Exploring the Inactivation of Foodborne Pathogens in Wheat Milling
Scott Jensen, REPCO; Rolando Gonzalez, The Acheson Group
Summary
Flour contaminated with pathogens has been the subject of multiple recalls in the recent past. Most flour sold in the marketplace is enriched with vitamin and mineral premixes. A study was conducted to evaluate the role of REPCO premixes in inactivating inoculated foodborne pathogens that could be introduced through premix addition. Results suggest a strong antimicrobial effect of all five vitamin premixes against the foodborne pathogens examined, and likely a synergistic inactivation effect due to combined low pH, low aw and the antimicrobial action of the vitamin and minerals that are part of their formulation.
Background and Significance
Flour is a raw, minimally processed product intended to be mixed with other ingredients and cooked before consumption. It is a low-water-content ingredient and typically does not support bacterial growth, however, Salmonella may occasionally contaminate grains and flour (Sperber et al., 2007). If uneven distribution of moisture in the products results in wet spots, Salmonella can grow. Exposure to water can create a microenvironment that is favorable to growth of Salmonella, which is a concern due to its persistence in dry conditions (Richter et al., 1993). Shiga toxin-producing Escherichia coli (STEC) has also been identified as a group of pathogens that can contaminate flour (Thorpe, 2004). The risk does not seem to be limited to certain facilities, crop years, or origin. Flour has been recalled during numerous years, by numerous companies/brands, over varied geographic origin (ADM-Pillsbury, King Arthur, ALDI, 2019; General Mills-Gold Medal, 2019; General Mills-Gold Meal, Signature Kitchens, 2016).
Intrinsic factors such as pH and water activity affect microbial growth and survivability. Intracellular pH must be maintained above some critical level at which proteins become irreversibly denatured and interference with microbial metabolism ensues, although acid tolerance response from bacteria can confer cross-protection to other environmental stressors. Controlling water activity (aw) has been used for centuries to preserve food because bacteria experience hyperosmotic shock when they are transferred to environments that have higher solute concentrations than are found in their cytoplasm. Various microbes have different aw requirements, with most bacteria preferring values above 0.93 and typically no growth observed below 0.86.
Characterization of antimicrobial effect
Heavy metals/minerals
Heavy metals are naturally occurring elements that comprise essential (e.g., Cu, Fe, Ni, and Zn) and nonessential metals (Cd, Hg, and Pb) (Dercia Santos et al., 2018). Intracellular accumulation of heavy metals has been found to cause direct and indirect effects in microorganisms (Ahalya et al., 2003; Nies, 1999). Dissolved ions indirectly affect bacteria by incorporating into bacterial proteins and thereby rendering proteins non-functional and/or malfunctioning. Because of high attraction of metal ions by proteins, it leads to increase in cellular concentrations and cell death. On the other hand, metal dissolution results in the formation of active radicals which affect bacteria directly, resulting in the rupture of the cell wall and death as described by Kumada et al. (2001). Thus, metals can show biocidal action even at low concentrations. Gram-negative and Gram-positive bacteria can exhibit different levels of tolerance to heavy metal ions and this difference could be attributed to the difference in the cell wall structure (Yasuyuki et al., 2010). Several examples of microbial toxicity by specific metal species have been described, and growth inhibition and cellular death can also be considered the outcome of a combination of different mechanisms (Lemire et al., 2013).
Vitamins
Ascorbic acid (vitamin C) exhibits antimicrobial effects that are species and concentration dependent. Przekwas et al. (2020) found the best antimicrobial activity of vitamin C against Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes in the concentration of 25 mg/mL (required for E. coli but effective as low as 0.25 mg/mL in the case of L. monocytogenes).
Vitamin D displays potent antibacterial effects against a multitude of both Gram-positive and Gram-negative foodborne pathogens, with a minimum inhibitory concentration (MIC) of 16 μg/mL (Golpour et al., 2019). Lipid-soluble compounds including vitamin D have been found to cause changes in the fluidity of the bacterial membrane, facilitating the penetration of other antimicrobial substances (Pretto et al., 2004; Nicolson et al., 1999) and thus potentially facilitating a synergistic effect (see Hurdle and synergistic effects section below).
Ahgilan et al. (2016) demonstrated the efficacy of a stand-alone riboflavin (vitamin B2) solution (1 g/mL) for inactivation of human foodborne pathogens. Pantothenol, a provitamin of pantothenic acid (vitamin B5) and analog of pantothenate, suppresses the growth of S. aureus (>32 mM or >1.47 mg/mL), S. epidermidis (>32 mM or >1.47 mg/mL), and S. saprophyticus (2 mM or 0.092 mg/mL).
Shahzad et al. (2018) showed in vitro that several vitamins exhibit synergistic antibacterial effect when combined with antimicrobial compounds. Final concentrations of 10 mg/mL for each water-soluble vitamin B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), B12 (methylcobalamin) and C (ascorbic acid), and 0.1 mg/mL for each fat-soluble vitamin A (retinol), D (cholecalciferol), E (α-tocopherol) and K (menadione) were used, with B1, B2, and B12 showing good synergism against Gram-positive human pathogens, and E and K being more effective against the Gram-negative bacteria.
Other antimicrobial compounds
Tricalcium phosphate (TCP) is a promising novel inorganic substrate for delivering antimicrobial metal ions because metal ions can be easily substituted for Ca2+ ions in the TCP structure (Matsumoto et al., 2009). Since some bacteria possess mechanisms for solubilization of TCP, this can potentially contribute synergistically to bacterial stasis or inactivation due to pH decrease in the growth environment (Yu et al., 2011).
Benzoyl peroxide, a flour bleaching agent, has long been known to also have broad-spectrum antimicrobial properties related to its role as an oxidizing agent and the generation of highly reactive oxygen radicals (FDA, 2007). Similarly, zinc ions (Zn2+) exhibit antimicrobial activity against various bacterial and fungal strains, including enteric bacterial pathogens. The partial dissolution of zinc oxide (ZnO) particles releases Zn2+ ions in aqueous suspension that contributes to the antimicrobial activity of ZnO (Pasquet et al., 2014).
Salts such as potassium iodide have been used to potentiate the antimicrobial effect of other compounds and technologies (Vecchio et al., 2015), and potassium chloride (KCl) has been shown to have an antimicrobial effect equivalent to that of sodium chloride (NaCl, common salt) on pathogenic bacterial species (Bidlas et al., 2008) with inhibition increasing above 1% all the way up to no growth observed at 12%.
Hurdle and synergistic effects
A novel strategy to improve pathogen inactivation efficacy is to utilize synergistic antimicrobial effects generated (Zhang et al., 2020). Hurdle technologies are developed based on the simultaneous use of multiple treatments and their effect can be additive or synergistic. Instead of setting one parameter to the extreme lit for growth, hurdle technology deoptimizes a variety of factors such that lower concentrations of each are needed to inhibit growth (Doyle and Buchanan, 2013). For example, a limiting aw of 0.85 or a limiting pH of 4.6 prevents the growth of foodborne pathogens. Hurdle technology is most effective when it combines two stressors that act by different mechanisms (e.g., pH and aw, pH and a compound with antimicrobial effect). Synergistic antimicrobial effects only refer to the combined hurdles or treatments that can result in intensified antimicrobial effect. Studies suggest that the sequence and timing of stresses can also play a role in enhancing microbial inactivation.
Regulatory considerations
While developing synergistic technologies composed of chemical compounds, attention must be paid to the maximum allowable concentration of a specific compound (Zhang et al., 2020). Although a higher dose of compounds during treatment usually brings higher efficiency, it is critical to consider their residual levels in the final products and ensure that it is below the maximum allowable limit (or “in accordance with good manufacturing practice” for GRAS substance) (U.S. Food and Drug Administration, 1977).
Application to food safety
A challenge study was conducted on five different REPCO products that are representative of different families of products created and sold by the Company. The study was aimed to determine if the premix products when inoculated at specific levels were able to support bacterial survival after a period of time, and whether when inoculated premix is mixed according to application rates with raw flour, the bacteria become viable over a period of time.
A third-party laboratory was hired to conduct a study to determine the fate of foodborne pathogens (Listeria monocytogenes, Salmonella, E. coli O157:H7) in REPCO vitamin mixes stored at 35°C for 3 days. Additionally, REPCO vitamin mixes that were inoculated and stored at 35°C for 3 days were mixed with sterile flours to assess potential survival of inoculated bacterial organisms.
A considerable reduction (>6 log CFU/g) in viable cell counts of each pathogen was observed within one hour of inoculation for all REPCO vitamin mixes. No viable cells were detected in any of the samples following 72 hours of sample storage at 35°C (Table 1). Similarly, no viable cells were detected from any of the inoculated REPCO vitamin mixes that were subsequently blended with sterile flour and further incubated at 35°C for 3 days prior to analysis (Table 2).
Table 1. Counts (log CFU/g) of Listeria monocytogenes, Salmonella and E. coli O157:H7 in various Repco blends after inoculation and incubated storage.
Table 2. pH and water activity (aw) of various Repco blends during incubated storage.
Table 3. Counts (log CFU/g) of Listeria monocytogenes, Salmonella and E. coli O157:H7 three days after mixing inoculated and incubated Repco blends with sterile flour.
Table 4. pH and water activity (aw) 3 days after mixing inoculated and incubated Repco blends with sterile flour.
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