Lifan Zhu

Mingzhong Wang

Yinsheng Xu

Xianghua Zhang

Ping Lu
email: lupingwh@whut.edu.cn

Dual effect of ZrO 2 on phase separation and crystallization in Li 2 O-Al 2 O 3 -SiO 2 -P 2 O 5 glasses

Keywords: ZrO 2, glass ceramic, nucleation, phase separation, crystallization

ZrO 2 is an effective nucleation agent for low-expansion lithium aluminum silicate (LAS) glass-ceramic (GC) with high Al 2 O 3 content. However, the effect of ZrO 2 is still not fully understand in LAS glasses with low contents of Al 2 O 3 and P 2 O 5 . In this work, the effect of ZrO 2 on the phase separation and crystallization of Li 2 O-Al 2 O 3 -SiO 2 -P 2 O 5 glasses were investigated. The results revealed that ZrO 2 significantly increased T g and the crystallization temperature of Li 2 SiO 3 and Li 2 Si 2 O 5 crystals. Li 3 PO 4 crystals precipitated preferentially in the glass containing 3.6 mol% ZrO 2 wherein Zr was stable in the network and no precipitation of ZrO 2 nanocrystals was observed. Moreover, the separation of phosphate-rich phases in the as-quenched glasses increased with the addition of ZrO 2 . The findings of the study revealed a dual role of ZrO 2 . First, ZrO 2 acted as a glass network former rather than a nucleation agent, increasing glass viscosity and the nucleation barrier of Li 2 SiO 3 through its strong network connectivity. Secondly, as Zr preferentially combined with NBO to form Si-O-Zr linkages, a sufficient amount of charge-balancing Li + ions existed in the network, which promoted the separation of phosphate-rich phases.

It indicated that the incorporation of ZrO 2 contributes to the activation of the nucleation role of P 2 O 5 , thus contribute to the formation of nanocrystals and fine microstructure of GCs.

Introduction

Since the pioneering work of Stookey in the 1950s, [START_REF] Stookey | Catalyzed crystallization of glass in theory and practice[END_REF] much attention has been directed toward the development of glass ceramics (GCs). These materials exhibit highly desirable properties [START_REF] Zanotto | A Bright Future for Glass-Ceramic[END_REF] which result from the combination of the advantages of their amorphous matrix and those of the microcrystalline phase dispersed throughout. The properties of GCs strongly depend on the nature and composition of their crystalline phases. Lithium aluminum silicate (LAS) GCs have been extensively studied owing to their low thermal expansion coefficients, high mechanical strength, and transparency characteristics. LAS glasses are widely used in fireplaces, telescope mirrors, and dental restorative materials. [START_REF] Beall | Nanophase glass-ceramics[END_REF][START_REF] Neuville | Nucleation, growth and phase separation: from research to applications[END_REF][START_REF] Huang | A novel high-strength lithium disilicate glass-ceramic featuring a highly intertwined microstructure[END_REF] Based on the phase diagram of LAS glass, crystals with different characteristics can be formed in situ by adjusting the glass composition. [START_REF] Arvind | Evolution of crystalline phases as a function of composition and dwell time in lithium aluminium silicate glass-ceramics[END_REF] In particular, the addition of nucleation agents such as ZrO 2 or P 2 O 5 , [START_REF] Guo | Nucleation and crystallization behavior of Li 2 O-Al 2 O 3 -SiO 2 system glass-ceramic containing little fluorine and no-fluorine[END_REF][START_REF] Höche | ZrTiO 4 crystallisation in nanosized liquid-liquid phase-separation droplets in glass-a quantitative XANES study[END_REF] which form abundant uniform nucleation sites, allow for controlled crystallization to be achieved.

The effects of nucleation agents on crystallization are significantly influenced by the composition of the original glass; e.g., P 2 O 5 promotes crystallization in LAS glasses with low Al 2 O 3 contents, however crystallization is inhibited for high Al 2 O 3 content. 9-11 31 P solid-state nuclear magnetic resonance (NMR) analyses have indicated that this behavior is related to changes in the structural environment of phosphorus. [START_REF] Bischoff | Phase evolution in lithium disilicate glass-ceramics based on non-stoichiometric compositions of a multi-component system: structural studies by 29 Si single and double resonance solid state NMR[END_REF][START_REF] Huang | Crystallization of a high-strength lithium disilicate glass-ceramic: An XRD and solid-state NMR investigation[END_REF] In LAS glasses with low Al 2 O 3 content, phosphorus is mainly present as orthophosphate segregated in the glass network accelerates the precipitation of amorphous orthophosphate phases which provides . and promotes phase separation and crystallization. With increasing Al 2 O 3 content, phosphorus combines with aluminum to form a POAl complex, and according to the aluminum avoidance principle (i.e., Loewenstein's Rule), phosphorus is evenly dispersed in the glass network, which inhibits crystallization. [START_REF] Lee | Al-O-Al and Si-O-Si sites in framework aluminosilicate glasses with Si/Al = 1: quantification of framework disorder[END_REF][START_REF] Lee | Extent of intermixing among framework units in silicate glasses and melts[END_REF] A similar dependence was observed for ZrO 2 , which acts as an effective nucleation agent for low-expansion LAS GCs with high Al 2 O 3 content, [START_REF] Kleebusch | A modified B 2 O 3 containing Li 2 O-Al 2 O 3 -SiO 2 glass with ZrO 2 as nucleating agent-Crystallization and microstructure studied by XRD and (S)TEM-EDX[END_REF] thereby decreasing the crystallization temperature and promoting the precipitation of the β-quartz solid solution (ss). It was reported that Zr-rich liquid-liquid phase-separation droplets are transformed into ZrO 2 nanocrystals, serving as nucleation sites for the β-quartz ss. [START_REF] Kleebusch | Phase formation during crystallization of a Li 2 O-Al 2 O 3 -SiO 2 glass with ZrO 2 as nucleating agent-An X-ray diffraction and (S)TEM-study[END_REF][START_REF] Kleebusch | The formation of nanocrystalline ZrO 2 nuclei in a Li 2 O-Al 2 O 3 -SiO 2 glass-a combined XANES and TEM study[END_REF] However, ZrO 2 inhibits crystallization in LAS glasses with low Al 2 O 3 content. Khalkhali et al. [START_REF] Khalkhali | Mechanical and Chemical Properties of Zr and P-Doped Lithium Disilicate Glass Ceramics in Dental Restorations[END_REF] reported that ZrO 2 significantly increases the crystallization temperature of the LAS glasses, the final GCs are coarse. Additionally, ZrO 2 was found to completely inhibit crystallization in stoichiometric Li 2 O-2SiO 2 glass and induce significant surface crystallization. [START_REF] Thieme | Nucleation and growth kinetics and phase analysis in zirconia-containing lithium disilicate glass[END_REF] For LAS glasses co-doped with ZrO 2 and P 2 O 5 , the effects of ZrO 2 are even more complex. Apel et al. [START_REF] Apel | Influence of ZrO 2 on the crystallization and properties of lithium disilicate glass-ceramics derived from a multi-component system[END_REF] reported that for a multi-component Li The crystalline phase and morphology of the heat-treated samples were analyzed by X-ray diffraction (XRD) and SEM, respectively. Because the effects of the nucleating agent strongly depend on the glass structure, Raman spectroscopy was used to investigate the origin of these effects from a structural perspective.

Experimental

Glass preparation

SiO 2 (99.98 %), Li 2 CO 3 (99.90 %), Al 2 O 3 (99.80 %), NH 4 H 2 PO 4 (≥99.0 %), and ZrO 2 (99.8 %) were used as starting materials. A standard melting-quenching technique was used to prepare glasses with nominal compositions (Table 1) of xZrO 2 -(100-x)(0.725SiO 2 -0.05Al 2 O 3 -0.215Li 2 O-0.01P 2 O 5 ) (x = 0, 1.8, 3.6 mol%), labeled as P1Z0, P1Z2, and P1Z4, respectively, and a P 2 O 5 -free glass with a composition of 73.5SiO 2 -5Al 2 O 3 -21.5Li 2 O (P0Z0; reference). For each composition, consecutively heated to 560, 620, 680, 700, 720, 750, 770, and 820 °C, while .

(90/10 wt%) crucible. The melt was quenched by pouring onto a copper plate and then immediately pressed by a second plate to maximize the cooling rate.

Glass compositions (Table 1) were determined by X-ray fluorescence spectroscopy (XRF, PANalytical Axios, Netherlands), while Li 2 O content was determined by atomic absorption spectrometry (AAS, CONTRAA-700, Analytik Jena, Germany). The relative errors of these two analytical methods were 0.05 and <0.01 mol%, respectively.

Heat treatment schedule

Differential scanning calorimetry (DSC) was used to determine the required heat treatment temperatures. As-quenched glasses were broken into small pieces prior to heat treatment and a heating rate of 10 °C/min was applied. All samples were quenched to 25 °C after heat treatment. The heat treatment conditions were listed in Table 2.

Schedule I. Nucleation. To investigate the effect of ZrO 2 on phase separation and nucleation, the samples were preheated within the typical range of nucleation temperatures, ~60 °C above the glass transition temperature (T g ).

P1Z0 was heated at 540 °C for 2 and 4 h, P1Z2 was heated at 560 °C for 2 and 4 h, and P1Z4 was heated at 580 °C for 2 and 4 h. Schedule II. Crystal growth. To study the effect of ZrO 2 on crystallization temperature, crystallization sequence, and crystallinity, P1Z0 was P1Z4 was similarly heated to 630, 680, 720, 750, 790, 820, 870, and 900 °C.

Samples were held at each temperature for 10 min to monitor the crystalline phase evolution, prior to the subsequent temperature step. Schedule Ⅲ. Primary phase evolution. To study the possible effect of ZrO 2 on the primary crystalline phase. P1Z0 was heated at 575 °C for 10, 30, and 60 min, while P1Z4 was heated at 650 °C for 10, 30, and 60 min.

Characterization

Glass granules (40 mg each) were subjected to DSC measurements (STA 449 F1, Netzsch, Germany) in the temperature range of 40-1000 °C at a heating rate of 10 °C/min in a flow of Ar. The obtained DSC curves were used to determine T g , onset of crystallization (T c ), and peak of crystallization (T p ) (Table 3). The error in the determination of T g , T c , and T p was estimated as ±2 °C.

Amorphous and crystalline phases were analyzed by XRD (PANalytical X'Pert Pro, Netherlands) using a Cu K α radiation source at 40 kV and 40 mA. Measurements were performed within the 2θ range of 10-80° at a scan rate of 2°/min and a step size of 0.013°/s. The crystallinities (X c ) of Schedule-I samples were calculated from the positions and relative intensities of their XRD peaks as follows: [START_REF] Zheng | Effects of P 2 O 5 and heat treatment on crystallization and microstructure in lithium disilicate glass ceramics[END_REF] % 100

a ci ci C      KI I I X , (1)
where I a and I ci are the integrated intensities of amorphous and crystalline phase peaks, respectively, while K is a constant that is related to the measurement conditions and glass composition (K = 0.963 for Li 2 Si 2 O 5 GCs).

Field-emission scanning electron microscopy (SEM; Hitachi S-4800, 5 kV, Japan) was used to observe phase separation before crystallization. The GC fracture surface was etched with 2 vol% aqueous HF for 25 s, cleaned by ultrasonication in deionized water for 10 min, and then sputter coated with platinum for 30 s to avoid the charging effect during SEM analysis.

Structural analysis was performed using a confocal laser Raman microscope (DXR, Thermo Electron, USA) equipped with a 532 nm laser operating at 100 mW.

The spectra of quenched glasses with cleaned surfaces were acquired at 25 °C in the wavenumber region of 100-1400 cm -1 . The signals at 850-1250 cm -1 were deconvoluted using Gaussian functions. Prior to deconvolution, spectra were normalized and the temperature and frequency dependency of the scattering intensity was corrected using a correction factor. [START_REF] Long | Raman spectroscopy[END_REF] 3. Results

Thermal properties

The DSC curves of as-quenched and preheated glasses are shown in Figure 1.

In the former case, the increase in T g with increasing ZrO 2 content was attributed to the increase in network connectivity. [START_REF] Matusita | Study on crystallization of glass by differential thermal analysis. Effect of added oxide on crystallization of Li 2 O-SiO 2 glasses[END_REF] For P1Z0 exothermic peaks were . observed at 636 and 744 °C which were shifted to higher temperatures for P1Z2.

Specifically, the peak at 636 °C shifted to 696 °C and a small peak appeared near 655 °C. The curve of P1Z4 showed only a small peak near 694 °C, indicating that ZrO 2 inhibited glass crystallization for this composition. It is unclear if peak observed at ~655 °C for P1Z2 represented glass crystallization, as it was less sharp than typical crystallization peaks. Similarly, the origin of the peak observed near 694 °C for P1Z4 was also uncertain.

Since preheating promoted crystallization, the exothermic peaks of preheated samples were broader and located at lower temperatures compared to the as-quenched samples. For P1Z2, after 2 h of preheating at 560 °C, the peak near 655 °C significantly decreased and was not present after 4 h of preheating.

In the case of P1Z4, preheating significantly reduced the intensity of the peak at approximately 694 °C and resulted in the appearance of an exothermic peak near 850 °C. Given that DSC is sensitive to the thermal history of samples and since fully crystallized samples do not exhibit crystallization peaks, the changes observed occurred during preheating.

Phase-separation behavior

The XRD patterns and Raman spectra of as-quenched and preheated glasses are shown in Figure 2. Excluding the P1Z0 sample preheated at 540 °C for 4 h, all XRD patterns featured only amorphous phase peaks, indicating the absence of crystalline phases (Figure 2A). Essentially, P1Z2 and P1Z4 did not crystallize .

during preheating and the small peak near 655 °C in the DSC curve of P1Z2 is not attributable to crystallization. The peaks of P1Z0, preheated at 540 °C for 4 h, were assigned to crystalline lithium metasilicate (Li 2 SiO 3 ; PDF No.

29-0829

) and this is further confirmed by the observation of peaks at 608 and 981 cm -1 in the corresponding Raman spectrum (Figure 2B). [START_REF] Richet | Melting and premelting of silicates: Raman spectroscopy and X-ray diffraction of Li 2 SiO 3 and Na 2 SiO 3[END_REF] The major preheating-induced change in the Raman spectra is observed near 950 cm -1 (black dashed line in Figure 2B). With increasing preheating duration, the bands of P1Z0 and P1Z2 (initially at 958 cm -1 ) shifted to 950 cm -1 and became sharper. In the case of P1Z4, after 4 h of preheating a sharp band at 950 cm -1 became more visible and was assigned to the characteristic [PO 4 ] group vibration [START_REF] Popović | Raman spectroscopic study of phase transitions in Li 3 PO 4[END_REF] . Thus, preheating mainly resulted in structural changes in the phosphorus environment, which was predicted to be more ordered from the accumulation of orthophosphate units, as indicated by the peak sharpening of the 950 cm -1 peak. [START_REF] Glatz | Different roles of phosphorus in the nucleation of lithium aluminosilicate glasses[END_REF] Further discussion of the Raman spectra of the as-quenched glasses is presented in section 3.5.

Morphology

The SEM micrographs of as-quenched and preheated samples are shown in Figure 3. Prior to analyzing sample morphology, the ability of HF to easily erode phosphate-rich and lesser polymerized phases was confirmed. The HF-eroded areas were found to be rich in phosphorus and lithium since phosphorus is easily separated from the network and forms isolated orthophosphate groups which spectra did not indicate nucleation and crystallization of Li 2 SiO 3 (Figure 2). . attract Li + ions. [START_REF] Mysen | The structural role of phosphorus in silicate melts[END_REF][START_REF] O'donnell | Effect of P 2 O 5 content in two series of soda lime phosphosilicate glasses on structure and properties-Part I: NMR[END_REF] All as-quenched glasses exhibited nanoscale granular or vermicular phase-separation structures with sizes of <20 nm. P1Z0 exhibited an interconnected morphology with closely clustered particles and poorly developed eroded ravines (Figure 3A). Compared to P1Z0, P1Z2 exhibited a partially interconnected morphology, with more pronounced eroded ravines and some isolated phase-separation droplets (Figure 3B); however, some traces of connectivity between droplets remained present. Conversely, P1Z4 featured a prominent phase-separation morphology with isolated droplets and clearly eroded ravines (Figure 3C). The addition of ZrO 2 affects the phase-separation morphology and results in an increased amount of separated phosphate-rich phases in as-quenched glasses.

With increased preheating time, the eroded ravines became more prominent in all cases, and isolated droplet phase-separation structures were observed after 4 h of preheating. As reported by Glatz et al., [START_REF] Glatz | Structural evolution at short and medium range distances during crystallization of a P 2 O 5 -Li 2 O-Al 2 O 3 -SiO 2 glass[END_REF] the separation of phosphate-rich phases is correlated to the nucleation of Li 2 SiO 3 in LAS glasses with low Al 2 O 3 content. Black erosion pits appeared between particles after 2 h of preheating (Figure 3D) and increased in size to ~30 nm after 4 h of preheating (Figure 3G). The erosion pits were associated with the Li 2 SiO 3 crystals observed in Figure 2A, which were easily eroded by HF because of their low degree of polymerization. [START_REF] Krüger | Nucleation kinetics of lithium metasilicate in ZrO 2 -bearing lithium disilicate glasses for dental application[END_REF] More eroded areas were observed as the preheating time was increased (Figures 3H and3I) and the corresponding XRD patterns and Raman . Furthermore, since HF mainly eroded phosphate-rich phases, it was concluded that preheating increased the content of these phases in P1Z2 and P1Z4. This behavior is consistent with the results of Raman spectroscopy (Figure 2B) which suggests that for P1Z2 and P1Z4, the preheating treatment induced changes in only the phosphorus environment. Therefore, the small peak near 655 °C in the DSC curve of P1Z2 (Figure 2B) was attributed to increased phase separation.

Crystallization and primary phase evolution

Based on the significant difference between the DSC curves of P1Z0 and P1Z4, the effect of ZrO The XRD patterns in Figure 5A reveal the first crystal phases to precipitate in P1Z0 and P1Z4 glasses. In P1Z0, precipitation preferentially resulted in Li 2 SiO 3 and Li 2 Si 2 O 5 crystals, as reported by Huang et al., [START_REF] Huang | Ex situ XRD, TEM, IR, Raman and NMR spectroscopy of crystallization of lithium disilicate glass at high pressure[END_REF] (at 609 and 981 cm -1 ). [START_REF] Richet | Melting and premelting of silicates: Raman spectroscopy and X-ray diffraction of Li 2 SiO 3 and Na 2 SiO 3[END_REF] The spectra of P1Z4 heated at 650 °C for 30 and 60 min exhibited a sharp signal at 951 cm -1 attributed to Li 3 PO 4 crystals with higher crystallinity. [START_REF] Kowada | Raman spectra of rapidly quenched glasses in the systems lithium borate-lithium silicate-lithium phosphate (Li 3 BO 3 -Li 4 SiO 4 -Li 3 PO 4 and Li 4 B 2 O 5 -Li 6 Si 2 O 7 -Li 4 P 2 O 7 )[END_REF] This band was also observed for P1Z0, although no Li 3 PO 4 peaks were observed in the corresponding XRD pattern, which may be owing to low crystallinity.

of the Q 2 , Q 3 , and Q 4 units, respectively. [START_REF] Mysen | The structural role of aluminum in silicate melts-a Raman spectroscopic study at 1 atmosphere[END_REF] The peak at 1004 cm -1 was assigned .

Glass structure

Figure 6 presents the Raman spectra of as-quenched glasses. The region between 400 and 600 cm -1 corresponds to the bending vibrations of the bridging oxygen (BO) bonds of SiO 4 tetrahedra. [START_REF] Ficheux | Structural evolution of high zirconia aluminosilicate glasses[END_REF] The band near 490 cm -1 indicated the presence of four-membered rings, while the long tail at lower frequencies was attributed to the stretching vibration of oxygen bonds in high-membered rings. [START_REF] Galeener | Planar Rings in Vitreous Silica[END_REF] The frequency changes in this region (marked with black arrows) are associated with silicate network deformation and ring statistic modification due to the presence of Zr atoms. [START_REF] Lu | Quantitative Structure-Property Relationship (QSPR) Analysis of ZrO 2 -Containing Soda-Lime Borosilicate Glasses[END_REF] The band near 601 cm -1 was attributed to the rocking motion of bonds to BO in structural units containing non-bridging oxygen (NBO), which is characteristic of defect bonds in the silicate network. [START_REF] Brawer | Raman spectroscopic investigation of the structure of silicate glasses (II). Soda-alkaline earth-alumina ternary and quaternary glasses[END_REF] With increasing ZrO 2 content, the intensity of this band (and hence the NBO content) decreased while the intensity of the band at 800 cm -1 remained nearly unchanged. For an enhanced illustration of the structural changes caused by ZrO 2 , the high-frequency, unresolved group of bands over the 850-1250 cm -1 range was fitted using 4-6

Gaussian peaks (Figure 7) and the extracted parameters are listed in Table 4.

Peaks within the region of 850-1250 cm -1 conventionally correspond to the stretching vibrations of the Si-O bonds within SiO 4 tetrahedra. These are often described as Q n units (where n is the number of BO atoms per tetrahedron), and provide important information on the glass structure. [START_REF] Mcmillan | Structural studies of silicate glasses and melts-applications and limitations of Raman spectroscopy[END_REF] For P1Z0, the peaks at 958, 1068, and 1152 cm -1 were assigned to the symmetric stretching vibrations thereby increasing the degree of polymerization of the glass network.

With .

as a T 2 vibrational mode of SiO 4 tetrahedra. [START_REF] Losq | The role of Al 3+ on rheology and structural changes in sodium silicate and aluminosilicate glasses and melts[END_REF] The peak at 957 cm -1 was attributed to the characteristic vibration of PO 4 tetrahedra, representing the structural environment of phosphorus in glass. [START_REF] Popović | Raman spectroscopic study of phase transitions in Li 3 PO 4[END_REF] The peak near 990 cm -1 observed for P1Z2 and P1Z4 was assigned to the Si-O-Zr vibration in borosilicate glass by comparison with the Raman spectrum of zektzerite (NaLiZrSi 6 O 15 ), in which Zr 4+ ions are six-fold coordinated. [START_REF] Quintas | ZrO 2 addition in soda-lime aluminoborosilicate glasses containing rare earths: Impact on the network structure[END_REF] This band was assigned to one Si atom linked to three Si and one Zr (i.e., a ZrO 6 octahedra) neighboring atoms by BO atoms with Li + and Na + as charge-balancing cations. Therefore, the peak near 990 cm -1 was denoted as Q 3 (Zr), while the peak at 1068 cm -1 was denoted as Q 3 (Li). Zr can also connect with Q 2 units to form Q 2 (Zr, Li) units. In contrast Si, Zr has a larger cationic radius (0.59 Å for Zr 4+ vs. 0.26 Å for Si 4+ ) and atomic weight, [START_REF] Shannon | Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides[END_REF] resulting in an elongation of the average T-O (T = Si, Zr) bond length. This accounts for the lower frequency of Q 3 (Zr) units compared to Q 3 (Li) units and the decrease in the frequency of Q 2 units.

Figure 8 shows the effects of ZrO 2 content on the relative areas of Raman bands, revealing that increasing ZrO 2 content resulted in a decrease in the combined area of Q 3 (Li)+Q [START_REF] Neuville | Nucleation, growth and phase separation: from research to applications[END_REF] and an increase in the area of Q 3 (Zr). The increase in the number of Q 3 (Zr) units reinforced the network as some Li + ions acted as charge-balancing cations of the ZrO 6 octahedra instead of engaging in network depolymerization through the formation of NBO in Q 3 (Li) units. This effect of ZrO 2 on glass structure compensated for the decrease in the amount of Q 4 units, .

increasing ZrO 2 content, the area of PO 4 peaks slightly increased, while the peak position remained largely unchanged. The areal changes of the Q 2 and T 2 units are likely due to variation in the relative number of other units. content, as reported previously. [START_REF] Khalkhali | Mechanical and Chemical Properties of Zr and P-Doped Lithium Disilicate Glass Ceramics in Dental Restorations[END_REF][START_REF] Apel | Influence of ZrO 2 on the crystallization and properties of lithium disilicate glass-ceramics derived from a multi-component system[END_REF] The DSC crystallization peak area is proportional to the remaining amount of residual glass, which depends on both the number of nuclei and the crystal growth rate. [START_REF] Zheng | Understanding Glass through Differential Scanning Calorimetry[END_REF] In the DSC curve of as-quenched P1Z0, the area of the first crystallization peak decreased after addition of contributed to this inhibitory effect. Indeed, the nucleation of Li 2 SiO 3 is known to be sensitive to kinetic parameters. [START_REF] Gaddam | The roles of P 2 O 5 and SiO 2 /Li 2 O ratio on the network structure and crystallization kinetics of non-stoichiometric lithium disilicate based glasses[END_REF] Considering the nucleation process, Li 2 SiO 3 is formed from both Q 2 and Q 3 units: 12

Q 2 (glass) ↔ Q 2 (crystal) 2Q 3 (glass) ↔ Q 2 (crystal) + Q 4 (glass) [disproportionation]
Based on the structural analysis, Zr can connect with Q 2 (Li) units to form Q 2 (Zr, Li) units and thus reduce the number of Q 2 (Li) units available to directly forming Li 2 SiO 3 . Moreover, the Si-O-Zr bond is stronger than Si-O-Li bond, and the disproportionation reaction is suppressed because the P1Z4 network features more Q 3 (Zr) units and fewer Q 3 (Li) units. Consequently, the barrier towards nucleation of Li2SiO3 increases and a higher nucleation temperature is required.

The crystallization of Li 2 SiO 3 was reported to be closely related to that of Li 2 Si 2 O 5 [START_REF] Höland | Studies of crystal phase formations in high-strength lithium disilicate glass-ceramics[END_REF] which explains the suppression of glass nucleation and the increase in the crystallization temperatures of Li 2 SiO 3 and Li 2 Si 2 O 5 with increasing ZrO 2 content.

The effects of heat treatment on the crystallinity of P1Z0 and P1Z4 are shown in Figure 9. The average grain size of Li 2 SiO 3 crystals precipitated in P1Z0 and P1Z4 was calculated using the Scherrer equation. During the early stage of crystallization, the crystallinity of P1Z4 was lower than that of P1Z0, while the grain size of Li 2 SiO 3 crystals in P1Z4 (~53 nm) exceeded that observed for with the proposed inhibitory effect of ZrO 2 on glass nucleation. Moreover, the highest crystallinity of P1Z4 was 18 % lower than that of P1Z0. Generally, when ZrO 2 is used as a nucleation agent in low-expansion LAS GCs, heterogeneous Zr-rich phase-separation droplets are preferentially formed during heat treatment which then gradually transform into ZrO 2 nanocrystals, providing heterogeneous nucleation sites. [START_REF] Kleebusch | The formation of nanocrystalline ZrO 2 nuclei in a Li 2 O-Al 2 O 3 -SiO 2 glass-a combined XANES and TEM study[END_REF] Herein, no ZrO 2 precipitation was observed during the crystallization of P1Z4. Based on the lower crystallinity of P1Z4 and the presence of numerous residual glass phases therein, the ZrO 2 content did not reach saturation. Notably, P1Z4 contains a large amount of Li 2 O which resulted in a lower degree of polymerization wherein ZrO 2 is more soluble. [START_REF] Kato | The corrosion of zircon and zirconia refractories by molten glasses[END_REF] Cormier et al. [START_REF] Cormier | Zr environment and nucleation role in aluminosilicate glasses[END_REF] suggested that the nucleation effect of Zr was caused by its distribution in the aluminosilicate matrix where direct connections between ZrO x polyhedra are required for ZrO 2 to function as a nucleation agent. The linkages between these ZrO x polyhedra increase the short-range order of the glass structure, leading to the separation of Zr-rich phases and the precipitation of ZrO 2 nuclei. However, the distribution of Zr is more regular in aluminosilicate glass networks with low degrees of polymerization. [START_REF] Frages | Structural environments of incompatible elements in silicate glass/melt systems-Zirconium at trace levels[END_REF] Recently, Cicconi et al. [START_REF] Cicconi | Nucleation mechanisms in a SiO 2 -Li 2 O-P 2 O 5 -ZrO 2 biomedical glass-ceramic: Insights on crystallisation, residual glasses and Zr 4+ structural environment[END_REF] reported the occurrence of the sub-nano Zr local clusters in a Zr-rich SiO 2 -Li 2 O-P 2 O 5 -ZrO 2 glass having a SiO 2 /Li 2 O molar ratio < 2, while the local environment of Zr 4+ is not dramatically changed during the different heat treatments, either when higher temperatures or longer times. This means that the connections between ZrO x polyhedra are unlikely to form in P1Z4, or even .

if Zr-rich clusters are formed, the absence of Zr-bearing crystals upon crystallisation of P1Z4 indicates that these aggregations do not evolve above the critical nucleus size. Consequently, ZrO 2 does not act as a nucleation agent in P1Z4.

Effect of ZrO 2 on phase-separation behavior

The separation of phosphate-rich phases or the formation of disordered Li 3 PO 4 phases was reported to occur prior to the crystallization of Li 2 SiO 3 , and highly crystalline Li 3 PO 4 was only formed at relatively higher temperatures. [START_REF] Bischoff | Phase evolution in lithium disilicate glass-ceramics based on non-stoichiometric compositions of a multi-component system: structural studies by 29 Si single and double resonance solid state NMR[END_REF][START_REF] Huang | Ex situ XRD, TEM, IR, Raman and NMR spectroscopy of crystallization of lithium disilicate glass at high pressure[END_REF] XRD and Raman spectroscopy revealed the preferential crystallization of Li 3 PO 4 in P1Z4 (Figure 5). The relatively higher content of phosphate-rich regions in P1Z4 facilitated the crystallization of Li 3 PO 4 , as the composition prerequisites were fulfilled. Combining the results of SEM analysis and Raman fitting, the results indicate that ZrO 2 can promote phase separation by facilitating the separation of phosphate-rich phases. While it is known that ZrO 2 promotes the phase separation of Zr-rich droplets in low-expansion LAS GCs, [START_REF] Kleebusch | Phase formation during crystallization of a Li 2 O-Al 2 O 3 -SiO 2 glass with ZrO 2 as nucleating agent-An X-ray diffraction and (S)TEM-study[END_REF][START_REF] Kleebusch | The formation of nanocrystalline ZrO 2 nuclei in a Li 2 O-Al 2 O 3 -SiO 2 glass-a combined XANES and TEM study[END_REF] there is no direct evidence that ZrO 2 promotes phase separation in Li 2 O-Al 2 O 3 -SiO 2 -P 2 O 5 -ZrO 2 glasses.

The SEM results of as-quenched samples (Figure 3A-C) indicates that phase separation occurs spontaneously in the original glass composition. As the ZrO 2 content increased, phosphate-rich phases increased and gradually connected, which caused silicon-rich regions to appear as isolated droplets (Figure 3B-C).

The kinetics of phosphate-rich phase separation is controlled by atomic coordination number of Zr may increase without sufficient chargebalancing .

diffusion, thus, the temperature and time of heat treatment significantly affect the evolution of phase separation. With the extension of heat treatment time, the separated phosphate-rich phases increase, which is consistent with that observed in Figure 3D-I. Notably, the development of phase separation of samples containing ZrO 2 is more obvious, which indicates that ZrO 2 increases the immiscibility of glass.

The connection between Zr and the glass network has been found in several glass systems (e.g., borosilicate and aluminosilicate). [START_REF] Ficheux | Structural evolution of high zirconia aluminosilicate glasses[END_REF][START_REF] Quintas | ZrO 2 addition in soda-lime aluminoborosilicate glasses containing rare earths: Impact on the network structure[END_REF] In these glass networks, Zr 4+ ions are nearly six-fold coordinated, and ZrO 6 octahedra share corners with SiO 4 or AlO 4 tetrahedra, [START_REF] Ferlat | Evidence for symmetric cationic sites in zirconium-bearing oxide glasses[END_REF][START_REF] Jollivet | An enhanced resolution of the structural environment of zirconium in borosilicate glasses[END_REF] as shown in Figure 10A. The origin of the promotional effect of ZrO 2 on the formation of phosphate-rich phases is illustrated by a structural scheme inspired by the models proposed by Quintas [START_REF] Quintas | ZrO 2 addition in soda-lime aluminoborosilicate glasses containing rare earths: Impact on the network structure[END_REF] and Greaves [START_REF] Greaves | EXAFS and the structure of glass[END_REF] (Figure 10B). As Zr enters the glass network, the solubility of phosphorus in the highly polymerized glass network decreases [START_REF] Mysen | The structural role of phosphorus in silicate melts[END_REF] and six-coordinated Zr is expected to preferentially bind to NBO. [START_REF] Frages | Structural environments of incompatible elements in silicate glass/melt systems-Zirconium at trace levels[END_REF] The formation of Si-O-Zr linkages transforms a large number of Li + ions, that are combined with NBO as modifying ions, into charge-balancing ions. Thus, the mobility of Li + ions are increased. The yellow arrows in Figure 10B show that phosphorus attracts the charge-balancing ions of ZrO 6 polyhedra, inducing the aggregation of Li + ions in the network. As P 5+ provides a stronger ionic field than Zr 4+ , the former ion has a stronger ability to attract charge-balancing ions, while the separation of Zr-rich droplets or precipitation of ZrO 2 crystals occurred.

Zr .

ions. The isolated PO 4 3-units mainly occur in depolymerized regions. The dotted lines in Figure 10B indicate the separation between the depolymerized and polymerized regions, indicating the separation of phosphate-rich phases. Lin et al. [START_REF] Lin | Composition dependent structure and elasticity of lithium silicate glasses: Effect of ZrO 2 additive and the . combination of alkali silicate glasses[END_REF] reported that in a binary lithium silicate glass, ZrO 2 promotes the separation of a depolymerized lithium-rich phase and a silicon-rich phase.

Considering the affinity between phosphorus and lithium, the formation of separated phosphate-rich phases was expected in this study.

Phase separation is closely related to glass nucleation and the crystallization processes. Pre-crystallization phase separation during heat treatment has been observed for many glasses containing nucleation agents. . Tables Table 1. Nominal (in parentheses) and actual glass compositions, mol%.

Glass

2

 2 Si 2 O 5 glass (containing minor amounts of Al 2 O 3 , K 2 O, and P 2 O 5 ), ZrO 2 did not precipitate as nanocrystal nuclei or form crystalline phases, but increased the glass viscosity and hindered the growth of Li 2 Si 2 O 5 crystals. Krüger et al. 22 studied the nucleation kinetics of a similar Li 2 Si 2 O 5 glass where it was assumed that ZrO 2 a 150 g batch was thoroughly mixed and melted at 1590 °C for 3 h in a Pt-Rh . numerous heterogeneous nucleation sites. The effects of ZrO 2 in multi-component Li 2 Si 2 O 5 -based glasses is not completely understood, particularly for glasses co-doped with ZrO 2 and P 2 O 5 . Elucidating the intrinsic role of the nucleation agent will enable improved control of the crystallization of GCs and further enhancement of their properties. Herein, we examined the effects of ZrO 2 on crystallization in multi-component Li 2 O-Al 2 O 3 -SiO 2 -P 2 O 5 glasses. Multi-stage thermal treatments were used to study the related phase-separation and crystallization behaviors.

  while Li 3 PO 4 crystals preferentially precipitated in P1Z4. The Raman spectrum of P1Z0 heated at 575 °C for 60 min (Figure 5B) exhibited bands of Li 2 Si 2 O 5 crystals 33 (at 414 and 1106 cm -1 ) and peaks corresponding to the Si-O-Si vibration in crystalline Li 2 SiO 3

4 . Discussion 4 . 1

 441 Effect of ZrO 2 on crystallization behavior DSC results indicate that glass crystallization was hindered by high ZrO 2

1. 8 mol%

 8 ZrO 2 . While, Li 2 SiO 3 and Li 2 Si 2 O 5 crystals precipitated preferentially in P1Z0, indicating the weakening of nucleation and crystal growth of Li 2 SiO 3 and Li 2 Si 2 O 5 with the addition of ZrO 2 . SEM and XRD data show that nucleation of Li 2 SiO 3 only occurred in P1Z0 during preheating, and no crystal nuclei were observed in P1Z2 and P1Z4 even after 4 h of preheating in the common nucleation temperature range. As shown in Figure 4, the crystallization temperature of Li 2 SiO 3 and Li 2 Si 2 O 5 increased by 100 °C. The addition of ZrO 2 increases the polymerization degree of glasses, resulting in higher viscosity, thus, it hinders atomic diffusion and the increases. kinetic barrier of crystallization 21, 22 Additionally, the change of glass structure upon ZrO 2 incorporation also P1Z0 (~21 nm). Thus, fewer Li 2 SiO 3 crystals precipitated in P1Z4, which agrees .

Figure 1 .

 1 Figure 1. DSC curves of as-quenched and preheated samples according to Schedule

Figure 2 .

 2 Figure 2. (A) XRD patterns and (B) Raman spectra of as-quenched and preheated

Figure 3 .

 3 Figure 3. Micrographs of (A-C) as-quenched [A: P1Z0; B: P1Z2, C: P1Z4] and (D-

Figure 4 .

 4 Figure 4. XRD patterns of (A) P1Z0 heat-treated from 560 to 820 °C for 10 min

Figure 5 .

 5 Figure 5. (A) XRD patterns and (B) Raman spectra of P1Z0 and P1Z4 heat-treated

Figure 6 .

 6 Figure 6. Raman spectra of as-quenched samples.

Figure 7 .

 7 Figure 7. Deconvoluted Raman spectra of (A) P0Z0, (B) P1Z0, (C) P1Z2, and (D)

Figure 8 .

 8 Figure 8. Effects of ZrO 2 content on the normalized relative areas of fitted

Figure 9 .

 9 Figure 9. Crystallinities (%) of P1Z0 and P1Z4 heat-treated according to

Figure 10 .

 10 Figure 10. (A) Stereoscopic structural diagram of the connections between ZrO 6

  Li x Al x Si 3-x O 6 ; PDF No. 31-0707), rapidly became more abundant above 720 °C, developed into the main crystalline phase of the GC, and then transformed into β-spodumene ss (Li x Al x Si 3-x O 6 ; PDF For P1Z4, Li 2 SiO 3 , Li 2 Si 2 O 5 , LiAlSi 4 O 10 , and β-quartz ss, crystals precipitated at relatively higher temperatures. However, LiAlSi 4 O 10 crystals did not disappear, and the transformation of β-quartz ss into β-spodumene ss was not observed in the investigated temperature range. Moreover, cristobalite (SiO 2 ; PDF No. 27-0605) crystals appeared at 720 °C and increased until 750 °C, then it decreased rapidly with the growth of Li 2 Si 2 O 5 . As it has been reported that cristobalite precipitated during the transformation from Li 2 SiO 3 into Li 2 Si 2 O 5 .[START_REF] Höland | Studies of crystal phase formations in high-strength lithium disilicate glass-ceramics[END_REF][START_REF] Huang | Ex situ XRD, TEM, IR, Raman and NMR spectroscopy of crystallization of lithium disilicate glass at high pressure[END_REF]

	No. 35-0794) at 820 °C. 31
	The XRD pattern of P1Z4 heated at 680 °C for 10 min exhibited peaks of Li 3 PO 4

2 on crystallization was mainly monitored for these compositions. During the heat treatment of P1Z0, both Li 2 SiO 3 (PDF No. 29-0829) and Li 2 Si 2 O 5 (PDF No. 40-0376) crystals precipitated at 620 °C and gradually grew until 680 °C (Figure 4A). Upon further heating, Li 2 Si 2 O 5 crystal content rapidly increased, and at 750 °C, Li 2 SiO 3 crystals disappeared. Petalite (LiAlSi 4 O 10 ; PDF No. 83-1470) crystals appeared at 700 °C, became slightly more apparent with heating to 750 °C, and then disappeared at 770 °C. Another aluminum-containing crystal phase, β-quartz ss (crystals at 33.9 and 36.5° (Figure 4B) that vanished as the crystallinity . increased.

  [START_REF] Glatz | Structural evolution at short and medium range distances during crystallization of a P 2 O 5 -Li 2 O-Al 2 O 3 -SiO 2 glass[END_REF][START_REF] Barry | The crystallizaion of glasses based on eutectic compositions in the system Li 2 O-Al 2 O 3 -SiO 2[END_REF][START_REF] Kleebusch | The evidence of phase separation droplets in the crystallization process of a Li 2 O-Al 2 O 3 -SiO 2 glass with TiO 2 as nucleating agent An X-ray diffraction and (S)TEM-study supported by EDX-analysis[END_REF] For example, when ZrO 2 and P 2 O 5 are used as nucleation agents, they easily ZrO 2 promoted the separation of phosphorus-rich phase from the original glass phase and thus promoted crystallization. Although these two effects seem contradictory, both originate from the structural role of ZrO 2 in glass, with the inhibitory effect on nucleation kinetics having the dominant role at high ZrO 2 content. The dual effect of ZrO 2 found in this work provided new insights into the roles of ZrO 2 and P 2 O 5 in low-Al 2 O 3 -content LAS GCs. The positive role of ZrO 2 on the crystallization was realized by the promoting effect on the separation of phosphorus-rich phase, rather than the nucleation of ZrO 2 itself. It indicated that appropriate ZrO 2 /P 2 O 5 ratio can minimize the inhibition of crystal growth and maximize the nucleation of P 2 O 5 , thus contribute to the formation of nanocrystals and fine microstructure of GCs.Structurally, we found that ZrO 2 had a dual effect. First, ZrO 2 inhibited crystallization by forming connections to the glass network that increased the viscosity and nucleation barrier of Li 2 SiO 3 , rather than resulting in the precipitation of ZrO 2 nanocrystals. Secondly, as Zr preferentially bonded to NBO, a sufficient amount of charge-balancing Li + ions existed in the network, which promoted the separation of phosphate-rich phases from the glass phase.

	was connected to the network, mainly affecting nucleation kinetics, increasing glasses and the development of phase separation during preheating. The
	glass viscosity, and inhibiting crystallization by forming strong network crystallization of Li 3 PO 4 was observed at a relatively lower temperature in P1Z4,
	aggregate in the glass matrix to form a short-range-ordered structure which promotes phase separation. The phase boundary between the phase-separation region and matrix features a lower interfacial free energy, while the sharp concentration gradient at the interface facilitates atomic diffusion. In this heated at 650 °C for 30 min, owing to its abundant phosphate-rich phases. Raman spectrum fitting showed that Zr was mainly connected to the glass network through Si-O-Zr linkages, which significantly enhanced network connectivity. Although this phase separation could improve glass crystallization, the dominant role of ZrO 2 was in the inhibition of glass crystallization. Thus, this study provides new insights into the roles of ZrO 2 and P 2 O 5 in low-Al 2 O 3 -content connections. Secondly, 5. Conclusion LAS GCs.
	study, ZrO 2 did not act as a nucleation agent but significantly promoted the The effect of ZrO 2 on the phase separation and crystallization in LAS glasses
	development of phase separation during preheating. In the DSC results, the with low Al 2 O 3 and P 2 O 5 contents was studied. Thermal analysis showed that with
	crystallization peaks of preheated samples shifted to relatively lower increasing ZrO 2 content, the crystallization peaks shifted to relatively higher
	temperatures and the curve of P1Z4 exhibited an additional exothermic peak near temperatures. For the glass containing 3.6 mol% ZrO 2 (P1Z4), the DSC curve was
	800 °C, which indicated enhanced glass crystallization. Therefore, in the case largely flat and a small crystallization peak was observed. The SEM and XRD
	of ZrO 2 and P 2 O 5 co-doped LAS GC, ZrO 2 exhibited a dual effect. First, no phase results revealed that ZrO 2 hindered nucleation and reduced crystallinity. Higher
	ZrO 2 content favored the separation of phosphate-rich phases in as-quenched
	. .

Table 2 .

 2 Heat treatment conditions.

		SiO 2	Al 2 O 3	P 2 O 5	Li 2 O	ZrO 2
	P1Z0	72.5(72.65)	5.1(5.00)	0.91(0.85)	20.1(21.50)	0(0)
	P1Z2	71.1(71.33)	4.9(4.91)	0.85(0.83)	19.9(21.13)	1.7(1.8)
	P1Z4	70.3(70.02)	4.9(4.82)	0.80(0.82)	19.5(20.74)	3.6(3.6)
	P0Z0	73.2(73.50)	4.8(5.00)	0.09(0)	20.5(21.50)	0(0)

.

Table 3 .

 3 Characteristic temperatures (±2 °C) determined by DSC, °C. 1, 2 and 3 in the subscripts of T cx and T px refer to the order of peaks.

	Glass	T g	T c1	T p1	T c2	T p2	T c3	T p3
	P1Z0*	480	594	636	714	744	-	-
	P1Z2*	501	-	655	674	696	740	769
	P1Z4	522	-	694	-	-	-	-

*

.

Table 4 .

 4 Raman shifts (cm -1 ), relative areas (%), and full-widths at half maximum (FWHM, cm -1 ) of peaks obtained after the deconvolution of Raman spectra.

		Q 2	T 2	Q 3	Q 4	PO 4	Q 3 (Zr)
	Raman shift
	P0Z0	956	1009	1071	1146	-	-
	P1Z0	958	1004	1068	1152	957	-
	P1Z2	938	1013	1064	1163	958	988
	P1Z4	930	1022	1067	1167	957	989
	Relative area
	P0Z0	12.41	10.88	58.11	18.67	-	-
	P1Z0	11.98	10.11	55.29	14.51	8.13	-
	P1Z2	12.64	4.73	56.75	7.08	8.67	10.12
	P1Z4	14.42	5.34	46.21	5.38	11.12	17.61
	FWHM
	P0Z0	66.5	53.1	86.1	100.3	-	-
	P1Z0	67.9	54.1	93.8	97.6	29.6	-
	P1Z2	64.4	44.1	111.7	82.9	32.8	44.1
	P1Z4	62.5	51.7	110.9	82.2	39.7	49.8

Acknowledgement This research study was funded by Xianning CSG Photovoltaic Glass Co., Ltd., [Grant No. XNGDJS2021082001]. The authors would also like to thank Dr. Jing Liu for SEM analyses performed at the State Key Laboratory of Advanced Technology for Materials Synthesis and Processing at Wuhan University of Technology, China.


Patricia Ballestra
email: ballest@u-bordeaux4.fr

Catherine Verret

Christian Cruz

Alain Largeteau

Gerard Demazeau

Abdelhamid El Moueffak

High pressure inactivation of Pseudomonas in black trufflecomparison with Pseudomonas fluorescens in tryptone soya broth

Keywords: high pressure, truffle, inactivation, Pseudomonas fluorescens, Pseudomonas

Pseudomonas is one of the most common genera in black Perigord truffle. Its inactivation by high pressure (100-500 MPa/10 min) applied on truffles at sub-zero or low temperatures was studied and compared with those of Pseudomonas fluorescens in tryptone soya broth. Pressurization of truffles at 300 MPa/4 • C reduced the bacterial count of Pseudomonas by 5.3 log cycles. Higher pressures of 400 or 500 MPa, at 4 • C or 20 • C, allowed us to slightly increase the level of destruction to the value of ca. 6.5 log cycles but did not permit us to completely inactivate Pseudomonas. The results showed a residual charge of about 10 CFU/g. Pressure-shift freezing of truffles, which consists in applying a pressure of 200 MPa/-18 • C for 10 min and then quickly releasing this pressure to induce freezing, reduced the population of Pseudomonas by 3.3 log cycles. The level of inactivation was higher than those obtained with conventional freezing. Endogenous Pseudomonas in truffle was shown to be more resistant to high pressure treatments than P. fluorescens used for inoculation of broths.

Introduction

Tuber melanosporum, well known as the Perigord black truffle, has important culinary and commercial value because of the flavors it imparts to foods [START_REF] Diaz | Truffle aroma characterization by headspace solid-phase microextraction[END_REF]. Truffles used as fresh products are minimally processed and stored at low temperature (4 • C), which permits preservation of their organoleptic properties and extends their shelf-life [START_REF] Saltarelli | Effect of storage on biochemical and microbiological parameters of edible truffle species[END_REF]. Truffles used for the production of a wide variety of food products such as oil, cheese, pasta and sauce are immediately frozen or sterilized in an autoclave at 100 • C for 3 h or at 120-130 • C for 30 min. Unfortunately, in these conditions, the sensory characteristics of the truffles are highly degraded [START_REF] Saltarelli | Effect of storage on biochemical and microbiological parameters of edible truffle species[END_REF][START_REF] Moueffak | Stabilization of black truffle of Perigord (Tuber melanosporum) by high pressure treatment[END_REF].

High pressure (HP) processing could be a promising alternative to stabilize black truffles [START_REF] Moueffak | Stabilization of black truffle of Perigord (Tuber melanosporum) by high pressure treatment[END_REF][START_REF] Ballestra | Effects of pressure-shift freezing on microorganisms and enzymes in black Perigord truffle (Tuber melanosporum)[END_REF][START_REF] Verret | The effect of high hydrostatic pressure on black truffle (Tuber melanosporum) flavour compounds[END_REF]. Generally, pressurization at refrigeration, ambient or moderate temperature allows inactivation of pathogenic and spoilage micro-organisms in foods with fewer changes in texture, color and flavor as compared with conventional technologies [START_REF] Torres | Commercial opportunities and research challenges in the high pressure processing of foods[END_REF]. A recent study showed that the treatment of black truffles by high pressure at low or sub-zero temperatures did not induce extensive modifications in major volatile compound concentrations [START_REF] Verret | The effect of high hydrostatic pressure on black truffle (Tuber melanosporum) flavour compounds[END_REF]. The present work focuses on the impact of such treatments on spoilage micro-organisms in truffles.

Microbial charge of freshly collected truffles is particularly extensive. The total mesophilic bacteria, including a large proportion of Pseudomonas, showed a range of 10 7 -10 8 CFU/g [START_REF] Barbieri | New evidence for bacterial diversity in the ascoma of ectomycorrhizal fungus Tuber borchii Vittad[END_REF][START_REF] Barbieri | Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation[END_REF][START_REF] Nazzaro | Evaluation of gamma rays influence on some biochemical and microbiological aspects in black truffles[END_REF]. Our objective was to study high pressure inactivation of Pseudomonas in black truffle (100-500 MPa/10 min) at sub-zero or low temperatures (-18 • C, 4 • C or 20 • C). Preliminary experiments were carried out on Pseudomonas fluorescens in suspension in tryptone soya broth.

Materials and methods

Fresh black truffles were harvested in Perigord (Dordogne, France). Fruit bodies were brushed, grated and mixed with great care. The strain used for preliminary experiments was Pseudomonas fluorescens CIP 69.13 (from the Institut Pasteur, Paris, France). The strain was grown in tryptone soya broth (TSB, Difco, Detroit, MI) at 37 • C for 18 h.

Samples consisting of 25 g of grated truffles or 25 mL of P. fluorescens suspensions were vacuum-packed in a sealed plastic bag and kept at 4 • C for a few hours before being hydrostatically pressurized in a 3-L stainless steel vessel using an hyperbar experimental apparatus from FRAMATOME (described previously in detail [START_REF] Ballestra | Effect of high-pressure treatment on polyphenoloxidase activity of the Agaricus bisporus mushroom[END_REF]). HP treatments were carried out at pressures ranging from 100 to 500 MPa. Temperatures of pressure-transmitting medium were -18 • C (only for pressurizations at 200 MPa), 4 • C or 20 • C. The treatment time was 10 min, excluding the pressure increase and release times.

HP treatments performed at 200 MPa/ -18 • C were followed by pressure release in 1 min 40 s, inducing significant ice nucleation in samples. Complete freezing was obtained by maintaining samples at -18 • C for a few hours before analyses. Such treatments are called pressure-shift freezing (PSF). In order to compare the effects of PSF with those of a classical freezing, some untreated samples (initially at 4 • C) were placed in deep freeze and stored at -18 • C for a few hours (the same time of storage as for pressurized samples).

Controls and samples pressurized at low temperature were kept at 4 • C for a few hours before analyses.

The number of surviving Pseudomonas was enumerated as CFU by surface plating on a cetrimide selective medium and counting after a 10 day incubation at 30 • C. Bacterial counts were subjected to analysis of variance using R software. Significance of differences was defined at p < 0.05.

Results and discussion

Effect of high pressures at low temperature

Table 1 shows the effect of high pressures at 4 • C or 20 • C on viable counts of Pseudomonas fluorescens in tryptone soya broth and Pseudomonas spp. in black truffles.

Pressurization of P. fluorescens suspensions at 300 MPa/4 • C led to the complete destruction of bacterial population. Between 100 and 300 MPa, the level of inactivation increased with pressure. At 200 MPa, it was higher at 4 • C than at 20 • C. These results are in accordance with those of Lopez-Caballero et al. [START_REF] Lopez-Caballero | Responses of Pseudomonas fluorescens to combined high pressure/temperature treatments[END_REF], indicating that the efficiency of HP in killing P. fluorescens decreased as the pressurization temperature increased between 5 • C and 35 • C. The application of high pressures on black truffles induced less extensive reductions of the Pseudomonas spp. population. Pressurization of truffles at 300 MPa/4 • C reduced the bacterial count of Pseudomonas by 5.3 log cycles. Higher pressures, 400 or 500 MPa, at 4 • C or 20 • C, allowed a slight increase in the level of destruction to the value of ca. 6.5 log cycles but did not permit complete inactivation of Pseudomonas. The results showed a residual charge of about 10 CFU/g.

Effect of high pressures at sub-zero temperature

PSF at 200 MPa and -18 • C led to the complete inactivation of P. fluorescens in tryptone soya broth (Table 2). The killing effect was considerable as compared with conventional freezing and higher than those obtained with pressurization at 200 MPa and 4 • C.

PSF was also able to inactivate endogenous Pseudomonas in black truffles. A 3.5 log reduction was obtained with PSF at 200 MPa and -18 • C, while no significant reduction was observed with conventional freezing (Table 2). However, it clearly appeared that endogenous Pseudomonas in truffle was more resistant to PSF than P. fluorescens inoculated in broths (as TSB) or foods (as smoked salmon mince, for example [START_REF] Picart | Microbial inactivation by pressure-shift freezing effects on smoked salmon mince inoculated with Pseudomonas fluorescens, Micrococcus luteus and Listeria innocua[END_REF]).

Even if some species of endogenous Pseudomonas may be naturally more resistant to pressure than P. fluorescens, it can be supposed that truffles exert a protective effect on endogenous Pseudomonas at the time of pressurization, more particularly at sub-zero temperatures.

Conclusion

Pressurization of black truffles at low or sub-zero temperatures permits us to significantly inactivate the endogenous Pseudomonas population. The ability of high pressure processing to stabilize black truffles has now to be confirmed by a preservation study.

Table 1 .

 1 Effect of high pressure at low temperature (4 • C or 20 • C/10 min) on viable counts of Pseudomonas fluorescens in tryptone soya broth and Pseudomonas spp. in black truffles. Values represent mean log 10 CFU/ml or CFU/g of unpressurized control samples (n = 12) and pressurized samples (n = 3) † No CFU was detected in 1 mL of cell suspension from each of the samples tested

		Pseudomonas fluorescens in	Pseudomonas spp. in
		tryptone soya broth	black truffles
		4 • C	2 0 • C	4 • C	2 0 • C
	Control	6.75 ± 0.21 a	6.75 ± 0.21 a	7.58 ± 0.30 d	7.58 ± 0.30 d
	100 MPa	6.75 ± 0.24 a	6.71 ± 0.23 a
	200 MPa	3.40 ± 0.28 b	4.81 ± 0.25 c
	300 MPa	ND †	ND	2.25 ± 0.31 e	1.63 ± 0.33 e,f
	400 MPa			1.28 ± 0.40 f	1.10 ± 0.38 f
	500 MPa			1.10 ± 0.37 f	1.33 ± 0.36 f
	Notes: Values with different superscript letters are significantly different (p < 0.05) ND, not detected

* *

Table 2 .

 2 Effect of pressure-shift freezing (PSF, i.e. pressurization at -18 • C/200 MPa/10 min and depressurization in 1 min 40 s inducing freezing) on viable counts of Pseudomonas fluorescens in tryptone soya broth and Pseudomonas spp. in black truffles.

		Pseudomonas fluorescens in	Pseudomonas spp. in
		tryptone soya broth	black truffles
	Control	6.75 ± 0.21 a	7.58 ± 0.30 c
	Classical freezing	5.15 ± 0.23 b	7.22 ± 0.31 c
	PSF	ND †	4.26 ± 0.32 d

*

Notes: Values with different superscript letters are significantly different (p < 0.05) * Values represent mean log 10 CFU/ml or CFU/g of unpressurized control samples (n = 12) and processed samples (n = 3) † No CFU was detected in 1 mL of cell suspension from each of the samples tested

Acknowledgements

This study was financially supported by the program FEDER, the Conseil général de la Dordogne and the Communauté d'Agglomération Périgourdine.