Analysis of the relationship between air cavities and Weissella viridescens in meat products and possibilities for air cavity elimination

Josef Kameník, Marta Dušková University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic

Abstract The cavities in cooked hams are not necessarily associated with bacterial contamination and the growth of bacteria. If we disregard the mechanical way of cavity formation, in which air gets into the meat and penetrates between the muscle fibres in pork, the bacterial origin of cavities is associated with shortcomings in the production process. A large number of genera and species of lactic acid bacteria (LAB) have been isolated from spoiled meat and meat products. Heat treatment plays an important role in the selection of bacteria that may be brought into the product with the used raw material (meat) or additives, or from the production environment. However, cooking is not always effective when it comes to thermo- tolerant vegetative bacteria. The very important species in meat processing is Weissella viridescens which may contribute to the spoilage of meat products. As weissellas are, to a certain extent, thermo-resistant, they could survive cooking process. In the scientific literature, isolated cases have been described in which weissellas contributed to the formation of cavities in cooked hams. However, LAB of the genus Leuconostoc were isolated from these products far more often than weissellas. W. viridescens has also been described as spoilers for other groups of meat products, such as hot smoked dry sausages.

Key Words: cooked meat products; cross contamination; lactic acid bacteria; Leuconostoc spp.; Weissella spp.

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Contents 1. Lactic acid bacteria: characteristics, occurrence and importance in foods………....…….. 3 2. Lactic acid bacteria: their occurrence and importance in meat…………………………… 7 2.1. Lactic acid bacteria and packaged meat……………………………………………… 8 2.2. The importance of selected genera of LAB in meat spoilage………………………. 11 3. Lactic acid bacteria: their occurrence and importance in cooked meat products……...... 13 3.1. Sources of lactic acid bacteria in products………………………………………….. 13 3.2. Lactic acid bacteria and comminuted meat products……………………………….. 14 3.3. Lactic acid bacteria and cooked hams……………………………………………… 16 4. Weissella viridescens in meat products……………………...……………. …………..... 19 4.1. Methods of identifying Weissella spp…………………………….………………… 21 4.2. The occurrence of Weissella spp…………………………………………………… 22 4.3. Weissella viridescens in meat products…………………………………………….. 22 5. Cavities inside whole-muscle meat products: their causes and significance……...…….. 30 5.1. Bacteria as a possible cause of cavities in whole-muscle meat products…………... 33 6. Conclusions………………………………………………………….…………………... 36 References…………………………………………………………………….……………... 37

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1. Lactic acid bacteria: characteristics, occurrence and importance in foods Lactic acid bacteria (LAB) are among the most widely studied bacterial groups at the present time. We can find them as agents of the spoilage of many foods and drinks, such as cooked meat products, mayonnaises, cheeses, salad dressings, beer, etc. Certain LAB may have virulence factors and belong among pathogens studied in human and veterinary medicine (e.g. certain representatives of the genus Streptococcus). The participation of LAB in the fermentation of foods of animal and plant origin is, however, far more widely known. They are one of most important groups of bacteria industrially and their use in fermentation processes has an extremely long history (Giraffa, 2014). The first starter culture was probably used for industrial purposes in Denmark, Germany and the USA in 1890 for the production of cheese and fermented milk (Holzapfel and Wood, 2014). LAB are a group of functionally related bacteria that are capable of producing lactic acid by homofermentative and/or heterofermentative metabolism (Ruiz et al., 2014). LAB are gram- positive bacteria of the phylum Firmicutes with a “low” G+C ratio (≤ 55 mol %) in the DNA (Holzapfel and Wood, 2014). The phylum Firmicutes takes in a number of classes. LAB are assigned to the class Bacilli. Two orders – Bacillales and Lactobacillales – belong to the class Bacilli. The order Lactobacillales currently has 6 families and 40 genera. This order is comprised of LAB characterised by enormous diversity. The six families are:

• Aerococcaceae (7 genera, e.g. Aerococcus)

• Carnobacteriaceae (16 genera, e.g. Carnobacterium)

• Enterococcaceae (7 genera, e.g. Enterococcus, Tetragenococcus, Vagococcus)

• Lactobacillaceae (3 genera, Lactobacillus, Paralactobacillus, Pediococcus)

• Leuconostocaceae (4 genera, e.g. Leuconostoc, Oenococcus, Weissella)

• Streptococcaceae (3 genera, Lactococcus, Lactovum, Streptococcus)

Bacterial groups that have a similar metabolism, but which are not phylogenetically related, are sometimes classified among LAB. A typical example is the genus Bifidobacterium, though it belongs to a different phylum – Actinobacteria. This phylum includes microorganisms with a G+C ratio in the DNA of ≥ 55 mol % (Stolaki et al., 2012). LAB are demanding microorganisms that require rich nutrients for growth, such as saccharides, amino acids, vitamins and minerals. Certain LAB require special growth factors, such as tomato juice, whey, etc. LAB decompose a large number of saccharides and related compounds by means of various metabolic pathways. ATP is generated by substrate-level phosphorylation and serves for the transport of dissolved substances across the cell membrane and for the purposes of biosynthesis. Environmental conditions influence the metabolic pathways used (Endo and Dicks, 2014). The phenotypic characteristics of LAB include a positive reaction to Gram staining, the absence of endospores, a negative oxidase and catalase

3 reaction (Leroi, 2010), a saccharide fermentation model, production of D(-) and L(+) lactic acid, aesculin and arginine hydrolysis, nitrate reduction, gelatin liquefaction, growth under various conditions of temperature, pH and NaCl content, and tolerance to oxygen. As LAB do not have a functional respiratory system, they have to obtain energy by the phosphorylation of suitable substrates (Wright and Axelsson, 2012). Two metabolic pathways exist in LAB depending on the fermentation of glucose – homofermentative and heterofermentative (Endo and Dicks, 2014). Homofermentative LAB use glycolysis – the Embden-Meyerhof-Parnas pathway (EMP, Figure 1). Species of LAB that exhibit a homofermentative metabolism produce more than 85 % of their lactic acid from hexoses (Salvetti et al., 2013). The EMP is characterised by the formation of fructose-1,6-diphosphate (FDP), which is split by the enzyme FDP aldolase into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). GAP (and also DHAP via GAP) is subsequently converted into pyruvic acid (Pot and Tsakalidou, 2009). Heterofermentative LAB use the phosphoketolase pathway (the pentose phosphate pathway, PPP, Figure 2). In addition to lactic acid, the heterofermentative pathway also produces a significant quantity of carbon dioxide and ethanol or acetic acid (Wright and Axelsson, 2012). The choice of metabolic pathway is determined at the family level (Endo and Dicks, 2014). The glycolytic pathway is used by representatives of the families Enterococcaceae, Lactobacillaceae and Streptococcaceae, with the exception of one group in the genus Lactobacillus. This pathway converts glucose into lactic acid (2 molecules of lactic acid to 1 molecule of glucose). Two molecules of ATP are generated from one molecule of glucose. Representatives of the family Leuconostocaceae and a number of species in the genus Lactobacillus metabolise glucose by the phosphoketolase pathway, during which one molecule of lactic acid, one molecule of CO2 and one molecule of ethanol are released from one molecule of glucose. The formation of ethanol from acetyl phosphate, during which NADH is oxidised into NAD+, is important in this pathway. This reaction is associated with the speed with which glucose is fermented, and thereby with the intensity of growth of heterofermentative LAB. LAB generally produce D(-) and L(+) lactic acid. The ratio at which these isomers are released is almost identical among strains of the same species and is considered one of the key characteristics for the classification of LAB into subgroups. Nevertheless, not all species respect this characteristic. LAB from the family Carnobacteriaceae produce lactic acid, formic acid, acetic acid and ethanol from glucose. CO2 is not produced, with the exception of Carnobacterium spp. The ratios of metabolites differ depending on the bacteria, though the main end products are lactic acid and formic acid.

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Figure 1: Glycolysis (Embden-Meyerhof-Parnas pathway). The diagram indicates the involvement of certain enzymes – a: glucokinase, b: fructose-1,6-diphosphate aldolase, c: lactate dehydrogenase (source: Endo and Dicks, 2014) The metabolism of disaccharides includes transport across the cell membrane either as free molecules or following phosphorylation. This is followed by cleavage into two monosaccharide molecules or a molecule of monosaccharide and a molecule of monosaccharide phosphate. Certain LAB prefer disaccharides to monosaccharides as growth substrates. Examples include the fermentation of lactose by dairy LAB or fermentation of maltose by dough LAB. LAB are divided into three groups according to the metabolic pathway used for the fermentation of glucose and the metabolism of pentoses (Endo and Dicks, 2014). The first group is defined as obligate homofermentative; these ferment glucose exclusively into lactic acid by a glycolytic pathway. They are not able to ferment pentoses and related compounds. Just a few species of the genus Lactobacillus belong to this group (known as Group I lactobacilli).

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Figure 2: Heterofermentation of glucose. The diagram indicates the involvement of certain enzymes – a: glucokinase, d: glucose-6-phosphate dehydrogenase, e: 6-phosphogluconate dehydrogenase, f: phosphoketolase, g: acetate kinase, h: acetaldehyde dehydrogenase, i: alcohol dehydrogenase, j: pentose isomerase, k: ribulokinase, l: transketolase, m: transaldolase (source: Endo and Dicks, 2014) The second group, facultative heterofermentative LAB, ferment glucose exclusively into lactic acid by a glycolytic pathway and ferment pentoses along with related compounds by a phosphoketolase pathway. This group includes, e.g., the genera Enterococcus, Lactococcus, Pediococcus and Streptococcus and Group II lactobacilli. Representatives of the family Carnobacteriaceae probably metabolise glucose into pyruvate using a glycolytic pathway, and pyruvate is subsequently converted into lactic acid, formic acid, acetic acid and ethanol with or without CO2. In view of their ability to metabolise pentoses they are classified as facultative heterofermentative bacteria. The third group, obligate heterofermentative LAB, metabolises glucose, pentoses and related compounds using a phosphoketolase pathway. The genera Leuconostoc, Oenococcus and Weissella and Group III lactobacilli are found in this group. LAB play two roles in the food industry. Their metabolism produces certain substances that help create an internal environment that leads to positive changes in fermented foodstuffs (dairy products, fermented sausages, fermented dough, sauerkraut, gherkins, pickled olives, kimchi, etc.). On the other hand, their growth and metabolic products can also cause

6 undesirable changes in foods leading to their spoilage (e.g. cooked meat products, packaged meat, beer).

2. Lactic acid bacteria: their occurrence and importance in meat Food spoilage induced by microorganisms is caused by their metabolism which releases substances leading to sensory changes in the product (Remenant et al., 2015). In the presence of oxygen (air), gram-negative aerobic bacteria capable of manifesting signs of spoilage in a relatively short period multiply rapidly in foods rich in nutrients. Preservative food packaging (a vacuum/VP/ or modified atmosphere/MAP) and refrigeration have been developed to suppress their growth (Pothakos et al., 2014). Bacteria colonise the surface of the carcass almost immediately during the course of the slaughter process. The body surface (skin and hair), the content of the digestive tract and the abattoir environment, including the staff and the tools they use, can all be sources of microbes (Petruzzelli et al., 2016). Cross contamination of the carcass surface, i.e. the meat, occurs not merely by direct contact with the outer surface of the skin during incorrect handling during carcass processing, but also through the air when dust particles or drops of water contaminated by bacteria from soiled hair or skin settle on the carcass surface (Okraszska- Lasica et al., 2012). The surface area increases with continuing cutting, portioning and slicing of the meat, thereby providing increasing opportunity for massive colonisation by microorganisms. The bacteria occurring most frequently on fresh meat are bacteria of the genera Acinetobacter, Pseudomonas, Brochothrix, Flavobacterium, Janthinobacterium, Psychrobacter, Moraxella, Staphylococcus and Micrococcus, as well as LAB and various species of the Enterobacteriaceae family (Jääskeläinen et al., 2016; Kaur et al., 2017). Representatives of the genus Pseudomonas and the family Enterobacteriaceae are associated with the metabolism of amino acids and lipids, while LAB and Brochothrix thermosphacta are associated with the metabolism of saccharides (Stellato et al., 2016). The character of meat spoilage and off-odours caused by the release of volatile substances – products of bacterial metabolism – are derived from this. Psychrotrophic LAB are present on meat stored at refrigeration temperatures along with other microbial groups and species. They often form a mixed community of species that dominates the overall bacterial population after a few days or weeks (Lucquin et al., 2012). LAB may be the cause, or are considered agents, of meat spoilage. Nevertheless, certain representatives of this group are also described as competitive microbiota that controls the growth of undesirable bacteria causing spoilage or alimentary disease. The role of LAB in microbial stability and meat quality is, for this reason, controversial. Pothakos et al. (2015a) consider LAB a microbial population that can release undesirable metabolites, with subsequent organoleptic deterioration of meat, though strains of certain species serve as bioprotective agents that, in contrast, suppress microorganisms causing spoilage. This means that large numbers of LAB in meat do not always lead to qualitative abnormalities.

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A large number of genera and species of LAB have been isolated from spoiled meat and meat products. The genera of LAB most often represented in microbial populations causing the spoilage of food in a modified atmosphere are Carnobacterium, Lactobacillus, Lactococcus, Leuconostoc and Weissella (Andreevskaya et al., 2015). The species of LAB occurring most often on beef meat are Lb. sakei and Leuc. mesenteroides. Other representatives of this group include Lb. curvatus, Leuc. carnosum and Carnobacterium divergens (Lucquin et al., 2012). Lb. sakei is considered a spoilage agent in vacuum-packed meat and meat packaged in a modified atmosphere. It is clear from the expert literature that only psychrotrophic LAB, such as the species Lb. sakei, Lb. curvatus, Lb. fuchuensis, Carn. divergens and Carn. maltaromaticum and Leuconostoc spp., can reach large numbers in MAP and VP meat stored at refrigeration temperatures. Species such as Lb. sakei and Lb. algidus have been isolated from vacuum-packed meat at 4 °C. If the temperature drops to 1 °C, dominance is obtained by Lactobacillus spp., Weissella spp. and Leuconostoc mesenteroides. The effect of temperature on the composition of microbiota is evident in this change under otherwise identical conditions (Doulgeraki et al., 2012).

2.1. Lactic acid bacteria and packaged meat Nieminen et al. (2011) tested changes to the population of LAB in minced meat at 6 °C packaged in an MAP (60 % O2, 25 % CO2 and 10 % residual air). The number of LAB reached 8.5–8.7 log CFU/g during the course of storage for a period of 10 days. Minced meat stored at 4 °C showed a population of LAB of 6.9–7.1 log CFU/g and a population of Brochothrix thermosphacta of 1.7–2.5 log CFU/g after 11 days. The number of bacteria increased still further after 19 days – to 8.6–8.8 log CFU/g for LAB and 7.8–7.9 log CFU/g for B. thermosphacta. In contrast, the number of representatives of the family Enterobacteriaceae did not exceed a value of 4.0 log CFU/g even after the 19 days. The species Lb. algidus, Lb. sakei, Leuc. gasicomitatum, Leuc. carnosum, Leuc. gelidum, Leuc. mesenteroides, Carn. divergens, Carn. maltaromaticum and Enterococcus raffinosus were isolated from the LAB population. Pennacchia et al. (2011) analysed 9 samples of beef sirloin of a portion size of 500 g. After the initial contamination was determined, the samples were divided into two groups – one group was vacuum-packed, the second stored under aerobic conditions at 4 °C. Carn. divergens, Brochothrix thermosphacta, Pseudomonas spp. and Psychrobacter spp. were present most frequently on the meat at the beginning of the experiment. B. thermosphacta, Pseudomonas spp. and Photobacterium spp. dominated after seven days of storage of unpackaged samples. Carn. divergens and Pseudomonas spp. were detected in vacuum- packed beef, with B. thermosphacta not isolated in any cases. The results were practically identical under aerobic conditions after 20 days as after 7 days – Pseudomonas spp. (though not in all samples), followed most frequently by B. thermosphacta, Carn. divergens and Photobacterium spp. The species Carn. divergens was present in all samples of vacuum- packed meat, Photobacterium spp. present in 8 cases and Lb. algidus in 7 cases. The population of Pseudomonas spp. in unpackaged samples after 20 days of storage amounted to

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5.6–8.1 log CFU/g. Sensory investigation determined that none of the samples stored under aerobic conditions were eatable after 7 days. Five of the nine samples of vacuum-packed beef slices were acceptable in sensory terms after 20 days, with the remaining 4 displaying an unacceptable aroma and appearance. While the LAB population amounted to 4.3–6.6 log CFU/g in the samples acceptable in sensory terms, it amounted to as much as 8.0 log CFU/g in the samples given a negative assessment. Jääskeläinen et al. (2016) analysed samples of beef meat that were vacuum-packed or packaged in a modified atmosphere (80 % O2, 20 % CO2) and stored at 6 °C. MAP favoured the growth of heterofermentative species and the amount of volatile organic substances was higher than it was for vacuum-packing. The LAB population reached a maximum after 9 days of storage (> 8 log CFU/g) and became the dominant microbiota in both VP and MAP meat. Sensory evaluation showed 2 samples of MAP beef to be spoiled after 9 days and 1 after 14 days of packaging. Samples of VP beef (all 3 samples) showed signs of spoilage after 26 days. Kameník et al. (2014) analysed fresh beef and pork (m. longissimus lumborum) packed in VP and MAP and stored at 2.0±0.5 °C. The total bacterial count on day 35 of storage of beef meat fell within a range of 6.3–7.8 log CFU/g with no significant differences between individual forms of packaging. The bacterial population in pork meat on day 21 fell within a range of 5.0–6.0 log CFU/g. Differences between the individual types of packaging were, however, seen in the representation of selected bacterial groups. From day 14 of storage, VP meat samples showed a LAB population two log higher than meat packed in a modified atmosphere. These differences were maintained for the duration of the experiment. Similar results were determined in vacuum-packed beef after 35 days of storage by Stella et al. (2013). In contrast, MAP enabled the growth of aerobic bacteria of the genus Pseudomonas (Kameník et al., 2014). Kaur et al. stored vacuum-packed lamb meat (bone-in hind shank) at –1.2 °C and 8 °C. Samples at –1.2 °C were taken on days 7, 10, 13, 16, 19 and 21 of packaging, while meat at 8 °C was sampled on days 42, 70, 77, 84, 93, 98, 115, 124 and 140 of packaging. The initial bacterial count ranged from 2.5 to 3.8 log CFU/cm2, increasing to around 7.7–8.5 log CFU/cm2 at the end of the storage period (Kaur et al., 2017). LAB increased from an initial state of 0.9–1.8 log CFU/cm2 to 6.8–8.1 log CFU/cm2. It goes without saying that the speed of increase differed according to the environmental temperature. The rate of growth was, in the case of the total bacterial count at 8 °C (0.46/day), around four times faster than at –1.2 °C (0.12/day). The rate of LAB growth was 0.56/day (8 °C) and 0.14/day (–1.2 °C). LAB became the dominant component of the bacterial population after 60 days of storage at –1.2 °C, while at 8 °C their number was 0.6–1.6 (average 0.8) log CFU lower than the total bacterial count. The samples did not show any signs of damage to the vacuum during storage, though abnormalities indicating a poor vacuum inside the packaging appeared after 19 days at 8 °C. Samples stored at 8 °C displayed signs of spoilage at 13–16 days; at –1.2 °C the shelf life of vacuum-packed lamb meat was 124 days (Kaur et al., 2017). Reid et al. (2017) tested the microbiota of VP meat. They packed 105 beef cuts obtained from one commercial cutting plant in Ireland and stored them for a period of 6 weeks. A meat

9 surface temperature was 3.6 °C immediately after packing, while the internal (core) temperature of the meat was 3.8 °C. After 2–3 weeks of storage, the temperature of the meat stabilised at –0.6 °C, with a fluctuation of the air temperature in cold storage from 1 °C to – 2.3 °C. The results of their microbial analysis are shown in Table 1.

Table 1: Development of microbiota in vacuum-packed beef during storage for a period of 6 weeks at a temperature of 1 to –2.3 °C (values in log CFU/cm2, the upper value represents the arithmetic average, the lower figures show the minimum and maximum values; source: Reid et al., 2017)

Week 0 1 2 3 4 5 6 G (hours) TMCp 2.46 3.05 4.03 4.97 5.43 6.51 6.52 75.2 1.26-3.63 2.33-3.46 3.35-4.42 4.28-5.56 4.50-6.28 6.14-6.70 6.16-6.79 TMCm 2.47 3.11 3.90 4.91 5.33 6.39 6.74 71.5 1.40-3.51 2.29-3.68 3.11-4.46 4.42-5.75 4.49-6.65 5.58-6.91 5.77-7.49 Enterob. ND ND -0.21 -0.06 0.10 2.13 1.75 174.5 ND ND -0.7-0.08 -0.4-0.36 -0.5-0.77 0.96-3.07 0.58-3.20 Pseudom. 2.12 2.76 3.62 3.51 4.02 4.65 4.42 132.6 0.76-3.22 1.62-3.70 2.76-4.15 3.08-4.25 3.09-4.51 4.35-4.81 4.17-4.79 LAB 1.79 2.25 3.72 4.68 5.14 6.70 6.23 68.8 1.26-2.55 1.35-3.37 3.15-4.42 4.19-5.60 4.30-6.44 5.42-8.03 5.56-6.86 Br. 1.09 1.82 2.64 3.80 4.14 4.68 4.76 83.2 therm. 0-1.69 1.13-2.34 1.91-3.10 2.88-4.74 3.25-5.55 3.94-5.12 3.77-5.29 Clostrid. 1.55 2.41 3.58 4.87 5.14 6.36 6.11 67.0 1.27-1.99 1.58-3.28 2.66-4.66 4.33-5.71 4.27-6.48 5.43-6.96 5.69-6.89

Note: TMCp = total microbial count of psychrophilic microorganisms; TMCm = total microbial count of mesophilic microorganisms; Enterob. = family Enterobacteriaceae; Pseudomon. = genus Pseudomonas; LAB = lactic acid bacteria; Br. therm. = Brochothrix thermosphacta; Clostrid. = genus Clostridium; G = generation time (in hours) An increase was seen in all groups of bacteria during the course of six weeks of storage in spite of a storage temperature of around 0 °C. This amounted to a log 4 increase in the TMC, with the bacterial concentration increasing from an initial population of 400–500 CFU/cm2 to values of around 5–6 million cells/cm2. LAB and the genus Clostridium made up a significant proportion of the microbiota in vacuum-packed beef. The shortest generation time, i.e. the time taken for a doubling of the number of cells to occur, was recorded for both these bacterial groups (Reid et al., 2017).

Säde et al. (2017) tested slices of beef shoulder packaged in an O2/CO2/70%/30% atmosphere (2–3 slices of meat to each package; weight 400 g). The meat was stored at 6 °C. A total of 6 batches of 3 packages, i.e. a total of 18 packages, were subjected to microbial analysis. Sampling and subsequent bacteriological analysis took place 2 days after packaging (collection A; n=6) and then 8–10 days after packaging (collection B; n=6); both collections were of samples within their shelf life. A third collection took place 10–12 days after packaging (collection C; n=6) and these packages of meat had already expired and were two days beyond their “use-by” date. The authors of the study focused on bacterial groups that play the greatest role in the spoilage of packaged meat. The results are depicted in Table 2.

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Table 2: The levels of bacterial groups in beef meat packaged in a modified atmosphere (O2/CO2: 70%/30%) and analysed 2, 8–10 and 10–12 days after packing (values expressed in log CFU/g as arithmetic average ± standard deviation; source: Säde et al., 2017)

Bacterial group time of sampling A B C TMC 3.98±0.64 7.94±0.44 8.02±0.63 Brochothrix spp. 2.72±0.58 6.55±0.71 6.19±1.66 Enterobacteriaceae 1.92±0.92 5.74±0.41 5.20±1.40 LAB 4.43±1.35 8.39±0.69 8.48±0.36 Note: A = collection 2 days after packing; B = collection 8–10 days after packing; C = collection 10–12 days after packing (2 days after expiry); TMC = total microbial count; LAB = lactic acid bacteria LAB dominated the microbiota of beef meat packed in a modified atmosphere for the duration of the analysis. Representatives of the genera Carnobacterium, Leuconostoc and Lactococcus predominated. A buttery flavour, which has been connected with signs of spoilage of packaged meat resulting from a high level of LAB growth, was detected during sensory analysis of samples of meat (collections B and C) (Säde et al., 2017). Representatives of the genus Brochothrix also made up a significant proportion of the microbiota present.

2.2. The importance of selected genera of LAB in meat spoilage

Species belonging to the genus Lactobacillus (e.g. Lb. sakei, Lb. curvatus, Lb. algidus, Lb. fuchuensis, Lb. oligofermentans) are associated with pronounced acidification, the release of compounds having a negative effect on aroma or the formation of stringy slime on poultry meat, marinated meat and minced beef and pork meat packaged in a vacuum or modified atmosphere (Pothakos et al., 2015a). The genus Leuconostoc (e.g. Leuc. gelidum, Leuc. carnosum, Leuc. mesenteroides) is often responsible for the formation of organic acids (e.g. acetic acid), the release of a buttery flavour, the formation of slime, blown of the packaging or a green discolouration in all kinds of meat and types of packaging. Representatives of the genus Carnobacterium (e.g. Carn. divergens, Carn. maltaromaticum) are extremely frequently isolated from beef, poultry and pork meat in packaging with a low proportion of oxygen. There are various signs of spoilage. Other genera and species involved include Weissella (in particular W. viridescens) and Lactococcus (e.g. Lc. piscium, Lc. raffinolactis), which were isolated from comminuted beef packed in a modified atmosphere or a vacuum, and certain species of the genus Enterococcus (e.g. Ent. viikkiensis, Ent. hermanniensis).

LAB do not exhibit an affinity for a specific substrate and are commonly resistant to the methods ordinarily used to extend the shelf life of meat. There is a difference between mesophilic LAB adapted to cold-store temperatures and strictly psychrophilic species that reproduce only within a certain range of temperatures. The first group, i.e. mesophilic LAB adapted to low temperatures (psychrotrophic), includes, for example, Lb. sakei, Lb. curvatus, Leuc. carnosum, Leuc. mesenteroides, Carnobacterium spp. and Weissella spp., and these LAB are identified as agents of spoilage in countries with a warm climate.

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The second category of strictly psychrophilic LAB unable to growth at 30 °C includes, for example, Leuc. gelidum subsp. gelidum, gasicomitatum and aenigmaticum, Lb. algidus, Lb. fuchuensis and Lc. piscium. According to the authors Andreevskaya et al. (2015), the only species of the genus Lactococcus described to date in food spoilage is Lc. piscium. It has been detected in samples of beef, poultry, pork and fish meat in an MAP, in which it represented the decisive microbiota at the end of the shelf life. Lc. piscium has been associated with meat spoilage and the spoilage of packed vegetable salads. Great attention has been devoted to these strictly psychrophilic LAB in the last decade in view of their importance in meat spoilage, their strong potential for spoilage, their absence of specificity and affinity to a certain type of meat, and their growth dynamics. This all makes them a priority among all the LAB contributing to spoilage (Pothakos et al., 2015a). The authors Lucquin et al. (2012) performed an analysis on samples of lean sliced beef meat – carpaccio. They obtained 214 isolates of LAB from 60 samples from the retail network (VP or MAP). Lb. sakei was isolated from 66 % of samples, Leuc. mesenteroides from 62 % and Leuc. carnosum from 55 %. In addition to these three representatives, they also managed to demonstrate the presence of another 6 species making up a minority share of the LAB microbiota specifically Lb. fuchuensis (25 %), Carn. divergens (22 %), Leuc. gasicomitatum (16 %), Lb. curvatus (10 %), Leuc. gelidum (8 %) and W. viridescens (1.6 %). These authors demonstrated that a VP is highly selective in relation to LAB, while a MAP is less discriminating and allows the growth of non-LAB agents of meat spoilage such as Brochothrix thermosphacta, Serratia spp. and Pseudomonas spp. VP carpaccio displays a dominant microbiota made up of the genus Lactobacillus. Lactobacilli have a stabilising effect in regard to microbial quality and enable a shelf life of as much as 14 days. Meat packed in a MAP, in contrast, is dominated by representatives of the genus Leuconostoc, and signs of meat spoilage appear after 6–7 days of storage. They also found a seasonal effect – Lb. sakei and Leuc. mesenteroides were more abundant in the summer months, while Lb. fuchuensis, Leuc. carnosum and Leuc. gasicomitatum were more abundant in the spring (the species B. thermosphacta was isolated most frequently in the autumn) (Lucquin et al., 2012). The spoilage of fresh meat is generally caused by the presence of volatile organic compounds of microbial origin, which are the cause of the changes in aroma in meat (off-odours). Volatile products of microbial catabolism include organic acids, volatile fatty acids, ethyl esters, sulphur compounds, ketones, aldehydes, alcohols, etc. Determining the spoilage potential of individual strains or microbial groups together is an extremely difficult problem. If microbial spoilage occurs, accompanied by changes in appearance (e.g. the formation of slime or discolouration) and/or changes in aroma, these signs are generally ascribed to the dominant microbiota, generally known as “specific spoilage organisms” (SSO). Nevertheless, other microbial groups may also contribute towards spoilage without necessarily directly causing unpleasant changes by means of their growth or metabolism. In reality, it is more likely that interactions occur between microbial species of various groups that result in the release of the molecules characteristic of spoilage. Further interactions may also take place between the molecules produced resulting in entirely

12 unpredictable and complex profiles with a view to the qualitative parameters of raw meat. In certain cases it is, for this reason, more correct to talk of “metabiotic spoilage association” if two or more microbial species are contributing towards spoilage and where the concept of SSO should be used for the determination of the group of bacteria whose interaction is leading to spoilage of the product (Pothakos et al., 2015a).

3. Lactic acid bacteria: their occurrence and importance in cooked meat products Heat treatment plays an important role in the selection of bacteria that may be brought into the product with the used raw material (meat) or additives, or from the production environment (Comi and Iacumin, 2012). However, cooking is not always effective when it comes to thermotolerant vegetative bacteria. Neither does it affect the secondary contamination that can occur while the product is being handled after cooking, especially during slicing and subsequent packaging (Vasilopoulos et al., 2010; Zagdoun et al., 2020). To protect cooked meat products and ensure their guaranteed shelf life, they are stored and distributed at refrigeration temperatures and packaged in a modified atmosphere or vacuum (Pothakos et al., 2012). Nevertheless, the shelf life of meat products is limited and sliced cooked hams packaged in a modified atmosphere or vacuum will last three to six weeks (Leroy et al., 2009). The major bacterial group associated with the spoilage of cooked meat products are LAB (Samelis et al., 2000; Vermeiren et al., 2005). Their growth is favoured by combinations of microaerophilic conditions in the product, the presence of sodium chloride and sodium nitrite, and a reduced water activity (Audenaert et al., 2010). It is not yet completely clear, however, whether the bacteria present in meat products come primarily from meat or from the environment, with contamination then occurring during handling (Dušková et al., 2017; Vasilopoulos et al., 2010). Cocolin et al. (2004) performed the microbiological analysis of fresh sausages in Italy. The products were tested immediately after production (Day 0) and then after 3, 6 and 10 days of storage at 4 °C. The LAB population grew from 3.86 log CFU/g (Day 0), to 5.11, 6.44 and finally 7.41 CFU/g on Day 10 of storage. The species successfully identified were Leuc. mesenteroides, Ent. casseliflavus, Lc. lactis subsp. lactis, Lb. casei and Lb. sakei.

3.1. Sources of lactic acid bacteria in products Hultman et al. (2015) analysed 195 samples from the environment of a meat processing plant, raw materials (fresh meat), semi-processed and final products (cooked comminuted emulsion- type products). LAB levels found in raw meat of 3.4 to 3.8 log CFU/g increased in semi- processed products to 4.2 ± 1.0 log CFU/g. The number of LAB in finished products at the end of their storage life ranged from values below the limit of detection to 9.7 log CFU/g. Most of the isolated strains belonged to the genus Leuconostoc, more specifically the species Leuc. mesenteroides, Leuc. pseudomesenteroides and Leuc. gelidum subsp. gasicomitatum. Other representatives of LAB were the species Lb. curvatus and Lc. lactis. According to the

13 authors, batter comminution (emulsification) in combination with the addition of sodium chloride and nitrites selects gram-positive bacteria, thanks to which LAB gain predominance (Hultman et al., 2015).

Spices are another potential source of LAB in the product. When Säde et al. (2016) analysed spices and dried vegetables, they found that 26 of 38 (68 %) samples of dried onion and garlic contained LAB > 4 log CFU/g, including two products whose LAB counts exceeded 6 log CFU/g. This finding probably reflected the minimal processing of these products. In contrast, samples of steam-treated products contained LAB less frequently (30 of 66, i.e. 45 %) and always in quantities of < 3.5 log CFU/g. However, LAB were not the only component of the bacterial population. The LAB level in steam-treated products was 1–2 log lower than the total aerobic bacterial count. Data from the literature shows that the bacterial community in spices consists mainly of aerobic sporogenic bacteria, especially bacilli. Of the 343 isolates obtained (from 104 samples of spices and dried vegetables), 61 % were identified as Weissella spp., 15 % as Pediococcus spp., 8 % as Enterococcus spp., 6 % as Leuconostoc spp. and 2 % as Lactobacillus spp. (Säde et al., 2016).

Of the isolated species, Leuc. citreum, Leuc. mesenteroides and W. confusa were involved in the spoilage of foods containing spices according to the data in the literature. However, other species such as Leuc. carnosum and Leuc. gelidum or W. viridescens were not ascertained (Säde et al., 2016). Jung et al. (2012) analysed garlic and green onion intended for the preparation of Korean kimchi. The LAB population in garlic samples varied between 8.8x104 and 1.3x108 CFU/ml, and in samples of green onion between 7.0x102 and 4.8x106 CFU/ml. The isolated strains were identified as Leuc. citreum, Leuc. mesenteroides, Leuc. lactis, W. cibaria, W. paramesenteroides, W. confusa and Lb. curvatus (garlic) and W. cibaria, W. paramesenteroides, Leuc. citreum, Leuc. mesenteroides, Lb. plantarum and Lc. lactis (green onion).

3.2. Lactic acid bacteria and comminuted meat products

The microbial population in sausages is most often comprised of the LAB species Lb. sakei, Lb. curvatus, Leuc. gelidum, Leuc. carnosum, Leuc. mesenteroides, Carn. piscicola, Carn. divergens and W. viridescens, and in some cases includes Br. thermosphacta, another gram- positive bacterium (Iacumin et al., 2014). Iacumin et al. (2014) investigated LAB populations in sausages of Italian origin, namely products that did not show signs of spoilage during storage at refrigeration temperatures together with products that showed sensory deviations indicative of the spoilage process. Most of the lactobacilli isolated from sausages with no sensory deviations were categorised as homofermentative. They comprised up to 70 % of the LAB population (Lb. sakei). The remaining 30 % were representatives of the genus Leuconostoc (Leuc. mesenteroides, Leuc. carnosum). In contrast, heterofermentative LAB (30 % Leuc. mesenteroides, 30 % Leuc. carnosum, 5 % W. viridescens, 5 % Carn. divergens, 5 % Ent.. faecalis) predominated in products with signs of spoilage. Strains of the species Lb. sakei and isolates belonging to the species Br. thermosphacta made up only 5 % and 20 %, respectively (Iacumin et al., 2014).

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Kameník et al. (2014) analysed a frankfurter-type product during storage within the shelf-life period and the production plant environment from the viewpoint of possible sources of contamination of meat products during their production. After cooking (minimum core temperature 70 °C/10 min.) and cooling to 4 °C, the sausages were packaged in a modified atmosphere (N2/CO2:70/30), weight of 1 package 500 g, and stored at 2 °C. Sampling and microbiological analysis focusing on the presence and amounts of LAB were performed immediately after packaging and then after 7, 14 and 21 days. Environmental swabs were taken in production areas (batter stuffing, cooking and cooling, packaging). The results of examination of frankfurter samples for the presence of LAB are shown in Table 3. Practically no LAB were detected immediately following production. After one week of storage, however, bacterial multiplication was observable on the surface, and after three weeks the LAB population reached more than 6 log CFU/cm2. In the case of surface microbiota, contamination most probably occurred during product handling or packaging. After 21 days, Leuc. carnosum, Lc. lactis and Lb. curvatus were successfully isolated from the sausage surface. After 2 weeks of storage, Leuc. mesenteroides was also present on the sausage samples. Positive LAB findings in the core of the product are likely to be comprised of thermoresistant strains that are able to survive heat treatment. However, the first two tests failed to detect LAB in the core of the product and their count did not increase until after 2 weeks of storage. After 21 days, the LAB population reached a level exceeding 5.7 log CFU/g. The strains detected in the sausage core were Lc. garvieae, Leuc. carnosum and Pediococcus pentosaceus. Table 3: Results of microbiological analysis for the presence of lactic acid bacteria (LAB) in samples of frankfurters packaged in a modified atmosphere and stored for 21 days at 2 °C (LAB count is given as log colony forming units per 1 cm2 in the case of surfaces or in 1 g in the analysis of the product core; source: Kameník et al., 2014)

product after after 7 days after 14 after 21 packaging days days frankfurters/surface < 1.70 1.85 < 1.70 6.61 frankfurters/core < 1.70 < 1.70 2.11 5.76

Swabs were taken from the production environment to identify possible sources of contamination. The aim was to determine the LAB species present in the environment, rather than their quantification. The results of species identification are shown in Table 4.

Table 4 shows that the production environment of the plant can be a source of LAB contamination for products manufactured there. However, in contrast to the final products (frankfurters surfaces and cores) from which only 6 LAB species were isolated, 11 LAB species were isolated from the production premises. Many species will probably not survive heat treatment, including smoking. Virtually all types of LAB found in frankfurters (with the exception of Leuc. carnosum) were also detected within the plant environment.

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Table 4: Results of microbiological analysis for the presence of lactic acid bacteria (LAB) in the production plant environment (source: Kameník et al., 2014) batter stuffing room cooling chambers packaging lines (work tables) (handling tables) Lb. curvatus/fructivorans, Aerococcus viridans Lb. fructivorans/curvatus, Leuc. citreum, Lc. garvieae*, Leuc. mesenteroides, Pd. Lb. brevis, Lb. plantarum, pentosaceus*, Lc. lactis, Lb. Lb. fermentum*, Lb. curvatus*, Lb. fermentum fructivorans/curvatus Lb – Lactobacillus, Leuc – Leuconostoc, Lc – Lactococcus, Pd – Pediococcus * isolate by MALDI-TOF MS with BioTyper log(score) ˂ 2.0 (probable identification at the genus level)

3.3. Lactic acid bacteria and cooked hams Cooked hams are prone to bacterial spoilage which causes undesirable effects, such as a decrease in the pH value, gas and slime formation, package swelling, discolouration and off- odours. The composition of the bacterial community depends on many factors, including packaging, gas composition, product composition, hygiene conditions during production and storage temperature (Raimondi et al., 2019).

The final stage in the production of cooked hams is cooking during which the core temperature reaches 70 °C, which kills the majority of vegetative bacteria. However, at the end of the shelf life, bacteria in cooked hams multiply rapidly, regardless of the combination of hygiene measures and the measures intended to extend shelf life, such as cooling, microaerophilic conditions and the presence of both salt and nitrite (Raimondi et al., 2019). Vasilopoulos et al. (2010) investigated cooked pork hams. After cooking at tmax. 72 °C, the bacterial count was below the limit of detection of < 2 log CFU/g. After the hams had been stored at 7 °C for 4 weeks, the surviving cells detected were multiplied on MRS agar to log 6.65 ± 1.15 (min. 4.08; max. 7.54) CFU/g. Almost half of the isolates were of the species Leuconostoc carnosum (Vasilopoulos et al., 2010). The same species was also found in cooked meat products by Pothakos et al. (2014). Most of the bacterial diversity in cooked hams analysed by Zagdoun et al. (2020) consisted of approximately 14 different species in various combinations, which together represented on average 98 % of the total relative bacterial count. The most abundant species in cooked hams were also present in raw meat after tumbling – Carn. divergens, Lb. sakei, Carn. maltaromaticum, Leuc. carnosum, Leuc. gelidum and Leuc. mesenteroides. Heat treatment eliminated only 50 % of the species present in the tumbled meat, mostly those that were subdominant. Dušková et al. (2016) performed an LAB-focused microbiological analysis of individual technological operations in the industrial production of cooked hams. In their experiment, they examined the raw material in the initial stage of production, i.e. at the slaughterhouse, and also in key areas in the processing rooms, i.e. the cutting area and the injection, tumbling, final slicing and packaging environments.

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In the study by Dušková et al. (2016), the LAB load was reduced from 4–5 log CFU/g of meat to practically zero during cooking (Experiments I and II). The source of LAB contamination was undoubtedly the raw meat. In Experiment I, in which pork of local origin from the processor’s own slaughterhouse was used, the initial meat contamination level was very low – in fact no LAB were isolated from the carcass or a portioned leg without prior enrichment in MRS broth. Higher LAB counts (approximately 2–3 log CFU/cm2) were found on the meat after brine injection. The fresh brine used had a very low bacterial population (1.73 log CFU/g) before the operation. After injection, the bacterial population increased to 3.32 log CFU/g. Thanks to the low level of injected meat contamination, the brine load remained low at all times, but it is clear that the brine was a source of meat contamination during injection. The role of brine in meat contamination in the production of cooked hams has been discussed by Comi and Iacumin (2012). In Experiment II (20 pork silverside samples: 10 samples of Czech origin from local slaughterhouses, 10 of German origin – imported), however, LAB counts on meat were much higher and ranged between 3 and 6 log CFU/cm2 (Dušková et al., 2016). Initial contamination levels of the meat used for cooked ham production determined the differences in LAB incidence on the meat prior to heat treatment. In Experiments I and II, the presence of LAB was detected at an amount of 2–5 log CFU/cm2 and 4–6 log CFU/cm2, respectively. The load from the environment increases at this stage. Swabs from the internal working spaces of tumblers during the production cycle revealed LAB levels of between 3.7 and 6.3 log CFU/cm2. The data obtained for the presence of LAB in meat after tumbling is in agreement with the results of Visilopoulos et al. (2010) who found an LAB population of 3.22 ± 1.08 log CFU/g at the same stage of the production process during the production of artisanal cooked hams in a small-scale facility in Belgium. After heat treatment, the LAB population fell below the detection limit. However, after subsequent storage at 7 °C, the surviving cells multiplied to a level of about 7 log CFU/g after 4 weeks (Vasilopoulos et al., 2010). In the Dušková et al. (2016) study, LAB were reduced from 4–5 log CFU/g of meat to practically zero during cooking. However, when samples were enriched in MRS broth, 4 samples were positive (Experiment I). In Experiment II of the presented work, the LAB population increased to a level of 7 log CFU/g during two-week storage of cooked hams at 2 ± 2 °C. The results obtained point to the fact that thermal treatment of products (core temperature 70 °C/10 min.) may kill bacteria or cause their sublethal injury. According to Wu (2008), injured cells are potentially as important as uninjured bacteria because they may resuscitate and then be able to grow normally. In cooked hams, they were able to multiply to the spoilage threshold, i.e. 7 log CFU/g, during two weeks of storage.

Further contamination from the environment occurs during subsequent slicing and packaging of the product. The source can be bacteria on the slicing machine, conveyors or work surfaces, or the product may be contaminated by employees. Samelis et al. (2000) found meat contamination at a level of 3 log CFU/g during product slicing. Similarly, in Experiment II a level of contamination of cooked ham slices of between 2.77 and 3.04 log CFU/g was demonstrated (Dušková et al., 2016). Only 2 samples out of 10 tested in Experiment I showed LAB contamination at a level of 2.18 log CFU/g. In the remaining 8 samples, LAB were below the limit of detection, though they were detected in all the samples tested following

17 enrichment in MRS broth at 15 °C. The LAB population increased during storage. The level of 7 log CFU/g, which is considered the spoilage threshold value (Matagaras et al., 2007; Pothakos et al., 2012), was reached after 3 and 2 weeks of storage in Experiments I and II, respectively. Leroy et al. (2009), however, consider product spoilage to be the moment at which a threshold value of 6 log CFU/g is reached, and this level was reached after only 2 weeks of storage even in Experiment I. The maximum value of the LAB population ranged from 7.85 to 8.88 log CFU/g and is in agreement with the data in the literature (Audenaert et al., 2010; Leroy et al., 2009; Matagaras et al., 2007; Vermeiren et al., 2005). As a result of sublethal cell injury (cooked ham after heat treatment) or very low incidence (pork leg after portioning – Experiment I) it was possible to isolate LAB from the examined samples only after enrichment in MRS broth. The levels were higher at an incubation temperature of 15 °C as compared with a culture temperature of 30 °C. Similarly, Pothakos et al. (2012) pointed out that a culture temperature of 30 °C does not provide an entirely objective picture of the microbiota present that causes the spoilage of food requiring refrigeration temperatures for storage. The most frequently present LAB on ham samples following heat treatment were those of the genus Leuconostoc, more specifically the species Leuc. carnosum, Leuc. mesenteroides and Leuconostoc gelidum (Dušková et al., 2016). These species were also isolated from the processing plant environment, from the cutting room (Leuc. gelidum) and production area (Leuc. carnosum, Leuc. mesenteroides), and the slicing and packaging section (Leuc. carnosum, Leuc. mesenteroides). Samelis et al. (2000) describe the predominance of leuconostocs among LAB in whole-muscle products in Greece, with a predominance of Leuc. mesenteroides (80 %) and Leuc. carnosum (10 %) in vacuum-packed slices. The authors attributed this dominance to the lower temperatures these products are exposed to as compared with sausages or smoked meats. Leuc. mesenteroides is considered the most common heterofermentative species of LAB, causing spoilage of whole-muscle meat products in Greece (Samelis et al., 2000). Both species have also been isolated by Comi and Iacumin (2012), and the occurrence of Leuc. carnosum in cooked hams is also described by Audenaert et al. (2010), Leroy et al. (2009) and Vasilopoulos et al. (2010). Vermeiren et al. (2005) consider Leuc. mesenteroides the most rapidly growing species in cooked hams. Other LAB species isolated from samples following heat treatment were Lb. sakei, Lb. curvatus and W. viridescens (Dušková et al., 2016). These species are also described as isolates from the same type of meat products (Audenaert et al., 2010; Comi and Iacumin, 2012). Like certain other authors (Comi and Iacumin, 2012; Samelis et al., 2000), Dušková et al. (2016) also failed to isolate members of the genus Carnobacterium, although these LAB are described as isolates from whole-muscle meat products (Audenaert et al., 2010; Leroy et al., 2009; Vasilopoulos et al., 2010). Comi and Iacumin (2012) attribute the absence of Carnobacterium bacteria to lower temperatures (< 12 ° C) which favour the selection of leuconostocs and Lb. sakei, which are more psychrotrophic as compared with Carnobacterium spp. The most frequently found bacterial genera in 16S rRNA analysis performed on cooked hams from several European countries at the beginning of testing were Lactobacillus, Pseudomonas, Acinetobacter, Brochothrix, Prevotella and Psychrobacter (Raimondi et al., 2019). The high diversity may probably have been due to the DNA of dead bacteria that did

18 not survive cooking, and was further increased by secondary contamination during slicing and packaging (Raimondi et al., 2019). During the shelf life period, the microbial population changed in terms of diversity loss and became simpler with an LAB dominance. Predominant genera were Carnobacterium, Lactobacillus, Leuconostoc and, less frequently, Weissella. Other bacteria found, besides the LAB group, were Vibrio rumoiensis and Brochothrix. An average of 2.9 ± 1.4 log CFU/g (median 2.6) was found at the beginning of testing. During the shelf life period, the LAB count increased (P <0.001), with the mean values reaching 7.7 ± 0.8 log CFU/g. The pH decreased during testing from 6.26 ± 0.28 to 5.74 ± 0.30 (P < 0.05). The most common isolated and identified LAB species were Lb. sakei, Leuc. carnosum, Lb. curvatus and Ent. gilvus (Raimondi et al., 2019).

4. Weissella viridescens in meat products Almost 70 years ago, Niven et al. (1954) described the negative action of heterofermentative lactobacilli in cooked meat products. Their attention was caught by agents of greening both on the surface and at the core of products. While surface colour changes were caused by bacteria contaminating the product only after cooking, the middle of the product went green due to microorganisms that survived the action of high temperatures. In their own experiment, they focused on testing the heat resistance of isolated lactobacilli. While strains obtained from surface changes did not survive heating to 65.56 °C (150 °F) for a period of just 10–12 minutes, lactobacilli from the core of the products withstood this temperature for 120 minutes. The thermotolerance of selected strains could be increased under laboratory conditions after repeated heating. Lörincz and Incze (1961) mention these heterofermentative lactic acid bacteria under the name Lactobacillus viridescens. The essence of the greening of meat products lies in the formation of hydrogen peroxide which oxidises nitrosomyochromogen, the colour pigment in meat products. L. viridescens was isolated from products after heating to 65 °C for 30 minutes or to 50 °C for 175 minutes. It was also detected in brine of a concentration of 10–14 % (Lörincz and Incze, 1961). Vrchlabský and Leistner (1971) also write about the heat resistance of L. viridescens. They cite data from the literature on the ability of a strain to survive in liver pâté at 95 °C for 10 minutes. Under laboratory conditions, they demonstrated the protective effect of NaCl at a concentration from 1.4 % to 3.4 % on the survival of L. viridescens during cooking. Products with an aw value from 0.985 to 0.975 provide these bacteria with better conditions for surviving higher temperatures. In 1993, Collins et al. focused on a group of unknown bacteria extremely similar to leuconostocs (known as Leuconostoc-like microorganisms) isolated from fermented Greek salamis. The new genus Weissella was proposed on the basis of the results of 16S rRNA gene sequence analysis and other preceding phylogenetic studies. This genus was named after German microbiologist Norbert Weiss who principally studied lactic acid bacteria, to which weissellas also belong (Collins et al., 1993). Fifteen species of weissellas were known in 2012: W. beninensis, W. ceti, W. cibaria, W. confusa, W. fabaria, W. ghanensis, W. halotolerans, W. hellenica, W. kandleri, W. kimchii (W. cibaria), W. koreensis, W. minor, W. paramesenteroides, W. soli, W. thailandensis and W. viridescens. In 2017, Kim et al. (2017)

19 listed 19 species, with the above species joined by W. diestrammenae, W. fabalis, W. oryzae and W. uvarum. As of 1 October 2020, the server lpsn.dsmz.de listed 25 species of weissellas, with those described above joined by W. bombi, W. cryptocerci, W. jogaejeotgali, W. muntiaci and W. sagaensis. Weissellas are part of the family Leuconostocaceae, the order Lactobacillales, the class Bacilli, the phylum Firmicutes and the domain Bacteria (Björkroth et al., 2002; Choi et al., 2002; Collins et al., 1993; De Bruyne et al., 2008; De Bruyne et al., 2010; Lee et al., 2002; Magnusson et al., 2002; Padonou et al., 2010; Tanasupawat et al., 2000; Vela et al., 2011). The macroscopic and microscopic morphology of weissellas is easily confused with that of other lactic acid bacteria, particularly leuconostocs and lactobacilli (Dušková et al., 2013). Five of the current weissellas species have been reclassified from the genus Lactobacillus (W. confusa, W. halotolerans, W. kandleri, W. minor and W. viridescens) and one from the genus Leuconostoc (W. paramesenteroides) (Kim et al., 2017). Weissellas are Gram-positive irregular short, even coccoid, rods with rounded ends occurring singly, in pairs or in short chains. Weissellas grow on MRS agar in small transparent colonies circular in shape with a slightly elevated profile; older cultures form a concentric structure (see Figs 3 and 4). They are facultative anaerobic microorganisms that grow rapidly under microaerophilic incubation conditions. They multiply at 15 °C, though not at 45 °C (with the exception of certain strains of W. confusa). They are non-spore-forming and usually non-motile, and oxidase and catalase negative. The species W. paramesenteroides may produce catalase at low glucose concentrations. Weissellas are capable of accumulating hydrogen peroxide under aerobic conditions, do not reduce nitrates and do not hydrolyse gelatin (Collins et al., 1993; Niven and Evans, 1957).

Figure 3: Microscopy of Weissella viridescens cells stained with the Gram stain (×1000 Magnification; source: Dušková et al., 2013)

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Figure 4: Culture of Weissella viridescens isolated from hot smoked dry sausage on MRS agar (Oxoid, UK) (source: Dušková et al., 2013).

4.1. Methods of identifying Weissella spp. Identification of representatives of the genus Weissella by phenotypic methods is not particularly easy. Differentiating weissellas from heterofermentative lactobacilli and leuconostocs is particularly problematic. Weissellas can be differentiated from homofermentative lactobacilli, pediococci, enterococci, lactococci and streptococci by the formation of gas during saccharide fermentation (Collins et al., 1993). Molecular biological methods can be applied with better results, such as the polymerase chain reaction (PCR) with the genus-specific primers Weissgrp and LWrev (Schillinger et al., 2008), during which weissellas form an amplicon of a size of 1200 bp. Jang et al. (2002) developed an amplified rDNA restriction analysis (ARDRA) method for the rapid identification of weissella species. They used restriction endonucleases MnlI, MseI and BceAI to cleave a product (of a size of 725 bp) specific for the genus Weissella. For the identification of the microbial diversity in Turkish dry fermented salamis of the sucuk type, Kesmen et al. (2012) used a polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE) for the V1 and V3 regions of 16S rDNA genes, and inter-repetitive PCR (rep-PCR) and the sequencing of genes 16S rDNA and 16S-23S rDNA of intergenic regions.

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Use of the MALDI-TOF MS method The rapid and precise identification of bacteria, including weissellas, is provided by a matrix- assisted laser desorption/ionisation time-of-flight mass spectrometry analyser (MALDI-TOF MS). This method combines the MALDI technique with a TOF analyser, in which the laser (most often a nitrogen UV laser or IR laser) acts on analyte molecules and the matrix mediates the transfer of energy. Bacterial proteins are not split, but merely ionised. The times of flight of the ions are measured in the TOF analyser. A mass spectrum of the proteome is created for each strain analysed (Carbonnelle et al., 2011; Fenselau and Demirev, 2001; Šedo et al., 2011).

4.2. The occurrence of Weissella spp. Bacteria of the genus Weissella may occur in extremely diverse environments. They are often isolated from plant materials, e.g. fresh vegetables, manioc and silage (Wang and Nishinno, 2008), cocoa beans (De Bruyne et al., 2008), meat and meat products (Collins et al., 1993; Comi and Iacumin, 2012; Han et al., 2010; Han et al., 2011; Kesmen et al., 2012; Milbourne, 1983; Patterson et al., 2010; Doulgeraki et al., 2012; Dušková et al., 2015; Raimondi et al., 2019), fish (Liu et al., 2009; Tanasupawat et al., 2000), kimchi (Choi et al., 2002; Jang et al., 2002; Lee et al., 2002), Malaysian specialities (Björkroth et al., 2002) and soil (Magnusson et al., 2002), and may in sporadic cases also occur in clinical materials from humans and animals. In these cases they are, of course, associated with other bacteria. They are generally considered non-pathogenic (Björkroth et al., 2002; Liu et al., 2009; Walter et al, 2001).

4.3. Weissella viridescens in meat products Thanks to their heterofermentative metabolism weissellas can contribute to the spoilage of foods. They ferment glucose into DL lactic acid, with the exception of W. paramesenteroides and W. hellenica, which produce D(–)-lactic acid (Collins et al., 1993). The very important species in meat processing is W. viridescens which may contribute to the spoilage of meat products. W. viridescens ferments glucose, mannose, fructose, maltose and sometimes saccharose, and in rare cases mannitol (Niven and Evans, 1957). W. viridescens can cause slime formation or green discolouration of meat. Slime formation begins with the growth of individual colonies on a wet surface, which later form a continuous layer of greenish slime. A green discolouration occurs in packaged meat products (frankfurters, smoked meat, vacuum-packed meat) in contact with oxygen. Weissella also causes a green discolouration inside smoked pork loin and frankfurters at a reduced redox potential that has enabled the accumulation of H2O2 (Marsden et al., 2009). Many studies focus in particular on the ability of weissellas to survive heat treatment and the possible increase to their heat resistance following repeated exposure to heat shock. The D60 value in lactic acid bacteria generally falls within a range of 0.25–0.66 min. (Franz and von Holy, 1996). Milbourne (1983) tested the survival of heating in W. viridescens in MRS broth.

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He found values of D65 = 23.5 min., D75 = 12 min. and D80 = 9.5 min. However, when he exposed weissellas to a number of consecutive heat shocks, the weissellas survived heating to 65 °C lasting 140 min. If weissellas manage to adapt gradually to high temperatures in the laboratory, then the same may also be true in practice, which poses a greater risk of the spoilage of meat products by thermoresistant strains of weissellas. When the survival of these strains in slices of ham was tested, the D65 values were higher than during testing in cultivation media. Peirson et al. (2003) also achieved higher D values in soft salami (D60 = 14.7 min.), though weissellas were sensitive to heating in ATP broth. Comi and Iacumin (2012) studied the spoilage of hams in which cavities formed in the muscle after heat treatment as a result of the growth of Weissella viridescens bacteria and production of CO2 during fermentation of the saccharides added to the brine used in the production of the hams from which these strains came. The spoiled hams also acquired a weak vinegary smell as weissellas also produce acetic acid during fermentation. These authors also studied the physiological properties of isolates of W. viridescens. They studied the growth of weissellas at 6 and 8% NaCl. A 6% NaCl environment did not affect the growth of weissellas, and 80% of strains even grew at 8% NaCl. Comi and Iacumin (2012) also found that W. viridescens is capable of growing at refrigeration temperatures (it has a generation time of 5 hours at 8 °C, 12 hours at 6 °C and 20 hours at 4 °C). Among the microbial culture contributing to the spoilage of ham, W. viridescens is the most resistant to the high-pressure process of ham production. It can survive the action of a pressure of 400 to 600 MPa at 22 °C for 10 min. W. minor also managed to survive these conditions, while W. paramesenteroides did not (Han et al., 2010; Han et al., 2011). Kameník et al. (2015) analysed the population of three different strains of W. viridescens following the simulation of cooking under laboratory conditions. While bacterial cells of the strain known as KAS 83 did not survive heating to 60 °C and a great reduction was seen following the action of 50 °C/5 mins, the remaining two strains (KAS 300 and KAS 399) displayed practically identical thermoresistant ability and viable cells could be found even following the action of 60 °C/15 min. However, no cells of the tested strains survived a temperature of 70 °C. The strain KAS 300 was used for the trial inoculation of the raw batter of hot smoked dry sausages. A total of 9 batches were prepared, differing in the amount of dextrose added or the density of the inoculum. The results of survival of W. viridescens KAS 300 in sausages under experimental conditions are shown in Table 5. It is clear from Table 5 that W. viridescens survived heat treatment of 70 °C/10 min. in the sausage batter environment, despite the fact that this thermal effect was entirely devitalising for bacterial cells under laboratory conditions. Although W. viridescens could not be demonstrated in batches with a lower inoculum (c1) following heat treatment, certain cells did survive and were subsequently capable of multiplying. A more concentrated inoculum (c2) enabled the survival of a W. viridescens population with a reduction of approximately 2 log. The glucose concentration did not influence the survival or intensity of multiplication of W. viridescens. The addition of glucose also did not have any effect on the pH values in the sausages during 2 weeks of ripening or during subsequent storage for a period of 4 weeks. In the period between week 1 and week 2, when the aw value fell beneath the prescribed value of

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0.93, the multiplication of W. viridescens continued in the samples of sausages with the exception of the control and batch c1 with a glucose concentration of 0.2 %. W. viridescens could not be demonstrated in these 2 batches in any of the samples taken. The results of the multiplication of W. viridescens in all the other batches demonstrated that these bacteria are not affected by the glucose concentration in the sausage batter (Kameník et al., 2015).

Table 5: The survival of W. viridescens during production of hot smoked dry sausages inoculated with the strain W. viridescens KAS 300 (source: Kameník et al., 2015). Number of W. viridescens Glucose (log CFU g-1) Designation concentration Before After After 1 After 2 After 6 (%) cooking cooking week weeks weeks C 0.3 <1.70 <1.70 <1.70 <1.70 n.p. 0 3.81 <1.70 <1.70 6.32 7.11 0.1 3.85 <1.70 <1.70 2.40 5.04 c1 0.2 3.77 <1.70 <1.70 <1.70 <1.70 0.3 3.74 <1.70 6.00 6.92 5.73 0 4.92 2.48 6.11 7.36 7.00 0.1 4.83 2.48 5.18 7.30 7.18* c2 0.2 4.88 1.70 6.40 7.20 7.08 0.3 4.95 2.45 4.58 6.81 7.80* -1 n.p. – not performed; C – control sample without W. viridescens; c1 – 3 log CFU g ; c2 – 4 log CFU g-1; * – sensory changes

No sensory abnormalities were recorded after two weeks of ripening. They did appear after subsequent 4-week storage, though only in batches prepared with a greater density of inoculum (c2) and with the addition of 0.1 and 0.3% glucose. A lighter colour on the slice and taste abnormalities of the “off-odour” type were evident in the analysed sausages. No sour taste could be shown in any of the tested batches of meat products (Kameník et al., 2015). Dušková et al. (2015) analysed the role played by W. viridescens in sensory changes in hot smoked dry sausages in the Czech Republic. LAB were detected in 12 of 39 samples (30.8 %) tested in group 1 (standard products from the retail network; in 4 cases only following prior enrichment in MRS broth) (Table 6). Sensory analyses of sausages obtained from the retail network (producers A–E) did not reveal any abnormalities in taste or aroma that would point to the spoilage process (results not shown). In samples positive for LAB (without enrichment) the number of CFU ranged from 2.00 to 6.43 log/g with an average value of 5.16 log CFU/g. A group of 48 samples from batches displaying sensory changes showed 30 positive findings (62.5 %), of which 4 samples only following prior enrichment. The LAB level ranged from 4.30 to 7.88 log CFU/g with an average value of 5.98 log CFU/g.

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Bacteria of the species W. viridescens were detected in 30 of the 42 samples positive for LAB (71.4 %), in 23 cases (53.5 %) as a LAB monoculture. LAB were demonstrated in the products of 7 of the 8 producers from the Czech Republic whose sausages were analysed in this study. All 6 samples from producer F were entirely negative and LAB were not demonstrated in these even following enrichment. W. viridescens was isolated from the sausages of all the other producers (Dušková et al., 2015). The species Lb. sakei was isolated from 6 of the 42 samples positive for LAB (14.3 %), of these as a LAB monoculture in 3 samples (7.1 %). This species was, however, demonstrated in the products of just 3 producers (C, G and H). Other LAB detected were Ent. faecium, Lc. lactis, Leuc. mesenteroides and others (Table 6).

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Table 6. The occurrence of LAB in hot smoked dry sausages from Producers A – H (source: Dušková et al., 2015). No. of samples / No. Count of LAB in LAB positive samples* Species of LAB in Group Producer of LAB positive samples (log CFU/g) LAB positive samples I I II E. faecalis, 1 A 15/5 nd , 5.53, nd, nd, nd II Lb.plantarum, Str.salivarius, B 4/1 6.15 III IV III IV W. viridescens, E. faecium, Lc. lactis, W. C 4/2 2.00 , 3.90 viridescens, Lb. sakei D 6/1 6.43 V V VI Lb. plantarum, Lc. lactis, Leuc. citreum, E. E 4/3 4.48 , 6.41, 6.34 casseliflavus, VI W. viridescens, E. faecium, F 6/0 - 5.94VII, 7.36VIII,ndIX, VIIW. viridescens, E. faecalis, VIIIW. viridescens, Lt. 2 A 7/7 IX 5.58, 6.51, 6.08, 5.72 garviae, E. faecium, 5.20X, 7.11, 7.26, X W. viridescens, Lb. sakei, Lb. mucosae G 12/6 6.23, 5.89, 5.67

XI 5.18, 5.48, 6.88 , 6.53, XILeuc.mesenteroides, Lb.sakei, XIIPd. pentosaceus, ndXII, 4.77XIII, 4.87XIV 5.71XV, XIII XIV XV H 29/17 Pd.pentosaceus,Leuc.citreum , Lb.sakei, Leuc. 7.88, 5.51, 6.53, 6.75, nd, mesenteroides,XVILb. sakei, XVII E. durans, XVIIIW. 4.30XVI, 5.70XVII, 4.89XVIII, ndXIX viridescens,Lb.fructivorans/curvatus, XIX Lb. sakei

1: sausages from the retail, 2: sausages with sensory deviations;* in samples without index only W. viridescens was detected; nd: under detection limit (positive result for LAB following enrichment); E. Enterococcus, Lb. Lactobacillus, Str. Streptococcus, W. Weissella, Lc. Lactococcus, Leuc. Leuconostoc, Pd. Pediococcus

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Table 7. The occurrence of LAB and Weissella viridescens in a factory environment (Producers A, F, H) (source: Dušková et al., 2015). No. of samples / No. of LAB positive No. of samples / No. of Wv positive Species of LAB in Producer samples samples LAB positive samples A 53/50I 53/0 I E. faecalis, E. faecium, Pd. acidilactici, Pd. pentosaceus, Lb. sakei, Lb. curvatus/ fructivorans, Lb. plantarum, Lb. paraplantarum, Lb. mucosae, Lb. amylovorus, Lb. brevis, Lb. versmoldensis, Lc. garvieae, Lc. lactis, Leuc. lactis, W. confusa

F 21/16II 21/0 II E. gilvus, Lb. spp., Lb. sakei, Lc. lactis, Lc. garviae, Lc. piscium, Leuc. pseudomesenteroides, Leuc. gelidum

H 10/9III 10/2 III Pd. pentosaceus, Lb. curvatus, Lb. sakei, Lb. garviae, Leuc. mesenteroides, Leuc. lactis E. Enterococcus, Lb. Lactobacillus, W. Weissella, Lc. Lactococcus, Leuc. Leuconostoc, Pd. Pediococcus

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Table 8. The occurence of LAB in rework and sausage Vysočina prepared with rework (producer H) (source: Dušková et al., 2015) Samples No. of samples/ Count of LAB in Species of LAB in LAB positive samples No. of LAB LAB positive samples -1 positive samples (log10 CFU.g ) Rework 4/4 6.15 Lb.sakei, 5.51 W.viridescens, Lb.sakei, 8.15 W.viridescens, Lb.sakei, 5.90 W.viridescens, Lb. fructivorans/curvatus Sausage Vysočina 4/4 7.41 Lb.sakei, prepared with rework 5.23 W.viridescens, Lb.sakei, Leuc.mesenteroides, 5.72 W.viridescens, Lb. curvatus/fructivorans, nd* Lb. sakei nd: under detection limit (positive result for Lb. sakei following enrichment).*Sample of sausage with rework following heating at 80 °C/10 min; Lb. Lactobacillus, W. Weissella, Lc. Lactococcus, Leuc. Leuconostoc.

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It can be concluded on the basis of the presence of LAB in undamaged final products that these species were capable of surviving heat treatment (the minimum heat effect stipulated by the legislation of 70 °C/10 min.). W. viridescens was demonstrated as the only LAB present in more than 50 % of cases in the analyses performed in the study by Dušková et al. (2015). The low aw value in this type of salami evidently further selects the LAB that survived heat treatment. In an effort to clarify the sources of contamination of sausage batter and, later, ready products, an analysis of the environment and samples of raw material (meat) was conducted at three producers for the presence of LAB, with an emphasis on the species W. viridescens (Table 7). In all cases, this involved merely testing for the presence of bacteria without quantitative determination. The species W. viridescens was discovered in just 2 out of 84 samples (swabs of the production environment and the surface of the raw material – meat), i.e. 2.4% of all swabs (Dušková et al., 2015). Two samples of the raw material (deboned pork shoulder) at the production facility of producer H were, however, positive. On the other hand, other species of LAB were detected in a total of 75 samples from the environment (including raw materials), i.e. 89.2% of samples were positive for LAB. This disparity (2.4% positive for W. viridescens and 89.2% positive for LAB) points to the fact that just a negligible number of LAB from the environment are capable of surviving in the batter of hot smoked dry sausages following heat treatment and subsequent ripening. The low level of detection of strains of the species W. viridescens in the environment and the relatively large level of detection in the batter of final sausages led the authors Dušková et al. (2015) to question where the W. viridescens in the batter for hot smoked dry sausages came from. The reason for this occurrence may have been the reworking of non-standard products in later production. A total of 4 samples of hot smoked dry sausages from producer H designated for reworking were analysed (Table 8). All contained LAB, with three samples being positive for the presence of W. viridescens and likewise three samples positive for L. sakei. Milbourne (1983) demonstrated earlier that thermoresistant strains of W. viridescens increase their resistance to heat following exposure to high temperatures. As soon as weissellas have survived heating in hot smoked dry sausages during ordinary cooking, the potential exists for a large number of bacteria to survive the thermal effect of 70 °C/10 min. during further reworking and subsequently be capable of improved multiplication. The result is then large numbers of CFU of W. viridescens in final products exceeding 6 log/g of product with the potential to induce sensory changes. Grünewald et al. (2007) draw attention to the growing trend towards the reworking of non-standard products in heat-treated products with the aim of saving production costs. One of the consequences of this step may be contamination of the prepared batter by bacteria that are capable of inducing product spoilage and that are characterised by higher heat resistance. Producer F was the only producer in whose hot smoked dry products no LAB were found even after enrichment. This producer was found not to have reworked non-standard products in any production of hot smoked dry sausages or other cooked meat products (Dušková et al., 2015). Raimondi et al. (2019) analysed samples of cooked hams (n = 11) sliced and packed in a modified atmosphere. The products came from 10 producers in six European countries. A few days after packaging, when the samples were analysed, the average LAB count was 2.9±1.4 log CFU/g (median 2.6 log CFU/g). During the storage period within the shelf life of the

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products the LAB count increased (P < 0.001) to 7.7±0.8 log CFU/g. A number of samples returned from the market due to sensory abnormalities showed similar LAB counts (7.7±0.9 log CFU/g). The pH values in the samples at the beginning of the experiment were 6.26±0.28, falling at the end of the shelf life to 5.74±0.30 (P < 0.05). Samples with sensory deviations showed similar, i.e. lower, pH values of 5.65±0.20. Isolated bacteria were identified by the RAPD- PCR method. The isolates belonged to 7 genera: Carnobacterium, Enterococcus, Lactobacillus, Leuconostoc, Staphylococcus, Streptococcus and Weissella, with closer differentiation into 17 species. Lb. sakei and Leuc. carnosum dominated. It is interesting that the pH value was lower at the end of the shelf life (5.59) in samples with a predominance of Lb. sakei than in samples with a dominance of Leuc. carnosum (5.80). The difference was statistically significant (P < 0.05). Staphylococci and streptococci were isolated from just a few samples at the beginning of the experiment, which testifies to initial contamination that was eventually overcome by LAB. In one sample at the end of the shelf life with no signs of spoilage, W. viridescens comprised 92% of the LAB population (8% L. sakei) (LAB count 7.8 log CFU/g; pH 6.0). W. viridescens comprised 2% of the population in another sample, likewise at the end of its shelf life, though showing signs of spoilage (pH 5.50; 7.2 log CFU/g); 98% of the LAB population in this sample was made up of Lb. sakei. W. viridescens was also isolated in one sample at the beginning of the experiment (pH value 7.09; 2.9 log CFU/g) and made up 19% of the LAB population (68% Leuc. carnosum, 13% Lb. sakei). W. viridescens was not isolated from another 30 samples of cooked ham (Raimondi et al., 2019). The finding of W. viridescens in a sample of cooked ham with no signs of spoilage and with a standard pH value may have meant the presence of an aerobic environment in the product. According to the study by Zotta et al. (2018), aerobic cultivation increases the resistance of cells and increases their biomass as a consequence of improved pH homeostasis, increased energy, synthesis of antioxidant enzymes and exhaustion of intracellular oxygen. In an experiment with various strains of the genera Lactobacillus, Leuconostoc and Weissella, the given authors found a negative dependence between the pH value and the volume of growth of bacterial calls in a growth medium during aerobic cultivation of all species of Weissella, i.e. there was no fall in pH in the medium. All the strains of the genus Weissella used showed better growth in aerobic conditions. The authors correlated this fact with the presence of genes for the aerobic metabolism of pyruvate (pox, ack).

5. Cavities inside whole-muscle meat products: their causes and significance The presence of cavities on the slice of cooked hams and other whole-muscle smoked meat products (Figure 5) is sometimes a cause of concern to meat producers, for whom the search for a means of rectifying the situation is a rather difficult one. The reason is that there seem to be more than one cause of the formation of such cavities and they are not necessarily associated with bacterial contamination and the growth of bacteria.

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Figure 5: Cavities in smoked meat may have different etiology At industrial meat-processing company A, samples of leg, shoulder and collar were separated immediately after brine injection and shock-frozen in a tunnel (-40 °C) to ensure the fixation of the pieces of meat. The same procedure was performed on raw meat before injection. Whole muscles were cut in a frozen state. Cavities were observed after cutting in the muscle following injection. These defects were not observed, however, in samples frozen before injection. No cavities were observed before injection in muscles after freezing. As fixation of the meat (freezing) was performed immediately after injection, the formation of cavities cannot have been associated with bacterial metabolism, but was of mechanical origin. The next stage of testing at concern A focused on confirming the finding that cavities arise in whole-muscle products during injection and not during tumbling. For this purpose, two samples of silverside were taken following injection, with the tumbling phase being omitted (the standard production procedure was observed in all other respects). The samples in which tumbling was omitted after injection were evaluated as highly porous, and more so than the samples produced in the standard way. The production managers at the given company A described one of the possible ways in which air (gas) gets into whole-muscle products. This company processes leg and shoulder of pork imported from abroad in the form of anatomically whole pieces on the bone and with the skin. The meat is deboned and prepared for production at a cutting plant. The workers on the cutting line use compressed air, which they inject between the skin and the meat of the leg or shoulder, to facilitate skinning. In order to test this possible cause, producer A supplied the University of Veterinary and Pharmaceutical Sciences in Brno with samples of pork topside obtained without the use of compressed air beneath the skin and samples of meat to which compressed air had been applied. The samples were vacuum-packed and cooked in a water bath at 75 °C to imitate the cooking of whole-muscle products. The results can be seen in Figures 6 and 7.

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Figure 6: Topside obtained by cutting leg of pork without injecting air between the skin and meat, following cooking at 75 °C/10 min.

Figure 7: Topside obtained by cutting leg of pork with injection of air between the skin and meat, following cooking at 75 °C/10 min. Air penetrates between the muscle fibres during this “mechanical” pathway of cavity formation in the meat or product. As can be seen in Figures 6 and 7, this may occur in the cutting room where air is evidently not deliberately applied directly to the meat. It is possible that the structure of the muscle fibres in pork meat may be affected by the pH value and processes taking place in the muscles during slaughter and shortly post mortem. The authors of this study are not aware of these phenomena having being studied previously by other authors in connection with the formation of cavities in whole-muscle products. Injectors may contribute to the further penetration of air into meat if they are adjusted inappropriately with air being injected into the meat instead of brine.

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5.1. Bacteria as a possible cause of cavities in whole-muscle meat products If we disregard the “mechanical” pathway of cavity formation, in which air gets into the meat and penetrates between the muscle fibres in pork meat, the “bacterial” origin of cavities is associated with shortcomings in the production process, i.e. with inadequate observation of the principles of good manufacturing practice. In essence, this involves the penetration of bacteria into the meat through injector needles along with the brine. Two elements are of key importance here. Firstly, the bacterial quality of the brine used, and secondly the temperature regime of the raw material (the meat used for the production of whole-muscle products) during the time from injection and tumbling to the moment of cooking, as well as the effectiveness of cooking itself and the rate of cooling of the final products following cooking. Let’s take a look now at both these two factors. The brine used in the production of cooked hams and other whole-muscle products contains components that promote the growth of many bacteria (saccharides, minerals and, in some cases, proteins). If the brine is not kept at refrigeration temperatures after preparation, there may be a risk of the multiplication of bacteria that have got into the brine during preparation. A far worse situation occurs when a producer using brine for injection does not use all the brine prepared during a single shift and keeps the remainder for the next day. Psychrotrophic bacteria can multiply and may then represent a significant source of contamination even if the brine is stored at refrigeration temperatures. Dušková et al. (2016) found an increase in the LAB count in brine during the injection process within a working shift lasting one hour (the injection process takes place automatically in the machines; brine that is not immediately injected into the meat circulates in the system and becomes contaminated with bacteria when it comes into contact with the meat) from an initial 1.7 log/g of fresh brine to 3.3 log CFU/g. Comi and Iacumin analysed a batch of 12 cooked hams prepared in small-scale artisanal production, in which signs of spoilage in the form of cavities inside the product appeared during the production process. The LAB count in the meat in the area immediately surrounding the cavities amounted to 7 log CFU/g, and as much as 8 log CFU/g in the juice (brine) released from the cooked hams. W. viridescens was isolated from the brine released from the cooked hams and from the cavities (Comi and Iacumin, 2012). The cited authors explained the origin of the cavities in the cooked hams by the injection of W. viridescens with the brine. The cited authors found W. viridescens at an amount of 200±100 CFU/g in samples of brine from the same producer. The increase in temperature during cooking between 15 and 32 °C for a period of around 3 hours during the production process then led to the multiplication of bacteria already present in the meat and the production of CO2 which caused the formation of the cavities. As weissellas are, to a certain extent, thermoresistant, they could survive cooking (72 °C/15 min. at the product core) and multiply during the fall in temperature from 32 to 15 °C, which took around 8 hours (Comi and Iacumin, 2012). The authors ruled out the growth of weissellas during tumbling and before cooking, since neither the environmental temperature (< 7 °C) nor the duration of this production phase (max. 12 hours) would allow W. viridescens to multiply. According to the sources in the literature, however, W. viridescens is not a bacterium occurring particularly frequently in cooked hams (Dušková et al., 2016; Raimondi et al., 2019) or other meat products (Geeraerts et al., 2018; Geeraerts et al., 2019). The reason for this may be its weaker adaptation to the low temperatures in meat products during the storage

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of final products at which other LAB species better adapted to cold conditions can multiply, particularly species of the genera Leuconostoc and Lactobacillus (Lb. sakei, Lb. curvatus). The species Leuc. gelidum has been described and isolated from vacuum-packed beef stored at refrigeration temperatures (Pothakos et al., 2015a). Based on 16S rRNA analysis, it is closely phylogenetically related to the species Leuc. inhae, Leuc. kimchii and Leuc. carnosum. All four species are psychrotrophic. Leuc. carnosum has also been isolated from packaged meat products stored at low temperatures, while the other two species are associated with the production of kimchi, a traditional Korean product prepared by the fermentation of vegetables. The species Leuc. gelidum was revised a few years ago, and 3 subspecies described: Leuc. gelidum subsp. gasicomitatum, Leuc. gelidum subsp. aenigmaticus and Leuc. gelidum subsp. gelidum (Jääskeläinen et al., 2015). The subspecies gasicomitatum and gelidum are known agents of the spoilage of packaged refrigerated foods, particularly meat and meat products. Johansson et al. (2011) state that Leuc. gasicomitatum was first described as an agent of the spoilage of raw marinated chicken meat in a modified atmosphere in 1997. In this case, bulging of the packaging was observed due to the production of CO2 after just 5 days of storage, in spite of the fact that the producer stated a shelf life of 14 days. Typical signs of spoilage induced by this species are the creation of dextran (slime), bulging of the packaging due to the formation of CO2, a sour or buttery aroma and greening (beef meat) or a yellow colouring (white sausages). Leuc. gelidum is an agent of meat spoilage (Pothakos et al., 2015b). Leuc. gelidum subsp. gasicomitatum has three alternative anaerobic pathways for the utilisation of pyruvate (Jääskeläinen et al., 2015). The accumulation of pyruvate is not advantageous to the growth and metabolism of bacteria. The three mentioned pathways are based on the enzymes pyruvate-dehydrogenase, lactate-dehydrogenase and α- acetolactate synthase (Johansson et al., 2011). α-acetolactate-synthase was activated if inosine or ribose served as a source of carbon, while the enzymes lactate-dehydrogenase and pyruvate-dehydrogenase were suppressed. This resulted in the formation of diacetyl/acetoin, as the unstable intermediate α-acetolactate was converted into acetoin by the enzyme α- acetolactate-decarboxylase. Psychrophilic leuconostocs do not grow at 30 °C or higher temperatures. Leuc. gelidum subsp. gasicomitatum, in particular, may gain an advantage from the addition of saccharides to marinades for the purpose of balancing a sour taste. The presence of haem iron in meat has been shown to enable this subspecies to use a respiratory metabolism (Zotta et al., 2018). The result is the release of a significant quantity of acetoin and diacetyl, while the speed of growth is also increased. Respiration is the key factor explaining why Leuc. gelidum subsp. gasicomitatum is so well adapted to meat packaged in a protective atmosphere with a large proportion of oxygen. Respiratory metabolism has not been shown in the other two subspecies of Leuc. gelidum. Pothakos et al. demonstrated the genus Leuconostoc in 12 samples of Belgian foods (representation 0.4–6.5%) and 24 samples from the environment (0.05–12.3%) (Pothakos et al., 2015b). The most common species of this genus were Leuc. gelidum and Leuc. mesenteroides. At the end of the shelf life, the psychrotrophic species Leuc. gelidum was isolated most frequently, at a proportion of 18.6–81.0% in a consortium of bacteria inducing

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spoilage. Other bacteria identified at this stage of the analysis were Lactobacillus spp., Pseudomonas spp., Lc. piscium, Brochothrix spp., Enterobacteriaceae and Weissella spp. Species identified in the environment were Leuc. gelidum (most abundant in the vegetable preparation room) and Lactobacillus spp. Lc. piscium, Leuc. mesenteroides and Lactobacillus spp. were detected in samples of water. Leuc. gelidum, Lc. piscium and Lactobacillus spp. were isolated from the walls of the cooler (Pothakos et al., 2015b). Geeraerts et al. (2018) isolated the species Leuc. carnosum, Leuc. gelidum subsp. gelidum, Leuc. gelidum subsp. gasicomitatum, Lb. sakei, Lb. curvatus, Carn. divergens, Carn. maltaromaticum and Carn. funditum from 42 samples of sliced poultry products packaged in a protective atmosphere. The isolates were dominated by the species Lb. sakei (32 %), Leuc. carnosum (27 %), Carn. divergens (19 %) and Carn. maltaromaticum (12 %).

Tab. 9: Selected growth parameters of W. viridescens and Leuc. mesenteroides on the meat product Morcilla de Burgos (source: Martins et al., 2020)

Species Temp (°C) Lag phase Cell growth Shelf life (h) (log CFU/g) (days) Weissella 5 4,55±0,59 10,38±0,37 4,2 viridescens 8 2,69±0,09 10,31±0,11 2,9 13 1,22±0,13 10,19±0,25 1,3 18 0,60±0,07 10,23±0,31 0,6 Leuconostoc 5 20,54±1,73 10,04±0,28 5,5 mesenteroides 8 12,44±0,91 10,10±0,28 3,5 13 5,99±0,40 10,10±0,18 1,6 18 3,74±0,36 10,71±0,45 1,0

Longhi et al. (2018) modelled the growth of W. viridescens following the artificial contamination of slices of cooked ham (pH 6.20±0.09; aw 0.975±0.002; salt 2.38±0.24 %). The authors used various combinations of the temperatures 4, 8, 12, 16, 20 and 25 °C in their experiment. The theoretical value for the minimum growth temperature in W. viridescens was beneath 0 °C, which testifies to possible growth at low refrigeration temperatures. In an environment in which other LAB species adapted to low temperatures exist, however, the growth of W. viridescens may be suppressed by competitor bacterial strains. In the cooked meat product Morcilla de Burgos, bacteria of the species Leuc. mesenteroides grew more quickly, with a more significant effect on sensory changes than was the case of W. viridescens (Diez et al., 2009). The same authors artificially inoculated the Morcilla de Burgos product with a cocktail of 5 strains of W. viridescens or 5 strains of Leuc. mesenteroides at a level of approximately 3 log CTU/g (Martins et al., 2020). The inoculated samples were vacuum packed and incubated at 5, 8, 13 and 18 °C. W. viridescens showed a shorter lag phase time, and thus faster growth, which was reflected in a shorter shelf life of the meat product (Table 9). Based on the available literature, it is clear that the growth of LAB can affect the environment of the meat product. If in cooked hams it is considered the fastest growing species of Leuc. mesenteroides (Vermeiren et al., 2005), in other products it is not so clear.

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The situation is different in the case of hot smoked dry sausages, which remain in air- conditioned chambers at temperatures > 10 °C after cooking. As the works by Dušková et al. (2015) and Kameník et al. (2015) have shown, W. viridescens can gain a dominance in this type of product and cause undesirable sensory changes.

6. Conclusions If producers want to prevent the formation of cavities in whole-muscle meat products they must, first and foremost, observe the principles of correct hygiene practice. This means: • Using only fresh raw materials with a low level of bacterial contamination; ideally with a maximum total bacterial count of 4 log CFU/cm2. • Always using fresh brine stored at a maximum temperature of 4 °C and using prepared brine during the course of a single working shift. Brine left over from the preceding day should never be used. • Devoting the appropriate attention to the cleaning of injection equipment (injectors), in particular needles and the blades of tenderisers, and taking apart and cleaning individual parts of machinery daily to prevent the formation of biofilms. • Observing low refrigeration temperatures at all stages of production. • Ensuring adequate and effective cooking of hams corresponding to the action of a temperature of 70 °C for a period of 10 min. Cooling hams as quickly as possible after cooking to ensure that the temperature phase 50–10 °C lasts as short a period as possible. • Ensuring the cold chain in the storage of final products following cooling. The cause of cavities inside whole-muscle products need not necessarily always be bacterial metabolism. The penetration of air during meat handling may also be a reason for it. It is not always easy to determine the exact cause, though bacterial contamination must always be ruled out.

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