Spoilage-related Microbiota Associated With Chilled Beef Stored in Air or Vakuum Pack
Introduction
Food is a complex, dynamic ecosystem, in which every component is continuously irresolute. Information technology is essential to recognise these changes to minimize unwanted development, such equally food spoilage, which is a naturally occurring procedure leading to undesirable modifications in sensory characteristics (appearance, texture, odour and season) and the absenteeism of acceptable qualities. This miracle determines non but economic losses, but also the lack of consumable foods. In fact, an excessive amount of food is wasted due to spoilage, even with modern preservation techniques (Gram et al., 2002; Remenant et al., 2015).
The Food and Agriculture Organization (FAO) of the United nations (United nations) and the World Health Arrangement (WHO) declare that one third of the nutrient produced for human consumption is wasted each year (FAO, 2011). Food rejection is mainly associated with spoilage and is characterized past any change which determines unacceptable products for the consumer (Koutsoumanis, 2009). The causes of the loss of adequate qualities may be concrete damage, chemical reactions, insect and rodent infestation and microbial growth (Gram et al., 2002; Ray and Bhunia, 2013). Despite refrigeration chains, chemic preservatives and the application of contempo techniques, information technology has been estimated that 25% of all food produced globally is wasted post harvest or post slaughter due to microbial spoilage, so that this is actually the most common cause of alterations in nutrient quality (Gram et al., 2002; Cenci-Goga et al., 2014).
Compared to a multitude of foodstuffs, meat represents one of the most perishable (Doulgeraki et al., 2012): first, for the presence of chemical and enzymatic activities, and 2nd, because information technology constitutes a perfect pabulum for the growth of a wide variety of microorganisms, especially every bit a result of its nutrient composition, loftier water content and moderate pH (Dave and Ghaly, 2011). Microbial growth, oxidation and enzymatic autolysis are the 3 basic mechanisms responsible for the spoilage of meat. In addition to lipid oxidation and enzyme reactions, meat spoilage is well-nigh always caused past microbial growth. The breakup of fat, protein and carbohydrates in meat results in the development of off-odours, off-flavours and slime formation, which determine disagreeable meat for human consumption (Ercolini et al., 2006; Nychas et al., 2008; Casaburi et al., 2015). The scientific community became interested in meat microbiology when meat products began to be shipped over long distances and when the spread of supermarkets in the 1950s changed consumers' habits (Nychas et al., 2008). Nowadays, products have been directed from local markets to international trade and set up-to-consume meat products accept unequivocally become function of modern diets. This new food culture requires high food quality and safety standards to be guaranteed for the unabridged commercial life of the product, with additional strict requisites to comply with in society to exist accepted for international trade. The stability of meat characteristics becomes the first essential step for nutrient producers to prevent undesirable modifications during the storage period. Many studies accept been conducted then far. Yet, some alterations on meat, such as ropy slime-germination on the surface of cooked meat products, are still persistent (Iulietto et al., 2014). Ropy filaments were found in vacuum packs and reported in Finland at the end of '80s and the cause was identified as the growth of certain psychrotrophic strains of lactic acid bacteria (LAB) (Korkeala et al., 1988). Even though several decades have passed, slime is all the same occasionally evident earlier the sell-by date, and consumers reject the products, every bit they find the appearance of the food unacceptable (Aymerich et al., 2002). To ostend the topicality of the problem, Pothakos et al. (2014) underlined the current spread of psychrotrophic LAB in Belgian nutrient processing environments, which led to unexpected spoilage in all kinds of packed and refrigerated foodstuffs in Northern Europe. Furthermore, as is easily understandable, ropy slime-forming bacteria make up one's mind huge financial losses for food producers in many countries (Korkeala et al., 1988; Aymerich et al., 2002).
Starting from the clarification of the general aspects of meat spoilage, the aim of this paper is to focus specifically on the particular aspects of meat alterations due to ropy slime-producing bacteria, from contamination sources to prevention strategies, in lodge to raise awareness to provide an effective reply for preventing the formation of ropy filaments on cooked meat products.
Shelf-life and microbial meat spoilage
The shelf-life of meat and meat products is the catamenia of time during which storage is possible and food retains its qualitative characteristics until the arrival of spoilage phenomena. The shelf-life of products is strongly linked to their deterioration, creating a borderline between an adequate and an unacceptable bacterial concentration, which determines off-odours, off-flavours and an undesirable appearance. These sensorial modifications are related to the number and types of microorganisms initially present and their subsequent growth. For meat products, the starting total microbiota is approximately x2-103 cfu gr-i, consisting of a huge variety of species (Ray and Bhunia, 2013).
The environmental conditions of the meat during the different steps in its production and trade create a specific ecological niche, which favours some microbial strains initially present in the meat or introduced by cross-contagion, whereas other strains are disadvantaged (Castellano et al., 2008; Nychas et al., 2008). The prevalence of a particular microbial strain depends on factors which persist during processing, transportation and storage. Storage at refrigeration temperatures limits the growth of simply ten% of the total microbiota and, when applicative, rut treatments remove the majority of vegetative cells. Therefore, shelf life may vary from days to several months and is strictly linked to postal service-processing recontamination. During storage, the ascendant microbiota tin crusade the deterioration and release of volatile compounds or slime formation; as a consequence, the product becomes unacceptable for human consumption (Gram et al., 2002; Kreyenschmidt et al., 2010).
Factors influencing shelf-life and spoilage of meat and meat products
The micro-organisms' power to abound in food is closely related to many factors, some of which are intrinsic in the substratum. Others are extrinsic, but all of them influence the development of the ecological environment (Cenci-Goga, 2012). The chief factors, which affect the shelf-life of meat products and favour some bacterial strains rather than others, are: packaging (aerobically, vacuum or modified atmosphere), storage temperature, the limerick of the products (presence of fatty, NaCl content, nitrites, awestward, pH) and other factors, such as antibacterial substances or biopreservatives (Nychas et al., 2008; Remenant et al., 2015) (Table one).
Intrinsic factors
Composition and antimicrobial hurdles
Meat represents a natural ecosystem in which the advantageous or disadvantageous weather condition determine the survival and growth of some specific strains. Micro-organisms need energy for their metabolism, essential substances which they cannot synthesize and components for the constitution of cells; all these necessary elements are nerveless from the surrounding food environment and their presence permit the effective survival of food-borne strains during the lag phase (Cenci-Goga, 2012). In general, meat is rich in protein, lipids, minerals and vitamins, merely poor in carbohydrates; this limerick provides an opportunity for some species instead of others with different nutrient requirements. After microbial death, intracellular enzymes tin catalyse some nutrient nutrients to simpler forms, which tin be exploited past other species. The presence of growth factors and natural or chemical inhibitors (additives such as nitrite) further select specific strains (Ray and Bhunia, 2013). All food substances which do non occur naturally or are environmental contaminants are mostly regarded as added. At that place are several categories within the broad class of added nutrient constituents. Nevertheless, a practical definition considers all the substances deliberately put into foods equally intentional substances and those which may get in past accident during processing as incidental. Among the starting time category of additives, antimicrobial agents are added to prevent bacterial contamination of nutrient, thus avoiding spoilage and poisoning processes caused past pathogens or their toxins (Cenci-Goga et al., 1996). The relatively recent increase in the interest in light-green consumerism has actually encouraged a renewal of scientific interest in natural approaches, such every bit the add-on of bioprotective cultures and natural antimicrobial compounds (essential oils, enzymes, bacteriocins) to meat products, in order to delay the growth of spoilage micro-organisms without interfering with the typical characteristics of the product (Burt, 2004). Plant-derived essential oils (EOs) are effluvious, oily liquids, obtained from institute material (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots) which take shown remarkable antimicrobial activity confronting spoilage and pathogenic microorganisms in meat and meat products. Essential oils originating from oregano, thyme, basil, marjoram, lemongrass, ginger and clove were investigated in vitro (Barbosa et al., 2009) and on meat products (Burt, 2004; Fratianni et al., 2010) and establish capable of affecting the growth and metabolic activity of foodborne microbiota (Skandamis and Nychas, 2001). Bacteriocins are microbial estrus-stable peptides, agile towards other bacteria (Gálvez et al., 2007); they are added as biopreservatives to improve the microbial stability and safety of arctic-stored fresh and cooked meat (Samelis et al., 2006). In the EU, nisin (E234), a polypeptide produced by Lactococcus lactis, and natamycin (E235), produced by Streptomyces natalensis, are currently the just commercially available bacteriocins (Ercolini et al., 2010; Doulgeraki et al., 2012).
Buffer chapters and pH
Meat pH also affects the selection of leaner; each species has an optimum and a range of pH for growth. During mail service slaughtering, muscle pH, normally decreases to v.iv-5.viii, while pH is >6 in meat coming from stressed animals (defined as dark, firm, dry meat) and in cooked meat products, such as sliced ham (Aymerich et al., 2002). The presence of adipose tissue and a loftier pH in meat determines a more rapid spoilage process due to a more rapid bacterial growth and consumption of nutrient (Ray and Bhunia, 2013).
Redox potential
The oxidation-reduction potential is a office of the pH, gaseous atmosphere and presence of reductants. It measures the potential difference, in a system generated by a coupled reaction, in which one substance is oxidized and a second substance is reduced simultaneously, in electrical units of millvolts (mV). The redox potential of a food is related to its chemical limerick, processing treatments and storage. Raw meat has an Eh (i.e., redox potential) of -200 mV, ground raw meat has an Eh of +225 mV and cooked meat a range of +90mV to -50mV (Cenci-Goga, 2012).
Water action
Water activity (aw) is the mensurate of the corporeality of h2o in a nutrient which is available for the growth of micro-organisms, including pathogens. It identifies the water available for conveying out enzymatic reactions, synthesizes cellular materials and takes part in other biochemical reactions. Raw meat has aw values of 0.98-0.99 and cooked meat approximately 0.94; those values allow the growth of nigh microorganisms (Aymerich et al., 2002). Stale products are usually considered shelf stable and are, therefore, ofttimes stored and distributed unrefrigerated. The feature of dried foods which makes them shelf stable is their low h2o activity. A water activity of 0.85 or below will prevent the growth and toxin production of pathogens, including Staphylococcus aureus and Clostridium botulinum. S. aureus grows at a lower h2o activity than other pathogens, and should, therefore, be considered the target pathogen for drying. Control of the drying process to prevent the growth and toxin product of pathogens, including S. aureus, in the finished production is critical to product safety if the production is distributed or stored unrefrigerated. Similarly, drying may not exist disquisitional for the safety of dried stored, refrigerated products, since refrigeration may be sufficient to prevent pathogen growth. Controlling pathogen growth and toxin formation by drying is all-time accomplished by: i) scientifically establishing a drying procedure that reduces the water activity to 0.85 or below; ii) designing and operating the drying equipment, so that every unit of product receives at to the lowest degree the established minimum process (Leonard, 2011).
Extrinsic factors
Packaging and gaseous temper
Packaging conditions and the gaseous composition of the atmosphere surrounding the meat greatly influence the composition of spoilage flora (Borch et al., 1996; Sechi et al., 2014; Rossaint et al., 2015). Aerobic storage conditions promote, higher up all, the growth of Pseudomonads (Rossaint et al., 2015). Pseudomonas spp., Acinetobacter spp., Moraxella spp. are considered the major source of meat deterioration in aerobically stored meat products at different temperatures from -i to 25°C. Members of the P. fluorescens group, together with the psychrotrophic P. fragi, P. ludensis and P. putida, are the nigh commonly isolates in aerobically packed, spoiled meat (Ercolini et al., 2006; Ercolini et al., 2010). The population of Pseudomonads at the arbitrary level of 107 CFU g-ane, has been attributed to the germination of slime and off-odours, peculiarly when the metabolism of nitrogenous compounds prevails over the fermentation of carbohydrates. Shewanella spp. is a genus closely related to Pseudomonas spp. and contributes significantly to spoiling food: S. putrefaciens is one of the predominant spoilers in chill-stored, vacuum-packed (VP) meat and high pH VP meat (Doulgeraki et al., 2012).
Packaging of meat under vacuum or COii modified atmosphere has resulted in extended shelf-life compared to traditional packaging atmospheric condition (Yost and Nattress, 2002). The utilise of CO2 and N2 extends the lag phase of aerobic microorganisms and promotes the growth of facultative and strict anaerobic species. This alter in packaging conditions determines a shift from aerobic leaner, such every bit Pseudomonas spp., to facultative anaerobic species, such every bit Brochotrix thermosphacta (Nychas et al., 2008) and lactic acid bacteria (Doulgeraki et al., 2012) (Table ii). Lactic acid bacteria are the predominant microflora of vacuum or CO2-modified temper packed products, representing dominant spoilage-causing bacteria (Yost and Nattress, 2000; Arvanitoyannis and Stratakos, 2012). In fact, the combination of micro-aerophilic weather and a reduced adue west inhibits gram-negative spoilage flora and favours the proliferation of LAB (Borch et al., 1996; Korkeala and Björkroth, 1997; Samelis et al., 2000b; Audenaert et al., 2010).
In addition, Modified Atmosphere Packaging (MAP) meats are afflicted by dynamics changes of headspace gases (headspace being the infinite in the bundle between the inside of the lid and the top of the food): COtwo concentration changes during storage in relation with meat absorption or evolution of COii, depending on initial headspace COtwo, temperature, packaging configuration and meat characteristics. CO2 would be adsorbed by the musculus and fatty tissue until saturation and its absorption determines a decrease in headspace volume in MAP until packages collapse (Zhao et al., 1995; Ercolini et al., 2006). Among Enterobacteriaceae, Serratia spp. is the most common genus isolated from MAP meat (Doulgeraki et al., 2012).
Storage temperature
Storage temperature affects the duration of the lag stage, the maximum specific growth rate and the final cell number (Doulgeraki et al., 2012). Lower refrigeration temperatures decrease bacterial growth and change the composition of the microbiota nowadays on meat: psychrotrophic bacteria could grow, either Gram-positive, such as LAB, or Gram-negative, such as Pseudomonas spp. (Doulgeraki et al., 2012), at chill temperature. In MAP and vacuum packed meat products, the dominance of lactic acid bacteria is as well maintained under refrigerated conditions. However, the growth rate is affected: Carnobacterium spp. prevails in a vacuum at -1.5°C, whereas homofermentative Lactobacillus spp. dominate at 4°C and 7°C (Ray and Bhunia, 2013). Among the Enterobacteriaceae, Hafnia alvei dominates at 4°C, and S. liquefaciens predominates at 1.5°C (Borch et al., 1996). Psychrophilic Clostridium spp. could be detected in vacuum-packed, chilled meat (Doulgeraki et al., 2012). Storage temperatures above x°C are non unusual and a shift in microbial populations can exist observed. Temperature abuse determines the growth of Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp (Koutsoumanis et al., 2006).
From these considerations, information technology is evident how important an accurate management of time/temperature can exist to control not only pathogen growth and toxin formation, but as well spoilage micro-organisms. Unwanted leaner growth and toxin germination as a upshot of the time/temperature abuse of nutrient products can cause consumer illness. Temperature corruption occurs when the product is allowed to remain a sufficient length of time at temperatures favourable to pathogen growth resulting in dangerous levels of pathogens or their toxins in the product (Cenci-Goga et al., 2005; Leonard, 2011; Cenci-Goga et al., 2014).
Alterations associated with spoilage
Since microbial survival follows different pathways depending on the many factors which occur, the detectable effects are multiple: visible growth (slime, colonies), textural changes (degradation of polymers) or off-odours and off-flavours (Borch et al., 1996; Gram et al., 2002; Nychas et al., 2008).
The characteristics of meat deteriorations depend on the availability of variable substrates: glucose, lactic acid, nitrogenous compounds and gratuitous amino acids present in meat, as the principal precursors of microbial metabolites responsible for spoilage (Nychas et al., 2008). Depending on the microbial species and their oxygen affinity, these compounds will produce dissimilar catabolic byproducts (Table 3).
Off odours and off flavours
The volatilome, the volatile fraction of the microbial catabolites, includes: sulphur compounds, ketones, aldehydes, organic acids, volatile fat acids, ethyl esters, alcohols, ammonia and other metabolites. Depending on their olfactory thresholds and the interaction between the volatile and not-volatile compounds, these molecules will bear upon the sensory quality of both fresh and cooked meat (Casaburi et al., 2015).
From aerobically stored meat, it is not infrequent to capeesh undesirable odours every bit putrid, cheesy, sulphuric, sweet and fruity (Borch et al., 1996). Off-odours are perceptible to consumers when the total bacterial count is between 10sevenCFU gr-1 and ten7.fiveCFU gr-one. Pseudomonas spp. and B. thermosphacta predominantly contribute to foul odours as a result of their metabolism (Nychas et al., 2008). When superficial contagion is nearly 10viiiCFU gr–1, the carbohydrates are depleted and Pseudomonaceae in association with psychrotrophic Gram-negatives, such as Moraxella spp., Alcaligenes spp, Aeromonas spp, Serratia spp., Pantoea spp., first using amino acids as sources of free energy. Nauseating odours are associated with free amino acids and nitrogen compounds (NHiii, indole, tryptophan). B. thermosphacta aerobic metabolism of glucose produces a foul-smelling odour, such equally acetoin and acetic acid (Koutsoumanis et al., 2006). Sulphur-containing compounds determine sulphuric odours, originating from hydrogen sulphide formed by Enterobacteriaceae and dimethyl sulphide by Pseudomonas spp. Cheesy odours are determined by acetoin/diacetyl and 3-methylbutanol formations produced past Enterobacteriaceae, B. thermosphacta and homofermentative Lactobacillus spp. (Casaburi et al., 2015).
The off-odor from vacuum and MA-packed meat is less intense and is represented past a sour, acid aroma equally a result of the spoilage acquired by lactic acid leaner, associated with the production of lactic- and acerb-acid during the logarithmic and stationary growth phase. The CO2 and O2 content affects the rate of consumption of glucose past B. thermosphacta. As a upshot, anaerobic metabolism produces less intense odours than aerobic metabolism, and so the utilise of a low concentration of oxygen on modified atmosphere packaging is better for maintaining adequate qualities (Pin et al., 2002). Shewanella spp. produces malodorant compounds, such every bit H2S in vacuum packaged meat (Gram et al., 2002; Doulgeraki et al., 2012).
Colour amending
The presence of bacterial patina on the surface of meat products is appreciable when the microbiota are between ten7.5-108CFU cm-2. Hydrogen sulphide, produced by L. sakei, H. alvei, Due south. putrefaciens, converts the muscle pigment to green sulphomyoglobin and its advent is a consequence of glucose consumption. Sulphomyoglobin is not formed in anaerobic atmospheres (Borch et al., 1996). Leuconostoc spp. and Leuconostoc- like microorganisms, such every bit Weissella viridescens, may crusade meat products to turn green, due to the formation of hydrogen peroxide, which oxidizes nitrosomyochromogen every bit the consequence of the exposure of meat to O2 (Dušková et al., 2013). S. putrefaciens may determine green discolouration in vacuum-packed meat (Doulgeraki et al., 2012). In addiction, among the factors affecting light-induced oxidative discoloration of cooked meat during the storage, the headspace volume directly influences the full amount of O2 available for the oxidation (Robertson, 2012).
Gas production
Clostridium spp. is responsible for the production of a big amount of gases (Htwo and CO2): vacuum-packed meat could be afflicted by blown pack spoilage, characterized past deformation of the pack due to the accumulation of a large amount of gases, putrid odours, the presence of exudates, extensive proteolysis, changes in pH and colour. This blazon of deterioration can occur in chilled, vacuum-packed meat, acquired by psychrophilic and psychrotrophic bacteria. Not only Clostridium spp. is responsible for blown pack (Yang et al., 2014), but LAB also play an important role in the product of the volatile, organic compounds found in the package headspace of spoiled meat (Hernandez-Macedo et al., 2012). CO2 concentration during the storage of packages is attributed to metabolic by-products of the heterofermentative lactobacilli and leuconostocs. It normally determines off-odours equally well.
Filaments and ropy slime
A high-incidence of ropy slime germination is found in vacuum-packed, cooked meat products, caused by the homofermentative Lactobacillus spp. and Leuconostoc spp. The stretchy, ropy slime are long, undesirable, polysaccharide ropes between the surface of the products and the casing or betwixt the slices (Figure 1). Slime production gives some bacteria an advantage, since it constitutes a protective layer to keep the bacteria moist (Bjorkroth and Korkeala, 1997a). W. viridescens may exist the crusade of ropy slime germination or meat turning greenish. Afterward the advent of individual colonies on a wet surface, a continuous layer of greenish slime is formed (Dušková et al., 2013).
Lactic acrid leaner associated with meat spoilage
Lactic acid bacteria are widespread in nature and in the environs of processing plants; they are unavoidably part of the contaminant flora of fresh meat subsequently slaughter, and also of cooked meat. They are generally regarded equally safe (GRAS) micro-organisms (Nychas et al., 2008; Ogier et al., 2008) with many applications in the food manufacture; in fact, under specific conditions, they compete efficiently with other micro-organisms for nutrients, and attain substantial, viable counts (Krockel, 2013). In food production, LAB are oft used for their desired effects, such as their awarding as a starter in meat to manufacture safe, high quality, fermented sausages or cooked meat products (Cenci-Goga et al., 2008, 2012; Zhao et al., 2014). Protective, bacteriocinogenic cultures institute a microbial ecosystem, typically associated with MAP and VP cooked meat, which prevents the multiplication of food-borne pathogens (Zhang and Holley, 1999).
Apart from their beneficial effects, some strains of lactic acid bacteria are the major spoilage bacteria in vacuum- and modified atmosphere-packed cooked meat products. In fact, they are indicated as Specific Spoilage Organisms (SSO), determining evident meat spoilage of products stored nether packaging conditions with an increased concentration of carbon dioxide (Nychas and Skandamis, 2005; Nychas et al., 2008; Koutsoumanis, 2009; Pothakos et al., 2014b).
The LAB well-nigh involved in meat spoilage consist of heterofermentative lactobacilli (Lactobacillus spp., mainly L. curvatus and L. sakei), heterofermentative leuconostocs (Leuconostoc spp.), Carnobacterium spp. (Hu et al., 2009) and, to a lesser extent, the homofermentative Lactobacillus spp. and Pediococcus spp. As a result of their metabolism, homofermentative LAB produce almost exclusively lactic acid, which is mild and palatable, whereas heterofermentative LAB produce a significant amount of undesirable catabolites, such as CO2 gas, ethanol, acetic-acrid, butanoic-acid and acetoin with consequent off-odours and visual furnishings, such as ropy slime formation and meat discolouration (Krockel, 2013).
As a consequence, LAB are responsible for some unusual alterations in meat: off-flavours, discolouration, gas production, a decrease in pH and slime formation, determining the spoilage of the products and reduction in shelf-life (Samelis et al., 2000a). Organoleptic modifications produced by LAB become appreciable after they have reached the stationary growth phase (Korkeala and Alanko, 1988; Korkeala et al., 1988): sourness (LAB produce lactic and acetic acrid during logarithmic and stationary phase of growth), gas formation (increase in CO2 concentration in packages during storage, attributed to metabolic by-products of the heterofermentative lactobacilli and Leuconostoc spp.), slime and grayness liquid (in some cases, the slime formation may be copious and unacceptable for selling; the amount increases with storage time and the appearance of the drip changes from transparent to white or grey) and ropy slime formation (Borch and Nerbrink, 1989; Bjorkroth and Korkeala, 1997b). A clear authorization of LAB is evident in MAP products at their sell-by appointment, under unlike temperature and atmospheric conditions (Champomier-Verges et al., 2001; Yost and Nattress, 2002; Ercolini et al., 2006). Non-LAB counts in MAP bolt, east.grand., cooked turkey breast, have been shown to be lower than 103CFU k-1 (Samelis et al., 2000a).
Ropy slime-producing lactic acid bacteria
Lactobacillus spp. and Leuconostoc spp. are almost the largest grouping which causes sensory changes, such as souring, the production of H2S, gas and slime. Furthermore, L. sakei and L. curvatus are the most frequent isolates, responsible for ropy slime-formation on the surface of meat products (Ray and Bhunia, 2013) (Table 4). Psychrotrophic strains are selected by the refrigerated conditions during meat processing; L. carnosum may be considered every bit the most typical psychrotrophic organism, also found frequently in artisan-type cooked MAP ham, determining defects of the products during a 3-week shelf-life (Bjorkroth et al., 1998; Vasilopoulos et al., 2008).
Ropy slime-producing lactobacilli belong to the atypical streptobacteria i.e., heterofermentative psychrotrophic lactobacilli. Atypical streptobacteria are characterized past their ability to grow at a lower temperature (two-4°C) than other streptobacteria.
Ropy slime producing leaner strains can survive on de Man Rogosa Sharpe Agar at temperatures below 0°C: the minimum growth temperature is beneath -1°C for lactobacilli and iv° for Leuconostoc spp., the maximum growth temperature fluctuates between 36.6°C and 39.8°C. (Korkeala and Björkroth, 1997; Sade et al., 2013).
This depression, minimum growth temperature allows these leaner to survive and compete with other bacteria in meat products and meat processing plants. Consequently, the use of low temperatures in the preparation and storage of meat products does not prevent the formation of ropy-slime, although refrigeration storage temperature determines a longer shelf-life of the product. The optimum temperature of growth is 30°C and such high temperatures are non unremarkably reached during the storage of meat products, in spite of temperature abuses (Korkeala et al., 1990).
Ropiness
Slime formation is due to the LAB secreting long-chain, loftier-molecular-mass, viscosifying or gelling exocellular polysaccharides into the environment. Extracellular polysaccharides or exopolysaccharides (EPS) are polysaccharides secreted outside the cell wall of the producing micro-organism. LAB synthesize a wide multifariousness of EPS: synthesis may occur extracellularly from sucrose by glucansucrases or intracellularly by glucosyltransferases from sugar nucleotide precursors (Ullrich, 2009).
2 forms of EPS are produced by lactic acrid bacteria: capsular polysaccharide (CPS) if they remain attached to the cells, or unattached and released into the environment as exopolysaccharides (EPS) (Hassan et al., 2007). Some strains are able to produce both forms of EPS, others only produce the unattached type. Nonetheless, strains producing only the capsular form accept not all the same been confirmed (Hassan et al., 2003; Ullrich, 2009). Ropiness is a term used to identify threads which can be drawn out from the surface of fermented milk by a needle. In addition, the term ropy has been used to describe strains producing EPS or ropiness. Therefore, LAB were distinguished as either ropy or not-ropy producers according to their ability to produce EPS (Hassan et al., 2007).
Hassan et al. (2003) divided lactic acid bacteria into four categories, related to EPS production: group I, sheathing-forming, ropy strains producing capsules and unattached ropy EPS; group II, sheathing-forming, not-ropy strains which produce capsules and possibly unattached EPS; group III, not-sheathing-forming, ropy strains; group IV, strains producing no or undetectable EPS. Depending on their limerick, EPS are divided into two classes: heteropolysaccharides (HePS) composed of dissimilar monosaccharides, such as galactose, glucose and rhamnose and homopolysaccharide (HoPS), containing simply one type of monosaccharide, either glucose (glucans) or fructose (fructans) (De Vuyst, 2011; Monsan et al., 2001). Leuconostoc spp. and some Lactobacillus spp. strains synthesize glucans and fructans from sucrose (Monsan, 2011; van Hijum, 2006). Notwithstanding, the formation of ropy slime is not inhibited by the absenteeism of sucrose in the meat product. Many different heteropolysaccharides (HePS) are secreted by LAB, depending on the sugar composition and molecular size (Degeest et al., 2001).
EPS production is associated with the protection of the prison cell against dessication, phage attacks, phagocytosis, antibiotics, toxic compounds, predation by protozoans and is involved in osmotic control, adhesion to surfaces and cellular recognition (Dudman, 1977; Ullrich, 2009). Slime production is influenced by the specific weather of packaging and storage temperature and is linked to biofilm formation, stress resistance and sucrose utilization of responsible strains (Aymerich et al., 2002; Ullrich, 2009).
In tardily 1980s a Finnish research group (Korkeala and Alanko, 1988; Korkeala et al., 1988) analysed the slime produced by ii different, homofermentative lactobacilli and a Leuconostoc strain, isolated from unlike ropy, vacuum-packed meat products: the slime had a molecular weight in the range of 70000-30000, determined past gel permeation chromatography (GPC), and contained glucose and galactose in a ratio of 10:1-10:2.
Isolation and identification
The identification of spoilage micro-organisms shows two dissimilar approaches: culture dependent and culture contained methods. The first procedure consists of the preliminary isolation and civilization of micro-organisms isolated from a food sample and the subsequent identification of a single, colony-forming unit of measurement on food and selective media (Figure 2). Culture independent approaches, on the other manus, practise not demand a preliminary culture, notwithstanding, strains can be detected straight on the nutrient sample via a Dna and RNA assay, which is also efficient for strains in a low concentration (Schirone and Visciano, 2014). From the 80s, ropy slime-producing leaner were identified past ways of selective media, and sugar fermentation was investigated with API fifty CHL and the sequencing of 16S ribosomal RNA (Korkeala et al., 1988). In industrial production plants, plate count methods are used in the microbial quality assessment of MAP meat products throughout the processing plant, in order to isolate meat-borne spoilage LAB strains on Plate Count Agar and de Man Rogosa Sharpe Agar media (Audenaert et al., 2010). For detailed information of the composition or the origin of the microbiota, phenotypic and/or molecular identification and typing of purified colonies is conducted (Audenaert et al., 2010). Molecular techniques in microbial ecology take changed the way of studying microbial diversity. In fact, they let rapid, reliable identification and typing of microorganisms, unremarkably by means of the detection of Dna polymorphisms between species or strains (Doulgeraki et al., 2012).
PCR-based, molecular typing methods allow differentiation at the species and intra-species level; the specificity of this approach is based on primer selections and amplification conditions (Randomly Ampliphied Polymorphic Deoxyribonucleic acid-PCR, Repetitive Extragenic Palindromic - PCR, Amplified Fragment Length Polymorphism) (Yost and Nattress, 2002; Casaburi et al., 2011). Yost and Natress (2000) divers a systematic arroyo to place lactic acrid bacteria associated with meat, to detect Carnobacterium spp., 50. curvatus, L. sakei and Leuconostoc spp past means of specific primers for Carnobacterium spp. and Leuconostoc spp., created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of L. curvatus and Fifty. sakei in multiplex PCR reactions.
Amidst the culture-independent approaches, PCR-denaturating gradient gel electrophoresis (PCR-DGGE) is a method to assess the biodiversity and population dynamics of microbiota occurring in different ecosystems, used in food microbiology to investigate bacterial successions in fermented nutrient or the composition of probiotic products (Temmerman et al., 2003; Masco et al., 2005). Among non PCR-based methods, the most promising is Restriction Enzyme Assay coupled with pulsed-field gel electrophoresis (REA-PFGE), which is used to obtain a strain-specific ring pattern for the monitoring of the succession of bacteria in meat during storage. Another method of pick, for taxonomy, is Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), which compares whole-cell protein patterns (Doulgeraki et al., 2012). Brake Fragment Length Polymorphism (RFLP) technique consists of the digestion of genomic DNA by specific enzymes and separation of obtained fragments on agarose gel. RFLP could be associated with PCR of specific sequences in presence of high interspecific polymorphisms. A specific application of RFLP, the and so-called Terminal restriction fragment length polymorphism (T-RFLP) is used to narrate psychrotrophic strains on MAP meat (Nieminen et al., 2011). The identification of causative agents of ropiness is carried out also through Ribotyping which is a method based on the analysis of ribosomal RNA where restriction enzymes provoke the formation of specific fragments of rRNA, determining a specific ribotypes for each strain (Bjorkroth and Korkeala, 1997a). Pulse Field Gel Electrophoresis (PFGE), finally, is a technique that allows the identification of loftier molecular weight molecules thanks to the electric field periodically modifying. The final consequence is a specific pulsotype for each strain (Bjorkroth et al., 1996)
Products involved
Nowadays, the consumption of cooked, sliced and packaged meat products, such every bit cooked ham, chicken and turkey breast, emulsion-style sausages (eastward.chiliad., frankfurters, luncheon meat) is increasing as a result of consumers' enhanced interest in low-calorie meat products (Hu et al., 2009). The majority of these products are sold under modified atmosphere (MAP) or vacuum-packed conditions and some of them are ready-to-eat' products (Audenaert et al., 2010). Their storage is under refrigeration with shelf-lives varying from days to several weeks. Modified atmosphere and vacuum packaging weather condition prolong the shelf-life of meat and favour the growth of psychrotrophic lactic acrid bacteria (Borch et al., 1996; Korkeala and Björkroth, 1997). During slicing and packaging, contagion may occur and psychrotrophic LAB may grow exponentially in the meat product, determining an alteration in the quality of the meat (Krockel, 2013).
The main categories of cooked meat products showing these contaminations are: grilled roast ham, cooked ham, archetype cooked ham, roast turkey chest, roast loin of pork. Even though the raw materials have unlike origins (pork or turkey), they follow a similar production process. Briefly, the main steps are: a careful pick of the meat, trimming, syringing after the preparation of the saline (a mix of water, spices, natural flavourings and additives), churning, cooking in controlled temperature ovens, where the temperature of the product must reach 70°C in the centre, cooling, vacuum-packaging process and pasteurization for several minutes at a temperature of 115°C. One time cooled, they are ready for distribution.
Sources of contamination
Since none of the ordinarily detected LAB species is highly heat-resistant and cooked meat products are heated to a core temperature of 68°-72°C, the bulk of the vegetative cells are killed at the processing plant (Vermeiren et al., 2005). LAB contamination may occur later on heat handling (Makela et al., 1992a; Makela et al., 1992b; Aymerich et al., 2002).
Mail service-cooking contamination takes place during chilling, handling, slicing and packaging, rather than via natural contaminants initially present on raw meat products determining MAP shelf-life (Borch et al., 1996). The potential contagion sources during the production process of MAP and VP cooked meat products are numerous: salt, spices and raw materials, but also the rooms, where products are stored before packaging. Not only materials collected from the surfaces of the processing rooms, only also air samples from the environment underlined the presence of lactic acid bacteria producing filaments (Makela et al., 1992a, 1992b). The origin of the contagion is also linked to the raw materials used, equally confirmed by the isolation of lactic acid bacteria from cooked sausages (Korkeala et al., 1990) and from samples taken from carcasses and raw meat establishments (Makela et al., 1992b). Information technology highlights the fact that lactic acrid bacteria tin exist transmitted through the air, by staff and via tools. Several authors have demonstrated re-contamination after the thermal processes following the handling of products (Mäkelä and Korkeala, 1987; Borch et al., 1988). The environs needs, therefore, to be thoroughly sanitized and a clear separation maintained betwixt raw and cooked products (Mäkelä and Korkeala, 1987).
Meat Spoilage: A Disquisitional Review of a Neglected Alteration Due to Ropy Slime Producing Bacteria
Published online:
17 Feb 2016
Effigy i. Stretchy filaments on the surface of cooked turkey breast.
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Figure i. Stretchy filaments on the surface of cooked turkey breast.
Meat Spoilage: A Disquisitional Review of a Neglected Alteration Due to Ropy Slime Producing Bacteria
Published online:
17 Feb 2016
Figure 2. Colonies of 50. mesenteroides on MSE agar.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/tjas20/2015/tjas20.v014.i03/ijas.2015.4011/20171122/images/medium/tjas_a_11667594_f0002_oc.gif"})
Effigy ii. Colonies of L. mesenteroides on MSE agar.
Table 1. Factors affecting the shelf-life of meat.
Table 2. Expected shelf-life of cooked meat products under refrigerated storage and dominant microbiota.
Tabular array 3. Meat spoilage: prevalent alterations detectable.
Tabular array 4. Ropy slime producing bacteria.
Prevention
In order to prevent the presence and growth of ropy slime-producers, there are various different approaches to consider. The rooms and equipment of meat processing plants act as sources of bacterial contamination and disinfection is a necessary procedure to minimize contagion of products with leaner. Therefore, temporal and spatial separation between raw meat and cooked products decreases the run a risk of cross-contamination (Audenaert et al., 2010). Not all detergents and sanitizers are effective in eliminating environmental contamination: in detail the apply of detergents and sanitizers with a depression concentration of hypochlorite is not recommended due to their proved inefficacy towards ropy slime-producing bacteria (Mäkelä et al., 1991). Concerning the utilize of advisable products for the in-depth hygiene of meat processing plants, cleaning and sanitizing have to be considered every bit primal procedures not but for avoiding pathogens contaminations but also for limiting the spoilage due to ropy slime producers. In food industry, detergent and sanitizer are used separately or in association. Detergents contain surfactants that reduce surface tensions between the soil and the surface while sanitizers are made of antimicrobial compounds able to reduce the microbiological contamination to an adequate level, according to local wellness regulations. Mäkelä et al. (1991) demonstrated that detergent-sanitizer (DS) products with different antimicrobial compounds (Na-dichloroisocyanurate at 0.06%, Na-hypoclorite at 0.017%, cocobenzyldimethyl ammonium chloride at 0.027% and Dimethylcoco ammonium betaine at 0.27%) were less constructive confronting ropy-slime producers than sanitizer (S) products used separately. In detail, applied sanitizer compounds were alkyldimethylbenzyl ammonium chloride (0.022% and 0.05%), alkyldimethyl ammonium chloride (0.014%), alkylmethylethylbenzyl ammonium chloride (0.022%), polyhexamethylene biguanide chloride (0.023%), Nahypochlorite (0.05%), paracetic acid (0.018%) and benzyldimethylalkyl ammonium chloride (0.1%). The lower effectiveness of detergent sanitizers was associated to the surface-active compounds which may modify the antimicrobial activity of the product. Consequently, in meat processing plants, it is better to use separately detergent and sanitizers than use combined detergents and sanitizers products. Fourth ammonium products and acid sanitizer with hydrogen peroxide are reported to be more than effective than products containing chlorine compounds and polyhexamethylene biguanide chloride (Makela et al., 1992a, 1992b). The prevention of unwanted meat processes must acquit in mind the rising interest of food producers and consumers in healthier nutrient production with fewer added substances. The new technologies of food preservation include non-thermal inactivation, such equally ionization radiation, high hydrostatic pressure and pulsed electric fields, active packaging, bio-preservation and natural antimicrobial compounds. The bio-preservation of meat could be the answer to this demand: in fact, it consists of the control of pathogenic and spoilage microbiota by competitive microflora and natural molecules. Bacteriocins, for example, are ribosomally-synthesized, antimicrobial peptides or proteins, which are active towards other leaner (Gálvez et al., 2007; Castellano et al., 2008). Bacteriocinogenic cultures and specific bacteriocins added to cooked meat are capable of preventing slime production (Aymerich et al., 2002). Nisin is a bacteriocin produced by L. lactis subsp. lactic and it inhibits the growth of Gram-positive organisms, including bacterial spores. Yet, it is not efficient against Gram-negative bacteria, fungi and yeast (Economou et al., 2009). It is non a toxic substance if it is ingested, it does non determine cross-resistance with medical antibody molecules and it is degraded by the intestinal tract (Kalschne et al., 2014). Nisin determines a significant inhibition of the growth of L. sakei on vacuum-packed sliced ham (Kalschne, 2014) with a shelf-life extension. Aymerich et al. (2002) demonstrated that Enterococcus faecium and 50. sakei, bacteriocin producers, prevent ropiness due to L. sakei, whereas nisin inhibits the activity of L. carnosum in cooked pork loin (Kalschne et al., 2014). In improver, these bacteriocins are oestrus-stable and resist to pasteurization. It is, therefore, possible to add bacteriocins to the meat before the cooking process. P. lactis produces pediocin, a bacteriocin constructive confronting Listeria spp. Yet, novel uses of this strain as a starter culture in some nutrient fermentations as well hypothesize the issue on strains of Gram-positive microorganisms (Kalschne et al., 2014). Bacteriocins are also involved in developing active packaging devices, creating an effective surface with antimicrobial furnishings. Bacteriocin-activated, plastic films for food packaging take been developed for the storage of hamburgers, hot dogs, frankfurters and cooked ham (Ercolini et al., 2010).
An alternative preservation method for the prevention of filaments is High Pressure Processing (HPP) for candy meat and meat products. Nearly vegetative microorganisms in meat samples are inactivated at a pressure of 400-600 MPa and HPP improves nutrient safety and prolongs the shelf-life of meat products. It could avoid the survival of bacterial strains responsible for ropiness on the surface of the production (Han et al., 2011).
Finally, food preservation through application of Ozone (O3) have been investigated, considering the bacterial inactivation adamant by the attacks on cellular constituents, avoiding cosmos of mutants, and leaving no dangerous chemic residuals. The reduction of Fifty. mesenteroides in make clean water was v log count (PPM O3 per 2 min of application) but direct application of ozone in food processing seems hardly feasible; the application on beef surface, in fact, resulted in depression activity towards Leuconostoc spp., Lactobacillus spp. and P. fluoresces, associated with discoloration and odour development (Pirani, 2010).
Conclusions
Meat spoilage and production shelf-life is an important challenge for all the experts gravitating around this area.
The spoilage due to ropy slime-formation has influenced the marketing of vacuum-packed meat products and the utilise of this applied science. The presence of ropy slime-producing leaner and their associated sensory abnormalities lead to high direct fiscal losses (waste matter product) and indirect (such as production selection, disinfection of contaminated surfaces and not-commitment at destination). Although food security is likely to be guaranteed, the macroscopic advent of the product at the time of packaging is particularly unpleasant, making it unsuitable for further processing or marketing. Food industries and productions must be supported by research, creating a potent link between discoveries and applications. Nowadays, ropy slime-germination on meat products represents a persistent problem, oftentimes ignored. It is, therefore, necessary to provide a basis as a starting point to find a benign solution.
Source: https://www.tandfonline.com/doi/full/10.4081/ijas.2015.4011
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