Numerous studies demonstrate the effectiveness of ozone nanobubbles to reduce bacterial counts on meat and to prolong its shelf life.
Food waste is a major global challenge. The United Nations estimates that 17% of world food production is wasted, with meat accounting for more than 20% of that loss. Microbial spoilage of meat occurs throughout the entire cold supply chain, including transport, processing or storage, rendering it unfit for sale or human consumption. Extending the shelf-life of meat and meat products is therefore a key goal for companies in the sector.
Antimicrobial sprays
Antimicrobial sprays are approved in Canada, the United States and the European Union to control meat-borne pathogens and meat spoilers post slaughter. Lactic acid, peracetic acid and chlorine-based compounds are commonly used to reduce bacterial contamination. Peracetic acid and chlorine-based compounds are commonly used for their efficiency and low cost. However, chlorine can create halogenated by-products and peracetic acid can cause occupational health risks. In addition, these conventional antimicrobials can lead to the development of microbial resistance.
Ozone as a disinfectant
The use of ozone as a disinfectant in the food industry is growing. In June 2001, the Food and Drug Administration (FDA) approved ozone, both in its gaseous and aqueous phases, as an antimicrobial agent for direct contact with food, including all meat products. Its antimicrobial effectiveness is related to its oxidizing capacity that inactivates microorganisms by the progressive oxidation of cell components. The reactivity of ozone leads to rapid degradation to molecular oxygen without leaving toxic by-products. However, ozone is unstable and also poses a risk to occupational health.
This limitation can be mitigated by combining ozone with nanobubble technology, i.e. nanometer-sized gas bubbles in a liquid. Unlike macro- and micro-sized bubbles, nanobubbles remain suspended in liquids for an extended period. They can be generated through various methods, with hydrodynamic cavitation being the most frequently used. Factors such as temperature, pressure and the type of dissolved gas influence both the stability and generation of nanobubbles.
Conventional aqueous ozone consists of macro- and micro-sized bubbles that have an irregular shape and size, leading to quick breakdown and ineffective cleaning. With a negligible buoyancy force, a solution of pure ozone nanobubbles will have minimal off-gassing, allowing for higher concentration solutions to be used in food processing facilities. Ozone nanobubbles provide a stable solution with a high concentration of dissolved ozone, ensuring uniform surface coverage and improved antimicrobial efficacy, while minimizing occupational health risks.
Application of ozone nanobubbles to food
Several articles have appeared in scientific journals that have dealt with the use of ozone nanobubbles on food. Ozone nanobubbles treatment resulted in a reduction of cell counts of Listeria monocytogenes by about 1 log (CFU/cm2) on the surfaces of apples, celery and lettuce without affecting the color of the fresh produce (“Application of ultra-fine bubble technology to reduce Listeria monocytogenes contamination of fresh produce principal investigator“, Upadhyay, Cent. Food SAF., 2021).
Parsley soaked with ozone nanobubble solutions showed increased firmness and reduced weight loss (“Ozone micro-nano bubble water preserves the quality of postharvest parsley”, Shi et al., Food Res. Int., 170-2023) In aquaculture applications, ozone nanobubbles significantly reduced the bacterial load of the fish pathogen Aeromonas veronii, and reduced the infection of Nile tilapia without adverse effects on the animals (“Ozone nanobubble treatments improve survivability of Nile tilapia (Oreochromis niloticus) challenged with a pathogenic multi-drug resistant Aeromonas hdrophila”, Dien et al. J. Fish Dis., 44 – 2021).
Recently, the application of ozone nanobubbles in controlling microbial spoilage of meat has been explored. The muscles of the animals are essentially sterile, but contamination occurs during slaughter when the carcass is converted to meat. Sources of contamination include microorganisms associated with the animals and microorganisms that reside in meat processing facilities. For example, conveyor belts were identified as the primary vector for the transmission of E. coli to beef after sanitation, while cutting tables and mesh gloves were identified as sources of contamination for meat spoilage bacteria, including Pseudomonas, Carno bacterium, and Yersinia. Bacteria survived even after sanitation programs.
The formation of biofilms by microbial communities on the surfaces of processing equipment or processing facility contributes to microbial persistence and contamination of meat. Even the strains of Escherichia coli O157:H7 and non-O157 formed biofilms on surfaces of food-contact equipment, thus reducing the effectiveness of sanitizers. In processing facilities, immersion or washes with antimicrobial agents are used to reduce the microbial load of carcasses and meat cuts. The application of sanitizers in combination with appropriate packaging methods and storage conditions alters the composition of meat microbiota and subsequently determines the organoleptic properties of meat products.
Common meat-spoiling bacteria include psychotropic species of Pseudomonas, Acinetobacter, Staphylococcus, and Psychrobacter, lactic acid bacteria, Enterobacteriaceae, and clostridia. Depending on storage conditions, these microorganisms produce enzymes that break down carbohydrates, proteins or lipids, resulting in off-odors, slime and discoloration. In vacuum-packaged meat products, strict aerobes are inhibited, while lactic acid bacteria including Carnobacterium, Latilactobacillus and Leuconostoc and Enterobacterales such as Serratia, Rahnella, Hafnia and Yersinia dominate the spoilage communities.
Ozonation above 20 ppm showed to substantially reduce the environmental microbiota in red meat processing plants, in particular of Brochothrix and Pseudomonas. The application of aqueous ozone to beef cuts apparently reduces E. coli O157:H7 by 0.85 log (CFU/cm 2). However, the disinfection capacity of ozone is limited by its instability and low water solubility. Integrating ozone in nano-sized bubbles in water improves its stability. In general, antimicrobials are applied to meat carcasses or primary cuts, in immersion systems, by low- or high-pressure washes or by spraying.
In the study entitled ‘Control of meat spoilage with ozone nanobubbles: Insights from laboratory model systems and commercial-scale treatments’, published in the International Journal of Food Microbiology last April, the immersion or spraying with ozone nanobubbles in laboratory model systems with aseptic pork samples inoculated with reconstituted meat microbiota prior to treatment was used. Additionally, pork samples processed by spraying of peracetic acid or ozone nanobubbles in a meat processing facility were analyzed.
In particular, the pork samples were surface-inoculated with a cocktail of common microorganisms associated with the meat spoilage, composed of Brochothrix thermosphacta, Latilactobacillus sakei, Leuconostoc gelidum, Carnobacterium maltaromaticum, Hafnia paralvei and Yersinia rohdei. Both freshly inoculated and stored pork samples were treated with the two sanitation agents, followed by differential enumeration of viable bacteria.
Ozone nanobubbles were comparable to peracetic acid solution, regardless of the initial inoculum concentration and sample type. The efficacy of ozone nanobubbles increased with increased solution volume and flow rate. Moreover, the sanitizing agents differentially impacted the members of the microbiota and changed the composition of tested strains during storage. The Gram-negative Y. rohdei and H. paralvei were more sensitive to peracetic acid than the Gram-positive strains. The use of ozone nanobubbles significantly reduced the presence of Vagococcus and Clostridium compared to control samples.
Conclusions
Ozone nanobubbles are a new tool to reduce bacterial counts on meat processing plants. The best use remains to be established: Immersion of cuts or carcasses, utensils, or equipment in ozone nanobubbles; spraying or washing of equipment, carcasses or cuts, and the use of ozone nanobubbles to minimize cross-contamination when chilling carcasses in water. Ozone nanobubble sprays, tested both in laboratory settings and commercial processing facilities, reduced the cell count of E. coli O157:H7 on fresh beef surfaces by about 1.5 log.
The effectiveness of ozone nanobubbles on meat samples may be improved by increasing the solution volume. Increasing the flow regime from laminar circulation to turbulent circulation increased the reduction of cells in biofilms. This finding underscores the importance of optimizing the flow rate and solution volume to maximize antimicrobial efficacy while minimizing water use. It was also seen that microbial interactions within the meat microbiome also impact on meat quality.
Ozone nanobubbles have shown to act on the composition of the microbiota in favour of non-altering bacteria. Ozone also demonstrated bactericidal and sporicidal effect on Clostridium perfringens. Controlling clostridia is challenging, as heat treatments or other intervention methods in the processing of fresh meat are insufficient to eliminate them. Taken together, ozone nanobubbles have proven to be effective and environmentally friendly disinfectants to control meat spoilage, thus reducing food waste and economic losses.
