Physical Methods for the Decontamination of Meat Surfaces

The market for minimally processed products is constantly growing due to consumer demand. Besides food safety and increased shelf life, nutritional value and sensory appearance also play a major role and have to be considered by the food processors. Therefore, the purpose of the review was to summarize recent knowledge about important alternative non-thermal physical technologies, including both those which are actually applied (e.g. high-pressure processing and irradiation) and those demonstrating a high potential for future application in raw meat decontamination (e.g. pulsed light UV-C and cold plasma treatment). The evaluation of the methods is carried out with respect to efficiency, preservation of food quality and consumer acceptance. It was evident that significantly higher bacterial reductions are achieved with gamma-ray, electron beam irradiation and high pressure, followed by pulsed light, UV-C and cold plasma, with ultrasound alone proving the least effective. As a limitation, it must be noted that sensory deviations may occur and that legal approvals may have to be applied for. In summary, it can be concluded that physical methods have the potential to be used for decontamination of meat surfaces in addition to common hygiene measures. However, the aim of future research should be more focused on the combined use of different technologies to further increase the inactivation effects by keeping meat quality at the same time.


Introduction
Until recently, contamination of poultry, pork or beef carcasses by zoonotic pathogens and of the deboned and further processed fresh meat derived thereof has been one of the most challenging problems in food hygiene worldwide. From an epidemiological point of view, research and risk management approaches are aimed at reducing the prevalence as well as the bacterial load of Campylobacter, Salmonella, Yersinia enterocolitica, pathogenic Escherichia coli and Listeria monocytogenes, which have been the main cause of human foodborne infections in the EU, with over 350,000 reported cases in 2018 [1]. In recent decades, different strategies and measures have been applied, mainly at the pre-harvest level, but, except for in the case of Salmonella in poultry, with varying degrees of success. Therefore, recently developed strategies aim to include the entire processing chain, including transportation, stunning and slaughtering, deboning and further meat processing. In the case of Campylobacter in poultry, strategies should mainly be focused on post-harvest levels in order to significantly reduce the number of cases in humans [2]. In this context, the impact of physical, chemical and biological decontamination technologies has been the subject of a number of studies which have focussed on the potential use of chemical decontamination. Although a number of these appear to be promising alternatives, only a few can legally be applied during meat processing, e.g. the use of lactic acid for the decontamination of beef carcasses in the EU or the chlorination of poultry carcasses in various countries outside the EU. Physical methods, particularly dry interventions, are considered to be fast, mild and residue-free, and have received more attention. In contrast to chemical procedures, they can be applied more broadly across the processing chain and in some cases permit the treatment of pre-packaged meat, as well as frozen meat. Gamma irradiation has been most extensively studied and has in recent decades been supplemented with other technologies such as ultrasound, electron beam, UV light and high hydrostatic pressure treatment, as well emerging technologies e.g. light pulses or cold atmospheric plasma. Other treatments, such as electrical stimulation or pulsed electric fields (PEF), are largely intended for use in the acceleration of meat processing (tenderisation, drying, curing) rather than for decontamination purposes, and are therefore not considered in this review.
The article presents an overview of recently acquired knowledge concerning the impact of physical technologies on the decontamination of carcasses and fresh meat surfaces, including minced meat. It refers mainly to the treatment of poultry, pork and beef, and considers the most relevant bacterial zoonotic pathogens.

Gamma Ray (γ-Ray) and Electron Beam (e-Beam) Irradiation
Food irradiation has already been applied for many decades and has been approved in around 60 countries around the world [3]. The exposure of food to ionizing radiation, either in form of electromagnetic energy (γ-ray) or charged particles (e-beam) can improve the microbial safety of food and extend its shelf life, even resulting in the sterility of the product [4]. Whilst the radioisotopes caesium-137 or cobalt-60 are used as source of γ-rays, e-beams are produced by a linear accelerator. Irradiation inactivates microorganisms directly by photoninduced single and double-stranded DNA breaks, and indirectly by DNA damage, which is induced by radiolysis products, e.g. hydroxyl radicals [4]. The antimicrobial properties of both forms of irradiation are comparable, but e-beams allow a higher dose rate (e-beam 10 3 -10 5 Gy/s; γ-ray 0.01-1 Gy/s), resulting in a shorter application time [5]. In contrast to ebeams, γ-rays penetrate deeper into the food matrix (60-80 cm vs. 8-10 cm) [6].
Complex life forms, which contain large DNA molecules, are affected by relatively low doses of less than 0.1 kGy, whilst simple life forms with smaller DNA such as bacteria (1.5-4.5 kGy) or spores (10-45 kGy) are inactivated at higher doses. In general, gram-negative bacteria are more sensitive to irradiation than gram-positive bacteria [4], but serotype and serovar variations have also been documented for E. coli [7] and Salmonella [8], respectively. Besides bacterial species, food composition (primarily water content), thickness and temperature also have an effect on irradiation efficiency. Frozen or dry foods need higher doses of γ-ray or e-beam [9], because low product temperatures reduce the diffusion of free radicals.
Irradiation has successfully been applied to meat products and raw meat for many years. Numerous studies (Table 1) of recent years deal with the reduction of pathogenic E. coli on meat using e-beams. For example, Amiri et al. 2019 [14], pointed out an E. coli O157 reduction >6 log, below the detection limit in camel meat, and [9] determined a reduction of >9 log units in chicken breast and ground beef by a dose of 3.0 kGy. Salmonella was reduced >1.9 log or 6 log in camel meat or beef using 1-5 kGy [8,14], respectively. γ-Ray treatment also resulted in high reductions, as demonstrated in the case of E. coli in ground chicken D 10 0.18-0.68 (about 2.5-7 log) at 1.5 kGy [13] and Campylobacter with a reduction of 5 log at 1 kGy of irradiation [12].
It should be noted that both technologies lead to sensory changes as the dose of radiation increases. In particular, lipid and protein oxidation were observed in meat and poultry [16,17]. Lipid oxidation may result in odour deviations and nutritional changes e.g. a decrease in unsaturated fatty acids [17]. For γ-ray irradiation, the alteration of vitamin content has also been reported [17].
Irradiated meat and meat products are already commercially available in some European countries, such as France, Belgium and the Netherlands. Processing plants also exist in Germany. The advantage of this technology is the possibility of treating frozen and packaged products. The International Atomic Energy Agency (IAEA), World Health Organization (WHO) and Food and Agriculture Organization (FAO) have confirmed that irradiation up to 10 kGy assures the safety of food without undesirable effects on human health [18].

Pulsed Light
Pulsed light (PL) treatment, inter alia, pulsed UV-light (PUV), intense pulsed light (IPL) or HIPL (high-intensity pulsed light) is characterized as another rapid and gentle decontamination technology [19]. Inert gas flash lamps (mostly xenonbased) are used to generate very short (μs) high power pulses of broad-spectrum light. PL has a similar spectrum to sunlight with wavelengths from 200 to 1100 nm and encompasses ultraviolet (UV), visible (VIS) and infrared (IR) light, with an enormous output in the UV range [20]. Flashes of light have a higher decontamination efficiency than the continuous application of UV-light because the energy incorporated is multiplied manifold [21,22].
The inactivation of microorganisms is a nonselective multitarget process, in which the photo-chemical effect is the most important mechanism. UV-C light is absorbed by DNA and pyrimidine dimers are formed, hindering DNA replication. The IR light component has a photo-thermal effect at higher powers [23], at which local overheating results in cell damage and cell ruptures [24]. The photo-physical effect is described by Takeshita et al. 2003 [25], who observed changes in cell shape and cell membrane, cytoplasmic damage and the leakage of intracellular compounds. The majority of publications reported a higher resistance of gram-positive bacteria in comparison to gram-negative bacteria [19] but strain-dependent susceptibility has also been observed [26]. The inactivation of fungal and bacterial spores differs and depends on the presence of pigments [27].
Actual reductions achieved on meat surfaces range from 0.9 log for Listeria monocytogenes on vacuum-packed beef carpaccio [28] to 4.39 log for Yersinia enterocolitica on pork skin [29] ( Table 2). Decontamination of skinless chicken thigh or breast has been reported of various pathogens at 2.09-3.0 log. The range of reduction on chicken thigh with skin was 1.82-1.96 log, depending on the conditions used (Table 2). Efficiency strongly depends on energy input (fluence J/cm 2 ), product surface and composition. Opaque and non-uniform surfaces limit the effect due to shading effects caused by pores and cracks [21]. High fat and protein content reduce treatment effectiveness due to increased UV absorption [19].
Effects on sensory behaviour are well documented [29,31,34,47,48]. Depending on the energy input and the kind of meat, colour and odour changes have been detected. Beef, pork and deer meat decreased in redness, whilst chicken colour increased in lightness at high fluences [33,48]. From the perspective of consumers, odour changes or off-flavours are a bigger issue. Significant changes have been described in the case of pork and chicken meat by Koch et al. 2019 [29] and McLeod et al. 2018 [31], even at low fluences. The offflavours may have been induced by the formation of ozone and nitrogen oxides which arise during treatment in the PL chamber [29]. Accelerated lipid peroxidation was indeed measured but only in low concentrations and this was not detected by panellists [29,31,34,47].
PL technology is a surface decontamination method, as the light penetrates only a few μm into the surface [23]. The main advantages are very short treatment times, the ability to inactivate bacterial spores, a lack of chemical residues and operation in batch or continuous mode. Although PL was approved by the FDA in 1996 for food disinfection, at present, it is only used in the food packaging industry, and not at a large scale.

Ultraviolet (UV-C) Light
UV-C (200-280 nm), a type of the ultraviolet light (100-400 nm), can help sterilize liquids, indoor air or surfaces [49][50][51]. Unfortunately, it is still underused in the food industry. The major goal of all studies has been to reduce pathogenic microorganisms in food products and extend shelf life without impairing freshness [35,42,44]. UV-C light generates photoproducts during treatment (pyrimidine pyrimidone (6-4) and pyrimidine dimers) resulting in damage to microbial DNA and proteins in living cells. DNA damage may involve the crosslinking of the strong hydrogen bonds between the nucleobases thymine-cytosine. DNA-transcription and replication are thus disturbed, which can disable repair processes, and cause mutations and cell death [52]. In relation to raw meat decontamination, several research groups have examined the potential of UV-C LEDs for the reduction of microbial load. For example, with doses of up to 2040 mJ/cm 2 , 0.56, 0.82 and CFU: colony-forming units D 10 : radiation dose [kGy] to reduce the exposed microbial population by 90% (one log10) CFU: colony-forming units; EHEC: Enterohaemorrhagic Escherichia coli; ESBL: Extended Spectrum Beta-Lactamase 0.95 log reductions were achieved for Yersinia enterocolitica on pork after 1, 7 and 14 days of storage (Table 2), respectively [42]. Similar results were reported at 1 J/cm 2 (1000 mJ/ cm 2 ) UV-C for Yersinia pestis on chicken breast filets and beef steaks [39].
In another study, greater reductions of up to 2.4, 1.8, 2.6 and 1.7 log were obtained for Salmonella Enteritidis, Listeria monocytogenes, Staphylococcus aureus and enterohaemorrhagic Escherichia coli, respectively, when chicken filets were irradiated with doses of up to 3 J/cm 2 at a distance of 6 cm [31]. In addition, combined treatments were investigated to improve microbial reductions on raw meat [45,53]. UV-C treatment at 590 mJ/ cm 2 reduced Listeria monocytogenes, Salmonella strains and Escherichia coli O157: H7 on fresh beef by 0.89, 1.08 and 1.19 log. Combination treatment with gaseous ozone significantly decreased the pathogens to 1.14, 1.33 and 1.42 log, respectively [40], whilst synergistic treatment of Listeria monocytogenes with UV-C light and phage on chicken breast resulted in a reduction of up to 2.04 log [44]. By contrast, only 0.8 log reduction of Listeria monocytogenes on chicken meat was observed after combined treatment with chlorine (200 mg/kg) and UV-C (300 mW/cm 2 ) [45]. Furthermore, most studies examined the impact of UV-C irradiation on organoleptic properties, such as the colour and texture of raw meat. The authors reported that there was minimal to zero influence on the quality parameters of the samples treated [40,42,45]. UV-C radiation can help reduce the microbes on the surface of the meat but not able to completely eliminate them. For this reason, it is recommended to use this method in combination with other decontamination technologies to improve the microbial safety of raw meat.

Cold Atmospheric Plasma
Non-thermal atmospheric plasma or cold plasma is another of many emerging food preservation technologies which can effectively reduce food-borne pathogens with only minimal detectable effects on product quality, or with none at all [54,55].
In recent years, many plasma-generating devices have been used in order to investigate their antibacterial potency on various food products [56][57][58][59]. In general, cold plasma consists of UV photons, excited atoms and molecules, electrons, ions, free radicals and reactive species (atomic oxygen, hydroxyl radicals, ozone, nitrogen oxides, singlet oxygen and superoxide), which have the ability to kill bacteria, viruses and fungi [60,61]. These compounds cause cell misfunction through the lesions in the membrane, the breaking of chemical bonds in the cell wall, intracellular disorder, loss of enzyme activity, denaturation of proteins and damage to RNA and DNA, which can lead to bacterial cell death [62][63][64][65][66][67]. Since 2015, several studies have significantly increased knowledge of cold plasma and its applications in the food industry. Treatment with dielectric-barrier discharge (DBD) cold plasma has been examined particularly intensively [54,68,69]. The DBD plasma system consists of a high voltage source, a high voltage electrode, a ground electrode and a dielectric barrier. The electrical discharge occurs between the electrodes through the application of an alternating current and high voltage [70]. This method can therefore reduce the number of microorganisms (≥ 2 log-units) on foods and food products within the package, avoiding re-contamination [68,71] (Table 3), respectively [54,72]. The shelf life of chicken breast filets has also been increased by up to 2 weeks by using cold plasma in modified atmosphere packaging (65% O 2 , 30% CO 2 and 5% N 2 ) stored at 4°C [75,76]. After 120 h, posttreatment with DBD (at 70 kV for 300 s) resulted in a reduction of approximately 1 log for Campylobacter jejuni and Salmonella Typhimurium on chicken breast filets stored at 4°C (P < 0.05) [69]. With the two-dimensional array of an integrated coaxial microhollow DBD device, it was possible to reduce the presence of Salmonella enterica Serovar Heidelberg by up to 3.7 log on chicken breast [68]. Combining this treatment with peracetic acid has proven successful for minimizing the presence of Salmonella Typhimurium on skinless chicken meat. Different hurdle interventions of peracetic acid (100-200 ppm) and atmospheric cold plasma (voltage 0-30 kV) were used to improve the antibacterial effect against pathogenic agents. Although this combined treatment did achieve significant inactivation of up to 5.3 log, it caused qualitative changes in the colours and moisture content of chicken meat [74•]. Direct treatment with plasma jet on raw chicken samples has been reported by Rossow et al. [57]. Argon and air were used as the feed gas to generate plasma by means of a jet. After 180 s, Campylobacter jejuni was reduced by around 2.50 log on skin and muscle.
Most studies, however, reported that changes in quality during plasma treatment were very limited [54,55,72,73,77]. This technology is environmentally friendly, with no chemical or organic residues, and has great potential. It could, for example, also be used to decontaminate equipment which has come into contact with meat or other foodstuffs, such as cutting tools [78]. It should be regarded as a future technology in the food industry for the enhancement of food safety.
Other authors used non-thermal plasma-activated water (PAW) to reduce microbial load on food [58, 74•]. PAW can be generated using two different methods. Activation occurs either in or above the water. In the case of the first method, cold plasma is discharged within bubbles, with the plasma source operating underwater [79]. In the case of the second method, the discharges occur in the space (3 mm) between four single plasma jets and the surface of the solution [80]. Generally, PAW has a low pH and contains reactive oxygen and nitrogen species (RONS,) which play a major role in disrupting microbes [81,82]. PAW has been used to reduce Salmonella Enteritidis on slices of beef by about 1 log level. To improve efficiency, the slices of beef were treated with different types of plasma-activated lactic acid. However, whilst this combined treatment resulted in a reduction in Salmonella load of up to 3.52 log [55], Escherichia coli and Staphylococcus aureus achieved only up to only 1.12, 0.86 log on skin and 1.33, 0.83 log on muscle respectively after treatment with PAW and ultrasound [58].

High Pressure
High pressure processing (HPP) is a non-thermal, residuefree technology and has been applied in the food industry for several decades. For application, the food is vacuumpackaged in a flexible and water-proof package and submitted to a pressure vessel to pressures generally ranging from 100 to 600 MPa, depending on the product. This takes a few minutes and is carried out at ambient temperatures [83]. In contrast to most other technologies, HPP treats the whole product, as the isostatic pressure affects the food product virtually instantaneously and uniformly, regardless of geometry and size [84].
Small molecules, such as vitamins and flavour compounds are unaffected, which is relevant to the taste and nutritional value of the product. HPP affects noncovalent bonds (electrostatic and hydrophobic interactions) therefore macromolecules such as proteins are subjected to changes in their tertiary and quaternary structures [85]. Consequently, cell structures are disrupted by protein denaturation, lipid conformation and enzyme inactivation, which promote the inactivation of microorganisms. Bacteria are generally more resistant than yeast and moulds and, with some exceptions, gram-positive bacteria are more resistant than gram-negative bacteria [86]. In addition, variations between strains in resistance to pressure have been demonstrated e.g. by Tamber [87••] in the case of Salmonella. Diverse studies have shown the efficiency of HPP in the reduction of pathogenic bacteria in raw poultry, beef and pork meat and organs (Table 4). Over the past 5 years, most studies were focused on the applicability to chicken meat and beef, with the focus being on various pathogenic E. coli strains, whereby high reductions of between 4 and > 7 log were demonstrated, some within periods of 4 min [97]. Additionally, Salmonella spp. and Listeria spp. were significantly reduced by 3.4-7.78 log after HPP treatment in chicken filet, depending on pressure and holding time. Generally, a higher pressure and longer pressure holding time increased the efficiency of the treatment, as shown for Salmonella and E. coli on chicken breast, where an increase from 200 to 300 MPa enhanced reduction to about 1-1.3 log and 0.3-1.4 log respectively when increasing the holding time by 5 min [93]. Using higher pressure can reduce the holding time, an important aspect for industrial applications. Unfortunately, increased pressure may accelerate the sensory deviations of the meat in terms of appearance, colour or texture. At >300 MPa, colour modifications were particularly observable in red and white meats. This is caused by myoglobin alterations and colours becoming browner in beef and lighter in pork and poultry [106]. Induced lipid oxidation was detected in fresh beef, poultry meat and pork at >300 MPa, most often occurring during subsequent storage [107].
HPP treatment enhances safety, extends the shelf life of meat and has good consumer acceptance [108]. The application is free from additives and can be used for packaged meat. Its efficiency can be further improved through combination with heat at approximately 60°C, the use of specific additives, or active packaging [107]. There are more than 300 HPP industrial facilities worldwide, with a share of 26% pressuretreated meat products [109]. Various HPP meat products, such as sliced and cooked ham, meat cuts or RTE products, are also commercially distributed in Europe.

Ultrasound
Ultrasound treatment or ultrasonication (US) is an emerging technology for diverse applications in food and nonfood areas which has been known of for some time. Ultrasound is defined as sound waves with frequencies higher than the upper limit of human hearing (20 kHz) and is therefore distinct from audible and infrasonic waves. In detail, US can be divided into power ultrasound (16-100 kHz), high-frequency ultrasound (100 kHz-1 MHz) and diagnostic ultrasound (1-10 MHz). US is already used in a variety of applications, e.g. measuring distances, cleaning, for sonography in medical imaging and in wastewater treatment. In food processing, it is used for the purposes of extraction, cleaning, emulsification and homogenisation. Because US is acoustic energy, ionizing and invasive effects can be excluded from consideration. Moreover, this technology uses a non-polluting form of mechanical energy and is therefore considered an emerging method for food processing which does not interfere with food quality, and which has high consumer acceptance.
Under exposure to US, compression and rarefaction are induced in the molecules of the medium in question. As a consequence of the pressure changes induced by the impingement of high-speed liquid jets and hydrodynamic shear forces, microbubbles are formed in liquid media, which expand and then implode. This phenomenon is called 'acoustic cavitation'. This mode of action can have various effects on living and nonliving materials. US is thus used in a variety of applications, including the antimicrobial treatment of food. For such purposes, it is assumed that the phenomenon of cavitation leads to the thinning of cell membranes, the generation of heat and the production of free radicals, which itself can damage cell membranes and DNA [110,111]. It has been shown that grampositive bacteria are more resistant to US than gram-negative bacteria due to their thicker cell walls [112,113].
Reviews show that the response to US of pathogenic and spoilage microorganisms in meat and meat products depends on many factors, such as acoustic energy and density, temperature and exposure time and product parameters such as pH, water activity, salt content and the presence of antimicrobials [114]. Although the antimicrobial effect of US is known, only a few studies consider it to be an alternative for the decontamination of carcasses and fresh meat surfaces.
Over the past decade, the majority of studies have used poultry meat and skin in order to examine the antimicrobial effect of US. In brief, various treatments were applied to artificially contaminated chicken meat, and showed no reduction, or only a slight reduction, in Campylobacter or Salmonella spp. [46,103,104,115,116] (Table 4). Similar results were obtained for Salmonella spp. and Staphylococcus aureus following a US bath, even after 50 min of treatment [105].
By contrast, it is evident that the antimicrobial effect of other non-thermal treatments, such as marination, chlorination, treatment with lactic acid, ethanol or with plasmaactivated water is enhanced if combined with US technology [58,103,[117][118][119].
Besides its antimicrobial effect, US is known to enhance various mass transfer processes, e.g. during the curing and pickling of meat. Furthermore, the use of US to trigger the tenderisation of meat has also been discussed [114]. All potential applications of US in the meat industry have been summarized in various review articles such as Alarcon-Rojo [120], Misra et al. [114] and Rosario et al. [121].
In conclusion, although data have shown that the treatment of raw meat surfaces using US alone can reduce bacterial load, the achieved reduction rates are low compared to those associated with other physical technologies. However, US can enhance antimicrobial impact in combination with biological or chemical treatments and is therefore a promising tool in terms of a combined hurdle concept for use during meat processing. In this context, future research and development are needed in order to implement the technology at an industrial scale. *Two-dimensional array of integrated, coaxial, microhollow, dielectric barrier discharge plasma CFU: colony-forming units

Conclusions
This review has examined recent research reports which focused on several non-thermal technologies (irradiation, pulsed light, UV-C light, cold plasma, high pressure and ultrasound), for use in reducing the presence of microorganisms on raw meat surfaces whilst minimizing loss of food quality. These treatments are environmentally friendly, free of chemicals and leave no residues, and one procedure alone is usually sufficient to decrease the pathogenic load on meat significantly. The time needed for the successful application of the different techniques varies; in the case of UV-C or pulsed light, the microorganisms on the product surface are reduced within seconds, but a much longer exposure time is required when using ultrasound. However, it has been shown that gramnegative pathogens often react more sensitively than grampositive pathogens and spores. For this reason, some authors suggest a combination of methods to achieve sterility effects, if necessary. However, more data are needed in order to optimize individual and synergistic treatments whilst maintaining the organoleptic properties and quality parameters of meat and meat products.
Additionally, food safety laws vary from country to country, so some technologies may not yet be permitted in some countries. Thus, it is not possible to deduce which method will prove to be the best.
In conclusion, it ought to be emphasized that the application of the decontamination procedures described above should be regarded as a supporting measure for combatting food-relevant infectious agents. Under no circumstances should they replace hygiene measures: good hygiene practice remains a priority.
Funding Open Access funding enabled and organized by Projekt DEAL.
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