 10.1186/s12866-025-04524-0. hal-05434411

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# Comparison of the biological activity of different probiotic strains under aerobic and anaerobic conditions

Received: 8 September 2025

Accepted: 30 October 2025

Published online: 18 December 2025

Cite this article as: Guilbaud J., Fadhlaoui K., Chalancon S. *et al.*
Comparison of the biological activity of different probiotic strains under aerobic and anaerobic conditions. *BMC Microbiol* (2025). https://doi.org/10.1186/s12866-025-04524-0

Justine Guilbaud, Khaled Fadhlaoui, Sandrine Chalancon, Eric Beyssac, Emmanuelle Laine & Ghislain Garrait

We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

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Comparison of the biological activity of different probiotic strains under
aerobic and anaerobic conditions

Justine Guilbaud¹, Khaled Fadhlaoui¹ ², Sandrine Chalancon ¹, Eric Beyssac¹ ³,
Emmanuelle Laine*¹ and Ghislain Garrait*¹³

¹Université Clermont Auvergne, UMR 454 MEDIS, Microbiologie Environnement Digestif et Santé, INRAE, Clermont-Ferrand, France

²Université Clermont Auvergne, CNRS, UMR 6023 CNRS-UCA, Laboratoire Microorganismes: Génome et Environnement LMGE, Clermont-Ferrand, France

³UMR 454 MEDIS, Microbiologie Environnement Digestif et Santé, Université Clermont Auvergne - INRAE, Galbiopharm Platform, Clermont-Ferrand, France

Abstract

Lactic acid bacteria (LAB) are widely recognized as probiotics due to their health-
promoting properties. Some strains can perform both aerobic metabolism and
fermentation, which may influence their biological functions. This study investigated the
impact of Lactobacilli metabolism on three specific biological functions: anti-
inflammatory, antioxidant, and antimicrobial effects, across eleven LAB strains. To this
end, growth profiles under aerobic and anaerobic condition were monitored by turbidity.
Antimicrobial potential of the probiotic strains was assessed against *Staphylococcus aureus*
and *Listeria monocytogenes* using extracellular or intracellular fractions of each probiotic
previously cultured aerobically or anaerobically. Antioxidant capacity was measured
indirectly through a colorimetric assay of iron II. Then anti-inflammatory potential was

<footnote>*: These authors contributed equally to this work</footnote>
evaluated in HT29 cells by cytokine quantification. For the first time, this article highlights that all strains studied enhanced their biological activity through fermentative metabolism. As expected, most strains inhibited *L. monocytogenes*. Among them, *Lactobacillus delbruekii* subsp. *lactis* showed the highest biological activity, followed by *Lacticaseibacillus rhamnosus* and *Limosilactobacillus mucosae*. These strains also reduced inflammation by 50 % and exhibited a high antioxidant capacity (585 ± 68 µmol/L). These results demonstrate interesting characteristics of probiotic strains, particularly under fermentative conditions. Their integration into probiotic formulation may provide valuable nutritional strategies to improve gut health and overall well-being.

**Keywords** LAB strain, aerobic/anaerobic cultivation, anti-inflammatory activity, oxidative stress, and antimicrobial capacity

**Backgrounds**

1. According to the World Health Organization (WHO), probiotics are defined as live microorganisms that, when administered in sufficient quantity, confer health benefits to the host (1). These microorganisms contribute to modulating the gut microbiota, thereby potentially improving host health through microbiota balance. However, one of the main challenges associated with the oral administration of probiotics lies in their ability to withstand the harsh physicochemical conditions of the gastrointestinal tract (low pH, digestive enzymes, bile salts) (2) prior to reach the colon. Moreover, depending on the probiotic strain used, the administered dose and the targeted pathology, these live microorganisms can have variable efficacy (3).

10. Probiotics are mainly available on the market as dietary supplements, and only a few are considered as Live Biotherapeutic Products or LBPs (e.g., ultralevure®, carbolevure®,
12 Lénia®, Lactéol®) and classified as medications. The distinction lies in the demonstrated
13 biological activity to treat or prevent a specific disease (4–7).

14 The most used probiotics include yeasts and bacteria, especially those belonging to the
15 genera *Lactobacilli*, *Bifidobacterium*, *Enterococcus* and *Saccharomyces* (8). Lactic acid bacteria
16 (LAB) are well documented for their diverse biological effect. Notably, LAB are known to
17 secrete bacteriocins, small molecules able to inhibit the growth of pathogens such as
18 *Listeria monocytogenes*, and *Staphylococcus aureus* (8,9). Pathogen colonisation at the
19 intestinal barrier can lead to an increase in reactive oxygen species, contributing to
20 oxidative stress and the onset of various pathologies. Additionally, LAB are also able to
21 stimulate immune response and produce antioxidant enzymes like catalase, superoxide
22 dismutase (SOD) or glutathione (GSH) (9).

23 Another key feature of LAB is their metabolic flexibility, allowing them to switch
24 between fermentation and aerobic metabolism. This metabolic adaptability is likely to
25 influence their biological properties and survival in different environments. However,
26 despite the recognized importance of metabolism in bacterial physiology, few studies
27 have directly examined how the metabolic state of LAB affects their functional
28 properties. In particular, the relationship between Lactobacilli metabolism and their
29 biological activities, such as anti-inflammatory effects, antioxidant potential, and
30 antimicrobial capacity, remains poorly understood.

31 Accordingly, the present study aims to clarify this relationship by investigating the impact
32 of Lactobacilli metabolism on three key biological functions: inflammation, oxidative
33 stress reduction, and antimicrobial activity, across eleven LAB strains. This work seeks to
34 provide a clearer understanding of how metabolic differences may modulate the
35 biological responses of *Lactobacilli*.
36 **Material and methods**

37 **Bacterial**

**strain**

All probiotics strains used in this study, including *Lactobacillus delbrueckii* subsp. *lactis* (L 110, DSMZ 20072), *Lactobacillus delbrueckii* subsp. *bulgaricus* (Lb 14, DSMZ 20081), *Lacticaseibacillus rhamnosus* (DSMZ 20021), *Limosilactobacillus fermentum* (Sib 28, DSMZ 20052), *Latilactobacillus sakei* subsp. *sakei* (TS, DSMZ 20017), *Lactiplantibacillus plantarum* subsp. *plantarum* (Lp 39, DSMZ 20174), *Lactiplantibacillus paraplantarum* (CST 10961, DSMZ 10667), *Pediococcus acidilactici* (B 213c, DSMZ 20284), *Ligilactobacillus salivarius* (H 066, DSMZ 20555), *Limosilactobacillus reuteri* (F 275, DSMZ 20016) and *Limosilactobacillus mucosae* (S 32, DSMZ 13345) were obtained from the DSMZ culture collection (Darmstadt, Germany). Theses strains were cultured in de Man, Rogosa, and Sharpe medium (MRS; Thermo Scientific, Waltham, Massachusetts, United States) at 37°C overnight under aerobic and anaerobic conditions. For anaerobic cultures, the same parameters were used with a supplementation of 0.05% (w/v) of L-cystein (Sigma-Aldrich, USA). Pathogenic strains of *S. aureus* (533 R4, DSMZ 20231, Darmstadt, Germany) and *L. monocytogenes* (53 XXIII, DSMZ 20600, Darmstadt, Germany) were cultured in Brain, Heart, Infusion medium (BHI; Thermo Scientific, Waltham, Massachusetts, United States), supplemented with 0.05% (w/v) L-cysteine (Sigma-Aldrich, USA), at 37°C overnight under anaerobic conditions.

**Growth Characteristics of Bacterial Strains**

To characterise the growth curve of each probiotic strain under both aerobic and anaerobic conditions, two types of base medium were prepared. The first medium, composing only of MRS, was placed in an aerobic vial. The second medium, which included MRS supplemented with 0.05% (w/v) L-cysteine (Sigma-Aldrich, USA), was
placed in an anaerobic vial. To do this, we first remove oxygen from the MRS environment, then maintain anaerobiosis via nitrogen flow. Each medium was inoculated with 10% (v/v) of respective probiotics culture under anaerobic or aerobic medium and incubated at 37°C. A single vial was prepared for each condition, and all experiments were performed in triplicate. Optical density was measured at 600 nm every hour, or every 30 minutes during the exponential growth phase to capture growth dynamics. Hourly optical density measurements did not require sample withdrawal, as absorbance was measured directly in the Pinocchio flask using a compatible spectrophotometer. Sampling was only performed at the final time point when the absorbance exceeded 2.00, the detection limit of the spectrophotometer; in this case, 500 µL of culture was withdrawn and diluted to a final volume of 2 mL in sterile MRS. Each vial had a total volume of 100 mL, of which 25 mL was filled with MRS. The maximum specific growth rate (µmax) was measured according to the following formula:

$$ \mu_{max} = \frac{(\ln abs2 - \ln abs1)}{(t2 - t1)} $$

Where µmax is expressed in hour⁻¹, t is expressed in hours and absorbance (abs) is unitless.

Subsequently, the generation time (G) was determined using the formula:

$$ G = \frac{\ln2}{\mu_{max}} $$

Where G is expressed in hours and µmax in hour⁻¹.

## Human cell culture

The human colon adenocarcinoma cell line HT29 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and was cultured in
Minimum Essential Medium (MEM; Gibco, Fisher: W3528A/ Invitrogen: 41) containing 4.5g/L glucose, supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. For all experiments, HT29 cells were incubated in a humidified incubator at 37°C with 5% CO₂.

*Anti-inflammatory activity of probiotic strains*

The experimental procedure was adapted from the method established by Lenoir (2020) (10). Briefly, HT29 cells were seeded into 12-well plates (5 x 10⁴ cells per well) in MEM supplemented with 10% (v/v) heat-inactivated FBS at 37 °C in a humidified incubator with 5% CO₂. The culture medium was changed every day. Experiments were initiated on day 7 post-seeding, once cells had reached 100% of confluence (approx. 1.83 x 10⁶ cells/well). On day 6, 24 h prior to culture with probiotics supernatant, the medium was replaced with MEM containing 5% heat-inactivated FBS and 1% glutamine. On the day of culture, the probiotic supernatant was added at a final concentration of 10% (v/v) in a total volume of 1 mL per well. Cells were simultaneously stimulated with Lipopolysaccharide (LPS) from *E. coli* O11: B4 (10 ng/ml; Sigma-Aldrich, USA) for 6 hours at 37°C under 5% CO₂. All experiments were performed in triplicate. Following the 6h of incubation, cell supernatants were collected and stored at -80 °C until analysis of IL-8 concentrations using ELISA kits (DY208-05, DuoSet, Elisa) according to the manufacturer's instructions.

*Cell viability assay*

Cell viability was assessed by flow cytometry using an Attune Nxt cytometer (Thermo Scientific, Waltham, Massachusetts, United States). After 6 hours of co-incubation, HT29 cells were detached with trypsin and then resuspended in Phosphate-
Buffered Saline (PBS; pH 7.2; Gibco), and 0.01% (v/v) propidium iodide (Thermo Scientific, Waltham, Massachusetts, United States) was added. Following a 5-minutes incubation in the dark at room temperature, the proportions of viable and non-viable cells were determined based on fluorescence intensity.

## Reducing power activity

The reducing power activity of LAB strains was determined according to the method described by Oyaizu (2003) (11). One millilitre of probiotics culture was centrifuged at 8000 x g for 5 min, and the resulted pellet was resuspended in 1 mL of PBS (pH 7.2; Gibco). The probiotic pellet was mechanically lysed using a bead beater (20 Hz, 2 cycles of 30 sec). Two hundred microliters of the lysate were mixed with 0.2 mL of 0.2 M sodium phosphate buffer (pH 6.6), and 0.2 mL of 1% potassium ferricyanide (Thermo Scientific). The reaction mixture was incubated at 50 °C for 20 min. After incubation, 0.2 mL of 10% trichloroacetic acid was added, and the mixture was centrifuged at 8000 × g for 10 min at 4 °C. Subsequently, 500 µL of the supernatant was mixed with 0.1 mL of 0.1% ferric chloride (Thermo Scientific) and 0.4 mL of distilled water. The final mixture was incubated for 10 min at room temperature. Absorbance was then measured at 700 nm using a spectrophotometer (BioTek Instruments, USA), and L-cysteine (Sigma-Aldrich) at 1000 µmol/L was used as the standard antioxidant for calibration.

## Antimicrobial activity

The pH of the probiotics cultures was adjusted to 7.0 using 5 M sodium hydroxide (NaOH; w/v). Cultures were then centrifuged at 8000 x g for 5 min. The supernatant was collected and stored, while the probiotic pellet was resuspended in PBS (pH 7.2; Gibco)
and wash twice. The probiotic pellet was mechanically lysed using a bead beater (20 Hz, 2 cycles of 30 sec) to obtain intracellular contents.

Pathogens strains were cultured in BHI medium supplemented with 0.1% of L-cysteine (Sigma-Aldrich). For the antimicrobial assays, pathogen cultures were supplemented with 0.1% (v/v) of either the probiotic supernatant or the probiotic lysate, previously sterilized by passage through a 0.22 µm filter. The control conditions were achieved by adding 0.1% MRS medium to the pathogen to mimic the extracellular matrix of probiotics and 0.1% of PBS medium to mimic the intracellular matrix of the probiotics. The cultures (probiotics/pathogens) were incubated overnight at 37°C. Bacterial growth inhibition was evaluated by measuring abs at 600 nm at both the start and the end of the incubation period.

## Statistics

All data were analysed using RStudio® software. Normality and homogeneity of variances was performed using the Shapiro-Wilk test. For normal samples with equal variances, a two-way ANOVA was performed. Multiple comparisons were performed using Tukey's post hoc test. Results are presented as mean ± Standard Error Mean (SEM), and differences were considered statistically significant at p < 0.05.

## Results

### Characterisation of probiotics strains

To initiate the comparative study of probiotics strains, we first evaluated their growth characteristics under both aerobic and anaerobic metabolic conditions. For each strain, the µmax was determined to assess the maximal biomass production. All cultures
were adjusted to the same initial concentration, regardless of the metabolic pathway studied. The generation time was also determined to better understand their growth rate. In general, the maximum specific growth rate was higher for the strains under aerobic conditions compared to anaerobic conditions (Table 1). This trend was particularly significant for *L. salivarius* and *L. fermentum*, with µmax values of 0.30 ± 0.02 and 0.41 ± 0.10 h⁻¹, respectively, for aerobic conditions compared to 0.20 ± 0.01 and 0.19 ± 0.01 h⁻¹ respectively for anaerobic conditions.

The G value varied notably between metabolic conditions. *L. bulgaricus* and *L. rhamnosus* exhibited significantly longer generation times under anaerobic conditions (16.93 ± 2.15 h and 5.23 ± 0.37 h, respectively) compared to aerobic conditions (8.35 ± 1.18 h and 3.41 ± 0.15 h, respectively), reflecting slower cell division in the absence of oxygen.

Regarding bacterial concentrations, most strains reached an average final density close to or above 9 log CFU/mL. Only *L. bulgaricus*, *L. lactis* and *L. mucosae* remained below this threshold in both conditions. Interestingly, *L. plantarum* achieved is significantly higher biomass under anaerobic conditions (9,85 ± 0,03 log CFU/mL) than under aerobic conditions (9,37 ± 0,03 log CFU/mL) suggesting enhanced fermentative efficiency. Conversely, *P. acidilactici* shows a significantly higher concentration under aerobic conditions compared to anaerobic conditions, with 9,82 ± 0,09 log CFU/mL versus 9,61 ± 0,05 log CFU/mL, reflecting a potentially more flexible or aerobic metabolism.

### Anti-inflammatory activity

The anti-inflammatory effect of the probiotics strains was evaluated using an *in vitro* co-stimulation model with LPS. The results presented in **Figure 1** are expressed as percentages relative to the inflammatory response of a control group treated with LPS alone (set at 100%). Values below 100 % indicate a reduction in IL-8 secretion and
therefore reflect anti-inflammatory activity. Most probiotics cultured under anaerobic conditions exhibited IL-8 levels lower than the control, suggesting a protective, anti-inflammatory effect. In contrast, several strains cultured under aerobic conditions elicited a pro-inflammatory response, with IL-8 levels exceeding 100 % of the control. However, the difference between the two conditions reached statistical significance only for *L. lactis*, which showed a notably larger anti-inflammatory effect in anaerobiosis.

Among the tested strains, *L. mucosae*, *L. bulgaricus* and *L. lactis* demonstrated the most potent anti-inflammatory activity under anaerobic conditions, reducing IL-8 secretion by 22 ± 11 %, 36 ± 15 % and 49 ± 13 %, respectively. Notably, *L. lactis* under anaerobic conditions induces a significantly lower inflammatory effect compared to *L. rhamnosus*, *L. paraplantarum* and *L. sakei* under aerobic conditions, which exhibited an increase in inflammation by 41 ± 37 %, 60 ± 34 % and 41 ± 49 %, respectively.

## Antioxidant activity

In our study, the antioxidant effect of each probiotic strain was evaluated by measuring their reductive activity, specifically their ability to reduce from $Fe^{3+}$/ ferricyanide complex to ferrous form. It is well known that sample reductive ability and oxidation resistance present linear correlation (12–14). The obtained results are presented in **Figure 2** and can be divided into three groups:

- The first group, with strong antioxidant potential (400–600 µmol/L), includes *L. rhamnosus*, *L. lactis* and *L. mucosae* under anaerobic conditions. These strains showed the highest reductive power, with values of 585 ± 68 µmol/L; 512 ± 19 µmol/L and 472 ± 55 µmol/L, respectively.

- The second group consists of strains with moderate antioxidant activity (200-400 µmol/L), included *L. plantarum*, *P. acidilactici* and *L. paraplantarum* under anaerobic conditions.
conditions; *L. mucosae* and *L. fermentum* under aerobic conditions and *L. bulgaricus* under both conditions. The highest value in this group was observed for *L. bulgaricus* under anaerobic conditions (389 ± 32 µmol/L), while the lowest came from *L. fermentum* under aerobic conditions (215 ± 32 µmol/L).

- The third group, comprising the least active strains (0-200 µmol/L), includes seven strains. Among these, *L. paraplantarum* displaying the lowest reducing power at 33 ± 6 µmol/L.

Interestingly, the highest antioxidant activities were consistently observed in *L. rhamnosus*, *L. lactis* and *L. mucosae*, all under anaerobic conditions. Indicated that strains studied have better antioxidant capacity through fermentation metabolism.

### Antimicrobial activity

To assess the antimicrobial activity of the probiotic strains tested in this study, two clinically relevant pathogens were selected: *Listeria monocytogenes* (Figure 3A and B) and *Staphylococcus aureus* (Figure 3C and D). To better understand the origin and functioning of the antimicrobial mechanism of probiotics, we separately analyzed the molecules synthesized but not secreted into the supernatant (intracellular compartment) as well as the molecules synthesized then secreted into the supernatant (extracellular part).

### *Listeria monocytogenes*

This pathogen appeared to be particularly sensitive to the probiotics tested. Both types of metabolism were compared for each compartment and each strain.

Although no significant differences were observed between control groups, a trend indicated that *L. monocytogenes* growth was lower in the intracellular fraction of probiotics cultured under aerobic conditions compared to anaerobic conditions. All intracellular
matrices of the strains, regardless of the metabolism observed, didn't significantly affect the growth of *L. monocytogenes*, except for the *L. bulgaricus* strains under anaerobic metabolism, where the growth of *L. monocytogenes* was significantly increased compared to the control. In addition, the inhibition of *Listeria monocytogenes* growth under anaerobic condition and quantified by absorbance (OD600) values, were : 2.31 ± 0.27 (*L. bulgaricus*), 1.28 ± 0.8 (*L. fermentum*), 0.98 ± 0.01 (*L. reuteri*), 0.96 ± 0.04 (*L. sakei*) and 1.12 ± 0.04 (*L. salivarius*), compared to better inhibition levels under aerobic conditions (1.01 ± 0.03; 0.87 ± 0.02; 0.75 ± 0.07; 0.71 ± 0.07 and 0.79 ± 0.02 respectively) **(Figure 3A)**. However, this observation is consistent with the trend observed in both control groups.

Regarding the extracellular fraction, nearly all strains demonstrated a significantly greater inhibitory effect under anaerobic conditions compared to both aerobic conditions and control. The only exceptions were *L. reuteri* and *L. salivarius*, which showed stronger inhibition under the aerobic condition (0.86 ± 0.04 and 0.90 ± 0.03, respectively) compared to anaerobic conditions (1.01 ± 0.03 and 1.08 ± 0.02, respectively) **(Figure 3B)**. No significant differences were observed for *L. rhamnosus* and *L. paraplantarum* between the two culture conditions.

*Staphylococcus aureus*

A similar inhibitory pattern was observed for *Staphylococcus aureus*. Under anaerobic conditions, the growth of *Staphylococcus aureus* is significantly lower than under aerobic conditions in the intracellular compartment for the control. For this compartment, all probiotic strains induce a significant reduction in the growth of *Staphylococcus aureus* under anaerobic conditions compared to aerobic conditions. However, only the intracellular compartment of *L. lactis* leads to a significant reduction in the growth of
*Staphylococcus aureus* compared to the control, with a value of 1.32 ± 0.06 against 1.84 ± 0.13 (Figure 3C). In contrast, *L. bulgaricus* and *L. paraplantarum* under anaerobic conditions, as well as *L. rhamnosus* under aerobic conditions, have a significantly lower impact on the reduction of *Staphylococcus aureus* growth compared to their respective controls.

For *Staphylococcus aureus* exposed to the extracellular compartment of probiotic strains, there are no differences compared to the control. Under aerobic conditions, probiotic strains don't significantly affect the growth of *Staphylococcus aureus*, which is not the case under anaerobic conditions. Indeed, under anaerobic conditions, *L. lactis*, *L. plantarum*, *L. rhamnosus*, *P. acidilactici*, *L. fermentum* and *L. salivarius* induce a significant reduction in the growth of *S. aureus* compared to the control value, with respective value of 1.59 ± 0.21; 1.94 ± 0.05; 2.38 ± 0.71; 1.91 ± 0.04; 1.94 ± 0.04 and 1.93 ± 0.04 against 3.02 ± 0.11 (Figure 3D). There is also a significantly greater reduction in the growth of *S. aureus* with *L. lactis*, *L. plantarum*, *P. acidilactici*, *L. fermentum* and *L. salivarius* under anaerobic conditions compared to aerobic conditions.

Additionally, each of these strains exhibited a significantly greater inhibitory effect under anaerobic versus aerobic conditions. These results demonstrate that anaerobic conditions can enhance the antimicrobial properties of LAB, particularly in the extracellular fraction, which may contain secreted bacteriocins or other antimicrobial metabolites that are differentially expressed depending on the metabolic state.

## Discussion

LAB strains are among the main bacteria used as probiotics and are well studied in the literature. They are particularly recognized for their numerous beneficial properties for
human health, including their ability to act on three main causes involved in many
diseases such as cardiovascular, neurodegenerative, metabolic or gastrointestinal diseases
(7). These bacteria are known to be facultative aerobes, but it is rare to find studies
comparing their probiotic power under both culture conditions (aerobic and
fermentation). Therefore, in order to better identify the most promising strains, eleven
LAB strains were selected and their antimicrobial, antioxidant and anti-inflammatory
activity under aerobic and under anaerobic conditions were assessed.

The growth characterization of bacterial strain was first assessed by focus on the
maximum specific growth rate, the generation time and the bacterial concentration. We
observed that most of the strains tested have a higher µmax rate under aerobic condition
compared to anaerobic conditions. Moreover, a slower cell division rate is observed under
anaerobiosis. This suggests a faster adaptation of these strains to oxygen-rich
environments so faster growth under aerobiosis compared to anaerobiosis. This can be
explained by the fact that the aerobic metabolism is more energetic than fermentation
(15,16). However, for the strains *L. plantarum* and *L. mucosae*, the results observed were
the opposite, with higher growth rate under anaerobic condition than under aerobiosis.
Indeed, Guidone *et al*. 2013 (17) demonstrate that several different strains of *L. plantarum*
had a significantly lower maximum specific growth rate under aerobiosis while other
prefer anaerobiosis state, as we find in our study. This could be due to the fact, for some
bacterial strains like *L. plantarum*, the enzymes involved in fermentation may be more
efficient than those involves in aerobic condition, which may favour growth under
anaerobic conditions (17,18). Moreover, Ianniello *et al*. 2016 (18) demonstrated that a
*Lactobacillus casei* N87 present a significant lower maximum specific growth rate under
aerobic condition compared to anaerobic conditions with respective values of 0.48 ± 0.0
h⁻¹ and 0.64 ± 0.01 h⁻¹. This study can explain the difference obtain in our results
concerning the bacterial concentrations reached at the end of the exponential state, for some of strain, the higher bacterial concentration is obtained under aerobiosis while for other it's obtained under anaerobiosis. So results better highlights strain-dependent metabolic preferences and underline the importance of growth conditions in modulating probiotic physiology, which may in turn influence their functional properties.

Then to explore the impact of different metabolic growth condition (anaerobiosis and aerobiosis) on functional capacity of our probiotic strain we first asses the anti-inflammatory capacity of our strains.

To do that, the dosage of IL-8 cytokine in an HT-29 cell model in co-stimulation with probiotic and LPS was assessed. It is interesting to note that for most of the strains, we observe an anti-inflammatory effect under anaerobic conditions, while under aerobic conditions it is rather a pro-inflammatory effect that is observed. This can be explained by the metabolism involved. In fact, under anaerobiosis, the production of lactate is greater than under aerobiosis. Moreover, this compound is known for its anti-inflammatory properties (19). Moreover, *L. rhamnosus* grown under anaerobic conditions reduced IL-8 secretion by approximately 17%, a more modest effect compared to the 56 % or the 33-34% reduction reported in previous study under anaerobic condition (20,21).

We also observe a stronger anti-inflammatory effect of *L. bulgaricus* and *L. fermentum* strain in our study compared to the other study which observe a reduction of IL-8 around 17 % and 1 % compared to the 36 % and 17 % that we found, both under anaerobic condition (20). Concerning *L. plantarum* and *L. salivarius*, we observe the opposite effect on inflammation compared to the literature. Effectively in our study we observe a proinflammatory effect of these two strains with increase inflammation of 18 % and 6 % respectively compared to a reduction of 5 % (anaerobiosis) and 25 % (aerobiosis) of inflammation in the study of Stöber *et al.* 2010 and Carbonne *et al.* 2023 (20,22). This
discrepancy may be attributed to differences in culture conditions, strain origin, or
experimental design. Effectively, in the Stöber *et al.* study 2010 (20), the inflammation of
HT29 cell is induced by EHEC strain and for the study of Carbone *et al.* 2023 (22),
inflammation was induced by TNF-α molecules. These findings support the hypothesis
that anaerobic metabolism enhances the anti-inflammatory properties of some probiotic
strains, potentially through the production of lactate which is less expressed under
aerobic growth.

To better explore the functional capacity of our strain under two different metabolisms (anaerobiosis and aerobiosis), we then assess their antioxidant ability. It is well established that certain probiotic strains can produce antioxidant compounds that help mitigate oxidative stress. Among these are catalase, superoxide dismutase (SOD) or glutathione (GSH), as described by Zotta et al. 2017 (9).

We observed a significantly higher reducing power under anaerobiosis than under
aerobiosis for all strains studied. The observed differences in antioxidant activity between
the two studied metabolic pathways could be attributed to the strain's production of
organic acids, particularly lactate, which is produced in greater amounts during
fermentation (23). Lactate can function as an antioxidant by being converted to pyruvate
via lactate dehydrogenase, releasing free electrons capable of reducing Fe³⁺ to Fe²⁺.
Additionally, lactate can lower the pH, thereby enhancing the solubility of iron (24).
Previous studies have confirmed that *L. rhamnosus* possesses genes encoding glutathione
and SOD enzymes, which may account for its enhanced reducing capability (9). In fact,
the reducing power observed for *L. rhamnosus* in our conditions (426.62 µmol/L) is
substantially higher than the 110 µmol/L previously reported by Sung-Ho *et al.* 2018 (25),
both under aerobic condition, possibly due to differences in experimental conditions or
strain-specific expression. In addition, it is well known that *L. sakei* and *L. plantarum* can
secrete some antioxidant enzyme such as SOD and NADH oxidase/peroxidase. Contrary to our findings, strains such as *L. delbrueckii* and *L. reuteri* are generally considered to have limited antioxidant defence mechanisms (9,26,27). By contrast, a study evaluates the reducing power of *L. delbrueckii lactis* at 1.108 (ABS) under aerobic condition compared to 0.509 (ABS) observe in our condition (12). These discrepancies further highlight the importance of evaluating antioxidant activity under controlled conditions and accounting for strain-specific metabolic variability.

Finally, the last probiotic activity evaluated was their antimicrobial properties under both metabolic conditions, anaerobiosis and aerobiosis.

Lactic acid bacteria (LAB) are known to produce antimicrobial peptides, commonly referred as bacteriocins. These molecules, which may be retained intracellularly or secreted into the extracellular environment, can partially or completely inhibit the growth of other bacterial species (28,29). To assess the capacity of our strains to secrete this type of molecule we chosen two well-known and greatly documented pathogens: *Listeria monocytogenes* and *Staphylococcus aureus*. The first one, known to be the reference to assess the antimicrobial activity of LAB strain because of its great sensibility to antimicrobial peptide secreted by LAB, represent a good control. The second one, was great studied with probiotic in general and represent in our case a control for more larger antimicrobial capacity because contrary to *Listeria monocytogenes*, this pathogen are more phenotypically different to LAB strain (28).

For both pathogens, the results demonstrated a stronger antimicrobial effect exerted by the extracellular compartment of probiotics. This finding suggests that the observed antimicrobial activity is indeed induced by bioactive compounds secreted by the strains. This conclusion is further supported by the fact that, in our experiments, the pH was
adjusted to exclude the possibility that the antimicrobial effect was merely due to acidification, since pH is known to influence pathogen growth (30).

Furthermore, we observed better inhibition against pathogens when probiotic strains were grown anaerobically. Regarding this difference, few explanations are available in the literature. One hypothesis could be explained by the oxidative stress present in aerobic conditions; this stress promotes the presence of ROS and can destabilized the metabolic pathway of LAB by decreasing the gene expression of bacteriocin or other antimicrobial compounds, resulting in a decrease in the production of antimicrobial molecules against pathogens. To explore this hypothesis in more detail, it might be interesting to compare the amount of these bacteriocins secreted in the two metabolisms.

Regarding the growth of *L. monocytogenes*, the observed results indicated a high response to the antimicrobial test for all strains studied confirming the result from the literature (28,29).

For *S. aureus*, in the literature it is indicated that *L. paraplantarum* under aerobic conditions exerted an antimicrobial effect against *S. aureus* and this is not observed in our study (31), this can be explained by the difference in the experimental protocol or by a concentration of each probiotic compartment insufficient to observe an inhibition of the pathogen.

## Conclusion

This study evaluated the biological activity of eleven lactic acid bacteria (LAB) strains under both aerobic and anaerobic conditions. Taken together, these findings indicate that conducting experiments under anaerobic conditions is important for a more accurate assessment of the functional potential of probiotic strains, as the colon is an anaerobic compartment and represent the primary site of bacterial colonization in the human body.
In fact, the results showed that anaerobic conditions significantly enhance the anti-inflammatory, antioxidant, and antimicrobial properties of several strains. Notably, *L. mucosae*, *L. lactis*, and *L. rhamnosus* demonstrated the strongest functional effects, particularly in reducing IL-8 secretion, increasing reducing power, and inhibiting the growth of *Listeria monocytogenes* and *Staphylococcus aureus*. The findings suggest that the metabolic state of probiotics plays a crucial role in their efficacy, and that anaerobic preconditioning could optimize their therapeutic potential. Further *in vivo* studies and molecular characterizations are needed to confirm these observations and elucidate underlying mechanisms. Combining high-performing strains such as *L. mucosae*, *L. rhamnosus* and *L. lactis* in a multi-strain formulation may offer synergistic effects on inflammation, oxidative stress and pathogen control. The development of next-generation probiotics could also benefit from pre-conditioning strains under anaerobic fermentation to optimize their bioactivity at the time of administration. Effectively, probiotics can be administered as freeze-dried powders within a gastro-protective carrier to enhance their health benefits in the colon. Ultimately, this study contributes to the growing understanding of how metabolic context shapes probiotic function, offering promising avenues for improving therapeutic applications and formulation strategies.

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**Declarations**
**Ethics approval and consent to participate**

Not applicable.

**Consent for publication**

Not applicable.

**Availability of data and materials**

All data and materials shall be made available on request to Mrs. Justine Guilbaud.

Email: justine.guilbaud0609@gmail.com.

**Competing interests**

The authors declare that they have no competing interests.

**Funding**

Not applicable.

**Authors' contributions**

JG, participated in the development of the study design, sample analysis, the collection, analysis and interpretation of the data and in the writing of the report. GG and EL participated in the development of the study design, samples analysis, the collection, analysis and interpretation of the data and in the editing of the report. KF and EB participated in the development of the study design and the editing of the report. SC participated in the collection of the study design. All authors read and approved the final manuscript.

**Acknowledgments**

Not applicable.
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Anti-inflammatory activity of probiotic strains under aerobic and anaerobic condition by measuring IL-8 concentration. The symbols indicate a statistically significant difference (P < 0.05) as determined by Tuckey test. All values are mean ± standard deviation; n=4. * aerobic conditions versus anaerobic conditions; # all conditions versus all conditions. * or # P < 0.05; ** or ## P < 0.01.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Antioxidant activity of probiotic strains as measured by reducing power activity. The letters at the bars and stars denote statistically significant difference (p < 0.05) as determined by Tuckey test. All values are mean ± standard deviation; n=4. * aerobic conditions versus anaerobic conditions. ** P < 0.01; **** P < 0.0001.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>(B)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>(C)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>(D)</figcaption></figure>

<figure><img src="image_7.png" /></figure>
**Figure 3.** Antimicrobial activity against *Listeria monocytogenes* of intracellular matrix of probiotics (A) and extracellular matrix (B), against *Staphylococcus aureus* of intracellular matrix of probiotics (C) and extracellular matrix (D). The letters at the bars and stars denote statistically significant difference (p < 0.05) as determined by Tuckey test. All values are mean ± standard deviation; n=3. * aerobic conditions versus anaerobic conditions. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Each control represents the pathogen in presence with 500 µL of MRS medium (for the extracellular matrix) or in presence with 500 µL of PBS medium (for the intracellular matrix).