# Influence of meat source, pH and production time on zinc protoporphyrin IX formation as natural colouring agent in nitrite-free dry fermented sausages

Hannelore de Maere, Sylvie Chollet, Jos de Brabanter, Chris Michiels, Hubert

Paelinck, Ilse Fraeye

► To cite this version:

Hannelore de Maere, Sylvie Chollet, Jos de Brabanter, Chris Michiels, Hubert Paelinck, et al. Influence of meat source, pH and production time on zinc protoporphyrin IX formation as natural colouring agent in nitrite-free dry fermented sausages. Meat Science, 2018, 135, pp.46-53. 10.1016/j.meatsci.2017.08.024. hal-02623763

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Influence of meat source, pH and production time on
zinc protoporphyrin IX formation as natural colouring agent in nitrite-free
dry fermented sausages

Hannelore De Maere¹, Sylvie Chollet², Jos De Brabanter³, Chris Michiels⁴, Hubert Paelinck¹,
Ilse Fraeye¹*

¹Research Group for Technology and Quality of Animal Products, Department M²S, member of Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven Technology Campus Ghent, Gebroeders De Smetstraat 1, B-9000 Ghent, Belgium

²ICV – Institut Charles Viollette, EA 7394, ISA, Univ. Lille 1, INRA, Univ. Artois, Univ. Littoral Côte d'Opale, 48 Boulevard Vauban, Lille F-59000, France

³Department of Electrical Engineering (ESAT), member of the division STADIUS, Stadius Centre for Dynamical Systems, Signal Processing and Data Analytics, Kasteelpark Arenberg 10 – box 2446, B-3001 Leuven, Belgium

⁴Centre for Food and Microbial Technology, Department M²S, member of Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 23 box 2457, B-3001 Leuven, Belgium

*corresponding author: Tel.: +32 9 331 66 17; fax: +32 9 265 87 24;
E-mail address: ilse.fraeye@kuleuven.be

Acknowledgements

This work was performed with financial support of internal funding of KU Leuven.
Influence of meat source, pH and production time on
zinc protoporphyrin IX formation as natural colouring agent in nitrite-free
dry fermented sausages

Hannelore De Maere¹, Sylvie Chollet², Jos De Brabanter³, Chris Michiels⁴, Hubert Paelinck¹,
Ilse Fraeye¹*

¹Research Group for Technology and Quality of Animal Products, Department M²S, member of Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven Technology Campus Ghent, Gebroeders De Smetstraat 1, B-9000 Ghent, Belgium

²ICV – Institut Charles Viollette, EA 7394, ISA, Univ. Lille 1, INRA, Univ. Artois, Univ. Littoral Côte d'Opale, 48 Boulevard Vauban, Lille F-59000, France

³Department of Electrical Engineering (ESAT), member of the division STADIUS, Stadius Centre for Dynamical Systems, Signal Processing and Data Analytics, Kasteelpark Arenberg 10 – box 2446, B-3001 Leuven, Belgium

⁴Centre for Food and Microbial Technology, Department M²S, member of Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 23 box 2457, B-3001 Leuven, Belgium

*corresponding author: Tel.: +32 9 331 66 17; fax: +32 9 265 87 24;
E-mail address: ilse.fraeye@kuleuven.be

Acknowledgements

This work was performed with financial support of internal funding of KU Leuven.
Influence of meat source, pH and production time on

zinc protoporphyrin IX formation as natural colouring agent in nitrite-free
dry fermented sausages

ABSTRACT

Nitrite is commonly used in meat products due to its plural technological advantages.
However, it is controversial because of its detrimental side effects on health. Within the
context of nitrite reduction, zinc protoporphyrin IX (Zn(II)PPIX) formation in meat products
as natural red colouring agent has been suggested. This investigation presents the evaluation
of naturally occurring pigments, namely Zn(II)PPIX, protoporphyrin IX (PPIX) and heme in
nitrite-free dry fermented sausages in function of time, meat source (pork, horsemeat and a
combination of both meat sources) and pH condition. In function of time, Zn(II)PPIX and
PPIX were formed and heme content decreased. Higher pH conditions promoted Zn(II)PPIX
and PPIX formation, whereas the influence of pH on heme was less clear. The use of
horsemeat also promoted Zn(II)PPIX formation. Moreover, even similar amounts were
formed when it was combined with pork. Product redness, however, could not be related to
Zn(II)PPIX formation.

**Keywords:** nitrite-free meat products; natural colouring; meat source; pH condition; production time
Influence of meat source, pH and production time on

zinc protoporphyrin IX formation as natural colouring agent in nitrite-free
dry fermented sausages

ABSTRACT

Nitrite is commonly used in meat products due to its plural technological advantages.
However, it is controversial because of its detrimental side effects on health. Within the
context of nitrite reduction, zinc protoporphyrin IX (Zn(II)PPIX) formation in meat products
as natural red colouring agent has been suggested. This investigation presents the evaluation
of naturally occurring pigments, namely Zn(II)PPIX, protoporphyrin IX (PPIX) and heme in
nitrite-free dry fermented sausages in function of time, meat source (pork, horsemeat and a
combination of both meat sources) and pH condition. In function of time, Zn(II)PPIX and
PPIX were formed and heme content decreased. Higher pH conditions promoted Zn(II)PPIX
and PPIX formation, whereas the influence of pH on heme was less clear. The use of
horsemeat also promoted Zn(II)PPIX formation. Moreover, even similar amounts were
formed when it was combined with pork. Product redness, however, could not be related to
Zn(II)PPIX formation.

**Keywords:** nitrite-free meat products; natural colouring; meat source; pH condition; production time
## 1. Introduction

Meat colour is considered to be an important quality parameter of meat and meat products, influencing consumer's buying decision. Myoglobin (Mb) is predominantly responsible for the colour of meat, although low levels of hemoglobin and other heme proteins may also contribute to it. Specifically, the conjugated heme molecule (iron protoporphyrin IX) is responsible for the ability of Mb to absorb visible light. This heme is located in a hydrophobic cleft of the protein where only small ligands, such as oxygen (O₂), nitric oxide (NO), carbon oxide, ... have ready access (Devine & Dikeman, 2004). Colour manifests itself in many different shades depending on the nature of ligand attached to iron and the oxidation state of iron. O₂ can only bind to iron in the ferrous redox state (Fe(II)) forming the cherry-red oxymyoglobin (OMb), in absence of O₂ no ligand is bound to Fe(II) whereby the purplish deoxymyoglobin (DMb) is formed, whereas water is bound to iron in the ferric redox state (Fe(III)) with formation of the brownish metmyoglobin (MMb) (Lindahl, 2005). The occurrence of these Mb forms depends on *e.g.* temperature and O₂ pressure. But also other parameters, such as Mb concentration, moisture and fat content have an effect on colour (Adamsen, Møller, Laursen, Olsen, & Skibsted, 2006).

Sodium nitrite is commonly used in meat products. Besides its antimicrobial (especially against *Clostridium botulinum* strains) and antioxidant properties, its contribution to an acceptable flavour and taste, nitrite is mainly used as colouring agent. After reduction of nitrite, Mb will form a complex with NO, resulting in the red pigment nitrosylmyoglobin (NOMb) (Honikel, 2008). Despite the many technological advantages, the addition of nitrite (E249, E250) is legally restricted to 150 mg/kg (expressed as NaNO₂/kg) in most meat products because of their detrimental side effects on health (Regulation (EC) No 1333/2008; lastly amended in 2015; Schuddeboom, 1993; Skibsted, 2011). In addition, the consumer
## 1. Introduction

Meat colour is considered to be an important quality parameter of meat and meat products, influencing consumer's buying decision. Myoglobin (Mb) is predominantly responsible for the colour of meat, although low levels of hemoglobin and other heme proteins may also contribute to it. Specifically, the conjugated heme molecule (iron protoporphyrin IX) is responsible for the ability of Mb to absorb visible light. This heme is located in a hydrophobic cleft of the protein where only small ligands, such as oxygen (O₂), nitric oxide (NO), carbon oxide, ... have ready access (Devine & Dikeman, 2004). Colour manifests itself in many different shades depending on the nature of ligand attached to iron and the oxidation state of iron. O₂ can only bind to iron in the ferrous redox state (Fe(II)) forming the cherry-red oxymyoglobin (OMb), in absence of O₂ no ligand is bound to Fe(II) whereby the purplish deoxymyoglobin (DMb) is formed, whereas water is bound to iron in the ferric redox state (Fe(III)) with formation of the brownish metmyoglobin (MMb) (Lindahl, 2005). The occurrence of these Mb forms depends on *e.g.* temperature and O₂ pressure. But also other parameters, such as Mb concentration, moisture and fat content have an effect on colour (Adamsen, Møller, Laursen, Olsen, & Skibsted, 2006).

Sodium nitrite is commonly used in meat products. Besides its antimicrobial (especially against *Clostridium botulinum* strains) and antioxidant properties, its contribution to an acceptable flavour and taste, nitrite is mainly used as colouring agent. After reduction of nitrite, Mb will form a complex with NO, resulting in the red pigment nitrosylmyoglobin (NOMb) (Honikel, 2008). Despite the many technological advantages, the addition of nitrite (E249, E250) is legally restricted to 150 mg/kg (expressed as NaNO₂/kg) in most meat products because of their detrimental side effects on health (Regulation (EC) No 1333/2008; lastly amended in 2015; Schuddeboom, 1993; Skibsted, 2011). In addition, the consumer
shows a strong desire to avoid all artificial food additives (so-called E-numbers) in the daily diet. In parallel, it is inconceivable to present a grey slice of meat product to the consumers.

As such, nitrite reduction is already some years a matter of interest, whereby colour formation in meat products without the use of nitrite or other artificial colouring agents is one of the challenges<fnref n="1" />.

In this context, zinc protoporphyrin IX (Zn(II)PPIX), a natural red pigment found in nitrite-free dry cured hams, has been investigated (Adamsen et al., 2006; Takenati, Mutsumi, Mizutani, Uebayashi, Numata, & Ohgari, 2007; Wakamatsu, Nishimura, & Hattori, 2004a).

The exact formation pathway of Zn(II)PPIX is still disputed, although it is nowadays generally assumed that Zn(II)PPIX originates from Mb whereby the iron in the heme moiety is replaced by zinc (Chau, Ishigaki, Kataoka, & Taketani, 2011; Takenati et al., 2007).

Enzymatic formation with ferrochelatase (FECH) includes both the removal of Fe(II) from heme and the insertion of zinc into protoporphyrin IX (PPIX) (Chau, Ishigaki, Kataoka, & Taketani, 2010). FECH activities have been detected in mammals, bacteria and yeast (Dailey, Dailey, Wu, Medlock, Wang, Rose, & Wang, 2000; Medlock, Swartz, Dailey, Dailey, & Lanzilotta, 2007). Wakamatsu, Okui, Ikeda, Nishimura, & Hattori (2004b) and Wakamatsu, Uemura, Odagiri, Okui, Hayashi, Hioki, Nishimura, Hattori (2009b), however, suggested a minor role of bacteria for the formation of Zn(II)PPIX in pork and dry cured ham. Formation of Zn(II)PPIX by endogenous FECH was first demonstrated by Wakamatsu et al. (2004b).

Additionally, also a non-enzymatic mechanism cannot be fully excluded, suggesting a parallel non-enzymatic and enzymatic formation of Zn(II)PPIX in meat products (Becker, Westermann, Hansson, & Skibsted, 2012).

Colour of nitrite-free dry cured hams is attributed mainly to the presence of Zn(II)PPIX (Wakamatsu et al., 2004a; Wakamatsu, Odagiri, Nishimura, & Hattori, 2009a). A steady

<footnote n="1">¹ We are well aware that omission of nitrite in dry fermented sausages results in reduced microbial safety, especially with regard to *Clostridium botulinum* which can cause food poisoning. However, further investigation on food safety was outside the scope of this work.</footnote>
76 shows a strong desire to avoid all artificial food additives (so-called E-numbers) in the daily diet. In parallel, it is inconceivable to present a grey slice of meat product to the consumers.
77
78 As such, nitrite reduction is already some years a matter of interest, whereby colour formation in meat products without the use of nitrite or other artificial colouring agents is one of the challenges<fnref n="1" />.
80

81 In this context, zinc protoporphyrin IX (Zn(II)PPIX), a natural red pigment found in nitrite-free dry cured hams, has been investigated (Adamsen et al., 2006; Takenati, Mutsumi, Mizutani, Uebayashi, Numata, & Ohgari, 2007; Wakamatsu, Nishimura, & Hattori, 2004a).
82
83 The exact formation pathway of Zn(II)PPIX is still disputed, although it is nowadays generally assumed that Zn(II)PPIX originates from Mb whereby the iron in the heme moiety is replaced by zinc (Chau, Ishigaki, Kataoka, & Taketani, 2011; Takenati et al., 2007).
84
85 Enzymatic formation with ferrochelatase (FECH) includes both the removal of Fe(II) from heme and the insertion of zinc into protoporphyrin IX (PPIX) (Chau, Ishigaki, Kataoka, & Taketani, 2010). FECH activities have been detected in mammals, bacteria and yeast (Dailey, Dailey, Wu, Medlock, Wang, Rose, & Wang, 2000; Medlock, Swartz, Dailey, Dailey, & Lanzilotta, 2007). Wakamatsu, Okui, Ikeda, Nishimura, & Hattori (2004b) and Wakamatsu, Uemura, Odagiri, Okui, Hayashi, Hioki, Nishimura, Hattori (2009b), however, suggested a minor role of bacteria for the formation of Zn(II)PPIX in pork and dry cured ham. Formation of Zn(II)PPIX by endogenous FECH was first demonstrated by Wakamatsu et al. (2004b).
87
88
89 Additionally, also a non-enzymatic mechanism cannot be fully excluded, suggesting a parallel non-enzymatic and enzymatic formation of Zn(II)PPIX in meat products (Becker, Westermann, Hansson, & Skibsted, 2012).
90
91
92
93
94
95
96
97 Colour of nitrite-free dry cured hams is attributed mainly to the presence of Zn(II)PPIX (Wakamatsu et al., 2004a; Wakamatsu, Odagiri, Nishimura, & Hattori, 2009a). A steady

<footnote n="1">¹ We are well aware that omission of nitrite in dry fermented sausages results in reduced microbial safety, especially with regard to *Clostridium botulinum* which can cause food poisoning. However, further investigation on food safety was outside the scope of this work.</footnote>
increase in redness intensity of Parma ham during processing could be seen and due to colour
differences between muscles with equal moisture losses, it was assumed that the increase in
redness could not only be a consequence of dehydration. Until now, however, no clear
relation between colour and Zn(II)PPIX formation in meat products could be demonstrated
(Adamsen et al., 2006; Parolari, Benedini, & Toscani, 2009). Degradation of heme may
complicate colour formation in the nitrite-free meat products (De Maere, Fraeye, De Mey,
Dewulf, Michiels, Paelinck, & Chollet, 2016a; Wakamatsu et al., 2009b).

In order to evaluate the formation of Zn(II)PPIX mainly pork has been used (Adamsen et al. 2006; Chau et al. 2010; Ishikawa, Kawabuchi, Kawakami, Sato, Numata, & Matsumoto, 2007; Wakamatsu et al. 2004a). In contrast, De Maere, Chollet, Claeys, Michiels, Govaert, De Mey, Paelinck, & Fraeye (2016b) compared different meat sources in their ability to form Zn(II)PPIX *in vitro* and related this pigment formation to several intrinsic parameters. This investigation revealed that endogenous enzymatic Zn(II)PPIX formation is species-dependent, whereby horsemeat, better than pork, showed very good ability to form Zn(II)PPIX and that zinc chelatase activity, followed by heme and zinc content, was the most important factor to explain the variation in Zn(II)PPIX formation between the investigated meat sources.

Furthermore, hardly any research focussed on Zn(II)PPIX formation in nitrite-free meat
products other than dry cured hams. In this respect, De Maere et al. (2016a) recently studied
Zn(II)PPIX formation in nitrite-free porcine dry fermented sausages. They found that
Zn(II)PPIX formation and product redness were significantly correlated. Zn(II)PPIX,
however, was only able to form at pH values higher than 4.9 and after an extensive drying
period up to 177 days, indicating that both pH and production time are crucial factors for its
formation.

Based on both recent studies, it was hypothesized that the use of horsemeat in the production of
dry fermented sausages may be a promising route to accelerate Zn(II)PPIX formation and
increase in redness intensity of Parma ham during processing could be seen and due to colour
differences between muscles with equal moisture losses, it was assumed that the increase in
redness could not only be a consequence of dehydration. Until now, however, no clear
relation between colour and Zn(II)PPIX formation in meat products could be demonstrated
(Adamsen et al., 2006; Parolari, Benedini, & Toscani, 2009). Degradation of heme may
complicate colour formation in the nitrite-free meat products (De Maere, Fraeye, De Mey,
Dewulf, Michiels, Paelinck, & Chollet, 2016a; Wakamatsu et al., 2009b).

In order to evaluate the formation of Zn(II)PPIX mainly pork has been used (Adamsen et al. 2006; Chau et al. 2010; Ishikawa, Kawabuchi, Kawakami, Sato, Numata, & Matsumoto, 2007; Wakamatsu et al. 2004a). In contrast, De Maere, Chollet, Claeys, Michiels, Govaert, De Mey, Paelinck, & Fraeye (2016b) compared different meat sources in their ability to form Zn(II)PPIX *in vitro* and related this pigment formation to several intrinsic parameters. This investigation revealed that endogenous enzymatic Zn(II)PPIX formation is species-dependent, whereby horsemeat, better than pork, showed very good ability to form Zn(II)PPIX and that zinc chelatase activity, followed by heme and zinc content, was the most important factor to explain the variation in Zn(II)PPIX formation between the investigated meat sources.

Furthermore, hardly any research focussed on Zn(II)PPIX formation in nitrite-free meat
products other than dry cured hams. In this respect, De Maere et al. (2016a) recently studied
Zn(II)PPIX formation in nitrite-free porcine dry fermented sausages. They found that
Zn(II)PPIX formation and product redness were significantly correlated. Zn(II)PPIX,
however, was only able to form at pH values higher than 4.9 and after an extensive drying
period up to 177 days, indicating that both pH and production time are crucial factors for its
formation.

Based on both recent studies, it was hypothesized that the use of horsemeat in the production of
dry fermented sausages may be a promising route to accelerate Zn(II)PPIX formation and
improve product redness. Therefore, the goal of this study was to examine the effect of meat
source on Zn(II)PPIX formation and relate the Zn(II)PPIX formation to colour development
in dry fermented sausages made of pork and horsemeat. By using horsemeat, however, a more
drastic pH decline could be expected upon fermentation, due to the presence of higher
concentrations of residual sugar. The concentration of glycogen and reducing sugars,
including glucose, in horsemeat *post mortem* have been reported as > 5 mg/ g and > 0.5 mg/
g, respectively (Gill, 2005). In pork, *post mortem* glycogen contents < 1.78 mg/ g were
reported (Choe, Choi, Lee, Shin, Ryu, Hong, & Kim, 2008). The expected strong pH decrease
upon fermentation could be disadvantageous for Zn(II)PPIX formation. Therefore, also dry
fermented sausages containing both horsemeat (having the advantage of high zinc chelatase
activity, as shown by De Maere et al., 2016b) and pork (having the advantage of a lower
amount of sugars, hence a higher pH upon fermentation) were included in the study.

In summary, this study presents the production of nitrite-free dry fermented sausages based on
pork, horsemeat and a 50/50 combination of pork and horsemeat at two different pH
conditions, whereby pigment and colour formation were evaluated in function of meat source,
pH and production time by linear mixed modelling.

141
improve product redness. Therefore, the goal of this study was to examine the effect of meat
source on Zn(II)PPIX formation and relate the Zn(II)PPIX formation to colour development
in dry fermented sausages made of pork and horsemeat. By using horsemeat, however, a more
drastic pH decline could be expected upon fermentation, due to the presence of higher
concentrations of residual sugar. The concentration of glycogen and reducing sugars,
including glucose, in horsemeat *post mortem* have been reported as > 5 mg/ g and > 0.5 mg/
g, respectively (Gill, 2005). In pork, *post mortem* glycogen contents < 1.78 mg/ g were
reported (Choe, Choi, Lee, Shin, Ryu, Hong, & Kim, 2008). The expected strong pH decrease
upon fermentation could be disadvantageous for Zn(II)PPIX formation. Therefore, also dry
fermented sausages containing both horsemeat (having the advantage of high zinc chelatase
activity, as shown by De Maere et al., 2016b) and pork (having the advantage of a lower
amount of sugars, hence a higher pH upon fermentation) were included in the study.

In summary, this study presents the production of nitrite-free dry fermented sausages based on
pork, horsemeat and a 50/50 combination of pork and horsemeat at two different pH
conditions, whereby pigment and colour formation were evaluated in function of meat source,
pH and production time by linear mixed modelling.

141
142

## 2. Material and methods

143

### 2.1. Dry fermented sausages

144

Nitrite-free dry fermented sausages were prepared as described in De Maere et al. (2016a).

145

In total, six treatments were made *in duplo*. The shoulder meat fractions originated from single homogeneous batches of pork, horsemeat or a 50/50 combination of both meat sources.

146

147

For each of the three meat source treatments, 0.00% and 0.70% dextrose was added to the meat batter in order to obtain two significantly different pH conditions during processing, a high and a low pH condition, respectively. An overview of the different nitrite-free dry fermented sausage preparations and the corresponding codes is given in Table 1.

148

149

150

151

### 2.2. Sampling

152

Core samples of sausages of each treatment were taken at different points in time during the production process, more specifically at production day (day 0), after the fermentation process (day 3), after the initial drying period which is normally the end of production for semi-dry Northern type dry fermented sausages (day 21), and during an extended drying period (day 42, 63, 84, 105, 126 and 168). General analyses for process monitoring, by means of weight losses, pH, dry matter (DM) and water activity ($a_w$), were performed immediately at each sampling day. Microbial analyses were performed at sampling days 0, 3 and 21. Also immediately after sampling, colour was measured and Zn(II)PPIX and/or PPIX formation was screened. Other samples were frozen at -24 °C until quantitative analysis was performed of PPIX, Zn(II)PPIX and total heme. The latter analyses were only performed at sampling days 0, 21, 63 and 168 (cf. *infra*). All measurements were done *in triplicate*, only colour was measured six times.

153

154

155

156

157

158

159

160

161

162

163
142

## 2. Material and methods

143

### 2.1. Dry fermented sausages

144

Nitrite-free dry fermented sausages were prepared as described in De Maere et al. (2016a).

145

In total, six treatments were made *in duplo*. The shoulder meat fractions originated from single homogeneous batches of pork, horsemeat or a 50/50 combination of both meat sources.

146

147

For each of the three meat source treatments, 0.00% and 0.70% dextrose was added to the meat batter in order to obtain two significantly different pH conditions during processing, a high and a low pH condition, respectively. An overview of the different nitrite-free dry fermented sausage preparations and the corresponding codes is given in Table 1.

148

149

150

151

### 2.2. *Sampling*

152

Core samples of sausages of each treatment were taken at different points in time during the production process, more specifically at production day (day 0), after the fermentation process (day 3), after the initial drying period which is normally the end of production for semi-dry Northern type dry fermented sausages (day 21), and during an extended drying period (day 42, 63, 84, 105, 126 and 168). General analyses for process monitoring, by means of weight losses, pH, dry matter (DM) and water activity ($a_w$), were performed immediately at each sampling day. Microbial analyses were performed at sampling days 0, 3 and 21. Also immediately after sampling, colour was measured and Zn(II)PPIX and/or PPIX formation was screened. Other samples were frozen at -24 °C until quantitative analysis was performed of PPIX, Zn(II)PPIX and total heme. The latter analyses were only performed at sampling days 0, 21, 63 and 168 (cf. *infra*). All measurements were done *in triplicate*, only colour was measured six times.

153

154

155

156

157

158

159

160

161

162

163
164 2.3. *Analysis*

165 2.3.1. General analyses

166 Weight losses, pH and aw were analysed as described in De Maere et al. (2016a).

167 2.3.2. Microbial count

168 Samples were aseptically homogenized with a stomacher (Masticor Classic 400, IUL
169 Instruments, Barcelona, Spain). Decimal dilution series were prepared with sterile ringer
170 solution (Oxoid, Basingstoke, England) and plated with a spiral plater (Eddy Jet, IUL
171 Instruments). Total aerobic count (TAC) was analysed on plate count agar (PCA, Merck,
172 Darmstadt, Germany) incubated at 26 °C for 48 hours, lactic acid bacteria (LAB) were
173 analysed on de Man, Rogosa and Sharpe agar (MRS, Merck) incubated with a double layer at
174 30 °C for 72 hours, Staphylococci on mannitol salt agar (MSA, Merck) incubated at 30 °C for
175 48 hours and *Enterobacteriaceae* on violet red bile glucose agar (VRBG, Biokar, Beauvais,
176 France) incubated with a double layer at 30 °C for 48 hours. Data are expressed as log colony
177 forming units (cfu) per gram meat sample.

178 2.3.3. Determination of PPIX, Zn(II)PPIX and total heme pigments

179 A screening method was used for the fast detection of the fluorescent Zn(II)PPIX and/ or PPIX
180 on transverse slices of meat products. Generally, the fluorescence emission obtained after
181 irradiation of meat slices with purple LED light of 420 nm in a darkened room was visualized
182 via image analysis. A darker picture is assumed to represent a higher amount of Zn(II)PPIX
183 and/ or PPIX. PPIX and Zn(II)PPIX were quantified simultaneously by means of High
184 Pressure Liquid Chromatography (HPLC) with fluorescence detection. Total heme content was
185 determined spectrophotometrically. These methods have been described extensively in earlier
186 published work by De Maere et al. (2016a).
164 2.3. *Analysis*

165 2.3.1. General analyses

166 Weight losses, pH and aw were analysed as described in De Maere et al. (2016a).

167 2.3.2. Microbial count

168 Samples were aseptically homogenized with a stomacher (Masticor Classic 400, IUL
169 Instruments, Barcelona, Spain). Decimal dilution series were prepared with sterile ringer
170 solution (Oxoid, Basingstoke, England) and plated with a spiral plater (Eddy Jet, IUL
171 Instruments). Total aerobic count (TAC) was analysed on plate count agar (PCA, Merck,
172 Darmstadt, Germany) incubated at 26 °C for 48 hours, lactic acid bacteria (LAB) were
173 analysed on de Man, Rogosa and Sharpe agar (MRS, Merck) incubated with a double layer at
174 30 °C for 72 hours, Staphylococci on mannitol salt agar (MSA, Merck) incubated at 30 °C for
175 48 hours and Enterobacteriaceae on violet red bile glucose agar (VRBG, Biokar, Beauvais,
176 France) incubated with a double layer at 30 °C for 48 hours. Data are expressed as log colony
177 forming units (cfu) per gram meat sample.

178 2.3.3. Determination of PPIX, Zn(II)PPIX and total heme pigments

179 A screening method was used for the fast detection of the fluorescent Zn(II)PPIX and/ or PPIX
180 on transverse slices of meat products. Generally, the fluorescence emission obtained after
181 irradiation of meat slices with purple LED light of 420 nm in a darkened room was visualized
182 via image analysis. A darker picture is assumed to represent a higher amount of Zn(II)PPIX
183 and/ or PPIX. PPIX and Zn(II)PPIX were quantified simultaneously by means of High
184 Pressure Liquid Chromatography (HPLC) with fluorescence detection. Total heme content was
185 determined spectrophotometrically. These methods have been described extensively in earlier
186 published work by De Maere et al. (2016a).
187 2.3.4. Colour measurements

188 A portable Miniscan EZ 4500L 45°/0° (Hunterlab, Murnau, Germany) with 8 mm viewing
189 area size, illuminant D65 and 10° standard observer was used to register the L*, a* and b*
190 values (based on CIE, 1976).

191 2.4. *Data analysis*

192 Differences in pH, weight losses and aw between the six different treatments at each sampling
193 day were assessed using a two-way ANOVA (Christensen, 2015) at a significance level of P
194 < 0.05. A Tukey correction was used to account for multiple testing (Hochberg & Tamhane,
195 1987) (IBM SPSS Statistics 21.0, Chicago, USA).

196 As already described in detail (De Maere et al., 2016a), Zn(II)PPIX, PPIX and total heme on
197 the one hand, and L*, a* and b* on the other hand, were analyzed using a linear mixed model
198 (Verbeke & Molenberghs, 2013) that included factors for meat source, pH condition, time and
199 their two-way and three-way interactions (SAS version 9.4 with SAS/STAT 14.1).

200
187 2.3.4. Colour measurements

188 A portable Miniscan EZ 4500L 45°/0° (Hunterlab, Murnau, Germany) with 8 mm viewing
189 area size, illuminant D65 and 10° standard observer was used to register the L*, a* and b*
190 values (based on CIE, 1976).

191 2.4. Data analysis

192 Differences in pH, weight losses and aw between the six different treatments at each sampling
193 day were assessed using a two-way ANOVA (Christensen, 2015) at a significance level of P
194 < 0.05. A Tukey correction was used to account for multiple testing (Hochberg & Tamhane,
195 1987) (IBM SPSS Statistics 21.0, Chicago, USA).

196 As already described in detail (De Maere et al., 2016a), Zn(II)PPIX, PPIX and total heme on
197 the one hand, and L*, a* and b* on the other hand, were analyzed using a linear mixed model
198 (Verbeke & Molenberghs, 2013) that included factors for meat source, pH condition, time and
199 their two-way and three-way interactions (SAS version 9.4 with SAS/STAT 14.1).

200
201 **3. Results and discussion**

202 **3.1. pH**

203 Means ± SE of pH are shown in Table 2. At day 0, no significant differences in pH of all
204 prepared meat batters were measured. This was expected as, despite the differences in
205 glycogen levels (Gill, 2005; Choe et al., 2008), similar ultimate pH values have already been
206 reported in literature (Devine & Dikeman, 2004; Gill, 2005; Litwinczuk, Florek, Skalecki, &
207 Litwinczuk, 2008). After fermentation, however, decreases of 0.40 and 1.10 pH units were
208 observed in the pork-high and pork-low treatments, respectively. Due to the presence of more
209 residual sugars (Gill, 2005), stronger decreases were obtained in the horse-high and horse-low
210 treatments, namely 0.95 and 1.26 pH units, respectively. For the combi-high and combi-low
211 treatments, intermediate decreases of 0.78 and 1.15 pH units were seen, respectively. For each
212 meat source, the differences in pH after fermentation between sausages based on addition of
213 different dextrose concentrations were statistically significant. It is important to stress,
214 however, that within the experimental setup, the actual pH values between the meat sources
215 vary, even within the treatment “high pH” or “low pH”. This must be kept in mind throughout
216 the interpretation of results obtained. During the further processing, more specifically at
217 production days 21, 42, 63, 84, 105, 126 and 168, the differences in pH between the two pH
218 conditions, but also between the different meat sources used, remained, despite the overall re-
219 increase of pH in function of production time as result of proteolysis (Toldra, 2008).

220 **3.2. Weight loss and aw**

221 Mean values ± SE of aw and weight losses are shown in Table 2. The use of different meat
222 sources for the preparation of nitrite-free dry fermented sausages and the obtained pH
223 conditions did not affect the weight losses up to day 105. From that day, however, higher
224 weight losses occurred in the horse-low treatment compared to the combi-low and pork-low
225 treatments, respectively. The use of different meat sources had also no clear influence on the
201 **3. Results and discussion**

202 **3.1. pH**

203 Means ± SE of pH are shown in Table 2. At day 0, no significant differences in pH of all
204 prepared meat batters were measured. This was expected as, despite the differences in
205 glycogen levels (Gill, 2005; Choe et al., 2008), similar ultimate pH values have already been
206 reported in literature (Devine & Dikeman, 2004; Gill, 2005; Litwinczuk, Florek, Skalecki, &
207 Litwinczuk, 2008). After fermentation, however, decreases of 0.40 and 1.10 pH units were
208 observed in the pork-high and pork-low treatments, respectively. Due to the presence of more
209 residual sugars (Gill, 2005), stronger decreases were obtained in the horse-high and horse-low
210 treatments, namely 0.95 and 1.26 pH units, respectively. For the combi-high and combi-low
211 treatments, intermediate decreases of 0.78 and 1.15 pH units were seen, respectively. For each
212 meat source, the differences in pH after fermentation between sausages based on addition of
213 different dextrose concentrations were statistically significant. It is important to stress,
214 however, that within the experimental setup, the actual pH values between the meat sources
215 vary, even within the treatment “high pH” or “low pH”. This must be kept in mind throughout
216 the interpretation of results obtained. During the further processing, more specifically at
217 production days 21, 42, 63, 84, 105, 126 and 168, the differences in pH between the two pH
218 conditions, but also between the different meat sources used, remained, despite the overall re-
219 increase of pH in function of production time as result of proteolysis (Toldra, 2008).

220 **3.2. Weight loss and aw**

221 Mean values ± SE of aw and weight losses are shown in Table 2. The use of different meat
222 sources for the preparation of nitrite-free dry fermented sausages and the obtained pH
223 conditions did not affect the weight losses up to day 105. From that day, however, higher
224 weight losses occurred in the horse-low treatment compared to the combi-low and pork-low
225 treatments, respectively. The use of different meat sources had also no clear influence on the
226 aw-decline during drying. For the sausages with high pH conditions, however, aw was
227 generally higher than those with low pH conditions except in those only made with pork.
228 Differences in aw might be explained by the coagulation of meat proteins at lower pH
229 conditions (Toldra, 2008). The reason why pH did not affect the pork treatments, could not be
230 explained. As a function of time, aw decreased gradually due to the persistent drying
231 conditions. Weight losses of the sausages increased, although more pronounced in the
232 beginning of the drying process. During the first 3 days, only a slight decrease of weight of
233 the sausages occurred, which can be attributed to the high relative humidity (95 % RH) during
234 fermentation.
235

3.3. *Screening of Zn(II)PPIX and/or PPIX formation as selection tool for the further quantification of natural pigments*

237 Figure 1 shows the red fluorescence emission of the six treatments at multiple time points
238 during the production process.
239

At day 0, only little and similar red fluorescence was observed for all treatments. After
239 fermentation, an overall slight increase in red fluorescence emission was seen, independent of
240 the meat source used and pH condition. The increased temperature of 24 °C during
241 fermentation, favouring FECH activity, probably plays a role here (Chau et al., 2011;
242 Wakamatsu, Okui, Hayashi, Nishimura, & Hattori, 2007). At day 21, clear fluorescence
243 appeared in the pork-high treatment, followed by the combi-high and the horse-high
244 treatments. Compared with day 3, no differences in fluorescence emission were seen for all
245 treatments with low pH conditions. Hence, higher pH values correspond with higher red
246 fluorescence emissions. At day 63, even higher red fluorescence emission could be seen in the
247 treatments with high pH conditions. However, a shift was seen, whereby darker pictures were
248 obtained in the combi-high treatment compared with the pork-high treatment, despite the
249 higher pH values of the latter. During the further production process, however, the differences
250
226

aw-decline during drying. For the sausages with high pH conditions, however, aw was generally higher than those with low pH conditions except in those only made with pork.

227

228

Differences in aw might be explained by the coagulation of meat proteins at lower pH conditions (Toldra, 2008). The reason why pH did not affect the pork treatments, could not be explained. As a function of time, aw decreased gradually due to the persistent drying conditions. Weight losses of the sausages increased, although more pronounced in the

229

230

231

232

beginning of the drying process. During the first 3 days, only a slight decrease of weight of the sausages occurred, which can be attributed to the high relative humidity (95 % RH) during fermentation.

233

234

235

### 3.3. Screening of Zn(II)PPIX and/or PPIX formation as selection tool for the further quantification of natural pigments

236

237

Figure 1 shows the red fluorescence emission of the six treatments at multiple time points during the production process.

238

239

At day 0, only little and similar red fluorescence was observed for all treatments. After fermentation, an overall slight increase in red fluorescence emission was seen, independent of the meat source used and pH condition. The increased temperature of 24 °C during fermentation, favouring FECH activity, probably plays a role here (Chau et al., 2011; Wakamatsu, Okui, Hayashi, Nishimura, & Hattori, 2007). At day 21, clear fluorescence appeared in the pork-high treatment, followed by the combi-high and the horse-high treatments. Compared with day 3, no differences in fluorescence emission were seen for all treatments with low pH conditions. Hence, higher pH values correspond with higher red fluorescence emissions. At day 63, even higher red fluorescence emission could be seen in the treatments with high pH conditions. However, a shift was seen, whereby darker pictures were obtained in the combi-high treatment compared with the pork-high treatment, despite the higher pH values of the latter. During the further production process, however, the differences

240

241

242

243

244

245

246

247

248

249

250
251 between the pork-high and horse-high treatments became less pronounced in function of time
252 (mainly because of a decreasing fluorescence emission in the cores of the pork-high treatment).

253 These observations could already indicate that our hypothesis, namely that producing nitrite-
254 free dry fermented sausages based on both pork and horsemeat could improve Zn(II)PPIX
255 formation due to the achievement of optimal pH values and zinc chelatase activities, was
256 promising.

257 From day 63, no differences in red fluorescence emission were seen for all sausages at low pH
258 condition in the core. However, clear changes with increasing red fluorescence emissions in
259 function of time were noted in the outer regions (periphery). This increase was most
260 pronounced in the pork-low treatment. In these samples, pH was measured in the periphery
261 (results not shown), showing higher pH values than those measured in the core. The
262 observation of more red fluorescence emission in the periphery compared to the core
263 corresponds to higher pH values in the periphery, despite the expected more aerobic
264 circumstances which was considered to inhibit Zn(II)PPIX formation in meat products
265 (Wakamatsu et al., 2004b; Wakamatsu et al., 2006).

266 The screening method offers the opportunity to easily and qualitatively assess the formation
267 of Zn(II)PPIX and/or PPIX. Based on the obtained observations, it was chosen only to
268 quantify Zn(II)PPIX, PPIX and total heme pigments at sampling day 0, 21, 63 and 168, as
269 these time points were assumed to deliver the most crucial information.

3.4. Evolution of Zn(II)PPIX, PPIX and total heme content in nitrite-free dry fermented
sausages based on different meat sources and at different pH conditions

272 The concentrations of Zn(II)PPIX, PPIX and total heme as a function of meat source, pH
273 condition and production time in nitrite-free dry fermented sausages, are presented in Table 3.

274 *Zn(II)PPIX analysis*
251 between the pork-high and horse-high treatments became less pronounced in function of time
252 (mainly because of a decreasing fluorescence emission in the cores of the pork-high treatment).

253 These observations could already indicate that our hypothesis, namely that producing nitrite-
254 free dry fermented sausages based on both pork and horsemeat could improve Zn(II)PPIX
255 formation due to the achievement of optimal pH values and zinc chelatase activities, was
256 promising.

257 From day 63, no differences in red fluorescence emission were seen for all sausages at low pH
258 condition in the core. However, clear changes with increasing red fluorescence emissions in
259 function of time were noted in the outer regions (periphery). This increase was most
260 pronounced in the pork-low treatment. In these samples, pH was measured in the periphery
261 (results not shown), showing higher pH values than those measured in the core. The
262 observation of more red fluorescence emission in the periphery compared to the core
263 corresponds to higher pH values in the periphery, despite the expected more aerobic
264 circumstances which was considered to inhibit Zn(II)PPIX formation in meat products
265 (Wakamatsu et al., 2004b; Wakamatsu et al., 2006).

266 The screening method offers the opportunity to easily and qualitatively assess the formation
267 of Zn(II)PPIX and/or PPIX. Based on the obtained observations, it was chosen only to
268 quantify Zn(II)PPIX, PPIX and total heme pigments at sampling day 0, 21, 63 and 168, as
269 these time points were assumed to deliver the most crucial information.

3.4. Evolution of Zn(II)PPIX, PPIX and total heme content in nitrite-free dry fermented
sausages based on different meat sources and at different pH conditions

272 The concentrations of Zn(II)PPIX, PPIX and total heme as a function of meat source, pH
273 condition and production time in nitrite-free dry fermented sausages, are presented in Table 3.

274 *Zn(II)PPIX analysis*
At day 0, no differences in initial Zn(II)PPIX content between all treatments was seen. Zn(II)PPIX formation occurred in the first 21 days of processing, in exception of the pork-low treatment. During the further production process, Zn(II)PPIX formation was observed in the horse-high, horse-low and combi-high treatments. However, no increase in Zn(II)PPIX could be seen in the pork-high treatment. This is in contrast to the results obtained in the earlier published work (De Maere et al., 2016a), whereby a remarkable formation of Zn(II)PPIX was seen after an extensive drying period of 177 days. It was stated that the factor time, potentially related to partial Mb denaturation, is of major importance for the formation of Zn(II)PPIX (Grossi, do Nascimento, Cardoso, & Skibsted, 2014; Paganelli, Grossi, Dores-Silva, Borges, Cardoso, & Skibsted, 2016). The reason why in this study the highly increased Zn(II)PPIX formation at a later stage in the production process did not occur in the nitrite-free porcine dry fermented sausages at a similar high pH level is not clear and requires further investigation. Possibly, variation in raw material (meat batch) or differences in starter culture development, resulting in differences in enzymatic activity (zinc chelatase or proteolytic activity causing Mb degradation) can be at the basis of this observation. Within the high pH treatments, Zn(II)PPIX formation was the poorest in the pork-high treatment, but showed to be equal in the horse-high and combi-high treatments. These results revealed that formation of Zn(II)PPIX in nitrite-free dry fermented sausages at the high pH condition can be significantly ameliorated if horsemeat is used. Moreover, a similar effect is obtained if only 50% of the meat was based on horsemeat.

In all cases, the pH condition of the sausages influenced the formation of Zn(II)PPIX, with significantly higher amounts at the highest pH conditions. This can be explained by the pH dependence of FECH activity, with pH optima around 5.5 for porcine FECH in meat-based models (Ishikawa, Yoshihara, Baba, Kawabuchi, Sato, Numata, & Matsumoto, 2006;
275

At day 0, no differences in initial Zn(II)PPIX content between all treatments was seen.

276

Zn(II)PPIX formation occurred in the first 21 days of processing, in exception of the pork-low

277

treatment. During the further production process, Zn(II)PPIX formation was observed in the

278

horse-high, horse-low and combi-high treatments. However, no increase in Zn(II)PPIX could

279

be seen in the pork-high treatment. This is in contrast to the results obtained in the earlier

280

published work (De Maere et al., 2016a), whereby a remarkable formation of Zn(II)PPIX was

281

seen after an extensive drying period of 177 days. It was stated that the factor time, potentially

282

related to partial Mb denaturation, is of major importance for the formation of Zn(II)PPIX

283

(Grossi, do Nascimento, Cardoso, & Skibsted, 2014; Paganelli, Grossi, Dores-Silva, Borges,

284

Cardoso, & Skibsted, 2016). The reason why in this study the highly increased Zn(II)PPIX

285

formation at a later stage in the production process did not occur in the nitrite-free porcine dry

286

fermented sausages at a similar high pH level is not clear and requires further investigation.

287

Possibly, variation in raw material (meat batch) or differences in starter culture development,

288

resulting in differences in enzymatic activity (zinc chelatase or proteolytic activity causing Mb

289

degradation) can be at the basis of this observation. Within the high pH treatments, Zn(II)PPIX

290

formation was the poorest in the pork-high treatment, but showed to be equal in the horse-high

291

and combi-high treatments. These results revealed that formation of Zn(II)PPIX in nitrite-free

292

dry fermented sausages at the high pH condition can be significantly ameliorated if horsemeat

293

is used. Moreover, a similar effect is obtained if only 50% of the meat was based on

294

horsemeat.

295

In all cases, the pH condition of the sausages influenced the formation of Zn(II)PPIX, with

296

significantly higher amounts at the highest pH conditions. This can be explained by the pH

297

dependence of FECH activity, with pH optima around 5.5 for porcine FECH in meat-based

298

models (Ishikawa, Yoshihara, Baba, Kawabuchi, Sato, Numata, & Matsumoto, 2006;
Wakamatsu et al., 2007). Specific pH optima for equine FECH, however, could not be found in literature.

Not included into the statistical experiment, but nevertheless worth mentioning, is that no significant differences in pH (at the majority of sampling days) were obtained between the pork-low and the horse-high treatments, which enables us to compare Zn(II)PPIX formation in sausages based on different meat sources at similar pH values. Remarkable differences in Zn(II)PPIX formation were observed between the pork-low and horse-high treatments. The horse-high treatment revealed more Zn(II)PPIX formation than the pork-low treatment.

Influence of meat source on Zn(II)PPIX formation has already been shown in De Maere et al., (2016b), whereby horsemeat showed better ability to form Zn(II)PPIX *in vitro* compared to pork, which was explained mainly by its higher zinc chelatase activity, but also by its higher heme and zinc content.

For all this, however, it is assumed that the influence of bacterial population on Zn(II)PPIX formation is minimal or similar. For both treatments, 9 log cfu/g on PCA agar plates, 9 log cfu/g on MRS agar plates and 6 log cfu/g on MSA agar plates were counted at day 21, indicating that the starter culture was present in equal amounts. *Enterobacteriaceae* did not exceed the quantification limit of 3.5 log cfu/g. Of course, more investigation about the influence of bacteria on the formation of Zn(II)PPIX should be done and was not included in this study.

### PPIX analysis

No differences in initial PPIX concentration were found. For the pork-high treatment, increasing PPIX concentrations were found up to day 63, but at day 168 a decreased concentration could be seen. PPIX formation occured during the first 21 days of processing and stabilized during the further drying period for the horse-high treatment. For the combi-high treatment, PPIX formation occured during the first 21 days of processing and stabilized
Wakamatsu et al., 2007). Specific pH optima for equine FECH, however, could not be found in literature.

Not included into the statistical experiment, but nevertheless worth mentioning, is that no significant differences in pH (at the majority of sampling days) were obtained between the pork-low and the horse-high treatments, which enables us to compare Zn(II)PPIX formation in sausages based on different meat sources at similar pH values. Remarkable differences in Zn(II)PPIX formation were observed between the pork-low and horse-high treatments. The horse-high treatment revealed more Zn(II)PPIX formation than the pork-low treatment.

Influence of meat source on Zn(II)PPIX formation has already been shown in De Maere et al., (2016b), whereby horsemeat showed better ability to form Zn(II)PPIX *in vitro* compared to pork, which was explained mainly by its higher zinc chelatase activity, but also by its higher heme and zinc content.

For all this, however, it is assumed that the influence of bacterial population on Zn(II)PPIX formation is minimal or similar. For both treatments, 9 log cfu/g on PCA agar plates, 9 log cfu/g on MRS agar plates and 6 log cfu/g on MSA agar plates were counted at day 21, indicating that the starter culture was present in equal amounts. *Enterobacteriaceae* did not exceed the quantification limit of 3.5 log cfu/g. Of course, more investigation about the influence of bacteria on the formation of Zn(II)PPIX should be done and was not included in this study.

### PPIX analysis

No differences in initial PPIX concentration were found. For the pork-high treatment, increasing PPIX concentrations were found up to day 63, but at day 168 a decreased concentration could be seen. PPIX formation occured during the first 21 days of processing and stabilized during the further drying period for the horse-high treatment. For the combi-high treatment, PPIX formation occured during the first 21 days of processing and stabilized
temporarily until a decrease was seen at day 168. No differences in PPIX formation between the different meat treatments were seen at day 21 and 63. But due to the decrease of PPIX in the pork-high and combi-high treatments at day 168, the concentration of PPIX was found to be higher in the horse-high treatment. At low pH conditions, PPIX formation did not occur in any meat treatment. Similar to Zn(II)PPIX formation, the pH condition of the sausages influenced the formation of PPIX, with significantly higher amounts at the highest pH conditions in all cases in exception of the combi treatment at day 168. An accumulation of PPIX at high pH conditions has already been noticed in porcine nitrite-free dry fermented sausages (De Maere et al., 2016a). In the current study, also an increased PPIX formation was seen, although not as pronounced as in previous study. In accordance to the results obtained for Zn(II)PPIX, a higher PPIX concentration was found in the horse-high treatment than in the pork-low treatment, having a similar pH evolution during processing.

## Total heme analysis

Depending on the meat source used, the meat batter showed clear differences in total heme amount, with the pork treatments having the lowest total heme concentrations, the horse treatments having the highest total heme concentrations and the combi treatments resulted in intermediate total heme concentrations. The higher concentrations of total heme in horsemeat compared to pork corresponds to what was already described in literature (De Maere et al., 2016b). During the production process, generally a decrease in total heme was seen (except for the combi-low treatment), although the course of this decrease differed between treatments, with no clear effect of meat source or pH treatment. A reduction of total heme concentrations has been already observed in dry cured ham and dry fermented sausages (Chasco, Lizaso, & Beriain, 1996; De Maere et al., 2016a; Wakamatsu et al., 2009b). The only exception was seen in the horse treatments, whereby a strong decrease in total heme was
temporarily until a decrease was seen at day 168. No differences in PPIX formation between the different meat treatments were seen at day 21 and 63. But due to the decrease of PPIX in the pork-high and combi-high treatments at day 168, the concentration of PPIX was found to be higher in the horse-high treatment. At low pH conditions, PPIX formation did not occur in any meat treatment. Similar to Zn(II)PPIX formation, the pH condition of the sausages influenced the formation of PPIX, with significantly higher amounts at the highest pH conditions in all cases in exception of the combi treatment at day 168. An accumulation of PPIX at high pH conditions has already been noticed in porcine nitrite-free dry fermented sausages (De Maere et al., 2016a). In the current study, also an increased PPIX formation was seen, although not as pronounced as in previous study. In accordance to the results obtained for Zn(II)PPIX, a higher PPIX concentration was found in the horse-high treatment than in the pork-low treatment, having a similar pH evolution during processing.

## Total heme analysis

Depending on the meat source used, the meat batter showed clear differences in total heme amount, with the pork treatments having the lowest total heme concentrations, the horse treatments having the highest total heme concentrations and the combi treatments resulted in intermediate total heme concentrations. The higher concentrations of total heme in horsemeat compared to pork corresponds to what was already described in literature (De Maere et al., 2016b). During the production process, generally a decrease in total heme was seen (except for the combi-low treatment), although the course of this decrease differed between treatments, with no clear effect of meat source or pH treatment. A reduction of total heme concentrations has been already observed in dry cured ham and dry fermented sausages (Chasco, Lizaso, & Beriain, 1996; De Maere et al., 2016a; Wakamatsu et al., 2009b). The only exception was seen in the horse treatments, whereby a strong decrease in total heme was
348

seen between day 21 and 63, but a re-increase at day 168. However, these findings could not

349

be explained.

350

Although a substitution reaction is assumed for the formation of Zn(II)PPIX, no clear

351

conclusion could be drawn about the decreasing heme pigments and the formation of

352

Zn(II)PPIX and PPIX. In this study, the sum of the three pigments generally decreased in

353

function of time, implying that more total heme breakdown occured than needed for the

354

substitution process alone.

355

### 3.5. Colour formation in nitrite-free dry fermented sausages based on different meat sources and at different pH conditions

356

357

Mean values of L*, a* and b* as a function of meat source, pH condition and production time

358

are shown in Table 4. In addition, analysis for a* was adjusted for Zn(II)PPIX, PPIX, total

359

heme and for all three simultaneously in order to relate the investigated pigments to the colour

360

measurements.

361

#### *L*\* analysis

362

Between the meat sources used, highly different results could be seen, with the pork

363

treatments having the highest L* values (being more bright), the horse treatments having the

364

lowest L* values (being more dark) and the combi treatments having intermediate L* values.

365

These differences remained during the further production process. During the first 21 days of

366

processing, a slight increase in L* value was observed in all treatments, which was significant

367

in case of the horse-low treatment. During further production, L* tended to decrease, which

368

can probably be attributed to a decrease in moisture content, resulting in a darker product.

369

Also no clear effect of pH on L* could be found, as the significant differences showed no

370

clear trend in this regard.

371

#### *a*\* analysis
348

seen between day 21 and 63, but a re-increase at day 168. However, these findings could not
be explained.

349

350

Although a substitution reaction is assumed for the formation of Zn(II)PPIX, no clear
conclusion could be drawn about the decreasing heme pigments and the formation of
Zn(II)PPIX and PPIX. In this study, the sum of the three pigments generally decreased in
function of time, implying that more total heme breakdown occured than needed for the
substitution process alone.

351

352

353

354

355

### 3.5. Colour formation in nitrite-free dry fermented sausages based on different meat sources and at different pH conditions

356

357

Mean values of L*, a* and b* as a function of meat source, pH condition and production time
are shown in Table 4. In addition, analysis for a* was adjusted for Zn(II)PPIX, PPIX, total
heme and for all three simultaneously in order to relate the investigated pigments to the colour
measurements.

358

359

360

361

#### *L*\* *analysis*

362

Between the meat sources used, highly different results could be seen, with the pork
treatments having the highest L* values (being more bright), the horse treatments having the
lowest L* values (being more dark) and the combi treatments having intermediate L* values.
These differences remained during the further production process. During the first 21 days of
processing, a slight increase in L* value was observed in all treatments, which was significant
in case of the horse-low treatment. During further production, L* tended to decrease, which
can probably be attributed to a decrease in moisture content, resulting in a darker product.
Also no clear effect of pH on L* could be found, as the significant differences showed no
clear trend in this regard.

363

364

365

366

367

368

369

370

371

#### *a*\* *analysis*
At day 0, the *a*\* values of the meat batter based on horsemeat were found, although not significant in case of the high pH treatment, higher than those based on pork. In contrast to *L*\*, the results of *a*\* were highly influenced by the fermentation process whereby the sausages evolved to a less red (decrease of *a*\*) colour, which can be attributed to the formation of higher concentrations of MMb (Adamsen et al., 2006). The more drastic decrease in *a*\* of the sausages based on horsemeat during fermentation could imply that higher amounts of MMb were formed, which could be a result of its higher Mb content (Feiner, 2006). For the pork and combi treatments, an increased *a*\* value was seen at the later phase of the production process, namely at sampling day 168. This was not the case for the horse treatments, which in contrast even decreased during further processing. Differences in *a*\* between the two pH conditions were mainly seen in the pork and combi treatments, with a higher *a*\* value at the highest pH condition. This was not seen for the horse treatments. The latter exhibited very low *a*\* values during production, in comparison with the sausages whereby pork was used as a meat source.

The linear mixed model also allowed to relate the investigated pigments with *a*\*. None of the pigments, however, was found to have an effect on *a*\* values. More specifically, no effect of Zn(II)PPIX on *a*\* (*P* = 0.9736) was found during production of nitrite-free dry fermented sausages based on different meat sources and at different pH conditions. Zn(II)PPIX formation occured mainly in the horse and combi treatments. Redness, however, was very poor in the horse treatments, was higher in the combi treatments and was the highest in the pork treatments. Also in earlier studies, it was not easy to relate the presence of Zn(II)PPIX to instrumental colour measurements in meat products (Adamsen et al., 2006; Parolari et al., 2009). In our previous study, *a*\* of the porcine sausages was significantly related to the content of Zn(II)PPIX (De Maere et al., 2016a), which suggested that Zn(II)PPIX influenced redness of the meat products. In the latter study, at the end of production a huge increase of
At day 0, the *a*\* values of the meat batter based on horsemeat were found, although not significant in case of the high pH treatment, higher than those based on pork. In contrast to *L*\*, the results of *a*\* were highly influenced by the fermentation process whereby the sausages evolved to a less red (decrease of *a*\*) colour, which can be attributed to the formation of higher concentrations of MMb (Adamsen et al., 2006). The more drastic decrease in *a*\* of the sausages based on horsemeat during fermentation could imply that higher amounts of MMb were formed, which could be a result of its higher Mb content (Feiner, 2006). For the pork and combi treatments, an increased *a*\* value was seen at the later phase of the production process, namely at sampling day 168. This was not the case for the horse treatments, which in contrast even decreased during further processing. Differences in *a*\* between the two pH conditions were mainly seen in the pork and combi treatments, with a higher *a*\* value at the highest pH condition. This was not seen for the horse treatments. The latter exhibited very low *a*\* values during production, in comparison with the sausages whereby pork was used as a meat source.

The linear mixed model also allowed to relate the investigated pigments with *a*\*. None of the pigments, however, was found to have an effect on *a*\* values. More specifically, no effect of Zn(II)PPIX on *a*\* (*P* = 0.9736) was found during production of nitrite-free dry fermented sausages based on different meat sources and at different pH conditions. Zn(II)PPIX formation occured mainly in the horse and combi treatments. Redness, however, was very poor in the horse treatments, was higher in the combi treatments and was the highest in the pork treatments. Also in earlier studies, it was not easy to relate the presence of Zn(II)PPIX to instrumental colour measurements in meat products (Adamsen et al., 2006; Parolari et al., 2009). In our previous study, *a*\* of the porcine sausages was significantly related to the content of Zn(II)PPIX (De Maere et al., 2016a), which suggested that Zn(II)PPIX influenced redness of the meat products. In the latter study, at the end of production a huge increase of
397 Zn(II)PPIX formation was seen at the highest pH condition, with Zn(II)PPIX concentrations
398 up to 125.69±5.66 nmol/g DM at day 177. In those sausages, an *a*\* value of 13.03±0.19 was
399 measured. In contrast, as discussed in section 3.4, this huge increase of Zn(II)PPIX formation
400 at the highest pH condition was not observed in the current study, the Zn(II)PPIX
401 concentration in porcine sausages at high pH was only 11.63±0.50 nmol/g DM at day 168.
402 Still, an *a*\* value of 12.74±0.46 was obtained, which is almost as high as the values obtained
403 in previous study. In contrast, the horse-high and combi-high treatments reached at day 168
404 Zn(II)PPIX concentrations of 59.51±2.47 nmol/g DM and 61.20±2.25 nmol/g DM,
405 respectively, but these concentrations did not result in increased *a*\* values. On the contrary,
406 *a*\* values were very low, especially in the case of sausages prepared with horsemeat.
407 Therefore it can be concluded that no relation could be found between the pigments quantified
408 in this study and redness in these dry fermented sausages, and that the Zn(II)PPIX formed did
409 not act as a natural coloring agent. Potentially, formation of MMb during fermentation,
410 especially in sausages prepared with horsemeat, may overrule the coloring effect of
411 Zn(II)PPIX.

412 *b*\* *analysis*

413 Differences in *b*\* values were found between the meat batters at day 0, with meat batters
414 based on pork showing the highest *b*\* values, meat batters based on horsemeat showing the
415 lowest *b*\* value and meat batters based on the 50/50 combination of both meat sources
416 showing intermediate values. However, these differences were rather small. Similar to the *a*\*
417 values, *b*\* values were also influenced by the fermentation process, whereby the sausages
418 evolved to a less yellow (decrease of *b*\*) colour. This can probably be attributed to the
419 formation of higher concentrations of MMb (Adamsen et al., 2006). Similarly as in case of the
420 *a*\* value, this decrease in *b*\* was again stronger for sausages prepared with horsemeat, which
421 may be related to its higher Mb content (Feiner, 2006), resulting in stronger formation of
397 Zn(II)PPIX formation was seen at the highest pH condition, with Zn(II)PPIX concentrations
398 up to 125.69±5.66 nmol/g DM at day 177. In those sausages, an *a*\* value of 13.03±0.19 was
399 measured. In contrast, as discussed in section 3.4, this huge increase of Zn(II)PPIX formation
400 at the highest pH condition was not observed in the current study, the Zn(II)PPIX
401 concentration in porcine sausages at high pH was only 11.63±0.50 nmol/g DM at day 168.
402 Still, an *a*\* value of 12.74±0.46 was obtained, which is almost as high as the values obtained
403 in previous study. In contrast, the horse-high and combi-high treatments reached at day 168
404 Zn(II)PPIX concentrations of 59.51±2.47 nmol/g DM and 61.20±2.25 nmol/g DM,
405 respectively, but these concentrations did not result in increased *a*\* values. On the contrary,
406 *a*\* values were very low, especially in the case of sausages prepared with horsemeat.
407 Therefore it can be concluded that no relation could be found between the pigments quantified
408 in this study and redness in these dry fermented sausages, and that the Zn(II)PPIX formed did
409 not act as a natural coloring agent. Potentially, formation of MMb during fermentation,
410 especially in sausages prepared with horsemeat, may overrule the coloring effect of
411 Zn(II)PPIX.

412 *b*\* *analysis*

413 Differences in *b*\* values were found between the meat batters at day 0, with meat batters
414 based on pork showing the highest *b*\* values, meat batters based on horsemeat showing the
415 lowest *b*\* value and meat batters based on the 50/50 combination of both meat sources
416 showing intermediate values. However, these differences were rather small. Similar to the *a*\*
417 values, *b*\* values were also influenced by the fermentation process, whereby the sausages
418 evolved to a less yellow (decrease of *b*\*) colour. This can probably be attributed to the
419 formation of higher concentrations of MMb (Adamsen et al., 2006). Similarly as in case of the
420 *a*\* value, this decrease in *b*\* was again stronger for sausages prepared with horsemeat, which
421 may be related to its higher Mb content (Feiner, 2006), resulting in stronger formation of
422

MMb during fermentation. In function of time, b* values of the pork treatments increased, but
increased and remained stable or decreased again for the horse and combi treatments. As a
result, b* values remained highest in the former treatments during the further production
process. However, the latter changes in b* values during processing were limited. Significant
pH effects were found, but no clear trend could be seen. It can be concluded that differences
in b* value were limited and could mainly be attributed to probable differences in MMb
formation during fermentation.

423

424

425

426

427

428

429

4. Conclusion

430

Zn(II)PPIX formation is significantly promoted by a higher pH and by the use of horsemeat.

Both effects can probably be attributed to higher zinc chelatase activity. However, Zn(II)PPIX contents obtained after long processing times were lower compared to a previous study based on pork. Due raw material variation and/or complex processes during fermentation and ripening, a stable, standardized formation of Zn(II)PPIX in nitrite-free dry fermented sausages cannot yet be guaranteed.

431

432

433

434

435

436

None of these pigments measured (Zn(II)PPIX, PPIX and heme) had an effect on the redness
(a* values) of the sausages. The higher concentrations of Zn(II)PPIX obtained in nitrite-free
dry fermented sausages based on horsemeat, but also by combining pork and horsemeat, did
not act as a natural colouring agent. This indicates that the redness of these sausages is
determined by other factors that were not quantified in this study, such as MMb content.
Therefore, a better insight in the factors determining colour of nitrite-free meat products
remains indispensable for meat industry for investigating the elimination of nitrite as
colouring agent in meat products.

437

438

439

440

441

442

443

444
422

MMb during fermentation. In function of time, b* values of the pork treatments increased, but increased and remained stable or decreased again for the horse and combi treatments. As a result, b* values remained highest in the former treatments during the further production process. However, the latter changes in b* values during processing were limited. Significant pH effects were found, but no clear trend could be seen. It can be concluded that differences in b* value were limited and could mainly be attributed to probable differences in MMb formation during fermentation.

423

424

425

426

427

428

429

## 4. Conclusion

430

Zn(II)PPIX formation is significantly promoted by a higher pH and by the use of horsemeat. Both effects can probably be attributed to higher zinc chelatase activity. However, Zn(II)PPIX contents obtained after long processing times were lower compared to a previous study based on pork. Due raw material variation and/or complex processes during fermentation and ripening, a stable, standardized formation of Zn(II)PPIX in nitrite-free dry fermented sausages cannot yet be guaranteed.

431

432

433

434

435

436

None of these pigments measured (Zn(II)PPIX, PPIX and heme) had an effect on the redness (a* values) of the sausages. The higher concentrations of Zn(II)PPIX obtained in nitrite-free dry fermented sausages based on horsemeat, but also by combining pork and horsemeat, did not act as a natural colouring agent. This indicates that the redness of these sausages is determined by other factors that were not quantified in this study, such as MMb content. Therefore, a better insight in the factors determining colour of nitrite-free meat products remains indispensable for meat industry for investigating the elimination of nitrite as colouring agent in meat products.

437

438

439

440

441

442

443

444
445 References

446 Adamsen, C. E., Møller, J. K. S., Laursen, K., Olsen, K., & Skibsted, L. H. (2006). Zn-porphyrin formation in cured meat products: Effect of added salt and nitrite. *Meat Science*, 72(4), 672-679.

449 Becker, E. M., Westermann, S., Hansson, M., & Skibsted, L. H. (2012). Parallel enzymatic and non-enzymatic formation of zinc protoporphyrin IX in pork. *Food Chemistry*, 130(4), 832-840.

452 Chasco, J., Lizaso, G., & Beriain, M. J. (1996). Cured colour development during sausage processing. *Meat Science*, 44(3), 203-211.

454 Chau, T. T., Ishigaki, M., Kataoka, T., & Taketani, S. (2010). Porcine Ferrochelatase: The relationship between iron-removal reaction and the conversion of heme to Zn-protoporphyrin. *Bioscience, Biotechnology, and Biochemistry*, 74(7), 1415-1420.

457 Chau, T. T., Ishigaki, M., Kataoka, T., & Taketani, S. (2011). Ferrochelatase catalyzes the formation of Zn-protoporphyrin of dry-cured ham via the conversion reaction from heme in meat. *Journal of Agricultural and Food Chemistry*, 59(22), 12238-1245.

460 Choe, J. H., Choi, Y. M., Lee, S. H., Shin, H. G., Ryu, Y. C., Hong, K. C., & Kim, B. C. (2008). The relation between glycogen, lactate content and muscle fiber type composition, and their influence on postmortem glycolytic rate and pork quality. *Meat Science*, 80(2), 355-362.

464 Christensen, R. (2015). *Analysis of Variance, Design, and Regression: Linear Modeling for Unbalanced Data, Second Edition*. Chapman & Hall/CRC Texts in Statistical Science.

466 Dailey, H. A., Dailey, T. A., Wu, C., Medlock, A. E., Wang, K., Rose, J. P., & Wang, B. (2000). Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters. *Cellular and Molecular Life Sciences*, 57, 1909-1926.

469 De Maere, H., Chollet, S., Claeys, E., Michiels, C., Govaert, M., De Mey, E., Paelinck, H., Fraeye, I. (2016b). *In vitro* zinc protoporphyrin IX formation in different meat sources related to potentially important intrinsic parameters. *Food and Bioprocess Technology*, 10(1), 131-142.

473 De Maere, H., Fraeye, I., De Mey, E., Dewulf, L., Michiels, C., Paelinck, H., & Chollet, S. (2016a). Formation of naturally occurring pigments during the production of nitrite-free dry fermented sausages. *Meat Science*, 114, 1-7.

476 Devine, C., & Dikeman, M. (2004). *Encyclopedia of Meat Sciences*. Oxford: Elsevier Acad. Press.

478 Feiner, G. (2006). *Meat products handbook: Practical science and technology*. Cambridge: Woodhead Publishing.
445 References

446 Adamsen, C. E., Møller, J. K. S., Laursen, K., Olsen, K., & Skibsted, L. H. (2006). Zn-porphyrin formation in cured meat products: Effect of added salt and nitrite. *Meat Science*, 72(4), 672-679.

449 Becker, E. M., Westermann, S., Hansson, M., & Skibsted, L. H. (2012). Parallel enzymatic and non-enzymatic formation of zinc protoporphyrin IX in pork. *Food Chemistry*, 130(4), 832-840.

452 Chasco, J., Lizaso, G., & Beriain, M. J. (1996). Cured colour development during sausage processing. *Meat Science*, 44(3), 203-211.

454 Chau, T. T., Ishigaki, M., Kataoka, T., & Taketani, S. (2010). Porcine Ferrochelatase: The relationship between iron-removal reaction and the conversion of heme to Zn-protoporphyrin. *Bioscience, Biotechnology, and Biochemistry*, 74(7), 1415-1420.

457 Chau, T. T., Ishigaki, M., Kataoka, T., & Taketani, S. (2011). Ferrochelatase catalyzes the formation of Zn-protoporphyrin of dry-cured ham via the conversion reaction from heme in meat. *Journal of Agricultural and Food Chemistry*, 59(22), 12238-1245.

460 Choe, J. H., Choi, Y. M., Lee, S. H., Shin, H. G., Ryu, Y. C., Hong, K. C., & Kim, B. C. (2008). The relation between glycogen, lactate content and muscle fiber type composition, and their influence on postmortem glycolytic rate and pork quality. *Meat Science*, 80(2), 355-362.

464 Christensen, R. (2015). *Analysis of Variance, Design, and Regression: Linear Modeling for Unbalanced Data, Second Edition*. Chapman & Hall/CRC Texts in Statistical Science.

466 Dailey, H. A., Dailey, T. A., Wu, C., Medlock, A. E., Wang, K., Rose, J. P., & Wang, B. (2000). Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters. *Cellular and Molecular Life Sciences*, 57, 1909-1926.

469 De Maere, H., Chollet, S., Claeys, E., Michiels, C., Govaert, M., De Mey, E., Paelinck, H., Fraeye, I. (2016b). *In vitro* zinc protoporphyrin IX formation in different meat sources related to potentially important intrinsic parameters. *Food and Bioprocess Technology*, 10(1), 131-142.

473 De Maere, H., Fraeye, I., De Mey, E., Dewulf, L., Michiels, C., Paelinck, H., & Chollet, S. (2016a). Formation of naturally occurring pigments during the production of nitrite-free dry fermented sausages. *Meat Science*, 114, 1-7.

476 Devine, C., & Dikeman, M. (2004). *Encyclopedia of Meat Sciences*. Oxford: Elsevier Acad. Press.

478 Feiner, G. (2006). *Meat products handbook: Practical science and technology*. Cambridge: Woodhead Publishing.
480
481

Gill, C. O. (2005). Safety and storage stability of horse meat for human consumption. *Meat Science*, **71**(3), 506-513.

482
483
484

Grossi, A. B., do Nascimento, E. S. P., Cardoso, D. R., & Skibsted, L. H. (2014). Proteolysis involvement in zinc-protoporphyrin IX formation during Parma ham maturation. *Food Research International*, **56**, 252-259.

485
486

Hochberg, Y., & Tamhane, A.C. (1987). *Multiple Comparison Procedures*. New York: Wiley.

487
488

Honikel, K.-O. (2008). The use and control of nitrate and nitrite for the processing of meat products. *Meat Science*, **78**(1-2), 68-76.

489
490
491

Ishikawa, H., Kawabuchi, T., Kawakami, Y., Sato, M., Numata, M., & Matsumoto, K. (2007). Formation of zinc protoporphyrin IX and protoporphyrin IX from oxymyoglobin in porcine heart mitochondria. *Food Science and Technology Research*, **13**(1), 85-88.

492
493
494

Ishikawa, H., Yoshihara, M., Baba, A., Kawabuchi, T., Sato, M., Numata, M., & Matsumoto, K. (2006). Formation of zinc protoporphyrin IX from myoglobin with pork loin extract. *Journal of the Faculty of Agriculture*, **51**(1), 93-97.

495

Lindahl, G. (2005). *Colour Characteristics of Fresh Pork*. Doctor's dissertation.

496
497
498

Litwinczuk, A., Florek, M., Skalecki, P., Litwinczuk, Z. (2008). Chemical composition and physicochemical properties of horse meat from the Longissimus. *Journal of Muscle Foods*, **19**, 223-236.

499
500

Mancini, R. A., & Hunt, M. C. (2005). Current research in meat color. *Meat Science*, **71**(1), 100-121.

501
502
503

Medlock, A., Swartz, L., Dailey, T. a, Dailey, H. a, & Lanzilotta, W. N. (2007). Substrate interactions with human ferrochelatase. *Proceedings of the National Academy of Sciences of the United States of America*, **104**(6), 1789-1793.

504
505
506

Paganelli, M. O., Grossi, A. B., Dores-Silva, P. R., Borges, J. C., Cardoso, D. R., & Skibsted, L. H. (2016). Limited proteolysis of myoglobin opens channel in ferrochelatase-globin complex for iron to zinc transmetallation. *Food Chemistry*, **210**, 491-499.

507
508
509

Parolari, G., Benedini, R., & Toscani, T. (2009). Color formation in nitrite-free dried hams as related to Zn-protoporphyrin IX and Zn-chelatase activity. *Journal of Food Science*, **74**(6), 413-418.

510
511

Schuddeboom, L. J. (1993). Nitrates and nitrites in foodstuffs. Council of Europe Press, Publishing and Documentation Service, ISBN 92-871-2424-6.

512
513

Skibsted, L. H. (2011). Nitric oxide and quality and safety of muscle based foods. *Nitric Oxide - Biology and Chemistry*, **24**(4), 176-183.
480
481

Gill, C. O. (2005). Safety and storage stability of horse meat for human consumption. *Meat Science*, **71**(3), 506-513.

482
483
484

Grossi, A. B., do Nascimento, E. S. P., Cardoso, D. R., & Skibsted, L. H. (2014). Proteolysis involvement in zinc-protoporphyrin IX formation during Parma ham maturation. *Food Research International*, **56**, 252-259.

485
486

Hochberg, Y., & Tamhane, A.C. (1987). *Multiple Comparison Procedures*. New York: Wiley.

487
488

Honikel, K.-O. (2008). The use and control of nitrate and nitrite for the processing of meat products. *Meat Science*, **78**(1-2), 68-76.

489
490
491

Ishikawa, H., Kawabuchi, T., Kawakami, Y., Sato, M., Numata, M., & Matsumoto, K. (2007). Formation of zinc protoporphyrin IX and protoporphyrin IX from oxymyoglobin in porcine heart mitochondria. *Food Science and Technology Research*, **13**(1), 85-88.

492
493
494

Ishikawa, H., Yoshihara, M., Baba, A., Kawabuchi, T., Sato, M., Numata, M., & Matsumoto, K. (2006). Formation of zinc protoporphyrin IX from myoglobin with pork loin extract. *Journal of the Faculty of Agriculture*, **51**(1), 93-97.

495

Lindahl, G. (2005). *Colour Characteristics of Fresh Pork*. Doctor's dissertation.

496
497
498

Litwinczuk, A., Florek, M., Skalecki, P., Litwinczuk, Z. (2008). Chemical composition and physicochemical properties of horse meat from the Longissimus. *Journal of Muscle Foods*, **19**, 223-236.

499
500

Mancini, R. A., & Hunt, M. C. (2005). Current research in meat color. *Meat Science*, **71**(1), 100-121.

501
502
503

Medlock, A., Swartz, L., Dailey, T. a, Dailey, H. a, & Lanzilotta, W. N. (2007). Substrate interactions with human ferrochelatase. *Proceedings of the National Academy of Sciences of the United States of America*, **104**(6), 1789-1793.

504
505
506

Paganelli, M. O., Grossi, A. B., Dores-Silva, P. R., Borges, J. C., Cardoso, D. R., & Skibsted, L. H. (2016). Limited proteolysis of myoglobin opens channel in ferrochelatase-globin complex for iron to zinc transmetallation. *Food Chemistry*, **210**, 491-499.

507
508
509

Parolari, G., Benedini, R., & Toscani, T. (2009). Color formation in nitrite-free dried hams as related to Zn-protoporphyrin IX and Zn-chelatase activity. *Journal of Food Science*, **74**(6), 413-418.

510
511

Schuddeboom, L. J. (1993). Nitrates and nitrites in foodstuffs. Council of Europe Press, Publishing and Documentation Service, ISBN 92-871-2424-6.

512
513

Skibsted, L. H. (2011). Nitric oxide and quality and safety of muscle based foods. *Nitric Oxide - Biology and Chemistry*, **24**(4), 176-183.
514 Takenati S., Mutsumi I., Mizutani A., Uebayashi M., Numata M., & Ohgari Y., K. S. (2007).

515 Heme synthase (Ferrochelatase) catalyzes the removal of iron from heme and

516 demetalation of metalloporphyrins. *Biochemistry*, **46**, 15054–15061.

517 Toldra, F. (2008). *Handbook of fermented meat and poultry*. Ames, Iowa: Wiley-Blackwell

518 Publishing.

519 Verbeke, G., & Molenberghs, G. (2013). *Linear Mixed Models for Longitudinal Data*.

520 Springer Series in Statistics.

521 Verordening (EG) Nr. 1333/2008 Van het Europees parlement en de raad van 16 december

522 2008 inzake levensmiddelenadditieven. (2008). *Publicatieblad van de Europese Unie*.

523 Wakamatsu, J., Nishimura, T., & Hattori, A. (2004a). A Zn-porphyrin complex contributes to

524 bright red color in Parma ham. *Meat Science*, **67**(1), 95–100.

525 Wakamatsu, J., Odagiri, H., Nishimura, T., & Hattori, A. (2009a). Quantitative determination

526 of Zn protoporphyrin IX, heme and protoporphyrin IX in Parma ham by HPLC. *Meat*

527 *Science*, **82**(1), 139–142.

528 Wakamatsu, J., Okui, J., Hayashi, N., Nishimura, T., & Hattori, A. (2007). Zn protoporphyrin

529 IX is formed not from heme but from protoporphyrin IX. *Meat Science*, **77**(4), 580–586.

530 Wakamatsu, J., Okui, J., Ikeda, Y., Nishimura, T., & Hattori, A. (2004b). Establishment of a

531 model experiment system to elucidate the mechanism by which Zn-protoporphyrin IX is

532 formed in nitrite-free dry-cured ham. *Meat Science*, **68**(2), 313–7.

533 Wakamatsu, J., Uemura, J., Odagiri, H., Okui, J., Hayashi, N., Hioki, S., Nishimura, T.,

534 Hattori, A. (2009b). Formation of zinc protoporphyrin IX in Parma-like ham without

535 nitrate or nitrite. *Animal Science Journal*, **80**(2), 198–205.
514 Takenati S., Mutsumi I., Mizutani A., Uebayashi M., Numata M., & Ohgari Y., K. S. (2007).

515 Heme synthase (Ferrochelatase) catalyzes the removal of iron from heme and

516 dematalation of metalloporphyrins. *Biochemistry*, **46**, 15054–15061.

517 Toldra, F. (2008). *Handbook of fermented meat and poultry*. Ames, Iowa: Wiley-Blackwell

518 Publishing.

519 Verbeke, G., & Molenberghs, G. (2013). *Linear Mixed Models for Longitudinal Data*.

520 Springer Series in Statistics.

521 Verordening (EG) Nr. 1333/2008 Van het Europees parlement en de raad van 16 december

522 2008 inzake levensmiddelenadditieven. (2008). *Publicatieblad van de Europese Unie*.

523 Wakamatsu, J., Nishimura, T., & Hattori, A. (2004a). A Zn-porphyrin complex contributes to

524 bright red color in Parma ham. *Meat Science*, **67**(1), 95–100.

525 Wakamatsu, J., Odagiri, H., Nishimura, T., & Hattori, A. (2009a). Quantitative determination

526 of Zn protoporphyrin IX, heme and protoporphyrin IX in Parma ham by HPLC. *Meat*

527 *Science*, **82**(1), 139–142.

528 Wakamatsu, J., Okui, J., Hayashi, N., Nishimura, T., & Hattori, A. (2007). Zn protoporphyrin

529 IX is formed not from heme but from protoporphyrin IX. *Meat Science*, **77**(4), 580–586.

530 Wakamatsu, J., Okui, J., Ikeda, Y., Nishimura, T., & Hattori, A. (2004b). Establishment of a

531 model experiment system to elucidate the mechanism by which Zn-protoporphyrin IX is

532 formed in nitrite-free dry-cured ham. *Meat Science*, **68**(2), 313–7.

533 Wakamatsu, J., Uemura, J., Odagiri, H., Okui, J., Hayashi, N., Hioki, S., Nishimura, T.,

534 Hattori, A. (2009b). Formation of zinc protoporphyrin IX in Parma-like ham without

535 nitrate or nitrite. *Animal Science Journal*, **80**(2), 198–205.
536 Table 1 Overview of the different nitrite-free dry fermented sausages treatments. Nitrite-free dry fermented sausages are based on different meat sources, namely
537 pork (pork), horsemeat (horse) and a 50/50 combination of both meat sources (combi). Different pH conditions are obtained by adding different concentrations of
538 dextrose to the meat batter, 0.00% (high) and 0.70% (low).

<table><thead><tr><th>meat source treatment</th><th>pH treatment</th><th>code</th></tr></thead><tbody><tr><td rowspan="2">Pork</td><td>0.00% dextrose</td><td>pork-high</td></tr><tr><td>0.70% dextrose</td><td>pork-low</td></tr><tr><td rowspan="2">horsemeat</td><td>0.00% dextrose</td><td>horse-high</td></tr><tr><td>0.70% dextrose</td><td>horse-low</td></tr><tr><td rowspan="2">50/50 combination pork and horsemeat</td><td>0.00% dextrose</td><td>combi-high</td></tr><tr><td>0.70% dextrose</td><td>combi-low</td></tr></tbody></table>

539

540
536 Table 1 Overview of the different nitrite-free dry fermented sausages treatments. Nitrite-free dry fermented sausages are based on different meat sources, namely

537 pork (pork), horsemeat (horse) and a 50/50 combination of both meat sources (combi). Different pH conditions are obtained by adding different concentrations of

538 dextrose to the meat batter, 0.00% (high) and 0.70% (low).

<table><thead><tr><th>meat source treatment</th><th>pH treatment</th><th>code</th></tr></thead><tbody><tr><td rowspan="2">Pork</td><td>0.00% dextrose</td><td>pork-high</td></tr><tr><td>0.70% dextrose</td><td>pork-low</td></tr><tr><td rowspan="2">horsemeat</td><td>0.00% dextrose</td><td>horse-high</td></tr><tr><td>0.70% dextrose</td><td>horse-low</td></tr><tr><td rowspan="2">50/50 combination pork and horsemeat</td><td>0.00% dextrose</td><td>combi-high</td></tr><tr><td>0.70% dextrose</td><td>combi-low</td></tr></tbody></table>

539

540
<table><caption>Table 2 pH (n = 6), aw (n = 6) and weight losses (n = 2) during the production of nitrite-free dry fermented sausages based on pork (pork), horsemeat (horse) and a 541 50/50 combination of both meat sources (combi). Different pH conditions are obtained by adding different concentrations of dextrose to the meat batter, 0.00% (high) 542 and 0.70% (low). 543</caption><thead><tr><th rowspan="2"></th><th rowspan="2">treatment</th><th colspan="9">production day</th></tr><tr><th>0</th><th>3</th><th>21</th><th>42</th><th>63</th><th>84</th><th>105</th><th>126</th><th>168</th></tr></thead><tbody><tr><td rowspan="6">pH<br>(-)</td><td>pork-high</td><td>5.36±0.01<sub>a,1</sub></td><td>4.96±0.01<sub>e</sub></td><td>5.22±0.02<sup>f</sup></td><td>5.41±0.01<sub>e</sub></td><td>5.68±0.04<sub>d</sub></td><td>5.60±0.02<sub>d</sub></td><td>5.68±0.06<sub>e</sub></td><td>5.78±0.02<sub>e</sub></td><td>6.03±0.10<sub>c</sub></td></tr><tr><td>pork-low</td><td>5.38±0.01<sub>a,1</sub></td><td>4.28±0.02<sub>b</sub></td><td>4.51±0.01<sub>d</sub></td><td>4.59±0.01<sub>c</sub></td><td>4.82±0.05<sub>b</sub></td><td>4.82±0.06<sub>b</sub></td><td>4.89±0.03<sub>b</sub></td><td>5.13±0.02<sub>c</sub></td><td>5.52±0.01<sub>b</sub></td></tr><tr><td>horse-high</td><td>5.40±0.01<sub>a,1</sub></td><td>4.45±0.01<sub>c</sub></td><td>4.43±0.02<sub>c</sub></td><td>4.55±0.02<sub>c</sub></td><td>4.76±0.02<sub>b</sub></td><td>4.88±0.04<sub>b</sub></td><td>5.06±0.03<sub>c</sub></td><td>5.17±0.04<sub>c</sub></td><td>5.47±0.02<sub>b</sub></td></tr><tr><td>horse-low</td><td>5.38±0.02<sub>a,1</sub></td><td>4.12±0.04<sub>a</sub></td><td>4.11±0.01<sub>a</sub></td><td>4.21±0.01<sub>a</sub></td><td>4.28±0.03<sub>a</sub></td><td>4.47±0.03<sub>a</sub></td><td>4.62±0.03<sub>a</sub></td><td>4.72±0.02<sub>a</sub></td><td>5.17±0.07<sub>a</sub></td></tr><tr><td>combi-high</td><td>5.39±0.02<sub>a,1</sub></td><td>4.61±0.01<sub>d</sub></td><td>4.78±0.02<sub>e</sub></td><td>4.99±0.01<sub>d</sub></td><td>5.30±0.08<sub>c</sub></td><td>5.31±0.03<sub>c</sub></td><td>5.45±0.03<sub>d</sub></td><td>5.54±0.03<sub>d</sub></td><td>5.83±0.04<sub>c</sub></td></tr><tr><td>combi-low</td><td>5.34±0.01<sub>a,1</sub></td><td>4.19±0.01<sub>a</sub></td><td>4.26±0.01<sub>b</sub></td><td>4.28±0.01<sub>b</sub></td><td>4.39±0.02<sub>a</sub></td><td>4.76±0.06<sub>b</sub></td><td>4.70±0.02<sub>a</sub></td><td>4.89±0.02<sub>b</sub></td><td>5.12±0.01<sub>a</sub></td></tr><tr><td rowspan="6">weight<br>loss<br>(%)</td><td>pork-high</td><td>0.00±0.00<sub>a,1</sub></td><td>4.02±0.19<sub>a,1</sub></td><td>15.37±0.87<sub>a,1</sub></td><td>24.87±1.37<sub>a,1</sub></td><td>29.82±1.65<sub>a,1</sub></td><td>33.33±2.17<sub>a,1</sub></td><td>36.27±2.09<sub>a,1</sub></td><td>36.76±2.11<sub>a,1</sub></td><td>40.14±2.11<sub>a,1</sub></td></tr><tr><td>pork-low</td><td>0.00±0.00<sub>a,1</sub></td><td>3.86±0.52<sub>a,1</sub></td><td>15.22±2.52<sub>a,1</sub></td><td>25.48±0.89<sub>a,1</sub></td><td>31.34±2.01<sub>a,1</sub></td><td>33.20±0.47<sub>a,1</sub></td><td>35.46±0.21<sub>a,1</sub></td><td>35.41±0.11<sub>a,1</sub></td><td>39.05±0.26<sub>a,1</sub></td></tr><tr><td>horse-high</td><td>0.00±0.00<sub>a,1</sub></td><td>3.00±0.22<sub>a,1</sub></td><td>15.37±1.18<sub>a,1</sub></td><td>27.58±0.43<sub>a,1</sub></td><td>32.43±0.56<sub>a,1</sub></td><td>35.65±0.36<sub>a,1</sub></td><td>39.02±0.26<sub>a,1</sub></td><td>39.23±0.55<sub>a,1</sub></td><td>43.28±0.84<sub>a,1</sub></td></tr><tr><td>horse-low</td><td>0.00±0.00<sub>a,1</sub></td><td>2.65±0.24<sub>a,1</sub></td><td>13.98±0.37<sub>a,1</sub></td><td>26.26±1.05<sub>a,1</sub></td><td>32.18±0.31<sub>a,1</sub></td><td>36.11±0.43<sub>a,1</sub></td><td>39.05±0.17<sub>a,3</sub></td><td>39.96±0.40<sub>a,3</sub></td><td>43.64±0.10<sub>a,3</sub></td></tr><tr><td>combi-high</td><td>0.00±0.00<sub>a,1</sub></td><td>3.14±0.10<sub>a,1</sub></td><td>15.53±0.51<sub>a,1</sub></td><td>26.97±0.11<sub>a,1</sub></td><td>30.46±0.05<sub>a,1</sub></td><td>33.44±0.49<sub>a,1</sub></td><td>36.97±0.02<sub>a,1</sub></td><td>38.28±0.34<sub>a,1</sub></td><td>41.95±0.54<sub>a,1</sub></td></tr><tr><td>combi-low</td><td>0.00±0.00<sub>a,1</sub></td><td>3.06±0.07<sub>a,1</sub></td><td>14.64±0.04<sub>a,1</sub></td><td>25.75±0.03<sub>a,1</sub></td><td>30.60±0.08<sub>a,1</sub></td><td>34.88±0.40<sub>a,1</sub></td><td>37.37±0.12<sub>a,2</sub></td><td>37.91±0.003<sub>a,2</sub></td><td>41.78±0.03<sub>a,2</sub></td></tr><tr><td rowspan="6">Aw<br>(-)</td><td>pork-high</td><td>0.963±0.001<sub>a,1</sub></td><td>0.960±0.001<sub>a</sub></td><td>0.948±0.001<sub>a,1</sub></td><td>0.933±0.002<sub>a,1</sub></td><td>0.916±0.002<sub>b,1</sub></td><td>0.890±0.002<sub>a,1</sub></td><td>0.882±0.002<sub>a,1</sub></td><td>0.874±0.001<sub>bc</sub></td><td>0.861±0.004<sub>a,2</sub></td></tr><tr><td>pork-low</td><td>0.961±0.001<sub>a,1</sub></td><td>0.962±0.001<sub>ab</sub></td><td>0.946±0.001<sub>a,1</sub></td><td>0.928±0.003<sub>a,1</sub></td><td>0.906±0.003<sub>a,1</sub></td><td>0.884±0.004<sub>a,1</sub></td><td>0.876±0.004<sub>a,12</sub></td><td>0.878±0.001<sub>c</sub></td><td>0.851±0.001<sub>a,2</sub></td></tr><tr><td>horse-high</td><td>0.965±0.002<sub>a,1</sub></td><td>0.965±0.001<sub>c</sub></td><td>0.952±0.001<sub>a,2</sub></td><td>0.935±0.001<sub>b,1</sub></td><td>0.914±0.001<sub>b,1</sub></td><td>0.890±0.001<sub>b,1</sub></td><td>0.883±0.001<sub>b,1</sub></td><td>0.874±0.001<sub>bc</sub></td><td>0.845±0.004<sub>b,1</sub></td></tr><tr><td>horse-low</td><td>0.962±0.001<sub>a,1</sub></td><td>0.964±0.0002<sub>bc</sub></td><td>0.950±0.0004<sub>a,2</sub></td><td>0.931±0.001<sub>a,1</sub></td><td>0.902±0.001<sub>a,1</sub></td><td>0.880±0.002<sub>a,1</sub></td><td>0.867±0.001<sub>a,1</sub></td><td>0.859±0.001<sub>a</sub></td><td>0.838±0.005<sub>a,1</sub></td></tr><tr><td>combi-high</td><td>0.965±0.001<sub>a,1</sub></td><td>0.965±0.0002<sub>c</sub></td><td>0.954±0.0003<sub>b,2</sub></td><td>0.936±0.001<sub>b,1</sub></td><td>0.916±0.001<sub>b,1</sub></td><td>0.903±0.002<sub>b,2</sub></td><td>0.888±0.002<sub>b,1</sub></td><td>0.876±0.002<sub>c</sub></td><td>0.850±0.002<sub>b12</sub></td></tr><tr><td>combi-low</td><td>0.964±0.001<sub>a,1</sub></td><td>0.960±0.001<sub>a</sub></td><td>0.950±0.001<sub>a,12</sub></td><td>0.930±0.002<sub>a,1</sub></td><td>0.909±0.001<sub>a,1</sub></td><td>0.890±0.001<sub>a,1</sub></td><td>0.878±0.001<sub>a,2</sub></td><td>0.869±0.001<sub>b</sub></td><td>0.842±0.002<sub>a12</sub></td></tr></tbody></table>

544 Data are expressed as means ± SE. If no interaction between meat source and pH condition, same letters indicate no significant differences (P < 0.05) between pH treatments 545 within sampling day and same numbers indicate no significant differences (P < 0.05) between meat sources within sampling day. If interaction, same letters indicate no 546 significant differences (P < 0.05) between all treatments within sampling day.
541 Table 2 pH (n = 6), aw (n = 6) and weight losses (n = 2) during the production of nitrite-free dry fermented sausages based on pork (pork), horsemeat (horse) and a 542 50/50 combination of both meat sources (combi). Different pH conditions are obtained by adding different concentrations of dextrose to the meat batter, 0.00% (high) 543 and 0.70% (low).

<table><thead><tr><th rowspan="2"></th><th rowspan="2">treatment</th><th colspan="9">production day</th></tr><tr><th>0</th><th>3</th><th>21</th><th>42</th><th>63</th><th>84</th><th>105</th><th>126</th><th>168</th></tr></thead><tbody><tr><td rowspan="6">pH<br>(-)</td><td>pork-high</td><td>5.36±0.01<sub>a,1</sub></td><td>4.96±0.01<sub>e</sub></td><td>5.22±0.02<sup>f</sup></td><td>5.41±0.01<sub>e</sub></td><td>5.68±0.04<sub>d</sub></td><td>5.60±0.02<sub>d</sub></td><td>5.68±0.06<sub>e</sub></td><td>5.78±0.02<sub>e</sub></td><td>6.03±0.10<sub>c</sub></td></tr><tr><td>pork-low</td><td>5.38±0.01<sub>a,1</sub></td><td>4.28±0.02<sub>b</sub></td><td>4.51±0.01<sub>d</sub></td><td>4.59±0.01<sub>c</sub></td><td>4.82±0.05<sub>b</sub></td><td>4.82±0.06<sub>b</sub></td><td>4.89±0.03<sub>b</sub></td><td>5.13±0.02<sub>c</sub></td><td>5.52±0.01<sub>b</sub></td></tr><tr><td>horse-high</td><td>5.40±0.01<sub>a,1</sub></td><td>4.45±0.01<sub>c</sub></td><td>4.43±0.02<sub>c</sub></td><td>4.55±0.02<sub>c</sub></td><td>4.76±0.02<sub>b</sub></td><td>4.88±0.04<sub>b</sub></td><td>5.06±0.03<sub>c</sub></td><td>5.17±0.04<sub>c</sub></td><td>5.47±0.02<sub>b</sub></td></tr><tr><td>horse-low</td><td>5.38±0.02<sub>a,1</sub></td><td>4.12±0.04<sub>a</sub></td><td>4.11±0.01<sub>a</sub></td><td>4.21±0.01<sub>a</sub></td><td>4.28±0.03<sub>a</sub></td><td>4.47±0.03<sub>a</sub></td><td>4.62±0.03<sub>a</sub></td><td>4.72±0.02<sub>a</sub></td><td>5.17±0.07<sub>a</sub></td></tr><tr><td>combi-high</td><td>5.39±0.02<sub>a,1</sub></td><td>4.61±0.01<sub>d</sub></td><td>4.78±0.02<sub>e</sub></td><td>4.99±0.01<sub>d</sub></td><td>5.30±0.08<sub>c</sub></td><td>5.31±0.03<sub>c</sub></td><td>5.45±0.03<sub>d</sub></td><td>5.54±0.03<sub>d</sub></td><td>5.83±0.04<sub>c</sub></td></tr><tr><td>combi-low</td><td>5.34±0.01<sub>a,1</sub></td><td>4.19±0.01<sub>a</sub></td><td>4.26±0.01<sub>b</sub></td><td>4.28±0.01<sub>b</sub></td><td>4.39±0.02<sub>a</sub></td><td>4.76±0.06<sub>b</sub></td><td>4.70±0.02<sub>a</sub></td><td>4.89±0.02<sub>b</sub></td><td>5.12±0.01<sub>a</sub></td></tr><tr><td rowspan="6">weight<br>loss<br>(%)</td><td>pork-high</td><td>0.00±0.00<sub>a,1</sub></td><td>4.02±0.19<sub>a,1</sub></td><td>15.37±0.87<sub>a,1</sub></td><td>24.87±1.37<sub>a,1</sub></td><td>29.82±1.65<sub>a,1</sub></td><td>33.33±2.17<sub>a,1</sub></td><td>36.27±2.09<sub>a,1</sub></td><td>36.76±2.11<sub>a,1</sub></td><td>40.14±2.11<sub>a,1</sub></td></tr><tr><td>pork-low</td><td>0.00±0.00<sub>a,1</sub></td><td>3.86±0.52<sub>a,1</sub></td><td>15.22±2.52<sub>a,1</sub></td><td>25.48±0.89<sub>a,1</sub></td><td>31.34±2.01<sub>a,1</sub></td><td>33.20±0.47<sub>a,1</sub></td><td>35.46±0.21<sub>a,1</sub></td><td>35.41±0.11<sub>a,1</sub></td><td>39.05±0.26<sub>a,1</sub></td></tr><tr><td>horse-high</td><td>0.00±0.00<sub>a,1</sub></td><td>3.00±0.22<sub>a,1</sub></td><td>15.37±1.18<sub>a,1</sub></td><td>27.58±0.43<sub>a,1</sub></td><td>32.43±0.56<sub>a,1</sub></td><td>35.65±0.36<sub>a,1</sub></td><td>39.02±0.26<sub>a,1</sub></td><td>39.23±0.55<sub>a,1</sub></td><td>43.28±0.84<sub>a,1</sub></td></tr><tr><td>horse-low</td><td>0.00±0.00<sub>a,1</sub></td><td>2.65±0.24<sub>a,1</sub></td><td>13.98±0.37<sub>a,1</sub></td><td>26.26±1.05<sub>a,1</sub></td><td>32.18±0.31<sub>a,1</sub></td><td>36.11±0.43<sub>a,1</sub></td><td>39.05±0.17<sub>a,3</sub></td><td>39.96±0.40<sub>a,3</sub></td><td>43.64±0.10<sub>a,3</sub></td></tr><tr><td>combi-high</td><td>0.00±0.00<sub>a,1</sub></td><td>3.14±0.10<sub>a,1</sub></td><td>15.53±0.51<sub>a,1</sub></td><td>26.97±0.11<sub>a,1</sub></td><td>30.46±0.05<sub>a,1</sub></td><td>33.44±0.49<sub>a,1</sub></td><td>36.97±0.02<sub>a,1</sub></td><td>38.28±0.34<sub>a,1</sub></td><td>41.95±0.54<sub>a,1</sub></td></tr><tr><td>combi-low</td><td>0.00±0.00<sub>a,1</sub></td><td>3.06±0.07<sub>a,1</sub></td><td>14.64±0.04<sub>a,1</sub></td><td>25.75±0.03<sub>a,1</sub></td><td>30.60±0.08<sub>a,1</sub></td><td>34.88±0.40<sub>a,1</sub></td><td>37.37±0.12<sub>a,2</sub></td><td>37.91±0.003<sub>a,2</sub></td><td>41.78±0.03<sub>a,2</sub></td></tr><tr><td rowspan="6">Aw</td><td>pork-high</td><td>0.963±0.001<sub>a,1</sub></td><td>0.960±0.001<sub>a</sub></td><td>0.948±0.001<sub>a,1</sub></td><td>0.933±0.002<sub>a,1</sub></td><td>0.916±0.002<sub>b,1</sub></td><td>0.890±0.002<sub>a,1</sub></td><td>0.882±0.002<sub>a,1</sub></td><td>0.874±0.001<sub>bc</sub></td><td>0.861±0.004<sub>a,2</sub></td></tr><tr><td>pork-low</td><td>0.961±0.001<sub>a,1</sub></td><td>0.962±0.001<sub>ab</sub></td><td>0.946±0.001<sub>a,1</sub></td><td>0.928±0.003<sub>a,1</sub></td><td>0.906±0.003<sub>a,1</sub></td><td>0.884±0.004<sub>a,1</sub></td><td>0.876±0.004<sub>a,12</sub></td><td>0.878±0.001<sub>c</sub></td><td>0.851±0.001<sub>a,2</sub></td></tr><tr><td>horse-high</td><td>0.965±0.002<sub>a,1</sub></td><td>0.965±0.001<sub>c</sub></td><td>0.952±0.001<sub>a,2</sub></td><td>0.935±0.001<sub>b,1</sub></td><td>0.914±0.001<sub>b,1</sub></td><td>0.890±0.001<sub>b,1</sub></td><td>0.883±0.001<sub>b,1</sub></td><td>0.874±0.001<sub>bc</sub></td><td>0.845±0.004<sub>b,1</sub></td></tr><tr><td>horse-low</td><td>0.962±0.001<sub>a,1</sub></td><td>0.964±0.0002<sub>bc</sub></td><td>0.950±0.0004<sub>a,2</sub></td><td>0.931±0.001<sub>a,1</sub></td><td>0.902±0.001<sub>a,1</sub></td><td>0.880±0.002<sub>a,1</sub></td><td>0.867±0.001<sub>a,1</sub></td><td>0.859±0.001<sub>a</sub></td><td>0.838±0.005<sub>a,1</sub></td></tr><tr><td>combi-high</td><td>0.965±0.001<sub>a,1</sub></td><td>0.965±0.0002<sub>c</sub></td><td>0.954±0.0003<sub>b,2</sub></td><td>0.936±0.001<sub>b,1</sub></td><td>0.916±0.001<sub>b,1</sub></td><td>0.903±0.002<sub>b,2</sub></td><td>0.888±0.002<sub>b,1</sub></td><td>0.876±0.002<sub>c</sub></td><td>0.850±0.002<sub>b12</sub></td></tr><tr><td>combi-low</td><td>0.964±0.001<sub>a,1</sub></td><td>0.960±0.001<sub>a</sub></td><td>0.950±0.001<sub>a,12</sub></td><td>0.930±0.002<sub>a,1</sub></td><td>0.909±0.001<sub>a,1</sub></td><td>0.890±0.001<sub>a,1</sub></td><td>0.878±0.001<sub>a,2</sub></td><td>0.869±0.001<sub>b</sub></td><td>0.842±0.002<sub>a12</sub></td></tr></tbody></table>

544 Data are expressed as means ± SE. If no interaction between meat source and pH condition, same letters indicate no significant differences (P < 0.05) between pH treatments 545 within sampling day and same numbers indicate no significant differences (P < 0.05) between meat sources within sampling day. If interaction, same letters indicate no 546 significant differences (P < 0.05) between all treatments within sampling day.
<table><caption>Table 3 Zn(II)PPIX, PPIX and total heme evolution (n = 2) during the production of nitrite-free dry fermented sausages, based on pork (pork), horsemeat (horse) and a 50/50 combination of both meat sources (combi) each at high and low pH conditions (addition of 0.00% and 0.70% dextrose, respectively)</caption><thead><tr><th rowspan="2"></th><th rowspan="2">treatment</th><th colspan="4">production day</th></tr><tr><th>0</th><th>21</th><th>63</th><th>168</th></tr></thead><tbody><tr><td rowspan="6">Zn(II)PPIX<br>(nmol/g DM)</td><td>pork-high</td><td>6.96±0.01<sub>a,1</sub></td><td><u>14.88±1.49<sub>a,2</sub></u></td><td><u>14.23±0.57<sub>a,2</sub></u></td><td><u>11.63±0.91<sub>a,12</sub></u></td></tr><tr><td>pork-low</td><td>6.55±0.14<sub>a,1</sub></td><td><u>8.56±0.08<sub>a,1</sub></u></td><td><u>8.20±0.19<sub>a,1</sub></u></td><td><u>6.78±0.01<sub>a,1</sub></u></td></tr><tr><td>horse-high</td><td>8.33±0.24<sub>a,1</sub></td><td><u>39.38±4.17<sub>b,2</sub></u></td><td><u>42.31±2.09<sub>b,2</sub></u></td><td><u>59.51±2.02<sub>b,3</sub></u></td></tr><tr><td>horse-low</td><td>8.74±0.13<sub>a,1</sub></td><td><u>20.92±0.23<sub>b,2</sub></u></td><td><u>23.49±0.98<sub>c,23</sub></u></td><td><u>26.15±2.34<sub>c,3</sub></u></td></tr><tr><td>combi-high</td><td>7.43±0.11<sub>a,1</sub></td><td><u>38.16±1.13<sub>b,2</sub></u></td><td><u>49.89±0.27<sub>c,3</sub></u></td><td><u>61.20±4.55<sub>b,4</sub></u></td></tr><tr><td>combi-low</td><td>7.48±0.03<sub>a,1</sub></td><td><u>19.00±0.93<sub>b,2</sub></u></td><td><u>17.29±1.19<sub>b,2</sub></u></td><td><u>16.93±0.33<sub>b,2</sub></u></td></tr><tr><td rowspan="6">PPIX<br>(nmol/g DM)</td><td>pork-high</td><td>3.49±0.004<sub>a,1</sub></td><td><u>8.57±0.88<sub>a,2</sub></u></td><td><u>12.49±0.86<sub>a,3</sub></u></td><td><u>6.21±2.73<sub>a,12</sub></u></td></tr><tr><td>pork-low</td><td>3.27±0.07<sub>a,1</sub></td><td><u>3.50±0.001<sub>a,1</sub></u></td><td><u>3.21±0.003<sub>a,1</sub></u></td><td><u>3.10±0.06<sub>a,1</sub></u></td></tr><tr><td>horse-high</td><td>3.68±0.04<sub>a,1</sub></td><td><u>10.29±0.70<sub>a,2</sub></u></td><td><u>10.73±0.48<sub>a,2</sub></u></td><td><u>10.59±0.18<sub>b,2</sub></u></td></tr><tr><td>horse-low</td><td>3.68±0.07<sub>a,1</sub></td><td><u>4.88±0.04<sub>a,1</sub></u></td><td><u>4.85±0.16<sub>a,1</sub></u></td><td><u>4.91±0.50<sub>ab,1</sub></u></td></tr><tr><td>combi-high</td><td>3.41±0.004<sub>a,1</sub></td><td><u>9.21±0.69<sub>a,2</sub></u></td><td><u>10.93±2.89<sub>a,2</sub></u></td><td><u>5.75±0.88<sub>a,1</sub></u></td></tr><tr><td>combi-low</td><td>3.44±0.01<sub>a,1</sub></td><td><u>5.06±0.07<sub>a,1</sub></u></td><td><u>4.59±0.16<sub>a,1</sub></u></td><td><u>5.77±0.74<sub>b,1</sub></u></td></tr><tr><td rowspan="6">total heme<br>(nmol/g DM)</td><td>pork-high</td><td><u>216.54±24.79<sub>a,2</sub></u></td><td>165.09±0.57<sub>a,1</sub></td><td>143.23±8.84<sub>a,1</sub></td><td>156.43±5.05<sub>a,1</sub></td></tr><tr><td>pork-low</td><td><u>187.71±6.58<sub>a,2</sub></u></td><td>161.15±2.77<sub>a,12</sub></td><td>153.62±4.53<sub>a,12</sub></td><td>132.95±5.91<sub>a,1</sub></td></tr><tr><td>horse-high</td><td>585.54±33.85<sub>c,4</sub></td><td>497.62±11.93<sub>c,3</sub></td><td><u>359.55±14.13<sub>c,1</sub></u></td><td>449.48±11.27<sub>c,2</sub></td></tr><tr><td>horse-low</td><td>565.87±1.62<sub>c,4</sub></td><td>492.89±13.62<sub>c,3</sub></td><td><u>307.81±4.35<sub>c,1</sub></u></td><td>437.40±4.16<sub>c,2</sub></td></tr><tr><td>combi-high</td><td><u>339.61±38.00<sub>b,2</sub></u></td><td><u>259.91±12.46<sub>b,1</sub></u></td><td>246.81±24.75<sub>b,1</sub></td><td><u>214.29±7.86<sub>b,1</sub></u></td></tr><tr><td>combi-low</td><td><u>290.60±14.22<sub>b,1</sub></u></td><td><u>296.81±5.19<sub>b,1</sub></u></td><td>254.11±5.74<sub>b,1</sub></td><td><u>267.03±15.71<sub>b,1</sub></u></td></tr></tbody></table>

Data are expressed as means ± SE. Same letters indicate no significant differences (P < 0.05) between meat source within sampling day and pH condition. Same numbers indicate no significant differences (P < 0.05) between sampling days within meat source and pH condition. If underlined, significant differences (P < 0.05) between pH condition within sampling day and meat source were obtained.

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<table><caption>Table 3 Zn(II)PPIX, PPIX and total heme evolution (n = 2) during the production of nitrite-free dry fermented sausages, based on pork (pork), horsemeat (horse) and a 50/50 combination of both meat sources (combi) each at high and low pH conditions (addition of 0.00% and 0.70% dextrose, respectively)</caption><thead><tr><th rowspan="2"></th><th rowspan="2">treatment</th><th colspan="4">production day</th></tr><tr><th>0</th><th>21</th><th>63</th><th>168</th></tr></thead><tbody><tr><td rowspan="6">Zn(II)PPIX<br>(nmol/g DM)</td><td>pork-high</td><td>6.96±0.01<sub>a,1</sub></td><td><u>14.88±1.49<sub>a,2</sub></u></td><td><u>14.23±0.57<sub>a,2</sub></u></td><td><u>11.63±0.91<sub>a,12</sub></u></td></tr><tr><td>pork-low</td><td>6.55±0.14<sub>a,1</sub></td><td><u>8.56±0.08<sub>a,1</sub></u></td><td><u>8.20±0.19<sub>a,1</sub></u></td><td><u>6.78±0.01<sub>a,1</sub></u></td></tr><tr><td>horse-high</td><td>8.33±0.24<sub>a,1</sub></td><td><u>39.38±4.17<sub>b,2</sub></u></td><td><u>42.31±2.09<sub>b,2</sub></u></td><td><u>59.51±2.02<sub>b,3</sub></u></td></tr><tr><td>horse-low</td><td>8.74±0.13<sub>a,1</sub></td><td><u>20.92±0.23<sub>b,2</sub></u></td><td><u>23.49±0.98<sub>c,23</sub></u></td><td><u>26.15±2.34<sub>c,3</sub></u></td></tr><tr><td>combi-high</td><td>7.43±0.11<sub>a,1</sub></td><td><u>38.16±1.13<sub>b,2</sub></u></td><td><u>49.89±0.27<sub>c,3</sub></u></td><td><u>61.20±4.55<sub>b,4</sub></u></td></tr><tr><td>combi-low</td><td>7.48±0.03<sub>a,1</sub></td><td><u>19.00±0.93<sub>b,2</sub></u></td><td><u>17.29±1.19<sub>b,2</sub></u></td><td><u>16.93±0.33<sub>b,2</sub></u></td></tr><tr><td rowspan="6">PPIX<br>(nmol/g DM)</td><td>pork-high</td><td>3.49±0.004<sub>a,1</sub></td><td><u>8.57±0.88<sub>a,2</sub></u></td><td><u>12.49±0.86<sub>a,3</sub></u></td><td><u>6.21±2.73<sub>a,12</sub></u></td></tr><tr><td>pork-low</td><td>3.27±0.07<sub>a,1</sub></td><td><u>3.50±0.001<sub>a,1</sub></u></td><td><u>3.21±0.003<sub>a,1</sub></u></td><td><u>3.10±0.06<sub>a,1</sub></u></td></tr><tr><td>horse-high</td><td>3.68±0.04<sub>a,1</sub></td><td><u>10.29±0.70<sub>a,2</sub></u></td><td><u>10.73±0.48<sub>a,2</sub></u></td><td><u>10.59±0.18<sub>b,2</sub></u></td></tr><tr><td>horse-low</td><td>3.68±0.07<sub>a,1</sub></td><td><u>4.88±0.04<sub>a,1</sub></u></td><td><u>4.85±0.16<sub>a,1</sub></u></td><td><u>4.91±0.50<sub>ab,1</sub></u></td></tr><tr><td>combi-high</td><td>3.41±0.004<sub>a,1</sub></td><td><u>9.21±0.69<sub>a,2</sub></u></td><td><u>10.93±2.89<sub>a,2</sub></u></td><td><u>5.75±0.88<sub>a,1</sub></u></td></tr><tr><td>combi-low</td><td>3.44±0.01<sub>a,1</sub></td><td><u>5.06±0.07<sub>a,1</sub></u></td><td><u>4.59±0.16<sub>a,1</sub></u></td><td><u>5.77±0.74<sub>b,1</sub></u></td></tr><tr><td rowspan="6">total heme<br>(nmol/g DM)</td><td>pork-high</td><td><u>216.54±24.79<sub>a,2</sub></u></td><td>165.09±0.57<sub>a,1</sub></td><td>143.23±8.84<sub>a,1</sub></td><td>156.43±5.05<sub>a,1</sub></td></tr><tr><td>pork-low</td><td><u>187.71±6.58<sub>a,2</sub></u></td><td>161.15±2.77<sub>a,12</sub></td><td>153.62±4.53<sub>a,12</sub></td><td>132.95±5.91<sub>a,1</sub></td></tr><tr><td>horse-high</td><td>585.54±33.85<sub>c,4</sub></td><td>497.62±11.93<sub>c,3</sub></td><td><u>359.55±14.13<sub>c,1</sub></u></td><td>449.48±11.27<sub>c,2</sub></td></tr><tr><td>horse-low</td><td>565.87±1.62<sub>c,4</sub></td><td>492.89±13.62<sub>c,3</sub></td><td><u>307.81±4.35<sub>c,1</sub></u></td><td>437.40±4.16<sub>c,2</sub></td></tr><tr><td>combi-high</td><td><u>339.61±38.00<sub>b,2</sub></u></td><td><u>259.91±12.46<sub>b,1</sub></u></td><td>246.81±24.75<sub>b,1</sub></td><td><u>214.29±7.86<sub>b,1</sub></u></td></tr><tr><td>combi-low</td><td><u>290.60±14.22<sub>b,1</sub></u></td><td><u>296.81±5.19<sub>b,1</sub></u></td><td>254.11±5.74<sub>b,1</sub></td><td><u>267.03±15.71<sub>b,1</sub></u></td></tr></tbody></table>

Data are expressed as means ± SE. Same letters indicate no significant differences (P < 0.05) between meat source within sampling day and pH condition. Same numbers indicate no significant differences (P < 0.05) between sampling days within meat source and pH condition. If underlined, significant differences (P < 0.05) between pH condition within sampling day and meat source were obtained.

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<table><caption>Table 4 Changes in L*, a* and b* (n = 2) during the production of nitrite-free dry fermented sausages, based on pork (pork), horsemeat (horse) and a 50/50 combination of both meat sources (combi) each at high and a low pH conditions (addition of 0.00% and 0.70% dextrose, respectively)</caption><thead><tr><th rowspan="2"></th><th rowspan="2">treatment</th><th colspan="4">production day</th></tr><tr><th>0</th><th>21</th><th>63</th><th>168</th></tr></thead><tbody><tr><td rowspan="5">L*</td><td>pork-high</td><td>59.39±1.60<sub>c,2</sub></td><td><u>58.31±1.36<sub>abc,12</sub></u></td><td>58.62±1.73<sub>b,12</sub></td><td><u>54.80±1.91<sub>b,1</sub></u></td></tr><tr><td>pork-low</td><td>59.67±0.48<sub>c,12</sub></td><td><u>61.30±0.09<sub>c,2</sub></u></td><td>59.53±0.20<sub>c,12</sub></td><td><u>57.50±0.10<sub>b,1</sub></u></td></tr><tr><td>horse-high</td><td><u>47.13±0.23<sub>a,12</sub></u></td><td>50.74±0.74<sub>a,2</sub></td><td><u>50.32±0.77<sub>a,2</sub></u></td><td>42.35±1.35<sub>a,1</sub></td></tr><tr><td>horse-low</td><td><u>45.05±1.49<sub>a,1</sub></u></td><td>51.93±0.06<sub>a,2</sub></td><td><u>48.06±0.08<sub>a,1</sub></u></td><td>46.92±0.95<sub>a,1</sub></td></tr><tr><td>combi-high</td><td>53.33±0.60<sub>b,1</sub></td><td>55.66±0.40<sub>b,1</sub></td><td>55.45±0.35<sub>b,1</sub></td><td><u>50.30±0.20<sub>b,1</sub></u></td></tr><tr><td></td><td>combi-low</td><td>52.87±1.07<sub>b,2</sub></td><td>55.99±0.94<sub>b,2</sub></td><td>55.48±0.41<sub>b,2</sub></td><td><u>49.02±1.28<sub>a,1</sub></u></td></tr><tr><td rowspan="5">a*</td><td>pork-high</td><td>15.36±0.52<sub>a,3</sub></td><td>10.10±0.24<sub>b,1</sub></td><td><u>10.70±0.75<sub>c,1</sub></u></td><td><u>12.74±0.90<sub>b,2</sub></u></td></tr><tr><td>pork-low</td><td>15.52±0.51<sub>a,3</sub></td><td>9.58±0.27<sub>c,1</sub></td><td><u>9.28±0.58<sub>c,1</sub></u></td><td><u>11.64±0.27<sub>c,2</sub></u></td></tr><tr><td>horse-high</td><td><u>18.64±0.05<sub>a,3</sub></u></td><td>6.18±0.34<sub>a,2</sub></td><td>3.21±0.02<sub>a,1</sub></td><td>3.70±0.48<sub>a,1</sub></td></tr><tr><td>horse-low</td><td><u>20.15±0.45<sub>b,3</sub></u></td><td>6.09±0.43<sub>a,2</sub></td><td>2.82±0.16<sub>a,1</sub></td><td>3.51±0.25<sub>a,1</sub></td></tr><tr><td>combi-high</td><td>17.10±0.16<sub>a,3</sub></td><td><u>9.55±0.32<sub>b,2</sub></u></td><td><u>7.46±0.13<sub>b,1</sub></u></td><td><u>9.91±0.36<sub>b,2</sub></u></td></tr><tr><td></td><td>combi-low</td><td>16.27±0.26<sub>a,3</sub></td><td><u>8.39±0.003<sub>b,2</sub></u></td><td><u>4.96±0.32<sub>b,1</sub></u></td><td><u>5.99±0.28<sub>b,1</sub></u></td></tr><tr><td rowspan="5">b*</td><td>pork-high</td><td>20.77±0.16<sub>c,4</sub></td><td><u>10.73±0.07<sub>c,1</sub></u></td><td>12.33±0.42<sub>c,2</sub></td><td><u>13.16±0.45<sub>c,3</sub></u></td></tr><tr><td>pork-low</td><td>20.84±0.35<sub>b,4</sub></td><td><u>8.45±0.06<sub>c,1</sub></u></td><td>10.78±0.42<sub>c,2</sub></td><td><u>11.19±0.22<sub>c,3</sub></u></td></tr><tr><td>horse-high</td><td><u>20.12±0.51<sub>a,3</sub></u></td><td>6.95±0.45<sub>a,1</sub></td><td>8.72±0.03<sub>a,2</sub></td><td><u>7.32±0.06<sub>a,1</sub></u></td></tr><tr><td>horse-low</td><td><u>20.63±0.22<sub>a,3</sub></u></td><td>6.20±0.31<sub>a,1</sub></td><td>7.53±0.39<sub>a,2</sub></td><td><u>8.11±0.41<sub>a,2</sub></u></td></tr><tr><td>combi-high</td><td><u>20.65±0.40<sub>b,3</sub></u></td><td>7.82±0.26<sub>b,1</sub></td><td>10.51±0.30<sub>b,2</sub></td><td>9.48±0.07<sub>b,2</sub></td></tr><tr><td></td><td>combi-low</td><td><u>19.68±0.16<sub>a,4</sub></u></td><td>6.99±0.26<sub>b,1</sub></td><td>8.97±0.29<sub>b,3</sub></td><td>8.66±0.17<sub>b,2</sub></td></tr></tbody></table>

Data are expressed as means ± SE. Same letters indicate no significant differences (P < 0.05) between meat source within sampling day and pH condition. Same numbers indicate no significant differences (P < 0.05) between sampling days within meat source and pH condition. If underlined, significant differences (P < 0.05) between pH condition within sampling day and meat source were obtained.

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564

565
<table><caption>Table 4 Changes in *L*, *a*\* and *b*\* (n = 2) during the production of nitrite-free dry fermented sausages, based on pork (pork), horsemeat (horse) and a 50/50 combination of both meat sources (combi) each at high and a low pH conditions (addition of 0.00% and 0.70% dextrose, respectively)</caption><thead><tr><th rowspan="2"></th><th rowspan="2">treatment</th><th colspan="4">production day</th></tr><tr><th>0</th><th>21</th><th>63</th><th>168</th></tr></thead><tbody><tr><td rowspan="5">L*</td><td>pork-high</td><td>59.39±1.60<sub>c,2</sub></td><td><u>58.31±1.36<sub>abc,12</sub></u></td><td>58.62±1.73<sub>b,12</sub></td><td><u>54.80±1.91<sub>b,1</sub></u></td></tr><tr><td>pork-low</td><td>59.67±0.48<sub>c,12</sub></td><td><u>61.30±0.09<sub>c,2</sub></u></td><td>59.53±0.20<sub>c,12</sub></td><td><u>57.50±0.10<sub>b,1</sub></u></td></tr><tr><td>horse-high</td><td><u>47.13±0.23<sub>a,12</sub></u></td><td>50.74±0.74<sub>a,2</sub></td><td><u>50.32±0.77<sub>a,2</sub></u></td><td>42.35±1.35<sub>a,1</sub></td></tr><tr><td>horse-low</td><td><u>45.05±1.49<sub>a,1</sub></u></td><td>51.93±0.06<sub>a,2</sub></td><td><u>48.06±0.08<sub>a,1</sub></u></td><td>46.92±0.95<sub>a,1</sub></td></tr><tr><td>combi-high</td><td>53.33±0.60<sub>b,1</sub></td><td>55.66±0.40<sub>b,1</sub></td><td>55.45±0.35<sub>b,1</sub></td><td><u>50.30±0.20<sub>b,1</sub></u></td></tr><tr><td rowspan="5">a*</td><td>pork-high</td><td>15.36±0.52<sub>a,3</sub></td><td>10.10±0.24<sub>b,1</sub></td><td><u>10.70±0.75<sub>c,1</sub></u></td><td><u>12.74±0.90<sub>b,2</sub></u></td></tr><tr><td>pork-low</td><td>15.52±0.51<sub>a,3</sub></td><td>9.58±0.27<sub>c,1</sub></td><td><u>9.28±0.58<sub>c,1</sub></u></td><td><u>11.64±0.27<sub>c,2</sub></u></td></tr><tr><td>horse-high</td><td><u>18.64±0.05<sub>a,3</sub></u></td><td>6.18±0.34<sub>a,2</sub></td><td>3.21±0.02<sub>a,1</sub></td><td>3.70±0.48<sub>a,1</sub></td></tr><tr><td>horse-low</td><td><u>20.15±0.45<sub>b,3</sub></u></td><td>6.09±0.43<sub>a,2</sub></td><td>2.82±0.16<sub>a,1</sub></td><td>3.51±0.25<sub>a,1</sub></td></tr><tr><td>combi-high</td><td>17.10±0.16<sub>a,3</sub></td><td><u>9.55±0.32<sub>b,2</sub></u></td><td><u>7.46±0.13<sub>b,1</sub></u></td><td><u>9.91±0.36<sub>b,2</sub></u></td></tr><tr><td rowspan="5">b*</td><td>pork-high</td><td>20.77±0.16<sub>c,4</sub></td><td><u>10.73±0.07<sub>c,1</sub></u></td><td>12.33±0.42<sub>c,2</sub></td><td><u>13.16±0.45<sub>c,3</sub></u></td></tr><tr><td>pork-low</td><td>20.84±0.35<sub>b,4</sub></td><td><u>8.45±0.06<sub>c,1</sub></u></td><td>10.78±0.42<sub>c,2</sub></td><td><u>11.19±0.22<sub>c,3</sub></u></td></tr><tr><td>horse-high</td><td><u>20.12±0.51<sub>a,3</sub></u></td><td>6.95±0.45<sub>a,1</sub></td><td>8.72±0.03<sub>a,2</sub></td><td><u>7.32±0.06<sub>a,1</sub></u></td></tr><tr><td>horse-low</td><td><u>20.63±0.22<sub>a,3</sub></u></td><td>6.20±0.31<sub>a,1</sub></td><td>7.53±0.39<sub>a,2</sub></td><td><u>8.11±0.41<sub>a,2</sub></u></td></tr><tr><td>combi-high</td><td>20.65±0.40<sub>b,3</sub></td><td>7.82±0.26<sub>b,1</sub></td><td>10.51±0.30<sub>b,2</sub></td><td>9.48±0.07<sub>b,2</sub></td></tr><tr><td rowspan="5">b*</td><td>pork-low</td><td><u>19.68±0.16<sub>a,4</sub></u></td><td>6.99±0.26<sub>b,1</sub></td><td>8.97±0.29<sub>b,3</sub></td><td>8.66±0.17<sub>b,2</sub></td></tr></tbody></table>

Data are expressed as means ± SE. Same letters indicate no significant differences (P < 0.05) between meat source within sampling day and pH condition. Same numbers indicate no significant differences (P < 0.05) between sampling days within meat source and pH condition. If underlined, significant differences (P < 0.05) between pH condition within sampling day and meat source were obtained.

565
<table><caption>Figure 1 Evolution of Zn(II)PPIX and/or PPIX during the production of nitrite-free dry fermented sausages based on pork (pork), horsemeat (horse) and a 50/50 combination of both meat sources (combi) using a fast screening method. Different pH conditions are obtained by adding different concentrations of dextrose to the meat batter, 0.00% (high) and 0.70% (low).</caption><thead><tr><th rowspan="2">meat source</th><th rowspan="2">pH treatment</th><th colspan="10">production day</th></tr><tr><th>0</th><th>3</th><th>21</th><th>42</th><th>63</th><th>84</th><th>105</th><th>26</th><th>168</th></tr></thead><tbody><tr><td rowspan="2">pork</td><td>high</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>low</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">horse</td><td>high</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>low</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">combi</td><td>high</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>low</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th rowspan="2">meat source</th><th rowspan="2">pH treatment</th><th colspan="10">production day</th></tr><tr><th>0</th><th>3</th><th>21</th><th>42</th><th>63</th><th>84</th><th>105</th><th>26</th><th>168</th></tr></thead><tbody><tr><td rowspan="2">pork</td><td>high</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>low</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">horse</td><td>high</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>low</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">combi</td><td>high</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>low</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Figure 1 Evolution of Zn(II)PPIX and/or PPIX during the production of nitrite-free dry fermented sausages based on pork (pork), horsemeat (horse) and a 50/50 combination of both meat sources (combi) using a fast screening method. Different pH conditions are obtained by adding different concentrations of dextrose to the meat batter, 0.00% (high) and 0.70% (low).