Weissella viridescens in meat products – a review

Although ubiquitous, bacteria of the Weissella genus are not given sufficient attention. Many members of the genus were originally classified as Leuconostoc or Lactobacillus. With the development of molecular methods, these phylogenetically closely related bacteria formed a separate group, the Weissella genus. Due to its heterofermentative metabolism, Weissella spp. may cause considerable damage particularly in the meat industry. Slime formation and greening of meat products are sensory defects for which the technologically important species Weissella viridescens is responsible. This article summarizes basic information about the influence of Weissella viridescens on meat processing. Lactic acid bacteria, Weissella spp., spoilage, greening, thermal resistance The negative effects of heterofermentative lactobacilli in cooked meat products were described almost 60 years ago by Niven et al. (1954). The researchers’ attention was drawn to the causative agents of greening, both on the surface and in the core of products. While colour changes of the surface were caused by bacteria contaminating the product only after cooking, the greening of the product core was due to the activity of bacteria that survived elevated temperatures. In their experiment, they paid particular attention to the testing of thermal resistance of the isolated lactobacilli. While strains collected from surface colorations did not survive exposure to the temperature of 65.56 °C (150 F) for only 10–12 min, lactobacilli in meat product cores survived that temperature for 120 min. Under laboratory conditions, it was possible to even increase thermotolerance of certain species by successive heating. Lörincz and Incze (1961) mentioned these heterofermentative lactic acid bacteria under the name of Lactobacillus viridescens. Collins et al. (1993) began to be interested in a group of unknown bacteria that very closely resembled leuconostocs (the so-called Leuconostoc-like microorganisms) isolated from fermented Greek salami. Based on the results of sequential analysis of the genes 16S rRNA and other previous phylogenetic studies, a new genus Weissella was proposed. The new genus was named after the German microbiologist Norbert Weiss, whose scientific interests focused mainly on lactic acid bacteria, which also include weissellas (Collins et al. 1993). At present, we recognize 15 species of weissellas: 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. Weissellas belong to the family Leuconostocaceae, order Lactobacillales, class Bacilli, phylum Firmicutes and domain Bacteria (Collins et al. 1993; Tanasupawat et al. 2000; Björkroth et al. 2002; Choi et al. 2002; Lee et al. 2002; Magnusson et al. 2002; De Bruyne et al. 2008; De Bruyne et al. 2010; Padonou et al. 2010; Vela et al. 2011). The aim of this paper was to summarize the data about the influence of Weissella viridescens on meat and meat products. ACTA VET. BRNO 2013, 82: 237–241; doi:10.2754/avb201382030237 Address for correspondence: MVDr. Josef Kameník, CSc., MBA Department of Meat Hygiene and Technology Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Palackého tř. 1/3, CZ-61242 Brno, Czech Republi Phone: +420 604 220 851 E-mail: kamenikj@vfu.cz http://actavet.vfu.cz/ Characteristic and occurrence of Weissella spp. Both macroscopic and microscopic morphology of weissellas is easily interchangeable with that of other representatives of lactic acid bacteria, in particular with the leuconostocs and lactobacilli. Weissellas are Gram-positive irregular short rods with rounded ends or coccoid rods, occurring singly, in pairs or in short chains (Plate I, Fig. 1). On de Man, Rogosa and Sharpe (MRS) agar, weissellas form small transparent colonies, circular in shape of a slightly elevated profile, older cultures form a concentric structure (Plates I, II, Figs 2 and 3). These microorganisms are optionally anaerobic, and grow rapidly under microaerophilic incubation. They multiply at 15 °C but not at 45 °C, with the exception of certain W. confusa strains. They are non-sporulating and mostly non-motile, and both oxidase and catalase negative. The W. paramesenteroides species is able to produce catalase at low glucose content. Under aerobic conditions, weissellas are capable of accumulating hydrogen peroxide, are unable to reduce nitrates and do not hydrolize gelatin (Niven and Evans 1957; Collins et al. 1993). Bacteria of the genus Weissella can exist in very diverse environments. They are frequently isolated from plant material, e.g. fresh vegetables, cassava and silage (Wang and Nishinno 2008), cacao beans (De Bruyne et al. 2008), as well as from meat and meat products (Milbourne 1983; Collins et al. 1993; Han et al. 2010; Patterson et al. 2010; Han et al. 2011; Comi and Iacumin 2012; Doulgeraki et al. 2012; Kesmen et al. 2012), fish (Tanasupawat et al. 2000; Liu et al. 2009), kimchi (Choi et al. 2002; Jang et al. 2002; Lee et al. 2002), specialties from Malaysia (Björkroth et al. 2002), soil (Magnusson et al. 2002) and, in isolated cases, they can also occur in clinical material of human or animal origin. In such cases, they are of course associated with other bacteria. Weissellas are generally considered as non-pathogenic (Walter et al. 2001; Björkroth et al. 2002; Liu et al. 2009). Methods of Weissella spp. identification Representatives of the Weissella genus are not easy to identify by means of phenotypic methods. It is particularly problematic to differentiate between weissellas and heterofermentative lactobacilli and leuconostocs. Weissellas can be distinguished from homofermentative lactobacilli, pediococci, enterococci, lactococci and streptococci by gas production during saccharide fermentation (Collins et al. 1993). Molecular biology techniques, such as polymerase chain reaction (PCR) with genus-specific primers Weissgrp and LWrev (Schillinger et al. 2008) during which an amplicon of 1200 bp is produced at weissellas (Plate II, Fig. 4) are more useful. To facilitate rapid identification of Weissella species, Jang et al. (2002) developed an amplified rDNA restriction analysis (ARDRA). They used restriction endonucleases MnlI, MseI and BceAI to cleave Weissella genusspecific product of 725 bp. For the identification of microbial diversity in Turkish dry fermented salami of the sucuk (sudžuk, soudjouk) type, Kesmen et al. (2012) used polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE) for V1 and V3 regions of genes 16S rDNA, and the interrepetitive PCR (rep-PCR) and the sequencing of genes 16S rDNA and 16S-23S rDNA of intergenic regions. Weissella viridescens in meat products Because of their heterofermentative metabolism, weissellas may also be involved in food spoilage. They ferment glucose to DL lactic acid, with the exception of W. paramesenteroides and W. hellenica that produce D(–)-lactic acid (Collins et al. 1993). The most important species in the meat processing industry is W. viridescens (formerly Lactobacillus viridescens), which may be involved in meat product spoilage. W. viridescens 238

The negative effects of heterofermentative lactobacilli in cooked meat products were described almost 60 years ago by Niven et al. (1954).The researchers' attention was drawn to the causative agents of greening, both on the surface and in the core of products.While colour changes of the surface were caused by bacteria contaminating the product only after cooking, the greening of the product core was due to the activity of bacteria that survived elevated temperatures.In their experiment, they paid particular attention to the testing of thermal resistance of the isolated lactobacilli.While strains collected from surface colorations did not survive exposure to the temperature of 65.56 °C (150 F) for only 10-12 min, lactobacilli in meat product cores survived that temperature for 120 min.Under laboratory conditions, it was possible to even increase thermotolerance of certain species by successive heating.Lörincz and Incze (1961) mentioned these heterofermentative lactic acid bacteria under the name of Lactobacillus viridescens.Collins et al. (1993) began to be interested in a group of unknown bacteria that very closely resembled leuconostocs (the so-called Leuconostoc-like microorganisms) isolated from fermented Greek salami.Based on the results of sequential analysis of the genes 16S rRNA and other previous phylogenetic studies, a new genus Weissella was proposed.The new genus was named after the German microbiologist Norbert Weiss, whose scientific interests focused mainly on lactic acid bacteria, which also include weissellas (Collins et al. 1993).At present, we recognize 15 species of weissellas: 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.Weissellas belong to the family Leuconostocaceae, order Lactobacillales, class Bacilli, phylum Firmicutes and domain Bacteria (Collins et al. 1993;Tanasupawat et al. 2000;Björkroth et al. 2002;Choi et al. 2002;Lee et al. 2002;Magnusson et al. 2002;De Bruyne et al. 2008;De Bruyne et al. 2010;Padonou et al. 2010;Vela et al. 2011).
The aim of this paper was to summarize the data about the influence of Weissella viridescens on meat and meat products.

Characteristic and occurrence of Weissella spp.
Both macroscopic and microscopic morphology of weissellas is easily interchangeable with that of other representatives of lactic acid bacteria, in particular with the leuconostocs and lactobacilli.Weissellas are Gram-positive irregular short rods with rounded ends or coccoid rods, occurring singly, in pairs or in short chains (Plate I, Fig. 1).On de Man, Rogosa and Sharpe (MRS) agar, weissellas form small transparent colonies, circular in shape of a slightly elevated profile, older cultures form a concentric structure (Plates I, II, Figs 2 and 3).These microorganisms are optionally anaerobic, and grow rapidly under microaerophilic incubation.They multiply at 15 °C but not at 45 °C, with the exception of certain W. confusa strains.They are non-sporulating and mostly non-motile, and both oxidase and catalase negative.The W. paramesenteroides species is able to produce catalase at low glucose content.Under aerobic conditions, weissellas are capable of accumulating hydrogen peroxide, are unable to reduce nitrates and do not hydrolize gelatin (Niven and Evans 1957;Collins et al. 1993).

Methods of Weissella spp. identification
Representatives of the Weissella genus are not easy to identify by means of phenotypic methods.It is particularly problematic to differentiate between weissellas and heterofermentative lactobacilli and leuconostocs.Weissellas can be distinguished from homofermentative lactobacilli, pediococci, enterococci, lactococci and streptococci by gas production during saccharide fermentation (Collins et al. 1993).Molecular biology techniques, such as polymerase chain reaction (PCR) with genus-specific primers Weissgrp and LWrev (Schillinger et al. 2008) during which an amplicon of 1200 bp is produced at weissellas (Plate II, Fig. 4) are more useful.To facilitate rapid identification of Weissella species, Jang et al. (2002) developed an amplified rDNA restriction analysis (ARDRA).They used restriction endonucleases MnlI, MseI and BceAI to cleave Weissella genusspecific product of 725 bp.For the identification of microbial diversity in Turkish dry fermented salami of the sucuk (sudžuk, soudjouk) type, Kesmen et al. (2012) used polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE) for V1 and V3 regions of genes 16S rDNA, and the interrepetitive PCR (rep-PCR) and the sequencing of genes 16S rDNA and 16S-23S rDNA of intergenic regions.

Weissella viridescens in meat products
Because of their heterofermentative metabolism, weissellas may also be involved in food spoilage.They ferment glucose to DL lactic acid, with the exception of W. paramesenteroides and W. hellenica that produce D(-)-lactic acid (Collins et al. 1993).
The most important species in the meat processing industry is W. viridescens (formerly Lactobacillus viridescens), which may be involved in meat product spoilage.W. viridescens ferments glucose, mannose, fructose, maltose, sometimes saccharose, and rarely also mannitol (Niven and Evans 1957).
W. viridescens may be the cause of slime formation or the greening of meat.Slime formation begins with individual colonies appearing on wet surface, to form a continuous layer of greenish slime later on.The underlying cause of the greening of meat products is the formation of hydrogen peroxide, which oxidizes nitrosomyochromogen, the colour pigment of meat products.Green discolouration of packaged meat products (sausages, smoked meat, vacuum-packaged meat) develops when the product is exposed to oxygen.Weissella is known to have been the cause of core greening of smoked pork loin and sausages with a lower redox potential which allowed for H 2 O 2 accumulation (Marsden et al. 2009).
Many studies investigated the capability of weissellas to survive heat treatment, and the possible increase in their thermal resistance after repeated exposure to thermal shock.W. viridescens has been isolated from products after heating at 65 °C for 30 min, or at 50 °C for 175 min.It has also been detected in brine at a 10-14% concentration (Lörincz and Incze 1961).Thermoresistance of W. viridescens was studied also by Vrchlabský and Leistner (1971).They quote a literary reference on the strain's ability to survive in liver pâté at 95 °C for 10 min.Under laboratory conditions, they demonstrated the protective effect of NaCl at concentrations in the 1.4-3.4% range on W. viridescens survival during cooking.Products with a w in the 0.985-0.975range thus offer better conditions for the survival of these bacteria at elevated temperatures.
D 60 values of a majority of lactic acid bacteria are around 0.25-0.66min (Franz and von Holy 1996).When Milbourne (1983) tested W. viridescens for survival in heated MRS broth, the D values that he obtained were 23.5 min (D 65 ), 12 min (D 75 ) and 9.5 min (D 80 ).However, after the weissellas had been exposed to a succession of thermal shocks, they were able to withstand the temperature of 65 °C for 140 min.If the Weissella can adapt itself to withstand high temperatures in the laboratory, it can do the same in processing facilities, which poses an increased risk of meat product spoilage by thermoresistant Weissella strains.D 65 values obtained from survival tests of these strains in sliced ham were higher than those obtained from tests with cultivation media.Peirson et al. (2003) also obtained higher D values from cooked sausage test (D 60 = 14.7 min), weissellas in ATP broth were sensitive to heat.Comi and Iacumin (2012) investigated spoilage in cooked hams where cavities in the muscle of hams were caused by the growth of Weissella viridescens and the CO 2 produced by the fermentation of carbohydrates added to the brine which was used for the production of the hams.Spoiled hams had a slight vinegar off-flavour because weissellas produce acetic acid during fermentation.The same authors also investigated physiological properties of W. viridescens isolates and studied weissellas growth in 6 and 8% NaCl.The 6% NaCl environment had no effect on the growth of weissellas, and 80% of strains grew even in 8% NaCl.Comi and Iacumin (2012) also found that Weissella viridescens was able to grow at refrigeration temperatures with generation times at 8 °C, 6 °C and 4 °C being 5, 12 and 20 h, respectively.
Among microbial cultures involved in ham spoilage, Weissella viridescens is the most resistant against high-pressure processing of hams.It can withstand pressures of 400 to 600 MPa at 22 °C for 10 min.W. minor was also able to survive under those conditions, whereas W. paramesenteroides was not (Han et al. 2010;Han et al. 2011).Patterson et al. (2010) demonstrated antimicrobial activity in W. viridescens strains against a broad range of microorganisms.This property could in the future be used to enhance microbiological safety of foods.The problem is, however, that not all of the tested strains demonstrated the inhibitory action against Listeria monocytogenes, Clostridium botulinum, E. coli and Salmonella spp.

Antimicrobial activity of W. viridescens
In conclusion, although weissellas may cause significant financial losses particularly in the meat processing industry, they do not belong among lactic acid bacteria that get the attention of either processors or service organisations.This opens up the area for new investigation that could help to prevent sensory defects caused by the bacterial genus of Weissella.

Fig. 3 .
Fig. 3.A detailed view of colonies of Weissella viridescens bacteria with concentric structure on de Man, Rogosa and Sharpe agar.