Generally, food additives are used in the commercial food industry to improve foods’ color, smell, and taste, enhance nutritional value, increase processing efficiency, and extend shelf life ^[1][2][1,2]. Food additives are a broad group of compounds that potentially improve food quality [3]. Traditional food additives include salt, sugar, herbs or spices, vinegar, sulfur dioxide, and colorants [3]. However, the demand for types of food additives has changed with more emphasis on alternative additives deemed acceptable as “clean-label” [4]. The clean-label market trend for foods has become increasingly important to consumers in Western countries, and the onset of the pandemic in 2020 provided further motivation for consumers to prioritize food ingredients that they perceive as supporting a healthy lifestyle [5]. There is no legal or regulatory definition of a “clean-label” [4]. Consumers may equate clean-labels with minimally processed foods with no artificial flavors, artificial colors, or synthetic additives [4].

Consequently, food ingredients viewed as “healthier” or providing health-like benefits have gained commercial interest in the retail marketplace. These ingredients include those identified as part of a “clean-label” type of classification. A broad definition of “clean-label” foods is those with no artificial additives and are less processed, with simple and short ingredient lists that the average consumer recognizes ^[6][7][6,7]. Reduced fat, sugar, and sodium contents may also fall under the term “clean-label” when contributing to improved health [7].

In general, consumers rely on informational statements on packages such as “no preservatives” or “reduced fat” rather than reading the ingredient statements; for example, it has been reported that 65% of respondents to surveys responded that they never checked ingredient labels for additives [8], and some estimate that only 5 to 10% of consumers read the ingredients [9]. Delgado-Pando et al. [10] believe that consumers associate clean-label products with those with a short list of nonsynthetic or familiar ingredients. The Institute of Food Technologists (IFT) has proposed a definition: “clean-label means making a product using as few ingredients as possible and making sure those ingredients are items that consumers recognize and think of as wholesome” [11]. Cao and Miao [12] surveyed consumer-perceived clean-label attributes and found that the top three were minimally processed, eliminated undesired ingredients and exercised humane treatment. In order of perceived importance, they were minimally processed, had familiar ingredients, eliminated undesired ingredients, used local ingredients, and demonstrated an absence of allergens [12].

Clean-labeling in meat products, particularly further processed meats such as ready-to-eat (RTE) meats, has also become an issue. Most antimicrobials that are applied to RTE meats tend to be broad-spectrum in their activities, so it would be expected that overall microbial ecology would be impacted by their administration [13]. This, in turn, could alter the extent of shelf life and subsequent development of quality deterioration identified as spoilage. Historically, this has been difficult to characterize with conventional microbial culture-based approaches due to the limitations and selectivity of most media used for cultivation and enumeration. 

2. Clean-Label Antimicrobials in the U.S.

2.1. Organic Acids

Organic acids and salts of acids are theorized to exhibit antimicrobial activity in their un-dissociated and uncharged forms by being able to cross the bacterial cell membrane ^[14][79], where they increase osmotic stress and disrupt biomolecule synthesis, leading to bacterial death ^[15][16][17][80,81,82]. Organic acids have shown promise as natural antimicrobials but may affect color and flavor changes in products ^[18][83]. The most used acids are acetic, citric, lactic, propionic, malic, succinic, and tartaric ^[19][20][84,85]. Their many advantages include GRAS (generally regarded as safe) status by the U.S. Food and Drug Administration (FDA), no limits on acceptable daily intake, low cost, and ease of use, but caution must be observed as some Salmonella strains can develop resistance to acidic conditions ^[21][86]. Sodium lactate, the salt of lactic acid, has been used as an antimicrobial in Chinese-style sausages; lower total aerobic and anaerobic plates were seen with 3% sodium lactate during a storage time of 12 weeks at 4 °C ^[22][87]. Sodium lactate was also shown to inhibit the growth of several pathogens in inoculated cooked ham ^[23][88]. When samples were stored at 4 °C, 8 °C, or 10 °C, growth rates of L. monocytogenes, E. coli, or Salmonella decreased significantly for both 1% and 2% sodium lactate at all temperatures. However, the inhibition was more significant at lower storage temperatures ^[23][88]. The shelf life of ground beef treated with sodium lactate increased from 8 to 15 days when stored at 4 °C; aerobic plate counts, psychrotrophic counts, Enterobacteriaceae, and lactic acid bacteria counts decreased significantly compared to untreated controls ^[24][89]. Sodium lactate also effectively delayed the growth of aerobic plate counts (approximately 1.5 to 2 log reduction) in ground pork meat ^[25][90]. Barmpalia et al. ^[26][91] treated pork bologna sausage with 1.8% sodium lactate with 0.25% sodium diacetate and found that total spoilage organisms were reduced as much as 5 logs compared to the control when stored at 4 °C. Mohan and Pohlman ^[27][92] studied the effects of nine organic acids (30 g/L) and peroxyacetic acid (2 g/L) as antimicrobial treatments for beef trimmings. In general, most organic acids were effective at reducing bacterial populations, but caprylic acid produced a considerable reduction in coliforms (4.78 log reduction), E. coli O157:H7 (4.73 log reduction), and aerobic plate count bacteria (2.48 log reduction) ^[27][92]. The use of organic acids and their salts extends the shelf life of meat products and increases their safety due to their strong inhibitory effect against pathogenic and spoilage bacteria ^[28][93]. However, it has become apparent that some microorganisms have the means to counteract the effects of such acid-based agents, allowing them to become resistant to antimicrobial activity and to be able to survive under severe acidic conditions ^[29][30][94,95], leading to the emergence of acid-tolerant foodborne pathogens ^[31][96].

2.2. Botanicals

Botanically derived compounds, especially polyphenols, have antimicrobial effects and could be incorporated into RTE meats to reduce spoilage and increase food safety ^[32][33][97,98]. Polyphenols have one or more aromatic rings and one or more -OH groups, giving them their antibacterial properties ^[18][83]. These plant-based compounds can be further divided into extracts (normally hydrophilic, obtained by aqueous extractions) and essential oils (lipophilic, obtained by distillation or extraction by organic solvents) ^[34][99]. Extraction and separation of these plant substances are performed to confirm antimicrobial effects ^[34][99], but using extracts rather than plant powders also has advantages. Bioactive molecules are concentrated in the extracts so they can be used at lower concentrations than powders; the extracts are more stable, accessible, and economical to ship ^[18][83]. McDonnell et al. ^[35][100] evaluated the addition of vinegar, lemon, and cherry powder blend (1.5%) against the growth of L. monocytogenes inoculated on the surface of cured ham and deli-style turkey breast. They detected no growth of L. monocytogenes during 12 weeks of storage at 4 °C. Red fruit extracts (plum, red grapes, and elderberries) also inhibited pathogens, including B. cereus, S. aureus, and E. coli, while also increasing the growth of probiotic bacteria ^[36][101]. Extracts obtained from green tea, stinging nettle, and olive leaves produced a greater than 1 log CFU/g reduction in the total viable count, resulting in shelf-life extension when used in a frankfurter-type sausage ^[37][102]. Fresh chicken sausages were treated with 1% lemongrass extract, which significantly reduced the counts of total psychrotrophic and aerobic mesophilic microorganisms compared to controls ^[38][103]. Similarly, lemongrass extract extended the shelf life of cooked and shredded chicken breast and inhibited Staphylococcus spp., Salmonella spp., and coliforms ^[39][104]. A broad range of essential oils (EOs) and their active compounds possess antimicrobial effects. They are widely used in meat production to prevent spoilage and inhibit the growth of foodborne pathogens ^[40][41][105,106]. Many of these are considered GRAS ^[41][42][106,107], and lemon balm, basil, clove, vanilla, thyme, coriander, and others are approved for food in the U.S. ^[43][108]. Oregano oil has been proven to inhibit spoilage bacteria, extend shelf life in meat ^[44][45][46][109,110,111], and possess activity against pathogens including Sal. Enteritidis ^[47][112], Sal. Typhimurium ^[45][48][110,113], St. aureus, and L. monocytogenes ^[49][114]. Essential oils often have a strong odor and flavor, which may impact consumer acceptance and limit their use in foods [12]. In addition to their sensory effects, botanical-based antimicrobials may not mix well in food matrices or may be inactivated by standard processing techniques ^[50][115]. Other considerations include variations in the effectiveness of extracts prepared at different times from different batches of plant material and the possibility of bacteria developing resistance to the compounds ^[51][116]. There have also been concerns about the safety of these compounds since there is a potential for plants to be contaminated with heavy metals ^[52][117], mycotoxins ^[53][118], or pesticides ^[54][119]. Hurdle technology should be explored with EOs combined with other compounds or processing methods to lessen the organoleptic effects of EOs. Some suggested techniques include the encapsulation of EOs in nanostructures, which should be explored to extend the shelf life and safety of RTE meats ^[50][115].

2.3. Digestible Films and Coatings

Digestible films, also more commonly referred to as edible films, are usually cast over an inert surface, dried, and then used on food products in the form of pouches, wraps, capsules, bags, or casings ^[55][120]. Edible coatings, on the other hand, are food-grade suspensions that are sprayed or spread over the surface of a product, or the product is dipped in the suspension; when the suspension dries, it forms a transparent thin layer over the food surface and becomes a part of the final product ^[56][121]. These products can extend the shelf life of RTE meats by acting as barriers to water vapor, oxygen, and carbon dioxide, or they may also be used to carry antimicrobials ^[55][120]. These edible films and coatings can be based on lipids, proteins, or carbohydrates ^[55][120].

2.3.1. Protein-Based Films and Coatings

The proteins for these products can be from both animal (casein, whey protein concentrate and isolate, collagen, gelatin, and egg albumin) and plant sources (corn, soybean, wheat, cottonseed, peanut, and rice) ^[55][120]. These protein-based films work well on hydrophilic surfaces and act as a barrier to oxygen and carbon dioxide but not water ^[57][122]. Adding hydrophobic materials such as beeswax or oils can improve the moisture barrier properties ^[58][59][60][123,124,125]. Collagen, keratin, and proteins from quinoa, milk, egg, whey, zein, and soy are widely used to make protein-based films ^[61][126]. Protein-based edible films have been investigated for salami ^[62][127], chicken breast ^[63][64][128,129], beef ^[65][66][130,131], and pork ^[67][132]. However, these films can potentially be degraded by innate enzymes in the meat or prove allergenic for some portion of the population ^[68][133].

2.3.2. Lipid-Based Films and Coatings

Lipids used in edible films and coating come from both animal and plant sources. For instance, vegetable oils such as peanut, coconut, or palm oil and animal-based products including lard, butter, and fatty acids have all been utilized ^[69][134]. Fat has been used as a food coating since the 16th century ^[70][135]. Waxes and oils have been used for years as protective coatings for fresh fruits and vegetables ^[71][136]. However, wax-based fat- and oil-based coatings can be too thick, inconsistent, and greasy, or the coatings may crack ^[72][137]. There can also be a waxy or rancid taste that limits their use ^[72][137]. Therefore, lipids are usually used in a multi-component system with proteins, starch, cellulose, and their derivatives ^[57][122].

2.3.3. Carbohydrate-Based Coatings and Films

Polysaccharides are carbohydrates used in coatings; they are relatively poor moisture barriers but have low permeability to O₂ and CO₂ and are resistant to fats and oils ^[73][138]. Cellulose, starch, pectin, seaweed extracts, gums, and chitosan can be used to make polysaccharide films ^[55][120]. These films and coatings extend the shelf life of meats by preventing dehydration, oxidative rancidity, and surface browning ^[55][120]. If these coatings are used on meat products that are smoked or steamed, the film dissolves and becomes part of the product, thus increasing yield, improving texture, and reducing loss of moisture ^[57][122]. Cellulose is the most abundant renewable resource on the planet, and cellulose derivatives are often used for edible films because they are biodegradable, tasteless, and odorless ^[74][75][76][139,140,141]. The most frequently utilized cellulose derivatives for edible films are methylcellulose (MC), carboxymethyl cellulose (CMC), and hydroxypropyl methylcellulose (HPMC) ^[77][142]. Films made from these cellulose derivatives are transparent and strong ^[78][79][143,144], and bioactive compounds can be added to impart antimicrobial properties ^[76][141]. Xie et al. ^[80][145] studied cellulose-based films with ZnO nanopillars as an antimicrobial packaging material. They concluded that these films possessed good mechanical properties, were excellent barriers to water and oxygen, and had optimal antimicrobial activity against both Gram-positive (St. aureus) and Gram-negative (E. coli) bacteria ^[80][145]. Starch is another abundant plant-derived polysaccharide considered cost-effective and forms desirable films ^[75][81][82][140,146,147]. Corn, potatoes, rice, cassava, tapioca, and sweet potatoes are the most common starch sources for edible films ^[83][148]. Radha Krishnan et al. ^[84][149] added clove and cinnamon essential oils to corn starch film, extending beef fillets’ shelf life. Grape juice incorporated into maize starch films increased the shelf life of chicken breast fillets ^[85][150]. Curcumin extract incorporated into a rice film increased the shelf life of chicken and fish stored at refrigeration temperatures ^[86][151]. Chitosan, a high-molecular-weight cationic polysaccharide obtained from the shells of crustaceans (mainly lobster and shrimp), exhibits a pronounced film-forming capacity and antimicrobial activities ^[76][87][88][141,152,153]. Chitosan-based films are transparent, flexible, tough, and very resistant to fat, oil, and oxygen but are highly sensitive to moisture ^[76][89][141,154]. Chitosan also possesses antimicrobial properties. Alaskan pollock sausages stored in chitosan–gelatin films had both Gram-positive and Gram-negative bacteria growth inhibited in the sausages over 42 days of storage ^[90][155]. Cyclodextrins (CDs) are water-soluble cyclic oligosaccharides made up of D-glucopyranoside units linked by α-1,4-glycosidic bonds ^[91][156]. The positions of hydroxyl groups in CDs allow the exterior to be hydrophilic while the interior is hydrophobic ^[92][157], enabling them to form inclusion complexes with hydrophobic molecules ^[93][94][158,159]. These CD inclusion complexes are used to maintain firmness and freshness and aid in water retention in fresh-cut fruits and vegetables ^[95][160]. Empty CDs are used to encapsulate detrimental volatile compounds inside their hollow cavity. Ethylene is the compound responsible for the ripening of fruits, but the control of ripening during shipping is desirable, and it has been successfully controlled by using CDs ^[96][161]. For example, α-CD complexes were used in one study to encapsulate ethylene to prevent the ripening of mangoes ^[97][162]. Antimicrobial compounds can also be encapsulated in CDs for slow release with various triggers. Eucalyptus essential oil was treated with CD to form an inclusion complex, and zein was added to enhance antimicrobial results; L. monocytogenes was reduced by 28.5% and S. aureus by 24.3% when using these films ^[98][163]. CD-based films have also been used to eliminate fish odors and preserve meat, juices, milk, beverages of all types, processed foods, ready-to-eat foods, and cheese ^[99][164]. As already noted, edible films produced from polysaccharides are suitable barriers to gas but inadequate for water vapors and have poor mechanical strength ^[73][138]. Protein-based films also have poor water vapor resistance but good mechanical strength ^[57][122]. However, lipids cannot be used to make edible films as they cannot form a cohesive structure and are used as edible coatings, primarily for fruits and vegetables, or combined with proteins or polysaccharides to make composite films ^[100][165]. Edible films have yet to be commercialized for various reasons, not the least of which is that edible films are not as strong as plastic films and do not elongate well ^[101][166]. Jeevahan et al. ^[101][166] have written an excellent article addressing the problems with the commercialization of edible films, addressing six problem areas: (a) functional properties, (b) film making and drying methods, (c) nanotechnology on edible films, (d) lack of knowledge, and (e) consumer acceptance.

2.4. Bacteriocins: Sources and Applications for RTE Meats in the U.S.

Bacteriocins are peptides produced by certain bacteria that can exhibit activity against Gram-positive and Gram-negative bacteria ^[102][103][167,168]. They are known to be safe for human consumption because they are inactivated by proteases in the gastrointestinal tract (GIT) without affecting the normal microbiota of the GIT ^[104][169]. An attractive feature is that target bacteria do not form resistance against them ^[105][170]. There are three different approaches to the use of bacteriocins in food: (1) direct addition of the bacteriocin into the product; (2) incorporation of the bacteriocin into an edible film; (3) inclusion of bacteriocin-producing cultures into the formula or in a coating ^[104][169]. Lactococcus lactis subsp. lactis produces a bacteriocin, nisin, which has been found to inhibit the growth of Clostridium and Bacillus ^[106][171]. Many countries, including the U.S., allow the use of nisin in food products such as milk, processed cheese, grated cheese, dairy products, canned vegetables, soups, and meat, as well as brewery products and mayonnaise ^{[107][108][109]}[172,173,174]. However, the use of nisin in meat is limited because it has low solubility in meat products, innate enzymes in the meat may destroy nisin, and it has shown limited effectiveness against several meat spoilage organisms ^[110][175]. Pediocin PA-1/AcH, produced by Pediococcus acidilactici, is another bacteriocin that can be used in dried sausages and fermented meat products ^{[103][105][110]}[168,170,175]. P. acidilactici MCH14, which produces pediocin PA-1, was incorporated into dry fermented sausages and was shown to inhibit the growth of both L. monocytogenes and Cl. perfringens ^[111][176]. Strain P. pentosaceus BCC3772, which produces pediocin PA-1/AcH, inhibited L. monocytogenes during the fermentation of a Thai pork sausage without significantly altering its odor or taste ^[112][177]. Pediocin and nisin were used in vacuum-packed sliced ham and reduced the counts of Lactobacillus sakei, an important spoilage organism ^[113][178]. Pediocin PA-1 minimizes the growth of spoilage microorganisms during the storage of meat products; it is active at low pH and acts synergistically with lactate or organic acids ^[114][179]. A bacteriocin produced by Leuconostoc mesenteroides ssp. mesenteroides IMAU: 10231 was incorporated into a fermented sausage product, reducing the growth of L. monocytogenes during 21 days of refrigerated storage ^[115][180]. This strain of Leuconostoc produces CO₂ during fermentation, which means the bacteriocin itself must be used rather than the bacteria being incorporated as a part of the culture ^[116][181]. However, a homofermentative lactic acid bacteria (LAB) that produces lactic acid instead of CO₂ could be used as a starter culture ^[104][169]. For example, Weissella paramesenteroides DX produces the bacteriocin weissellin A. When this organism is grown in oxygen, sodium nitrite inhibits the bacteriocin production ^[116][181]. However, in anaerobic conditions such as those found when fermenting meat sausages, sodium nitrite does not inhibit the production of bacteriocin, making this organism an optimal candidate as a starter culture ^[116][181]. The bacteriocin BacFL31 produced by Enterococcus faecium sp. FL 31 inhibited the growth of spoilage microorganisms, L. monocytogenes, and Sal. Typhimurium in ground turkey meat during refrigerated storage ^[117][182]. Direct application of bacteriocins in the meat matrix reduces the antimicrobial activity of some other bacteriocins ^[118][183]. To avoid reductions in the efficacy of other bacteriocins, an alternative application method is to incorporate the bacteriocin into the food packaging material to serve as an active packaging component, therefore reducing the direct contact of the bacteriocin with the food product ^[104][169]. This approach allows the use of a smaller quantity of bacteriocins and controlled release, as the activity is on the product’s surface ^[119][184]. Over the past ten years, nisin has been widely utilized as an active packaging component ^[120][185]. Nisin used in various films and coatings has been shown to reduce the growth of L. monocytogenes, B. thermosphacta, Enterobacteriaceae, and spoilage LAB in raw meat, sliced ham, and ground beef ^{[65][121][122][123]}[130,186,187,188]. Pediocin incorporated into cellulose-based packages also reduced the growth of L. monocytogenes in sliced ham, turkey, and beef ^[124][125][189,190]. Marcos et al. ^[126][191] demonstrated the increased safety of sliced ham by delaying or reducing the growth of L. monocytogenes using enterocins incorporated into alginate, zein, or polyvinyl alcohol-based biodegradable film. Despite the success of nisin and pediocin, the use of bacteriocins in foods may be limited by their poor solubility and the uneven distribution and partitioning in the food matrix ^[104][127][169,192]. Their narrow spectrum of activity means that specific spoilage organisms must be identified and bacteriocins discovered that inactivate these bacteria ^[128][193]. Purification of bacteriocins can be difficult and expensive ^[128][193].

2.5. Bacteriophage for RTE Meats in the U.S.

Bacteriophages (phages), viruses that infect bacteria, are ubiquitous in the world, existing everywhere that there are bacteria ^{[129][130][131]}[194,195,196]. Bacteriophages have been studied for use as antimicrobials to increase food safety ^[132][197]. Since bacteriophages are specific for a particular bacterium, most studies in RTE meats and bacteriophages have been conducted on pathogens, especially L. monocytogenes ^[133][198]. The U.S. Food and Drug Administration (FDA) has approved two bacteriophage preparations, using phages P100 and LMP-102, as food ingredients to control L. monocytogenes ^[134][199]. Alves et al. ^[135][200] incorporated bacteriophage IBB-PF7A in a sodium alginate film to limit meat spoilage caused by Pseudomonas fluorescens. The number of Ps. fluorescens organisms decreased by 2 Logs CFU/cm² during the first two days of refrigerated storage and then only dropped by 1 Log CFU/cm² over the next five days ^[135][200]. This highlights one of the problems with using bacteriophage in food products: their stability ^[135][200]. A more significant problem is the specificity of phages ^[133][198]. To target specific spoilage organisms, there is a need to definitively identify most, if not all, members of the microbial community of meat and meat products, especially regarding spoilage. Another limitation is that phages cannot be combined with other methods because the other techniques would inactivate the phages ^[136][201]. There are two types of bacteriophages, lytic and lysogenic, which also challenge their use ^[130][195]. Lytic phages take over the protein synthesis of the target cell and produce more phages, which then burst the target cell to infect other bacteria ^[130][195]. Lysogenic phages, on the other hand, integrate themselves into the host cell’s chromosome, making lytic phages the most suitable for food applications ^[130][136][195,201]. Bacteriophages cannot diffuse throughout food and are limited to surface application ^[136][201].