Hassan Safa To cite this version · 2020-05-26 · Hassan Safa To cite this version: Hassan Safa....

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HAL Id: tel-01312196 https://tel.archives-ouvertes.fr/tel-01312196 Submitted on 4 May 2016 HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Réduction combinée en chlorure de sodium et en matière grasse animale lors de la fabrication du saucisson sec : effets sur les propriétés physicochimiques et les réactions biochimiques en lien avec la production aromatique et les attributs sensoriels Hassan Safa To cite this version: Hassan Safa. Réduction combinée en chlorure de sodium et en matière grasse animale lors de la fabrication du saucisson sec : effets sur les propriétés physicochimiques et les réactions biochimiques en lien avec la production aromatique et les attributs sensoriels. Alimentation et Nutrition. Université Blaise Pascal - Clermont-Ferrand II, 2016. Français. NNT: 2016CLF22668. tel-01312196

Transcript of Hassan Safa To cite this version · 2020-05-26 · Hassan Safa To cite this version: Hassan Safa....

Page 1: Hassan Safa To cite this version · 2020-05-26 · Hassan Safa To cite this version: Hassan Safa. Réduction combinée en chlorure de sodium et en matière grasse animale lors de

HAL Id: tel-01312196https://tel.archives-ouvertes.fr/tel-01312196

Submitted on 4 May 2016

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

Réduction combinée en chlorure de sodium et enmatière grasse animale lors de la fabrication du

saucisson sec : effets sur les propriétés physicochimiqueset les réactions biochimiques en lien avec la production

aromatique et les attributs sensorielsHassan Safa

To cite this version:Hassan Safa. Réduction combinée en chlorure de sodium et en matière grasse animale lors de lafabrication du saucisson sec : effets sur les propriétés physicochimiques et les réactions biochimiquesen lien avec la production aromatique et les attributs sensoriels. Alimentation et Nutrition. UniversitéBlaise Pascal - Clermont-Ferrand II, 2016. Français. �NNT : 2016CLF22668�. �tel-01312196�

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Page 15: Hassan Safa To cite this version · 2020-05-26 · Hassan Safa To cite this version: Hassan Safa. Réduction combinée en chlorure de sodium et en matière grasse animale lors de

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1 TeRiFiQ: Combining Technologies to achieve significant binary Reductions in Sodium, Fat and Sugar content in everyday foods whilst optimizing their nutritional Quality (Convention de subvention n °289397, www.terifiq.eu)

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Chapitre 4: Discussion générale

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Page 174: Hassan Safa To cite this version · 2020-05-26 · Hassan Safa To cite this version: Hassan Safa. Réduction combinée en chlorure de sodium et en matière grasse animale lors de

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Chapitre 4: Discussion générale

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ORIGINAL PAPER

Effect of Combined Salt and Animal Fat Reductionson Physicochemical and Biochemical ChangesDuring the Manufacture of Dry-Fermented Sausages

Hassan Safa1 & Philippe Gatellier1 & André Lebert2 &

Laurent Picgirard3& Pierre-Sylvain Mirade1

Received: 13 April 2015 /Accepted: 13 July 2015 /Published online: 23 July 2015# Springer Science+Business Media New York 2015

Abstract Reducing salt and fat contents in dry-fermentedsausages could benefit consumer health. This study aimed toquantify, from an experimental design, the effects of salt andfat contents and combined salt and fat reductions on the time-course of several physicochemical (product weight loss, meanwater activity and pH values) and biochemical (proteolysis,lipolysis and lipid and protein oxidations) parameters.Statistical analyses found that time, salt and fat contents hada very significant impact on weight loss and aw and that timeand salt content (not fat content) had a significant impact onpH. Biochemical results indicated that proteolysis was salt-content-dependent and amplified by combined salt and fatreductions. Intensity of lipolysis was mainly dependent onfat content. Lipid and protein oxidations were more intensein higher-fat formulations. Combined salt and fat reductions indry-fermented sausages increased acidification, weight lossesand aw, leading to more proteolysis, less lipolysis and lessoxidation. Sensory studies are now required to investigateconsumer acceptability of these healthier sausages.However, the present results constitute a valuable set of datafor helping professionals wishing to reduce salt and fat con-tents in dry-fermented sausages.

Keywords Dry-fermented sausage . Combined salt and fatreductions . Physicochemical properties . Proteolysis .

Lipolysis . Lipid and protein oxidations

Introduction

Meat and meat products are very important elements inhuman diet as they are especially rich in proteins, vitaminsand minerals (Josquin et al. 2012). Dry fermentation anddry curing are centuries-old techniques of meat preserva-tion, and dry-processed meats have become typical, authen-tic and very popular products across Europe. To illustrate,Spain produces about 200,000 t of dry-fermented sausageper year (Martin-Sanchez et al. 2011) while France pro-duces another 110,000 t (Hoz et al. 2004). Dry-fermentedsausages are made to different recipes using different tech-nologies in different countries and local regions, but all aregenerally prepared from raw meats consisting of about two-thirds lean meat and one-third fat. In general, the lean meatcomes from different animal sources such as pork and beef,whereas the fat is almost always pig backfat. The termBdry-fermented sausage^ corresponds to raw meats pre-served under the effect of salting, lactic fermentation anddrying. These products are manufactured by mincing rawmeat and pork backfat added with 5 % of spices, curingagents—chiefly sodium chloride—additives and starter cul-ture. The meat batter thus obtained is pushed into artificialor natural casings before undergoing fermentation, steamingand drying processes (Olivares et al. 2009).

Excessive consumption of salt, especially sodium chloride,in human diet is strongly related to increased blood pressureand consequently cardiovascular disease (Gelabert et al.2003), and to certain cancers, including stomach cancer(Stollewerk et al. 2012). To limit the development of these

* Pierre-Sylvain [email protected]

1 INRA, UR370 Qualité des Produits Animaux,63122 Saint-Genès-Champanelle, France

2 Institut Pascal, UMR6602 UBP/CNRS/IFMA, 24 avenue desLandais, BP80026, 63171 Aubière Cedex, France

3 ADIV, 10 rue Jacqueline Auriol, ZAC Les Gravanches,63039 Clermont-Ferrand Cedex 2, France

Food Bioprocess Technol (2015) 8:2109–2122DOI 10.1007/s11947-015-1563-3

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human diseases, several health organizations in industrializedcountries worldwide recommend limiting salt consumption to6 g per day (e.g., US Department of Health and Human Services2005). These organizations also prone reducing fat intake to lessthan 30 % of total calories to limit the development of serioushealth disorders like obesity, high blood cholesterol and coronaryheart diseases which are highly related to excessive intake of fat-rich foods, especially saturated fatty acids (SFAs) and cholesterol(Ansorena and Astiasarán 2004; Ruiz-Capillas et al. 2012).Given that dry-fermented sausages contain high levels of sodiumand SFA, from a health standpoint, it would be beneficial toreduce the salt and fat contents in this family of meat products.However, salt and fat play key functional ingredients in dry-fermented sausagemanufacture. For instance, salt ensures micro-biological safety by decreasing water activity (aw), as well asacting on the drying process by decreasing weight losses. It hasalso been reported that salt affects biochemical reactions such asproteolysis, lipolysis, oxidation and fermentation that are in-volved in the development of the final sensory attributes ofdry-fermented sausages, such as texture, flavour and appearance.On the other hand, fat in meat products is considered a valuablesource of energy, fatty acids and vitamins, and biochemical reac-tions affecting the lipid fraction are also involved in the develop-ment of the final sensory attributes of dry-fermented sausages,such as flavour, juiciness and texture. Several studies have fo-cussed on the effect of reducing salt or fat content on physico-chemical properties (weight losses, aw and pH) and biochemicalreactions (proteolysis, lipolysis and lipid and protein oxidations).For instance, Mora-Gallego et al. (2013) recently reported thatsalt slows the drying process by affecting the binding water ca-pacity of protein and that reducing fat content accelerates productweight losses. Furthermore, Roseiro et al. (2008) showed thatsalt-reduced dry-fermented sausages showed higher aw valuesthan control batches. Likewise, Olivares et al. (2010) showedthat reducing fat content in dry-fermented sausages led to slightlyhigher end-of-process aw. The literature on pH is far less consis-tent. Corral et al. (2013) did not find significant difference be-tween pH values of salt-reduced and control dry-fermented sau-sages, whereas Roseiro et al. (2008) reported higher fermentationrates in salt-reduced dry-fermented sausages. With respect to fatcontent, Olivares et al. (2010) found a faster pH decline in low-fat sausages whereas Muguerza et al. (2002) did not find anyeffect of fat level on pH value. Other authors have focussed onthe effect of salt content and fat content on the biochemicalreactions in meat products. Harkouss et al. (2014), for example,reported higher proteolytic activity when salt is reduced in dry-cured ham. Several authors have found higher proteolysis rates insalt-reduced dry-fermented sausages (Armenteros et al. 2012).Likewise, the literature reports higher lipolysis rates in driedsalted meat products (Armenteros et al. 2012; Quintanilla et al.1996; Stahnke 1995). Several studies have reported that NaCl isable to accelerate the oxidation process (Jin et al. 2013), whereasother authors find that salt plays an antioxidant role. For example,

Rhee et al. (1983) reported that lipid oxidation is inhibited inground pork meat prepared with high NaCl concentrations.Some authors also observed a significant increase of lipid oxida-tion in sodium-reduced dry-fermented sausage formulationscompared to the traditional one, when using calcium chlorideto partially replace sodium chloride (Flores et al. 2005; Zanardiet al. 2010). Reducing fat content logically reduces the extent oflipolysis and the production of lipid oxidation products. Sanchez-Zapata et al. (2013) highlighted a positive impact of adding tigernut fibre as a source of fibre and walnut oil as a source of unsat-urated fatty acids during the manufacture of a traditional Spanishdry-cured fermented sausage. However, all the above cited stud-ies focussed on the impact of separate reductions in salt or fatcontent during dry-fermented sausage manufacture. To ourknowledge, few studies have addressed a combined reductionin salt and fat.

Therefore, the objective of this study was to use an exper-imental design to quantify the effect of salt content, fat contentand combined salt and fat reductions on the time-course ofseveral physicochemical properties and on the biochemicalreactions that occur during the manufacture of dry-fermentedsausages.

Materials and Methods

Manufacture of Dry-Fermented Sausages

To investigate the effects of time, fat content, salt content andtheir interactions on physicochemical and biochemical chang-es during the processing of dry-fermented sausages, aDoehlert design (Doehlert 1970) was established on the basisof two factors: initial animal fat content in the range 8.4–21 %and initial salt content in the range 2.0–2.8 %. The first factor,i.e., fat content, was studied at five levels: 8.4, 11.6, 14.7, 17.9and 21.0 %, while the second factor, i.e., salt content, wasstudied at three levels: 2.0, 2.4 and 2.8 %. In this study, settingup a Doehlert design leads to seven formulations of dry-fermented sausages with different amounts of salt and fat, asindicated in Table 1. We added a control fabrication numberedBeight^ in which dry-fermented sausages were manufacturedfrom a 2.8% initial salt content and a 21% initial fat content tobe used as baseline reference for the other seven fabrications.

For each formulation in Table 1, about 30 dry-fermentedsausages were manufactured as per the following procedure.Raw pork meat, i.e., pork shoulder and pork backfat, waspurchased from a local distributor (DISTRIPORC,Clermont-Ferrand, France). On receipt of the pork meat, weverified the pH, moisture and water activity (aw) of the porklean as pH=5.98, moisture=73.5% and aw=0.97. Pork shoul-ders were defatted and cut into small parallelepipeds. For eachformulation, the corresponding amount of defatted porkshoulder and backfat was weighed, ground to 6-mm diameter

2110 Food Bioprocess Technol (2015) 8:2109–2122

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and mixed with the corresponding amount of salt, a set ofadditives and a starter culture corresponding to a mid-acidification kinetic starter. The starter culture (MF55,Biovitec, Lissieu, France) was prepared at 100 kg/L concen-tration and added to each formulation at 10 g/kg. In each meatbatter, we added dextrose (5 g/kg), potassium nitrate(0.3 g/kg), potassium erythorbate (0.5 g/kg), black pepper(2 g/kg), garlic powder (0.5 g/kg) and finally a solution ofstarters (10 g/kg). The meat batter was then stuffed into 50-mm-diameter collagen casings. The raw sausages, weighing450 g and about 20 cm in length, were then plunged in aPenicillium nalgiovensis solution to cover their surface duringthe drying stage. All products were steamed for 4 days at24 °C and 70 % relative humidity (RH) and then dried for25 days at 13 °C and 70 % RH in the same ripening room.

For the eight batches of Table 1, three sausages were taken atdays 1, 2, 5, 7, 21 and 29 of drying to evaluate the time-coursepatterns of chemical composition, physicochemical parameters(aw, weight loss, pH) and biochemical parameters (proteolysis,fermentation, protein oxidation, lipid oxidation). As the experi-ments were cumbersome to set up, the biochemical parameterswere ultimately only assessed at four timepoints, i.e., days 1, 7,21 and 29. In addition, for biochemical and basic chemical anal-yses, all the dried products were individually treated with liquidnitrogen, ground down into fine powder to minimize problemstied to heterogeneity of sampling in subsequent analysis andstored at −80 °C until analysis.

Chemical Composition of Dry-Fermented Sausages

Moisture

Moisture was determined by drying about 1.5 g of powdered-down sample at 80±2 °C in a temperature-controlled chamber(Model FT127U, Firlabo, France) until constant weight,which took at least 48 h (adapted from Norm NF V 04401).

Moisture content was expressed on a total matter basis(kg H2O/kg TM). All moisture measurements were performedin six replicates.

Salt Content

Salt content was measured using 2 g of powdered-down sam-ple. The sample was homogenized (Ultra-Turrax system, Ika,Germany) with 20 mL of ultrapure water. After a 3-h restperiod, the homogenate was centrifuged at 11,300g for10 min at room temperature (MiniSpin Plus, Eppendorf,France). The supernatant was recovered, diluted in ultrapurewater and run through an ion chromatography system (850professional IC, Metrohm France SAS, France) to systemati-cally measure chloride ion and sodium ion contents. The chlo-ride ion or sodium ion values were then used to calculate anequivalent NaCl content (%). All the details on this measure-ment technique developed in the laboratory can be found inBombrun (2013). All salt content measurements were per-formed in six replicates.

Fat Content

Fat content was determined on powdered-down samplesbased on the method of Folch et al. (1957) but usingdichloromethane/ethanol (2:1) instead of chloroform/methanol (2:1) as solvent. Total lipids from 0.5 g of samplewere extracted with 50 mL of solvent. The organic phase(dichloromethane) containing total lipids was separated using10 mL of salt solution at 0.73 %, after centrifugation at 2000gfor 5 min, at 4 °C. The extract obtained was evaporated in avacuum evaporator and weighed to determine total lipid con-tent. All fat content measurements were performed in sixreplicates.

Physicochemical Analysis of Dry-Fermented Sausages

Weight Loss

Throughout the dry-fermented sausage drying period, nineproducts from each batch arranged on the same bar wereweighed together practically every day to determine the kinet-ics of weight loss. Weight loss was expressed as percentage ofinitial weight.

Water Activity (aw)

Water activity (aw) was measured at 20 °C with a laboratoryaw meter (aw Sprint TH-500, Novasina, Switzerland).Preliminary tests performed to measure aw individually onthe three sausages of each formulation showed that therewas no significant difference between the three values

Table 1 Details of experiments giving all the formulations of dry-fermented sausages performed in the present study

Experiments Animal fat content (%) Sodium chloridecontent (%)

S1 14.7 2.4

S2 21 2.4

S3 17.9 2.8

S4 8.4 2.4

S5 11.6 2.0

S6 17.9 2.0

S7 11.6 2.8

S8 control 21 2.8

This list was built from a Doehlert design on the basis of two factors:initial animal fat content in the range 8.4–21% total matter and initial saltcontent in the range 2.0–2.8 % total matter

Food Bioprocess Technol (2015) 8:2109–2122 2111

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(<0.001 aw unit). Water activity values were therefore deter-mined on a mixture of the three sausages for each formulation.

pH

pH was determined using 1 g of powdered-down sample ho-mogenized (Ultra-Turrax system, Ika, Germany) with 10 mLof ultrapure water. pHwasmeasured conventionally with a pHmeter (InLab427, Mettler Toledo, France) calibrated withstandard solutions of pH 4 and pH 7. All pH measurementswere performed in nine replicates.

Biochemical Analysis of Dry-Fermented Sausages

Proteolysis

The proteolysis index (PI) of each powdered-down sample ofdry-fermented sausages was determined using a fluorescencemethod based on the reaction of fluorescamine with N termi-nal α-amino groups of peptides in TCA soluble fractions(Harkouss et al. 2012). In this method, PI is defined as thepercentage of the ratio of amino group content to total proteincontent. All PI measurements were performed in ninereplicates.

Lipolysis

The degradation of fat into fatty acids was quantified by de-termining the acid value of fat (Norm NF T 60–204) in sam-ples of dry-fermented sausages, at day 29. Briefly, total freefatty acids from 25 g of sample were solubilized in a solventmix of ether/ethanol. Total free fatty acids were determinedquantitatively by potassium hydroxide (0.1 N) in the presenceof a colour indicator (phenolphthalein). Before neutralization,phenolphthalein (acid medium) is colourless. Under basicconditions (beyond neutralization), it colours pink. The num-ber of equivalents of potassium hydroxide poured is equal tothe number of equivalents of acid present in the sample, andacid value is the mass of potassium hydroxide, in milligrams,required to neutralize 1 g of fat. All these measurements wereperformed in three replicates.

Lipid Oxidation

Lipid oxidation was quantified in powdered-down samples ofdry-fermented sausages by determining hydrosoluble Schiffbases (HSBs) using the procedure reported in Gatellier et al.(2009). For this purpose, the aqueous phase obtained to deter-mine fat content was collected to quantify HSB levels. Astandard curve of commercial Schiff bases (quinine) was pre-pared in parallel. Fluorescence of each point of the standardcurve and fluorescence of each sample were measured with aspectrofluorometer (FP 8300, Jasco France, France) at a 370-

nm excitation wavelength (excitation slit, 10 nm), a 470-nmemission wavelength (emission slit, 10 nm) and a 3-s integra-tion time. A linear standard curve of quinine was plotted, andsample HSB levels were expressed as micromoles per kilo-gram of meat versus quinine equivalent. All lipid oxidationmeasurements were performed in nine replicates.

Protein Oxidation

Protein oxidation was assessed in powdered-down samples ofdry-fermented sausages by determining free thiol groups ofcysteine residues from Ellman’s assay using 2,2′-dithiobis(5-nitropyridine) (DTNP) as reagent (Morzel et al. 2006). In al-kaline solution, DTNP binds to the anionic free thiol groups ofcysteine residues to form a complex that absorbs at 386 nm.The results are expressed in nanomoles of bound DTNP permilligram of protein. All protein oxidation measurementswere performed in nine replicates.

Statistical Analyses

To make the results easier to interpret and the figures easier toread, a specific statistical treatment called hierarchical clusteranalysis (HCA) (Chrétien and Szymoniak 1987; Wishart1969) was applied to all measured raw values. HCA consistsin clustering samples that lead to similar results on a givenparameter, thereby creating classes of dry-fermented sausages.HCA was calculated using Ward’s method on STATISTICA10-V2014 software. In addition, when a class of sausages isformed, the values of the parameter corresponding to the classare calculated, at each time point, by averaging the values ofall the same-class sausages.

Statistical analyses of the results were completed by anal-ysis of variance (ANOVA) using STATISTICA 10-V2014software. The objective was to assess the effect of each factor(time, fat content, salt content and salt content × fat contentinteraction) on each variable measured in this study. WhenANOVA found a significant effect (p<0.05), post hoc proce-dures were used: Multiple comparisons among means wereexamined by the Tukey test to determine the level of signifi-cance between groups.

Results and Discussion

Effects on Chemical Composition of Dry-FermentedSausages

Drying dry-fermented sausages globally leads to a reductionin in-product water content due to water evaporation from theproduct surface and, in turn, to a fat and salt concentration thatincreases fat and salt contents, respectively. We therefore ranchemical analysis of moisture, salt content and fat content at

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four timepoints, i.e., day 1, day 7, day 21 and day 29(end of drying) to track and trend the time-course ofthese parameters. Table 2 shows the measured valuesof moisture, salt content and fat content in these dry-fermented sausages.

Moisture

Analysis of Table 2 confirms that in-product moisture de-creases globally with drying, with mean values decreasingfrom initial values of 59.9–68.1 % at day 1 to final values of36.9–43.6 % at day 29 (Table 2). The lowest final water con-tent values were obtained for the two 21 % animal fat formu-lations (S2 and S8, i.e., the control) and for S6 correspondingto a high-fat but low-salt formulation (17.9 % fat and 2.0 %salt, respectively). Note that at day 1, in-product water content

values of all formulations were lower than the water contentmeasured at reception of the fresh lean meat that was not dried(73.5 %), probably as a result of the pork backfat, curingagents (NaCl, nitrite) and additives added to the pork leanwhen preparing the meat batter. Indeed, at day 1, the highermoisture contents were measured on the three formulationscontaining less than 12 % animal fat, and the highest value(68.1 %) was even obtained for the lowest-fat formulation(S4).

It can be concluded that the respective proportions of leanmeat and pork backfat logically influence moisture valuesduring dry-fermented sausage manufacture (Table 2). For anidentical drying process, the highest moisture valuesexpressed as percentage of total matter are obtained for thelow-fat products.

Salt Content

Analysis of Table 2 logically indicates a net increase in in-product salt content with drying. Note that fresh pork lean isknown to naturally contain small amounts of sodium and chlo-ride ions; here we measured a 0.17 % equivalent of NaClcontent in the fresh pork lean. At day 1, measured salt contentvalue ranged from 2.34 to 3.13 % depending on formulation(Table 2), whereas intended salt content corresponded to thelevels of the Doehlert design: 2.0, 2.4 and 2.8 %. This impliesthat real salt content values are globally higher than intendedvalues. Accurate analysis between real and intended salt con-tent shows that discrepancies ranged from 0.18 % (S8) to0.52 % (S2) depending on formulation, with a mean discrep-ancy of 0.37 %. This underlines just how difficult it is to addexactly the intended amount of NaCl to meat batters withvarying proportions of lean meat and fat, even at pilot-scalelevel. At day 29, the final NaCl content values ranged from6.26 to 7.64 % depending on experiment and on the weightloss experienced by the dry-fermented sausages. The highestfinal NaCl content value was measured for the control (S8)that initially contained about 2.98 % NaCl (day 1) and thatcounted one of the lowest final water contents measured(60.8 %, Table 2).

Regarding in-product salt content, it can be concluded thatthe measured salt content values at day 1 were still higher thanthe intended values, probably as a result of the natural pres-ence of sodium and chloride ions in lean pork meat andvery probably as a result of the real difficulty in per-fectly adjusting the amount of salt added during themeat batter preparation. However, the formulations withthe higher intended salt content values neverthelesscontained more salt than the others once the meat bat-ters were prepared, thus a priori lowering the impact ofthis observed discrepancy on the physicochemical andbiochemical results subsequently obtained.

Table 2 Time-course evolution of chemical composition (moisture,sodium chloride and animal fat contents) measured in dry-fermentedsausages for the eight formulations of Table 1

Experiments Day 1 Day 7 Day 21 Day 29

Moisture content (% total matter)

S1 62.5±0.8c 57.7±0.7c 46.1±0.6bc 43.6±1.0a

S2 61.1±0.7b 54.8±0.6de 41.3±0.8e 37.4±0.8c

S3 59.9±0.5a 55.6±0.8d 43.4±1.2de 41.1±0.6b

S4 68.1±0.6f 62.9±1.0a 49.8±0.9a 40.7±1.0b

S5 66.0±0.5e 59.8±1.2b 48.1±0.3ab 39.7±0.7b

S6 61.7±0.5bc 51.7±0.7f 42.0±0.5e 38.2±0.5c

S7 64.8±0.3d 59.3±0.6b 45.4±0.3cd 40.4±0.7b

S8 control 60.8±0.6ab 54.1±0.5e 42.8±1.0e 36.9±0.7c

Sodium chloride content (% total matter)

S1 2.86±0.50bcd 3.76±0.30c 5.77±0.41cd 6.54±0.60bcd

S2 2.92±0.25abc 4.35±0.15ab 5.93±0.12c 6.56±0.23bcd

S3 3.13±0.30a 4.5±0.40a 6.76±0.40a 7.15±0.50ab

S4 2.84±0.23cd 3.77±0.29c 6.27±0.20b 6.86±0.20bc

S5 2.34±0.17de 3.62±0.13d 5.61±0.18d 6.26±0.13d

S6 2.44±0.13cde 3.64±0.14d 5.66±0.14d 6.39±0.20cd

S7 3.08±0.14ab 4.23±0.16b 5.71±0.15cd 6.34±0.16cd

S8 control 2.98±0.16ab 4.33±0.20ab 6.57±0.17ab 7.64±0.23a

Animal fat content (% total matter)

S1 14.3±0.2c 17.7±1.3b 22.8±1.0c 25.6±0.4d

S2 18.9±0.2ab 23.3±0.6a 31.1±1.6a 34.0±1.4ab

S3 17.1±0.4b 19.8±1.0b 25.7±1.6b 29.5±1.6c

S4 8.0±0.3e 10.8±1.3d 14.7±0.2e 16.5±0.4f

S5 11.2±0.2d 14.2±1.1c 19.7±0.2d 20.8±0.4e

S6 18.1±0.4b 22.7±0.8a 27.8±1.6b 31.0±1.8bc

S7 12.0±0.2d 14.5±1.4c 19.3±1.4d 21.0±1.4e

S8 control 20.6±0.5a 25.2±1.1a 31.9±1.3a 35.0±1.3a

Measured values were the means±standard deviation calculated from sixindependent determinations. Values not bearing common superscriptsdiffered significantly (p<0.05)

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Total Fat Content

Analysis of Table 2 confirms that animal fat concentrates withdrying. Like for salt content, discrepancies emerged betweenmeasured in-product total lipid content and the intended fat con-tent fixed by the Doehlert design, but the order is respected,meaning that the formulations with the highest intended fat con-tents have the highest measured total lipid contents, and viceversa (Table 2). For example, formulations corresponding to S2and S8 for which intended fat content was 21% really contained18.9 and 20.6% total lipids, respectively, at day 1. For the 17.9%fat formulations (S3 and S6), the discrepancy in total lipid con-tent was −0.8 and +0.2 %, respectively. Moreover, 8.0 % totallipid content was measured in the specific case of formulation S4where 8.4 % fat content was planned. Finally, discrepancy intotal lipid content ranged from −1.1 to +0.2 % depending onexperiment investigated, again underlining the real difficulty inperfectly adjusting the amount of added fat during meat batterpreparation. The formulations can be sorted as a function of theirreal total lipid content in the same order, from the beginning (day1) until the end (day 29) of the drying process. However, fortu-nately, the formulations with the higher intended fat contentvalues really contained more fat than the others, once the meatbatters were prepared, thus a priori lowering the impact of thisobserved discrepancy on the results subsequently obtained.

Effect on Physicochemical Parameters in Dry-FermentedSausages

Time-Course of Weight Loss

Figure 1a charts weight loss kinetics during the ripening pro-cess. First of all, from day 1, all the dry-fermented sausageslose weight corresponding to the water lost through evapora-tion during steaming and ripening. HCA applied to all weightloss values led to the formation of three classes of formula-tions, as shown in Fig. 1a. The first class, corresponding to thelowest weight loss (about 43 % at day 29), is composed ofexperiments S1, S2, S3, S6 and S8 (control). The two otherclasses strongly diverge from the first class from day 5, andthey pool the three lowest-fat formulations that generatehigher water losses from the sausages. These two classes stayvery close up to day 14 and thus separate the three lowest-fatformulations as a function of their relative NaCl content; thehighest-salt formulation (S7) led to lower weight loss (45.4 %at day 29) than the two other formulations (S4 and S5) whichare less salty (2.4 and 2.0%, respectively) for which water lossreached 49 % at the end of drying. Thus, Fig. 1a shows thatlean dry-fermented sausages globally lose more water than thefattiest products.

Furthermore, these results were confirmed by the ANOVAand Tukey tests (Table 3) that highlighted very highly signif-icant effects of time, backfat and salt content (p<0.001) but no

significant effect of the interaction between salt and fat content(p>0.05). These results are in line with several studies de-signed to assess the impact of reducing salt or fat content onthe sensory qualities of these kinds of meat products (Corralet al. 2014; Liaros et al. 2009), but disagree with Corral et al.(2013) who did not find any differences in weight losses forslow-fermented dried sausages prepared with different levelsof salt (2.7 and 2.26%). However, the salt effect observed herewas for a higher level of salt reduction (−30%) combinedwithhigh fat reduction. Regarding the effect of fat content on prod-uct water loss, Olivares et al. (2010) failed to find differencesin water loss between high and low-fat sausages, but theystudied dried slow-fermented sausages slow-ripened for63 days, whereas here where we applied high fermentationand faster drying.

Time-Course of Mean Water Activity Values

Figure 1b charts the kinetics of mean in-product aw valuesmeasured for the eight formulations of Table 1. Generallyspeaking, the drying process led to a reduction in water con-tent and to a concentration of salt into the matrix, and thus to adecrease in mean aw value.

HCA applied to aw values led to the formation of threeclasses of formulations (Fig. 1b). The class corresponding tothe lowest mean values of aw, with variations from 0.958 atday 1 to 0.886 at day 29, is formed by batches S3, S7 and S8,i.e., the three highest-salt formulations containing at least11.6 % animal fat. The class corresponding to the highest in-product mean aw values, with variations from 0.963 at day 1 to0.901 at day 29, is formed by batches S4, S5 and S6, i.e., thetwo lowest-NaCl formulations (S5 and S6) and the lowest-fatformulation (containing just 8.4 % animal fat) (S4). The thirdclass is formed by experiments S1 and S2 which contain amoderate NaCl content (2.4 %) and a fairly high (>14.7 %)animal fat content. This class presented an intermediate pat-tern in terms of time-course of mean in-product aw, withvalues ranging from 0.960 at day 1 to 0.897 at day 29. Notethat all the final mean in-product aw values are below 0.92 andthat aw values below 0.92 are considered safe in terms ofListeria monocytogenes growth capacity (Ingham et al. 2004).

In a similar way to the time-course of weight loss, thestatistical analyses performed on the mean aw values, i.e.,ANOVA and Tukey tests, showed very highly significant ef-fects of time, salt content and fat content (p<0.001) but nosignificant effect of the interaction between salt and fat content(p>0.05) (Table 3). Literature data on the effect of fat contenton aw values is inconsistent. Some authors have reported thatreducing fat content during dried sausage manufacture led tohigher final aw values (Corral et al. 2014; Gómez and Lorenzo2013) or higher aw values at the fermentation stage (Olivareset al. 2010) whereas others did not find any significant differ-ence in aw values between low and high-fat dried sausages

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0

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Experiment S7 (45.4% at day 29)

Experiments S4-S5 (49.0% at day 29)

Experiments S1-S2-S3-S6-S8 control (42.9% at day 29)

A

a

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Experiments S1-S2 (0.899 at day 29)

Experiments S4-S5-S6 (0.901 at day 29)

Experiments S3-S7-S8 control (0.886 at day 29)

0.88

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Experiments S5-S6 (5.25 at day 29)

Experiments S1-S2-S3-S4-S7-S8 control (5.40 at day 29)

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6.5

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Fig. 1 Time-course of threephysicochemical parametersinvestigated for the eightformulations of dry-fermentedsausages given in Table 1: aweight losses, b mean wateractivity (aw) values and c pHvalues. Hierarchical clusteranalysis was performed on rawvalues in order to pool dry-fermented sausage formulationspresenting similar patterns.Values not bearing commonsuperscripts differed significantly(p<0.05)

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manufactured with cereal and fruit fibres (García et al. 2002).Concerning salt content, several studies have mentioned thatreducing salt logically led to higher aw values (Olesen et al.2004; Roseiro et al. 2008). In contrast, other authors did notfind any difference in final aw values of dried sausages madewith different levels of salt. For example, Corral et al. (2013)found significant differences in aw values after 9 days but,surprisingly, not at the end of processing. However, Corralet al. (2014) reported that a combined reduction of salt andfat contents affected sausage quality by producing an increasein final aw values.

Concerning the time-course of mean aw values, the HCA-based results presented here logically showed a strong impactof salt content and also an effect of fat content. Indeed, mod-ifying the fat content of the meat batter modifies the salt con-centration in the lean part of the batter, and thus the wateractivity value. From a water activity perspective, reducingfat content in dry-fermented sausages provokes the same in-crease on aw as reducing salt content. So, binary reductions infat and salt contents may prove detrimental from a safetystandpoint if the products are not sufficiently dried afterwards.

Time-Course of pH Values

Figure 1c charts the time-course of pH values. All formula-tions followed a normal time-course of pH values with astrong decrease in pH during the first week of process, from5.91 to 5.93 at day 1 to a minimum of about 5.0 at day 7,

corresponding to intense acidification due to the action of thelactic acid bacteria (LAB) added during sausage manufacture.This period is characterized by an exponential growth phase ofLAB that fermented sugar substrate into lactic acid. Beyondday 7, pH values increased progressively until the end of thedrying process as a result of an array of phenomena includinga strong decrease in LAB acidifying action due to the deple-tion of sugar substrate, the transformation of lactic acid intoother chemical substances, consumption of lactic acid bymoulds (Flores et al. 2004) and/or the production of alkalinemolecules due to proteolytic mechanisms (Ordóñez et al.1999).

HCA applied to the pH values of all the formulations led tothe formation of two distinct classes differentiated accordingto NaCl content of the formulations (Fig. 1c). Lower pHvalues were obtained for the two lowest-salt formulations,i.e., S5 and S6 that contain only 2.0 % NaCl, probably dueto more intense LAB activity. The second class pooled all theother formulations that contain at least 2.4 % NaCl. The dif-ference between the two classes resided in the fact that a moreintense acidification was observed for the two lowest-salt for-mulations during the first week of process (with 0.10 pH unitless at day 7); this discrepancy persisted as pH values in-creased over the next 2 weeks of process, before growingslightly stronger during the last week of process to peak at0.15 pH units at day 29. However, all these pH values, espe-cially the lowest and final values, are fully representative ofwhat classically happens during French dry-fermented

Table 3 Details of statistical analyses: A –Analysis of variance andB –Post hoc Tukey procedure, performed on the values of physicochemical(weight loss, aw, pH) and biochemical (proteolysis, lipolysis and lipid and

protein oxidations) parameters measured in the dry-fermented sausagesfor all the formulations of Table 1

A – Analysis of variance (ANOVA)

Weight losses aw pH Proteolysis (proteolysisindex)

Lipolysis (acidityvalue)

Lipid oxidation(HSB)

Protein oxidation(thiol group)

Time *** *** *** *** (−) *** ***

Salt *** *** ns *** ** ns ns

Fat *** *** *** *** *** *** ***

Salt*Fat ns ns ns *** ns ns ns

B – Post hoc procedure (Tukey test)

S1 19.0a 0.940de 5.45±0.34a 4.80±1.25b 8.1±0.33ab 3.70±1.07b 26.64±1.19b

S2 18.9a 0.939cd 5.43±0.35 a 4.75±1.22b 10.4±0.26cd 3.87±1.11cd 21.80±1.21a

S3 18.7a 0.935bc 5.41±0.34a 4.39±0.92a 9.0±0.13b 3.82±1.10bc 26.67±0.83b

S4 22.3b 0.943de 5.41±0.31a 4.89±1.28b 7.4±0.30a 3.51±0.86a 30.16±0.53d

S5 21.6b 0.944e 5.33±0.37b 5.45±1.51d 7.9±0.25ab 3.52±0.89a 28.60±0.62c

S6 20.5ab 0.944e 5.31±0.36b 5.17±1.38c 9.7±0.35c 3.83±1.08bc 26.32±0.6b

S7 21.0b 0.935bc 5.41±0.33a 4.34±0.96a 7.5±0.45a 3.50±0.94a 28.72±1.46c

S8 control 18.6a 0.933a 5.40±0.34a 4.25±0.81a 10.0±0.38c 3.96±1.26d 21.81±0.64a

ANOVAwas run to test the effects of time, salt content, fat content and the interaction salt content × fat content on all the parameters studied. Significanceis noted as follows: ns (p>0.05), * (p<0.05), ** (p<0.01), *** (p<0.001), and (−) corresponds to a factor not studied. ATukey test was applied on themeans±standard deviation calculated from all independent determinations and for all timepoints. Values not bearing common superscripts differedsignificantly (p<0.05)

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sausage manufacture (Rason et al. 2007). Note that in the dry-ing process, low aw values and the pH values at fermentationstage (day 7) and at the end of ripening (day 29) obtained in thisstudy are considered enough to ensure the microbial safety ofdry-fermented sausages (Papadima and Bloukas 1999).

ANOVA and Tukey test (Table 3) showed very highly sig-nificant effects of time and fat content (p<0.001). On theother hand, salt content and the interaction between salt andfat content appeared non-significant (p>0.05). There is exten-sive research on the effect of reducing salt or fat content on pHvalues in dry-fermented sausages. For example, Olesen et al.(2004) and Stahnke (1995) reported lower pH values at fer-mentation stage in sausages made with lower amounts ofNaCl. In contrast, Corral et al. (2013) did not find a significanteffect on pH evolution nor on final pH value when NaCl wasreduced by 16 % or substituted by 16 % KCl in slow-fermented sausages. The literature reports conflicting data onthe effect of fat content on pH values. For instance, somestudies did not find differences in pH values when comparinghigh- and low-fat dried sausages (García et al. 2002; Liaroset al. 2009; Papadima and Bloukas 1999). In contrast, Olivareset al. (2010) highlighted a faster pH decline in the case of fat-reduced dried sausages. Finally, Corral et al. (2014) did notfind any significant difference in pH values between controlsausages and sausages made with combined reduced-fat andreduced-salt formulations.

Our results (Fig 1c) show that combined salt and animal fatreductions during the manufacture of dry-fermented sausagesled to a fully normal time-course of pH values, with strongacidification during the first week of process followed by aprogressive increase in pH value.

Effect on Biochemical Parameters in Dry-FermentedSausages

During dry-fermented sausage manufacture, and especiallyduring the fermentation and ripening stages, the meat proteinand lipid components undergo enzymatic and chemical mod-ifications. The enzymatic modifications involved are chieflyproteolysis and lipolysis. These biochemical reactions are ofhigh interest because they shape the development of sensoryattributes of dry-fermented sausages, such as texture, juicinessand aroma.

Time-Course of Proteolysis

Figure 2 charts the time-course of proteolysis index. All eightformulations produced the same pattern of behaviour interms of PI time-course. On the first day of process, pro-teolytic enzyme activity is already visible, which is quitelogical because proteolytic enzymes are highly active at atemperature of about 24 °C (Harkouss et al. 2014).Proteolysis index (PI) ranged between 2.9 and 3 % at

day 1 and reached 4.6–5.9 % at day 7 depending onformulation. From day 7, rate of proteolysis decreasednoticeably, probably due to a non-optimal pH value forproteolytic enzyme activity (García-Garrido et al. 2000)due to the intense lactic acidification occurring at thatpoint, as shown in Fig. 1c. Beyond day 21, very surpris-ingly, PI values decreased for all formulation classes(Fig. 2). To be sure that this decrease was not due to aproblem with the fluorescent technique used, we measuredPI in some samples with the classic Kjeldhal methodconsisting in calculating the percentage ratio of non-protein nitrogen content to total nitrogen content. Thisclassical PI measurement method confirmed the decreasein PI values over the last week of the drying process (datanot shown). This ultimately tends to prove that some endproducts of proteolysis had disappeared, likely consumedby microorganisms present in the dry-fermented sausagesat that point in time, and so could not be detected by themeasurement techniques used.

Figure 2 shows that HCA formed three classes of formula-tions. In-depth analysis of results highlighted that the formu-lations are perfectly classified as a function of their respectiveNaCl content, thus further confirming the inhibitory effect ofsalt content on proteolytic enzyme activity. Indeed, the firstclass that led to the lowest PI values is formed by the three2.8 % NaCl formulations (S3, S7 and control S8), the secondclass by the 2.4 % NaCl formulations (S1, S2 and S4) and thethird class by the 2.0 % NaCl formulations (S5 and S6). Atday 21, PI values were 5.2, 6.3 and 6.7 %, respectively, ac-cording to class/salt content considered.

Moreover, ANOVA and Tukey test (Table 3) highlighted,for the first time, very highly significant effects of all thefactors tested, i.e., time, salt content, fat content and also theinteraction between salt and fat content (p<0.001).However, no marked effect of fat content is visible inFig. 2. Nevertheless, when comparing experiments S5and S6 that belong to the same class, finer-grained anal-ysis of the raw results revealed that proteolysis wasslightly more intense in S5 than in S6, with day 21 PIvalues of 6.8 and 6.6 %, respectively. This probablyresulted from a lower fat content (11.6 vs. 17.9 %) thatprovoked a more marked salt dilution and thus a lowersalt concentration in the lean part of the meat batter thatcontained the greater quantity of lean pork meat (i.e.,experiment S5). These results are in good agreementwith Armenteros et al. (2009, 2012).

The present results (Fig. 2) confirmed that proteolysisin dry-fermented sausages was mainly governed by saltcontent but also highlighted that combined salt and an-imal fat reductions could amplify proteolytic enzymeactivity. So, reducing the salt and fat content increasesproteolysis; this can prove detrimental to the final tex-ture of the end products.

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Lipolysis of End Products

Applying HCA on the acidity values (lipolysis) determined atday 29 for all eights formulations led to the formation of twodistinct classes as a function of animal fat content regardless ofNaCl content. One class pooled the four formulations forwhich fat content was at most equal to 14.7 % and the otherclass pooled the 17.9 % animal fat and 21 % animal fat for-mulations. Mean acidity value was significantly higher for theformulations containing over 17 % animal fat, at 9.8 vs.7.6 mg KOH per gram of product fat.

ANOVA showed that salt content significantly affected(p<0.01) rate of lipolysis in dry-fermented sausages and thatfat content was a highly significant factor strongly impactingrate of lipolysis (p<0.001), unlike the interaction between saltand fat content (p>0.05). Olivares et al. (2011) also cited astrong effect of fat content on lipolysis and on the generationof free fatty acids. These authors reported that slow-fermentedsausages initially containing 30% pork backfat showed higherrates of lipolysis than dried products made with lower porkbackfat content (10 and 20 %). Furthermore, several authorshave reported a more intense lipolysis in salt-reduced driedsausages (Stahnke 1995).

To conclude on lipolysis, our data suggests that the inten-sity of this biochemical phenomenon is strongly dependent onfat content and also influenced by salt content, but withoutsignificant interaction between these two factors, unlike forproteolysis.

Time-Course of Lipid and Protein Oxidations

Lipid and protein oxidations that occur throughout the manu-facture of dry-fermented sausages are also involved in the

elaboration of the taste, flavour and odour of these kinds ofdried cured products (Corral et al. 2013).

Lipid Oxidation

Figure 3a charts the time-course of lipid oxidation quantifiedby determining hydrosoluble Schiff base (HSB) values.However, lipid oxidation was first measured by the thiobarbi-turic acid reactive substances (TBARS) method (data notshown). A biphasic curve was obtained for all eight formula-tions, with TBARS value decreasing with time (mg MDA/kgof sample) beyond day 1, which is not a logical pattern andindicates that the TBARS quantification method was ill-suitedto accurate assessment of lipid oxidation in the present study.This prompted us to assess lipid oxidation based on the fluo-rescence of HSB. Schiff bases are formed in meat duringstorage and cooking (Gatellier et al. 2009). They correspondto the binding of the aldehydic products of lipid peroxidationto free amino groups of proteins. The polarity of Schiff basesdepends on their composition. It was observed in raw meatthat the aqueous fluorophores were responsible for up to 72 %of total fluorescence intensity, whereas cooking reversed thepercentages to reach 36 % in the polar phase and 64 % in theapolar phase (Gatellier et al. 2009).

Figure 3a shows the results of HCA applied to HSB valuesof all formulations. Contrary to the TBARS method (data notshown), HSB values increased observably as a function oftime, meaning that lipid oxidation logically increased withtime. Applying HCA led to classify the formulations into threedistinct classes as a function of fat content; higher fat contentmeans higher HSB values and thus higher lipid oxidation. Thefirst class is formed of the three formulations containing atmost 11.6 % animal fat (experiments S4, S5 and S7), thesecond class is formed of the three formulations containing

0

2

4

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8

1 7 21 29

Pro

teo

lysi

s in

dex

in d

ry-f

erm

ente

d s

ausa

ges

(%

)

Time (days)

Experiments S3-S7-S8 control (4.8% at day 29)

Experiments S1-S2-S4 (5.2% at day 29)

Experiments S5-S6 (5.8% at day 28)

aaa

e

c

b

gf

d

e

d

c

Fig. 2 Time-course ofproteolysis index investigated forthe eight formulations of dry-fermented sausages given inTable 1. Hierarchical clusteranalysis was performed on rawvalues in order to pool dry-fermented sausage formulationspresenting similar patterns. Foreach class of dried sausages,values were means±standarddeviation calculated from all theindependent measurement valuesof each experiment constitutingthe class. Values not bearingcommon superscripts differedsignificantly (p<0.05)

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between 14.7 and 17.9 % animal fat (experiments S1, S3 andS6), and the third class gathered the two 21 % animal fatformulations (experiments S2 and control S8). Figure 3ashows that these three classes differ in terms of lipid oxidation,especially at day 21 but less so at the end of product drying(day 29) and not really early on in the process (days 1 and 7)

ANOVA and Tukey tests (Table 3) indicated very highlysignificant effects of time and animal fat content (p<0.001) onlipid oxidation but no significant effect of salt content, nor theinteraction between salt and fat content (p>0.05). These re-sults are in line with Liaros et al. (2009) and Soyer et al. (2005)who reported that low-fat sausage presented lower lipid oxi-dation levels than control sausages. Olivares et al. (2011) alsoreported higher TBARS values in high-fat sausages comparedto low-fat sausages. The literature is less clear on the effect ofsalt on lipid oxidation. For example, very recently, Corral et al.(2015) reported higher TBARS values and higher abundanceof volatile compounds derived from lipid oxidation in salt-

reduced dried sausages, thus confirming their previous results(Corral et al. 2013). However, Jin et al. (2013) reported thathigh salt levels led to higher oxidation by decreasing antiox-idant enzyme activity and releasing iron from heme-bindingprotein. Furthermore, Corral et al. (2015) recently reportedhigher lipid oxidation levels in salt-and-fat-reduced sausages.

Protein Oxidation

Protein oxidation was assessed by quantifying free-thiol-group content. Indeed, thiol groups of cysteine are very sen-sitive to the oxidative process and their oxidation leads to theformation of disulfide bridges. Note that protein oxidation ismaximal when free thiol group content is minimal.

HCA-based results, shown in Fig. 3b, led to the formationof three classes of formulations, once again classified as afunction of their respective fat content. Maximal protein oxi-dation was observed for the two 21 % animal fat formulations

0

1

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3

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6

Hyd

roso

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le S

chif

f b

ase

con

ten

t

(μ m

ola

r/kg

dry

-fer

men

ted

sau

sag

e)

Time (days)

Experiments S4-S5-S7 (4.8 μmolar/kg at day 29)

Experiments S1-S3-S6 (5.2 μmolar/kg at day 29)

Experiments S2-S8 control (5.2 μmolar/kg at day 29)

aaa

bbb

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d

c

ff

e

A

0

10

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1 7 21 29

1 7 21 29

Fre

e-th

iol-

gro

up

co

nte

nt

(nm

ol/m

g p

rote

in)

Time (days)

Experiments S4-S5-S7 (28.4 nmol/mg at day 29)

Experiments S1-S3-S6 (25.9 nmol/mg at day 29)

Experiments S2-S8 control (21.0 nmol/mg at day 29)

c

b

a

d

bc

b bb

bcbc

dd

B

Fig. 3 Time-course of twooxidation reactions investigatedfor the eight formulations of dry-fermented sausages of Table 1: alipid oxidation quantified bydetermining hydrosoluble Schiffbase (HSB) content and b proteinoxidation quantified bydetermining free thiol groupcontent. Hierarchical clusteranalysis was performed on rawvalues in order to pool dry-fermented sausage formulationspresenting similar patterns. Foreach class of dried sausages,values were means±standarddeviation calculated from all theindependent measurement valuesof each experiment constitutingthe class. Values not bearingcommon superscripts differedsignificantly (p<0.05)

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(experiments S2 and S8). Minimal protein oxidation wasfound for the three formulations containing at most 11.6 %animal fat (experiments S4, S5 and S7). An intermediate classof experiments S1, S3 and S6 was formed from the formula-tions containing either 14.7 % animal fat or 17.9 % animal fat.However, since no clear change in protein oxidation appearswith time, beyond day 1 (Fig. 3b), protein oxidation seems tooccur rapidly, maybe directly during the meat batter prepara-tion, without subsequent intensification.

Like for lipid oxidation, ANOVA and Tukey test (Table 3)showed very highly significant effects of time and fat content(p<0.001) on protein oxidation, but without any significanteffect of salt content, nor the interaction between salt and fatcontent (p>0.05). Protein and lipid oxidations are linked bythe fact that lipid oxidation produces free radicals that, in turn,drive protein oxidation. Therefore, maximal protein oxidationoccurred in high-fat formulations. The present results are ingood agreement with Fuentes et al. (2014) who recently re-ported a strong impact of fat level on protein oxidation.Regarding the effect of salt content on protein oxidation,Kanner et al. (1991) concluded that salt level increased thesusceptibility of myofibril to carbonylation and the pro-oxidant activity of iron.

The present results indicate that lipid and protein oxida-tions in dry-fermented sausages are mainly affected by fatlevel but not by salt level in the range studied here (2.0–2.8 % NaCl) nor by the combined salt and animal fat reduc-tions. The fact that reducing fat level decreases both theseoxidative phenomena can potentially affect the final aromaof these products.

Conclusions

A series of eight fabrications of dry-fermented sausages wasdesigned to investigate the effect of reducing salt and animalfat contents on the time-course of physicochemical parameterssuch as pH, weight loss and aw and biochemical parameterssuch as proteolysis, lipolysis, lipid oxidation and protein oxi-dation. Analysis of the time-course of chemical compositionin dry-fermented sausages confirmed that product dryingglobally leads to a reduction in in-product water content dueto water evaporation from the product surface and, in turn, to afat and salt concentration that increases fat and salt contents,respectively. Statistical analyses of the physicochemical pa-rameters measured for dry-fermented sausages showed veryhighly significant impacts of time, salt and animal fat contentson weight loss and aw values but only of time and salt contenton pH values. Indeed, modifying the fat content of the meatbatter modifies the salt concentration in the lean part of thebatter, and thus the water activity value. Regarding aw, reduc-ing fat content in the products provokes the same increase inwater activity as reducing salt content. So, binary reductions

in fat and salt content may prove detrimental from a safetystandpoint if the products are not sufficiently dried. The resultson proteolysis confirmed that this biochemical phenomenonin dry-fermented sausages was mainly governed by salt con-tent and highlighted that it can be amplified by a combinedreduction in fat and salt contents. Our experimental data alsoindicated that intensity of lipolysis was mainly dependent onfat content and that lipid and protein oxidations were moreintense in high-fat formulations.

Combined salt and fat reductions during the manufacture ofdry-fermented sausages globally affect end product quality byincreasing acidification, weight losses and aw values. Thesecombined reductions also accelerate proteolysis and deceler-ate lipolysis and oxidative mechanisms, which can negativelyaffect the final sensory attributes of dry-fermented sausages.Additional, sensory studies are thus necessary to investigatethe final product quality and consumer acceptability of low-salt and low-fat dry-fermented sausages. Furthermore, it willalso be important from an industrial point of view to obtainaccurate data on the potential application of new technologiesin dried sausage production, such as partial substitution ofNaCl by potassium chloride and adding vegetable oil rich inmono-unsaturated fatty acids, in an effort to reduce end prod-uct content of SFA by 60 % and salt content by 30 % in linewith nutritional health recommendations.

Acknowledgments The research leading to these results receivedfunding from the European Union Seventh Framework Programme(FP7/2007-2013) under grant agreement No. 289397 (TeRiFiQ project).This paper is part of the thesis of Hassan Safa, whoworks for this researchprogram. The authors thank R. Favier, R. Agouninessouk, S. Portanguen,and the ADIV staff for their technical assistance, and ATT for proofread-ing the manuscript.

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