Thermal pasteurization techniques have been around since Louis Pasteur, and there is no question they’re effective. But unfortunately, thermal pasteurization can alter the taste, color or texture of many foods and beverages. Plus, some products simply don’t lend themselves to thermal treatments, either because of their physiology or because they’re marketed as high-end foods and beverages that must be treated in a way so they will still be considered organic or all natural—without damaging their natural state.

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A year ago, FE looked at four types of non-thermal pasteurization: high-pressure processing (HPP), ultrasonic, pulsed light and irradiation. The article delved further into HPP and bacteriophages, ozone and chemical treatments and their ability to sterilize food contact surfaces and equipment. In many cases, more than one non-thermal solution may need to be applied to achieve the required pathogen kill step or level of inactivation.


HPP: More adoption, reliability and capacity

The best part of HPP is that it not only kills Listeria, it also inactivates common food spoilage organisms. “HPP is an effective kill step for vegetative pathogens such as E. coli, Listeria, Salmonella and Campylobacter,” says Glenn Hewson, partner at GoToMarket LLC. Because HPP also inactivates spoilage organisms such as yeast and molds, its use extends shelf life by two times or more over other preservation methods, which extend distribution, he adds.

“HPP can kill off all sorts of vegetative microorganisms, both gram negative and positive,” says Francisco Purroy, technical sales manager, Hiperbaric, SA. “In general, gram-positive [bacteria] are more resistant than gram negative. And, in general, coccus genres or shapes are more resistant than bacillus genres or shapes. For example, Staphylococcus aureus is more resistant than Listeria monocytogenes. It’s hard to get more than two- to three-log reductions of S. aureus with HPP, whereas it’s typical to see five- to six-log reductions of Listeria with it.”

“Gram-positive spore formers are not inactivated in current HPP applications for pasteurization,” says Errol Raghubeer, senior VP of microbiology and food technology, Avure Technologies. Foods that lend themselves to HPP pasteurization include RTE and ready-to-cook meats, ready meals, fruits and vegetables, juices and smoothies, soups and sauces, wet salads and dips, dairy products, seafood and shellfish, according to Raghubeer.

HPP is suited to all sorts of food and beverage products with high Aw (water activity) values, adds Purroy. But, it is not suitable for dry products such as spices and dried nuts, and it requires adequate packaging.

“HPP has no effect on covalent bonds,” says Raghubeer. Therefore, it doesn’t change the taste of food. As for texture, HPP can improve viscosity and creaminess, based on the carbohydrate and protein content in the particular food application, while with color, its effects are typically product and packaging dependent. In terms of nutrition, HPP does not affect vitamins, minerals and other bioactive compounds, continues Raghubeer.

“HPP is considered to be a ‘what goes in is what comes out’ technology,” says Hewson. “Since it is a non-thermal process, it protects both organoleptic and nutritional properties, which has been proven in the super-premium juice, guacamole and salsa categories. In some cases, HPP can actually work to enhance flavors. Consequently, product formulation may need to be looked at when converting existing products to HPP from other preservation methods.”

While HPP has little, if any, effect on cooked meat, it can create small changes to raw meat or fish. “HPP causes gelification or texturization of the proteins, hence changing color [paler] and texture [gelified],” explains Hiperbaric’s Purroy. However, if the meat has already been stabilized in a cooking process, HPP will not cause any further changes.

In the last 10 years, HPP has become more efficient, affordable and available. According to Purroy, in 2005, the best-selling Hiperbaric system was a 300-liter machine capable of processing 800kg (1,764 lbs.) per hour. Today, Hiperbaric has a 525-liter machine with a capacity in excess of 2,700kg (5,952 lbs.) per hour. “Maintenance costs also are always being reduced,” adds Purroy. “More customers are looking to automate and integrate their HPP lines, so these lines are becoming more effective and cheaper in the long run.”

Obviously, not all processors are able to purchase their own HPP equipment. “A company’s annual capacity [lbs. or bottles per year] helps determine whether to purchase equipment or use a toll processor,” observes Lisa Pitzer, Avure marketing director. “Some companies have capital funds and production floor space, while others choose not to make the capital investment.” Some companies set up their own lines to run their products.  If they have left-over capacity, they’ll run a tolling operation as well.

In addition, new HPP tolling facilities are coming online in the US. For example, in April, Universal Pasteurization Company LLC announced the opening of a 170,000-sq.-ft. facility in Malvern, PA. The facility offers HPP using state-of-the-art, 525-liter HPP machines, cold storage and other value-added services for food companies in the Northeast. According to Melanie Galloway, Universal Pasteurization CEO, the facility will eventually have over 9,000 slots of refrigerated space as well as two HPP lines, with the capacity to add up to four more HPP machines.

Currently, Avure has 11 tolling facilities for food and beverage processors in the US and 20 worldwide, with more scheduled to start up this year. Hiperbaric has more than 25 tolling locations across the US.

 

Can bacteria become resistant to phages?

The important issue of resistance is often raised whenever phage therapy or the treatment of food is discussed. To avoid extinction, bacteria have evolved to be able to escape and balance phage predation at some level, says Dirk de Meester, Micreos food safety business development manager. Bacteria can protect themselves against bacteriophage attack through a variety of resistance mechanisms.

Spontaneous mutations by bacteria can sometimes also result in phage insensitivity. A famous experiment demonstrated that such mutations occur at the same rate in a bacterial population whether phages are present or not, says de Meester. However, mutations are mostly detrimental, and any effects related to phage resistance may disappear when the phage pressure is relieved, either by reversion to the wild type (i.e., back mutation) or because remaining wild-type cells replace the mutant cells due to their greater fitness.

These phenomena have also been observed in phage treatment of foods. Apparently unstable mutation featuring aberrant cell shapes, followed by reversion after a few generations, has been reported. In one study, a mutation rate of 1-10 million was calculated to occur during phage treatment. In this case, where phage was applied on food to eradicate pathogens, such a mutation rate did not have any measurable effect on overall efficacy, simply because the number of bacterial cells per weight unit was too low to give rise to significant numbers of phage-resistant mutant bacteria, says de Meester.

“Considering a realistic situation with low-level contamination, any intervention strategy that reduces the bacterial burden by more than two orders of magnitude will significantly enhance food safety,” explains de Meester. “However, a decrease of 2 log10 also means that, statistically, 1 percent of the cells will be missed. These will survive and may eventually grow to higher numbers.  But if you start with a low number of cells, along with modest additional bacterial growth, this regrowth should still give rise to only 1 percent as many bacteria as would have been present, given growth without the initial reduction—a situation that is quite different from bacterial growth to the stationary-phase densities that can be observed in the laboratory.” 

Thus, it is evident that a theoretically possible mutant survival frequency of 1 in 1-10 million will be irrelevant in those cases where food is treated with phage and subsequently leaves the production facility. If phage-resistant bacterial mutants do not escape into an environmental niche where phage-selective pressure is high, they cannot be expected to present any danger to the long-term efficacy of phage-based intervention.

Phage suppliers, however, are ahead of the game in dealing with resistance issues. “Bacterial resistance can eventually develop, but Intralytix proprietary technology enables us to formulate bacteriophage preparations that significantly reduce this risk,” says Alexander Sulakvelidze, Intralytix chief scientist. “Moreover, if and when resistance becomes an issue, we have the capabilities to update our phage products rapidly to restore their full efficacy.”



Bacteriophages—selective inactivating

Vaccinations can reduce the amount of bacteria animals bring into the processing plant, which, in turn, can lessen the shedding of bacteria by live animals and the transmission of bacteria during and after slaughter and in the processing/packaging stages. Decreased bacterial loads in the plant make it easier for non-thermal measures like bacteriophages to inactivate the bacteria more quickly, potentially reducing human infections.

In 2013, research comparing the use of bacteriophages to physical and chemical treatments showed bacteriophages were effective in reducing bacterial counts of Salmonella Enteritidis and Typhimurium on poultry skin that had been intentionally inoculated for testing purposes. According to the study, “bacteriophages were found effective at a multiplicity of infection [MOI] of 10 at both 37°C and 25°C in liquid medium, with no significant reductions taking place when MOI was less than 10. [MOI is defined as the ratio of agents to the number of target cells present in a defined space.] When samples of chicken skin experimentally contaminated with 1x105 CFU/cm2 of Salmonella Enteritidis were treated with phage cocktail or chemical agents, similar reductions of about 1 log CFU/cm2 were observed. These data suggest that bacteriophages can be employed as an alternative agent to reduce Salmonella Enteritidis contamination of poultry carcasses in industrial conditions.”1

A 2005 study (“Use of lytic bacteriophages to reduce Salmonella Enteritidis in experimentally contaminated chicken cuts”)  demonstrated that “Salmonella CFUs were reduced in the bacteriophage-treated cuts on days 3, 6 and 9 post-treatment when compared to their non-treated counterparts.”2

Bacteriophages target specific pathogenic bacteria such as Salmonella, but specific phages have also been developed to combat Listeria monocytogenes and E. coli O157:H7. The use of bacteriophages is neither a sterilization nor pasteurization process, says Alexander Sulakvelidze, Intralytix chief scientist. “It is a novel ‘green’ technology that offers a safer and healthier approach to food safety. It does not change or compromise freshness, and the food retains its organic, unprocessed flavors, texture and nutritional value.”

Phages pose no environmental threat and use no chemicals. When dealing with countries that require imports to have no added chemicals, the use of phages can be an advantage to processors. “Our phage products are very simple to use,” states Sulakvelidze. “The customer dilutes them with tap water as indicated on the label and applies [the diluted solution] to the surface of the food by simple spraying it on. No specialized or expensive equipment is required. It is a straightforward, easy, safe application.”

“With phages, it’s all about getting proper coverage,” offers Dirk de Meester, Micreos food safety business development manager. “Challenge tests conducted with phages at low-level inoculations have proven to be extremely effective.  Two-log reductions have the potential to protect products at different stages from slaughter to packaging.”


Ozone outside and in

In 1957, UDSA approved ozone in gaseous form for the storage of meat. In 1975, FDA recognized ozone treatment to be a GMP for the bottled water industry,  recommending 0.1 part per million (0.1mg/l) of ozone in an enclosed system for at least five minutes. A panel of experts from the food science, ozone technology and other related fields declared ozone to be generally recognized as safe (GRAS) for use in food processing. In 2001, FDA recognized ozone as an antimicrobial agent suitable for use in food processing and agricultural production. USDA put out a final rule on ozone in 2002 under FSIS Directive 7120.1, Safe and suitable ingredients used in the production of meat and poultry.

Although ozone in a gaseous form can be applied to meat, high levels of it (in excess of 500 ppm) may affect the color or odor of meat, so it’s important to balance ozone concentration with bacteria kill rates. Typically, meat can tolerate 100 ppm of gaseous ozone exposure for up to 30 minutes, and 200 ppm concentrations have been shown to kill 95 percent or more of E. coli.3

Ozone dissolved in water has a typical half life of about 20 minutes, which can be both a plus and a minus. Because it breaks apart so quickly, ozone leaves behind no trace in the food or beverage, assuming an extremely high concentration isn’t used, which could affect colors or odors. Also, due to ozone’s instability in a water solution, it loses efficacy as it breaks down into oxygen. However, keeping the water cold and/or at freezing temperatures will retain it in solution much longer.

In the seafood industry, the combination of ozonated water and ice is used, which retains ozone for longer periods of time. According to Lynn Rayner, CEO of Acadian Fishermen’s Co-op on Prince Edward Island, Canada, ozone lowers bacterial counts that are tracked not only by food inspectors but customers as well. (For more on this, see FE’s Tech Flash, November 12, 2013.) In addition to using ozone on the seafood itself, the co-op uses it as a cleanup tool in the plant, making floors less slippery and safer for employees.

Ozonated water also has been used to wash/rinse fruit. In addition, Food Microbiology magazine reported a study found ozone could achieve a five-log reduction of E. coli in four minutes at the lowest pH of apple juice; at the juice’s highest pH, it took 18 minutes to achieve the same reduction.4

Ozone has also been tested in the production of an RTE, preservative-free ginger root product. Ozone was dissolved in water at a pH of 3.0, due to the acidic nature of the seasoning liquid used. Ginger roots were submerged in the seasoning liquid with ozone feeding for 15 and 20 minutes. The bacteria, mold and yeast counts in the product were less than 10 CFU/per gram, for a sterilization rate greater than 99.9 percent. The hygiene index of the RTE ginger product met the standards for pickles as stipulated under GB 2714-2003.5


Chemical treatments move from equipment to food

Some chemical treatments originally designed for equipment surface applications with permissible food contact have also been, or are being, approved for use either on the surface of food or mixed in it. One example is the cationic surfactant Ethyl Lauric Arginate, more commonly known as LAE. “LAE is typically applied as a surface treatment to kill bacteria on contact by disrupting the cell membrane of both gram-negative and positive bacteria,” says Brendon Baer, A&B Ingredients technical sales representative. “A GRAS compound, LAE is considered a processing aid and as a recognized pathogen intervention.”

LAE achieved GRAS status from FDA in September 2005 and was recognized by EFSA as “a fatty acid that can enter normal fatty acid metabolism.” EFSA also concluded LAE presents no danger of being genotoxic in mammalian cells and is devoid of reproductive and developmental toxicity.6

 “LAE can be used to reduce or eliminate Listeria, Salmonella, E. coli and Campylobacter,” adds Baer. “Listeria is the most common target pathogen for LAE. Log reductions typically range from three to four. Other organisms and spoilage mechanisms [e.g., Lactobacillus and Leuconostoc] typically see around two-log reductions.”

LAE is most widely used in RTE meat products, but its usage is expanding into fresh produce, nuts, raw meat products and cheeses. LAE is applied at extremely low levels and doesn’t affect the taste, texture, color, aroma, mouthfeel or nutritional characteristics of food. In addition, the action mechanism is not pH dependent, so it is a much more gentle process than other surface antimicrobials, says Baer.

LAE typically requires spraying equipment to deliver the antimicrobial directly onto the product surface or finished product container, says Baer. Accurate spraying technology is very important to minimize the cost of the antimicrobial vs. the upfront equipment costs, which have proven to be hurdles in the past. However, low-cost spray technology continues to evolve and makes the process more cost effective. “The options we now have available allow us to work with customers to hit any price point and still keep waste minimal,” concludes Baer.

LAE is the primary ingredient of A&B Ingredients’ CytoGuard antimicrobial product, which has a broad spectrum of antimicrobial efficacy and works over a pH range of three to seven. It has a high partition coefficient of greater than 10, which means the product concentrates in the water phase of products where most bacterial action occurs.


Pure nanomaterials vs. ionic metals

Silver nanoparticles have been showing up in antimicrobial coatings, textiles, keyboards, kids’ toys, baby blankets, socks, towels, toothpaste, wound dressings and biomedical devices. While there is no debate about silver’s efficacy in killing bacteria, the issue is what do we know about ingesting silver or nanoparticles of any substance?

FDA’s approach to regulating nanotechnology allows engineered materials to enter the food supply as GRAS substances without the agency’s knowledge. “Because GRAS notification is voluntary, and companies are not required to identify nano-materials in their GRAS substances, FDA has no way of knowing the full extent to which engineered materials have entered the US food supply as part of GRAS substances,” says the GAO study, “FDA Should Strengthen Its Oversight of Good Ingredients Determined to Be Generally Recognized as Safe (GRAS).” This is in contrast to the rules in Canada and the EU, which require that all engineered nanomaterials be submitted for scrutiny before entering the marketplace.

While there are probably no silver nanoparticles in today’s food chain, National Center for Toxicological Research (NCTR) scientists have found that silver nanoparticles, administered by oral gavage, caused size- and dose-dependent changes in the gastrointestinal microbiota and the gut-associated immune response of rats, with smaller nanoparticles demonstrating the greatest antimicrobial behavior.7 The question remains: What are the long-term effects of ingested silver nanoparticles? And a second question: If the use of these particles doesn’t have to be indicated on the label, how do we know if we’re ingesting them?

Fortunately, one way around this problem entirely is through the use of ionic silver, an antimicrobial technology based on silver dihydrogen citrate or SDC for short. “SDC is distinguished by being non-toxic and offering 24-hour residual protection when used to disinfect food contact surfaces,” explains Hank Lambert, CEO, PURE Bioscience, Inc. “SDC products are not nano-silver, but silver ions suspended in citric acid. SDC utilizes a completely different, patented technology that is not found in any other silver products.” According to Lambert, using less silver to create SDC eliminates the safety and environmental issues commonly associated with nano-silver and other silver materials.

“We have developed a formulation that can be used in direct food contact applications as a processing aid or intervention on poultry, produce and meats,” adds Lambert. “We are in the process of obtaining the necessary regulatory approvals for these direct food applications. Our testing has demonstrated that when SDC is applied to raw poultry during online processing, the presence of Salmonella is reduced to below detection levels.”

Lambert says preliminary testing also shows that, when used as a wash or rinse on fresh produce during processing, SDC is far more effective than chlorine and other chemistries in eliminating E. coli, Listeria and Salmonella. SDC does not have any impact on the composition, sensory or appearance characteristics of the food to which it is applied.

A copper-based solution is another approach that uses metallic ions to kill bacteria. CMS Technology’s PoultrypHresh liquid additives can be used with or without chlorine to kill Salmonella and Campylobacter bacteria in poultry carcass rinse applications. By controlling the pH of the solution, the product serves as a processing aid at multiple intervention points during secondary processing within the poultry industry.

The pHresh line withstands the high temperatures required in scalding and uses a dual inhibition mechanism that couples a buffered acid with a copper compound. The antibacterial system is stable over a wide range of temperatures, and all its ingredients are considered GRAS, according to John Meccia, CMS Technology CEO. For more information on PoultrypHresh, see pages 167 to 168 in this issue

To get the desired kill/inactivation rates, processors may need to combine solutions. But one thing is clear: Many well-tested and proven applications are available, and there are many potential applications that have yet to be attempted.


References:

  1. “Use of bacteriophages to reduce Salmonella in chicken skin in comparison with chemical agents,” Food Research International, Elsevier ScienceDirect; June 2013.
  2. “Use of lytic bacteriophages to reduce Salmonella Enteritidis in experimentally contaminated chicken cuts,” Revista Brasileira de Ciência Avícola; Fiorentin, Vieira, Barioni; Rev. Bras. Cienc. Avic. Vol.7 no.4 Campinas October/December 2005.
  3. “Efficacy of Gaseous Ozone Against Generic E. coli in Ground Beef,” Joel Leusink and George Kraft, Ozone Solutions, Inc., www.ozonesolutions.com.
  4. “Ozone could inactivate E. coli in apple juice, finds study,” Food Production Daily, May 14, 2010. Accessed from the Internet 5-14-2015.
  5. “Non-thermal Ozone Pasteurization of a Ginger Root Product,” Fujian Journal of Agricultural Sciences 2012, Vol. 27 Issue 4. Accessed from the Internet 5-14-2015.
  6. “Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food,” The EFSA Journal, April 17, 2007, 511, 1-27.
  7. “Effects of subchronic exposure of silver nanoparticles on intestinal microbiota and gut-associated immune responses in the ileum of Sprague-Dawley rats;” Williams K, Milner J, Boudreau MD, Gokulan K, Cerniglia CE, Khare S; Pub-Med; May 2015.


For more information:

Glenn Hewson, GoToMarket LLC, 760-943-8931, glenn.hewson@gmail.com

Francisco Purroy, Hiperbaric, +34  947473874, (US: 305-639-9770), f.purroy@hiperbaric.com, www.hiperbaric.com

Errol Raghubeer, Avure Technologies, 614-255-6633, errol.raghubeer@avure.com, www.avure-hpp-foods.com

Lisa Pitzer, Avure Technologies, 614-255-6633, lisa.pitzer@avure.com, www.avure-hpp-foods.com

Alexander Sulakvelidze, Intralytix, 877-489-7424, asulakvelidze@intralytix.com, www.intralytix.com

Dirk de Meester, Micreos Food Safety, +31 317 421 414, d.demeester@micreos.com, www.micreosfoodsafety.com

Brandon Baer, A&B Ingredients, 973-227-1390, bbaer@abingredients.com, www.abingredients.com

Hank Lambert, PURE Bioscience Inc., 619-596-8600, hlambert@purebio.com, www.purebio.com

John Meccia, CMS Technology, 212-205-6171, jmeccia@cmstechnology.com, www.cmstechnology.com