Chloé Modugno

Souhir Kmiha

Hélène Simonin
email: helene.simonin@agrosupdijon.fr

Chedia Aouadhi

Esther Diosdado Cañizares

Emilie Lang

Stéphane André

Slah Mejri

Abderrazak Maaroufi

Jean-Marie Perrier-Cornet

High pressure sensitization of heat-resistant and pathogenic foodborne spores to nisin

Keywords: Bacillus, Clostridium, psychrotrophic, highly-heat-resistant, germination

Introduction

When exposed to unfavorable environmental conditions, several bacterial species, mostly Bacillus and Clostridia, can enter a metabolically dormant state by forming endospores.

Endospores (here after called "spores") have exceptional resistance properties against high temperatures, radiation, desiccation, pressure and toxic chemicals. They can survive under starvation for years before germinating and forming vegetative cells again. Spore-forming bacteria play an important role in food spoilage and foodborne disease [START_REF] Wells-Bennik | Bacterial Spores in Food: Survival, Emergence, and Outgrowth[END_REF]. High-heat treatments, such as sterilization or Ultra-High-Temperature (UHT) treatments, are needed to inactivate spores in food products. Such treatments are usually effective to reduce spores to sufficiently low numbers; however, various species of sporeformers, such as Bacillus sporothermodurans, are able to survive commercial wet heat sterilization [START_REF] André | Thermophilic spore-forming bacteria isolated from spoiled canned food and their heat resistance. Results of a French ten-year survey[END_REF][START_REF] Huemer | Thermal Death Kinetics of Spores of Bacillus sporothermodurans Isolated from UHT Milk[END_REF]). In addition, non-sterilized and low-acid food, such as refrigerated ready-to-eat-food, can be contaminated by bacterial spores, namely, from psychrotrophic species such as non-proteolytic Clostridium botulinum species [START_REF] Mills | Factors affecting microbial spoilage and shelf-life of chilled vacuum-packed lamb transported to distant markets: A review[END_REF]). As a consequence, preservative ingredients are often required to ensure their safety.

The high-pressure (HP) process is used worldwide as an alternative to heat pasteurization or as an additional decontamination step in packaged food products. However, similar to many inactivation technologies, HP alone is not effective on bacterial spore destruction (Black et al., 2007a). If HP at a temperature lower than 50 °C can hardly inactivate spores, it has long been known that HP initiates germination of the spores of Bacillus sp. and Clostridium sp.

when applied between 200 -400 MPa [START_REF] Doona | Effects of High Pressure Treatment on Spores of Clostridium species[END_REF][START_REF] Reineke | Mechanisms of endospore inactivation under high pressure[END_REF].

HP has also been shown to act synergistically with nisin to inactivate spore-forming bacteria of various species. In the abundant literature on the subject, the experimental conditions tested are very different from one study to another. The tested species are different; the nisin concentrations are not always expressed in the same units and vary from 0.2 to more than 2000 IU / mL. Finally, the contact between spores and nisin is not performed under the same conditions. In most studies reporting a synergy, nisin is added at concentrations ranging from 120 to 500 IU/mL to the spore suspension before a combined heat and HP treatment [START_REF] Aouadhi | The combined effect of nisin, moderate heating and high hydrostatic pressure on the inactivation of Bacillus sporothermodurans spores[END_REF][START_REF] Black | The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk[END_REF][START_REF] Gao | Assessment of Clostridium perfringens Spore Response to High Hydrostatic Pressure and Heat with Nisin[END_REF][START_REF] Kalchayanand | Inactivation of bacterial spores by combined action of hydrostatic pressure and bacteriocines in roast beef[END_REF]Sokolowska et al., 2012). Rare studies were interesting in adding nisin in the spore recovery medium after HP. Lopez-Pedemonte et al. (2003) show that adding nisin at 15600 UI/mL in two-pressure-cycle-HP treated cheeses increased the inactivation of Bacillus cereus and inhibited the regrowth during 15 days. And [START_REF] Roberts | Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin[END_REF] found that an HP treatment (400 MPa, 70 °C and low pH) renders spores of Bacillus coagulans more sensitive to nisin present in the recovery medium.

The objective of the present work was to investigate the effect of the combination of HP and nisin on the inactivation of relevant food pathogenic and spoilage spore species in nutrient media to clarify the conditions allowing the synergy. Bacillus sporothermodurans, B. pumilus, and B. licheniformis were chosen for their high resistance to temperature and their frequent implication in UHT and raw milk spoilage [START_REF] Aouadhi | Incidence and characterisation of aerobic spore-forming bacteria originating from dairy milk in Tunisia[END_REF][START_REF] Klijn | Genotypical and phenotypical characterization of Bacillus sporothermodurans strains, surviving UHT sterilisation[END_REF][START_REF] Kmiha | Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia[END_REF]. Clostridium sp. and B. weihenstephanensis were chosen regarding their potential pathogenicity and ability to contaminate meat products and for growing at refrigerated temperatures [START_REF] Mazuet | An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham[END_REF][START_REF] Thorsen | Characterization of emetic Bacillus weihenstephanensis, a new cereulideproducing bacterium[END_REF].

For this purpose, low nisin concentrations (≤50IU/mL), which highlight the synergy, were added to the spore suspensions during HP treatments at 20 and 50 °C and/or after treatment in the recovery medium to identify when inhibition occurs. Furthermore, without this being proved, it is generally admitted that HP germination is the underlying mechanism of the synergy between nisin and HP. Thus, our aim was also to clarify whether spore germination is necessary for the action of nisin on HP treated spores.

Material and methods

Bacterial strains, growth and sporulation conditions

Bacillus weihenstephanensis KBAB4 (provided by the Institut National de Recherche Agronomique, Unité SQPOV, Avignon, France), Bacillus licheniformis, Bacillus pumilus (both isolated from UHT Tunisian milk), Bacillus sporothermodurans (isolated from raw Tunisian milk) [START_REF] Kmiha | Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia[END_REF] and Clostridium sp. BAFF C3DSM 1985 were used as target strains in this study. As reported on the DSMZ website, this strain is, based on a partial 16S rDNA sequence, closely related to Clostridium botulinum type E, strain ATCC 23387 (Curators or the DSMZ n.d.).

Table 1 presents the conditions applied to produce the bacterial spores used in this study.

For Bacillus species, cultures were obtained by inoculating 2-3 fresh colonies into 50 mL of nutrient broth. After incubation (see Table 1), 0.4 mL of the culture was spread on sporulation Petri dishes (∅: 145 mm; h: 20 mm).

Sporulation was monitored by phase contrast microscopy and spores were harvested when more than 95% of phase bright spores were observed. Spores were harvested by flooding the agar plates with cold sterile distilled water and scraping the agar surface with a sterile cell spreader. Spore suspensions were centrifuged (3600 g, 15 min, 4 °C) and spores were suspended in 0.2 µm filtrated 70% ethanol (filter: cellulose acetate, Sartorius, France; ethanol: Elvetec, France) for 1 h to inactivate vegetative cells [START_REF] Koransky | Use of ethanol for selective isolation of sporeforming microorganisms[END_REF]. Spores were then washed three times by successive centrifugation (3600 g, 15 min, 4 °C) and suspension in sterile distilled water at 4 °C. Spores were enumerated by CFU determination on their respective enumeration media (EM). The concentration of the final spore suspensions was standardized at 10 8 CFU/mL. Suspensions were stored at 4 °C for a maximum of one month.

For Clostridium sp., a subculture was obtained by adding 0.1 mL of stock cultures previously stored at -80 °C to 9 mL of LYBHI (Table 1) in an anaerobic Hungate tube incubated for 24 h at 30 °C. A culture was obtained by diluting 0.5 mL of subculture into 10 mL of fresh LYBHI in an anaerobic Hungate tube. After incubation, 0.2 mL of this culture was inoculated into sporulation Petri dishes (Table 1) for sporulation. After one month at 30 °C under anaerobic conditions, the spores were harvested, purified and enumerated as previously described for Bacillus spp. strains. The concentration of the final spore suspensions was standardized at 10 7 CFU/mL. Suspensions were stored at 4 °C and stored for up to one month.

Effect of nisin on spore cultivability

Nisin stock solution in 0.02 mol/L HCl was prepared from nisin powder (10 6 IU/g, Sigma-Aldrich, France) and sterilized by 0.2 µm filtration (cellulose acetate, Sartorius, France).

Nisin stock solution was stored at 4 °C for a maximum of one week until use. The nisin level of the stock solution was confirmed by a plate diffusion assay using Micrococcus flavus DSM 1790, as described by [START_REF] Tramer | Estimation of nisin in foods[END_REF]. Following a protocol adapted from [START_REF] Stewart | Sensitivity of spores of Bacillus subtilis and Clostridium sporogenes PA 3679 to combinations of high hydrostatic pressure and other processing parameters[END_REF], nisin stock solution was added to tempered agar enumeration medium (Table 1) to achieve final concentrations ranging from 0 IU/mL to 200 IU/mL [START_REF] Stewart | Sensitivity of spores of Bacillus subtilis and Clostridium sporogenes PA 3679 to combinations of high hydrostatic pressure and other processing parameters[END_REF]. Spore suspensions were serially diluted and plated on their respective enumeration media supplemented with nisin (Table 1). Plates were incubated for 48 h (temperatures indicated in Table 1), and colonies were counted. The spore cultivability was plotted against the nisin concentration, and concentrations inducing less than -1.5 logs (CFU/mL) spore count reduction were selected for treatment combining HP and nisin.

Therefore, 50 IU/mL was chosen as the nisin concentration for all the Bacillus spp., and 20 IU/mL was chosen for Clostridium sp.

HP treatments in buffer and measurement of germination

HP tests were performed in a 700 MPa vessel (Top Industrie, France) with a double-walled metal pressure chamber of 20 cm 3 (working temperature: -20 °C / 90 °C). Water was used as the pressure-transmission fluid. The internal temperature of the pressure chamber was maintained by a water bath (Minisat 240, Huber, Germany) connected to the double wall of the pressure chamber and monitored by a Pt100 thermocouple (Omega, USA). The temperature of the water bath was set to limit the adiabatic heating to +5 °C above the desired temperature set point. The pressure and temperature during HP treatment were recorded using the instruNet World (iW) software. The compression rate was 3 MPa/s and the decompression was nearly instantaneous (< 3 sec).

Samples were prepared by a 90% dilution of the initial spore suspensions into 0.11 mol/L 2-(N-morpholino) ethane sulfonic acid (MES) buffer at pH 6.1 (Sigma Aldrich, France) to reach a final concentration of approximatively 10 7 CFU/mL for Bacillus spp. and 10 6 CFU/mL for Clostridium sp.. MES buffer was chosen because its pH varies only slightly with temperature and pressure (∆pKa/ °C = -0.011; pH varies from 5.5 to 6.5 between 10 and 1000 MPa at 25°C) [START_REF] Bruins | Buffer selection for HP treatment of biomaterials and its consequences for enzyme inactivation studies[END_REF]. Then, 0.5 mL of each spore suspensions in MES was heat-sealed into polyethylene pouches (polyethylene transfer pipet, Dominique Dutscher). For each strain, zero-time samples were taken to determine the initial concentration of the spores before treatment.

Pouches were placed in the HP vessel 5 min before treatment to let the temperature equilibrate. Samples were treated at 500 MPa for 10 min, at 20 °C or 50 °C and immediately immersed in iced water.

After treatment, samples were divided into two equal volumes, one of which was treated at 80 °C for 10 min in a water bath to inactivate germinated spores. The two volumes were then serially diluted and 100 µL of each dilution were plated on the sample enumeration media (Table 1). The fraction of spores induced in germination by HP was calculated as the difference between the logarithmic counts of in pressure-and heat-treated (NHPT) and pressure-treated (NHP) portions of the samples (log10(NHPT)-log10(NHP)).

HP treatment with nisin

Immediately before HP treatment, spore suspensions were diluted to approximately 10 7 CFU/mL for Bacillus spp. and 10 6 CFU/mL for Clostridium sp. in MES buffer supplemented or not supplemented with nisin at 50 UI/mL or 20 UI/mL for Bacillus spp. and Clostridium sp. respectively. Immediately after HP treatment, spore suspensions were centrifuged at 11200 g for 5 min at 4 °C, and spores were re-suspended in PBS (phosphate buffered saline, Sigma-Aldrich, France). Spores were then plated both on their respective EM (Table 1) and on EM supplemented with nisin at 20 or 50 IU/mL for Clostridium sp. and Bacillus spp.

respectively. After 48 h of incubation (see temperatures in Table 1), colonies were counted.

As a control for the effect of nisin alone, non-HP-treated samples were plated on EM + nisin.

HP treatments in nutrient media

UHT skim milk (Lactel, Lactalis, France) was used as a nutrient medium for the HP treatment of B. pumilus, B. sporothermodurans and B. licheniformis strains.

For B. weihenstephanensis and Clostridium sp., a liquid medium was formulated to suit the physicochemical properties of cooked ham brine (pH, aw, sugars and salt contents). The composition of this Medium Modelling Ham (MMH) was adapted from the composition of typical organic ham brine. The MMH is composed of 17.6 g/L of sodium chloride (Sigma Aldrich, France); 10 g/L of peptone from porcine heart (Sigma-Aldrich, France); 5 g/L of autolytic yeast extract (Biokar, France); 2.3 g/L of D-lactose monohydrate (Sigma-Aldrich, France); 0.7 g/L of Sucrose (Sigma-Aldrich, France); and 0.11 mol/L MES buffer qs 1 L (Sigma-Aldrich). The pH was adjusted to 6.1 before autoclaving.

Nitrites, which are usually added as preservatives in ham brine, were omitted to focus only on the effects of HP and nisin on the spores.

For HP treatment without nisin, spores were suspended in their respective nutrient media In the case of HP treatment with nisin, the bacteriocin was present during HP treatment and into the recovery medium..

Statistical analysis

All experiments were independently performed 3 times. The effect of the factors on spore inactivation after HP treatment was evaluated for each strain by analysis of variance (ANOVA) using R software (R development core team, 2008). Significance was considered to be when the p-value was equal to or less than 0.05. In this case, a Tukey's HSD (Honest Significant Difference) test was performed to observe significant differences among the conditions.

In the case of HP treatment in combination with nisin, the sum of the independent effects of HP and nisin was compared with the effect of the treatment combining HP and nisin. When the effect of the combined treatments was significantly higher than the addition of the two independent effects, a synergy was attained. A Student's t-test using a 5% level of significance (p < 0.05) was used to detect differences between the sum of the HP-germinated and inactivated spore fractions and the total inactivation provided by the application of nisin during and after HP.

Results and Discussion

Effect of HP and nisin on spore inactivation in the buffer

Spores were suspended in MES buffer and treated with nisin. Nisin concentrations used were 50 IU/mL for Bacillus spp. and 20 IU/mL for Clostridium sp.

Nisin was added at different stages of treatment, creating two groups of samples:

-Nisin present only during HP (plating on EM without nisin) -Nisin present during HP and added into the plating medium at the same concentration.

The results obtained are shown in Fig. 1. The effect of nisin alone on the spores (striped bars) represents the inhibition of spores by only plating on the EM + nisin. As expected, nisin at 50 IU/mL and 20 IU/mL induced approximately 1 log inhibition of Bacillus spp. and Clostridium sp. spores respectively (that is what the nisin concentration was determined for). Regarding the effect of HP alone, it can be noticed that pressure resistance of spores is strongly related to the temperature of the treatment. While spores treated at 20 °C did not show any significant inactivation (<1 log for all the strains), the viability of the spores of B. weihenstephanensis and B. pumilus processed at 50 °C was strongly affected (approx. 4 log inactivation for the two strains).

Spores of psychrotrophic species, such as Clostridium sp. and Bacillus weihenstephanensis are poorly heat-resistant [START_REF] Garcia | Comparative analysis of Bacillus weihenstephanensis KBAB4 spores obtained at different temperatures[END_REF][START_REF] Juneja | Thermal Resistance of Nonproteolytic Type B and Type E Clostridium botulinum Spores in Phosphate Buffer and Turkey Siurry[END_REF] while Bacillus pumilus, Bacillus licheniformis and Bacillus sporothermodurans are highly heat-resistant spores isolated from UHT milk [START_REF] Kmiha | Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia[END_REF]. Our results confirm the absence of a link between thermo-and baroresistance according to what has been reported in the rare literature on the subject [START_REF] Margosch | Comparison of pressure and heat resistance of Clostridium botulinum and other endospores in mashed carrots[END_REF][START_REF] Nakayama | Comparison of pressure resistances of spores of six bacillus strains with their heat resistances[END_REF]. Three groups of spore species clearly appear according to their baroresistance at a temperature ≤ 50 °C, in order of MPa was related to the germination induction during HP treatment and the subsequent resistance loss. At this pressure level, this induction was thought to be driven through spoVA channel activation and Ca-DPA release in B. subtilis [START_REF] Reineke | Mechanisms of endospore inactivation under high pressure[END_REF]). The B. subtilis spoVA proteins are composed of 7 subunits, including the SpoVAC subunit, which has recently been identified as a mechanical stress sensitive channel for DPA release [START_REF] Velásquez | Bacillus subtilis spore protein SpoVAC functions as a mechanosensitive channel[END_REF]. B. pumilus and B. licheniformis also have genes from the same spoVA subunits as B. subtilis [START_REF] Paredes-Sabja | Germination of spores of Bacillales and Clostridiales species: mechanisms and proteins involved[END_REF]), but it is not known for the other two species of Bacillus studied here. Moreover, the lytic enzymes of the cortex can be denatured by HP at 500 MPa, which consequently inhibits the second stage of germination [START_REF] Wuytack | Comparative study of pressure-induced germination of Bacillus subtilis spores at low and high pressures[END_REF]).

Thus, baroresistance differences could be attributed to varying sensitivities of DPA spoVA channels and lytic enzymes of the cortex in different Bacillus species. Regarding Clostridium spores, there are important differences in the germination mechanisms of Bacillus spores and certain Clostridium species. HP has recently been shown to cause the release of DPA by some Clostridium species [START_REF] Doona | Effects of High Pressure Treatment on Spores of Clostridium species[END_REF]). However, our results show that the effect of HP up to 600 MPa (results not shown) at the moderate temperature on the inactivation of Clostridium sp. remains low. This corresponds to what is already reported in the literature on C. botulinum type E [START_REF] Reddy | inactivation of Clostridium botulinum type E spores by High Pressure Processing[END_REF].

When nisin was present during HP, a slightly significant increase in spore inactivation was found at 20 °C and 50 °C for B. sporothermodurans and B. licheniformis (p < 0.01). In comparison with the effects of HP alone, the addition of nisin during HP provided a 1 log supplementary inactivation for both strains. Nisin addition during HP also provided a significant and synergistic increase in the inactivation of B. pumilus only at 20 °C (inactivation: 2.5 log; p < 0.001). In contrast, nisin addition during HP did not significantly improve B. weihenstephanensis and Clostridium sp. inactivation, regardless of the temperature. The same applied for B. pumilus at 50 °C. However, the presence of nisin during HP and into the plating medium induced a significant, synergistic effect on the reduction of the outgrowth of B. sporothermodurans, B. licheniformis and B. weihenstephanensis, both at 20 °C and 50 °C (p < 0.001). For Clostridium sp. treated at 50 °C and B. pumilus treated at 20 °C, a significant further reduction in spore outgrowth was provided by nisin addition during HP and into the plating medium (p < 0.01), resulting in the addition of the independent effects of HP and nisin. First, regarding the effect of HP alone, we noticed that inactivation was reduced when spores were treated in nutrient media, in comparison with the inactivation obtained after HP treatment in MES buffer. This reduction was significant for B. pumilus and B. weihenstephanensis at 50 °C for which the inactivation was reduced by 2.5 log and 2 log respectively. The protective effect of MMH on B. weihenstephanensis could be due to the presence of sodium chloride and sugars, inducing lower water activity (MMH aw: 0.97) [START_REF] Daryaei | Microbiological Aspects of High-Pressure Processing of Food: Inactivation of Microbial Vegetative Cells and Spores[END_REF]. A slight protective effect was also noticed for spores treated in milk. A possible explanation for this may be the natural baroprotective effect of milk, due to its high content in calcium and magnesium (Black et al. 2007b;[START_REF] Hauben | Protective effect of calcium on inactivation of Escherichia coli by high hydrostatic pressure[END_REF].

Effect of HP and nisin on spore inactivation in nutrient media

Second, the combination of HP and nisin gave similar results in nutrient media and in buffer. Indeed, a significant, synergistic effect was obvious for all the strains when nisin was present during HP and in the plating medium (p < 0.001), except for Clostridium sp. However, the outgrowth inhibition of Bacillus spp. was lower than after HP treatments in buffer.

Particularly, the inhibition of B. licheniformis at 20 °C was significantly reduced from -5 log in buffer to -3.5 log in milk (p < 0.01). This could be due to nisin adsorption on milk proteins, resulting in a decrease in its antimicrobial activity [START_REF] Lakamraju | Nisin adsorption and exchange with selected milk proteins at silanized silica surfaces[END_REF].

Nevertheless, a high synergy was found in both buffer and nutrient media for B. sporothermodurans, B. pumilus, B. licheniformis and B. weihenstephanensis, leading to a reduction of spore outgrowth of over 4 logs for all the Bacillus spp. strains.

Recovery of Bacillus and Clostridium spore growth was inhibited in the presence of nisin [START_REF] Egan | Bacteriocins: Novel Solutions to Age Old Spore-Related Problems?[END_REF][START_REF] Hofstetter | Effects of nisin and reutericyclin on resistance of endospores of Clostridium spp. to heat and high pressure[END_REF]. The inhibitory effect of nisin occurred rapidly (<5 minutes) after the initiation of germination. It appeared that nisin prevented spores from becoming metabolically active by disrupting the establishment of their membrane potential [START_REF] Gut | Inhibition of Bacillus anthracis spore outgrowth by nisin[END_REF]. Nisin binds to lipid II for both spores and vegetative cells, but for spores, this is not sufficient to induce membrane pore formation and thus inhibits growth recovery [START_REF] Gut | Mechanism of Inhibition of Bacillus anthracis Spore Outgrowth by the Lantibiotic Nisin[END_REF]. The formation of pores could also be due to the binding of nisin to the thiol groups of membrane proteins which become accessible only at the time of spore germination [START_REF] Morris | Identification and characterization of some bacterial membrane sulfhydryl groups which are targets of bacteriostatic and antibiotic action[END_REF]. It is thus commonly admitted that the synergy between HP and nisin is due to the action of nisin on HP-germinated spores which appeared more sensitive to nisin than spore germinated in the growth medium. Some authors also hypothesized HP-induced physical damages to the spore outer layers (coat and cortex) allow nisin molecules access to their site of action without germination [START_REF] Aouadhi | The combined effect of nisin, moderate heating and high hydrostatic pressure on the inactivation of Bacillus sporothermodurans spores[END_REF]. We thus measured the number of HP-germinated spores to clarify whether spore germination is a necessary condition for the action of nisin on HP treated spores.

Effect of HP on spore inactivation and germination induction

We hypothesized that spores, whose germination was initiated by HP, became sensitive to nisin. We thus measured the HP-induced germination rate of the spores. Spores of B. licheniformis, B. pumilus, B. sporothermodurans, and B. weihenstephanensis in suspension in MES buffer were treated by HP at 500 MPa for 10 min at 50 °C or 20 °C. Spores induced in germination (at least first stage) after HP were immediately inactivated at 80 °C for 10 min. First, the major phenomenon observed after HP at 50 °C is the spore HP inactivation, while the germination without HP-inactivation represented less than 1 log for all the strains. In contrast, after HP treatment at 20°C, a large proportion of spores was germinated and not inactivated by HP.

Second, the cumulative HP-inactivated and germinated spore fraction are significantly lower than the total inhibition provided by the treatment combining HP and nisin for all the treatments, except B. pumilus and B. licheniformis at 50 °C (p < 0.05). For these two treatments, the spores inhibited by the combination of HP and nisin were equivalent those inactivated by a combination of HP and thermal treatment.

With the hypothesis that heat treatment after HP (80°C, 10 min.) inactivates all the HP induced-germinated spores, i.e. spore having released DPA after HP, the results presented in Fig. 3 clearly indicate that nisin sensitization does not simply come from spores induced in germination by HP. However, our results do not allow concluding if the spores are only reversibly inhibited by the combination of HP and nisin or if they are irreversibly inactivated in the presence of nisin in their growth medium.

Nevertheless, HP induced a modification or an alteration of the spores which sensitized spores to nisin without necessarily inducing germination.

HP could target spore coat or cortex. Rare studies show that spores with a damaged cortex are more sensitive to nisin [START_REF] Delves-Broughton | Applications of the bacteriocin, nisin[END_REF][START_REF] Rao | The Synergistic Effect of High Pressure CO(2) and Nisin on Inactivation of Bacillus subtilis Spores in Aqueous Solutions[END_REF].

Functionally, the spore coat constitutes an initial barrier to large molecules, such as peptidoglycan-lytic enzymes, which would otherwise have access to the spore cortex [START_REF] Leggett | Bacterial spore structures and their protective role in biocide resistance[END_REF]. The spore coat has also been identified as a critical resistance layer against many chemicals, especially oxidizing agents, such as nitrogen peroxide, ozone, chlorine and hypochlorite. In contrast, smaller molecules, such as the spore germinants, must presumably pass through this barrier. Nisin is a cationic peptide with a molar weight of 3300 Da, and its potential interaction with the spore coat is unknown.

Finally, the inhibition of spores by nisin after HP could be due to the modification of the properties of some inner membrane proteins, resulting in a better binding of nisin to sulfhydryl groups. Indeed, HP acts generally by promoting the unfolded form of the proteins, which are less voluminous than the native form. Pressures between 100 MPa and 500 MPa have been shown to increase the number and reactivity of sulfhydryl groups by unfolding the protein structure and exposing the interior sulfhydryl groups [START_REF] Funtenberger | High Pressure Promotes -Lactoglobulin Aggregation through SH/S-S Interchange Reactions[END_REF][START_REF] Zhang | Chemical forces and water holding capacity study of heat-induced myofibrillar protein gel as affected by high pressure[END_REF].

Conclusion

In conclusion, this work reports an important sporostatic effect of combining an HP treatment at 500 MPa and the addition of nisin into the recovery medium for several food-borne Bacillus species. The inhibition is significant even if the HP treatment is carried out at 20 °C and with a concentration of nisin as lower as 50 IU/ml while the regulatory concentration in Europe is 120 to 500 IU/mL. Furthermore, we demonstrated that the inhibition of growth

shown by the combination of HP and nisin does not need that spores are induced in germination by HP. The underlying mechanism needs further investigation, which is currently underway with Bacillus subtilis to bring new elements of understanding a: BHI vit: Brain Heart Infusion (Sigma-Aldrich, France) 37 g/L supplemented with1 mg/L of vitamin B12 (Sigma-Aldrich, France) [START_REF] Klijn | Genotypical and phenotypical characterization of Bacillus sporothermodurans strains, surviving UHT sterilisation[END_REF]. BHI vit + agar: BHI vit supplemented with 15 g/L of agar (pH: 7,2 ±0,2). b: CM (Complex Medium): meat extract (Biokar, France) 10 g/L; yeast extract (Biokar, France) 2 g/L, MnSO4 (Sigma-Aldrich, France) 0.04 g/L [START_REF] André | Thermophilic spore-forming bacteria isolated from spoiled canned food and their heat resistance. Results of a French ten-year survey[END_REF]. c: LYBHI: Brain Heart Infusion (Sigma-Aldrich, France) 37 g/L, supplemented with 5 g/L of yeast extract (Biokar, France), 5 g/L of hemin (Sigma-Aldrich, France), 1 g/L of cellobiose (Sigma-Aldrich, France), 1 g/L of Maltose (Sigma-Aldrich, France); 0.5 g/L of L-cysteine (Sigma-Aldrich, France). The pH was adjusted to 7.4 before autoclaving. d: CMA (Complex Medium Agar): CM supplemented with 15 g/L of agar (Biokar, France). e: VSFR: meat liver glucose agar with 2 g/L yeast extract (Biokar, France) (pH: 7.4±0,2). f: BCP: Bromocresol Purple Dextrose Tryptone Agar (Biokar, France) (pH: 7.0±0,2).

Legend of table and figures

(

  MMH for B. weihenstephanensis and Clostridium sp. and skim milk for B. pumilus, B. sporothermodurans and B. licheniformis), treated by HP at 500 MPa at 50 °C or 20 °C for 10 min and plated on EM for CFU determination.

  increasing baroresistance: B. weihenstephanensis; B. pumilus > B. licheniformis; B. sporothermodurans > Clostridium sp. It is well known that inactivation by HP beyond 400

B

  . licheniformis, B. pumilus, and B. sporothermodurans in suspension in skim milk and B. weihenstephanensis, and Clostridium sp. in suspension in MMH were treated by HP at 500 MPa for 10 min at 20 °C or 50 °C. Nisin was added to the spore suspensions just before HP treatment to reach 20 IU/mL for Clostridium sp. and 50 IU/mL for Bacillus spp. to simulate the presence of nisin in HP-treated food products, nisin was also added into the plating medium. Results are shown in Fig. 2.

Fig. 3

 3 Fig. 3 presents the HP-inactivated spore fraction (dark grey bars), the HP-germinated spores fraction (light grey bars) and the total spore inhibition provided by the addition of nisin during HP and into the plating medium (black bars). The cumulated inactivation (dark and light grey bars) represent the inactivation obtained after pressure and temperature treatments.

Fig 1 .

 1 Fig 1. Effect of nisin addition on high pressure inactivation of spores (500 MPa, 10 min). Treatment in MES buffer. Nisin concentrations: 50 IU/mL for Bacillus spp. 20 IU/mL for Clostridium sp. Striped bars: effect of nisin alone; Grey bars: effect of HP treatment at 20°C; Black bars: effect of HP treatment at 50°C. DL: Detection limit. Error bars represent SD calculated from independent triplicates. The letters represent a significant difference (p<0.05) obtained with Tukey's HSD (Honest Significant Difference) test.

Fig 2 .

 2 Fig 2. Effect of nisin addition on high pressure inactivation of spores (500 MPa, 10 min). Treatment in nutrient media. Spores of B. weihenstephanensis and Clostridium sp. were treated in MMH; spores of B. sporothermodurans; B. pumilus and B. licheniformis were treated in milk. Nisin concentrations: 50 IU/mL for Bacillus spp. 20 IU/mL for Clostridium

Fig 3 .

 3 Fig 3. Inactivated and germinated spores after HP treatment at 500 MPa -10 min:

Table 1 .

 1 Culture, sporulation and enumeration conditions

Table 1 . Culture, sporulation and enumeration conditions

 1

	Strain		Culture	Sporulation	Enumeration
		Medium	Incubation	Medium Incubation Medium Incubation
	B. sporothermodurans BHI vit a	37 °C	CMA d	37 °C	BHI vit +	37 °C
			24 h		7 days	agar a	24 h
	B. licheniformis	BHI vit a	37 °C	CMA d	37 °C	BHI vit +	37 °C
			24 h		7 days	agar a	24 h
	B. pumilus	BHI vit a	37 °C	CMA d	37 °C	BHI vit +	37 °C
			24 h		7 days	agar a	24 h
	B. weihenstephanensis CM b	30 °C	CMA d	30 °C	BCP f	30 °C
			until 0.4 < OD600 <		7 days		48 h
			0.6
	Clostridium sp.	LYBHI c	30 °C	VSFR e	30 °C	VSFR e	30 °C
			24 h		1 month		48 h

Funding

This work is a part of the project ANR-14-CE20-0004, funded by the French Agence Nationale de la Recherche (ANR). This work is also supported by the the Regional Council of Bourgogne -Franche Comté, the "Fonds Européen de DEveloppement Régional (FEDER)"

Conflict of Interest

None of the authors declare a conflict of interest.


Giorgio Varesco
email: giorgio.varesco@univ-st-etienne.fr

Loïc Espeit
email: l.espeit@univ-st-etienne.fr

Léonard Féasson

Thomas Lapole
email: thomas.lapole@univ-st-etienne.fr

Vianney Rozand
email: vianney.rozand@univ-st-etienne.fr

Léonard Feasson
email: leonard.feasson@univ-st-etienne.fr

activation characteristics of knee extensors in very old men

Keywords: aging, rate of force development, rate of EMG rise, neuromuscular performance, knee extensors

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

Introduction

The ability to quickly produce a high amount of force is strongly related to the completion of daily activities and can be further associated with the risk of falling in older adults [START_REF] Thompson | Age related differences in maximal and rapid torque characteristics of the leg extensors and flexors in young, middle-aged and old men[END_REF][START_REF] Klass | Age-related decline in rate of torque development is accompanied by lower maximal motor unit discharge frequency during fast contractions[END_REF][START_REF] Aagaard | Mechanical muscle function, morphology, and fiber type in lifelong trained elderly[END_REF]. A useful tool to assess the capacity to rapidly increase force is the rate of force development (RFD), considered to be more sensitive than maximal isometric force (Fmax) to detect changes in neuromuscular function in clinical and old populations [START_REF] Maffiuletti | Rate of force development: physiological and methodological considerations[END_REF][START_REF] Rodríguez-Rosell | Physiological and methodological aspects of rate of force development assessment in human skeletal muscle[END_REF]. Another advantage of RFD is the possibility to differentiate various phases of the force-time curve to better detect performance changes after a training protocol or in clinical vs healthy populations [START_REF] Maffiuletti | Rate of force development: physiological and methodological considerations[END_REF][START_REF] Andersen | Influence of maximal muscle strength and intrinsic muscle contractile properties on contractile rate of force development[END_REF][START_REF] Folland | Human capacity for explosive force production: neural and contractile determinants[END_REF]. This analysis can be further implemented by studying the rise in the EMG signal. The rate of EMG rise (RER) has been used to evaluate the rate of muscle activation concurrent to the rise in force in order to account for the neural factors that contribute to RFD [START_REF] Aagaard | Increased rate of force development and neural drive of human skeletal muscle following resistance training[END_REF][START_REF] Clark | Muscle performance and physical function are associated with voluntary rate of neuromuscular activation in older adults[END_REF][START_REF] Thompson | Age-related changes in the rate of muscle activation and rapid force characteristics[END_REF].

Healthy older adults present lower peak RFD than young adults for various upper and lower limb muscle groups, including the knee extensors [START_REF] Izquierdo | Maximal strength and power characteristics in isometric and dynamic actions of the upper and lower extremities in middle-aged and older men[END_REF][START_REF] Mau-Moeller | Age-related changes in neuromuscular function of the quadriceps muscle in physically active adults[END_REF], plantar flexors [START_REF] Thompson | Age-related changes in the rate of muscle activation and rapid force characteristics[END_REF], dorsiflexors [START_REF] Klass | Age-related decline in rate of torque development is accompanied by lower maximal motor unit discharge frequency during fast contractions[END_REF] and elbow extensors [START_REF] Bellumori | Age-related decline in the rate of force development scaling factor[END_REF]. Conflicting results were reported when RFD was normalized to Fmax: while some studies showed greater values in young vs older adults [START_REF] Klass | Age-related decline in rate of torque development is accompanied by lower maximal motor unit discharge frequency during fast contractions[END_REF][START_REF] Thompson | Age-related changes in the rate of muscle activation and rapid force characteristics[END_REF][START_REF] Mau-Moeller | Age-related changes in neuromuscular function of the quadriceps muscle in physically active adults[END_REF][START_REF] Korhonen | Aging, muscle fiber type, and contractile function in sprint-trained athletes[END_REF], others did not [START_REF] Thompson | Age related differences in maximal and rapid torque characteristics of the leg extensors and flexors in young, middle-aged and old men[END_REF][START_REF] Thelen | Effects of Age on Rapid Ankle Torque Development[END_REF]. The discrepancy in the results could be due to different time intervals examined and different techniques used to normalize RFD by Fmax (e.g. some author calculated RFD on the force-time curve, while others used %MVC on the x-axis and measured the RFD value at the point when the force reached 10%, 30% or 50% of the MVC [START_REF] Thompson | Age-related changes in the rate of muscle activation and rapid force characteristics[END_REF]). However, it remains unclear whether the age-related decrease in RFD capacity is only due to the decrease in Fmax. For instance, RFD may be influenced more than Fmax by changes at the motor units level (i.e. loss of fast-type motor units [START_REF] Hepple | Innervation and neuromuscular control in ageing skeletal muscle[END_REF]), or neural impairments (i.e. motor unit discharge rate and doublets [START_REF] Klass | Age-related decline in rate of torque development is accompanied by lower maximal motor unit discharge frequency during fast contractions[END_REF]).

Regarding the methodology, it has to be noted that a large part of the available literature measured RFD during maximal isometric voluntary contractions (MVC), asking the participants to exert isometric contractions as strong and as fast as possible (e.g. [START_REF] Thompson | Age-related changes in the rate of muscle activation and rapid force characteristics[END_REF][START_REF] Izquierdo | Maximal strength and power characteristics in isometric and dynamic actions of the upper and lower extremities in middle-aged and older men[END_REF][START_REF] Mau-Moeller | Age-related changes in neuromuscular function of the quadriceps muscle in physically active adults[END_REF]).

Instead, recent recommendations suggest to perform specific impulsive isometric contractions as fast as possible to investigate RFD separately from Fmax [START_REF] Maffiuletti | Rate of force development: physiological and methodological considerations[END_REF]. The rigor in the instructions is essential, as Sahaly et al. [START_REF] Sahaly | Maximal voluntary force and rate of force development in humans -Importance of instruction[END_REF] showed a significant improvement in peak RFD when participants were instructed to push "fast" rather than "hard and fast". Consequently, RFD analyzed during MVC could be suboptimal and may not represent the maximal capacity to rapidly increase force. Furthermore, considering that older adults exhibit greater variability in force measurements compared to young adults [START_REF] Hunter | The aging neuromuscular system and motor performance[END_REF], measuring RFD during an MVC may lead to inconsistent results and a misinterpretation of the age-related changes in RFD.

In this scenario, also the age group of participants is determinant: several cross-sectional and longitudinal studies focusing on the advanced stages of human life documented a sudden acceleration in the decline of neuromuscular function starting from the ninth decade [START_REF] Mitchell | Sarcopenia, dynapenia, and the impact of advancing age on human skeletal muscle size and strength; a quantitative review[END_REF][START_REF] Venturelli | Skeletal Muscle Function in the Oldest-Old: The Role of Intrinsic and Extrinsic Factors[END_REF].

However, RFD has been mainly studied in individuals aged between 60 and 80 years and only one study focused on the RFD performance in octogenarians [START_REF] Bellumori | Age-related decline in the rate of force development scaling factor[END_REF]. In their study, Bellumori, Jaric, & Knight [START_REF] Bellumori | Age-related decline in the rate of force development scaling factor[END_REF] analyzed the absolute peak RFD from MVC of the elbow extensors and index finger abductors in very old adults and observed a ~50% reduction in RFD compared to young adults. Considering that the age-related impairments in neuromuscular performance are typically greater in the lower limb compared to upper limb muscles [START_REF] Mitchell | Sarcopenia, dynapenia, and the impact of advancing age on human skeletal muscle size and strength; a quantitative review[END_REF][START_REF] Frontera | Aging of skeletal muscle: a 12-yr longitudinal study[END_REF] with functional repercussions on balance and autonomy [START_REF] Mcgregor | It is not just muscle mass: A review of muscle quality, composition and metabolism during ageing as determinants of muscle function and mobility in later life[END_REF], investigating the capacity to rapidly increase force of the lower limb muscles is particularly relevant in very old adults. Identifying the mechanisms of the possible impairment in RFD may help to design specific interventions to preserve the neuromuscular function in very old age.

In the present study, we aimed to investigate the age-related changes in RFD characteristics and RER of knee extensors (KE) between healthy young (<30 years) and active very old men (>80 years). We hypothesized that 1) both absolute and relative (to Fmax) RFD characteristics of the KE would be considerably reduced in the octogenarians compared to the young adults during specific impulsive contractions and 2) RER of the vastus lateralis would be lower in very old adults.

Methods

2.1.Participants

Twelve recreationally active males of 24±4 years (range 20 -33 yr, 182±6 cm, 76±8 kg) and fifteen active very old men of 82±1 years (range 80 -83 yr, 168±6 cm, 71±9 kg) volunteered to participate in the experiment and gave their written consent. Very old participants were selected from the PROgnostic indicator OF cardiovascular and cerebrovascular events (PROOF) cohort, which is a prospective longitudinal cohort of old adults started in 2001 [START_REF] Barthélémy | Autonomic nervous system activity and decline as prognostic indicators of cardiovascular and cerebrovascular events: The 'PROOF' study: Study design and population sample. Associations with sleep-related breathing disorders: The 'SYNAPSE' study[END_REF].

Individual physical activity of the participants from this cohort is regularly assessed using a wrist wearable accelerometer (wGT3X-BT, ActiGraph, Pensacola, USA) and analyzed using ActiLife software (v6.13.4, ActiGraph, Pensacola, USA). Considering those data, all the very old men were considered as physically active (8840±1972 steps.day -1 , [START_REF] Tudor-Locke | Utility of Pedometers for Assessing Physical Activity[END_REF]). All participants were free from musculoskeletal or neurological disorders. Before the experimental session, participants were asked to avoid strenuous exercises for at least 24 hours. All the experimental procedures were approved by the Comité de Protection des Personnes Sud-Est 1 (France), and the Agence Nationale Sanitaire et du Médicament (ANSM-France) and was performed in accordance with the Declaration of Helsinki (ClinicalTrials.gov, Identifier: NCT02675192).

2.2.Mechanical recording and electromyography

Participants were seated upright on a custom-built chair with hip angle set at 120° (180° corresponds to full extension) and knee angles set at 90° of flexion (Figure 1A). Knee extensors force of the right leg was assessed using a calibrated force transducer (Omega Engineering Inc., Stamford, CT, USA). Extraneous movements of the upper body were minimized by three belts across the thorax and waist. The force transducer was firmly attached above the malleoli. Force was sampled at 2 kHz and recorded without analogical or digital filter using a Power-Lab data acquisition system (16/30-ML880/P, ADInstruments, Bella Vista, Australia). Passive resting force was subtracted from the signal so that the baseline was set at 0 N. Electromyographic (EMG) activity was recorded from the vastus lateralis (VL) muscle of the right leg using pairs of self-adhesive Ag/AgCl surface electrodes (recording diameter = 10 mm, Kendall MediTrace foam electrode, MA) placed in bipolar configuration over the muscle belly with an interelectrode (center to center) distance of 30 mm. Electrodes were placed following the SENIAM project recommendations. A reference electrode was placed on the right patella. Prior to electrode placement, the skin was shaved, abraded and cleaned with alcohol swab to lower the impedance (<5 kΩ, 87v, Fluke Corp., Everett, WA, USA). EMG and force signals were digitized simultaneously at the same sampling rate (2 kHz) by PowerLab System (16/30-ML880/P, AD Instruments, Bella Vista, Australia). Force and EMG data were analyzed offline using Labchart 8 software (ADInstruments). EMG signals were amplified (gain = 500) using an octal bio-amplifier (ML138, AD Instruments) with a bandwidth frequency range of 10-500 Hz obtained through a band-pass filter. Prior to analysis, data were rectified and smoothed using a moving rootmean-square (RMS) with a time constant interval of 50 ms [START_REF] Aagaard | Increased rate of force development and neural drive of human skeletal muscle following resistance training[END_REF].

2.3.Experimental protocol

All the participants were clearly informed on the modality of the test, and an experimenter provided practical demonstration if required. Participants performed a standardized warm-up consisting in 10 isometric contractions of the knee extensors at increasing intensities until reaching 90% of their perceived Fmax. Warm-up was followed by familiarization with the experimental procedures: participants were instructed to extend their knee (in isometric condition) "as fast as possible" in order to assess the RFD variables [START_REF] Maffiuletti | Rate of force development: physiological and methodological considerations[END_REF]. Familiarization consisted in 5 to 10 non-successive impulsive contractions, allowing participant to feel comfortable with the procedures. Successively, 5-min of rest were allowed before asking participants to perform two 5-s MVC separated by at least 60 s of rest. If the difference between the two MVCs was greater than 5%, participants performed a third MVC. In order to perform MVC, participants were asked to contract "as hard as possible", strong verbal encouragements and visual feedback were provided. After a 3-min rest, participants performed a series of eight isometric contractions (0.5-1.5 s; Figure 1B) separated by 30 s of rest. Strong verbal encouragement was provided. In case of countermovement, i.e. quick flexion prior to the knee extension, determined from a force drop of 2 N below the baseline, or if the force attained was less than 70% of the Fmax [START_REF] Folland | Human capacity for explosive force production: neural and contractile determinants[END_REF][START_REF] Ruiter | Initial phase of maximal voluntary and electrically stimulated knee extension torque development at different knee angles[END_REF], the trial was repeated again in order to obtain eight reliable contractions.

2.4.Data analysis

All data were analyzed offline using Labchart 8 Software (ADInstruments). The Fmax and associated RMS-EMG signal (EMGmax) were defined as the average of the highest 0.5-s window among the best MVC. Repeatability of MVC was tested using a bivariate correlation.

The maximal rate of force development (RFDmax) was determined as the maximal value of the force-time derivative curve for each RFD contraction. Absolute RFDmax was used to identify the best five impulsive contractions retained and then averaged for further analysis, while the others were discarded (Figure 1B). To assess the influence of within-subject variability among young and very old adults, the coefficient of variation (CV) was calculated for each participant and compared between groups. Time to RFDmax (tRFDmax) and force level at 25, 50, 100 and 200 ms from the onset of the force (see below) were identified. Then, the mean RFD was calculated at three time intervals of 0-50 ms (RFD0-50), 50-100 ms (RFD50-100), 100-200 ms (RFD100-200) and 0-200 ms (RFD 0-200) from the linear slope of the force-time curve [START_REF] Aagaard | Increased rate of force development and neural drive of human skeletal muscle following resistance training[END_REF] (Figure 1C). The onset of the force development was defined automatically as the point at which the level of force exceeded three standard deviations over the average resting baseline [START_REF] Ruiter | Initial phase of maximal voluntary and electrically stimulated knee extension torque development at different knee angles[END_REF]. This method has been chosen because it provides a valid and reliable method to determine the onset of the contraction when low-noise dynamometers (~1 N) are used [START_REF] Maffiuletti | Rate of force development: physiological and methodological considerations[END_REF]. All RFD parameters are presented in both absolute values and expressed as percentage of Fmax.

The rate of EMG rise (RER) was calculated as the linear slope of the RMS EMG-time curve from two different onsets: the first onset was set 70 ms prior to the force onset as suggested by Aagaard et al. [START_REF] Aagaard | Increased rate of force development and neural drive of human skeletal muscle following resistance training[END_REF], and the second onset was visually identified from the rise in the RMS EMG-time curve. RER was analyzed at time intervals of 0-30 ms (RER30), 0-50 ms (RER50) and 0-75ms (RER75). All EMG signals were normalized to EMGmax [START_REF] Thompson | Age-related changes in the rate of muscle activation and rapid force characteristics[END_REF]. Electromechanical delay was measured from the RMS EMG onset visually identified to the force signal onset.

(2-column fitting image) measures (r = 0.98, P<0.001) and significantly lower (37%) for the very old men compared to the young men (P<0.001). The associated EMGmax was also lower for the older adults (27%, P=0.42). Absolute RFDmax was greater in the young men (35%, P<0.001), however the agerelated difference was not observed when normalized to the maximal force (Figure 2A, P=0.78). Normalized RFD was not influenced by the between-subject variability, as CV was similar between young (9±6%) and very old men (12±6%, P=0.21).

Results

Due

(2-column fitting image) It can be noticed on Figure 2A that two outliers from the very old group may have influenced the mean. Nevertheless, the statistical analysis was also performed without these two participants and the results remained unchanged. Finally, no age-related differences were observed for the tRFDmax (P=0.25).

Of those two outliers, one very old man showed higher Fmax (554 N) and RFD (12409 N*s -1 ) than some young men. From the onset of the contraction, there were significant age-related differences in the absolute force level at every time point and were 23.1±8.5 N vs 12.6±10. Average RFD were greater for the young than the very old men at every time interval (Table 1). When normalized to the Fmax, average RFD were 3.8±1.2% (0-50 ms), 6.1±0.9% (50-100 ms) and 3.0±0.5% (100-200 ms) for young men and 2.5±1.7% (0-50 ms), 4.8±2.7% (50-100 ms) and 2.5±1.0% (100-200 ms) for very old men (Figure 2B). Significance was found only at the 0-50 ms time interval (P=0.04), while no differences were observed at 50-100 ms (P=0.16) and 100-200 ms (P=0.20). Finally, significant difference was found at the 0-200 ms time interval between young (4.2±0.7%) and very old men (3.5±0.9%; P=0.04).

RER of the VL, expressed as %EMGmax, exhibited different results depending on the onset considered (Figure 4). Data obtained with the RER onset 70 ms prior to the force onset showed significantly lower RER during the 0-30 ms time interval in the young men (P=0.04), while no age-related differences were observed during the 0-50 ms (P=0.26) and 0-75 ms time intervals (P=0.36; Figure 4A). The opposite trend was observed when RER onset was visually set at the point of RMS EMG rise, RER was greater for the young than the very old

Discussion

The aim of the present study was to assess the age-related impairments in rapid force production capacity and rate of muscle activation of the KE. We reported reduced Fmax and absolute RFDmax in active octogenarians compared to young men. For every time interval of the force-time curve, absolute force and RFD values were considerably lower in very old men, as initially hypothesized. Contrary to our hypothesis, no age-related difference in RFDmax was observed when normalized to Fmax. However, normalized RFD values in the early phase of the contractions (0-50 ms) were lower in very old compared to young men.

This age-related difference disappeared in the successive phases of the contraction. )

R E R 3 0 R E R 5 0 R E R 7 5 R E R 3 0 R E R 5 0 R E R 7
R E R 3 0 R E R 5 0 R E R 7 5 R E R 3 0 R E R 5 0 R E R 7

A B

Regarding RER, when the onset was defined manually on the RMS EMG-time curve, RER was significantly lower for very old men at every time intervals.

Although investigating the capacities of the KE is functionally relevant in old and very old age [START_REF] Mitchell | Sarcopenia, dynapenia, and the impact of advancing age on human skeletal muscle size and strength; a quantitative review[END_REF][START_REF] Laukkanen | Muscle strength and mobility as predictors of survival in 75-84-year-old people[END_REF], the literature regarding RFD performance with aging for this muscle group is limited. Thompson and colleagues [START_REF] Thompson | Age related differences in maximal and rapid torque characteristics of the leg extensors and flexors in young, middle-aged and old men[END_REF] observed ~28% lower Fmax and absolute RFDmax measured during isometric KE MVC in old (67 yr) compared to young adults (25 yr), while Mau Moeller et al. [START_REF] Mau-Moeller | Age-related changes in neuromuscular function of the quadriceps muscle in physically active adults[END_REF] reported even greater age-related impairments (Fmax, ~40%; RFDmax, ~65%) between young (25 yr) and old (70 yr) men during the same task. In agreement with the previous studies, we reported a ~36% lower absolute Fmax and RFDmax in very old men compared to young men. During the rapid isometric contractions, the absolute force level of very old men was significantly lower than the young men at every time point, and this difference persisted when normalized to Fmax. These results confirm the marked impairment in the capacity to rapidly increase the level of force with very old age that accompany the decline in maximal force production capacity [START_REF] Venturelli | Skeletal Muscle Function in the Oldest-Old: The Role of Intrinsic and Extrinsic Factors[END_REF].

Interestingly, no age-related differences were observed in RFDmax when normalized to Fmax, which could indicate similar factors are responsible for the decline of those two parameters.

Maximal force and rapid force development are both affected by sarcopenia and the consequent muscle remodeling process with aging [START_REF] Gueugneau | Skeletal Muscle Lipid Content and Oxidative Activity in Relation to Muscle Fiber Type in Aging and Metabolic Syndrome[END_REF]. Only one very old man was more performant in both Fmax and RFDmax than some young men. This is in agreement with previous investigations showing no age-related change in normalized RFD in plantar flexors [START_REF] Thelen | Effects of Age on Rapid Ankle Torque Development[END_REF], dorsiflexors [START_REF] Thelen | Effects of Age on Rapid Ankle Torque Development[END_REF], leg extensors [START_REF] Thompson | Age related differences in maximal and rapid torque characteristics of the leg extensors and flexors in young, middle-aged and old men[END_REF] and leg flexors [START_REF] Thompson | Age related differences in maximal and rapid torque characteristics of the leg extensors and flexors in young, middle-aged and old men[END_REF]. However, other studies reported a reduction in the relative RFDmax of plantar flexors [START_REF] Thompson | Age-related changes in the rate of muscle activation and rapid force characteristics[END_REF], dorsiflexors [START_REF] Klass | Age-related decline in rate of torque development is accompanied by lower maximal motor unit discharge frequency during fast contractions[END_REF] and leg extensors [START_REF] Mau-Moeller | Age-related changes in neuromuscular function of the quadriceps muscle in physically active adults[END_REF][START_REF] Korhonen | Aging, muscle fiber type, and contractile function in sprint-trained athletes[END_REF]. In contrast with the previous investigations, in the present study Fmax and RFD were obtained during different contractions and giving specific instructions, according to the most recent recommendations [START_REF] Maffiuletti | Rate of force development: physiological and methodological considerations[END_REF][START_REF] Rodríguez-Rosell | Physiological and methodological aspects of rate of force development assessment in human skeletal muscle[END_REF], obtaining good within-subject reliability of the RFD measures in both groups. Consequently, different muscles involved, data analysis and protocols adopted may partially explain the discrepancies in the literature. The majority of the studies analyzed RFD during MVC, while we used specific impulsive isometric contractions. As we asked our participants to push "as hard as possible" during MVC rather than "hard and fast", we could not compare RFD during MVC and the impulsive contractions. Other than methodological factors, the high level of physical activity of our participants may influenced age-related differences in RFD. In the present study, the very old participants were healthy community-dwelling volunteers, and classified as "active" according to the number of steps/day measured (threshold defined at 7000 steps/day between active and sedentary very old men [START_REF] Ce | How Many Steps / day are Enough ? For Older Adults and Special Population[END_REF]). These healthy individuals may not represent the overall population over 80 years, but rather those with greater lifespan physical activity behaviors and/or with biological superiority [START_REF] Porter | Aging of human muscle : structure , function and adaptability[END_REF]. The natural selection bias of healthy participants over 80 years (alive, autonomous, without motor or neural impairments, etc…) could also explain why the agerelated declines in performance observed in previous cross-sectional and longitudinal studies were less important than what expected in the ninth decade from linear extrapolation models [START_REF] Aniansson | Muscle morphology, enzymatic activity, and muscle strength in elderly men: A follow-up study[END_REF][START_REF] Greig | The quadriceps strength of healthy elderly people remeasured after eight years[END_REF]. Moreover, physical activity has important implications in the age-related changes in RFD. For example, Aagaard et al. [START_REF] Aagaard | Mechanical muscle function, morphology, and fiber type in lifelong trained elderly[END_REF] showed that chronic exposure to strength training preserves RFDmax at a similar level in young and old individuals, and Caserotti et al. [START_REF] Caserotti | Explosive heavy-resistance training in old and very old adults: Changes in rapid muscle force, strength and power[END_REF] reported similar performances after 12-weeks of heavy-resistance training in old (60-65 years) and very old women (80-89 years). Further investigations should compare normalized RFD of very old men with different levels of physical activity to clarify the relative contribution of maximal force decline, aging and physical activity level in RFD performance.

The normalized 0-200 ms time interval was used to provide a more representative indicator of the age-related RFD changes, and was significantly lower in the very old men. This reflects an overall impairment in the capacity of rapid force generation with aging that has been evidenced at every time point on the force-time curve. To better illustrate RFD differences between groups, different time epochs were analyzed and normalized to Fmax.

The average normalized RFD values were significantly lower only in the early phase of the contraction (0-50 ms), but not in the successive time intervals (50-100 ms and 100-200 ms), contrary to the absolute RFD values and the normalized force values. The non-significant difference in the middle time epochs regarding RFD data could be explained by the higher between-subject variability in very old men. Despite higher variability, the difference at 0-50 ms was great enough to reach statistical significance. In other words, the force increment in this short time period (50 ms) was greater for the young men, which determined a difference in the relative RFD between groups. This could suggest that different neural strategies could be adopted during aging to compensate the progressive loss in RFD capacity, still evident in the 0-50 ms time interval. This could be partially supported by our results on time to reach RFDmax, which was similar between groups. Similar results were obtained by Thompson et al.

[10] and Mau-Moeller et al. [START_REF] Mau-Moeller | Age-related changes in neuromuscular function of the quadriceps muscle in physically active adults[END_REF]. Thompson et al. [START_REF] Thompson | Age-related changes in the rate of muscle activation and rapid force characteristics[END_REF] found a difference between young and old adults in the plantar flexors at time intervals of 0-50 ms and 100-200 ms. However, these authors didn't report the time interval 50-100 ms. Thus, it seems that the early phase of the contraction is determinant for the age-related impairment in absolute RFD capacity of the leg extensors. Previous investigations associated the first phase of the contraction (0-50 ms) to the rate of muscle activation. For instance, a recent study of Gerstner et al. [START_REF] Gerstner | Neural and Muscular Contributions to the Age-Related Reductions in Rapid Strength[END_REF] on the plantar flexors found similar rate of torque development capacity between young and old men (~69 yr) in the firsts 50 ms of contraction, while it was reduced in the late interval (100-200 ms).

These authors suggested that while factors like poorer muscle quality, the inability to sustain a high rate of muscle activation and reduced level of physical activity could influence the late interval of contraction in old men, muscle activation could reflect changes in the early time interval. To provide further insight, we quantified using the rate of EMG rise (RER). We set two different RMS EMG onset for the RER analysis. With the EMG onset fixed 70 ms prior to force onset [START_REF] Aagaard | Increased rate of force development and neural drive of human skeletal muscle following resistance training[END_REF], RER was significantly higher in very old compared to young men only in the early phase of the contraction (0-30ms), while no age-related differences were observed at 0-50 ms and 0-75ms. However, the electromechanical delay was significantly different between groups, and only the electromechanical delay of the very old men was close to the fixed value of 70 ms suggested by Aagaard et al. [START_REF] Aagaard | Increased rate of force development and neural drive of human skeletal muscle following resistance training[END_REF]. Thus, setting the onset visually on the RMS EMG-time curve seems to be the best option when two different populations are compared. In the present study, when the RMS EMG onset was set manually, RER was significantly lower for very old men for all the time intervals considered. The longer electromechanical delay in the very old group compared to the young, despite in small part dependent from the electrode placement, indicate a deficit in the transmission and conversion of the voluntary drive to force. The reduced capacity to rapidly activate the muscle suggests that impairments occur also in the voluntary drive, e.g. the generation of doublet discharge, the decrease in discharge frequency and/or the inability to maintain high discharge frequencies, leading to different or less efficient activation pattern with aging [START_REF] Klass | Age-related decline in rate of torque development is accompanied by lower maximal motor unit discharge frequency during fast contractions[END_REF][START_REF] Mitchell | Rate of Muscle Activation in Power-and Endurance-Trained Boys[END_REF]. To interpret those results, it has been shown that the age-related slowing of muscle contractile kinetics, the progressive reduction in type II muscle fibers [START_REF] Mitchell | Sarcopenia, dynapenia, and the impact of advancing age on human skeletal muscle size and strength; a quantitative review[END_REF] and changes in the muscle architecture [START_REF] Gerstner | Neural and Muscular Contributions to the Age-Related Reductions in Rapid Strength[END_REF] are muscle dependent and could influence the EMG signal. Limiting our analysis to the VL and recognizing the limitations of surface EMG measurements [START_REF] Farina | Neural Control of Movement: The Extraction of Neural Strategies from the Surface {EMG}[END_REF],

further evidences are needed to clarify the specific role of neural vs contractile mechanisms involved in the loss of RFD across different muscles.

Conclusions

The present study demonstrated that rapid force development capacity is impaired in active octogenarians. However, the age-related difference is minimized when RFD is normalized by the maximal force of each individual. The results suggest that muscle force and RFD have common mechanisms determining neuromuscular changes associated with healthy aging. The impairments in RFD are accentuated in the early phase of the contraction, which has been associated in the literature with the rate of muscle activation. Further studies are needed to establish whether similar levels of Fmax or RFDmax are related to similar motor function in very old age.

Conflicts of interest

No conflicts of interests, financial or otherwise are declared by the authors.

Figure 1 .

 1 Figure 1. Example of the force recording during the isometric impulsive contraction for two

  analysis Data are reported as mean ± SD in the text, the table and the figures. Assumption of normality (Shapiro-Wilk test) was tested for all variables and outliers were determined using a step of 1.5 x Interquartile range from the Turkey's Hinges. Intraclass correlation coefficient (ICC, Two-way mixed model), standard error of the measurement (SEM), average CV and repeated-measure analysis of variance (ANOVA) were used to assess reliability of RFD measures[START_REF] Weir | Quantifying Test -Retest Reliability Using the Intraclass Correlation Coefficient and the SEM[END_REF] on both groups pooled together and separated. Independent samples T-tests were used to compare Fmax, EMGmax, and CV between groups. Mann-Withney U Tests were used to compare time to RFDmax, absolute and relative RFDmax. Two-way ANOVAs were performed with age as between-subjects factor and time as within-subjects factor for all the remaining force and EMG-derived variables. Sphericity (Mauchly test) were tested for all dependent variables. If the assumption of sphericity was violated, the correction of Greenhouse-Geisser was applied. When significant main effects were observed, Bonferroni's test was used for post-hoc analysis. Significance was set as P<.05 for all tests. Statistical analysis was conducted using IBM SPSS software (Version 23.0, SPSS, Inc, Chicago, IL).

Figure 2 .

 2 Figure 2. Normalized RFD performance of young vs very old adults. A: Differences in the

  Fmax= maximal force; EMGmax: maximal EMG; RFD= rate of force development; RFDmax=maximal rate of force development; tRFDmax= time to the maximal rate of force development. a = from an outlier; *= P<0.05; #= P<0.01.

Figure 3 .

 3 Figure 3. Differences between young men (empty circles) and very old men (black squares)

  time interval (RER30: P=0.003; RER50: P<0.001; RER75: P=0.007; Figure4B). Electromechanical delay was longer in the very old men (76.8±27.5 ms) than in the young men (37.4±0.9 ms; P<0.001).

Figure 4 .

 4 Figure 4. Comparison between young (white) and very old (black) men in the RER

  to technical issues, the EMG signals for two very old men were unusable and thus discarded. Thus, EMG parameters are presented for 13 very old men. Absolute values for Fmax, EMGmax, RFD, RFDmax and tRFDmax are presented in Table1. Reliability of RFD

	measurement was excellent either when both groups were pooled together (ICC=0.98,
	CI=0.97-0.99; SEM=893.87 N*s -1 ; CV=11±6%; ANOVA: F(4,104)=0.84, P=.50) and when the
	young (ICC=0.96, CI=0.91-0.98; SEM=998.78 N*s -1 ; CV=12±6%; ANOVA: F(4,44)=2.10,
	P=.10) and very old men (ICC=0.98, CI=0.96-0.99; SEM=810.80 N*s -1 ; CV=9±6%;

ANOVA: F(4,56)=0.27, P=.90) were analyzed separately. Fmax was consistent between

Table 1 .

 1 Absolute force, RFD and EMG data.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.