ORIGINAL PAPER

A Study of the Effects of pH and Water Activityon the N-Nitrosopiperidine Formation in a Protein-BasedLiquid System

Eveline De Mey & Johan Viaene & Bieke Dejaegher & Hannelore De Maere &

Lore Dewulf & Hubert Paelinck & Yvan Vander Heyden & Ilse Fraeye

Received: 28 October 2013 /Accepted: 25 December 2013# Springer Science+Business Media New York 2014

Abstract To estimate the risk of N-nitrosopiperidine (NPIP)formation from piperidine in dry fermented sausages, theinfluences of pH and water activity (aw) were investigatedusing two protein-based liquid systems. In the first system(NaCl system), sodium chloride solutions (0-30 %) were usedto reduce the aw (between 0.99 and. 0.79) at two pH values(pH 4.0 and 5.0). At pH 4.0, reducing aw through the additionof salt significantly decreased the level of NPIP from 30.8±2.1 to 6.2±0.2 μg/ml. However, these extreme NaCl concen-trations do not exist in dry fermented sausages (only ca. 3 %).A second system (polyethylene glycol (PEG) system), inwhich PEG was added to reduce aw, was also developed. Arotatable central composite design (RCCD) was used to eval-uate the influences of pH (3.0–7.0), aw (0.80–0.99) and incu-bation time (1.3–98.7 h) on the response NPIP in the PEGsystem. A quadratic polynomial model was built to describethe response behaviour as a function of the factors examined.The response surface plots showed a significant increase inNPIP levels at longer incubation times, higher aw and lowerpH. Within the experimental domain, at 79 h, pH 3.8 and aw0.952, maximum NPIP levels of 110.0 and 113.6 μg/ml werepredicted and measured, respectively. The model

demonstrated the importance of controlling the pH and awduring the production of the sausages.

Keywords Acidification .Water activity .N-Nitrosamine .

Dry fermented sausage . Response surface methodology

Introduction

N-Nitrosamines are carcinogenic contaminants in food, andtheir formation is often related to the presence of nitrite and/ornitrate and (secondary) amines. In foodstuff, such as dryfermented meat products, both precursors can be present,since their production is characterized by the use of nitrite ornitrate as a curing agent, while the fermentation and ripeningstages provide optimal circumstances (time and temperature)for the growth of decarboxylating microflora. Themanufacturing of these food products results in an accumula-tion of biogenic amines, mainly tyramine, putrescine andcadaverine (Maijala et al. 1995; Mendes et al. 2001; De Meyet al. 2013a).

In the literature, it has been suggested that the biogenicamine cadaverine can be converted to N-nitrosopiperidineduring food processing (Warthesen et al. 1975). Cadaverineis deaminated and cyclized to piperidine prior to nitrosation(Shalaby 1996). However, in a previous study (De Mey et al.2013b), we demonstrated that the presence of an excess ofcadaverine during the production of nitrite-cured dryfermented sausage did not increase N-nitrosopiperidine(NPIP) levels. Elevated heating temperatures are necessaryto convert cadaverine to NPIP (Drabik-Markiewicz et al.2011). Nevertheless, several studies demonstrate the presenceof NPIP in unheated meat products, such as dry fermentedsausages (DeMey et al. 2013a;Mavelle et al. 1991; Ellen et al.1986). In these cases, the occurrence of NPIP in the sausagescan be related to the addition of black pepper (Yurchenko and

E. De Mey (*) :H. De Maere : L. Dewulf :H. Paelinck : I. FraeyeResearch Group for Technology and Quality of Animal Products,Department M2S, Member of Leuven Food Science and NutritionResearch Centre (LFoRCe), KU Leuven Campus Gent (KAHOSint-Lieven), Gebroeders De Smetstraat 1, 9000 Ghent, Belgiume-mail: eveline.demey@kuleuven.be

J. Viaene : B. Dejaegher :Y. Vander HeydenDepartment of Analytical Chemistry and PharmaceuticalTechnology, Center for Pharmaceutical Research (CePhaR),Vrije Universiteit Brussel—VUB, Laarbeeklaan 103,1090 Brussels, Belgium

H. De MaereFood Quality Laboratory, ISA, Boulevard Vauban 48, 59046 LilleCedex, France

Food Bioprocess TechnolDOI 10.1007/s11947-013-1249-7

Mölder 2007). Piperine, the main pungent compound of thiswidely used spice, can be cleaved at the amidic bond resultinginto the nitrosatable alkaloid piperidine (Shenoy andChoughuley 1992).

In contrast to cooked meat products, dry sausagesundergo a fermentation process causing a decrease ofpH from approximately 5.7 to values between 4.5 and5.5 (Toldrá 2007). This can promote the N-nitrosamineformation since the nitrosation of secondary amines re-quires an acidic environment (pH<5) (Challis andKyrtopoulos 1977). However, at pH values above 4, theaddition of moderate concentrations of sodium chloride(max. 12 %) inhibited the formation of N-nitrosamines(Rywotycki 2002; Hildrum et al. 1975; Theiler et al.1981). The inhibitory effect of sodium chloride has beenexplained by its contribution to the ionic strength of themedium, which influences the reaction between nitriteand secondary amines (Bulushi et al. 2009). While dryfermented sausages contain a certain amount of sodiumchloride (ca. 3 %), the product is also characterized by areduced water activity. The obtained aw levels are theresult of the combined action of sodium chloride and theremoval of water during drying. However, in the litera-ture, no evidence was found that a reduced aw, apartfrom the influence of sodium chloride, inhibits thenitrosation reaction.

Response surface methodology (RSM), initially de-scribed by Box and Wilson (1951), can be used forthe modeling and analysis of a response, which isinfluenced by several variables. Although RSM is main-ly used for the optimization of industrial processes(Ahmadi 2005; Saxena et al. 2009) and analytical pro-cedures (Hossain et al. 2011), it is also applied to studythe formation of toxic contaminants in food products,e.g. heterocyclic aromatic amines (Dundar et al. 2012;Gibis 2007) and acrylamide (Lasekan and Abbas 2011).The advantage of RSM is the possibility to test severalvariables simultaneously by means of an experimentaldesign, such as a central composite design (CCD). Fromthe experimental results, a model can be built whichestimates the relationship between the variables and theresponse.

The aim of this study was to estimate the influences ofpH and water activity on the NPIP formation during theproduction of dry fermented sausages. For that purpose,two systems were built. In the first system (NaCl system),at two pH values (pH 4.0 and 5.0), sodium chloride wasadded to reduce aw, prior to measuring the NPIP levels after72 h. The second system contained mixtures of polyethyl-ene glycol (PEG) and water to reduce the water activity.RSM was used to evaluate the influences of pH (3.0–7.0),aw (0.80–0.99) and incubation time (1.3–98.7 h) on theNPIP concentration in the PEG system.

Materials and Methods

Preparation of the Protein-Based Systems

Two types of liquid systems (25 ml) were prepared in plastictest tubes (VWR International, West Chester, PA, USA). Thefirst system (NaCl system) consisted of brain-heart broth(BHI, 37 g/l, Merck, Darmstadt, Germany) dissolved in water.In order to reduce the water activity, sodium chloride (VWRInternational) was added to the test tubes in the range of 0 to30 g/100 ml, resulting in aw values between 0.990 and 0.790,respectively. The test tubes were sterilized in a bench-topautoclave (Systec D-150, Wettenberg, Germany), and aftercooling, piperidine (100 mg/l) was added. The initial pH ofthe mixture (pH 7.5) was reduced by the addition of 300 and450 μl lactic acid (90 %, VWR International) in order toobtain pH values of 5 and 4, respectively. Finally, sodiumnitrite (150 mg/l, VWR International) was added just beforeincubation at 26 °C for 72 h. Experiments using the NaClsystem were done in quadruple.

The second system (PEG system) was prepared bydissolving BHI in a mixture of water and polyethyleneglycol 200 (PEG, Merck). In order to reduce the wateractivity, the ratio of PEG/water was varied, rangingfrom 0:25 (v/v) to 15:10 (v/v), resulting in aw valuesbetween 0.990 and 0.800. Similar to the NaCl system,the test tubes were sterilized and piperidine and sodiumnitrite were added. The pH of the samples was alteredby the addition of 200–900 μl lactic acid, to cover thepH range of 3.0–7.0. The levels of aw and pH of thePEG systems, according the experimental design (asdescribed below), are given in Table 1.

Measurement of pH and aw

The pH was measured by immersing the glass pH electrode(Knick Portamess, Elscolab, Terschuur, The Netherlands) inthe liquid samples. The water activity (aw) was determinedusing an electronic hygrometer (AquaLab, Decagon Devices,Pullman, WA, USA).

Table 1 Independent factors and their levels for the CCD. Experimentalset-up of the PEG system

Factor Symbol Factor coding

−1.68 −1 0 +1 +1.68

pH x1 3.0 3.8 5.0 6.2 7.0

aw x2 0.800 0.839 0.895 0.952 0.990

Time (h) x3 1.3 21.0 50.0 79.0 98.7

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N-Nitrosopiperidine Determination

N-Nitrosopiperidine was analysed according to the N-nitrosa-mine method of Drabik-Markiewicz et al. (2011) with somemodifications. The liquid protein samples (25 ml) were spikedwith 0.02 μg/mlN-nitrosodipropylamine (NDPA, Sigma-Aldrich, St. Louis, MO, USA) and mixed with 200 ml 3 NKOH (VWR International). The volatile N-nitrosamines werethen extracted from the samples by means of vacuum distilla-tion (Heidolph Laborota 4010 digital, Schwabach, Germany).After the addition of 4 ml 37 % HCl (VWR International), thedistillate (150 ml) was extracted three times with 50 ml ofdichloromethane (DCM). Subsequently, the extract was con-centrated to 100 μl in a Kuderna-Danish apparatus (Sigma-Aldrich). For the detection and quantification of NPIP, a gaschromatograph coupled to a thermal energy analyser (GC-TEA, Thermo Electron Corporation) was used. The extracts(5 μl) were injected on a packed column (10 % Carbowax20M+2 % KOH on ChromosorbWAW, 80/100 mesh, 1.8 m,2 mm i.d., Varian, Middelburg, The Netherlands), and achromatographic separation was carried out by using argonas carrier gas (25 ml/min). The injection port was set at175 °C, and the oven temperature was increased from 110 to180 °C at 5 °C/min. The temperatures of the interface andpyrolizer of the TEAwere set at 250 and 500 °C, respectively.

Data Analysis and Response Surface Design

In the NaCl system, the influences of the independent vari-ables, NaCl concentration and pH on the NPIP formation wereevaluated by a two-way analysis of variance (ANOVA), withTukey’s honestly significant difference criterion as a post hoctest.

The formation of NPIP in the PEG system was studiedusing RSM. A three-factor, five-level rotatable central com-posite design (RCCD) (Box et al. 1978) was used. The factorsand their levels are given in Table 1. The design consisted of15 experiments (Table 2) and was executed in triplicate. Toestimate the response surface, the experimental data werefitted to a quadratic polynomial equation:

Y ¼ b0 þ b1x1 þ b2x2 þ b3x3 þ b12x1x2 þ b13x1x3þ b23x2x3 þ b11x1

2 þ b22x22 þ b33x3

2

ð1Þ

in which the NPIP concentration (response Y) was correlat-ed to the factors pH (x1), aw (x2) and time (x3) and with b0 asthe intercept, b1, b2 and b3 as the linear, b12, b13 and b23 asthe interaction and b11, b22 and b33 as the the quadraticcoefficients. The model was evaluated by the Fisher testvalue (F value), the coefficient of multiple determination(R2) and the adequacy (paired t test of the experimental andfitted predicted data). The influences of the variables were

evaluated by an ANOVA. A three-way ANOVA was usedfor the PEG system (independent variables pH, aw andincubation time).

For all statistics, significance was determined at the 5 %significance level (α=0.05). The software MATLAB 5.3 (TheMath-Works, Natick, MA, USA) and PASW Statistics 20.0.0(SPSS, Armonk, NY, USA) were used for all statistical andgraphical analyses.

Results and Discussion

Influence of the Sodium Chloride Concentration in the NaClSystem

During the production of dry fermented sausages, a dryingstep is included to achieve the desired water activity andflavour characteristics. Depending on the drying period andthe sausage diameter, the water activity of the sausages usuallydecreases from 0.96 to 0.82–0.90 (Toldrà 2002). In the NaClsystem, the water activity was reduced from 0.990 to 0.790 bythe addition of increasing concentrations of sodium chloride(0–30 %) (Table 3). At a given aw value, the NPIP concentra-tions at pH 4.0 and 5.0 were always found to be different. Bothat pH 4.0 and 5.0, a significant effect of aw, caused by thevarying sodium chloride concentrations, is also seen on theNPIP formation. At pH 4.0, high NPIP levels were measuredat aw levels of 0.935 or higher (10 % NaCl or lower). When

Table 2 Central composite design, performed on the PEG system, tostudy the influence of three factors on the NPIP concentrations

Exp. no. Factor level NPIP (μg/ml)

pH (x1) aw (x2) Time (x3) Yexpa Ypred Yexp − Ypred

1 −1 −1 −1 36.3±1.4 30.8 5.5±1.5

2 +1 −1 −1 9.9±0.4 5.8 4.1±0.4

3 −1 +1 −1 61.6±5.3 59.1 2.5±5.3

4 +1 +1 −1 12.3±7.0 20.0 -7.7±7.0

5 −1 −1 +1 94.0±7.1 78.6 15.4±7.1

6 +1 −1 +1 33.9±2.4 28.6 5.2±2.4

7 −1 +1 +1 113.6±0.7 110.0 3.6±0.7

8 +1 +1 +1 48.2±2.5 45.9 2.2±2.5

9 −1.68 0 0 63.4±8.1 75.8 -12.4±8.1

10 +1.68 0 0 2.5±0.7 1.0 1.5±0.7

11 0 −1.68 0 16.0±1.3 30.3 -14.2±1.3

12 0 +1.68 0 71.9±2.1 68.6 3.3±2.1

13 0 0 −1.68 21.1±5.0 20.0 1.1±5.5

14 0 0 +1.68 69.9±8.4 81.9 -12.0±8.4

15 0 0 0 30.6±5.7 28.7 1.9±5.7

Yexp experimental value, Ypred predicted value, Yexp − Ypred residueaMean ± standard deviation (n=3)

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the NaCl concentration increased (to 20 % or more) and thusthe aw was reduced to 0.868 or lower, the NPIP concentrationsdecreased significantly. Compared to the results obtained atpH 4.0, the NPIP formation at pH 5.0 was significantly lowerat high aw values. The NPIP concentrations were again alsoreduced by increasing salt concentrations, but the effect wasless pronounced at pH 5.0. As a result, at reduced aw levels,the NPIP concentrations were higher at the highest investigat-ed pH. Previously, an inhibitory effect of NaCl (up to 12%) onnitrosamine formation has been reported (Rywotycki 2002;Hildrum et al. 1975; Theiler et al. 1981). Our results confirmthis observation. However, it is important to note that it isunclear to what extent the observed effects can be attributed toan increase in ionic strength, or a reduction in aw. In dryfermented sausages, the low aw value results from, on theone hand, a low amount of NaCl (ca. 3 %) and, on the otherhand, removal of water. The high NaCl concentrations (up to30 %) used in the model system, in order to achieve low awlevels, do not reflect conditions in dry fermented sausage.Therefore, in order to study only the effect of reducing thewater activity in dry fermented sausages and avoiding extremeionic effects of salts, as sodium chloride, an alternative addi-tive was used.

Response Surface Plots of the PEG System

Because of its good water solubility and stability to acidand high temperatures (Chen et al. 2005), PEG is oftenused to reduce the water activity in experimental models(Hallsworth and Magan 1999; Martinez et al. 2001).Although an inhibitory effect of PEG on colon cancer,initiated by N-nitrosamines and heterocyclic amines, wasdemonstrated by Corpet et al. (2000), the choice ofPEG 200 to reduce the water activity of the liquid

model (PEG system) is justifiable since the anti-tumoreffects are only related to high molecular weight PEGs,such as PEG 8000, while no chemical properties, whichcan influence the nitrosation reaction, are known.

In this experiment, the formation of NPIP was stud-ied as a function of pH, water activity and time. For thepurpose of identifying the region with the highest NPIPformation, a quadratic model was applied. The experi-mental data, obtained from a RCCD set-up, are shownin Table 2 and allowed the development of a quadraticmodel (Eq. 1), where the NPIP concentration (Y) isexpressed as a function of pH (x1), aw (x2) and time(x3). The resulting model is as follows:

Y ¼ 28:66−22:25x1 þ 11:40x2 þ 18:42x3−3:53x1x2−6:24x1x3 þ 0:78x2x3 þ 3:45x21 þ 7:36x22 þ 7:90x23

ð2Þ

The regression model was highly significant (F=43.6,p<0.001), and the total variance was highly explained(R2=0.918). The adequacy of the model was checked bythe comparison of the experimentally obtained and thepredicted values. The experimental (Yexp) and predictedvalues (Ypred), together with the residues (Yexp−Ypred), aregiven in Table 2. As no significant difference was foundbetween the experimental and predicted values, the ad-equacy of the model was confirmed. Therefore, themodel can be employed for the description of theNPIP formation in the liquid protein-based system.

In order to easily interpret the effects and interactions ofthe variables, as statistically analysed by ANOVA(Table 4), the model (Eq. 2) was visualized by responsesurface plots (Fig. 1). However, one should realize thatthese plots only visualize a small part of the entire responsesurface which is situated in a four-dimensional space. Tovisualize, one factor is to be kept constant. For this factor,the response surfaces at −1.68, 0 and +1.68 were evaluatedand a similar behaviour was seen. Therefore, the surfaces atlevels 0 were plotted and discussed.

Table 3 The NPIP formation (micrograms per millilitre) at different pHlevels after 72 h of incubation, influenced by the NaCl concentration ofthe NaCl system

Added NaCl (g/100 ml) aw NPIP

pH 4 pH 5

0 0.990 30.8±2.1a,c 18.0±2.5b,c

10 0.935 28.1±1.3a,c 17.3±0.7b,c

20 0.868 7.1±0.6a,d 17.7±2.2b,c

30 0.790 6.2±0.2a,d 11.5±0.8b,d

Different letters (a, b) in the same row indicate significant differences(p<0.05) among the pH levels. Different letters (c, d) in same columnindicate significant differences (p<0.05) among the NaCl concentrations.Data are given as mean ± standard deviation (n=4)

Table 4 Results of thethree-way ANOVAanalyses on the NPIPconcentrations, with x1(pH), x2 (aw), x3 (time)

*p<0.05 (significant)

Factor F p

x1 202* 0.000*

x2 63* 0.000*

x3 144* 0.000*

x1x2 8.7* 0.006*

x1x3 27* 0.000*

x2x3 0.43 0.517

x1x2x3 3.3 0.077

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In Fig. 1a, the response surface plot is given whichrepresents the effect of pH (x1) and time (x3) at a constantaw of 0.895 (x2=0). It seems evident that a longer incu-bation time results in higher NPIP formation. Moreover, adecrease of pH significantly increases the NPIP concentra-tions. As a consequence, maximum NPIP formation wasdetected at the lowest pH level of the design and thehighest time. This observation is in accordance to thestudy of Mirvish (1975), which situated the pH optimumfor the nitrosation of piperidine at ca. pH 3.0.Nevertheless, it is important to note that the solutions usedin the PEG system were sterilized before incubation,preventing biological degradation of the N-nitrosaminesformed. In contrast, degradation of NPIP was observedin dry fermented sausages (De Mey et al. 2013b), whichcan probably be attributed to microbial activity in themeat products (Hauser and Heiz 1978).

In Fig. 1b, the effects of aw (x2) and time (x3) are presentedat pH 5 (x1=0). The highest NPIP concentrations were ob-served after the longest incubation time (x3=1.68), which wasmore than 4 days, and at the highest water activity (x2=1.68).In other words, a decrease of the water activity of the model,by means of increasing amounts of PEG, significantlyinhibited the formation of NPIP. Moreover, the inhibitoryeffect of decreasing aw is less pronounced at aw values below0.895 (x2=0) because in that domain, the NPIP formation isalready low. Since no effects of PEG on the N-nitrosamineformation are known, it can be assumed that only the wateractivity is influencing the nitrosation reaction of piperidine.As a consequence, the inhibition of N-nitrosamine formationin food products is presumably not only attributed to the ioniceffects of the added chloride. As shown in the PEG system,decreasing the water activity, without increasing the NaClconcentration, results also in an inhibitory effect on the N-nitrosamines formation. It thus can be concluded that thedrying step in the production of dry fermented sausage is notonly enhancing the microbial safety but also can be consideredimportant in the inhibition of NPIP.

Figure 1c represents the response surface plot where theeffects of pH (x1) and aw (x2) on the NPIP concentration (Y) ata constant incubation time of 50 h (x3=0) are given. As alreadydiscussed, higher NPIP concentrations can be observedwhen thepH decreases and water activity increases. The highest NPIPconcentrations can be found in the system with pH 3.0 (x1=−1.68) and aw 0.990 (x2=1.68). Moreover an interaction(Table 4) between pH and aw occurred. The effect of a pHdecrease is larger when the water activity is high, while theNPIP concentrations during the acidification of an environmentwith low aw increase less rapidly (Fig. 1c). Similar effects wereseen in theNaCl system, although there, the NPIP concentrationsat high pH and low aw were higher than at low pH and low aw

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Fig. 1 Response surface plot for the effects of a pH (x1) and time (x3) atthe aw (x2) level 0 (0.895), baw (x2) and time (x3) at the pH (x1) level 0 (5.0)and c pH (x1) and aw (x2) at the time (x3) level 0 (50 h) on the NPIPformation in the PEG system

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(disordinal interaction). Here, the interaction effects between awand pH are much more important (Table 3).

From the above discussion and from the prediction of gridpoints (Fig. 2), it is observed that the highest NPIP concen-trations are predicted at low pH values and at high values foraw and time.

Implications for the Production of Dry Fermented Sausages

In this study, the systems were all prepared with the additionof 100 mg/l piperidine and 150 mg/l sodium nitrite.Consequently, since equimolar amounts of both precursorsare necessary to form NPIP, piperidine was the limiting pre-cursor. As a result, a maximum of 134 μg/ml NPIP could beformed in the system when a 100 % conversion would occur.Within the experimental domain of the design, a maximum of110.0 μg/ml (or a yield of 82 %) was predicted after anincubation time of 79 h at a pH of 3.8 and an aw value of0.952. As can be seen in Table 2, the experimental maximumof 113.6 μg/ml NPIP was measured at the same conditions.

Fortunately, such high yields do not occur in dry fermentedsausages. Firstly, the amount of piperidine will be less since itis mainly introduced by the addition of pepper in the sausage.In Table 5, the amounts of pepper, commonly added to somedry fermented sausage types, are given. Even the rather pep-pered sausages, e.g. 5.0 g/kg of pepper in Napoli sausages,will not contain high amounts of piperidine. Since 1 g ofpepper contains ca. 11 mg piperidine (De Mey et al. 2013c),approximately 55 mg piperidine may be present in 1 kg ofmeat batter. Secondly, the added amount of sodium nitrite,legally restricted to a maximum 150 mg per kg meat product

(European Parliament and the Council of the European Union2006), will firstly react with various compounds of the meatsuch as heme, sulfhydryl/thiol residues of non-heme proteinsand lipid derivatives and can even be converted to nitrousgases (Pegg and Shahidi 2000). As a result, only small resid-ual nitrite levels (below 5 mg/kg) can be detected in the dryfermented sausages (De Mey et al. 2013b). Consequently,only low amounts of both precursors are present. Moreover,after 3 days of fermentation, the pH decreases from approxi-mately 5.7 to values commonly between 4.4 and 5.6 (Table 5).Due this acidification, the nitrosation is slightly favoured.Nevertheless, subsequently, the sausages are dried and the

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Fig. 2 Predicted results for thegrid points resulting in the highestNPIP concentrations

Table 5 The pH, aw and pepper content of some dry fermented sausagetypes

Type Black + whitepepper (g/kg)

pH (min–max range) aw(mean)

Napoli (Italy) 5.0+0.0a 5.0–5.1f 0.92f

Salami (North Belgium) 0.0+2.8b 4.6–5.6f 0.91f

Chorizo (Spain) 1.0+0.0c 4.6–5.6f 0.89f

Rosette (France) 0.0+1.5d,e,g 4.4–5.3f 0.90f

Cervelat (Germany) 3.7+0.0c 4.9f 0.94f

aMoretti et al. (2004)b Ravyts et al. (2008)c Toldrà (2007)d Thévenot et al. (2005)eMontet et al. (2009)f De Mey et al. (2013a)g Amount in French sausages is not particular for Rosette

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aw decreases from ca. 0.96 to values preferably between 0.82and 0.90, whereby the nitrosation is inhibited. In conclusion,the conversion to NPIP will be minute since the precedingresults demonstrate that the pH and aw optima for NPIPformation can be avoided during the production of thesausages.

Conclusions

At both pH levels (pH 4.0 and 5.0), the NPIP formation wasreduced by the addition of high amounts of NaCl. However, itwas unclear to what extent these effects could be attributed tothe higher ionic strength or the lower aw. In contrast, the use ofPEG in the system could, without introducing high salt con-centrations, efficiently decrease the water activity similar todry fermented sausages.

Three major conclusions could be drawn from the responsesurface plots, derived from the quadratic model based on theexperimental design experiments applied on the PEG system.Firstly, the NPIP concentrations increased during incubation,which is probably related to the lack of microbial degradationin the sterile environment of the liquid system. Secondly,based on our results, the NPIP formation is maximal atpH 3.0. This is in accordance with the results of Mirvish(1975), which determined the pH optimum of around 3.0.Thirdly, the NPIP formation can be inhibited by reducing thewater activity.

The results obtained in the systems contribute to a betterunderstanding of the combined effect of pH and water activityon the NPIP formation during the production of dry fermentedsausages. The model simulations clearly show that the pH andaw significantly influence the formation of NPIP. In conclu-sion, as long as the water activity is low and decreasing, andthere is no extreme acidification during the fermentation, it isnot likely that the addition of sodium nitrite and piperidine(from pepper) will form a major risk for the formation ofcarcinogenic NPIP in dry fermented sausages.

Acknowledgments This work was performed in the framework of theMeCagrO2 project “Safe products, sustainable processes and employ-ment increased attractiveness for companies from the 2 Seas agro-foodArea”. The document reflects the authors’ views. The INTERREG IVA 2Seas Programme Authorities are not liable for any use that may be madeof the information contained therein.

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