drying/ripening which occurs at temperatures of 10–15°C. (Iacumin et al., 2009; Asefa et al., 2011; Rodriguez et al., 2012; Rodriguez et al., 2015; Zadravec et al., 2019), conditions never encountered during the dry-ageing of beef.

Mycotoxins can be produced, all-be-it at very low concentrations and very slowly by Penicillium spp. at temperatures as low as 0–1°C on fruits, corn and other crops (Table 4) but there are no reports of this occurring in meat.

<table><caption>**Table 4:** A summary of mycotoxin production by Penicillium spp. at chill temperatures</caption><thead><tr><td>Organism</td><td>Mycotoxin</td><td>Food</td><td>Mycotoxin production at temperature</td><td>Reference</td></tr></thead><tbody><tr><td>Penicillium expansum</td><td>Patulin</td><td>Fruits</td><td>0°C</td><td>Buchanan et al. (1974)</td></tr><tr><td>Penicillium spp.</td><td>Penicillic acid</td><td>Various agricultural commodities (corn, white rice, barley...)</td><td>1°C</td><td>Ciegler and Kurtzman (1970)</td></tr><tr><td>Penicillium martensii</td><td>Penicillic acid</td><td>Corn</td><td>≤ 1°C</td><td>Kurtzman and Ciegler (1970)</td></tr><tr><td>Penicillium expansum</td><td>Patulin</td><td>Apples</td><td>0°C</td><td>Sommer et al. (1974)</td></tr></tbody></table>

The Meat and Livestock Australia (MLA) review published in 2019 concluded that Penicillium spp. and Aspergillus spp. are not capable to produce mycotoxins on dry-aged meat at temperatures between –0.5°C and 3°C (MLA, 2019). This conclusion was based on 4 key references, 3 of which describe the production of mycotoxins in food (ICMSF, 1996; Hocking and Pitt, 2003; Pitt and Hocking, 2009) and conclude that of 3 genera of concern for human health, specifically, Penicillium, Aspergillus and Fusarium, only Penicillium and Aspergillus spp. are found on meat and neither are capable of producing mycotoxins at temperatures between –0.5°C and 3°C.

The fourth publication was the CSIRO report published in 2018 (Olivier, 2018), which stated that there was no evidence that moulds found on red meat are capable of producing mycotoxins at temperatures between –0.5°C and 3°C and a RH of 75–85%. In addition to the low temperature, mycotoxin production would be further inhibited by the low aw in dry-aged beef (Olivier, 2018).

PrimeSafe (Victoria, Australia) recommend an air temperature in the dry-ageing chamber of 1–3°C if the dry-ageing process takes less than 14 days and –0.5°C to 1.0°C if > 14 days but less than 35 days, with a RH of 75–85% and an air velocity of 0.2–0.5 m/s.

### 3.4.2. Spoilage bacteria

Fresh meat is highly perishable as the pH, $a_w$ and high nutrient content support bacterial growth (Woraprayote et al., 2016). Meat spoilage defects and associated bacteria are shown in Table 5.

<table><caption>**Table 5:** The main spoilage defects and causal bacteria (adapted from Nychas et al., 2007)</caption><thead><tr><td>Defect</td><td>Meat product</td><td>Causal bacteria</td></tr></thead><tbody><tr><td>Slime</td><td>Fresh meat</td><td>Pseudomonads, lactic acid bacteria (former Lactobacillus spp.), Enterococci, Weissella spp. and Brochothrix spp.</td></tr><tr><td>Hydrogen peroxide greening</td><td>Aerobically stored fresh meat</td><td>Weissella spp., Leuconostoc spp., Enterococci and Lactobacilli</td></tr><tr><td>Hydrogen sulfide greening</td><td>Vacuum-packed fresh meat</td><td>Shewanella spp. and Clostridium spp.</td></tr><tr><td>Sulfide odour</td><td>Vacuum-packed fresh meat</td><td>Clostridium spp. and Hafnia spp.</td></tr><tr><td>Cheesy or dairy odour</td><td>Vacuum-packed fresh meat</td><td>Brochothrix thermosphacta</td></tr><tr><td>Bone taint</td><td>Carcasses</td><td>Clostridium spp. and Enterococci</td></tr><tr><td>Souring</td><td>Vacuum-packed meats</td><td>Lactic acid bacteria, Enterococci, Micrococci, Bacilli and Clostridium spp.</td></tr></tbody></table>
drying/ripening which occurs at temperatures of 10–15°C. (Iacumin et al., 2009; Asefa et al., 2011; Rodriguez et al., 2012; Rodriguez et al., 2015; Zadravec et al., 2019), conditions never encountered during the dry-ageing of beef.

Mycotoxins can be produced, all-be-it at very low concentrations and very slowly by Penicillium spp. at temperatures as low as 0–1°C on fruits, corn and other crops (Table 4) but there are no reports of this occurring in meat.

<table><caption>**Table 4:** A summary of mycotoxin production by Penicillium spp. at chill temperatures</caption><thead><tr><td>Organism</td><td>Mycotoxin</td><td>Food</td><td>Mycotoxin production at temperature</td><td>Reference</td></tr></thead><tbody><tr><td>Penicillium expansum</td><td>Patulin</td><td>Fruits</td><td>0°C</td><td>Buchanan et al. (1974)</td></tr><tr><td>Penicillium spp.</td><td>Penicillic acid</td><td>Various agricultural commodities (corn, white rice, barley...)</td><td>1°C</td><td>Ciegler and Kurtzman (1970)</td></tr><tr><td>Penicillium martensii</td><td>Penicillic acid</td><td>Corn</td><td>≤ 1°C</td><td>Kurtzman and Ciegler (1970)</td></tr><tr><td>Penicillium expansum</td><td>Patulin</td><td>Apples</td><td>0°C</td><td>Sommer et al. (1974)</td></tr></tbody></table>

The Meat and Livestock Australia (MLA) review published in 2019 concluded that Penicillium spp. and Aspergillus spp. are not capable to produce mycotoxins on dry-aged meat at temperatures between –0.5°C and 3°C (MLA, 2019). This conclusion was based on 4 key references, 3 of which describe the production of mycotoxins in food (ICMSF, 1996; Hocking and Pitt, 2003; Pitt and Hocking, 2009) and conclude that of 3 genera of concern for human health, specifically, Penicillium, Aspergillus and Fusarium, only Penicillium and Aspergillus spp. are found on meat and neither are capable of producing mycotoxins at temperatures between –0.5°C and 3°C.

The fourth publication was the CSIRO report published in 2018 (Olivier, 2018), which stated that there was no evidence that moulds found on red meat are capable of producing mycotoxins at temperatures between –0.5°C and 3°C and a RH of 75–85%. In addition to the low temperature, mycotoxin production would be further inhibited by the low aw in dry-aged beef (Olivier, 2018).

PrimeSafe (Victoria, Australia) recommend an air temperature in the dry-ageing chamber of 1–3°C if the dry-ageing process takes less than 14 days and –0.5°C to 1.0°C if > 14 days but less than 35 days, with a RH of 75–85% and an air velocity of 0.2–0.5 m/s.

### 3.4.2. Spoilage bacteria

Fresh meat is highly perishable as the pH, $a_w$ and high nutrient content support bacterial growth (Woraprayote et al., 2016). Meat spoilage defects and associated bacteria are shown in Table 5.

<table><caption>**Table 5:** The main spoilage defects and causal bacteria (adapted from Nychas et al., 2007)</caption><thead><tr><td>Defect</td><td>Meat product</td><td>Causal bacteria</td></tr></thead><tbody><tr><td>Slime</td><td>Fresh meat</td><td>Pseudomonads, lactic acid bacteria (former Lactobacillus spp.), Enterococci, Weissella spp. and Brochothrix spp.</td></tr><tr><td>Hydrogen peroxide greening</td><td>Aerobically stored fresh meat</td><td>Weissella spp., Leuconostoc spp., Enterococci and Lactobacilli</td></tr><tr><td>Hydrogen sulfide greening</td><td>Vacuum-packed fresh meat</td><td>Shewanella spp. and Clostridium spp.</td></tr><tr><td>Sulfide odour</td><td>Vacuum-packed fresh meat</td><td>Clostridium spp. and Hafnia spp.</td></tr><tr><td>Cheesy or dairy odour</td><td>Vacuum-packed fresh meat</td><td>Brochothrix thermosphacta</td></tr><tr><td>Bone taint</td><td>Carcasses</td><td>Clostridium spp. and Enterococci</td></tr><tr><td>Souring</td><td>Vacuum-packed meats</td><td>Lactic acid bacteria, Enterococci, Micrococci, Bacilli and Clostridium spp.</td></tr></tbody></table>
The main spoilage bacteria of aerobically stored meat are *Pseudomonas* spp., Enterobacteriaceae (e.g. *Hafnia alvei*, *Serratia liquefaciens* and *Pantoea agglomerans*), LAB and *Brochothrix thermosphacta*, all of which are capable of growth at refrigeration temperatures in the presence of oxygen (Nychas et al., 2007; Wang et al., 2017). However, LAB and *B. thermosphacta* are not major contributors to the spoilage of meat when stored aerobically (Reid et al., 2017). Moreover, LAB may be supressed during the dry-ageing process (Mikami et al., 2021).

Under anaerobic conditions (e.g. in vacuum packs) *Enterobacteriaceae*, LAB and *B. thermosphacta* (all facultative anaerobes) will spoil meat as will psychrophilic and psychrotrophic *Clostridium* spp. (Ercolini et al., 2006; Hungaro et al., 2016). Yeasts and moulds may also cause meat spoilage, especially at the lower *a*<sub>w</sub> conditions found in dry-aged beef (Capouya et al., 2020). During the wet-ageing of beef, pork and lamb, when the primals are vacuum packaged and therefore under anaerobic conditions, LAB and to a lesser extent *Br. thermosphacta* become the dominant spoilage organisms (Russo et al., 2006; Stanborough et al., 2017). It is therefore expected that LAB counts would be higher on wet-aged as compared to dry-aged beef and this has been reported by several authors (Parrish et al., 1991; Li et al., 2013; Berger et al., 2018). In contrast, Campbell et al. (2001) obtained higher LAB counts on dry-ageing beef when compared to wet-ageing after 14 days.

Compared to other spoilage bacterial groups, psychrotrophic species within the former genus of *Lactobacillus* (currently named *Lactilactobacillus sakei*, *Lactilactobacillus curvatus*, *Levilactobacillus brevis* etc. Zheng et al., 2020) and other LAB (such as *Leuconostoc* spp., *Weissella* spp. and *Carnobacterium* spp.) are considered strong competitors under reduced oxygen availability and are frequently reported as the predominant spoilage microbiota under vacuum and modified atmosphere packaging (Doulgeraki et al., 2012). The psychrotrophic *Br. thermosphacta* can be an important proportion of the spoilage microbiota and occasionally become the dominant microorganism (Borch et al., 1996; Doulgeraki et al., 2012). Generally, the growth of *Br. thermosphacta* is likely to be similar to or faster than LAB in the presence of oxygen (e.g. in O<sub>2</sub> containing MAP or in VP that uses a film with low barrier properties) but is more sensitive to carbon dioxide (CO<sub>2</sub>) as compared to LAB (Devlieghere and Debevere, 2000; Mejlholm and Dalgaard, 2013). In vacuum packaged wet-aged meat, the composition of the gaseous phase changes during the storage. Residual oxygen decreases while carbon dioxide increases, which gradually becomes a selective factor favouring CO<sub>2</sub>-tolerant lactic acid bacteria (Borch et al., 1996). In meat stored under anaerobic chill conditions, it has also been reported that *Br. thermosphacta* on meat does not compete well against LAB (Sakala et al., 2002; Russo et al., 2006; Doulgeraki et al., 2012; McSharry et al., 2021).

TVCs are often used as indicators to monitor microbial shelf-life with the end of shelf-life reached when the counts reach a concentration of 5–7 log<sub>10</sub> CFU/g or cm<sup>2</sup>. Several studies have reported TVC, LAB as well as yeast and mould counts on dry-aged beef cuts after trimming. The TVC ranged from approximately 2–9 log<sub>10</sub> CFU/g or cm<sup>2</sup>, the LAB counts from approximately 5–7 log<sub>10</sub> CFU/g or cm<sup>2</sup> while yeasts and moulds ranged from not detected to 7 log<sub>10</sub> CFU/g or cm<sup>2</sup> (Table 6).

Gowda et al. (2022) provide TVC data for beef steaks made from dry-aged meat, which ranged from < 1 to 7.4 log<sub>10</sub> CFU/cm<sup>2</sup>. Significantly lower numbers of psychrotrophic TVC, Pseudomonads and *Br. thermosphacta* were found on steaks compared to the corresponding loins before trimming. However, they detected *Enterobacteriaceae* more frequently (40%) with higher maximum counts (7.4 log<sub>10</sub> CFU/cm<sup>2</sup>) on trimmed steaks as compared to the untrimmed loin crust (31%; 4.3 log<sub>10</sub> CFU/cm<sup>2</sup>), suggesting cross-contamination during trimming had occurred. In addition, *Listeria* spp. were detected on 10% (3/30) of steaks, but not on any of the 13 loin surfaces sampled. Herman et al. (2016) reported TVC, *Pseudomonas* spp., *Br. thermosphacta* and LAB counts on aerobically stored dry-aged beef of 4.7, 4.8, 3.0, and 3.9 log<sub>10</sub> CFU/cm<sup>2</sup> after 3 days at 4°C. The corresponding counts on vacuum packaged produce were 7.6, 5.5, 4.9 and 7.0 log<sub>10</sub> CFU/cm<sup>2</sup> after 10 days at the same temperature.

Even when correctly chilled, wet-aged beef, pork and lamb may be subject to blown pack spoilage (BPS), resulting in the production of gas (mainly carbon dioxide but also small amounts of hydrogen sulfide), causing severe pack distension/failure, offensive odours and a metallic sheen on the meat (Bolton, 2015). BPS is caused by psychrophilic *Clostridium estertheticum* and psychrotolerant *Clostridium gasigenes*. Other *Clostridium* spp. such as *Clostridium algidicarnis*, *Clostridium frigoris*, *Clostridium bowmanii*, *Clostridium frigidicarmis* and *Clostridium ruminantium* may also cause spoilage without gas production (Broda et al., 1999, Broda et al., 2000; Adam et al., 2010; Cavill et al., 2011).
The main spoilage bacteria of aerobically stored meat are *Pseudomonas* spp., Enterobacteriaceae (e.g. *Hafnia alvei*, *Serratia liquefaciens* and *Pantoea agglomerans*), LAB and *Brochothrix thermosphacta*, all of which are capable of growth at refrigeration temperatures in the presence of oxygen (Nychas et al., 2007; Wang et al., 2017). However, LAB and *B. thermosphacta* are not major contributors to the spoilage of meat when stored aerobically (Reid et al., 2017). Moreover, LAB may be supressed during the dry-ageing process (Mikami et al., 2021).

Under anaerobic conditions (e.g. in vacuum packs) *Enterobacteriaceae*, LAB and *B. thermosphacta* (all facultative anaerobes) will spoil meat as will psychrophilic and psychrotrophic *Clostridium* spp. (Ercolini et al., 2006; Hungaro et al., 2016). Yeasts and moulds may also cause meat spoilage, especially at the lower *a*<sub>w</sub> conditions found in dry-aged beef (Capouya et al., 2020). During the wet-ageing of beef, pork and lamb, when the primals are vacuum packaged and therefore under anaerobic conditions, LAB and to a lesser extent *Br. thermosphacta* become the dominant spoilage organisms (Russo et al., 2006; Stanborough et al., 2017). It is therefore expected that LAB counts would be higher on wet-aged as compared to dry-aged beef and this has been reported by several authors (Parrish et al., 1991; Li et al., 2013; Berger et al., 2018). In contrast, Campbell et al. (2001) obtained higher LAB counts on dry-ageing beef when compared to wet-ageing after 14 days.

Compared to other spoilage bacterial groups, psychrotrophic species within the former genus of *Lactobacillus* (currently named *Lactilactobacillus sakei*, *Lactilactobacillus curvatus*, *Levilactobacillus brevis* etc. Zheng et al., 2020) and other LAB (such as *Leuconostoc* spp., *Weissella* spp. and *Carnobacterium* spp.) are considered strong competitors under reduced oxygen availability and are frequently reported as the predominant spoilage microbiota under vacuum and modified atmosphere packaging (Doulgeraki et al., 2012). The psychrotrophic *Br. thermosphacta* can be an important proportion of the spoilage microbiota and occasionally become the dominant microorganism (Borch et al., 1996; Doulgeraki et al., 2012). Generally, the growth of *Br. thermosphacta* is likely to be similar to or faster than LAB in the presence of oxygen (e.g. in O<sub>2</sub> containing MAP or in VP that uses a film with low barrier properties) but is more sensitive to carbon dioxide (CO<sub>2</sub>) as compared to LAB (Devlieghere and Debevere, 2000; Mejlholm and Dalgaard, 2013). In vacuum packaged wet-aged meat, the composition of the gaseous phase changes during the storage. Residual oxygen decreases while carbon dioxide increases, which gradually becomes a selective factor favouring CO<sub>2</sub>-tolerant lactic acid bacteria (Borch et al., 1996). In meat stored under anaerobic chill conditions, it has also been reported that *Br. thermosphacta* on meat does not compete well against LAB (Sakala et al., 2002; Russo et al., 2006; Doulgeraki et al., 2012; McSharry et al., 2021).

TVCs are often used as indicators to monitor microbial shelf-life with the end of shelf-life reached when the counts reach a concentration of 5–7 log<sub>10</sub> CFU/g or cm<sup>2</sup>. Several studies have reported TVC, LAB as well as yeast and mould counts on dry-aged beef cuts after trimming. The TVC ranged from approximately 2–9 log<sub>10</sub> CFU/g or cm<sup>2</sup>, the LAB counts from approximately 5–7 log<sub>10</sub> CFU/g or cm<sup>2</sup> while yeasts and moulds ranged from not detected to 7 log<sub>10</sub> CFU/g or cm<sup>2</sup> (Table 6).

Gowda et al. (2022) provide TVC data for beef steaks made from dry-aged meat, which ranged from < 1 to 7.4 log<sub>10</sub> CFU/cm<sup>2</sup>. Significantly lower numbers of psychrotrophic TVC, Pseudomonads and *Br. thermosphacta* were found on steaks compared to the corresponding loins before trimming. However, they detected *Enterobacteriaceae* more frequently (40%) with higher maximum counts (7.4 log<sub>10</sub> CFU/cm<sup>2</sup>) on trimmed steaks as compared to the untrimmed loin crust (31%; 4.3 log<sub>10</sub> CFU/cm<sup>2</sup>), suggesting cross-contamination during trimming had occurred. In addition, *Listeria* spp. were detected on 10% (3/30) of steaks, but not on any of the 13 loin surfaces sampled. Herman et al. (2016) reported TVC, *Pseudomonas* spp., *Br. thermosphacta* and LAB counts on aerobically stored dry-aged beef of 4.7, 4.8, 3.0, and 3.9 log<sub>10</sub> CFU/cm<sup>2</sup> after 3 days at 4°C. The corresponding counts on vacuum packaged produce were 7.6, 5.5, 4.9 and 7.0 log<sub>10</sub> CFU/cm<sup>2</sup> after 10 days at the same temperature.

Even when correctly chilled, wet-aged beef, pork and lamb may be subject to blown pack spoilage (BPS), resulting in the production of gas (mainly carbon dioxide but also small amounts of hydrogen sulfide), causing severe pack distension/failure, offensive odours and a metallic sheen on the meat (Bolton, 2015). BPS is caused by psychrophilic *Clostridium estertheticum* and psychrotolerant *Clostridium gasigenes*. Other *Clostridium* spp. such as *Clostridium algidicarnis*, *Clostridium frigoris*, *Clostridium bowmanii*, *Clostridium frigidicarmis* and *Clostridium ruminantium* may also cause spoilage without gas production (Broda et al., 1999, Broda et al., 2000; Adam et al., 2010; Cavill et al., 2011).
<table><caption>**Table 6:** TVC, LAB, and yeasts and mould counts on dry-aged beef and wet-aged beef, pork and lamb</caption><thead><tr><th rowspan="2">Temperature (°C)<br>Time (days) in<br>brackets</th><th colspan="3">Microbial counts (log<sub>10</sub> cfu/g or cm<sup>2</sup>)</th><th rowspan="2">Reference</th></tr><tr><th>TVC</th><th>LAB</th><th>Yeasts (Y) and<br>moulds (M)</th></tr></thead><tbody><tr><td colspan="5"><strong>Dry-aged beef</strong></td></tr><tr><td>1-4 (3)</td><td>2-2.5</td><td>0</td><td>0</td><td>Ryu et al. (2018)</td></tr><tr><td>1-4 (25)</td><td>4-4.2</td><td>3.6-4.3</td><td>4.5-5.8</td><td>Ryu et al. (2018)</td></tr><tr><td>2 (21)</td><td>3.4</td><td>2.4</td><td>&lt; 2.0</td><td>da Silva et al. (2021)</td></tr><tr><td>2 (42)</td><td>2.8</td><td>1.4</td><td>-</td><td>Ribeiro et al. (2021b)</td></tr><tr><td>2.2 (21-28)</td><td>3.5</td><td>1.3</td><td>ND</td><td>DeGeer et al. (2009)</td></tr><tr><td>2.2 (21-28)</td><td>2.9</td><td>1.1</td><td>-0.66</td><td>DeGeer et al. (2009)</td></tr><tr><td>2.9 (35)</td><td>4.6</td><td>&lt; 3.0</td><td>-</td><td>Mikami et al. (2021)</td></tr><tr><td>2.9 (10)</td><td>5.2 (fat)<br>6.4 (lean)</td><td>0.98 (fat)<br>2.8 (lean)</td><td>Y: 2.5 (fat); 3.7 (lean)<br>M: 0.16 (fat); 0.01 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>2.9 (21)</td><td>6.9 (fat); 8.8<br>(lean)</td><td>2.3 (fat); 3.2<br>(lean)</td><td>Y: 4.2 (fat); 5.7 (lean)<br>M: 0.3 (fat); 0.7 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>4 (28)</td><td>4.3</td><td>1.8</td><td>Y: 2.6, M: 2.9</td><td>Lee H.J. et al. (2018)</td></tr><tr><td>4 (14)</td><td>5.8</td><td>3.8</td><td>Y: 5.6, M: 5.4</td><td>Gudjonsdottir et al.<br>(2015)</td></tr><tr><td>4 (21)</td><td>6.3</td><td>3.6</td><td>Y:5.9, M:5.8</td><td>Gudjonsdottir et al.<br>(2015)</td></tr><tr><td>2.2 (21-28)</td><td>5.5</td><td>1.3</td><td>0.4</td><td>DeGeer et al. (2009)</td></tr><tr><td>2.2 (21-28)</td><td>2.6</td><td>1.4</td><td>ND</td><td>DeGeer et al. (2009)</td></tr><tr><td>2.9 (10)</td><td>4.7 (fat); 5.3<br>(lean)</td><td>1.06 (fat); 2.01<br>(lean)</td><td>Y: 2.3 (fat); 2.7 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>2.9 (21)</td><td>6.2 (fat); 6.6<br>(lean)</td><td>2.6 (fat); 4.4<br>(lean)</td><td>Y: 3.9 (fat); 4.9 (lean)<br>M: 0.3 (fat); 0.11 (lean)</td><td>Li et al. (2014)</td></tr><tr><td colspan="5"><strong>Wet-aged beef</strong></td></tr><tr><td>2 (21)</td><td>6.3</td><td>6.5</td><td>&lt; 2.0</td><td>da Silva et al. (2021)</td></tr><tr><td>2(35)</td><td>2.4-6.7</td><td>1.8-6.2</td><td>-</td><td>Reid et al. (2017)</td></tr><tr><td>2 (42)</td><td>3.5</td><td>2.9</td><td>-</td><td>Ribeiro et al. (2021b)</td></tr><tr><td>2.9 (10)</td><td>4.44 (fat)<br>3.28 (lean)</td><td>2.61 (fat)<br>2.27 (lean)</td><td>Y: 1.11 (fat)<br>0.37 (lean)<br>M: 0.21 (fat)<br>0.24 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>2.9 (21)</td><td>5.76 (fat); 5.87<br>(lean)</td><td>4.13 (Fat); 5.34<br>(lean)</td><td>Y: 0.67 (fat); 0.51 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>2.9 (21)</td><td>2.4</td><td>1.7</td><td>Y: 0.01,<br>M: 0.01</td><td>Li et al. (2013)</td></tr><tr><td>3 (10)</td><td>-</td><td>-</td><td>-</td><td>Vásquez et al. (2009)</td></tr><tr><td>3 (15)</td><td>-</td><td>-</td><td>-</td><td>Vásquez et al. (2009)</td></tr><tr><td>3 (23)</td><td>-</td><td>-</td><td>-</td><td>Vásquez et al. (2009)</td></tr><tr><td>3 (31)</td><td>6.15</td><td>-</td><td>-</td><td>Vásquez et al. (2009)</td></tr><tr><td>(3 or 10)</td><td>-</td><td>1.54</td><td>-</td><td>Vieira et al. (2009)</td></tr><tr><td>4 (14)</td><td>2.65</td><td>5.26</td><td>Y: 3.52,<br>M: 2.74</td><td>Gudjonsdottir et al.<br>(2015)</td></tr></tbody></table>
<table><caption>**Table 6:** TVC, LAB, and yeasts and mould counts on dry-aged beef and wet-aged beef, pork and lamb</caption><thead><tr><th rowspan="2">Temperature (°C)<br>Time (days) in<br>brackets</th><th colspan="3">Microbial counts (log<sub>10</sub> cfu/g or cm<sup>2</sup>)</th><th rowspan="2">Reference</th></tr><tr><th>TVC</th><th>LAB</th><th>Yeasts (Y) and<br>moulds (M)</th></tr></thead><tbody><tr><td colspan="5"><strong>Dry-aged beef</strong></td></tr><tr><td>1-4 (3)</td><td>2-2.5</td><td>0</td><td>0</td><td>Ryu et al. (2018)</td></tr><tr><td>1-4 (25)</td><td>4-4.2</td><td>3.6-4.3</td><td>4.5-5.8</td><td>Ryu et al. (2018)</td></tr><tr><td>2 (21)</td><td>3.4</td><td>2.4</td><td>&lt; 2.0</td><td>da Silva et al. (2021)</td></tr><tr><td>2 (42)</td><td>2.8</td><td>1.4</td><td>-</td><td>Ribeiro et al. (2021b)</td></tr><tr><td>2.2 (21-28)</td><td>3.5</td><td>1.3</td><td>ND</td><td>DeGeer et al. (2009)</td></tr><tr><td>2.2 (21-28)</td><td>2.9</td><td>1.1</td><td>-0.66</td><td>DeGeer et al. (2009)</td></tr><tr><td>2.9 (35)</td><td>4.6</td><td>&lt; 3.0</td><td>-</td><td>Mikami et al. (2021)</td></tr><tr><td>2.9 (10)</td><td>5.2 (fat)<br>6.4 (lean)</td><td>0.98 (fat)<br>2.8 (lean)</td><td>Y: 2.5 (fat); 3.7 (lean)<br>M: 0.16 (fat); 0.01 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>2.9 (21)</td><td>6.9 (fat); 8.8<br>(lean)</td><td>2.3 (fat); 3.2<br>(lean)</td><td>Y: 4.2 (fat); 5.7 (lean)<br>M: 0.3 (fat); 0.7 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>4 (28)</td><td>4.3</td><td>1.8</td><td>Y: 2.6, M: 2.9</td><td>Lee H.J. et al. (2018)</td></tr><tr><td>4 (14)</td><td>5.8</td><td>3.8</td><td>Y: 5.6, M: 5.4</td><td>Gudjonsdottir et al.<br>(2015)</td></tr><tr><td>4 (21)</td><td>6.3</td><td>3.6</td><td>Y:5.9, M:5.8</td><td>Gudjonsdottir et al.<br>(2015)</td></tr><tr><td>2.2 (21-28)</td><td>5.5</td><td>1.3</td><td>0.4</td><td>DeGeer et al. (2009)</td></tr><tr><td>2.2 (21-28)</td><td>2.6</td><td>1.4</td><td>ND</td><td>DeGeer et al. (2009)</td></tr><tr><td>2.9 (10)</td><td>4.7 (fat); 5.3<br>(lean)</td><td>1.06 (fat); 2.01<br>(lean)</td><td>Y: 2.3 (fat); 2.7 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>2.9 (21)</td><td>6.2 (fat); 6.6<br>(lean)</td><td>2.6 (fat); 4.4<br>(lean)</td><td>Y: 3.9 (fat); 4.9 (lean)<br>M: 0.3 (fat); 0.11 (lean)</td><td>Li et al. (2014)</td></tr><tr><td colspan="5"><strong>Wet-aged beef</strong></td></tr><tr><td>2 (21)</td><td>6.3</td><td>6.5</td><td>&lt; 2.0</td><td>da Silva et al. (2021)</td></tr><tr><td>2(35)</td><td>2.4-6.7</td><td>1.8-6.2</td><td>-</td><td>Reid et al. (2017)</td></tr><tr><td>2 (42)</td><td>3.5</td><td>2.9</td><td>-</td><td>Ribeiro et al. (2021b)</td></tr><tr><td>2.9 (10)</td><td>4.44 (fat)<br>3.28 (lean)</td><td>2.61 (fat)<br>2.27 (lean)</td><td>Y: 1.11 (fat)<br>0.37 (lean)<br>M: 0.21 (fat)<br>0.24 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>2.9 (21)</td><td>5.76 (fat); 5.87<br>(lean)</td><td>4.13 (Fat); 5.34<br>(lean)</td><td>Y: 0.67 (fat); 0.51 (lean)</td><td>Li et al. (2014)</td></tr><tr><td>2.9 (21)</td><td>2.4</td><td>1.7</td><td>Y: 0.01,<br>M: 0.01</td><td>Li et al. (2013)</td></tr><tr><td>3 (10)</td><td>-</td><td>-</td><td>-</td><td>Vásquez et al. (2009)</td></tr><tr><td>3 (15)</td><td>-</td><td>-</td><td>-</td><td>Vásquez et al. (2009)</td></tr><tr><td>3 (23)</td><td>-</td><td>-</td><td>-</td><td>Vásquez et al. (2009)</td></tr><tr><td>3 (31)</td><td>6.15</td><td>-</td><td>-</td><td>Vásquez et al. (2009)</td></tr><tr><td>(3 or 10)</td><td>-</td><td>1.54</td><td>-</td><td>Vieira et al. (2009)</td></tr><tr><td>4 (14)</td><td>2.65</td><td>5.26</td><td>Y: 3.52,<br>M: 2.74</td><td>Gudjonsdottir et al.<br>(2015)</td></tr></tbody></table>
<table><thead><tr><th rowspan="2">Temperature (°C)<br>Time (days) in<br>brackets</th><th colspan="3">Microbial counts (log<sub>10</sub> cfu/g or cm<sup>2</sup>)</th><th rowspan="2">Reference</th></tr><tr><th>TVC</th><th>LAB</th><th>Yeasts (Y) and<br>moulds (M)</th></tr></thead><tbody><tr><td>4 (21)</td><td>2.95</td><td>6.11</td><td>Y: 3.48,<br>M: 3.00</td><td>Gudjonsdottir et al.<br>(2015)</td></tr><tr><td>4°C (2–21)</td><td>5.5 ± 0.12</td><td>5.5 ± 0.12</td><td>–</td><td>Bhattacharjee et al.<br>(2011)</td></tr><tr><td colspan="5"><strong>Wet-aged pork</strong></td></tr><tr><td>2 (7)</td><td>1.1–1.2</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><td>2 (14)</td><td>1.8–2.7</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><td>2 (21)</td><td>2.5–4.5</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><td>2 (28)</td><td>3.3–6.4</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><td>2 (28) + 4.5 (1–3)</td><td>1–2.5</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><td colspan="5"><strong>Wet-aged lamb</strong></td></tr><tr><td>-1.5 ± 0.5 (21)</td><td>5.2</td><td>2.6</td><td>Y: 2.4, M: &lt; 1</td><td>Zhang et al. (2021)</td></tr><tr><td>2 (14)</td><td>1.2</td><td>–</td><td>–</td><td>McKenna et al. (2003)</td></tr><tr><td>2 (20)</td><td>3.5</td><td>–</td><td>–</td><td>McKenna et al. (2003)</td></tr><tr><td>5 ± 2 (5)</td><td>2.42</td><td>–</td><td>–</td><td>Constantino et al.<br>(2012)</td></tr><tr><td>5 ± 2 (9)</td><td>3.01</td><td>–</td><td>–</td><td>Constantino et al.<br>(2012)</td></tr></tbody></table>

### 3.4.3. Bacterial pathogens on meat

Beef, pork and lamb carcasses may be contaminated with pathogens from the hide/fleece/skin and digestive tracts/faeces of the animals and from the slaughter and processing environment. These include STEC, *Salmonella* spp., *Campylobacter* spp., enteropathogenic *Yersinia* spp., *L. monocytogenes*, *Clostridium* spp. and *Staphylococcus aureus*. Of these, *L. monocytogenes*, *Yersinia* spp. and non-proteolytic *C. botulinum* are psychrotrophic pathogens and may grow at the chilled conditions encountered during the ageing of meat.

#### 3.4.3.1. Shiga toxin-producing *E. coli*

Phillips et al. (Phillips et al., 2012) reported that 0.1% of beef primals for dry-ageing were contaminated with STEC. These bacteria were not detected on dry-aged beef in a study by Ribeiro et al. (2021b) but were present on wet-aged products. Furthermore, Ryu et al. (2018) failed to detect *Escherichia coli* O157:H7 during the 3rd, 25th, 40th, 50th, and 60th day of dry-ageing beef originated from eight carcasses. Tittor et al. (2011) examined the efficacy of dry and wet-ageing of beef on *E. coli* O157:H7 on lean and fat beef tissues. After inoculation of the beef samples to approximately 6.0 log₁₀ CFU/cm², one set was spray chilled with water at 10°C for 15 min and then for 1 min every 17 min for 17 h before vacuum packaging and storage for 28 days. Dry-ageing was performed at 3°C, RH 80% with an air velocity of 0.25 m/s for a similar time. The final *E. coli* O157:H7 count on the dry and wet-aged beef was 1.0 and 3.7 log₁₀ CFU/cm², respectively. The authors concluded that dry and wet-ageing could be considered as pathogen control points in the beef chain. Ingham et al. (2010) observed a mean decrease of 0.8 log₁₀ of *E. coli* O157:H7 after 6 days of dry-ageing at 4°C and 92% RH. Van Damme et al. (2022) observed a decrease of *E. coli* O157:H7 of more than 3 log₁₀ after 42 days of dry-ageing at 2–6°C and 75–85% RH under all conditions, although for certain replicates this 3-log₁₀ reduction was already observed as early as 14 days.

#### 3.4.3.2. *Salmonella* spp.

Ribeiro et al. (2021b) investigated differences in microbiological safety and quality between beef (*Longissimus dorsi*) that was wet-aged and dry-aged for 30 days and reported that *Salmonella* spp. was not isolated from either dry or wet-aged beef samples. Moreover, Ryu et al. (2018) failed to detect *Salmonella* serovars during the 3rd, 25th, 40th, 50th, and 60th day of dry-ageing beef originated from eight carcasses. Knudsen et al. (2011) investigated the survival of different *Salmonella* strains on beef
<table><thead><tr><th rowspan="2">Temperature (°C)<br>Time (days) in<br>brackets</th><th colspan="3">Microbial counts (log<sub>10</sub> cfu/g or cm<sup>2</sup>)</th><th rowspan="2">Reference</th></tr><tr><th>TVC</th><th>LAB</th><th>Yeasts (Y) and<br>moulds (M)</th></tr></thead><tbody><tr><td>4 (21)</td><td>2.95</td><td>6.11</td><td>Y: 3.48,<br>M: 3.00</td><td>Gudjonsdottir et al.<br>(2015)</td></tr><tr><td>4°C (2–21)</td><td>5.5 ± 0.12</td><td>5.5 ± 0.12</td><td>–</td><td>Bhattacharjee et al.<br>(2011)</td></tr><tr><th colspan="5">Wet-aged pork</th></tr><tr><td>2 (7)</td><td>1.1–1.2</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><td>2 (14)</td><td>1.8–2.7</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><td>2 (21)</td><td>2.5–4.5</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><td>2 (28)</td><td>3.3–6.4</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><td>2 (28) + 4.5 (1–3)</td><td>1–2.5</td><td>–</td><td>–</td><td>Holmer et al. (2009)</td></tr><tr><th colspan="5">Wet-aged lamb</th></tr><tr><td>-1.5 ± 0.5 (21)</td><td>5.2</td><td>2.6</td><td>Y: 2.4, M: &lt; 1</td><td>Zhang et al. (2021)</td></tr><tr><td>2 (14)</td><td>1.2</td><td>–</td><td>–</td><td>McKenna et al. (2003)</td></tr><tr><td>2 (20)</td><td>3.5</td><td>–</td><td>–</td><td>McKenna et al. (2003)</td></tr><tr><td>5 ± 2 (5)</td><td>2.42</td><td>–</td><td>–</td><td>Constantino et al.<br>(2012)</td></tr><tr><td>5 ± 2 (9)</td><td>3.01</td><td>–</td><td>–</td><td>Constantino et al.<br>(2012)</td></tr></tbody></table>

### 3.4.3. Bacterial pathogens on meat

Beef, pork and lamb carcasses may be contaminated with pathogens from the hide/fleece/skin and digestive tracts/faeces of the animals and from the slaughter and processing environment. These include STEC, *Salmonella* spp., *Campylobacter* spp., enteropathogenic *Yersinia* spp., *L. monocytogenes*, *Clostridium* spp. and *Staphylococcus aureus*. Of these, *L. monocytogenes*, *Yersinia* spp. and non-proteolytic *C. botulinum* are psychrotrophic pathogens and may grow at the chilled conditions encountered during the ageing of meat.

#### 3.4.3.1. Shiga toxin-producing *E. coli*

Phillips et al. (Phillips et al., 2012) reported that 0.1% of beef primals for dry-ageing were contaminated with STEC. These bacteria were not detected on dry-aged beef in a study by Ribeiro et al. (2021b) but were present on wet-aged products. Furthermore, Ryu et al. (2018) failed to detect *Escherichia coli* O157:H7 during the 3rd, 25th, 40th, 50th, and 60th day of dry-ageing beef originated from eight carcasses. Tittor et al. (2011) examined the efficacy of dry and wet-ageing of beef on *E. coli* O157:H7 on lean and fat beef tissues. After inoculation of the beef samples to approximately 6.0 log₁₀ CFU/cm², one set was spray chilled with water at 10°C for 15 min and then for 1 min every 17 min for 17 h before vacuum packaging and storage for 28 days. Dry-ageing was performed at 3°C, RH 80% with an air velocity of 0.25 m/s for a similar time. The final *E. coli* O157:H7 count on the dry and wet-aged beef was 1.0 and 3.7 log₁₀ CFU/cm², respectively. The authors concluded that dry and wet-ageing could be considered as pathogen control points in the beef chain. Ingham et al. (2010) observed a mean decrease of 0.8 log₁₀ of *E. coli* O157:H7 after 6 days of dry-ageing at 4°C and 92% RH. Van Damme et al. (2022) observed a decrease of *E. coli* O157:H7 of more than 3 log₁₀ after 42 days of dry-ageing at 2–6°C and 75–85% RH under all conditions, although for certain replicates this 3-log₁₀ reduction was already observed as early as 14 days.

#### 3.4.3.2. *Salmonella* spp.

Ribeiro et al. (2021b) investigated differences in microbiological safety and quality between beef (*Longissimus dorsi*) that was wet-aged and dry-aged for 30 days and reported that *Salmonella* spp. was not isolated from either dry or wet-aged beef samples. Moreover, Ryu et al. (2018) failed to detect *Salmonella* serovars during the 3rd, 25th, 40th, 50th, and 60th day of dry-ageing beef originated from eight carcasses. Knudsen et al. (2011) investigated the survival of different *Salmonella* strains on beef
portions during 14 days of cooling. In a conventional refrigerator (at 1 ± 2°C and RH varying between 70% and 100%), reduction rates varied between -0.216 to -0.113 log₁₀/day, thus requiring 4.6 and 8.9 days to decrease by 1 log₁₀. The authors observed significant differences between strains, including a relatively lower survivability of S. Dublin strains as compared to S. Typhimurium DT104 and S. Enteritidis PT8 and PT4. In the Tittor et al. (2011) study reported above, *Salmonella* decreased from 6.0 log₁₀ CFU/cm² on the dry and wet-aged beef to 1.3 and 2.7 log₁₀ CFU/cm², respectively. Van Damme et al. (2022) also observed a reduction of at least 3 log₁₀ after 42 days of dry-ageing.

### 3.4.3.3. *L. monocytogenes*

Phillips et al. (2012) reported that 0.1% of beef primals for dry-ageing were contaminated with *L. monocytogenes*. However, Ribeiro et al. (2021b) found that these bacteria were not detected after dry-ageing but were present on wet-aged beef. These authors suggested that the dehydration of the beef during the dry-ageing process inhibits bacterial survival thus reducing the occurrence of microbiological hazards. Gowda et al. (2022) also failed to detect *L. monocytogenes* on dry-aged beef in 15 companies in Belgium. Furthermore, Hulankova et al. (2018b) failed to detect *Listeria* spp. in the samples originating from 27 beef carcasses before ageing and in samples that were dry-aged for up to 36 days. Ryu et al. (2018) similarly failed to detect *L. monocytogenes* on the 3rd, 25th, 40th, 50th, and 60th day of dry-ageing beef originated from eight carcasses. da Silva et al. (2019) artificially inoculated *L. innocua* ATCC 33090 on loin pieces (1.5 kg each; 20 × 13 × 8 cm) from two boneless loins (*M. longissimus lumborum*). The samples were dry-aged in refrigeration chambers at 2 ± 1°C and 8 ± 1°C with 75 ± 2% RH and an air velocity of 2 ± 0.5 m/s for up to 42 days. The highest reduction rate occurred in the first seven days of ageing. The time needed to reach the first decimal reduction of *L. innocua* was 4.7 days at 2°C and 4 days at 8°C. The time required to achieve a 3 log₁₀ reduction was estimated to be 69 and 33 days at 2 and 8°C, respectively. After trimming, 67% of the samples dry-aged at 2°C and 33% dry-aged at 8°C were *L. innocua* positive. The authors concluded that increasing the dry-ageing time and/or temperature would further decrease *L. innocua* counts. Van Damme et al. (2022) mostly observed a significant decrease of *L. monocytogenes* over time when ageing beef loins at 2–6°C and 75–85% RH. The observed reduction varied between 1 and > 3 log₁₀ after 42 days of ageing. Nevertheless, *L. monocytogenes* numbers significantly increased on lean meat on one of the loins (2°C, 85% RH, pH 6.21), resulting in a 1 log₁₀ higher number at day 42 than the initial inoculation level, suggesting that *L. monocytogenes* may grow under certain dry-ageing conditions.

### 3.4.3.4. Enteropathogenic Yersinia spp.

Pigs are the main reservoirs of pathogenic *Y. enterocolitica*, and pork and pork products are major sources for human infection (Petsios et al., 2016). Bolton et al. (2013) reported a 0.52% prevalence in Irish pigs, while higher rates of 37% and 93% have been found in Belgium (Van Damme et al., 2010) and Spain (Martínez et al., 2011), respectively. The prevalence on pork carcasses ranges from 0.3% (Gürtler et al., 2005) to 39.7% of carcass surfaces post-evisceration (Van Damme et al., 2015). *Y. enterocolitica* can grow at temperatures of between -2°C to 42°C with an optimum of 28–29°C and will multiply faster than *L. monocytogenes* at chill temperatures (Gill and Reichel, 1989). The minimum pH for growth is between 4.2 and 4.4 while the minimum $a_w$ is 0.96 (Stern et al., 1980).

### 3.4.3.5. Campylobacter spp.

The European Union One Health 2020 Zoonoses Report reported that of the 86 pork and 134 bovine samples tested, 8.5 and 5.1% were positive for *Campylobacter*, respectively (EFSA and ECDC, 2021). Whyte et al. (2004) detected these bacteria in 7/221 (3.2%) of beef, 10/197 (5.1%) of pork and 31/262 (11.8%) of lamb samples. Thépault et al. (2018) reported a 69% prevalence of thermophilic *Campylobacter* in French cattle at slaughterhouse level. The prevalence in adult cattle was 39%, while 99.4% of calves were contaminated. *C. jejuni* was the most prevalent species with prevalence of 37.3% and 98.5%, respectively. The prevalence of *C. coli* was 3% in adult cattle and 12.5% in calves. Recently, Espunyes et al. (2021) isolated *Campylobacter* from 23.3% of cattle and 7.7% of sheep, with *Campylobacter jejuni* being the most frequent species. However, *Campylobacter* do not grow outside the host and are sensitive to desiccation during the chilling of beef, pork and lamb carcasses and reduced prevalence and counts are obtained after this stage of processing (Narvaez-Bravo et al., 2017).

### 3.4.3.6. Clostridium spp.

Bovine, ovine and porcine faeces often contain *Clostridium* spp. (Russell et al., 2021). These authors used PCR methods to confirm that these were cluster 1 clostridia, which includes *C. botulinum*,
portions during 14 days of cooling. In a conventional refrigerator (at 1 ± 2°C and RH varying between 70% and 100%), reduction rates varied between -0.216 to -0.113 log₁₀/day, thus requiring 4.6 and 8.9 days to decrease by 1 log₁₀. The authors observed significant differences between strains, including a relatively lower survivability of S. Dublin strains as compared to S. Typhimurium DT104 and S. Enteritidis PT8 and PT4. In the Tittor et al. (2011) study reported above, *Salmonella* decreased from 6.0 log₁₀ CFU/cm² on the dry and wet-aged beef to 1.3 and 2.7 log₁₀ CFU/cm², respectively. Van Damme et al. (2022) also observed a reduction of at least 3 log₁₀ after 42 days of dry-ageing.

### 3.4.3.3. *L. monocytogenes*

Phillips et al. (2012) reported that 0.1% of beef primals for dry-ageing were contaminated with *L. monocytogenes*. However, Ribeiro et al. (2021b) found that these bacteria were not detected after dry-ageing but were present on wet-aged beef. These authors suggested that the dehydration of the beef during the dry-ageing process inhibits bacterial survival thus reducing the occurrence of microbiological hazards. Gowda et al. (2022) also failed to detect *L. monocytogenes* on dry-aged beef in 15 companies in Belgium. Furthermore, Hulankova et al. (2018b) failed to detect *Listeria* spp. in the samples originating from 27 beef carcasses before ageing and in samples that were dry-aged for up to 36 days. Ryu et al. (2018) similarly failed to detect *L. monocytogenes* on the 3rd, 25th, 40th, 50th, and 60th day of dry-ageing beef originated from eight carcasses. da Silva et al. (2019) artificially inoculated *L. innocua* ATCC 33090 on loin pieces (1.5 kg each; 20 × 13 × 8 cm) from two boneless loins (*M. longissimus lumborum*). The samples were dry-aged in refrigeration chambers at 2 ± 1°C and 8 ± 1°C with 75 ± 2% RH and an air velocity of 2 ± 0.5 m/s for up to 42 days. The highest reduction rate occurred in the first seven days of ageing. The time needed to reach the first decimal reduction of *L. innocua* was 4.7 days at 2°C and 4 days at 8°C. The time required to achieve a 3 log₁₀ reduction was estimated to be 69 and 33 days at 2 and 8°C, respectively. After trimming, 67% of the samples dry-aged at 2°C and 33% dry-aged at 8°C were *L. innocua* positive. The authors concluded that increasing the dry-ageing time and/or temperature would further decrease *L. innocua* counts. Van Damme et al. (2022) mostly observed a significant decrease of *L. monocytogenes* over time when ageing beef loins at 2–6°C and 75–85% RH. The observed reduction varied between 1 and > 3 log₁₀ after 42 days of ageing. Nevertheless, *L. monocytogenes* numbers significantly increased on lean meat on one of the loins (2°C, 85% RH, pH 6.21), resulting in a 1 log₁₀ higher number at day 42 than the initial inoculation level, suggesting that *L. monocytogenes* may grow under certain dry-ageing conditions.

### 3.4.3.4. Enteropathogenic Yersinia spp.

Pigs are the main reservoirs of pathogenic *Y. enterocolitica*, and pork and pork products are major sources for human infection (Petsios et al., 2016). Bolton et al. (2013) reported a 0.52% prevalence in Irish pigs, while higher rates of 37% and 93% have been found in Belgium (Van Damme et al., 2010) and Spain (Martínez et al., 2011), respectively. The prevalence on pork carcasses ranges from 0.3% (Gürtler et al., 2005) to 39.7% of carcass surfaces post-evisceration (Van Damme et al., 2015). *Y. enterocolitica* can grow at temperatures of between -2°C to 42°C with an optimum of 28–29°C and will multiply faster than *L. monocytogenes* at chill temperatures (Gill and Reichel, 1989). The minimum pH for growth is between 4.2 and 4.4 while the minimum $a_w$ is 0.96 (Stern et al., 1980).

### 3.4.3.5. Campylobacter spp.

The European Union One Health 2020 Zoonoses Report reported that of the 86 pork and 134 bovine samples tested, 8.5 and 5.1% were positive for *Campylobacter*, respectively (EFSA and ECDC, 2021). Whyte et al. (2004) detected these bacteria in 7/221 (3.2%) of beef, 10/197 (5.1%) of pork and 31/262 (11.8%) of lamb samples. Thépault et al. (2018) reported a 69% prevalence of thermophilic *Campylobacter* in French cattle at slaughterhouse level. The prevalence in adult cattle was 39%, while 99.4% of calves were contaminated. *C. jejuni* was the most prevalent species with prevalence of 37.3% and 98.5%, respectively. The prevalence of *C. coli* was 3% in adult cattle and 12.5% in calves. Recently, Espunyes et al. (2021) isolated *Campylobacter* from 23.3% of cattle and 7.7% of sheep, with *Campylobacter jejuni* being the most frequent species. However, *Campylobacter* do not grow outside the host and are sensitive to desiccation during the chilling of beef, pork and lamb carcasses and reduced prevalence and counts are obtained after this stage of processing (Narvaez-Bravo et al., 2017).

### 3.4.3.6. Clostridium spp.

Bovine, ovine and porcine faeces often contain *Clostridium* spp. (Russell et al., 2021). These authors used PCR methods to confirm that these were cluster 1 clostridia, which includes *C. botulinum*,
C. sporogenes, C. tetani, C. perfringens, as well as Clostridioides difficile, and spoilage organisms, C. estertheticum and C. gasigenes (Song et al., 2004). Previous research reported C. estertheticum and C. gasigenes in 17.9% and 25.4% of bovine faecal samples, respectively (Moschonas et al., 2009). A more recent study found C. difficile in 7% of bovine faeces as well as in 13% of ovine faeces (Marcos Lázaro et al., 2021). However, the same study failed to detect C. difficile in beef and lamb products at retail. As Clostridium spp. are strict anaerobes, growth and/or toxin production during the dry-ageing of beef is unexpected but could occur in vacuum packaged beef, pork and lamb during wet-ageing if chilled conditions were not strictly maintained. Of most concern is C. botulinum with types A, B, E and F associated with human botulism. These belong to two groups that differ mainly with respect to proteolysis. Type A strains are proteolytic, type E are non-proteolytic while B and F have a mixture. Proteolytic strains do not grow below 10°C, while non-proteolytic strains may grow at temperatures above approximately 3°C (Lynt et al., 1982; Carter and Peck, 2015; Brunt et al., 2020).

In their report on non-proteolytic C. botulinum and vacuum and modified atmosphere packaged foods, published in 2020, the Advisory Committee on the Microbiological Safety of Food (ACMSF, 2020) found that there was no evidence of outbreaks having been caused by VP/MAP fresh, chilled meats within the scope of the study: beef, pork and lamb. Indeed, none of the outbreaks associated with any commercial chilled food were caused by correctly stored products. There had been only eight studies on fresh, chilled meat and the experimental approaches used varied markedly, including inoculum size, sample size, methodology for assessing risk (growth or toxin formation), analytical methods and sensitivity of assay. The studies demonstrated variation in the time to toxin formation within and between meat species and it was not possible to draw general conclusions from the studies regarding growth and toxin production of C. botulinum in fresh, chilled meats. The ACMSF subgroup reviewed new evidence to assess the safety of increasing the shelf life of vacuum packaged fresh beef, lamb and pork from 10 days (as stated in Food Safety Agency guidance of 2017 updated on 2020, FSA, 2020a,b) to 13 days. A new series of challenge tests were undertaken with six products representative of the UK market, including meats with both short and long ageing periods and using a temperature profile of < 3°C for 1 day, followed by 5°C for 1 day, 2 h at 22°C (to simulate transport by consumers) and then at 8°C for the remaining period (to simulate domestic storage). According to the study (Peck et al., 2020), toxin was not detected in beef or lamb stored for up to 50 and 35 days, respectively; while in one of the two fresh chilled pork products, toxin was detected at 35 days but not after 25 days. These results provided some evidence about the potential production of non-proteolytic C. botulinum toxin in vacuum packaged fresh meat when stored at 8°C, though it did not occur within the organoleptic shelf-life.

### 3.4.3.7. Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacterium that is commonly found on the skin and nasal membranes of production animals, humans and other mammals (Pugazhendhi et al., 2020). It is an opportunistic pathogen, causing a range of illnesses including urinary tract infections, toxic shock syndrome, and food poisoning. However, the main food safety issue is Staphylococcal food poisoning, a food-borne intoxication caused by staphylococcal enterotoxins. The limits for staphylococcal enterotoxin production are temperatures of between 10°C and 48°C, a pH range of 4-9.6, and NaCl concentrations of 0-10% (Zeaki et al., 2019). Thus, as meat is aged at temperatures below 10°C, enterotoxins are not produced during this process.

Prevalence rates for S. aureus of 64%, 20% and 17% were reported in raw beef, sheep and lamb samples at retail with an overall prevalence of 21% (Şanlıbaba, 2022). Similar studies in the United States of America, Japan and Poland isolated this bacterium from 28%, 33% and 68%, respectively (Hiroi et al., 2012; Krupa et al., 2014; Carrel et al., 2017). Although information on growth on meat products is limited, Phillips et al. (2012) reported that 9% of beef primals for dry-ageing were contaminated with S. aureus and Yu et al. (2020) predicted these bacteria would grow on vacuum packaged beef achieving 6 log₁₀ CFU/g after 100 h at 25°C. In contrast, Ryu et al. (2018) failed to detect S. aureus on the 3rd, 25th, 40th, 50th and 60th day of dry-ageing beef, originated from eight carcasses.

### 3.4.4. Effect of dry and wet-ageing on bacteria

There are relatively few studies on the effect of dry and wet-ageing beef on the concentrations of specific pathogens and the data that is available is summarised in Table 7. Interestingly dry-ageing caused a decrease in E. coli O157:H7, Salmonella spp. and L. innocua (as a surrogate for
C. sporogenes, C. tetani, C. perfringens, as well as Clostridioides difficile, and spoilage organisms, C. estertheticum and C. gasigenes (Song et al., 2004). Previous research reported C. estertheticum and C. gasigenes in 17.9% and 25.4% of bovine faecal samples, respectively (Moschonas et al., 2009). A more recent study found C. difficile in 7% of bovine faeces as well as in 13% of ovine faeces (Marcos Lázaro et al., 2021). However, the same study failed to detect C. difficile in beef and lamb products at retail. As Clostridium spp. are strict anaerobes, growth and/or toxin production during the dry-ageing of beef is unexpected but could occur in vacuum packaged beef, pork and lamb during wet-ageing if chilled conditions were not strictly maintained. Of most concern is C. botulinum with types A, B, E and F associated with human botulism. These belong to two groups that differ mainly with respect to proteolysis. Type A strains are proteolytic, type E are non-proteolytic while B and F have a mixture. Proteolytic strains do not grow below 10°C, while non-proteolytic strains may grow at temperatures above approximately 3°C (Lynt et al., 1982; Carter and Peck, 2015; Brunt et al., 2020).

In their report on non-proteolytic C. botulinum and vacuum and modified atmosphere packaged foods, published in 2020, the Advisory Committee on the Microbiological Safety of Food (ACMSF, 2020) found that there was no evidence of outbreaks having been caused by VP/MAP fresh, chilled meats within the scope of the study: beef, pork and lamb. Indeed, none of the outbreaks associated with any commercial chilled food were caused by correctly stored products. There had been only eight studies on fresh, chilled meat and the experimental approaches used varied markedly, including inoculum size, sample size, methodology for assessing risk (growth or toxin formation), analytical methods and sensitivity of assay. The studies demonstrated variation in the time to toxin formation within and between meat species and it was not possible to draw general conclusions from the studies regarding growth and toxin production of C. botulinum in fresh, chilled meats. The ACMSF subgroup reviewed new evidence to assess the safety of increasing the shelf life of vacuum packaged fresh beef, lamb and pork from 10 days (as stated in Food Safety Agency guidance of 2017 updated on 2020, FSA, 2020a,b) to 13 days. A new series of challenge tests were undertaken with six products representative of the UK market, including meats with both short and long ageing periods and using a temperature profile of < 3°C for 1 day, followed by 5°C for 1 day, 2 h at 22°C (to simulate transport by consumers) and then at 8°C for the remaining period (to simulate domestic storage). According to the study (Peck et al., 2020), toxin was not detected in beef or lamb stored for up to 50 and 35 days, respectively; while in one of the two fresh chilled pork products, toxin was detected at 35 days but not after 25 days. These results provided some evidence about the potential production of non-proteolytic C. botulinum toxin in vacuum packaged fresh meat when stored at 8°C, though it did not occur within the organoleptic shelf-life.

### 3.4.3.7. Staphylococcus aureus

*Staphylococcus aureus* is a Gram-positive bacterium that is commonly found on the skin and nasal membranes of production animals, humans and other mammals (Pugazhendhi et al., 2020). It is an opportunistic pathogen, causing a range of illnesses including urinary tract infections, toxic shock syndrome, and food poisoning. However, the main food safety issue is Staphylococcal food poisoning, a food-borne intoxication caused by staphylococcal enterotoxins. The limits for staphylococcal enterotoxin production are temperatures of between 10°C and 48°C, a pH range of 4–9.6, and NaCl concentrations of 0–10% (Zeaki et al., 2019). Thus, as meat is aged at temperatures below 10°C, enterotoxins are not produced during this process.

Prevalence rates for S. aureus of 64%, 20% and 17% were reported in raw beef, sheep and lamb samples at retail with an overall prevalence of 21% (Şanlıbaba, 2022). Similar studies in the United States of America, Japan and Poland isolated this bacterium from 28%, 33% and 68%, respectively (Hiroi et al., 2012; Krupa et al., 2014; Carrel et al., 2017). Although information on growth on meat products is limited, Phillips et al. (2012) reported that 9% of beef primals for dry-ageing were contaminated with S. aureus and Yu et al. (2020) predicted these bacteria would grow on vacuum packaged beef achieving 6 log₁₀ CFU/g after 100 h at 25°C. In contrast, Ryu et al. (2018) failed to detect S. aureus on the 3rd, 25th, 40th, 50th and 60th day of dry-ageing beef, originated from eight carcasses.

### 3.4.4. Effect of dry and wet-ageing on bacteria

There are relatively few studies on the effect of dry and wet-ageing beef on the concentrations of specific pathogens and the data that is available is summarised in Table 7. Interestingly dry-ageing caused a decrease in E. coli O157:H7, Salmonella spp. and L. innocua (as a surrogate for
*L. monocytogenes*). Van Damme et al. (2022) reported a 1.0 log₁₀ increase or a 3.0 log₁₀ decrease in *L. monocytogenes* depending on the dry-ageing conditions and beef muscle used. While a decrease in bacterial pathogens during dry-ageing may be due to the lowering of the aw, this would not explain the decreases in *E. coli* O157 and *Salmonella* spp. on wet-aged beef reported by Tittor et al. (2011). It should be noted that these findings are specific to the conditions used during ageing and further investigation is required before a more comprehensive conclusion on the fate of specific pathogens during meat ageing can be drawn.

<table><caption>**Table 7:** The change in the concentrations of specific pathogenic bacteria under different conditions of dry and wet-ageing of beef</caption><thead><tr><th rowspan="2">Conditions</th><th rowspan="2">Pathogen</th><th colspan="2">Log<sub>10</sub></th><th rowspan="2">Reference</th></tr><tr><th>Increase</th><th>Decrease</th></tr></thead><tbody><tr><td colspan="5"><strong>Dry-ageing</strong></td></tr><tr><td>3°C, RH 80%, air velocity of 0.25 m/s for 28 days</td><td><i>E. coli</i> O157:H7</td><td>–</td><td>4.8</td><td>Tittor et al. (2011)</td></tr><tr><td>4°C, RH 92% for 6 days</td><td><i>E. coli</i> O157:H7</td><td>–</td><td>0.8</td><td>Ingham et al. (2010)</td></tr><tr><td>2–6°C, RH 75–85% for 42 days</td><td><i>E. coli</i> O157:H7</td><td>–</td><td>3.5–> 5.0</td><td>Van Damme et al. (2022)</td></tr><tr><td>3°C, RH 80%, air velocity of 0.25 m/s for 28 days</td><td><i>Salmonella</i> spp.</td><td>–</td><td>4.7</td><td>Tittor et al. (2011)</td></tr><tr><td>2–6°C, RH 75–85% for 42 days</td><td><i>Salmonella</i> spp.</td><td>–</td><td>2.6–4.0</td><td>Van Damme et al. (2022)</td></tr><tr><td>2 ± 1°C, RH 75 ± 2%, air velocity of 2 ± 0.5 m/s for up to 42 days</td><td><i>L. innocua</i> ATCC 33090<sup>(1)</sup></td><td>–</td><td>2.4</td><td>da Silva et al. (2019)</td></tr><tr><td>8 ± 1°C, RH 75 ± 2%, air velocity of 2 ± 0.5 m/s for up to 35 days</td><td><i>L. innocua</i> ATCC 33090</td><td>–</td><td>3.4</td><td>da Silva et al. (2019)</td></tr><tr><td>2–6°C and RH 75–85%, for 42 days</td><td><i>L. monocytogenes</i></td><td>–</td><td>1.0–3.3</td><td>Van Damme et al. (2022)</td></tr><tr><td>2°C, RH 85% for 42 days</td><td><i>L. monocytogenes</i></td><td>1.0</td><td>1.1–1.8</td><td>Van Damme et al. (2022)</td></tr><tr><td colspan="5"><strong>Wet-ageing</strong></td></tr><tr><td>3°C, RH 80%, air velocity of 0.25 m/s for 28 days</td><td><i>E. coli</i> O157:H7</td><td>–</td><td>2.2</td><td>Tittor et al. (2011)</td></tr><tr><td>3°C, RH 80%, air velocity of 0.25 m/s for 28 days</td><td><i>Salmonella</i> spp.</td><td>–</td><td>2.2</td><td>Tittor et al. (2011)</td></tr></tbody></table>

## 3.5. TOR3: impact of dry-ageing and wet-ageing of meat on the load of public health-relevant microbiological hazards and spoilage bacteria, when compared to standard fresh meat

### 3.5.1. Selection of relevant microorganisms for the different scenarios

In addition to the ability to grow and/or produce toxin at the chilled temperatures used during meat ageing, the criteria used to select bacteria for the modelling studies included the availability of suitable predictive models to estimate growth under dry and wet-aged meat conditions, including the relevant $a_w$ values (in case of dry-ageing, Figure 4) and the atmosphere conditions (aerobic for dry-ageing and anaerobic for vacuum packaged wet-ageing).

Moreover, the psychrotrophic pathogenic and spoilage bacteria showing the highest growth rates were considered for the assessment, as representative of the worst-case scenario. The pathogenic bacteria *L. monocytogenes*, *Y. enterocolitica* and, for wet-ageing only, non-proteolytic *Clostridium* spp. were therefore selected. Mesophilic pathogens (e.g. *Salmonella* and *E. coli*) were not included in the assessment. *Pseudomonas* and LAB were the main spoilage bacterial groups (as discussed in Section 3.4.2) that satisfied the criteria for dry and wet-ageing, respectively.

Figure 4 shows the behaviour of growth rate at 4°C for different $a_w$ values for the different bacteria considered according to predictive models available at ComBase portal and FSSP. LAB and *L. monocytogenes* are the most resistant bacteria to $a_w$ reduction, as the reduction of the growth rate due to the decrease of $a_w$ was considerably lower as compared to *Pseudomonas*, *Y. enterocolitica* and

<footnote>(1): *L. innocua* ATCC 33090 was used as a surrogate for *L. monocytogenes*.</footnote>
*L. monocytogenes*). Van Damme et al. (2022) reported a 1.0 log₁₀ increase or a 3.0 log₁₀ decrease in *L. monocytogenes* depending on the dry-ageing conditions and beef muscle used. While a decrease in bacterial pathogens during dry-ageing may be due to the lowering of the aw, this would not explain the decreases in *E. coli* O157 and *Salmonella* spp. on wet-aged beef reported by Tittor et al. (2011). It should be noted that these findings are specific to the conditions used during ageing and further investigation is required before a more comprehensive conclusion on the fate of specific pathogens during meat ageing can be drawn.

<table><caption>**Table 7:** The change in the concentrations of specific pathogenic bacteria under different conditions of dry and wet-ageing of beef</caption><thead><tr><th rowspan="2">Conditions</th><th rowspan="2">Pathogen</th><th colspan="2">Log<sub>10</sub></th><th rowspan="2">Reference</th></tr><tr><th>Increase</th><th>Decrease</th></tr></thead><tbody><tr><td colspan="5"><strong>Dry-ageing</strong></td></tr><tr><td>3°C, RH 80%, air velocity of 0.25 m/s for 28 days</td><td><i>E. coli</i> O157:H7</td><td>–</td><td>4.8</td><td>Tittor et al. (2011)</td></tr><tr><td>4°C, RH 92% for 6 days</td><td><i>E. coli</i> O157:H7</td><td>–</td><td>0.8</td><td>Ingham et al. (2010)</td></tr><tr><td>2–6°C, RH 75–85% for 42 days</td><td><i>E. coli</i> O157:H7</td><td>–</td><td>3.5–> 5.0</td><td>Van Damme et al. (2022)</td></tr><tr><td>3°C, RH 80%, air velocity of 0.25 m/s for 28 days</td><td><i>Salmonella</i> spp.</td><td>–</td><td>4.7</td><td>Tittor et al. (2011)</td></tr><tr><td>2–6°C, RH 75–85% for 42 days</td><td><i>Salmonella</i> spp.</td><td>–</td><td>2.6–4.0</td><td>Van Damme et al. (2022)</td></tr><tr><td>2 ± 1°C, RH 75 ± 2%, air velocity of 2 ± 0.5 m/s for up to 42 days</td><td><i>L. innocua</i> ATCC 33090<sup>(1)</sup></td><td>–</td><td>2.4</td><td>da Silva et al. (2019)</td></tr><tr><td>8 ± 1°C, RH 75 ± 2%, air velocity of 2 ± 0.5 m/s for up to 35 days</td><td><i>L. innocua</i> ATCC 33090</td><td>–</td><td>3.4</td><td>da Silva et al. (2019)</td></tr><tr><td>2–6°C and RH 75–85%, for 42 days</td><td><i>L. monocytogenes</i></td><td>–</td><td>1.0–3.3</td><td>Van Damme et al. (2022)</td></tr><tr><td>2°C, RH 85% for 42 days</td><td><i>L. monocytogenes</i></td><td>1.0</td><td>1.1–1.8</td><td>Van Damme et al. (2022)</td></tr><tr><td colspan="5"><strong>Wet-ageing</strong></td></tr><tr><td>3°C, RH 80%, air velocity of 0.25 m/s for 28 days</td><td><i>E. coli</i> O157:H7</td><td>–</td><td>2.2</td><td>Tittor et al. (2011)</td></tr><tr><td>3°C, RH 80%, air velocity of 0.25 m/s for 28 days</td><td><i>Salmonella</i> spp.</td><td>–</td><td>2.2</td><td>Tittor et al. (2011)</td></tr></tbody></table>

## 3.5. TOR3: impact of dry-ageing and wet-ageing of meat on the load of public health-relevant microbiological hazards and spoilage bacteria, when compared to standard fresh meat

### 3.5.1. Selection of relevant microorganisms for the different scenarios

In addition to the ability to grow and/or produce toxin at the chilled temperatures used during meat ageing, the criteria used to select bacteria for the modelling studies included the availability of suitable predictive models to estimate growth under dry and wet-aged meat conditions, including the relevant $a_w$ values (in case of dry-ageing, Figure 4) and the atmosphere conditions (aerobic for dry-ageing and anaerobic for vacuum packaged wet-ageing).

Moreover, the psychrotrophic pathogenic and spoilage bacteria showing the highest growth rates were considered for the assessment, as representative of the worst-case scenario. The pathogenic bacteria *L. monocytogenes*, *Y. enterocolitica* and, for wet-ageing only, non-proteolytic *Clostridium* spp. were therefore selected. Mesophilic pathogens (e.g. *Salmonella* and *E. coli*) were not included in the assessment. *Pseudomonas* and LAB were the main spoilage bacterial groups (as discussed in Section 3.4.2) that satisfied the criteria for dry and wet-ageing, respectively.

Figure 4 shows the behaviour of growth rate at 4°C for different $a_w$ values for the different bacteria considered according to predictive models available at ComBase portal and FSSP. LAB and *L. monocytogenes* are the most resistant bacteria to $a_w$ reduction, as the reduction of the growth rate due to the decrease of $a_w$ was considerably lower as compared to *Pseudomonas*, *Y. enterocolitica* and

<footnote>(1): *L. innocua* ATCC 33090 was used as a surrogate for *L. monocytogenes*.</footnote>
non-proteolytic *C. botulinum*. For *L. monocytogenes*, an overlap for growth and inactivation was noted at $a_w$ values below 0.96.

<figure><img src="image_9.png" /><figcaption>**Figure 4:** Growth rate (as square root of $\mu_{max}$) at 4°C as a function of $a_w$ of relevant pathogenic and spoilage bacteria according to predictive models (input value for pH = 6), CB: ComBase; FSSP: Food Spoilage and Safety Predictor. Dotted line represents the square root of the inactivation rate ($k_{max}$ multiplied by 10 to present on the same scale) according to the non-thermal survival model of ComBase</figcaption></figure>

### 3.5.2. Development of scenarios to evaluate

#### 3.5.2.1. Development of scenarios to evaluate in ToR3 and ToR4

To address ToR3, a range of different scenarios were developed for dry-aged beef processes as well as wet-aged and standard fresh meat processes for beef, pork and lamb. These scenarios are based on the scientific literature and information provided in the answers to the questionnaire (see ToR1). The scenarios, distributions and associated parameters used in the predictive modelling are summarised in the Tables D.1–D.4 in Appendix D. Wet-ageing is assumed to be the standard process used for standard fresh meat preparation, with the times being up to 14 days for beef (Table D.1), 4 days for pork (Table D.3) and 4 days for lamb (Table D.4).

#### 3.5.2.2. The changing pH, T and $a_w$ values during dry-ageing of beef

Despite the lack of information available in the literature about the drying profile of the meat surface during beef ageing, four references were found reporting the change of $a_w$ on the meat surface during the ageing process of beef aged at different target temperature and RH conditions (da Silva et al., 2018; Smaldone et al., 2019; Panella-Riera et al., 2021; Bover Cid et al., 2022). Five realistic case-examples of specific combinations of temperature, $a_w$ and pH were obtained as shown in Figure 5. These examples are within the wide range of variability covered in the scenarios described in Section 3.5.2.1 (Tables D.1–D.4 in Appendix D)
non-proteolytic *C. botulinum*. For *L. monocytogenes*, an overlap for growth and inactivation was noted at $a_w$ values below 0.96.

<figure><img src="image_8.png" /><figcaption>**Figure 4:** Growth rate (as square root of $\mu_{max}$) at 4°C as a function of $a_w$ of relevant pathogenic and spoilage bacteria according to predictive models (input value for pH = 6), CB: ComBase; FSSP: Food Spoilage and Safety Predictor. Dotted line represents the square root of the inactivation rate ($k_{max}$ multiplied by 10 to present on the same scale) according to the non-thermal survival model of ComBase</figcaption></figure>

### 3.5.2. Development of scenarios to evaluate

#### 3.5.2.1. Development of scenarios to evaluate in ToR3 and ToR4

To address ToR3, a range of different scenarios were developed for dry-aged beef processes as well as wet-aged and standard fresh meat processes for beef, pork and lamb. These scenarios are based on the scientific literature and information provided in the answers to the questionnaire (see ToR1). The scenarios, distributions and associated parameters used in the predictive modelling are summarised in the Tables D.1–D.4 in Appendix D. Wet-ageing is assumed to be the standard process used for standard fresh meat preparation, with the times being up to 14 days for beef (Table D.1), 4 days for pork (Table D.3) and 4 days for lamb (Table D.4).

#### 3.5.2.2. The changing pH, T and $a_w$ values during dry-ageing of beef

Despite the lack of information available in the literature about the drying profile of the meat surface during beef ageing, four references were found reporting the change of $a_w$ on the meat surface during the ageing process of beef aged at different target temperature and RH conditions (da Silva et al., 2018; Smaldone et al., 2019; Panella-Riera et al., 2021; Bover Cid et al., 2022). Five realistic case-examples of specific combinations of temperature, $a_w$ and pH were obtained as shown in Figure 5. These examples are within the wide range of variability covered in the scenarios described in Section 3.5.2.1 (Tables D.1–D.4 in Appendix D)
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

**Case 1**
Bover-Cid et al (2022)
Target T = 0 °C
Target RH= 78 %

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

**Case 2**
Bover-Cid et al (2022)
Target T = 3 °C
Target RH= 60 %

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

**Case 3**
Panella-Riera et al.
(2021)
Target T = 2 °C
Target RH= 70 %

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

**Case 4**
Smaldone et al (2019)
Target T = 0 °C
Target RH= 68-70 %
(actual temperature not recorded)\nAging time truncated

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

**Case 5**
da Silva et al (2018)
Target T = 2 ± 1 °C
Target RH= 75 % ± 2 %
(actual temperature not recorded)
(pH values did not changed)

**Figure 5:** Measured, pH and $a_w$ values during dry-ageing of beef for different process conditions (target temperature and RH) reported in the literature used as realistic examples. For case 4 and 5 temperature represent the target value (represented as dotted lines)

### 3.5.3. Log₁₀ increase of microorganisms during dry-ageing of beef and prolonged wet-ageing of beef, pork and lamb compared to standard fresh meat preparation

To assess the potential impact of dry-ageing of beef and wet-ageing on beef, pork and lamb under selected current practices, on the load of public health-relevant microbiological hazards and spoilage bacteria as compared to standard fresh meat and to identify the most relevant bacteria, the potential log₁₀ increases were estimated based on input parameters for the different meat types
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

**Case 1**
Bover-Cid et al (2022)
Target T = 0 °C
Target RH= 78 %

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

**Case 2**
Bover-Cid et al (2022)
Target T = 3 °C
Target RH= 60 %

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

**Case 3**
Panella-Riera et al.
(2021)
Target T = 2 °C
Target RH= 70 %

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

**Case 4**
Smaldone et al (2019)
Target T = 0 °C
Target RH= 68-70 %
(actual temperature
not recorded)\nAging time truncated

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

**Case 5**
da Silva et al (2018)
Target T = 2 ± 1 °C
Target RH= 75 % ± 2 %
(actual temperature
not recorded)
(pH values did not
changed)

**Figure 5:** Measured, pH and $a_w$ values during dry-ageing of beef for different process conditions (target temperature and RH) reported in the literature used as realistic examples. For case 4 and 5 temperature represent the target value (represented as dotted lines)

### 3.5.3. Log₁₀ increase of microorganisms during dry-ageing of beef and prolonged wet-ageing of beef, pork and lamb compared to standard fresh meat preparation

To assess the potential impact of dry-ageing of beef and wet-ageing on beef, pork and lamb under selected current practices, on the load of public health-relevant microbiological hazards and spoilage bacteria as compared to standard fresh meat and to identify the most relevant bacteria, the potential log₁₀ increases were estimated based on input parameters for the different meat types
(Tables D.1–D.4). In this ToR, the effects of inactivation, rate of drying and competition are not included. The potential effects of these factors are shown in Section 3.4.4 when evaluating ToR4.

For wet-aged beef, based the median of the mean growth rates during standard fresh meat preparation (14 days), the predicted log₁₀ increase of *L. monocytogenes* after 49 days of wet-ageing is around 4 log₁₀, and 7 log₁₀ for LAB, whereas practically no growth is predicted for non-proteolytic *C. botulinum*. The corresponding minimum log₁₀ increases were approximately 0.5, 0, and 3, for *L. monocytogenes*, non-proteolytic *C. botulinum* and LAB, respectively (Figure 6).

<figure><img src="image_25.png" /><figcaption>**Figure 6:** Predicted growth (log₁₀ increase) of selected pathogens and spoilage bacteria during wet-ageing of beef. The solid lines are the minimum (blue), median (black), and maximum (red) log₁₀ increases during ageing based on constant mean growth rates. These growth rates were estimated from the variable growth rates during the standard fresh meat preparation (dotted vertical line). Light grey area indicates the min and max variable range of log₁₀ increases and dark grey area the 5 and 95 percentile variable range</figcaption></figure>

The predicted log₁₀ increases for the other ageing and meat types are shown in Figures 7–9. The predicted median log₁₀ increases at the end of ageing (i.e. 77 days) in dry-aged beef were around 5.2, 6.2, and 10 (assumed max log₁₀ increase) log for *L. monocytogenes*, LAB and *Pseudomonas*, respectively. In comparison, Van Damme et al. (2022) observed that mean concentrations of *Pseudomonas* after 42 days of dry-ageing increased from 2.1 log₁₀ CFU/cm² to 4.0 log₁₀ CFU/cm² (i.e. 1.9 log₁₀ increase considering the mean concentrations) on adipose tissue with a maximum concentration of 7.4 log₁₀ CFU/cm² (i.e. max 5.3 log₁₀ increase). The reported concentrations were smaller on lean tissue. For *L. monocytogenes* a 1 log₁₀ increase after 42 days was only observed for one replicate of four at 2°C, 85% RH, whereas at other conditions (2°C and 6°C, 75 or 85% RH) varying rates of reductions were observed resulting in 1 to approximately 3 log₁₀ decrease (Van Damme et al., 2022).
(Tables D.1–D.4). In this ToR, the effects of inactivation, rate of drying and competition are not included. The potential effects of these factors are shown in Section 3.4.4 when evaluating ToR4.

For wet-aged beef, based the median of the mean growth rates during standard fresh meat preparation (14 days), the predicted log₁₀ increase of *L. monocytogenes* after 49 days of wet-ageing is around 4 log₁₀, and 7 log₁₀ for LAB, whereas practically no growth is predicted for non-proteolytic *C. botulinum*. The corresponding minimum log₁₀ increases were approximately 0.5, 0, and 3, for *L. monocytogenes*, non-proteolytic *C. botulinum* and LAB, respectively (Figure 6).

<figure><img src="image_24.png" /><figcaption>**Figure 6:** Predicted growth (log₁₀ increase) of selected pathogens and spoilage bacteria during wet-ageing of beef. The solid lines are the minimum (blue), median (black), and maximum (red) log₁₀ increases during ageing based on constant mean growth rates. These growth rates were estimated from the variable growth rates during the standard fresh meat preparation (dotted vertical line). Light grey area indicates the min and max variable range of log₁₀ increases and dark grey area the 5 and 95 percentile variable range</figcaption></figure>

The predicted log₁₀ increases for the other ageing and meat types are shown in Figures 7–9. The predicted median log₁₀ increases at the end of ageing (i.e. 77 days) in dry-aged beef were around 5.2, 6.2, and 10 (assumed max log₁₀ increase) log for *L. monocytogenes*, LAB and *Pseudomonas*, respectively. In comparison, Van Damme et al. (2022) observed that mean concentrations of *Pseudomonas* after 42 days of dry-ageing increased from 2.1 log₁₀ CFU/cm² to 4.0 log₁₀ CFU/cm² (i.e. 1.9 log₁₀ increase considering the mean concentrations) on adipose tissue with a maximum concentration of 7.4 log₁₀ CFU/cm² (i.e. max 5.3 log₁₀ increase). The reported concentrations were smaller on lean tissue. For *L. monocytogenes* a 1 log₁₀ increase after 42 days was only observed for one replicate of four at 2°C, 85% RH, whereas at other conditions (2°C and 6°C, 75 or 85% RH) varying rates of reductions were observed resulting in 1 to approximately 3 log₁₀ decrease (Van Damme et al., 2022).
<figure><img src="image_26.png" /><figcaption>**Figure 7:** Predicted $log_{10}$ increases of selected pathogens and spoilage bacteria during dry-ageing of beef. The solid lines are the minimum (blue), median (black) and maximum (red) $log_{10}$ increases during ageing based on constant mean growth rates. These growth rates were estimated from the variable growth rates during the first 14 days (standard fresh meat preparation time). The comparison for standard meat is the grey shaded areas in the corresponding graphs in Figure 6</figcaption></figure>

In wet-aged pork, the greatest potential median $log_{10}$ increase after 28 days was predicted for *Y. enterocolitica*, at approximately 6.2 $log_{10}$, whereas the median log predicted increases was around 2 and 3.2 $log_{10}$ for *L. monocytogenes* and LAB, respectively (Figure 8). In wet-aged lamb, the predicted median potential log increases after 21 days of wet-ageing were approximately 2 and 3 $log_{10}$ for *L. monocytogenes* and LAB, respectively (Figure 9).
<figure><img src="image_25.png" /><figcaption>**Figure 7:** Predicted $log_{10}$ increases of selected pathogens and spoilage bacteria during dry-ageing of beef. The solid lines are the minimum (blue), median (black) and maximum (red) $log_{10}$ increases during ageing based on constant mean growth rates. These growth rates were estimated from the variable growth rates during the first 14 days (standard fresh meat preparation time). The comparison for standard meat is the grey shaded areas in the corresponding graphs in Figure 6</figcaption></figure>

In wet-aged pork, the greatest potential median $log_{10}$ increase after 28 days was predicted for *Y. enterocolitica*, at approximately 6.2 $log_{10}$, whereas the median log predicted increases was around 2 and 3.2 $log_{10}$ for *L. monocytogenes* and LAB, respectively (Figure 8). In wet-aged lamb, the predicted median potential log increases after 21 days of wet-ageing were approximately 2 and 3 $log_{10}$ for *L. monocytogenes* and LAB, respectively (Figure 9).
<figure><img src="image_27.png" /><figcaption>**Figure 8:** Predicted log₁₀ increases of selected pathogens and spoilage bacteria during wet-ageing of pork. The solid lines are the minimum (blue), median (black) and maximum (red) log₁₀ increases during prolonged ageing based on constant mean growth rates. These growth rates were estimated from the variable log₁₀ increases during the standard fresh meat preparation time (dotted vertical line). Light grey indicates the min and max variable range of log₁₀ increases and dark grey the 5 and 95 variable percentile range</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>**Figure 9:** Predicted log₁₀ increases of selected pathogens and spoilage bacteria during wet-ageing of lamb. The solid lines are the minimum (blue), median (black) and maximum (red) log₁₀ increases during prolonged ageing based on constant mean growth rates. These growth rates were estimated from the variable log₁₀ increases during the standard fresh meat preparation time (dotted vertical line). Light grey indicates the min and max variable range of log₁₀ increases and dark grey the 5 and 95 variable percentile range</figcaption></figure>
<figure><img src="image_26.png" /><figcaption>**Figure 8:** Predicted log₁₀ increases of selected pathogens and spoilage bacteria during wet-ageing of pork. The solid lines are the minimum (blue), median (black) and maximum (red) log₁₀ increases during prolonged ageing based on constant mean growth rates. These growth rates were estimated from the variable log₁₀ increases during the standard fresh meat preparation time (dotted vertical line). Light grey indicates the min and max variable range of log₁₀ increases and dark grey the 5 and 95 variable percentile range</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>**Figure 9:** Predicted log₁₀ increases of selected pathogens and spoilage bacteria during wet-ageing of lamb. The solid lines are the minimum (blue), median (black) and maximum (red) log₁₀ increases during prolonged ageing based on constant mean growth rates. These growth rates were estimated from the variable log₁₀ increases during the standard fresh meat preparation time (dotted vertical line). Light grey indicates the min and max variable range of log₁₀ increases and dark grey the 5 and 95 variable percentile range</figcaption></figure>
In comparison, the range of predicted log₁₀ increases of the microbiological hazards and spoilage bacteria during standard fresh meat preparation are indicated by the shaded area in Figures 6, 8 and 9. The median predicted log₁₀ increases during standard fresh meat preparation for *L. monocytogenes* and LAB varied between 1 and 2 for wet-aged beef.

Figure 66. The corresponding log₁₀ increases in dry-aged beef after 14 days were similar, except for *Pseudomonas* with a predicted log₁₀ increase of around 3.0 (Figure 7). In lamb and pork after 4 days, the predicted log₁₀ increases were around 0.5 to 1, with a maximum predicted log₁₀ increase for *Yersinia* of around 1 (Figures 8 and 9). A summary of the range of predicted log₁₀ increases for pathogens and spoilage bacteria during standard fresh meat preparation and ageing is shown in Appendix G.

Thus, the predictions show that ageing beyond the durations used for standard fresh meat could potentially impact on the load of microbiological hazards and spoilage bacteria under current practices. The estimated minimum predicted log₁₀ increases over time indicate that equivalent loads compared to the range of loads on meat after standard fresh meat preparation times can be achieved, i.e. y-values of blue lines in figures are lower or equal to the upper grey areas, at least for a part, if not all, of the extended ageing time evaluated. In some cases, e.g. *Y. enterolitica* in wet-aged pork, the ageing time appears to be at most around 10–15 days (Figure 8). In ToR4, the conditions resulting in similar log₁₀ increases as standard meat preparation for the relevant pathogens and spoilage bacteria are evaluated. It should be noted that the model predictions in ToR3 are considered overestimations of log₁₀ increases since the effect of competition and inactivation are not included. Additionally, for dry-ageing the rate of drying is also important. Moreover, the range of variability during standard fresh meat preparation to be used for the determination of equivalence will also affect the outcome of ToR4. The impact of these factors is evaluated in Section 3.4.4.

The predictions discussed above apply to the full range of conditions considered in the scenarios developed in Section 3.5.2.1 and provide the predicted minimum and the maximum log₁₀ increase values, but not the relative frequency or probability that these values could occur within the current practices of meat ageing. Although this type of information could not be collected from the scientific literature or the questionnaire, specific cases of meat ageing process conditions were developed (Section 3.5.2.2) as realistic examples of the dynamic combination of ageing temperature, $a_w$ and pH on the meat surface during ageing.

Figure 10 shows the result of the simulation of the growth of *L. monocytogenes* (as log₁₀ increase) together with the changes of $a_w$ and pH of the meat surface and the temperature during the beef dry-ageing process for each case example. The potential growth of *L. monocytogenes* varied depending on the specific ageing profiles, from a 1 to 2 log₁₀ increase in cases 1 and 4, that kept temperature at 0°C and case 5 corresponding to the fastest and more intense drying among the assessed examples. The highest log₁₀ increase was predicted for case 2, in which the temperature was 3°C. The magnitude of the simulated growth of these realistic case examples fell within the range of potential log₁₀ increase of *L. monocytogenes* of the overall scenarios shown above (Figure 7).
In comparison, the range of predicted log₁₀ increases of the microbiological hazards and spoilage bacteria during standard fresh meat preparation are indicated by the shaded area in Figures 6, 8 and 9. The median predicted log₁₀ increases during standard fresh meat preparation for *L. monocytogenes* and LAB varied between 1 and 2 for wet-aged beef.

Figure 66. The corresponding log₁₀ increases in dry-aged beef after 14 days were similar, except for *Pseudomonas* with a predicted log₁₀ increase of around 3.0 (Figure 7). In lamb and pork after 4 days, the predicted log₁₀ increases were around 0.5 to 1, with a maximum predicted log₁₀ increase for *Yersinia* of around 1 (Figures 8 and 9). A summary of the range of predicted log₁₀ increases for pathogens and spoilage bacteria during standard fresh meat preparation and ageing is shown in Appendix G.

Thus, the predictions show that ageing beyond the durations used for standard fresh meat could potentially impact on the load of microbiological hazards and spoilage bacteria under current practices. The estimated minimum predicted log₁₀ increases over time indicate that equivalent loads compared to the range of loads on meat after standard fresh meat preparation times can be achieved, i.e. y-values of blue lines in figures are lower or equal to the upper grey areas, at least for a part, if not all, of the extended ageing time evaluated. In some cases, e.g. *Y. enterolitica* in wet-aged pork, the ageing time appear to be at most around 10–15 days (Figure 8). In ToR4, the conditions resulting in similar log₁₀ increases as standard meat preparation for the relevant pathogens and spoilage bacteria are evaluated. It should be noted that the model predictions in ToR3 are considered overestimations of log₁₀ increases since the effect of competition and inactivation are not included. Additionally, for dry-ageing the rate of drying is also important. Moreover, the range of variability during standard fresh meat preparation to be used for the determination of equivalence will also affect the outcome of ToR4. The impact of these factors is evaluated in Section 3.4.4.

The predictions discussed above apply to the full range of conditions considered in the scenarios developed in Section 3.5.2.1 and provide the predicted minimum and the maximum log₁₀ increase values, but not the relative frequency or probability that these values could occur within the current practices of meat ageing. Although this type of information could not be collected from the scientific literature or the questionnaire, specific cases of meat ageing process conditions were developed (Section 3.5.2.2) as realistic examples of the dynamic combination of ageing temperature, $a_w$ and pH on the meat surface during ageing.

Figure 10 shows the result of the simulation of the growth of *L. monocytogenes* (as log₁₀ increase) together with the changes of $a_w$ and pH of the meat surface and the temperature during the beef dry-ageing process for each case example. The potential growth of *L. monocytogenes* varied depending on the specific ageing profiles, from a 1 to 2 log₁₀ increase in cases 1 and 4, that kept temperature at 0°C and case 5 corresponding to the fastest and more intense drying among the assessed examples. The highest log₁₀ increase was predicted for case 2, in which the temperature was 3°C. The magnitude of the simulated growth of these realistic case examples fell within the range of potential log₁₀ increase of *L. monocytogenes* of the overall scenarios shown above (Figure 7).
<figure><img src="image_29.png" /><figcaption>**Figure 10:** Predicted growth of *L. monocytogenes* (red) for different realistic cases of dynamic profiles of $a_w$ (orange), pH (grey) and temperature (blue) during dry-ageing of beef under specific target temperature and relative humidity (RH) conditions found in the scientific literature (Bover Cid et al., 2022 for case 1 and 2; Panella-Riera et al., 2021 for case 3; Smaldone et al., 2019 for case 4; da Silva et al., 2018 for case 5)</figcaption></figure>
<figure><img src="image_28.png" /><figcaption>**Figure 10:** Predicted growth of *L. monocytogenes* (red) for different realistic cases of dynamic profiles of $a_w$ (orange), pH (grey) and temperature (blue) during dry-ageing of beef under specific target temperature and relative humidity (RH) conditions found in the scientific literature (Bover Cid et al., 2022 for case 1 and 2; Panella-Riera et al., 2021 for case 3; Smaldone et al., 2019 for case 4; da Silva et al., 2018 for case 5)</figcaption></figure>
### 3.6. TOR4: conditions during the production of dry-aged and wet-aged meat and possible further storage that would result in a similar or lower load of the relevant microbiological hazards and spoilage bacteria as compared to standard fresh meat before consumption (i.e. at the end of the shelf-life)

#### 3.6.1. Scenario analysis of ageing processes – Approach and scenarios evaluated

In ToR4, the conditions resulting in similar $log_{10}$ increases as for standard fresh meat preparation are identified. Since temperature and duration are the variables that may be directly controlled, the analysis was focused on the impact of these factors on the potential $log_{10}$ increase of the fastest growing pathogenic and spoilage bacteria, respectively. Since $log_{10}$ increase depends on time and temperature equivalent conditions must entail shorter times or lower temperatures, unless maximum bacterial concentrations have already been achieved during the standard fresh meat preparation time However, $a_w$ and pH also have an impact on the equivalent time and temperatures. Figures 10 and 11 illustrates this by showing the time and temperature conditions during wet-ageing of beef that corresponds to predicted $log_{10}$ increases of L. monocytogenes that are the same or lower than the mean $log_{10}$ increase during standard fresh beef preparation, i.e. here a 1 $log_{10}$ increase. For instance, at a pH of 5.1, equivalent conditions are observed at 1 to 4°C for up to 49 days and at 5°C for up to 29 days of ageing (at the range of $a_w$-values evaluated in the scenario), whereas at a pH of 5.9 the corresponding highest temperature is 1°C and the ageing only at 22 days (presumedly at the lower $a_w$ values evaluated, Figure 11a). Thus, at the higher temperatures long duration is only possible at lower values for $a_w$ and especially, pH (Figure 11).
### 3.6. TOR4: conditions during the production of dry-aged and wet-aged meat and possible further storage that would result in a similar or lower load of the relevant microbiological hazards and spoilage bacteria as compared to standard fresh meat before consumption (i.e. at the end of the shelf-life)

#### 3.6.1. Scenario analysis of ageing processes – Approach and scenarios evaluated

In ToR4, the conditions resulting in similar $log_{10}$ increases as for standard fresh meat preparation are identified. Since temperature and duration are the variables that may be directly controlled, the analysis was focused on the impact of these factors on the potential $log_{10}$ increase of the fastest growing pathogenic and spoilage bacteria, respectively. Since $log_{10}$ increase depends on time and temperature equivalent conditions must entail shorter times or lower temperatures, unless maximum bacterial concentrations have already been achieved during the standard fresh meat preparation time However, $a_w$ and pH also have an impact on the equivalent time and temperatures. Figures 10 and 11 illustrates this by showing the time and temperature conditions during wet-ageing of beef that corresponds to predicted $log_{10}$ increases of L. monocytogenes that are the same or lower than the mean $log_{10}$ increase during standard fresh beef preparation, i.e. here a 1 $log_{10}$ increase. For instance, at a pH of 5.1, equivalent conditions are observed at 1 to 4°C for up to 49 days and at 5°C for up to 29 days of ageing (at the range of $a_w$-values evaluated in the scenario), whereas at a pH of 5.9 the corresponding highest temperature is 1°C and the ageing only at 22 days (presumedly at the lower $a_w$ values evaluated, Figure 11a). Thus, at the higher temperatures long duration is only possible at lower values for $a_w$ and especially, pH (Figure 11).
<figure><img src="image_30.png" /><figcaption>**Figure 11:** The impact of the range of $a_w$-values at fixed pH-values (a) and the range of pH-values at fixed $a_w$-values (b) on the equivalent temperature and ageing time conditions. These temperatures and times correspond to predicted $\log_{10}$ increases of *L. monocytogenes* in wet beef being equal or lower than the mean $\log_{10}$ increase during standard fresh meat preparation</figcaption></figure>

Taking the range of predicted median $\log_{10}$ increases of the microbiological hazards and spoilage bacteria evaluated in ToR3, as well as the potential effect of overestimation arising from not considering competition and the rate of drying, it was decided to evaluate equivalence in terms of 0.5, 1, 2, 3 and 4 predicted $\log_{10}$ increases.
<figure><img src="image_29.png" /><figcaption>**Figure 11:** The impact of the range of $a_w$-values at fixed pH-values (a) and the range of pH-values at fixed $a_w$-values (b) on the equivalent temperature and ageing time conditions. These temperatures and times correspond to predicted $\log_{10}$ increases of *L. monocytogenes* in wet beef being equal or lower than the mean $\log_{10}$ increase during standard fresh meat preparation</figcaption></figure>

Taking the range of predicted median $\log_{10}$ increases of the microbiological hazards and spoilage bacteria evaluated in ToR3, as well as the potential effect of overestimation arising from not considering competition and the rate of drying, it was decided to evaluate equivalence in terms of 0.5, 1, 2, 3 and 4 predicted $\log_{10}$ increases.
### 3.6.2. Dry-ageing – conditions to achieve similar or lower log increase of pathogens or spoilage bacteria before consumption

The relationship between the temperature and the maximum dry-ageing time that would result in log₁₀ increases equivalent to the log₁₀ increases that represent standard fresh meat preparation, i.e. between 0.5 and 4 log₁₀, are shown in Figures 12 and 13 for *L. monocytogenes* and LAB. Point estimates of the maximum time or maximum temperature (and the corresponding temperatures and times) conditions that would achieve the different log₁₀ increases considered equivalent to standard fresh beef preparation is shown for different target bacteria in Appendix F, Table F.1. The importance of the pH and a_w during ageing is illustrated by the different equivalent conditions resulting using different scenarios for these parameters. The scenarios used for the predictions are based on scenarios with minimum, median, or maximum values of pH and a_w during standard fresh meat preparation. For example, assuming that equivalence is represented by a 1 log₁₀ increase of *L. monocytogenes*, the maximum ageing time would be at least 77 days (at 5°C), the maximum time evaluated, in the minimum scenario, and 20 days (at 0°C) in the maximum scenario (Figure 12). Similarly, if an ageing time of 35 days is required, ageing according to the predictive model need to take place at 0.5°C, 1.25°C, 2.25°C, 3°C or 3.75°C if 0.5, 1, 2, 3, or a 4-log₁₀ increase, respectively, is considered for equivalence in the medium scenario for pH and aw. The required ageing temperatures results from the predictive model and the small differences between scenarios reflect the great impact of temperature on the predictions. Taking observed temperature variations during ageing into consideration it is difficult to control temperature to this extent under practical conditions. The pH and more especially the aw can be variable during dry-ageing, and controllable to some extent. The impact of drying rate is evaluated in Section 3.6.4.3.

<figure><img src="image_31.png" /></figure>
### 3.6.2. Dry-ageing – conditions to achieve similar or lower log increase of pathogens or spoilage bacteria before consumption

The relationship between the temperature and the maximum dry-ageing time that would result in log₁₀ increases equivalent to the log₁₀ increases that represent standard fresh meat preparation, i.e. between 0.5 and 4 log₁₀, are shown in Figures 12 and 13 for *L. monocytogenes* and LAB. Point estimates of the maximum time or maximum temperature (and the corresponding temperatures and times) conditions that would achieve the different log₁₀ increases considered equivalent to standard fresh beef preparation is shown for different target bacteria in Appendix F, Table F.1. The importance of the pH and a_w during ageing is illustrated by the different equivalent conditions resulting using different scenarios for these parameters. The scenarios used for the predictions are based on scenarios with minimum, median, or maximum values of pH and a_w during standard fresh meat preparation. For example, assuming that equivalence is represented by a 1 log₁₀ increase of *L. monocytogenes*, the maximum ageing time would be at least 77 days (at 5°C), the maximum time evaluated, in the minimum scenario, and 20 days (at 0°C) in the maximum scenario (Figure 12). Similarly, if an ageing time of 35 days is required, ageing according to the predictive model need to take place at 0.5°C, 1.25°C, 2.25°C, 3°C or 3.75°C if 0.5, 1, 2, 3, or a 4-log₁₀ increase, respectively, is considered for equivalence in the medium scenario for pH and aw. The required ageing temperatures results from the predictive model and the small differences between scenarios reflect the great impact of temperature on the predictions. Taking observed temperature variations during ageing into consideration it is difficult to control temperature to this extent under practical conditions. The pH and more especially the aw can be variable during dry-ageing, and controllable to some extent. The impact of drying rate is evaluated in Section 3.6.4.3.

<figure><img src="image_30.png" /></figure>
<figure><img src="image_32.png" /><figcaption>**Figure 12:** Relationship between ageing temperature and maximum time for dry-ageing of beef corresponding to different log₁₀ increases of *L. monocytogenes* considered equivalent to standard beef ageing under the assumption of three scenarios of pH and *a*<sub>w</sub>, i.e. maximum (pH = 6.2; *a*<sub>w</sub> = 0.99), median (pH = 5.85; *a*<sub>w</sub> = 0.955) or minimum scenarios (pH = 5.5; *a*<sub>w</sub> = 0.92), and a maximum ageing time of 77 days</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>**Figure 12:** Relationship between ageing temperature and maximum time for dry-ageing of beef corresponding to different log₁₀ increases of *L. monocytogenes* considered equivalent to standard beef ageing under the assumption of three scenarios of pH and a<sub>w</sub>, i.e. maximum (pH = 6.2; a<sub>w</sub> = 0.99), median (pH = 5.85; a<sub>w</sub> = 0.955) or minimum scenarios (pH = 5.5; a<sub>w</sub> = 0.92), and a maximum ageing time of 77 days</figcaption></figure>
<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>
<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>
<figure><img src="image_35.png" /><figcaption>**Figure 13:** Relationship between ageing temperature and maximum time for dry-ageing of beef corresponding to different log₁₀ increases of LAB considered equivalent to standard beef ageing under the assumption of three scenarios of pH and a<sub>w</sub>, i.e. maximum (pH = 6.2; a<sub>w</sub> = 0.99), median (pH = 5.85; a<sub>w</sub> = 0.955) or minimum scenarios (pH = 5.5; a<sub>w</sub> = 0.92), and a maximum ageing time of 77 days</figcaption></figure>

### 3.6.3. Wet-ageing – conditions to achieve similar or lower log increase of pathogens or spoilage bacteria before consumption

The relationship between ageing temperature and maximum wet-ageing time that would result in log₁₀ increases equivalent to the different log₁₀ increases considered to represent standard fresh meat preparation, i.e. between 0.5 and 4 log, are shown in Figures 14, 16, 18 for *L. monocytogenes* and Figures 15, 17, 19 for LAB. Point estimates of the maximum time or maximum temperature (and the corresponding temperatures and times) conditions that would achieve different log₁₀ increases considered equivalent to standard fresh meat preparation of beef, pork and lamb are shown for different bacteria in Appendix F, Table F.2. The importance of the pH and a<sub>w</sub> during ageing is illustrated by the different equivalent conditions resulting using different scenarios for these parameters. For instance, assuming that equivalence in pork is represented by a 1 log₁₀ increase of *L. monocytogenes*, the maximum ageing time would be 28 days (at 3°C) in the minimum scenario, and 26 days (at 0°C) in the maximum scenario (Table F.2). For wet-aged beef, if an ageing time of 35 days is desirable, ageing according to the predictive model needs to take place at 0.5°C, 1.6°C, 2.6°C or 3.4°C if 1, 2, 3, or a 4-log₁₀ increase of *L. monocytogenes*, respectively, is considered for equivalence in the medium scenario for pH and aw (Figure 14). Considering spoilage bacteria and wet-ageing of beef, assuming that equivalence is represented by a 3-log₁₀ increase of LAB, the maximum ageing time would be at least 49 days (at 3°C), the maximum time evaluated, in the minimum scenario, and 36 days (at 0°C) in the maximum scenario (Figure 15). Similarly, if an ageing time of 35 days is desirable, the lowest achievable log₁₀ increase according to the predictive model in the medium scenario is a 3-log₁₀ increase. To allow 35 days ageing time, ageing need to take place at 0.7°C or 1.6°C if a 3-, or a 4-log₁₀ increase, respectively, is considered for equivalence in the medium scenario for pH and aw (Figure 15).
<figure><img src="image_34.png" /><figcaption>**Figure 13:** Relationship between ageing temperature and maximum time for dry-ageing of beef corresponding to different log₁₀ increases of LAB considered equivalent to standard beef ageing under the assumption of three scenarios of pH and a<sub>w</sub>, i.e. maximum (pH = 6.2; a<sub>w</sub> = 0.99), median (pH = 5.85; a<sub>w</sub> = 0.955) or minimum scenarios (pH = 5.5; a<sub>w</sub> = 0.92), and a maximum ageing time of 77 days</figcaption></figure>

### 3.6.3. Wet-ageing – conditions to achieve similar or lower log increase of pathogens or spoilage bacteria before consumption

The relationship between ageing temperature and maximum wet-ageing time that would result in log₁₀ increases equivalent to the different log₁₀ increases considered to represent standard fresh meat preparation, i.e. between 0.5 and 4 log, are shown in Figures 14, 16, 18 for *L. monocytogenes* and Figures 15, 17, 19 for LAB. Point estimates of the maximum time or maximum temperature (and the corresponding temperatures and times) conditions that would achieve different log₁₀ increases considered equivalent to standard fresh meat preparation of beef, pork and lamb are shown for different bacteria in Appendix F, Table F.2. The importance of the pH and a<sub>w</sub> during ageing is illustrated by the different equivalent conditions resulting using different scenarios for these parameters. For instance, assuming that equivalence in pork is represented by a 1 log₁₀ increase of *L. monocytogenes*, the maximum ageing time would be 28 days (at 3°C) in the minimum scenario, and 26 days (at 0°C) in the maximum scenario (Table F.2). For wet-aged beef, if an ageing time of 35 days is desirable, ageing according to the predictive model needs to take place at 0.5°C, 1.6°C, 2.6°C or 3.4°C if 1, 2, 3, or a 4-log₁₀ increase of *L. monocytogenes*, respectively, is considered for equivalence in the medium scenario for pH and aw (Figure 14). Considering spoilage bacteria and wet-ageing of beef, assuming that equivalence is represented by a 3-log₁₀ increase of LAB, the maximum ageing time would be at least 49 days (at 3°C), the maximum time evaluated, in the minimum scenario, and 36 days (at 0°C) in the maximum scenario (Figure 15). Similarly, if an ageing time of 35 days is desirable, the lowest achievable log₁₀ increase according to the predictive model in the medium scenario is a 3-log₁₀ increase. To allow 35 days ageing time, ageing need to take place at 0.7°C or 1.6°C if a 3-, or a 4-log₁₀ increase, respectively, is considered for equivalence in the medium scenario for pH and aw (Figure 15).
<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>
<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>
<figure><img src="image_38.png" /><figcaption>**Figure 14:** Relationship between ageing temperature and maximum time for wet-ageing of beef corresponding to different log₁₀ increases of *L. monocytogenes* considered equivalent to standard beef ageing under the assumption of three scenarios of pH and *a*<sub>w</sub>, i.e. maximum (pH = 5.9; *a*<sub>w</sub> = 0.99), median (pH = 5.5; *a*<sub>w</sub> = 0.98) or minimum scenarios (pH = 5.1; *a*<sub>w</sub> = 0.97), and a maximum ageing time of 49 days</figcaption></figure>

<figure><img src="image_39.png" /></figure>
<figure><img src="image_37.png" /><figcaption>**Figure 14:** Relationship between ageing temperature and maximum time for wet-ageing of beef corresponding to different log₁₀ increases of *L. monocytogenes* considered equivalent to standard beef ageing under the assumption of three scenarios of pH and *a*<sub>w</sub>, i.e. maximum (pH = 5.9; *a*<sub>w</sub> = 0.99), median (pH = 5.5; *a*<sub>w</sub> = 0.98) or minimum scenarios (pH = 5.1; *a*<sub>w</sub> = 0.97), and a maximum ageing time of 49 days</figcaption></figure>

<figure><img src="image_38.png" /></figure>
<figure><img src="image_40.png" /><figcaption>**Figure 15:** Relationship between ageing temperature and maximum time for wet-ageing of beef corresponding to different log₁₀ increases of LAB considered equivalent to standard beef ageing under the assumption of three scenarios of pH and a<sub>w</sub>, i.e. maximum (pH = 5.9; a<sub>w</sub> = 0.99), median (pH = 5.5; a<sub>w</sub> = 0.98) or minimum scenarios (pH = 5.1; a<sub>w</sub> = 0.97), and a maximum ageing time of 49 days</figcaption></figure>
<figure><img src="image_39.png" /><figcaption>**Figure 15:** Relationship between ageing temperature and maximum time for wet-ageing of beef corresponding to different log₁₀ increases of LAB considered equivalent to standard beef ageing under the assumption of three scenarios of pH and a<sub>w</sub>, i.e. maximum (pH = 5.9; a<sub>w</sub> = 0.99), median (pH = 5.5; a<sub>w</sub> = 0.98) or minimum scenarios (pH = 5.1; a<sub>w</sub> = 0.97), and a maximum ageing time of 49 days</figcaption></figure>
<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>
<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>
<figure><img src="image_43.png" /><figcaption>**Figure 16:** Relationship between ageing temperature and maximum time for wet-ageing of pork corresponding to different log₁₀ increases of *L. monocytogenes* considered equivalent to standard pork ageing under the assumption of three scenarios of pH and *a*<sub>w</sub>, i.e. maximum (pH = 6.3; *a*<sub>w</sub> = 0.99), median (pH = 5.85; *a*<sub>w</sub> = 0.97) or minimum scenarios (pH = 5.4; *a*<sub>w</sub> = 0.95), and a maximum ageing time of 28 days</figcaption></figure>

<figure><img src="image_44.png" /></figure>
<figure><img src="image_42.png" /><figcaption>**Figure 16:** Relationship between ageing temperature and maximum time for wet-ageing of pork corresponding to different log₁₀ increases of *L. monocytogenes* considered equivalent to standard pork ageing under the assumption of three scenarios of pH and *a*<sub>w</sub>, i.e. maximum (pH = 6.3; *a*<sub>w</sub> = 0.99), median (pH = 5.85; *a*<sub>w</sub> = 0.97) or minimum scenarios (pH = 5.4; *a*<sub>w</sub> = 0.95), and a maximum ageing time of 28 days</figcaption></figure>

<figure><img src="image_43.png" /></figure>
<figure><img src="image_45.png" /><figcaption>**Figure 17:** Relationship between ageing temperature and maximum time for wet-ageing of pork corresponding to different log₁₀ increases of LAB considered equivalent to standard pork ageing under the assumption of three scenarios of pH and a<sub>w</sub>, i.e. maximum (pH = 6.3; a<sub>w</sub> = 0.99), median (pH = 5.85; a<sub>w</sub> = 0.97) or minimum scenarios (pH = 5.4; a<sub>w</sub> = 0.95), and a maximum ageing time of 28 days</figcaption></figure>
<figure><img src="image_44.png" /><figcaption>**Figure 17:** Relationship between ageing temperature and maximum time for wet-ageing of pork corresponding to different log₁₀ increases of LAB considered equivalent to standard pork ageing under the assumption of three scenarios of pH and a<sub>w</sub>, i.e. maximum (pH = 6.3; a<sub>w</sub> = 0.99), median (pH = 5.85; a<sub>w</sub> = 0.97) or minimum scenarios (pH = 5.4; a<sub>w</sub> = 0.95), and a maximum ageing time of 28 days</figcaption></figure>
<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>
<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>
<figure><img src="image_48.png" /><figcaption>**Figure 18:** Relationship between ageing temperature and maximum time for wet-ageing of lamb corresponding to different log₁₀ increases of *L. monocytogenes* considered equivalent to standard pork ageing under the assumption of three scenarios of pH and *a*<sub>w</sub>, i.e. maximum (pH = 5.9; *a*<sub>w</sub> = 0.99), median (pH = 5.7; *a*<sub>w</sub> = 0.97) or minimum scenarios (pH = 5.5; *a*<sub>w</sub> = 0.95), and a maximum ageing time of 21 days</figcaption></figure>
<figure><img src="image_47.png" /><figcaption>**Figure 18:** Relationship between ageing temperature and maximum time for wet-ageing of lamb corresponding to different log₁₀ increases of *L. monocytogenes* considered equivalent to standard pork ageing under the assumption of three scenarios of pH and a<sub>w</sub>, i.e. maximum (pH = 5.9; a<sub>w</sub> = 0.99), median (pH = 5.7; a<sub>w</sub> = 0.97) or minimum scenarios (pH = 5.5; a<sub>w</sub> = 0.95), and a maximum ageing time of 21 days</figcaption></figure>
<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>
<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>
<figure><img src="image_51.png" /><figcaption>**Figure 19:** Relationship between ageing temperature and maximum time for wet-ageing of lamb corresponding to different log₁₀ increases of LAB considered equivalent to standard pork ageing under the assumption of three scenarios of pH and a<sub>w</sub>, i.e. maximum (pH = 5.9; a<sub>w</sub> = 0.99), median (pH = 5.7; a<sub>w</sub> = 0.97) or minimum scenarios (pH = 5.5; a<sub>w</sub> = 0.95), and a maximum ageing time of 21 days</figcaption></figure>

### 3.6.4. Impact of factors on predictions

#### 3.6.4.1. Competition

The impact of competition from LAB on the potential growth of *L. monocytogenes* was evaluated for different scenarios of initial concentrations of LAB (Table 3). The initial concentration of *L. monocytogenes* is also provided to allow calculation of the predicted log₁₀ increase, which was used in other evaluations to quantify the potential growth. There was a clear impact of competition as compared to no competition (No LAB in Table 8) already at a low LAB concentration of 1 log₁₀ CFU/g, and especially for the upper percentiles. The predicted 95th-percentile was below 100 CFU/g when the LAB concentration was higher than 3 log₁₀ CFU/g but not when it was 2 log₁₀ CFU/g (Table 8).

<table><caption>**Table 8:** *L. monocytogenes* predicted final concentrations (log₁₀ CFU/g) after wet-ageing depending on initial level of LAB bacteria assuming competition according to the Jameson effect. Conditions for the modelling are shown in Table 3</caption><thead><tr><th rowspan="2">Scenario</th><th rowspan="2">Stage</th><th colspan="5">L. monocytogenes concentrations (log<sub>10</sub> CFU/g)</th></tr><tr><th>Mean</th><th>Median</th><th>75 percentile</th><th>95 percentile</th><th>Maximum</th></tr></thead><tbody><tr><td>Initial concentration<sup>(a)</sup></td><td>Start fresh meat preparation/ ageing</td><td>-1.27</td><td>-1.32</td><td>-0.97</td><td>-0.45</td><td>0.65</td></tr><tr><td>No LAB</td><td>End ageing</td><td>0.80</td><td>0.71</td><td>1.40</td><td>2.57</td><td>5.04</td></tr><tr><td>1 log<sub>10</sub> CFU/g</td><td></td><td>0.76</td><td>0.70</td><td>1.35</td><td>2.38</td><td>4.65</td></tr><tr><td>2 log<sub>10</sub> CFU/g</td><td></td><td>0.69</td><td>0.64</td><td>1.25</td><td>2.18</td><td>4.33</td></tr><tr><td>3 log<sub>10</sub> CFU/g</td><td></td><td>0.52</td><td>0.47</td><td>1.01</td><td>1.86</td><td>3.71</td></tr><tr><td>4 log<sub>10</sub> CFU/g</td><td></td><td>0.21</td><td>0.18</td><td>0.69</td><td>1.47</td><td>3.08</td></tr></tbody></table>

<footnote>(a): LMOV = rnorm, mean = -1.40, sd = 0.55, rtrunc = TRUE, linf = -2, lsup = 1.</footnote>
<figure><img src="image_50.png" /><figcaption>**Figure 19:** Relationship between ageing temperature and maximum time for wet-ageing of lamb corresponding to different $log_{10}$ increases of LAB considered equivalent to standard pork ageing under the assumption of three scenarios of pH and $a_w$, i.e. maximum (pH = 5.9; $a_w$ = 0.99), median (pH = 5.7; $a_w$ = 0.97) or minimum scenarios (pH = 5.5; $a_w$ = 0.95), and a maximum ageing time of 21 days</figcaption></figure>

### 3.6.4. Impact of factors on predictions

#### 3.6.4.1. Competition

The impact of competition from LAB on the potential growth of *L. monocytogenes* was evaluated for different scenarios of initial concentrations of LAB (Table 3). The initial concentration of *L. monocytogenes* is also provided to allow calculation of the predicted $log_{10}$ increase, which was used in other evaluations to quantify the potential growth. There was a clear impact of competition as compared to no competition (No LAB in Table 8) already at a low LAB concentration of 1 $log_{10}$ CFU/g, and especially for the upper percentiles. The predicted 95th-percentile was below 100 CFU/g when the LAB concentration was higher than 3 $log_{10}$ CFU/g but not when it was 2 $log_{10}$ CFU/g (Table 8).

<table><caption>**Table 8:** *L. monocytogenes* predicted final concentrations ($log_{10}$ CFU/g) after wet-ageing depending on initial level of LAB bacteria assuming competition according to the Jameson effect. Conditions for the modelling are shown in Table 3</caption><thead><tr><th rowspan="2">Scenario</th><th rowspan="2">Stage</th><th colspan="5">L. monocytogenes concentrations (log<sub>10</sub> CFU/g)</th></tr><tr><th>Mean</th><th>Median</th><th>75 percentile</th><th>95 percentile</th><th>Maximum</th></tr></thead><tbody><tr><td>Initial concentration<sup>(a)</sup></td><td>Start fresh meat preparation/ ageing</td><td>-1.27</td><td>-1.32</td><td>-0.97</td><td>-0.45</td><td>0.65</td></tr><tr><td>No LAB</td><td>End ageing</td><td>0.80</td><td>0.71</td><td>1.40</td><td>2.57</td><td>5.04</td></tr><tr><td>1 log<sub>10</sub> CFU/g</td><td></td><td>0.76</td><td>0.70</td><td>1.35</td><td>2.38</td><td>4.65</td></tr><tr><td>2 log<sub>10</sub> CFU/g</td><td></td><td>0.69</td><td>0.64</td><td>1.25</td><td>2.18</td><td>4.33</td></tr><tr><td>3 log<sub>10</sub> CFU/g</td><td></td><td>0.52</td><td>0.47</td><td>1.01</td><td>1.86</td><td>3.71</td></tr><tr><td>4 log<sub>10</sub> CFU/g</td><td></td><td>0.21</td><td>0.18</td><td>0.69</td><td>1.47</td><td>3.08</td></tr></tbody></table>

<footnote>(a): LMOV = rnorm, mean = -1.40, sd = 0.55, rtrunc = TRUE, linf = -2, lsup = 1.</footnote>
### 3.6.4.2. Inactivation

The effect of including inactivation in the assessment of the microbial behaviour during meat ageing was evaluated for *L. monocytogenes* on dry-ageing of beef using different predictive models. The conditions used for the evaluation are shown in Table 3. Inactivation was evaluated under the assumption of no-growth conditions, or independent of growth conditions (all times). The predicted log₁₀ increase of *L. monocytogenes* during dry-ageing of beef based on variable temperature, pH, a<sub>w</sub>, and duration with or without inactivation considered in the modelling is shown in Table 9. The greatest impact of inactivation was with the model obtained from Van Damme data assuming inactivation occurring in parallel with growth. Under this scenario there was a mean decrease of 0.26 log₁₀, i.e. corresponding to approximately a halving of the *L. monocytogenes* concentration. The observed log₁₀ reduction after 42 days ranged between approximately 1 to more than 3 logs (Van Damme et al., 2022) contrary to our predictions. However, a log₁₀ increase was also observed in some of the dry-ageing meat samples in that study. The Coroller et al. (2012) model employs parameters that depend on the environmental conditions so the most reasonable outcome from that model is the 'all times-prediction'. The impact of inactivation with this scenario was a decrease around 0.1 log₁₀ except for the maximum log₁₀ increase compared to the prediction with no inactivation (Table 9).

<table><caption>**Table 9:** Predicted log₁₀ change of *L. monocytogenes* during dry-ageing of beef. Log₁₀ increase is shown for two different inactivation models under the assumption of inactivation occurring all times or only under no-growth conditions</caption><thead><tr><th rowspan="2">Scenario</th><th rowspan="2">Model</th><th colspan="6">L. monocytogenes log<sub>10</sub> change</th></tr><tr><th>Mean</th><th>Median</th><th>5th percentile</th><th>75th percentile</th><th>95th percentile</th><th>Maximum</th></tr></thead><tbody><tr><td>No inactivation</td><td>-</td><td>1.02</td><td>0.84</td><td>0</td><td>1.49</td><td>2.68</td><td>7.68</td></tr><tr><td>Inactivation, all times</td><td>Van Damme</td><td>-0.26</td><td>-0.37</td><td>-1.55</td><td>0.27</td><td>1.36</td><td>4.95</td></tr><tr><td>Inactivation, when no growth is predicted</td><td>Van Damme</td><td>0.61</td><td>0.56</td><td>-1.27</td><td>1.35</td><td>2.66</td><td>7.68</td></tr><tr><td>Inactivation, all times</td><td>Coroller</td><td>0.89</td><td>0.72</td><td>-0.13</td><td>1.36</td><td>2.52</td><td>7.39</td></tr><tr><td>Inactivation, when no growth is predicted</td><td>Coroller</td><td>0.97</td><td>0.81</td><td>-0.12</td><td>1.47</td><td>2.68</td><td>7.68</td></tr></tbody></table>

### 3.6.4.3. Rate of drying

Two studies, cases 2 and 5 of the realistic examples (Section 3.3.2.2), reporting temperature, a<sub>w</sub> and pH during dry-ageing of beef were used to evaluate the effect of the rate of drying. These data were used in Section 3.5.3 as input data in the *L. monocytogenes* growth model to predict the log₁₀ increase during dry-ageing. To illustrate the effect of the rate of drying, i.e. the decrease of a<sub>w</sub>, the a<sub>w</sub>-data were used here as reported and the temperature and pH were set at 2.5°C and 5.5, respectively. The rate of drying is dependent on temperature, but temperature and pH were similar in these case studies. Using the original a<sub>w</sub> case study data and the same pH and T, the effect of different rates of a<sub>w</sub> decrease results in a difference in predicted log₁₀ increase of about 1 log₁₀ after 42 days of ageing (Figure 20).
<figure><img src="image_51.png" /></figure>

### 3.6.4.2. Inactivation

The effect of including inactivation in the assessment of the microbial behaviour during meat ageing was evaluated for *L. monocytogenes* on dry-ageing of beef using different predictive models. The conditions used for the evaluation are shown in Table 3. Inactivation was evaluated under the assumption of no-growth conditions, or independent of growth conditions (all times). The predicted log₁₀ increase of *L. monocytogenes* during dry-ageing of beef based on variable temperature, pH, a<sub>w</sub>, and duration with or without inactivation considered in the modelling is shown in Table 9. The greatest impact of inactivation was with the model obtained from Van Damme data assuming inactivation occurring in parallel with growth. Under this scenario there was a mean decrease of 0.26 log₁₀, i.e. corresponding to approximately a halving of the *L. monocytogenes* concentration. The observed log₁₀ reduction after 42 days ranged between approximately 1 to more than 3 logs (Van Damme et al., 2022) contrary to our predictions. However, a log₁₀ increase was also observed in some of the dry-ageing meat samples in that study. The Coroller et al. (2012) model employs parameters that depend on the environmental conditions so the most reasonable outcome from that model is the 'all times-prediction'. The impact of inactivation with this scenario was a decrease around 0.1 log₁₀ except for the maximum log₁₀ increase compared to the prediction with no inactivation (Table 9).

<table><caption>**Table 9:** Predicted log₁₀ change of *L. monocytogenes* during dry-ageing of beef. Log₁₀ increase is shown for two different inactivation models under the assumption of inactivation occurring all times or only under no-growth conditions</caption><thead><tr><th rowspan="2">Scenario</th><th rowspan="2">Model</th><th colspan="6">L. monocytogenes log<sub>10</sub> change</th></tr><tr><th>Mean</th><th>Median</th><th>5th percentile</th><th>75th percentile</th><th>95th percentile</th><th>Maximum</th></tr></thead><tbody><tr><td>No inactivation</td><td>-</td><td>1.02</td><td>0.84</td><td>0</td><td>1.49</td><td>2.68</td><td>7.68</td></tr><tr><td>Inactivation, all times</td><td>Van Damme</td><td>-0.26</td><td>-0.37</td><td>-1.55</td><td>0.27</td><td>1.36</td><td>4.95</td></tr><tr><td>Inactivation, when no growth is predicted</td><td>Van Damme</td><td>0.61</td><td>0.56</td><td>-1.27</td><td>1.35</td><td>2.66</td><td>7.68</td></tr><tr><td>Inactivation, all times</td><td>Coroller</td><td>0.89</td><td>0.72</td><td>-0.13</td><td>1.36</td><td>2.52</td><td>7.39</td></tr><tr><td>Inactivation, when no growth is predicted</td><td>Coroller</td><td>0.97</td><td>0.81</td><td>-0.12</td><td>1.47</td><td>2.68</td><td>7.68</td></tr></tbody></table>

### 3.6.4.3. Rate of drying

Two studies, cases 2 and 5 of the realistic examples (Section 3.3.2.2), reporting temperature, a<sub>w</sub> and pH during dry-ageing of beef were used to evaluate the effect of the rate of drying. These data were used in Section 3.5.3 as input data in the *L. monocytogenes* growth model to predict the log₁₀ increase during dry-ageing. To illustrate the effect of the rate of drying, i.e. the decrease of a<sub>w</sub>, the a<sub>w</sub>-data were used here as reported and the temperature and pH were set at 2.5°C and 5.5, respectively. The rate of drying is dependent on temperature, but temperature and pH were similar in these case studies. Using the original a<sub>w</sub> case study data and the same pH and T, the effect of different rates of a<sub>w</sub> decrease results in a difference in predicted log₁₀ increase of about 1 log₁₀ after 42 days of ageing (Figure 20).
<figure><img src="image_52.png" /><figcaption>**Figure 20:** The predicted log₁₀ increase of *L. monocytogenes* during dry-ageing of beef depending on the change of aw over time. Temperature was assumed to be 2.5°C and pH 5.5. The black line is case study 2 and the red line case study 5</figcaption></figure>

### 3.6.4.4. Trimming

One study reported numbers on dry-aged loin surfaces and freshly trimmed steaks originating from the same loins (Gowda et al., 2022). On average, numbers on the steak surface were 0.8–1.0 log₁₀ lower than on the loin surface before trimming for total psychrotrophic aerobic bacteria, *Pseudomonas* spp., and *Br. thermosphacta*. Moulds were detected on 4 out of 13 loins and only 3 out of 30 steaks. However, numbers are very variable after trimming, and occasionally high numbers are found on steaks. Moreover, *Enterobacteriaceae* were detected on 4 out of 13 loins and 12 out of 30 steaks, in numbers up to 7.4 log₁₀ CFU/cm². *Listeria* spp. were found on 3 steaks but not on any of the loins. Although trimming can potentially reduce bacterial/mould contamination, numbers are generally only slightly lower than numbers on the dry-aged loins, and additional (environmental) contamination may occur during trimming.

Due to the limited data that are available for assessing the effect of trimming, this step in the production of dry-aged beef was not included in the models. Trimming could result in an increase or decrease of pathogens. When trimming is performed hygienically, it will result in a decrease of pathogens, assuming that there is no internal contamination of the meat.

### 3.6.4.5. Storage

Due to the lack of data about the storage conditions of dry-aged, wet-aged and standard beef, pork and lamb, the growth of pathogens/spoilage bacteria during storage was not predicted.

The aw of trimmed dry-aged beef steaks is similar to the aw of standard fresh beef. Assuming that the pH is also similar, the growth of *Listeria monocytogenes* on dry-aged meat during storage is expected to be equal or lower than that on standard fresh meat. However, differences in storage (time, temperature, packaging) and background microbiota may result in different bacterial counts on standard fresh meat, dry-aged beef and wet-aged meat.
<figure><img src="image_52.png" /><figcaption>**Figure 20:** The predicted log₁₀ increase of *L. monocytogenes* during dry-ageing of beef depending on the change of aw over time. Temperature was assumed to be 2.5°C and pH 5.5. The black line is case study 2 and the red line case study 5</figcaption></figure>

### 3.6.4.4. Trimming

One study reported numbers on dry-aged loin surfaces and freshly trimmed steaks originating from the same loins (Gowda et al., 2022). On average, numbers on the steak surface were 0.8–1.0 log₁₀ lower than on the loin surface before trimming for total psychrotrophic aerobic bacteria, *Pseudomonas* spp., and *Br. thermosphacta*. Moulds were detected on 4 out of 13 loins and only 3 out of 30 steaks. However, numbers are very variable after trimming, and occasionally high numbers are found on steaks. Moreover, *Enterobacteriaceae* were detected on 4 out of 13 loins and 12 out of 30 steaks, in numbers up to 7.4 log₁₀ CFU/cm². *Listeria* spp. were found on 3 steaks but not on any of the loins. Although trimming can potentially reduce bacterial/mould contamination, numbers are generally only slightly lower than numbers on the dry-aged loins, and additional (environmental) contamination may occur during trimming.

Due to the limited data that are available for assessing the effect of trimming, this step in the production of dry-aged beef was not included in the models. Trimming could result in an increase or decrease of pathogens. When trimming is performed hygienically, it will result in a decrease of pathogens, assuming that there is no internal contamination of the meat.

### 3.6.4.5. Storage

Due to the lack of data about the storage conditions of dry-aged, wet-aged and standard beef, pork and lamb, the growth of pathogens/spoilage bacteria during storage was not predicted.

The aw of trimmed dry-aged beef steaks is similar to the aw of standard fresh beef. Assuming that the pH is also similar, the growth of *Listeria monocytogenes* on dry-aged meat during storage is expected to be equal or lower than that on standard fresh meat. However, differences in storage (time, temperature, packaging) and background microbiota may result in different bacterial counts on standard fresh meat, dry-aged beef and wet-aged meat.
## 3.7. TOR5: to recommend additional good hygiene practices specific to the production and storage of dry-aged and wet-aged meat, as compared to those relevant for the production and storage of standard fresh meat

### 3.7.1. Food safety assurance

The food safety of standard fresh meat, dry-aged beef and wet-aged meat is assured through the development and implementation of hazard analysis and critical control point (HACCP) and prerequisite programme (PRP) activities, including good hygiene practice (GHP). HACCP targets specific hazards using CCPs. These are defined as a step at which a control can be applied and is essential to prevent or eliminate a hazard or reduce it to an acceptable level (Codex Alimentarius, 2001 (Joint Fao/Who Codex Alimentarius Commission F and Agriculture Organization of the United Nations WHO, 2001)). Each CCP has critical limits (e.g. temperature) and this parameter(s) is constantly monitored with any breach requiring corrective action. GHP is a standardised way of operating which ensures that foodstuffs are produced safely and hygienically.

The HACCP and PRP should be applied at all stages along the meat chain including slaughter, carcass dressing, chilling, fresh meat preparation, dry or wet-ageing and during subsequent storage in catering or retail (Ninios et al., 2014). Indeed, Regulation (EC) No 853/2004 lays down specific hygiene requirements that must be implemented by food businesses handling food of animal origin at all stages of the food chain including the specific hygiene requirements for premises and operations such as production, handling, processing, storage and distribution.

A full description of the HACCP and PRP used in meat plants, butcher shops and/or restaurants is beyond the scope of this Opinion. Moreover, as standard fresh meat preparation and wet-aged meat differ only in the time applied, the control activities for these processes are similar. This section will therefore primarily focus on the current recommendations in the scientific and grey literature for hygienically producing dry-ageing beef, including international guidance on the conditions required during the dry-ageing of beef to minimise bacterial growth and/or prevent mycotoxin production. The use of the predictive modelling outputs in ToR4 to develop critical limits (in terms of time and temperature) for dry-ageing beef and wet-ageing beef, pork and lamb that ensures the growth of pathogenic and spoilage bacteria is no higher than that obtained on standard fresh meat is also presented. Finally, any additional hazards arising from the preparation of minced meat or MSM from dry or wet-aged meat are discussed.

### 3.7.2. Hygienically producing dry-aged beef

Although limited information is available, the following practices have been reported as contributing to the hygienic production of dry-aged beef (Asefa et al., 2010; Perry, 2012; López-Gómez et al., 2013; Park et al., 2018; Rezende-de-Souza et al., 2021).

1) Use fresh meat of good microbial quality. Dark, firm and dry (DFD) meat must not be used as the higher pH would facilitate pathogen growth during the ageing process. Thus, all primals and sub-primals intended for dry-ageing should be checked to ensure the pH is in the usual range of 5.5 to 6.2 and inspected for overall condition.

2) Dry-ageing should be undertaken in a dedicated purpose-built room or chamber, thus avoiding cross contamination from other meat products, and the primals/sub-primals should be aged for the correct period of time. Dry-aged meat products should be fully traceable.

3) The beef should not be placed in the chamber until the required temperature and RH have been achieved (to minimise the opportunity for pathogen growth) and the specific conditions of time, temperature, RH and airflow should be strictly applied and continuously monitored.

4) The meat cuts should be hung from the bone to prevent internal contamination and they should be separated with sufficient space between them to facilitate air flow. If using a shelf, it should be sufficiently perforated to allow for effective air flow and the meat cuts should be turned regularly, using hygienic methods.

5) The highest airflow should be used at the start of the ageing process to facilitate early crust development and reduce the surface aw, thereby restricting bacterial growth. As a general rule, the ageing time should be as short as required to achieve the desired organoleptic properties as longer storage will allow for growth of pathogenic and spoilage bacteria.
## 3.7. TOR5: to recommend additional good hygiene practices specific to the production and storage of dry-aged and wet-aged meat, as compared to those relevant for the production and storage of standard fresh meat

### 3.7.1. Food safety assurance

The food safety of standard fresh meat, dry-aged beef and wet-aged meat is assured through the development and implementation of hazard analysis and critical control point (HACCP) and prerequisite programme (PRP) activities, including good hygiene practice (GHP). HACCP targets specific hazards using CCPs. These are defined as a step at which a control can be applied and is essential to prevent or eliminate a hazard or reduce it to an acceptable level (Codex Alimentarius, 2001 (Joint Fao/Who Codex Alimentarius Commission F and Agriculture Organization of the United Nations WHO, 2001)). Each CCP has critical limits (e.g. temperature) and this parameter(s) is constantly monitored with any breach requiring corrective action. GHP is a standardised way of operating which ensures that foodstuffs are produced safely and hygienically.

The HACCP and PRP should be applied at all stages along the meat chain including slaughter, carcass dressing, chilling, fresh meat preparation, dry or wet-ageing and during subsequent storage in catering or retail (Ninios et al., 2014). Indeed, Regulation (EC) No 853/2004 lays down specific hygiene requirements that must be implemented by food businesses handling food of animal origin at all stages of the food chain including the specific hygiene requirements for premises and operations such as production, handling, processing, storage and distribution.

A full description of the HACCP and PRP used in meat plants, butcher shops and/or restaurants is beyond the scope of this Opinion. Moreover, as standard fresh meat preparation and wet-aged meat differ only in the time applied, the control activities for these processes are similar. This section will therefore primarily focus on the current recommendations in the scientific and grey literature for hygienically producing dry-ageing beef, including international guidance on the conditions required during the dry-ageing of beef to minimise bacterial growth and/or prevent mycotoxin production. The use of the predictive modelling outputs in ToR4 to develop critical limits (in terms of time and temperature) for dry-ageing beef and wet-ageing beef, pork and lamb that ensures the growth of pathogenic and spoilage bacteria is no higher than that obtained on standard fresh meat is also presented. Finally, any additional hazards arising from the preparation of minced meat or MSM from dry or wet-aged meat are discussed.

### 3.7.2. Hygienically producing dry-aged beef

Although limited information is available, the following practices have been reported as contributing to the hygienic production of dry-aged beef (Asefa et al., 2010; Perry, 2012; López-Gómez et al., 2013; Park et al., 2018; Rezende-de-Souza et al., 2021).

1) Use fresh meat of good microbial quality. Dark, firm and dry (DFD) meat must not be used as the higher pH would facilitate pathogen growth during the ageing process. Thus, all primals and sub-primals intended for dry-ageing should be checked to ensure the pH is in the usual range of 5.5 to 6.2 and inspected for overall condition.

2) Dry-ageing should be undertaken in a dedicated purpose-built room or chamber, thus avoiding cross contamination from other meat products, and the primals/sub-primals should be aged for the correct period of time. Dry-aged meat products should be fully traceable.

3) The beef should not be placed in the chamber until the required temperature and RH have been achieved (to minimise the opportunity for pathogen growth) and the specific conditions of time, temperature, RH and airflow should be strictly applied and continuously monitored.

4) The meat cuts should be hung from the bone to prevent internal contamination and they should be separated with sufficient space between them to facilitate air flow. If using a shelf, it should be sufficiently perforated to allow for effective air flow and the meat cuts should be turned regularly, using hygienic methods.

5) The highest airflow should be used at the start of the ageing process to facilitate early crust development and reduce the surface aw, thereby restricting bacterial growth. As a general rule, the ageing time should be as short as required to achieve the desired organoleptic properties as longer storage will allow for growth of pathogenic and spoilage bacteria.
6) The chamber used for dry-ageing of the beef must be carefully cleaned and disinfected between batches including racks, shelves, etc. Moreover, the chamber should be empty during cleaning to avoid cross-contamination with chemical and physical contaminants.

7) Air conditioning refrigeration system components such as evaporative coolers, evaporative condensers and cooling towers should be designed to facilitate effective cleaning and disinfection.

8) Thermometers, relative humidity probes and other equipment monitoring the set conditions in the chamber must be routinely calibrated close to the range at which they are used.

9) The air leaving the evaporator, returning to the evaporator and coming in contact with the beef should be filtered or ultraviolet (UV) treated to eliminate microbial contamination thereby minimising cross contamination.

Trimming of the crust should be carried out in a hygienic manner immediately after removal from the chamber avoiding perforating the meat below the crust. This activity should be performed in an area separate to those used for fresh meat.

The trimming area should be air-conditioned to avoid cross contamination from other meat areas and have dedicated equipment that is hygienically maintained.

The dry crust should not be used for the preparation of other products as it contains most of the microbial contamination unless subsequently treatments such as heat or high pressure are applied to eliminate any pathogens present.

### 3.7.3. Preventing mycotoxin production during the dry-ageing of beef

Mould growth and mycotoxin production during the dry-ageing of beef have been discussed in Section 3.4.1. Based on the limited available literature, the formation of mycotoxins below 5°C is considered very unlikely (Olivier, 2018). Moreover, due to the reduction in aw on the dry-aged meat surface, the probability of moulds producing mycotoxins is further reduced. The Meat and Livestock Australia (MLA, 2019) concluded that using dry-ageing conditions limited to temperatures of -0.5 to 3.0°C with an RH of 75 to 85% for 14-35 days was unlikely to allow for mycotoxin production.

These conditions are also supported by PrimeSafe (Victoria, Australia) who recommend an air temperature in the dry-ageing chamber of -0.5 to 1.0°C (although > 1 to 3°C could be acceptable where the ageing process lasts 7-14 days), a RH of 75 to 85% and an air velocity of 0.2-0.5 m/s for 14-35 days.

### 3.7.4. Using the outputs of ToR4 to establish the critical time and temperature limits for dry-ageing beef and wet-ageing beef, pork and lamb

The modelling analysis undertaken in ToR4 may be used to estimate equivalence in terms of specific bacterial growth during dry-ageing (Section 3.4.2) and wet-ageing (Section 3.4.3). Dry-ageing of beef for 35 days, for example, at a temperature of 3°C will not result in a higher log10 increase in the concentration of *L. monocytogenes* than an assumed 2-log10 increase in standard fresh beef. Thus, using these parameters (3°C for no more than 35 days) the FBO can ensure the growth of *L. monocytogenes* is no higher than would be achieved on standard fresh meat (used as the baseline). In a second example, based on the predictive modelling in ToR4, wet-ageing of beef for 35 days at a temperature of 2°C will not result in a higher log10 increase in the concentration of *L. monocytogenes* than an assumed 2-log10 increase in standard fresh beef. Thus, the FBO can use this time-temperature combination to ensure that the wet-aged beef does not have a higher concentration of *L. monocytogenes* (and all else being equal, present a higher risk to the consumer) than standard fresh beef.

### 3.7.5. Bactericidal treatments for dry and wet-aged meat and consumer information as a PRP

Experimental studies have shown that hot acid solutions (Blagojevic et al., 2015) or high-pressure processing (Witte et al., 2020) will reduce the bacterial load on the trimmings resulting from dry-aged meat (Blagojevic et al., 2015; Witte et al., 2020). However, other considerations, such as effect on the organoleptic qualities of the meat and cost under commercial conditions may inhibit application. Both dry and wet-aged meats are usually cooked before consumption and if cooked thoroughly this final preparation step will eliminate the bacterial non-spore-forming pathogens such as *Salmonella* spp. and STEC. However, aged meats may on occasion be insufficiently cooked or eaten raw
6) The chamber used for dry-ageing of the beef must be carefully cleaned and disinfected between batches including racks, shelves, etc. Moreover, the chamber should be empty during cleaning to avoid cross-contamination with chemical and physical contaminants.

7) Air conditioning refrigeration system components such as evaporative coolers, evaporative condensers and cooling towers should be designed to facilitate effective cleaning and disinfection.

8) Thermometers, relative humidity probes and other equipment monitoring the set conditions in the chamber must be routinely calibrated close to the range at which they are used.

9) The air leaving the evaporator, returning to the evaporator and coming in contact with the beef should be filtered or ultraviolet (UV) treated to eliminate microbial contamination thereby minimising cross contamination.

Trimming of the crust should be carried out in a hygienic manner immediately after removal from the chamber avoiding perforating the meat below the crust. This activity should be performed in an area separate to those used for fresh meat.

The trimming area should be air-conditioned to avoid cross contamination from other meat areas and have dedicated equipment that is hygienically maintained.

The dry crust should not be used for the preparation of other products as it contains most of the microbial contamination unless subsequently treatments such as heat or high pressure are applied to eliminate any pathogens present.

### 3.7.3. Preventing mycotoxin production during the dry-ageing of beef

Mould growth and mycotoxin production during the dry-ageing of beef have been discussed in Section 3.4.1. Based on the limited available literature, the formation of mycotoxins below 5°C is considered very unlikely (Olivier, 2018). Moreover, due to the reduction in aw on the dry-aged meat surface, the probability of moulds producing mycotoxins is further reduced. The Meat and Livestock Australia (MLA, 2019) concluded that using dry-ageing conditions limited to temperatures of -0.5 to 3.0°C with an RH of 75 to 85% for 14-35 days was unlikely to allow for mycotoxin production.

These conditions are also supported by PrimeSafe (Victoria, Australia) who recommend an air temperature in the dry-ageing chamber of -0.5 to 1.0°C (although > 1 to 3°C could be acceptable where the ageing process lasts 7-14 days), a RH of 75 to 85% and an air velocity of 0.2-0.5 m/s for 14-35 days.

### 3.7.4. Using the outputs of ToR4 to establish the critical time and temperature limits for dry-ageing beef and wet-ageing beef, pork and lamb

The modelling analysis undertaken in ToR4 may be used to estimate equivalence in terms of specific bacterial growth during dry-ageing (Section 3.4.2) and wet-ageing (Section 3.4.3). Dry-ageing of beef for 35 days, for example, at a temperature of 3°C will not result in a higher log10 increase in the concentration of *L. monocytogenes* than an assumed 2-log10 increase in standard fresh beef. Thus, using these parameters (3°C for no more than 35 days) the FBO can ensure the growth of *L. monocytogenes* is no higher than would be achieved on standard fresh meat (used as the baseline). In a second example, based on the predictive modelling in ToR4, wet-ageing of beef for 35 days at a temperature of 2°C will not result in a higher log10 increase in the concentration of *L. monocytogenes* than an assumed 2-log10 increase in standard fresh beef. Thus, the FBO can use this time-temperature combination to ensure that the wet-aged beef does not have a higher concentration of *L. monocytogenes* (and all else being equal, present a higher risk to the consumer) than standard fresh beef.

### 3.7.5. Bactericidal treatments for dry and wet-aged meat and consumer information as a PRP

Experimental studies have shown that hot acid solutions (Blagojevic et al., 2015) or high-pressure processing (Witte et al., 2020) will reduce the bacterial load on the trimmings resulting from dry-aged meat (Blagojevic et al., 2015; Witte et al., 2020). However, other considerations, such as effect on the organoleptic qualities of the meat and cost under commercial conditions may inhibit application. Both dry and wet-aged meats are usually cooked before consumption and if cooked thoroughly this final preparation step will eliminate the bacterial non-spore-forming pathogens such as *Salmonella* spp. and STEC. However, aged meats may on occasion be insufficiently cooked or eaten raw
(da Silva et al., 2019) to achieve a specific desired flavour (Lee et al., 2021), to increase the tenderness, decrease the cooking loss (Latorre et al., 2019) or simply in error (Suman et al., 2014). Raw meat products, such as, carpaccio or raw kibbeh, are popular in different regions and should include a warning about the risk associated with consumption.

### 3.7.6. Specific considerations for minced meat of MSM preparation from dry or wet-aged meat

As previously stated, when chilled (not frozen) meat is used for minced meat it must be prepared either within no more than 6 days after the slaughter of the ungulates, or within 15 days in the case of boned, vacuum-packed beef and veal (Point 2(b) of Chapter III to Section V of Annex III to Regulation (EC) No 853/2004). Before the production of mechanically separated meat, the maximum storage period of the (chilled) raw material can be no more than 7 days when derived from the on-site slaughterhouse and 5 days in other cases (Point 3(a) and 4(a) of Chapter III to Section V of Annex III to Regulation (EC) No 853/2004). Thus, meat that is aged for more than 14 days or more is not currently allowed to be used for the production of minced meat or MSM. These regulations are based on the assumption that older meat will have higher concentrations of bacteria due to growth during chilled storage and some of these may be pathogenic to humans.

Minced meat and MSM may be considered to present a higher food safety risk to consumers as bacteria on the meat surface are mixed into the core during mincing and MSM preparation, thereby increasing the probability of survival if the meat product is not thoroughly cooked. Thus, limiting the opportunity for bacterial growth by restricting the time between slaughter and minced meat or MSM preparation would seem a sensible approach. However, there is limited and contradictory data as to whether longer (ageing) times necessarily results in higher bacterial counts. James et al. (2006) reviewed the available scientific evidence and concluded there was no scientific basis for the restrictions on the age of meat used to produce minced meat. A second review by James and James (2012) concluded that total viable, *E. coli* and *Enterobacteriaceae* counts from meat after mincing do not increase with the length of storage time prior to mincing. Beef, pork, and lamb (from the UK and New Zealand) were aged for up to 59, 18, 26, 67 days, respectively, after slaughter, before being minced. Total viable counts (TVC) in minced meat varied between 1 and 5.9 log10 CFU/g for beef, between 2.6 and 8.0 log10 CFU/g for pork, and between 2.7 and 8.5 for lamb and there was no correlation between the bacterial concentration in the minced meat and the duration of ageing. In contrast, Garner et al. (2014) reported a mean TVC of 2.9 log10 CFU/g in beef patties prepared from meat matured for 7 days which increased to 3.9–4.7 log10 CFU after 21 days and 6.4–7.2 log10 CFU after 42 days.

Regardless of the bacterial counts, if the minced meat or MSM are thoroughly cooked any bacterial pathogens such as STEC, *Salmonella* or *L. monocytogenes* will be killed. Thus, using meat that is aged for longer periods than applied to standard fresh meat and/or trimmings from dry-aged meat should not present a higher risk to the consumer if proper cooking procedures are consistently applied.

## 4. Conclusions

**ToR1: AQ1:** What are the practices and processes used by meat FBOs and restaurants in the EU for the dry-ageing of beef and the wet-ageing of beef, pork and lamb? Specifically, what are the processing conditions (e.g. time, temperature and RH) and the associated intrinsic factors (e.g. pH and $a_w$) of meat surface for each of these processes?

* In the EU, FBOs and restaurants usually dry-age beef aerobically in dedicated chambers at 1–4°C and a RH of 75–85% for 21–35 days. Scientific studies are in broad agreement with the majority undertaken at 0–4°C, a RH of 70–80% and an airflow of 0.5–2.5 m/s for 14–35 days.

* Wet-ageing is an anaerobic process, with vacuum packaged primal and sub-primal cuts vacuum packaged and stored at 0–4°C for 14–49 days (beef), 0–4°C for 4–6 days (pork) or –1 to 5°C for 7–77 days (lamb) in meat plants. The conditions used in scientific studies were similar with most using temperatures of 0.6–4°C, a RH of 75–85% and an air flow of 0.2–7.0 m/s for 21–35 days.

* The shelf-life for unpacked dry-aged beef steaks was typically 4 days but ranged from 2 to 10 days. If packaged in modified atmosphere the shelf-life was 5 days and if vacuum packed the shelf-life increased to 18 days but ranged from 5 to 30 days. The shelf-life for wet-aged beef, pork and lamb was 3–5 days for unpacked steaks/chops and 10–14 days for vacuum packaged products. All of the above assume adequate chilled storage conditions at 0–4°C.
(da Silva et al., 2019) to achieve a specific desired flavour (Lee et al., 2021), to increase the tenderness, decrease the cooking loss (Latorre et al., 2019) or simply in error (Suman et al., 2014). Raw meat products, such as, carpaccio or raw kibbeh, are popular in different regions and should include a warning about the risk associated with consumption.

### 3.7.6. Specific considerations for minced meat of MSM preparation from dry or wet-aged meat

As previously stated, when chilled (not frozen) meat is used for minced meat it must be prepared either within no more than 6 days after the slaughter of the ungulates, or within 15 days in the case of boned, vacuum-packed beef and veal (Point 2(b) of Chapter III to Section V of Annex III to Regulation (EC) No 853/2004). Before the production of mechanically separated meat, the maximum storage period of the (chilled) raw material can be no more than 7 days when derived from the on-site slaughterhouse and 5 days in other cases (Point 3(a) and 4(a) of Chapter III to Section V of Annex III to Regulation (EC) No 853/2004). Thus, meat that is aged for more than 14 days or more is not currently allowed to be used for the production of minced meat or MSM. These regulations are based on the assumption that older meat will have higher concentrations of bacteria due to growth during chilled storage and some of these may be pathogenic to humans.

Minced meat and MSM may be considered to present a higher food safety risk to consumers as bacteria on the meat surface are mixed into the core during mincing and MSM preparation, thereby increasing the probability of survival if the meat product is not thoroughly cooked. Thus, limiting the opportunity for bacterial growth by restricting the time between slaughter and minced meat or MSM preparation would seem a sensible approach. However, there is limited and contradictory data as to whether longer (ageing) times necessarily results in higher bacterial counts. James et al. (2006) reviewed the available scientific evidence and concluded there was no scientific basis for the restrictions on the age of meat used to produce minced meat. A second review by James and James (2012) concluded that total viable, *E. coli* and *Enterobacteriaceae* counts from meat after mincing do not increase with the length of storage time prior to mincing. Beef, pork, and lamb (from the UK and New Zealand) were aged for up to 59, 18, 26, 67 days, respectively, after slaughter, before being minced. Total viable counts (TVC) in minced meat varied between 1 and 5.9 log10 CFU/g for beef, between 2.6 and 8.0 log10 CFU/g for pork, and between 2.7 and 8.5 for lamb and there was no correlation between the bacterial concentration in the minced meat and the duration of ageing. In contrast, Garner et al. (2014) reported a mean TVC of 2.9 log10 CFU/g in beef patties prepared from meat matured for 7 days which increased to 3.9–4.7 log10 CFU after 21 days and 6.4–7.2 log10 CFU after 42 days.

Regardless of the bacterial counts, if the minced meat or MSM are thoroughly cooked any bacterial pathogens such as STEC, *Salmonella* or *L. monocytogenes* will be killed. Thus, using meat that is aged for longer periods than applied to standard fresh meat and/or trimmings from dry-aged meat should not present a higher risk to the consumer if proper cooking procedures are consistently applied.

## 4. Conclusions

**ToR1: AQ1:** What are the practices and processes used by meat FBOs and restaurants in the EU for the dry-ageing of beef and the wet-ageing of beef, pork and lamb? Specifically, what are the processing conditions (e.g. time, temperature and RH) and the associated intrinsic factors (e.g. pH and $a_w$) of meat surface for each of these processes?

* In the EU, FBOs and restaurants usually dry-age beef aerobically in dedicated chambers at 1–4°C and a RH of 75–85% for 21–35 days. Scientific studies are in broad agreement with the majority undertaken at 0–4°C, a RH of 70–80% and an airflow of 0.5–2.5 m/s for 14–35 days.

* Wet-ageing is an anaerobic process, with vacuum packaged primal and sub-primal cuts vacuum packaged and stored at 0–4°C for 14–49 days (beef), 0–4°C for 4–6 days (pork) or –1 to 5°C for 7–77 days (lamb) in meat plants. The conditions used in scientific studies were similar with most using temperatures of 0.6–4°C, a RH of 75–85% and an air flow of 0.2–7.0 m/s for 21–35 days.

* The shelf-life for unpacked dry-aged beef steaks was typically 4 days but ranged from 2 to 10 days. If packaged in modified atmosphere the shelf-life was 5 days and if vacuum packed the shelf-life increased to 18 days but ranged from 5 to 30 days. The shelf-life for wet-aged beef, pork and lamb was 3–5 days for unpacked steaks/chops and 10–14 days for vacuum packaged products. All of the above assume adequate chilled storage conditions at 0–4°C.
* Commercial data on the pH and $a_w$ is lacking. The data published in the scientific literature (based on samples usually taken at the start and/or end of the ageing process) reported that the surface pH of the beef was usually 5.5-5.9 and the $a_w$ 0.95-0.99. The corresponding pH values for wet-aged beef, pork and lamb were 5.1-5.9, 5.4-6.3 and 5.5-5.9, respectively. The $a_w$ values for wet-aged meat ranged from 0.93 to 0.99, regardless of species.

**ToR2: AQ2:** What are the relevant microbiological hazards and spoilage bacteria that occur and which of these can grow and/or produce toxins during the dry-ageing of beef and the wet-ageing of beef, pork and lamb and on the subsequently stored product, including in minced meat or MSM prepared from the aged meat?

* The pathogenic bacteria that may be present on dry-aged beef and/or wet-aged beef, pork and lamb include Shiga toxin-producing *E. coli* (STEC) (more common in beef), *Salmonella* spp., *Staphylococcus aureus*, *L. monocytogenes*, enterotoxigenic *Yersinia* spp. (usually pork), *Campylobacter* spp. and *Clostridium* spp. However, only *L. monocytogenes* and *Y. enterocolitica* are capable of growth on the meat surface under chilled (0-4°C) conditions. All of these pathogenic bacteria are relevant as although some may not grow under the conditions of ageing, they may survive dry-ageing and wet-ageing processes and may therefore be present in minced meat or MSM prepared from these meats.

* The spoilage bacteria include Pseudomonads, the former *Lactobacillus* spp., *Micrococcus* spp., *Enterococcus* spp., *Weissella* spp., *Brochothrix* spp., *Leuconostoc* spp., *Enterococcus* spp., *Lactococcus* spp., *Shewanella* spp., *Bacillus* spp. and *Clostridium* spp., many of which are capable of growth during the ageing process. *Pseudomonas* spp. are the main spoilage bacteria on dry-aged beef while *Lactobacillus* spp. and psychrotrophic/psychrophilic *Clostridium* spp. are the main spoilage bacteria of wet-aged beef, pork and lamb.

* *Thamnidium* spp., *Pilaira anomala*, *Debaryomyces hansenii*, *Aspergillus* spp., *Cladosporium* spp., *Trametes gibbosa*, *Alternaria alternate*, *Polyporales* spp., *Nectriaceae* spp., *Penicillium roquefortii*, *Pleosporaceae* spp., *Ascomycota* spp., *Colletotrichum acutatum*, *Podospora anserine*, *Penicillium bialowiezense*, *Candida* spp. and *Penicillium polonicum* have all been detected on dry-aged beef. Moulds such as *Aspergillus* spp. and *Penicillium* spp. may grow on beef during the dry-ageing process. These moulds may produce mycotoxins on fruit, grain and other crops at low temperatures, but this has not been reported for meat products. It was judged 60-90% certain that a meat surface temperature of -0.5 to 3.0°C, with a RH of 75-85% and an airflow of 0.2-0.5 m/s will prevent mycotoxin production at least up to 35 days.

**ToR3: AQ3:** What is the increase in the relevant microorganisms (from AQ2) during the dry-ageing of beef and the wet-ageing of beef, pork and lamb (from AQ1) and during subsequent storage, as compared to 'standard fresh meat'?

* Relevant values for the intrinsic (pH and $a_w$) and extrinsic (temperature) factors, as well as ageing time, were identified and used to develop scenarios covering current practices for dry-ageing of beef, and wet-ageing of beef, pork and lamb. Scenario parameters were used as input in predictive models to estimate the potential $\log_{10}$ increases of pathogenic and spoilage bacteria during the preparation of standard fresh meat and ageing of meat.

* Standard fresh meat from beef, pork and lamb is not defined in the legislation, and was defined here as typically anaerobic maturation for up to 14 days (beef) or 4 days (pork and lamb).

* Other parameters in the standard fresh meat preparation such as temperature, pH and $a_w$ vary between producers and thus, the $\log_{10}$ increase during standard fresh meat preparation is variable depending on conditions of the process and the dynamic properties of the meat during the process. In addition, there is uncertainty associated with both the predicted $\log_{10}$ increases during ageing and standard fresh meat preparation, due to parameter, model and scenario uncertainties.

* Based on the predicted growth, the main microbiological hazard to consider is *L. monocytogenes* for all meat types, and for pork also *Y. enterocolitica*. The main spoilage bacteria are LAB for all meat types, and, for dry-aged beef, also psychrotrophic *Pseudomonas*.

* Considering the scenarios of temperature, pH and $a_w$ resulting in minimum, median and maximum growth rate of *L. monocytogenes*, the predicted ranges of $\log_{10}$ increases of *L. monocytogenes* were estimated (Appendix G).
* Commercial data on the pH and $a_w$ is lacking. The data published in the scientific literature (based on samples usually taken at the start and/or end of the ageing process) reported that the surface pH of the beef was usually 5.5-5.9 and the $a_w$ 0.95-0.99. The corresponding pH values for wet-aged beef, pork and lamb were 5.1-5.9, 5.4-6.3 and 5.5-5.9, respectively. The $a_w$ values for wet-aged meat ranged from 0.93 to 0.99, regardless of species.

**ToR2: AQ2:** What are the relevant microbiological hazards and spoilage bacteria that occur and which of these can grow and/or produce toxins during the dry-ageing of beef and the wet-ageing of beef, pork and lamb and on the subsequently stored product, including in minced meat or MSM prepared from the aged meat?

* The pathogenic bacteria that may be present on dry-aged beef and/or wet-aged beef, pork and lamb include Shiga toxin-producing *E. coli* (STEC) (more common in beef), *Salmonella* spp., *Staphylococcus aureus*, *L. monocytogenes*, enterotoxigenic *Yersinia* spp. (usually pork), *Campylobacter* spp. and *Clostridium* spp. However, only *L. monocytogenes* and *Y. enterocolitica* are capable of growth on the meat surface under chilled (0-4°C) conditions. All of these pathogenic bacteria are relevant as although some may not grow under the conditions of ageing, they may survive dry-ageing and wet-ageing processes and may therefore be present in minced meat or MSM prepared from these meats.

* The spoilage bacteria include Pseudomonads, the former *Lactobacillus* spp., *Micrococcus* spp., *Enterococcus* spp., *Weissella* spp., *Brochothrix* spp., *Leuconostoc* spp., *Enterococcus* spp., *Lactococcus* spp., *Shewanella* spp., *Bacillus* spp. and *Clostridium* spp., many of which are capable of growth during the ageing process. *Pseudomonas* spp. are the main spoilage bacteria on dry-aged beef while *Lactobacillus* spp. and psychrotrophic/psychrophilic *Clostridium* spp. are the main spoilage bacteria of wet-aged beef, pork and lamb.

* *Thamnidium* spp., *Pilaira anomala*, *Debaryomyces hansenii*, *Aspergillus* spp., *Cladosporium* spp., *Trametes gibbosa*, *Alternaria alternate*, *Polyporales* spp., *Nectriaceae* spp., *Penicillium roquefortii*, *Pleosporaceae* spp., *Ascomycota* spp., *Colletotrichum acutatum*, *Podospora anserine*, *Penicillium bialowiezense*, *Candida* spp. and *Penicillium polonicum* have all been detected on dry-aged beef. Moulds such as *Aspergillus* spp. and *Penicillium* spp. may grow on beef during the dry-ageing process. These moulds may produce mycotoxins on fruit, grain and other crops at low temperatures, but this has not been reported for meat products. It was judged 60-90% certain that a meat surface temperature of -0.5 to 3.0°C, with a RH of 75-85% and an airflow of 0.2-0.5 m/s will prevent mycotoxin production at least up to 35 days.

**ToR3: AQ3:** What is the increase in the relevant microorganisms (from AQ2) during the dry-ageing of beef and the wet-ageing of beef, pork and lamb (from AQ1) and during subsequent storage, as compared to 'standard fresh meat'?

* Relevant values for the intrinsic (pH and $a_w$) and extrinsic (temperature) factors, as well as ageing time, were identified and used to develop scenarios covering current practices for dry-ageing of beef, and wet-ageing of beef, pork and lamb. Scenario parameters were used as input in predictive models to estimate the potential $\log_{10}$ increases of pathogenic and spoilage bacteria during the preparation of standard fresh meat and ageing of meat.

* Standard fresh meat from beef, pork and lamb is not defined in the legislation, and was defined here as typically anaerobic maturation for up to 14 days (beef) or 4 days (pork and lamb).

* Other parameters in the standard fresh meat preparation such as temperature, pH and $a_w$ vary between producers and thus, the $\log_{10}$ increase during standard fresh meat preparation is variable depending on conditions of the process and the dynamic properties of the meat during the process. In addition, there is uncertainty associated with both the predicted $\log_{10}$ increases during ageing and standard fresh meat preparation, due to parameter, model and scenario uncertainties.

* Based on the predicted growth, the main microbiological hazard to consider is *L. monocytogenes* for all meat types, and for pork also *Y. enterocolitica*. The main spoilage bacteria are LAB for all meat types, and, for dry-aged beef, also psychrotrophic *Pseudomonas*.

* Considering the scenarios of temperature, pH and $a_w$ resulting in minimum, median and maximum growth rate of *L. monocytogenes*, the predicted ranges of $\log_{10}$ increases of *L. monocytogenes* were estimated (Appendix G).
* Under current practices, ageing of meat has an impact on the load of microbiological hazards and spoilage bacteria compared to standard fresh meat preparation. The extent and direction of the impact depend on the conditions of ageing, the properties of the meat, and the presence of competing microorganisms.

* Ageing under defined and controlled conditions can achieve similar or lower loads of microbiological hazards and spoilage bacteria than the variable $log_{10}$ increases predicted for standard fresh meat preparation.

**ToR4: AQ4:** What are the conditions for producing, handling (including trimming, cutting, packaging, etc.) and storing dry-aged beef and wet-aged beef, pork and lamb to ensure similar or lower counts/load/concentrations of pathogenic microorganisms, spoilage bacteria and, if relevant, mycotoxins at the end of shelf-life as compared to standard fresh meat?

* As the $log_{10}$ increase in standard fresh meat varies depending on the conditions used, the conditions for meat ageing to achieve equivalent growth are provided for a given $log_{10}$ increase in standard fresh meat.

* Based on the predicted $log_{10}$ increases during standard fresh meat preparation, conditions of time and temperature during ageing that result in an increase of up to 0.5, 1, 2, 3 or 4 $log_{10}$ of pathogenic or spoilage bacteria were evaluated using developed relationships between predicted time and temperature under different scenarios of pH and $a_w$.

* The impact of sources of uncertainties due to, for instance the effects of microbial competition between spoilage and pathogenic bacteria (wet-ageing), inactivation (dry-ageing), trimming and storage, and the variable and dynamic (over time) pH and $a_w$ was considered and assessed using informal EKE.

* Examples of conditions ensuring an equivalent $log_{10}$ increase of *L. monocytogenes* as the most relevant pathogen under the scenario of medium pH and $a_w$ in the meat are assessed as:

* Dry-ageing beef:

* It was judged 80-90% certain that dry-ageing of beef for 35 days at a temperature of 3°C will not result in a higher $log_{10}$ increase in the concentration of *L. monocytogenes* than an assumed $2-log_{10}$ increase in standard fresh beef.

* Wet-ageing beef

* It was judged 66-90% certain that wet-ageing of beef for 35 days at a temperature of 2°C will not result in a higher $log_{10}$ increase in the concentration of *L. monocytogenes* than an assumed $2-log_{10}$ increase in standard fresh beef.

* Wet-ageing pork

* It was judged 66-90% certain that wet-ageing of pork for 10 days at a temperature of 3°C will not result in a higher $log_{10}$ increase in the concentration of *L. monocytogenes* than an assumed $1-log_{10}$ increase in standard fresh pork.

* Wet-ageing lamb

* It was judged 66-90% certain that wet-ageing of lamb for 10 days at a temperature of 3°C will not result in a higher $log_{10}$ increase in the concentration of *L. monocytogenes* than an assumed $1-log_{10}$ increase in standard fresh lamb.

**ToR5: AQ5:** What additional control actions including prerequisite programme (GHP and GMP) and CCPs could be employed to minimise the prevalence and/or concentration of pathogenic and spoilage bacteria and mycotoxin formation (if relevant) on dry and wet-aged meat?

* Twelve currently used or recommended PRP activities are provided that should be used to prevent contamination and minimise the growth of pathogens and spoilage bacteria.

* Conditions for the dry-ageing of beef that prevent mycotoxin production are provided in the answers to Tor2 (3rd bullet point).

* The only difference between standard fresh beef, pork and lamb and wet-aged beef, pork and lamb is the time used for ageing. The same prerequisite activities and HACCP required for standard fresh meat should therefore be applied for wet-aged meat. Moulds do not grow on wet-aged meat and mycotoxin production is therefore not an issue.
* Under current practices, ageing of meat has an impact on the load of microbiological hazards and spoilage bacteria compared to standard fresh meat preparation. The extent and direction of the impact depend on the conditions of ageing, the properties of the meat, and the presence of competing microorganisms.

* Ageing under defined and controlled conditions can achieve similar or lower loads of microbiological hazards and spoilage bacteria than the variable $log_{10}$ increases predicted for standard fresh meat preparation.

**ToR4: AQ4:** What are the conditions for producing, handling (including trimming, cutting, packaging, etc.) and storing dry-aged beef and wet-aged beef, pork and lamb to ensure similar or lower counts/load/concentrations of pathogenic microorganisms, spoilage bacteria and, if relevant, mycotoxins at the end of shelf-life as compared to standard fresh meat?

* As the $log_{10}$ increase in standard fresh meat varies depending on the conditions used, the conditions for meat ageing to achieve equivalent growth are provided for a given $log_{10}$ increase in standard fresh meat.

* Based on the predicted $log_{10}$ increases during standard fresh meat preparation, conditions of time and temperature during ageing that result in an increase of up to 0.5, 1, 2, 3 or 4 $log_{10}$ of pathogenic or spoilage bacteria were evaluated using developed relationships between predicted time and temperature under different scenarios of pH and $a_w$.

* The impact of sources of uncertainties due to, for instance the effects of microbial competition between spoilage and pathogenic bacteria (wet-ageing), inactivation (dry-ageing), trimming and storage, and the variable and dynamic (over time) pH and $a_w$ was considered and assessed using informal EKE.

* Examples of conditions ensuring an equivalent $log_{10}$ increase of *L. monocytogenes* as the most relevant pathogen under the scenario of medium pH and $a_w$ in the meat are assessed as:

* Dry-ageing beef:

* It was judged 80–90% certain that dry-ageing of beef for 35 days at a temperature of 3°C will not result in a higher $log_{10}$ increase in the concentration of *L. monocytogenes* than an assumed $2-log_{10}$ increase in standard fresh beef.

* Wet-ageing beef

* It was judged 66–90% certain that wet-ageing of beef for 35 days at a temperature of 2°C will not result in a higher $log_{10}$ increase in the concentration of *L. monocytogenes* than an assumed $2-log_{10}$ increase in standard fresh beef.

* Wet-ageing pork

* It was judged 66–90% certain that wet-ageing of pork for 10 days at a temperature of 3°C will not result in a higher $log_{10}$ increase in the concentration of *L. monocytogenes* than an assumed $1-log_{10}$ increase in standard fresh pork.

* Wet-ageing lamb

* It was judged 66–90% certain that wet-ageing of lamb for 10 days at a temperature of 3°C will not result in a higher $log_{10}$ increase in the concentration of *L. monocytogenes* than an assumed $1-log_{10}$ increase in standard fresh lamb.

**ToR5: AQ5:** What additional control actions including prerequisite programme (GHP and GMP) and CCPs could be employed to minimise the prevalence and/or concentration of pathogenic and spoilage bacteria and mycotoxin formation (if relevant) on dry and wet-aged meat?

* Twelve currently used or recommended PRP activities are provided that should be used to prevent contamination and minimise the growth of pathogens and spoilage bacteria.

* Conditions for the dry-ageing of beef that prevent mycotoxin production are provided in the answers to Tor2 (3rd bullet point).

* The only difference between standard fresh beef, pork and lamb and wet-aged beef, pork and lamb is the time used for ageing. The same prerequisite activities and HACCP required for standard fresh meat should therefore be applied for wet-aged meat. Moulds do not grow on wet-aged meat and mycotoxin production is therefore not an issue.
* The modelling analysis undertaken in ToR4 (Sections 3.4.2 and 3.4.3) provides the time and temperature combinations that could be used for dry-ageing of beef and wet-ageing of beef, pork and lamb to ensure bacterial growth is equivalent or less than that which would be achieved during standard fresh meat preparation. This approach could be used by FBOs to further enhance the safety of aged meat products.

* It is currently not possible to conclude on the relative food safety of minced meat and MSM prepared from dry or wet-aged meat, as compared to standard fresh meat due to the lack of information on the impact of the time between slaughter, chilled storage and minced meat or MSM preparation on bacterial growth and the limited microbiological data on bacterial counts in minced meat and MSM prepared from aged meat (more than 14 days). However, if the minced meat (including trimmings from dry-aged beef) or MSM are thoroughly cooked any vegetative bacterial pathogens such as STEC, *Salmonella* or *L. monocytogenes* will be eliminated.

**5. Recommendations**

* Research is required to establish the exact conditions under which moulds such as *Aspergillus* spp. and *Penicillium* spp. produce mycotoxins including challenge studies on dry-aged meat using a range of different combinations of temperature, RH, airflow and time that achieve varying combinations of surface temperature, *a*<sub>w</sub> and pH. The information generated should inform future food safety control systems for the dry-ageing of beef.

* Research is also required on the effect of the time between slaughter and minced meat or MSM preparation on the bacterial counts, including psychrotrophic and psychrophilic pathogenic bacteria. This could inform a risk assessment of any additional risks associated with minced meat and MSM prepared from dry-aged beef or wet-aged beef, pork or lamb, which would, in turn, facilitate a review of current legislation (Regulation (EC) No 853/2004).

* Challenge tests are required to assess the growth of pathogens such as *L. monocytogenes* during different conditions of dry-ageing of beef and wet-ageing of beef, pork and lamb and during subsequent storage to validate the predictions in ToR3 and ToR4.

**References**

ACMSF, 2020. The safety and shelf-life of vacuum and modified atmosphere packed chilled foods with respect to non-proteolytic *Clostridium botulinum*. Acta Biologica Szegediensis. Available online: http://www2.sci.u-szeged.hu/ABS

Adam KH, Flint SH and Brightwell G, 2010. Psychrophilic and psychrotrophic clostridia: sporulation and germination processes and their role in the spoilage of chilled, vacuum-packaged beef, lamb and venison. International Journal of Food Science and Technology, 45, 1539–1544. https://doi.org/10.1111/j.1365-2621.2010.02320.x

Adams MR, Little CL and Easter MC, 1991. Modelling the effect of pH, acidulant and temperature on the growth rate of *Yersinia enterocolitica*. Journal of Applied Bacteriology, 71, 65–71. https://doi.org/10.1111/j.1365-2672.1991.tb04664.x

Ahnstrom ML, Seyfert M, Hunt MC and Johnson DE, 2006. Dry aging of beef in a bag highly permeable to water vapour. Meat Science, 73, 674–679. https://doi.org/10.1016/j.meatsci.2006.03.006

Anon, 2006. Technology plays a key role. Fleischwirtschaft, 86:43-44Baranyi J, Pin C and Ross T, 1999. Validating and comparing predictive models. International Journal of Food Microbiology, 48, 159–166. https://doi.org/10.1016/S0168-1605(99)00035-5

Asefa DT, Kure CF, Gjerde RO, Omer MK, Langsrud S, Nesbakken T and Skaar I, 2010. Fungal growth pattern, sources and factors of mould contamination in a dry-cured meat production facility. International Journal of Food Microbiology, 140, 131–135. https://doi.org/10.1016/j.ijfoodmicro.2010.04.008

Asefa DT, Kure CF, Gjerde RO, Langsrud S, Omer MK, Nesbakken T and Skaar I, 2011. A HACCP plan for mycotoxigenic hazards associated with dry-cured meat production processes. Food Control, 22, 831–837. https://doi.org/10.1016/j.foodcont.2010.09.014

Baranyi J and Roberts TA, 1994. A dynamic approach to predicting bacterial growth in food. International Journal of Food Microbiology, 23, 277–294. https://doi.org/10.1016/0168-1605(94)90157-0

Baranyi J, Pin C and Ross T, 1999. Validating and comparing predictive models. International Journal of Food Microbiology, 48, 159–166. https://doi.org/10.1016/S0168-1605(99)00035-5

Barrera O, Rodríguez-Calleja JM, Santos JA, Otero A and García-López M-L, 2007. Effect of different storage conditions on *E. coli* O157:H7 and the indigenous bacterial microflora on lamb meat. International Journal of Food Microbiology, 115, 244–251. https://doi.org/10.1016/j.ijfoodmicro.2006.10.053

Battilani P, Palumbo R, Giorni P, Dall'Asta C, Dellafiora L, Gkrillas A, Toscano P, Crisci A, Brera C, De Santis B, Cammarano R, Seta M, Campbell K, Elliot C, Venâncio A, Gonçalves A, Terciolo C and Oswald I, 2020. Mycotoxin mixtures in food and feed: holistic, innovative, flexible risk assessment modelling approach: MYCHIF. EFSA Supporting Publications 2020:EN-1757. https://doi.org/10.2903/sp.efsa.2020.EN-1757
* The modelling analysis undertaken in ToR4 (Sections 3.4.2 and 3.4.3) provides the time and temperature combinations that could be used for dry-ageing of beef and wet-ageing of beef, pork and lamb to ensure bacterial growth is equivalent or less than that which would be achieved during standard fresh meat preparation. This approach could be used by FBOs to further enhance the safety of aged meat products.

* It is currently not possible to conclude on the relative food safety of minced meat and MSM prepared from dry or wet-aged meat, as compared to standard fresh meat due to the lack of information on the impact of the time between slaughter, chilled storage and minced meat or MSM preparation on bacterial growth and the limited microbiological data on bacterial counts in minced meat and MSM prepared from aged meat (more than 14 days). However, if the minced meat (including trimmings from dry-aged beef) or MSM are thoroughly cooked any vegetative bacterial pathogens such as STEC, *Salmonella* or *L. monocytogenes* will be eliminated.

## 5. Recommendations

* Research is required to establish the exact conditions under which moulds such as *Aspergillus* spp. and *Penicillium* spp. produce mycotoxins including challenge studies on dry-aged meat using a range of different combinations of temperature, RH, airflow and time that achieve varying combinations of surface temperature, *a*<sub>w</sub> and pH. The information generated should inform future food safety control systems for the dry-ageing of beef.

* Research is also required on the effect of the time between slaughter and minced meat or MSM preparation on the bacterial counts, including psychrotrophic and psychrophilic pathogenic bacteria. This could inform a risk assessment of any additional risks associated with minced meat and MSM prepared from dry-aged beef or wet-aged beef, pork or lamb, which would, in turn, facilitate a review of current legislation (Regulation (EC) No 853/2004).

* Challenge tests are required to assess the growth of pathogens such as *L. monocytogenes* during different conditions of dry-ageing of beef and wet-ageing of beef, pork and lamb and during subsequent storage to validate the predictions in ToR3 and ToR4.

## References

ACMSF, 2020. The safety and shelf-life of vacuum and modified atmosphere packed chilled foods with respect to non-proteolytic *Clostridium botulinum*. Acta Biologica Szegediensis. Available online: http://www2.sci.u-szeged.hu/ABS

Adam KH, Flint SH and Brightwell G, 2010. Psychrophilic and psychrotrophic clostridia: sporulation and germination processes and their role in the spoilage of chilled, vacuum-packaged beef, lamb and venison. International Journal of Food Science and Technology, 45, 1539–1544. https://doi.org/10.1111/j.1365-2621.2010.02320.x

Adams MR, Little CL and Easter MC, 1991. Modelling the effect of pH, acidulant and temperature on the growth rate of *Yersinia enterocolitica*. Journal of Applied Bacteriology, 71, 65–71. https://doi.org/10.1111/j.1365-2672.1991.tb04664.x

Ahnstrom ML, Seyfert M, Hunt MC and Johnson DE, 2006. Dry aging of beef in a bag highly permeable to water vapour. Meat Science, 73, 674–679. https://doi.org/10.1016/j.meatsci.2006.03.006

Anon, 2006. Technology plays a key role. Fleischwirtschaft, 86:43-44Baranyi J, Pin C and Ross T, 1999. Validating and comparing predictive models. International Journal of Food Microbiology, 48, 159–166. https://doi.org/10.1016/S0168-1605(99)00035-5

Asefa DT, Kure CF, Gjerde RO, Omer MK, Langsrud S, Nesbakken T and Skaar I, 2010. Fungal growth pattern, sources and factors of mould contamination in a dry-cured meat production facility. International Journal of Food Microbiology, 140, 131–135. https://doi.org/10.1016/j.ijfoodmicro.2010.04.008

Asefa DT, Kure CF, Gjerde RO, Langsrud S, Omer MK, Nesbakken T and Skaar I, 2011. A HACCP plan for mycotoxigenic hazards associated with dry-cured meat production processes. Food Control, 22, 831–837. https://doi.org/10.1016/j.foodcont.2010.09.014

Baranyi J and Roberts TA, 1994. A dynamic approach to predicting bacterial growth in food. International Journal of Food Microbiology, 23, 277–294. https://doi.org/10.1016/0168-1605(94)90157-0

Baranyi J, Pin C and Ross T, 1999. Validating and comparing predictive models. International Journal of Food Microbiology, 48, 159–166. https://doi.org/10.1016/S0168-1605(99)00035-5

Barrera O, Rodríguez-Calleja JM, Santos JA, Otero A and García-López M-L, 2007. Effect of different storage conditions on *E. coli* O157:H7 and the indigenous bacterial microflora on lamb meat. International Journal of Food Microbiology, 115, 244–251. https://doi.org/10.1016/j.ijfoodmicro.2006.10.053

Battilani P, Palumbo R, Giorni P, Dall'Asta C, Dellafiora L, Gkrillas A, Toscano P, Crisci A, Brera C, De Santis B, Cammarano R, Seta M, Campbell K, Elliot C, Venâncio A, Gonçalves A, Terciolo C and Oswald I, 2020. Mycotoxin mixtures in food and feed: holistic, innovative, flexible risk assessment modelling approach: MYCHIF. EFSA Supporting Publications 2020:EN-1757. https://doi.org/10.2903/sp.efsa.2020.EN-1757
Benech N, Aguiar S and Grinspan GA, 2021. Monitoring ageing in beef samples using surface wave elastography: a feasibility study. Journal of Food Engineering, 307, 110647. https://doi.org/10.1016/j.jfoodeng.2021.110647

Bennett JW and Klich M, 2003. Mycotoxins. Microbiology and Molecular Biology Reviews, 16, 497–516. https://doi.org/10.1128/CMR.16.3.497-516.2003

Berger J, Kim YHB, Legako JF, Martini S, Lee J, Ebner P and Zuelly SMS, 2018. Dry-aging improves meat quality attributes of grass-fed beef loins. Meat Science, 145, 285–291. https://doi.org/10.1016/j.meatsci.2018.07.004

Beriain MJ, Goni MV, Indurain G, Sarries MV and Insausti K, 2009. Predicting *Longissimus dorsi* myoglobin oxidation in aged beef based on early post-mortem colour measurements on the carcass as a colour stability index. Meat Science, 81, 439–445. https://doi.org/10.1016/j.meatsci.2008.09.009

Bhattacharjee P, Panigrahi S, Lin D, Logue CM, Sherwood JS, Doetkott C and Marchello M, 2011. A comparative qualitative study of the profile of volatile organic compounds associated with *Salmonella* contamination of packaged aged and fresh beef by HS-SPME/GC-MS. Journal of Food Science and Technology-Mysore, 48, 1–13. https://doi.org/10.1007/s13197-010-0138-6

Blagojevic B, Antic D, Adzic B, Tasic T, Ikonic P and Buncic S, 2015. Decontamination of incoming beef trimmings with hot lactic acid solution to improve microbial safety of resulting dry fermented sausages – a pilot study. Food Control, 54, 144–149. https://doi.org/10.1016/j.foodcont.2015.01.047

Bodnaruk PW and Draughon FA, 1998. Effect of packaging atmosphere and pH on the virulence and growth of *Yersinia enterocolitica* on pork stored at 4°C. Food Microbiology, 15, 129–136. https://doi.org/10.1006/fmic.1997.0150

Bolton DJ, Ivory C and McDowell D, 2013. A small study of *Yersinia enterocolitica* in pigs from birth to carcass and characterisation of porcine and human strains. Food Control, 33, 521–524. https://doi.org/10.1016/j.foodcont.2013.03.039

Bolton DJ, Carroll J and Walsh D, 2015. A four-year survey of blown pack spoilage C lostridium estertheticum and C lostridium gasigenes on beef primal cuts. Letters in Applied Microbiology, 61, 153–157.

Borch E, Kant-Muermans ML and Blixt Y, 1996. Bacterial spoilage of meat and cured meat products. International Journal of Food Microbiology, 33, 103–120. https://doi.org/10.1016/0168-1605(96)01135-X

Bover-Cid S, Jofré A, Possas A, Comaposada J, Brun A, Zomeño C, Masferrer G and Panella-Riera N, 2022. Assessing the microbiological safety of beef aging process: Impact of temperature and relative humidity conditions. Poster presented at the 27th International ICFMH Conference: FoodMicro2022 (ref. P6.7); 28–31 August 2022, Athens Greece.

Broda DM, Lawson PA, Bell RG and Musgrave DR, 1999. *Clostridium frigidicarnis* sp. nov., a psychrotolerant bacterium associated with 'blown pack' spoilage of vacuum-packed meats. International Journal of Systematic Bacteriology, 49 Pt 4, 1539–1550. https://doi.org/10.1099/00207713-49-4-1539

Broda DM, Lawson PA, Bell RG and Musgrave DR, 2000. *Clostridium gasigenes* sp. nov., a psychrophile causing spoilage of vacuum-packed meat. International Journal of Systematic and Evolutionary Microbiology, 50 Pt 1, 107–118. https://doi.org/10.1099/00207713-50-1-107

Brunt J, van Vliet AHM, Stringer SC, Carter AT, Lindström M and Peck MW, 2020. Pan-Genomic Analysis of *Clostridium botulinum* Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment. Toxins, 12, 306. https://doi.org/10.3390/toxins12050306

Buchanan RL and Bagi LK, 1999. Microbial competition: effect of *Pseudomonas fluorescens* on the growth of *Listeria monocytogenes*. Food Microbiology, 16, 523–529. https://doi.org/10.1006/fmic.1998.0264

Buchanan JR, Sommer NF, Fortlage RJ, Maxie EC, Mitchell FG and Hsieh DPH, 1974. Patulin from *Penicillium expansum* in stone fruits and pears. Journal of the American Society for Horticultural Science, 99, 262–265.

Campano SG, Kotula AW and Kinsman DM, 1985. Antibacterial nature of molds isolated from aged beef. Journal of Food Protection, 48, 699–701. https://doi.org/10.4315/0362-028X-48.8.699

Campbell RE, Hunt MC, Levis P and Chambers E, 2001. Dry-aging effects on palatability of beef *longissimus* muscle. Journal of Food Science, 66, 196–199. https://doi.org/10.1111/j.1365-2621.2001.tb11315.x

Capouya R, Mitchell T, Clark DI, Clark DL, Bass P, Capouya RD and Bass PD, 2020. A survey of microbial communities on dry-aged beef in commercial meat processing facilities. Meat and Muscle Biology, 4.

Cardoso TAB, Bridi AM, Fagan EP, Tarsitano MA, Bolfe FC, Perez LM and Prohmann PEF, 2012. Quality of aged meat from Charolais vs Nellore cattle. Ciências Agrárias, Londrina, 33(suplemento 2), 3123–3132. https://doi.org/10.5433/1679-0359.2012v33Supl2p3123

Carrel M, Zhao C, Thapaliya D, Bitterman P, Kates AE, Hanson BM and Smith TC, 2017. Assessing the potential for raw meat to influence human colonization with *Staphylococcus aureus*. Scientific Reports, 7, 10848. https://doi.org/10.1038/s41598-017-11423-6

Carter AT and Peck MW, 2015. Genomes, neurotoxins and biology of *Clostridium botulinum* Group I and Group II. Research in Microbiology, 166, 303–317. https://doi.org/10.1016/j.resmic.2014.10.010

Cavill L, Renteria-Monterrubio AL, Helps CR and Corry JEL, 2011. Detection of cold-tolerant clostridia other than *Clostridium estertheticum* in raw vacuum-packed chill-stored meat. Food Microbiology, 28, 957–963. https://doi.org/10.1016/j.fm.2011.01.003

Ciegler A and Kurtzman CP, 1970. Penicillic acid production by blue-eye fungi on various agricultural commodities. Applied Microbiology, 20, 761–764. https://doi.org/10.5433/1679-0359.2012v33Supl2p3437
Benech N, Aguiar S and Grinspan GA, 2021. Monitoring ageing in beef samples using surface wave elastography: a feasibility study. Journal of Food Engineering, 307, 110647. https://doi.org/10.1016/j.jfoodeng.2021.110647

Bennett JW and Klich M, 2003. Mycotoxins. Microbiology and Molecular Biology Reviews, 16, 497-516. https://doi.org/10.1128/CMR.16.3.497-516.2003

Berger J, Kim YHB, Legako JF, Martini S, Lee J, Ebner P and Zuelly SMS, 2018. Dry-aging improves meat quality attributes of grass-fed beef loins. Meat Science, 145, 285-291. https://doi.org/10.1016/j.meatsci.2018.07.004

Beriain MJ, Goni MV, Indurain G, Sarries MV and Insausti K, 2009. Predicting *Longissimus dorsi* myoglobin oxidation in aged beef based on early post-mortem colour measurements on the carcass as a colour stability index. Meat Science, 81, 439-445. https://doi.org/10.1016/j.meatsci.2008.09.009

Bhattacharjee P, Panigrahi S, Lin D, Logue CM, Sherwood JS, Doetkott C and Marchello M, 2011. A comparative qualitative study of the profile of volatile organic compounds associated with *Salmonella* contamination of packaged aged and fresh beef by HS-SPME/GC-MS. Journal of Food Science and Technology-Mysore, 48, 1-13. https://doi.org/10.1007/s13197-010-0138-6

Blagojevic B, Antic D, Adzic B, Tasic T, Ikonic P and Buncic S, 2015. Decontamination of incoming beef trimmings with hot lactic acid solution to improve microbial safety of resulting dry fermented sausages - a pilot study. Food Control, 54, 144-149. https://doi.org/10.1016/j.foodcont.2015.01.047

Bodnaruk PW and Draughon FA, 1998. Effect of packaging atmosphere and pH on the virulence and growth of *Yersinia enterocolitica* on pork stored at 4°C. Food Microbiology, 15, 129-136. https://doi.org/10.1006/fmic.1997.0150

Bolton DJ, Ivory C and McDowell D, 2013. A small study of *Yersinia enterocolitica* in pigs from birth to carcass and characterisation of porcine and human strains. Food Control, 33, 521-524. https://doi.org/10.1016/j.foodcont.2013.03.039

Bolton DJ, Carroll J and Walsh D, 2015. A four-year survey of blown pack spoilage C lostridium estertheticum and C lostridium gasigenes on beef primal cuts. Letters in Applied Microbiology, 61, 153-157.

Borch E, Kant-Muermans ML and Blixt Y, 1996. Bacterial spoilage of meat and cured meat products. International Journal of Food Microbiology, 33, 103-120. https://doi.org/10.1016/0168-1605(96)01135-X

Bover-Cid S, Jofré A, Possas A, Comaposada J, Brun A, Zomeño C, Masferrer G and Panella-Riera N, 2022. Assessing the microbiological safety of beef aging process: Impact of temperature and relative humidity conditions. Poster presented at the 27th International ICFMH Conference: FoodMicro2022 (ref. P6.7); 28-31 August 2022, Athens Greece.

Broda DM, Lawson PA, Bell RG and Musgrave DR, 1999. *Clostridium frigidicarnis* sp. nov., a psychrotolerant bacterium associated with 'blown pack' spoilage of vacuum-packed meats. International Journal of Systematic Bacteriology, 49 Pt 4, 1539-1550. https://doi.org/10.1099/00207713-49-4-1539

Broda DM, Lawson PA, Bell RG and Musgrave DR, 2000. *Clostridium gasigenes* sp. nov., a psychrophile causing spoilage of vacuum-packed meat. International Journal of Systematic and Evolutionary Microbiology, 50 Pt 1, 107-118. https://doi.org/10.1099/00207713-50-1-107

Brunt J, van Vliet AHM, Stringer SC, Carter AT, Lindström M and Peck MW, 2020. Pan-Genomic Analysis of *Clostridium botulinum* Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment. Toxins, 12, 306. https://doi.org/10.3390/toxins12050306

Buchanan RL and Bagi LK, 1999. Microbial competition: effect of *Pseudomonas fluorescens* on the growth of *Listeria monocytogenes*. Food Microbiology, 16, 523-529. https://doi.org/10.1006/fmic.1998.0264

Buchanan JR, Sommer NF, Fortlage RJ, Maxie EC, Mitchell FG and Hsieh DPH, 1974. Patulin from *Penicillium expansum* in stone fruits and pears. Journal of the American Society for Horticultural Science, 99, 262-265.

Campano SG, Kotula AW and Kinsman DM, 1985. Antibacterial nature of molds isolated from aged beef. Journal of Food Protection, 48, 699-701. https://doi.org/10.4315/0362-028X-48.8.699

Campbell RE, Hunt MC, Levis P and Chambers E, 2001. Dry-aging effects on palatability of beef *longissimus* muscle. Journal of Food Science, 66, 196-199. https://doi.org/10.1111/j.1365-2621.2001.tb11315.x

Capouya R, Mitchell T, Clark DI, Clark DL, Bass P, Capouya RD and Bass PD, 2020. A survey of microbial communities on dry-aged beef in commercial meat processing facilities. Meat and Muscle Biology, 4.

Cardoso TAB, Bridi AM, Fagan EP, Tarsitano MA, Bolfe FC, Perez LM and Prohmann PEF, 2012. Quality of aged meat from Charolais vs Nellore cattle. Ciências Agrárias, Londrina, 33(suplemento 2), 3123-3132. https://doi.org/10.5433/1679-0359.2012v33Supl2p3123

Carrel M, Zhao C, Thapaliya D, Bitterman P, Kates AE, Hanson BM and Smith TC, 2017. Assessing the potential for raw meat to influence human colonization with *Staphylococcus aureus*. Scientific Reports, 7, 10848. https://doi.org/10.1038/s41598-017-11423-6

Carter AT and Peck MW, 2015. Genomes, neurotoxins and biology of *Clostridium botulinum* Group I and Group II. Research in Microbiology, 166, 303-317. https://doi.org/10.1016/j.resmic.2014.10.010

Cavill L, Renteria-Monterrubio AL, Helps CR and Corry JEL, 2011. Detection of cold-tolerant clostridia other than *Clostridium estertheticum* in raw vacuum-packed chill-stored meat. Food Microbiology, 28, 957-963. https://doi.org/10.1016/j.fm.2011.01.003

Ciegler A and Kurtzman CP, 1970. Penicillic acid production by blue-eye fungi on various agricultural commodities. Applied Microbiology, 20, 761-764. https://doi.org/10.5433/1679-0359.2012v33Supl2p3437