Imène Ferroukhi

Cécile Bord

Christophe Chassard

Julie Mardon
email: julie.mardon@vetagro-sup.fr

Effect of salt reduction or partial substitution with calcium lactate on the quality of a blue-veined cheese

Keywords: Blue-veined cheese, sodium, dry-salt, sensory profile, bacterial community

Reducing sodium in food has become a global health concern. In this study we investigated the impact of sprinkling and substitution of salt by calcium lactate during the dry salting of blue-veined cheese. Analyses included physicochemical and biochemical composition, microbial counts, 16rDNA gene metabarcoding analyses and a sensory profile.

Salt substitution induced a significant sodium reduction of 40% while sprinkling did not significantly reduce salt (-7%). The mineral migration, B-vitamin evolution and other biochemical parameters were not impacted. No differences in flavour, aroma and odour were detected. However, calcium lactate affected the cheese texture with less hardness and sandy rind. These methods provide new opportunities to reduce salt in dry-salted cheeses, but it is necessary to investigate their effects on other cheese matrixes.

Introduction

Currently, it is obvious that high dietary sodium chloride intake is linked to adverse health effects [START_REF] Who | Strategy for sodium-salt substitution: On the relationship between hypertension and dietary intake of cations[END_REF]. Sodium intake is associated with high blood pressure which causes 7.5 million deaths annually, the equivalent of about 12.8% of all deaths worldwide. It is a major risk factor for cardiovascular diseases CVD, which in itself is the leading cause of deaths globally [START_REF] Who | Strategy for sodium-salt substitution: On the relationship between hypertension and dietary intake of cations[END_REF]. The World Health Organization has proposed to reduce sodium intake by 30% in order to reach the guideline of 2g/day (i.e., 5 g of salt/day) by 2025 [START_REF] Who | Strategy for sodium-salt substitution: On the relationship between hypertension and dietary intake of cations[END_REF]. In France, mean daily salt intakes range from 7 to 10 g of salt and are well in excess of dietary needs (ANSES, 2012). Given that 75% of sodium intake comes from processed foods (Brown et al., 2009), reducing the salt content in these foods is an important lever.

Cheese is well-known sources of sodium, especially blue cheeses, with levels as high as 4% of NaCl (ANSES, 2012). A recent study showed that 40 g of blue-veined cheese could contribute to 23% of the recommended salt intake [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF]. However, reducing sodium without loss of cheese quality is a challenge due to the specific properties of sodium in flavour, functionality and shelf life extension of cheeses [START_REF] Guinee | Salting and the role of salt in cheese[END_REF]. Moreover, these cheeses can contain an attractive nutritional composition. [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF] highlighted interesting calcium and vitamin B contents in a blue-veined cheese. It was demonstrated that 40 g of Bleu d'Auvergne PDO cheese contributed to 11% of adequate intake for B12 [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF]. A link between B vitamins levels and microorganisms has also been established. Except for this last study, no data exists on the evolution of these vitamins in cheese matrix reduced in salt. With increasing demand for more nutritious foods, it is important to improve knowledge of the evolution of the B-vitamin content of dairy products according to different producing factors [START_REF] Leblanc | B-Group vitamin production by lactic acid bacteriacurrent knowledge and potential applications[END_REF].

Numerous attempts have been made by the scientific community and cheese industry to develop an acceptable low-sodium cheese [START_REF] Bansal | Reduced-sodium cheeses: Implications of reducing sodium chloride on cheese quality and safety[END_REF]. Alternative approaches to achieve sodium-reduced foods include simply reducing NaCl levels or partially replacing NaCl with sodium substitutes, such as KCl, CaCl 2 , MgCl 2 [START_REF] Chavhan | Effect of potassium-based emulsifying salts on sensory, textural, and functional attributes of low-sodium processed Mozzarella cheese[END_REF][START_REF] Dugat-Bony | Effect of sodium chloride reduction or partial substitution with potassium chloride on the microbiological, biochemical and sensory characteristics of semi-hard and soft cheeses[END_REF][START_REF] Grummer | Manufacture of reduced-sodium Cheddar-style cheese with mineral salt replacers[END_REF][START_REF] Møller | Erratum to: Manufacture and biochemical characteristics during ripening of Cheddar cheese with variable NaCl and equal moisture content[END_REF] . . In the literature, the reduction of added salt is mainly concerned with Cheddar cheese [START_REF] Møller | Erratum to: Manufacture and biochemical characteristics during ripening of Cheddar cheese with variable NaCl and equal moisture content[END_REF][START_REF] Rulikowska | The impact of reduced sodium chloride content on Cheddar cheese quality[END_REF]. There is a lack of knowledge about salt reduction in blue-veined cheeses which are usually dry salted on the surface. Salt substitution is another interesting practice widely reported contributing to the decrease of salt intake by consumers [START_REF] Bansal | Reduced-sodium cheeses: Implications of reducing sodium chloride on cheese quality and safety[END_REF][START_REF] Grummer | Manufacture of reduced-sodium Cheddar-style cheese with mineral salt replacers[END_REF]. Although KCl has been shown effective in reducing the sodium content of various cheeses [START_REF] Grummer | Manufacture of reduced-sodium Cheddar-style cheese with mineral salt replacers[END_REF][START_REF] Katsiari | Reduction of sodium content in Feta cheese by partial substitution of NaCl by KCl[END_REF], adverse effects on the quality of Cheddar have been reported [START_REF] Gomes | Manufacture of low-sodium Minas fresh cheese: Effect of the partial replacement of sodium chloride with potassium chloride[END_REF][START_REF] Lindsay | Effect of Sodium/Potassium (1:1) Chloride and Low Sodium Chloride Concentrations on Quality of Cheddar Cheese1[END_REF]. The substitution of NaCl by MgCl 2 or CaCl 2 has related to off flavours and a decrease in acceptability of Cheddar cheese [START_REF] Lindsay | Effect of Sodium/Potassium (1:1) Chloride and Low Sodium Chloride Concentrations on Quality of Cheddar Cheese1[END_REF]. Moreover, some of these substitute salts are all acidifying compounds [START_REF] Remer | Influence of nutrition on acid-base balance-Metabolic aspects[END_REF]. KCl-substituted products should be consumed cautiously by consumers with kidney disease because of the risk of hyperkalaemia [START_REF] Berthet | Nutrition et insuffisance rénale chronique[END_REF]. Within this context, organic calcium salts have been used to reduce the acidforming potential of blue-veined cheeses [START_REF] Gore | Calcium lactate as an attractive compound to partly replace salt in blue-veined cheese[END_REF]. This study found that a partial replacement of NaCl by calcium lactate induced a 19% reduction in Na content in Fourme d'Ambert cheese. However, this research mentioned the use of brining prior to dry salting on the surface. In an exploratory work, different techniques to reduce dry salt on the surface of blue-veined cheese were proposed by [START_REF] Ferroukhi | Exploring alternative salting methods to reduce sodium content in blue-veined cheeses[END_REF]. These authors reported that a substitution of salt by calcium lactate and the sprinkling of a controlled amount of NaCl revealed their technological relevance but deserved to be improved and reproduced in the traditional two-step salting process of blue cheese. Nevertheless, this research mentioned the use of brining before dry salting on the surface. More recently, [START_REF] Ferroukhi | Exploring alternative salting methods to reduce sodium content in blue-veined cheeses[END_REF] demonstrated that a substitution of salt by 30% calcium lactate was technologically relevant but deserves to be improved in the salting process of blue cheese.

Moreover, during the ripening of cheeses, various biochemical and microbiological mechanisms are involved [START_REF] Mcsweeney | Biochemistry of cheese ripening[END_REF], contributing, together with the complexity of the matrix, to the formation of the nutritional and organoleptic properties of blue-veined cheeses. So far, rare works have examined the evolution of these phenomena within a salt reduction context in blue cheeses dry salted on the surface.

Within an important health context, alternatives salting methods are required to reduce sodium in surface dry-salted blue cheeses. Thus, we hypothesise that modifications of the dry surface salting method through a decrease in the quantity of applied salt and a partial replacement of salt by calcium lactate might be relevant alternative techniques to reduce salt content in blue-veined cheeses. The objective of this research was to study the effect of two salt reduction treatments on physicochemical, biochemical, microbiological and sensory quality of this cheese during ripening.

MATERIALS AND METHODS

Blue cheese production

Blue cheeses were produced under standard process conditions according to the specifications for Bleu d'Auvergne PDO [START_REF] Inao | Institut national de l'origine et de la qualité[END_REF]. The raw cow's milk was pasteurised (72 °C, 30s) and standardized for fat content. After calcium chloride addition, the milk then was matured (36 °C, 45 min) and inoculated with a starter cultures (Lactococcus lactis subsp.

Cremoris, Lactococcus lactis subsp. Lactis, Streptococcus thermophiles) (Danisco, Danemark), Penicillium roqueforti (SAS LIP, France) and a rennet extract (Caglificio Clerici, Italy). The resulting coagulum was cut vertically and horizontally and then stirred to form the 'cap' of the coagulum grains (3 cm 3 ). The curds were drained in the moulds with several turnings. At day 2 after coagulation, the curds were double dry-salted according to the various salting treatments tested. Six days after the manufacture, the cheeses were pricked. This step aims to create air channels in the core of the cheese to enable the Penicillium to develop.

Ripening was carried out over 34 days at 8 °C and 95% RH.

Salting Treatments and Sampling

Salting process

As commonly practised in Bleu d'Auvergne cheese, salting process was done by rubbing fine dry salt (Esco, Germany) on the surface of the cheese in two steps. To obtain anhydrous salts, all the fine salt (0.20-0.23 mm) was previously dried (102 °C, 24h). Surface Three cheeses per salting method and per ripening time were collected from the same production batch. The salting treatment (A) represented the control cheese and consisted of a conventional salting method by rubbing the cheese in excess dry salt on the surface. The second salting treatment (B) was a sprinkling of 100 g of fine salt on the cheese surface with a regular rubbing and turning of the cheese. The third salting treatment (C) was a partial substitution of NaCl with 30% calcium lactate (Merck KGaA, Germany). In order to obtain mixtures of salts of homogeneous granulometry with Ca salts, calcium lactate in powder form (0.11-0.15 mm) was also dried and then mixed, ground with NaCl and put into resealable bags. All salting treatments were repeated identically the next day.

Sampling

The sampling plan is presented in Figure S 1, (II). A total of 36 Bleu d'Auvergne cheeses were produced. Three blocks of cheeses (2.5 ± 0.1 kg) were sampled per treatment at 6, 13, 21 and 34 days of ripening (corresponding to d6, d13, d21 and d34). The rind and core were separated from each cheese. All samples were ground, homogenised and stored at -20 °C until analysis, except for cheeses collected at 34 days of ripening dedicated to sensory analysis, that were stored at 4 °C.

Cheese composition

Physico-chemical and biochemical analyses

All samples were analysed in triplicate according to the standards of the International Organization for Standardization (AFNOR, 2022). We measured pH, dry matter content, fat content as a proportion of dry matter, total, soluble and non-protein nitrogen contents (SN and NPN), sodium and calcium contents and lactate content at various stages of maturation, according to reference methods (for more information, see the Supplementary Methods). The ratios SN:TN and NPN:TN were used as cheese proteolysis indicators. We measured concentrations of vitamin B2, B6, B9 and B12 in the cheeses throughout ripening as previously described [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF].

Microbiological Analyses

The different microorganism flora levels were estimated over all ripening periods. Cheese samples (25 g) were collected aseptically into sterile sample bags (BagMixer CC, France). The initial suspension and further decimal dilutions (10 -7 ) were prepared in 250mL of sterile peptone water. Total aerobic mesophilic bacteria were enumerated as per standard NF EN ISO 4833-2:2013. Leuconostoc populations were counted on Mayeux-Sandine-Elliker agar (MSE, Mayeux et al., 1962) Lactococcus on M17 agar as described by Terzaghi and Sandine (1975) and Lactobacillus on MRS agar according to De MAN et al. (1960).

Yeasts and moulds were counted as per standard NF V 08 - 059. Enterobacteriaceae were determined on violet red bile glucose agar (VRBG) according to NF V08-054:2009. All microbiological measurements were done in triplicate (for more information, see the Supplementary Methods).

Bacterial community structure using 16S rRNA gene metabarcoding

The impact of salt reduction on the bacterial community was analysed from samples collected at 6 and 34 days of ripening. As mentioned in a previous study [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF], cheeses produced with pasteurised milk did not show large differences in bacterial populations. DNA extraction from the cheese core and PCR amplification of the V3-V4 region of the 16S rRNA gene were performed as previously described [START_REF] Caron | Strong effect of Penicillium roqueforti populations on volatile and metabolic compounds responsible for aromas, flavor and texture in blue cheeses[END_REF] (for more information, see the Supplementary Methods). Amplicon data from high-throughput sequencing was analysed using the rANOMALY pipeline [START_REF] Theil | rANOMALY: AmplicoN wOrkflow for Microbial community AnaLYsis[END_REF]. Taxonomic assignment of bacterial sequences was based on two databases, DAIRYdb v2.0 and SILVA 138, keeping the assignment with the highest confidence or the deepest taxonomic rank [START_REF] Meola | DAIRYdb: A manually curated reference database for improved taxonomy annotation of 16S rRNA gene sequences from dairy products[END_REF]. The filtered ASVs count table was used to perform statistical analyses.

Sensory Analysis

Ten subjects were recruited from the trained panel of the sensory analysis laboratory of the VetAgro Sup Institute and trained according to standard ISO 8586-1:1993. They were very familiar with blue cheeses and had good experience in sensory analysis. The sensory evaluation was performed using a sensory profile according to the NF ISO 13299:2016 method. The descriptive analysis evaluates the aspect, aroma, texture and taste properties of cheeses at 34 days of ripening.

Training sessions were conducted before the evaluation sessions wherein the panellists tasted the produced cheeses and other mouldy cheeses. The participants generated a large number of attributes to describe the blue cheeses tasted. After statistical analysis, a set of 29 attributes was selected and divided into 5 items: appearance, texture, taste, odour and aroma according to [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF]. The performance of the panel was validated during this session in order to control-check for repeatability, discriminative capacity, and consensus according to the guidelines in NF ISO 8586-1:1993. Then, the sensory profile was carried out on cheeses ripened for 34 days and sampled using the method in Figure S 1, (III). Slices of cheese (50 g) and removed rind (2 mm) were presented on plates coded with random 3-digit numbers. A vertically-cut slice (rind and core) of each cheese was also presented to the panel to have a representative analysis of the appearance of the cheeses. All cheeses were served at 20 ± 1 °C, presented monadically and distributed according to a Williams Latin Square designed to take into account the first effect of order and carry-over. Scoring was done using a 10-cm linear scale, anchored from 0 (very weak) to 10 (very intense), to record the intensity of the sensory attributes. Between each sample, panellists were asked to rinse their mouth with unsalted crackers then tepid water to remove fatty residuals. There were two evaluation sessions led in a computerized booth according to ISO standard 8589:2010. Data was collected using Tastel software® (version 2019; ABT Informatique, Rouvroy-sur-Marne, France).

Data Analysis

All results were reported as mean ± standard deviation. A Shapiro-Wilk test (p <0.05) was used to check the normality of the data. Two-way mixed-model ANOVA (salting treatment*ripening time) with interaction was carried out on biochemical and microbiological data and a post-hoc comparison (Fisher's test) were applied and differences were considered significant when p <0.05. All statistical analysis of biochemical data was performed using XLSTAT software 2020 (Addinsoft, Paris, France). In addition, a principal component analysis (PCA) was also performed on the mean of all data using a Pearson correlation. For sensory data, a two-way mixed-model ANOVA (product and assessor) with interaction was carried out on the sensory data, with the assessor as random effect and product as fixed effect, at 95% of confidence levels. Tukey's test was used for multiple comparison tests on the product means, for each attribute, when means were considered different (P <0.05). A hierarchical cluster analysis on the PCA means was performed to estimate the homogeneous groups. The rANOMALY interface was used to investigate the diversity of bacterial communities in the cheeses studied at 6 and 34 days of ripening. Species evenness and richness (α-diversity) of the cheese was evaluated using Shannon, Simpson and InvSimpson indexes to determine evenness and the Observed and Chao1 indexes to determine richness.

The compositional dissimilarity between samples (β-diversity) was assessed by the Bray-Curtis method. Differences in the dispersion of bacterial profiles from the centroid between the two ripening times were tested by Tukey's HSD test (P <0.05).

Results

Physicochemical and Biochemical Characteristics

The effect of lowering applied salt on the compositional and physicochemical evolution throughout ripening of Bleu d'Auvergne cheese is presented in Table 1. The dry matter increased during the ripening time in all three cheese models. At d34, the calcium lactate-substituted cheese C had a slightly lower dry matter content (53.79%) than the sprinkled cheese B (54.39%) and the control cheese A (55.05%). An increase in pH in the core and rind of the three cheese models was also observed (P<0.0001) (Figure S 2). The pH values reached 6.02, 6.31 and 6.07 at the end of ripening in the core of A, B and C cheeses, respectively. A decreasing pH gradient from the rind to the core of the cheeses was observed during ripening. This gradient was greater at 34 days of ripening, particularly for A and C cheeses with 0.83 and 0.86 points respectively. Concerning nitrogen matter, the salting methods had no impact on crude protein and total nitrogen. In contrast, L-lactate levels decreased during maturation (P<0.0001). The quantity of D-lactate was negligible. However, the crude protein and total nitrogen contents increased during the ripening of the three cheese models, reaching an average crude protein and total nitrogen content of 19.47 and 3.04 g/100g at d 34, respectively.

Non-protein nitrogen (NPN) and soluble nitrogen (SN) also increased during ripening in the core of all cheeses. At d 13 and d 21, the NPN and SN values appeared to be higher in the salt-reduced cheeses B and C. At the end of ripening the NPN and SN values were stabilised in the three cheeses with an average value of 0.789 and 2.143 g/100g, respectively. Furthermore, all cheeses were characterised by an increasing level of proteolysis during ripening (Figure S 3). At d 21, the SN:TN and the NPN:TN ratios (reflecting primary and secondary proteolysis, respectively) were higher in the salt-reduced cheeses B and C than in the A cheese. The level of proteolysis plateaued at d34 in all cheeses.

Salt content increased in the cheeses core during the ripening period, reaching 2.87% in A cheese, 2.67% in B cheese and 1.73% in C cheese. At d34, the sprinkling technique reduced salt content by 7% compared to the control cheese, which was not significant. In contrast, the substitution of salt with calcium lactate reduced salt content by 40% compared to the control cheese (<0.0001).

The evolution of minerals (Figure 1. Evolution of sodium and calcium content of the cheese core and rind during ripening according to three salting methods (mean ± standard deviation of three cheeses). showed an increase of sodium in the cheese core and a decrease in the cheese rind (P<0.001). The sodium content in the rind of cheese A (1443.33 mg /100g) was higher than in cheeses B (1085.00 mg /100g) and C (684.50mg /100g). On the other hand, the calcium content in the rind increased with higher content at d 34 (P<0.0001) in cheese C (1240.00 mg /100g) compared to A (642.50 mg /100g) and B (790 mg/ 100g). In cheeses core, the calcium evolution during ripening was not noticeable. The calcium levels at d 34 were 613.50, 547.00 and 582.83 mg /100g in the core of A, B and C cheeses respectively. A similar pattern was obtained by estimating the studied parameters on dry matter.

In the present study, the impact of reducing salt on vitamin B2, B6, B9 and B12 changes during ripening was investigated. The trends represented in Figure 2 revealed that the applied salting methods had no effect on B-vitamin composition of the blue cheeses (P>0.05).

However, the ripening time affected the evolution of these vitamins (P<0.0001). Vitamin B2 and B6 contents increased significantly during the ripening period in all cheeses. The average B2 vitamin content of cheeses at d 34 was about 0.38 mg/ 100g. A lower B6 content (0.07 mg/ 100g) was observed in B cheese at d 34. The concentrations of B9 and B12 increased between 6 and 13 days of ripening, afterwards their content decreased to reach an average value of 22.92 µg /100g for B9 and 1.15 µg /100g for B12 in the blue cheeses at the end of ripening.

Microbial and 16S rRNA gene Metabarcoding Analyses

Cheese samples were analysed at each ripening time using classical culture-based counts (Table 2). In parallel, 16S rRNA gene metabarcoding was performed to gain further insight into the bacterial community composition. Finally, 16S rRNA gene Metabarcoding analysis was applied at the beginning was used at the beginning (d 6) and end of ripening (d 34) to assess the impact of salt reductions on the overall microbial community activity. For all bacterial populations studied, the salting method did not induce a major impact on bacterial evolution compared to the ripening time. The total microflora count at the end of the ripening process was higher in B (10.18 log cfu/g) and C (9.42 log cfu/g) cheeses than in A control cheese (8.71 log cfu/g). Enterobacteriaceae counts showed no impact of salting treatments.

The salt reduction methods did not produce important changes in the count of lactic acid bacteria (LAB) in the cheese core. Leuconostoc population tended to increase with the ripening time. Yeast and moulds increased during ripening in all cheeses, without major differences between the control and the NaCl-reduced cheeses.

Sequencing of the 16S rRNA (V3-V4 regions) of the samples generated a total of 715303 reads that were assigned to 18 ASVs. Taxonomic assignment of each ASV was possible up to the species level (or group of species sharing nearly identical sequences) in most cases. The results of the 16S rRNA gene metabarcoding revealed that the composition of the bacterial communities was similar to the three salting conditions (Table 3). A predominance of Lactococcus and Streptococcus species in all cheeses were observed with a raw abundance sum of 419585 and 249397 reads, respectively. Sub-dominant species of Leuconostoc Mesenteroides (31238 reads), Brevibacterium (7568 reads), Brachybacterium (2410 reads) and Lactobacillus (3852 reads) were also present in all cheeses. Community richness and diversity were assessed for each salting method at 6 and 34 days of ripening using four alpha diversity parameters (Observed, Chao1, Shannon and Simpson). The results in Table S 1 showed that the salting techniques had no impact on the richness and diversity of the bacterial community at 6 and 34 days of ripening (p>0.05). The mean richness indices (Observed and Chao1) of blue cheeses were 19.333 for both ripening times. The mean Shannon and Simpson indices of the cheeses were 1.337 and 0.637 at 6 days and 1.273 and 0.583 at 34 days, respectively. Species dissimilarity between the salting methods at d 6 and d 34 was assessed based on beta diversity using the Bray-Curtis method. The results revealed that the same species were present in all cheeses at both 6 days (p=0.886) and 34 days (p=0.444) of ripening (data not shown).

Sensory Characteristics

Significant differences in texture and marbling were highlighted by the panel between the A, B and C cheeses. The sensory profile results showed a significant (P <0.05) product effect for 8 sensory attributes over 28 total attributes (Figure 3). The reliability of the panel was also demonstrated by a significant product × assessor interaction (P <0.05) only for two sensory attributes (lactic aroma and cavities-quantity_C). Surprisingly, no significant salty taste and bitterness difference was observed between cheeses. The observation plot (Figure 3, A andB) and the dendrogram from the agglomerative hierarchical clustering (Figure 3,C) revealed that the calcium lactate substituted cheese constituted a distinctive cluster from the sprinkled and control cheeses. The A and B cheeses were perceived as being significantly firmer than the C cheese, which was characterised by a creamier texture in the core and sandy in the rind. On the other hand, marbling appeared to be more pronounced in C cheese than in A and B cheeses.

Discussion

Reducing salt in the diet is becoming a global concern (WHO, 2021). Given the major role of salt in cheese production, it is necessary to examine the impact of salt reduction in the changes in physico-chemical, biochemical and microbiological parameters and the consequences that may have on sensory characteristics. To date, the studies conducted in this way have been mainly interested in hard and semi-hard cheeses [START_REF] Gomes | Manufacture of low-sodium Minas fresh cheese: Effect of the partial replacement of sodium chloride with potassium chloride[END_REF][START_REF] Grummer | Manufacture of reduced-sodium Cheddar-style cheese with mineral salt replacers[END_REF][START_REF] Lindsay | Effect of Sodium/Potassium (1:1) Chloride and Low Sodium Chloride Concentrations on Quality of Cheddar Cheese1[END_REF]. We decided to provide comparable data on blueveined cheeses, which are very little studied but rather popular with consumers. The present research investigated the impact of salt reduction by sprinkling method and partial replacement of NaCl by 30% calcium lactate on all physico-chemical, biochemical, microbiological, and sensory aspects of blue-veined cheeses during ripening.

Biochemical and physicochemical changes in blue cheese matrix under salt reduction conditions

The mineral exchange observed in the current work between the rind and the core (Naion inflow and Ca-ion outflow) has been demonstrated in previous studies [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF][START_REF] Guinee | Salting and the role of salt in cheese[END_REF]. This reaction is induced by the application of dry salt on the surface, which causes an exudation of the mineral-laden water, while salt (sodium) moves into the cheese matrix [START_REF] Guinee | Salting and the role of salt in cheese[END_REF][START_REF] Graet | Migration des macro et oligo-éléments dans un fromage à pâte molle de type Camembert[END_REF]. The increasing pH gradient found was negatively correlated (r= -0.73, p<0.05) to a decreasing lactate content. According to [START_REF] Mcsweeney | Biochemistry of cheese ripening[END_REF], the bacteria on the surface metabolise lactate into CO 2 and O 2 , thereby deacidifying the cheese surface and creating a pH gradient developed from the centre to the rind. As the surface pH increases, Ca and phosphates move irreversibly from the core to the cheese surface. The same pattern has been found in other studies [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF][START_REF] Gore | Calcium lactate as an attractive compound to partly replace salt in blue-veined cheese[END_REF][START_REF] Graet | Migration des macro et oligo-éléments dans un fromage à pâte molle de type Camembert[END_REF]. The pH gradient is thus responsible for an accumulation of these minerals on the cheese surface which is even more important with the application of calcium lactate, impacting the texture of the cheese rind. Indeed, in the present work, the calcium lactate-substituted cheeses had the highest calcium content in the rind. However, there was no calcium contribution to the cheese core as found by [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF] and [START_REF] Gore | Calcium lactate as an attractive compound to partly replace salt in blue-veined cheese[END_REF] in blue-veined-cheeses, suggesting that the calcium brought by the salt mixture remained on the cheese surface.

In this work, a technological relevance of the sprinkling and calcium lactate substitution methods was demonstrated during the dry salting of blue-veined cheese in two steps. This is consistent with the previous findings of [START_REF] Ferroukhi | Exploring alternative salting methods to reduce sodium content in blue-veined cheeses[END_REF]. However, these authors reported a 45% decrease in sodium in cheese with the sprinkling technique, contrary to what was obtained in the present paper (-7%). This can be attributed to the double salting applied here and conventionally in the salting of blue cheeses, unlike the exploratory study of [START_REF] Ferroukhi | Exploring alternative salting methods to reduce sodium content in blue-veined cheeses[END_REF]. In addition, we can hypothesise that the quantity of salt sprinkled in two steps was not sufficiently decreased to allow a marked reduction of sodium in the cheese matrix, and the wet surface of the blue cheeses retains a large proportion of salt. Calcium lactate was an effective salt substitute as already reported in another blue-veined cheese [START_REF] Gore | Calcium lactate as an attractive compound to partly replace salt in blue-veined cheese[END_REF]. The significant decrease of sodium caused by the double salting the cheese with the mixture of salt and 30% calcium lactate has supported the assumptions made by [START_REF] Ferroukhi | Exploring alternative salting methods to reduce sodium content in blue-veined cheeses[END_REF] . We can suppose that the application of calcium lactate on the surface would prevent some of the salt from penetrating, thus leading to a reduction in sodium content.

Blue-veined cheeses are known to have a high proteolysis rate [START_REF] Mcsweeney | Biochemistry of cheese ripening[END_REF]. In our experience, the increase in proteolysis, positively correlated with the increase in the mould count and pH (r=0.881 for moulds, r=0.909 for pH, p<0.05), corroborated the literature data [START_REF] Cantor | Chapter 37-Blue Cheese[END_REF][START_REF] Dugat-Bony | The effect of reduced sodium chloride content on the microbiological and biochemical properties of a soft surface-ripened cheese[END_REF][START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF]. As described in Bleu d'Auvergne cheese [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF]) and other blue-veined cheeses [START_REF] Fernandez-Salguero | Proteolysis of Cabrales cheese and other European blue vein cheese varieties[END_REF][START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF][START_REF] Prieto | Picón Bejes-Tresviso blue cheese: An overall biochemical survey throughout the ripening process[END_REF], a high and increasing rate of proteolysis marked the three cheeses produced (A, B and C). The rapid proteolysis of saltreduced cheeses suggested that moisture conditions in salt-reduced cheeses accelerated the proteolytic activity of moulds, particularly at 21 days of ripening. A similar effect was reported in Minas cheese [START_REF] Gomes | Manufacture of low-sodium Minas fresh cheese: Effect of the partial replacement of sodium chloride with potassium chloride[END_REF] and soft cheeses substituted with potassium chloride [START_REF] Dugat-Bony | Effect of sodium chloride reduction or partial substitution with potassium chloride on the microbiological, biochemical and sensory characteristics of semi-hard and soft cheeses[END_REF]. At 34 days, there was no difference between the control cheese and the reduced salt cheeses.

In addition, the impact of reducing salt on the water-soluble vitamin composition of blue-veined cheeses during ripening was investigated. We have highlighted in a previous study that Bleu d'Auvergne cheese may contain interesting quantities of B vitamins [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF]. Nevertheless, before this study, no data were available on the evolution of water-soluble vitamins during the ripening of blue-veined cheeses. The salt reduction applied here did not affect the levels of vitamins B2, B6, B9 and B12 and this is relevant for the nutritional value of these cheeses. Indeed, B vitamins have a number of major roles in metabolic processes such as energy production and red blood cell formation [START_REF] Leblanc | B-Group vitamin production by lactic acid bacteriacurrent knowledge and potential applications[END_REF]. B vitamins, normally present in many foods, are easily removed or destroyed during food processing, so inadequate intakes are common in many populations.

The average results obtained here for vitamin B2 and B6 content were comparable to those found in other blue-veined cheeses [START_REF] Altangerel | The determination of water-soluble vitamins and in vitro digestibility of selected Czech cheeses[END_REF][START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF][START_REF] O'brien | Chapter 24-Nutritional Aspects of Cheese[END_REF]. The pyridoxine and riboflavin contents depend largely on the milk's initial composition and the cheese-making technology. Moreover, it has been reported that blue cheeses, with a high proteolysis rate, contain a higher content of B vitamins [START_REF] Gregory | Section D. Nutritive value of milk and milk products. Water-soluble vitamins in milk and milk products[END_REF]. Regarding folates, the level of this vitamin in the cheeses seems lower than those found in blue-veined cheeses such as Roquefort or Stilton [START_REF] O'brien | Chapter 24-Nutritional Aspects of Cheese[END_REF].

The increase in folate content and subsequent decrease after 13 days of ripening may result from synthesis followed by consumption of folate by cheese microbiota. For instance, most authors claim that Streptococcus thermophilus normally produce folates whereas Lactobacillus delbrueckii subsp. bulgaricus would be a folate consumer [START_REF] Leblanc | B-Group vitamin production by lactic acid bacteriacurrent knowledge and potential applications[END_REF], so the selection of adequate combination of strains is essential to develop fermented foods with increased vitamin concentrations. Attractive amounts of vitamin B12 were also found in the three cheeses (control and salt-reduced cheeses) at the end of the ripening process. A previous investigation demonstrated that a 40 g portion of Bleu d'Auvergne could significantly contribute to the nutritional requirements of vitamin B12 [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF].

Impacts of Reducing Salt on Sensory Properties

Salt has a key role in the development of sensory characteristics during the ripening of cheese and its decrease can affect cheese organoleptic and functional quality [START_REF] Bansal | Reduced-sodium cheeses: Implications of reducing sodium chloride on cheese quality and safety[END_REF][START_REF] Guinee | Salting and the role of salt in cheese[END_REF]. The sensory analysis of this work revealed no difference in flavour, odour and aroma of the salt-reduced cheeses and the control cheese. Similar observations were reported during a flash profile in a previous investigation [START_REF] Ferroukhi | Exploring alternative salting methods to reduce sodium content in blue-veined cheeses[END_REF]. Reducing the added salt content in cheeses is often related to the appearance of bitterness probably caused by a high proteolysis indexes [START_REF] Møller | Erratum to: Manufacture and biochemical characteristics during ripening of Cheddar cheese with variable NaCl and equal moisture content[END_REF]. In spite of the increasing proteolysis that was observed in experimental cheeses, there was no difference in bitterness between the control and salt-reduced cheeses, presumably owing to the stabilisation of this phenomenon at the end of the ripening process.

The significant salt reduction in the cheese substituted with calcium lactate (-40%) did not change the flavour. This result is consistent with observations made on Fourme d'Ambert cheese [START_REF] Gore | Calcium lactate as an attractive compound to partly replace salt in blue-veined cheese[END_REF]. These authors concluded that calcium lactate could substitute NaCl up to 75% without affecting the bitterness and salinity of the cheeses. On the other hand, in this same study, Fourme d'Ambert cheeses had previously received salt by brining.

Here, the reduction of salt in the dry salting of blue cheeses showed that using calcium lactate could impact the texture of the cheeses. In general, it has been reported that low sodium cheeses tend to produce softer and creamier cheeses [START_REF] Guinee | Salt in Cheese: Physical, Chemical and Biological Aspects[END_REF]. Here, the effect of salt reduction methods on the sensory quality of blue cheeses was less important as compared to the salt reduction of Cheddar cheese [START_REF] Lindsay | Effect of Sodium/Potassium (1:1) Chloride and Low Sodium Chloride Concentrations on Quality of Cheddar Cheese1[END_REF][START_REF] Rulikowska | The impact of reduced sodium chloride content on Cheddar cheese quality[END_REF]. The nature of the calcium lactate (white powder) remaining on the surface may explain the sandy texture of the rind of B cheese. The same observation was reported for Fourme d'Ambert [START_REF] Gore | Calcium lactate as an attractive compound to partly replace salt in blue-veined cheese[END_REF]. Furthermore, calcium lactate applied to the cheese surface may block the moisture release. Hence, the softer texture of the calcium lactate substituted cheese is probably due to a more significant presence of moisture (reflected by the slight lower dry matter for calcium lactate substituted cheese) and casein hydration. As reported by [START_REF] Guinee | Salting and the role of salt in cheese[END_REF] and [START_REF] Johnson | The Melt and Stretch of Cheese. The dairy pipeline center for dairy research[END_REF], salt increases the water-holding capacity of cheese matrix by increasing casein hydration, matrix volume and casein solubilisation. Consequently, the reduction in casein hydration decreased the ability of the cheese matrix to resist deformation, resulting in a decrease in hardness.

On the other hand, the sensory analysis also indicated that the Ca-lactate substituted cheese contained more marbling than the other cheeses. This doesn't necessarily mean a higher growth of Penicillium roqueforti, but could be related to the heterogeneity of the matrix of a blue-veined cheese, which leads to heterogeneous cheese slices served during the testing [START_REF] Ferroukhi | Exploring alternative salting methods to reduce sodium content in blue-veined cheeses[END_REF].

Impacts of Reducing Salt on Microbial Growth

A significant salt lowering in cheeses is often associated with an alteration of their microbiological quality [START_REF] Guinee | Salt in Cheese: Physical, Chemical and Biological Aspects[END_REF]. The microbial data highlighted a total absence of Enterobacteriacae. Given that the presence of these bacteria is recognised as an indicator of contamination and hygiene in cheeses [START_REF] Mladenović | Enterobacteriaceae in food safety with an emphasis on raw milk and meat[END_REF], this may suggest that the salting treatments applied did not alter the sanitary quality of the cheeses. These confirmed the results obtained by [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF]. Contrary to the reported increase of Pseudomonas in semi-hard and soft-surface ripened cheeses reduced in salt [START_REF] Dugat-Bony | The effect of reduced sodium chloride content on the microbiological and biochemical properties of a soft surface-ripened cheese[END_REF], 2019), in this study applied reduction methods did not induce an increase of spoilage microbiota.

The cheeses reduced in salt had more total microflora at the end of the ripening process, probably owing to more favourable biochemical conditions (moisture, pH, salt.. etc.). In essence, the applied sodium lowering treatments did not have a major impact on bacterial evolution in the produced blue cheeses. The rapid growth of yeasts and moulds observed was a typical feature of blue-veined cheeses, previously reported in Bleu d'Auvergne [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF], Gorgonzola [START_REF] Gobbetti | Microbiology and biochemistry of gorgonzola cheese during ripening[END_REF] and Roquefort cheese [START_REF] El Dairouty | Chemical and microbiological changes in Roquefort style cheese during ripening[END_REF] for example. The activity and growth of this fungus is related to some environmental conditions inside the cheese such as prickling (oxygen in cavities) and salt level (r=0.622, p<0.05) [START_REF] Caron | Strong effect of Penicillium roqueforti populations on volatile and metabolic compounds responsible for aromas, flavor and texture in blue cheeses[END_REF]. Our data revealed that reducing salt by 7% and 40% in this research did not interfere with the growth and activity of this species.

Penicillium roqueforti is used in blue cheeses as a secondary starter for flavour production, mostly through proteolysis and lipolysis during ripening [START_REF] Cantor | Chapter 37-Blue Cheese[END_REF][START_REF] Caron | Strong effect of Penicillium roqueforti populations on volatile and metabolic compounds responsible for aromas, flavor and texture in blue cheeses[END_REF]. The correlation found between increased proteolysis and mould growth confirmed the high proteolytic activity of Penicillium roqueforti. Through a combination of intra-and extracellular enzymes, this fungus is able to degrade most proteins. The resulting peptides contribute to cheese flavour, and their breakdown into amino acids further influences cheese aromas and the proliferation of other microorganisms [START_REF] Mcsweeney | Biochemistry of cheese ripening[END_REF]).

Among the bacterial populations studied, lactic acid bacteria (LAB) did not show a large variation with salt reduction. Comparable findings have been made in Halloumi cheese [START_REF] Kamleh | The effect of substitution of sodium chloride with potassium chloride on the physicochemical, microbiological, and sensory properties of Halloumi cheese[END_REF], and other semi-hard and soft cheeses substituted with potassium chloride [START_REF] Dugat-Bony | Effect of sodium chloride reduction or partial substitution with potassium chloride on the microbiological, biochemical and sensory characteristics of semi-hard and soft cheeses[END_REF]. In contrast, LAB counts were consistently higher in the salt reduced Cheddar cheese [START_REF] Rulikowska | The impact of reduced sodium chloride content on Cheddar cheese quality[END_REF][START_REF] Schroeder | Reduction of Sodium Chloride in Cheddar Cheese: Effect on Sensory, Microbiological, and Chemical Properties1[END_REF]. This group of microorganisms can influence the ripening process and the sensory characteristics of cheeses due to the production of different organic acids and flavour compounds [START_REF] Mcsweeney | Biochemistry of cheese ripening[END_REF].

As expected, starter bacteria (Lactococcus and Streptococcus) inoculated into the milk during manufacture were highly dominant over the other species. As reported in a previous study [START_REF] Ferroukhi | Functional changes in Bleu d'Auvergne cheese during ripening[END_REF], Lactococcus species were positively correlated with B2 and B6 contents (r>0.960, p<0.05) in contrast to a negative correlation with Streptococcus species (r<-0.950, p<0.05) in all produced cheeses. It is of great interest to highlight that the salt reductions tested in this work did not seem to interfere in the activity of certain microorganisms involved in the metabolism of these nutrients. However, depending on the bacterial strain, the metabolism of vitamins can be different and remains to be investigated.

CONCLUSION

In conclusion, in this paper we proposed new methods to reduce Na content in a blueveined cheese. In practice, sprinkling and partial substitution of salt by calcium lactate appear to be suitable alternatives to conventional dry-salting methods. The cheeses obtained using a 30% substitution of salt with calcium lactate contained 40% less sodium than the control cheese. The minor effect of salt sprinkling on sodium content suggests that the salting process should be improved by decreasing the amount of salt applied. Regarding the sensory aspect, the main changes concerned textural modifications of the cheeses brought by the substitution of calcium lactate. All other organoleptic qualities remained unchanged with the proposed salt reduction techniques. The impacts of the salt reduction procedures proposed in this research on the physicochemical, biochemical and microbiological criteria of the cheeses were insignificant, suggesting a preservation of intrinsic properties of the cheeses. This paper highlights that salt reduction did not affect the nutritional quality of blue-veined cheeses in terms of B vitamins. The B-vitamin results also provide interesting data that were lacking in the literature and that can assist in the nutritional optimization of cheese. In prospect, it would be interesting to investigate these salting methods on another cheese dry salted on the surface to estimate their impact on the quality of the ripened cheese.

Figures Captions

Figure 1. Evolution of sodium and calcium content of the cheese core and rind during ripening according to three salting methods (mean ± standard deviation of three cheeses).

A: control cheese salted by rubbing the cheese in excess dry salt on the surface.; B: Reduction of salt by sprinkling of 100 g of fine salt on the cheese surface with a regular rubbing and turning of the cheese.; C: Reduction of salt by a partial substitution of NaCl with 30% calcium lactate. The figure shows an increase in the amount of sodium in the cheese core and a decrease in the rind with the three salting modalities. Simultaneously the calcium content decreases in the core and increases in the rind of the cheeses with a high level in the cheese substituted with calcium lactate. a-g Values with the same letter are statistically homogeneous groups on the basis of a two-way ANOVA ('ripening time*salting method' interaction). Cheeses salted by sprinkling 100 g of fine salt, C: Cheeses salted with a mix of NaCl and 30% of calcium lactate, W: Cheeses unmoulded before salting. Sampling of the rind and core was performed to follow the biochemical and physico-chemical parameters during the ripening of the cheeses. For each ripening time, triplicates of cheeses were produced by salting method.

Figure S 2.

Evolution of pH of the cheese core and rind during ripening according to three salting methods (mean ± standard deviation of three cheeses). The figures show an increasing pH in the rind and core of the manufactured cheeses. The pH of the rind was higher than the pH of the core creating an increasing pH gradient from core to rind cheese. a-h Values with the same letter are statistically homogeneous groups on the basis of a two-way ANOVA ('ripening time*salting method' interaction).

Figure S 3.

Evolution of proteolysis of the cheese core during ripening according to three salting methods (mean ± standard deviation of three cheeses). Primary and secondary proteolysis increased throughout the ripening of all cheeses. The level of proteolysis was higher in the salt-reduced cheeses at 21 days of ripening and then stabilised at the end of ripening. a-e Values with the same letter are statistically homogeneous groups on the basis of a two-way ANOVA ('ripening time*salting method' interaction).

TABLES

Table 1. Evolution of the composition of the cheese core during ripening according to three salting methods (mean ± standard deviation of three cheeses). a-h Means in a column sharing common superscripts are similar as tested by Fisher test (Two-way ANOVA, P <0.05).

1 Expressed as g/100 g.

2 Expressed as mg/100 g.

Table 2. Microbiological counts (log cfu/g) of cheese core samples during ripening (mean ± standard deviation of three cheeses).

A: control cheese salted by rubbing the cheese in excess dry salt on the surface.; B: Reduction of salt by sprinkling of 100 g of fine salt on the cheese surface with a regular

rubbing and turning of the cheese.; C: Reduction of salt by a partial substitution of NaCl with 30% calcium lactate. Brachybacterium_species 1,02±0.00 b 0,58±0.00 b 0,41±0.00 a 0,50±0.01 ab 0,35±0.00 a 0,31±0.00 a Brevibacterium_species 2,00±0.01 b 1,06±0.00 ab 0,82±0.00 a 1,08±0.01 ab 0,65±0.00 a 0,64±0.00 a Lactobacillus_species 0,11±0.00 ab 0,19±0.00 bc 0,18±0.00 bc 0,39±0.00 c 0,07±0.00 a 1,03±0.01 d Enterobacteriaceae_species 0,11±0.00 a 0,08±0.00 a 0,09±0.00 a 0,14±0.00 a 0,06±0.00 a 0,07±0.00 a Lentilactobacillus_species 0,13±0.00 b 0,06±0.00 a 0,06±0.00 a 0,05±0.00 a 0,05±0.00 a 0,04±0.00 a Romboutsia_species 0,01±0.00 b 0,01±0.00 ab 0,01±0.00 ab 0,00±0.00 a 0,01±0.00 ab 0,01±0.00 ab Acinetobacter_johnsonii 0,02±0.00 a 0,00±0.00 a 0,00±0.00 a 0,01±0.00 a 0,01±0.00 a 0,00±0.00 a A: control cheese salted by rubbing the cheese in excess dry salt on the surface.; B: Reduction of salt by sprinkling of 100 g of fine salt on the cheese surface with a regular

rubbing and turning of the cheese.; C: Reduction of salt by a partial substitution of NaCl with 30% calcium lactate. the instructions, cheese samples (1 g) were homogenised in a 100 mL volumetric flask with distilled water heated to 60°C, with stirring for 20 min or until complete dispersion.

Microbiological Analyses

We estimated the different microorganism flora levels over all ripening periods in order to assess the effect of salt reduction on the evolution of the microorganisms. Cheese samples (25 g)

were collected aseptically into sterile sample bags and homogenised with 250 mL of peptone water for 2 min in a stomacher (BagMixer CC, France). Total aerobic mesophilic bacteria number was counted after three days at 30 °C on plate count agar (PCA) as per standard NF EN ISO 4833-2:2013. Leuconostocs were counted on Mayeux-Sandine-Elliker agar (MSE, Mayeux et al., 1962) after 2 days at 30 °C. Lactococcus were counted on M17 agar as described by Terzaghi and Sandine (1975) after 2 days at 30 °C. Lactobacillus were enumerated on Man Rogosa Sharpe agar (MRS) incubated at 30 °C for 3 days according to De MAN et al. (1960).

Yeasts and moulds were counted on Oxytetracycline-Glucose-Yeast Extract Agar (OGA) as per standard NF V 08 - 059. The medium was supplemented with oxytetracycline before plating and incubated at 25 °C for 5 days. Enterobacteria were enumerated on violet red bile glucose (VRBG) incubated in anaerobic conditions during 24h according to NF V08-054:2009. All media were purchased from Biokar Diagnostics (Biokar, Beauvais, France). All microbiological measurements were done in triplicate.

  dry-salting of Bleu d'Auvergne cheese was conducted under three treatments (Figure S 1, I).

Figure 2 .

 2 Figure 2. Evolution of B-vitamins content of the cheese core and rind during ripening according to three salting methods (mean ± standard deviation of three cheeses). A: control cheese salted by rubbing the cheese in excess dry salt on the surface.; B: Reduction of salt by sprinkling of 100 g of fine salt on the cheese surface with a regular rubbing and turning of the cheese.; C: Reduction of salt by a partial substitution of NaCl with 30% calcium lactate. The figure demonstrates a similar trend in the evolution of B vitamins in three salting treatments. Vitamins B2 and B6 increased during ripening while vitamin B9 and B12 tended to decrease. a-g Values with the same letter are statistically homogeneous groups on the basis of a two-way ANOVA ('ripening time*salting method' interaction).

Figure 3 .

 3 Figure 3. Principal component analysis performed on sensory profile: plot of principal axes F1 -F2. A: control cheese salted by rubbing the cheese in excess dry salt on the surface.; B: Reduction of salt by sprinkling of 100 g of fine salt on the cheese surface with a regular rubbing and turning of the cheese.; C: Reduction of salt by a partial substitution of NaCl with 30% calcium lactate. (A) Correlation circle, (B) Representation of cheeses, (C) Dendrogram derived from hierarchical classification ascendant performed on the first three components of principal component analysis, obtained from sensory profile. The dashed line shows the level of truncation. In the correlation circle (A), only 8 descriptors out of 28 studied had a significant 'product' effect. The representation (B) shows an opposition of cheese substituted with calcium lactate compared to control and sprinkled cheese. This is confirmed by the homogeneous groups created in the dendrogram (C), where A and B cheeses from the same group differentiated on C cheese.

Figure S 1 .

 1 Figure S 1. Sampling plan. Sampling of cheeses during the ripening periods (I). Sampling method for the rind and core of Bleu d'Auvergne cheese (II). Sampling method for the sensory analysis (III). A: Standard cheese salted in excess of salt by a traditional method, B:

a- d Figure 4 .

 d4 Figure 4. Evolution of sodium and calcium content of the cheese core and rind during ripening according to three salting methods (mean ± standard deviation of three cheeses). a-g Values with the same letter are in statistically homogeneous groups on the basis of a two-way ANOVA ('ripening time*salting method' interaction).

Figure 5 .

 5 Figure5. Evolution of B-vitamins content of the cheese core and rind during ripening according to three salting methods (mean ± standard deviation of three cheeses). a-f Values with the same letter are in statistically homogeneous groups on the basis of a two-way ANOVA ('ripening time*salting method' interaction).

Figure 6 .

 6 Figure 6. Principal component analysis performed on sensory profile: plot of principal axes F1 -F2. (A) Correlation circle, (B) Representation of cheeses, (C) Dendrogram derived from hierarchical classification ascendant performed on the first three components of principal component analysis, obtained from sensory profile. The dashed line shows the level of truncation.

Figure S 1 .

 1 Figure S 1. Sampling plan. Sampling of cheeses during the ripening periods (I). Sampling method for the rind and core of Bleu d'Auvergne cheese (II). Sampling method for the sensory analysis (III). A: Standard cheese salted in excess of salt by a traditional method, B: Cheeses salted by sprinkling 100 g of fine salt, C: Cheeses salted with a mix of NaCl and 30% of calcium lactate, W: Cheeses unmoulded before salting. Sampling of the rind and core was performed to follow the biochemical and physico-chemical parameters during the ripening of the cheeses. For each ripening time, triplicates of cheeses were produced by salting method.

Figure S 2 .

 2 Figure S 2. Evolution of pH of the cheese core and rind during ripening according to three salting methods (mean ± standard deviation of three cheeses). a-h Values with the same letter are in statistically homogeneous groups on the basis of a two-way ANOVA ('ripening time*salting method' interaction).

Figure S 3 .

 3 Figure S 3. Evolution of proteolysis of the cheese core during ripening according to three salting methods (mean ± standard deviation of three cheeses). a-e Values with the same letter are in statistically homogeneous groups on the basis of a two-way ANOVA ('ripening time*salting method' interaction).

Table 3 .

 3 Sum of relative abundance of each species produced blue-cheese cores (mean ± standard deviation of three cheeses).

	a-h Means in a column sharing common superscripts are similar as tested by Fisher test (Two-way ANOVA, P <0.05).
	ND : No detected

ACKNOWLEDGEMENTS

This work was supported by FEDER (European Regional Development Funds) in the framework of the call for proposals Pack Ambition Recherche 2018 in the Auvergne-Rhône-Alpes region (France) and it was also co-financed by the syndicats interprofessionnels du Bleu d'Auvergne (SIRBA) et de la Fourme d'Ambert (SIFAM). We thank the Pôle Fromager AOP Massif Central for their invaluable assistance.

We thank S. Alvarez, K. Fayolle and D. Guerinon, R. Lavigne (UMR 545 Fromage, Vetagro Sup), for their valuable technical assistance. We also thank B. Desserre for biomolecular analysis help and S. Theil for the bioinformatics support.

Declarations of competing interest

The authors declare that they have not known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supplementary Data

Supplementary Materials

Physico-chemical and biochemical analysis

Standard physicochemical measurements of the cheeses were performed: All samples for analysis had been previously ground and stored at -20 °C. Cheese pH was measured using a penetration pH electrode CG 840 (Schott, Mainz, Germany). The dry matter (DM) was analysed by desiccation. In compliance with the standard NF ISO 5534:2004, a weighed test portion mixed with sand was dried by heating it for 24 h at 102 °C. The dried test portion was then weighed to determine the mass loss. Fat content was determined using the HEISS method according to the procedure described in standard NF V 04-287:2019. In a cheese butyrometer the proteins were first dissolved with a combination of acetic acid and perchloric acid and then separated from the fat by centrifugation. The fat content was read directly on the butyrometric scale with correction equation.

Total nitrogen matter (TNM) was determined by the Kjeldahl method in compliance with the standards NF ISO 8968-1:2014. About 1g of the sample was mineralised with a mix of concentrated sulfuric acid and potassium sulfate. Copper sulfate was used as a catalyst to convert the organic nitrogen present to ammonium sulfate. Then excess sodium hydroxide was added to the cooled mineralization to release ammonia which was steam distilled into excess boric acid solution.

Hydrochloric acid titration was performed to calculate the nitrogen content from the amount of ammonia produced. Total Nitrogen content was converted in crude protein by a factor of 6.38. Calcium (Ca) and sodium (Na) contents were measured by inductively coupled plasma optical emission spectroscopy according to NF EN 16943:2017. The samples were first digested with nitric acid (60%) and hydrochloric acid (30%). After nebulisation, the aerosol was directed to a high frequency induced argon plasma, in which the elements were atomised and excited for irradiation. Sodium content was converted into salt percent. All measurements were performed in triplicate. Lactate levels were measured by enzymes assay kit (Megazyme, Ireland). As described in