A Novel Device Reduces Spoilage from Aerobic Bacterial Growth in Refrigerated Apples
by Bill Misner, PhD
Online publication only. . .

Abstract
Objective: To simultaneously assess the rate of aerobic bacteria, yeast-mold proliferation count in 2 red delicious apple sections from a parent apple specimen, with and without proximal exposure to a packet of Zeolite-coated Potassium Permanganate (Z-KMnO₄). (Note: This is a plastic louvered container enclosed sachet filled with Potassium Permanganate, KMnO₄.)
Design, Sample, Location: Following 28-days' refrigeration in separate compartments, two "Red Delicious" apple samples excised from a single specimen were reduced to 1:1000 solutions then incubated for 36 and 72 hours to determine aerobic bacteria and yeast-mold counts respectively, in Northeastern Washington. One sample was exposed to an antimicrobial mineral compound, Z-KMnO₄, while the other sample was isolated (compartment-separated) from the antimicrobial Z-KMnO₄ mineral compound.
Results: Harvested apples predictably release ethylene gas, which acts as a ripening hormone. As ripening progresses, commensal environmental microbes proliferate in samples exposed to air. Eating over-ripened, microbe-infected spoiled produce has been associated with severe gastric stress, compromised immunity, and allergic reactions. The spoilage rate of stored produce is dependent upon time, temperature, viscosity, pH, humidity, space, and nutrient access. Refrigeration in temperatures below 50 degrees F and under 45% humidity delays the rate at which produce deteriorates by inhibiting ethylene gas ripening hormone release, hence the rate at which aerobic bacteria, yeast, and mold grows in produce. Zeolite-coated Potassium Permanganate (Z-KMnO₄) placed proximal to refrigerated produce reduces ripening rate, inhibits microbial growth, and spoilage. Identifying which microbes and how many are inactivated by packets of Z-KMnO₄ in refrigerator compartments, until now, has not been identified. A randomly selected Red Delicious apple sample was refrigerated for 28-days and enclosed adjacent to a single Z-KMnO₄ packet. This sample, following incubation, yielded 10,000 aerobic bacteria per milliliter count. Simultaneously, an apple section from the same apple was refrigerated for 28-days without Z-KMnO₄ exposure. This sample yielded a count of 10,000,000 aerobic bacteria per milliliter. From the same Red Delicious apple sample, both sections after being refrigerated for 28-days surprisingly yielded an equal ratio of 100,000 yeast/mold per milliliter in the sample with Z-KMnO₄, and 100,000 yeast/mold per milliliter in the sample isolated from exposure to Z-KMnO₄.
Conclusion: This shows that a single packet (Z-KMnO₄) stored with a refrigerated apple sample reduced the aerobic bacteria (AB) proliferation growth count by a factor of 1000, (10,000 AB Z-KMnO₄) as compared to 10,000,000 AB non-exposed sample). However, Z-KMnO₄ exposure did not reduce yeast/mold growth in either sample (100,000 Z-KMnO₄ as compared to 100,000 in non-exposed sample). Given the cost to health from incidental consumption of spoiled produce, reducing the rate of aerobic bacteria growth in produce is a nutrient-protective intervention with disease-preventative implications. This single experimental study is conclusively limited and therefore requires more extensive research in order to explain or confirm the evidence reported.

Introduction
Ethylene Gas (C₂H₄), a hydrocarbon, is a natural product of plant metabolism and is produced by all tissues of plants as a hormone, increasing the rate of aging and ripening properties. Harvested apples release significant amounts of ethylene, a hormone-ripening agent. During the ripening stage of fruit, ethylene gas accelerates the maturity process. Ethylene gas (released at a very high rate from apples) accelerates ripening, loss of chlorophyll, abortion of plant parts, stem shortening, abscission of plant parts, and epinasty (Han JH, 2003).

Apple Rate of Ethylene Production

Table V. How Ethylene Gas Affects Produce
(Ethylene production and sensitivity levels in selected fresh produce)

Following harvest, apple produce commences to release ripening-hormone ethylene gas. Over-ripening reduces the shelf life of apples, and so the reduction of ethylene is necessary. Ethylene gas removers include potassium permanganate (KMnO₄), zeolite, clay, bentolite, alumino-silicate and active carbon. Other sources of ethylene include ripening fruit, rotting vegetation, exhaust from internal combustion engines/heaters, smoke (including cigarettes), welding, and natural gas leaks (Han JH, 2003).

As fruit ripens, the fluid-juice within the fruit body supports aerobic bacteria and yeast/mold proliferation. Food-borne illnesses may occur due to incidental consumption of commercial, nonpasteurized ("fresh" or "unpasteurized") fruit juices (Matthys AW). Nonpasteurized fruit juice has been associated with numerous food-borne illness outbreaks since the 1920s. Disease syndromes have included salmonellosis, typhoid fever, cryptosporidiosis, Escherichia coli-related diarrhea, and hemolytic uremia (Parish 1997).

Apple Juice-associated Food Poisoning Outbreaks

Aerobic bacteria, E. coli 0157:H7, L. monocytogenes, and Salmonella proliferate in apple juice. Escherichia coli 0157:H7, Listeria monocytogenes, and Salmonella have been detected in samples of apple, orange, pineapple, and white grape juice concentrates even after 12 weeks of storage at -23 degrees C (Oyarzabal et al., 2003). Yeast, mold fungi, lactic-acid bacteria, and cocci log growth rate parallels deterioration of fruit juice represented by spore mesophyll aerobes of the subtilis-mesentericus type (Slovachevskaia et al., 1988). Pasteurization of apple cider is therefore a validated treatment for ensuring adequate destruction of E. coli 0157:H7, Salmonella spp., and L. monocytogenes (Mak et al., 2001, Teo et al., 2001).

Preventing microbial infection of whole fruit is unavoidable. In three field studies, samples of unwashed apples (drops and picked), washed apples, and freshly pressed cider were presumptively analyzed for total coliforms, E. coli, and enterococci using qualitative and/or quantitative methods. Drop apples were more likely than picked apples to be contaminated with E. coli (26.7% vs. 0%) and enterococci (20% vs. 0%). Washing had little effect on coliform populations and in one field study was associated with increased numbers. Total coliform populations in cider ranged from <1 CFU/ml to >738 most probable number/ml, depending on the enumeration method used and the sample origin. E. coli was not recovered from washed apples or cider, but enterococci were present on 13% of washed apple samples. The qualitative coliform method successfully detected these bacteria on apples and in cider. Based on its exclusively fecal origin, good survival in apple cider, and association with drop apples, research concludes that E. coli is the most useful organism for confirming apple and cider sanitation (Lang et al., 1999).

Reducing either the regeneration rate of commensal microbe proliferation as apples ripen or reducing the rate of ripening without toxically affecting the nutrient profile of the fruit using Zeolite-coated Potassium Permanganate (Z-KMnO₄) in an enclosed packet was examined. Potassium Permanganate (KMnO₄) is a mild antiseptic/astringent, with antimicrobial properties (Anderson 2003). One study quantified the inactivation of the endotoxin derived from Escherichia coli 055:B5 by Potassium Permanganate (KMnO₄) used as an oxidant in drinking water treatment and was shown to inactivate 1.0 endotoxin units (EU)/mLh derived from Escherichia coli 055:B5 (Anderson et al., 2003). It was reported that potassium permanganate inactivated 90–100% of Pasteurella multocida strains (Karaivanov 1976, Brown et al., 1978). The Salmonaella enzyme was observed to be oxidized/inactivated by exposure to Potassium Permanganate (Roberts et al., 1975). Even the highly resistant virus of Creutzfeldt Jakob disease, exposed to a preparation of potassium permanganate, is either inactivated or inhibited (Uysal & Kaaden 1993).

In a preliminary investigation, I excised 2 Red Delicious apple sections from the same apple and refrigerated them separately for 7 days. One sample was exposed to Zeolite-Coated Potassium Permanganate (Z-KMnO₄) and the other sample was not exposed to Z-KMnO₄. The Z-KMnO₄ sample presented no outward visible spoilage/deterioration effects while the non-exposed sample displayed significant observable deterioration. What then was the numerical antimicrobial effect from Z-KMnO₄ on aerobic bacteria and yeast/mold growth rate?

In the present investigation, I repeated the preliminary experiment, but extended refrigerated storage from 7 to 28 consecutive days. Taken from a parent apple specimen, one apple section was exposed to Z-KMnO₄ but not the other. Aerobic bacteria growth rate in the Z-KMnO₄ exposed sample was significantly inhibited (by a factor of 1000 X (10,000 AB/ml Z-KMnO₄ sample to 10,000,000 AB/ml non-exposed sample). It was further observed that Z-KMnO₄ did not inhibit Yeast/Mold (YM) growth. Both samples following 28 days refrigeration yielded no significant difference in YM count/ml (Z-KMN04 = 100,000/ml YM: non-exposed sample = 100,000/ml YM).

Materials and Methods
Red Delicious Apples. Random selected produce was purchased from a Supermarket, Safeway Food & Drug, 1616 Northwest Blvd., Spokane, Washington, 99205.

Aerobic Bacteria and Yeast/Mold Test System Kits. Biosan Laboritories donated a SaniCheckAB and a SaniCheckYM Test System For Counting Aerobic Bacteria and Yeast/Mold. Biosan Laboratories, Inc., 1950 Tobsal Court, Warren Michigan, 48091–1351.

Zeolite-coated Potassium Permanganate (Z-KMnO₄) packets were donated by WayChem INC, P.O. Box 1450, 1101 Main Street, Evanston, Wyoming, 82931.

Design
A single Red Delicious apple was randomly selected from a dozen specimens. The outside of the apple was washed thoroughly with detergent soap then washed again with a 1% solution of hydrogen peroxide (H₂O₂) to inactivate transient microbes from the exterior skin surfaces. The parent apple was sectioned into 25-gram and 28-gram samples, respectively. Sterile technique was employed with all utensils prior to placing samples inside two enclosed containers, one with a 7.5-gram packet of Z-KMnO₄ and one without. Both containers were simultaneously stored in opposite refrigerator produce drawers. Each refrigerated compartment retained a consistent 45° F at 40% humidity for this 28-day storage period. After 28-days, each slice was blended in a distilled water 1:10 solution then further triturated to 1:1000 solution. Each solution's microbial count was determined by application of a SaniCheckAB and SaniCheckYM test pad to each sample. Each sample was incubated at 25–30°C (77–86°F) for either 36 hours for aerobic bacteria count or 72 hours for Yeast/Mold count, respectively.

Results
When the phenolic compound in each apple sample was exposed to air, a predictable MaillardReaction, browning of the fruit occurred at a more rapid rate in the control sample than in the VCPP sample (See Table I) or pictured example from a Gala Apple sample. In isolated refrigeration compartments, aerobic bacteria growth within an enclosed Red Delicious apple sample was inversely associated with proximal effective exposure to a single 7.5-gram packet containing Z-KMnO₄. The aerobic bacteria count following 28-days refrigeration, as determined by a SaniCheckAB Test, was 10,000/ml in the Z-KMnO₄ enclosed sample. However, the aerobic bacteria count in the other apple sample not exposed to Z-KMnO₄ was 10,000,000/ml. The Yeast/Mold count following 28 days refrigeration in these samples, as determined by a SaniCheckYM Test, was 100,000/ml in both Z-KMnO₄ & non-exposed samples, respectively. Aerobic bacteria growth rate was inhibited by a factor of 1000 in an apple sample exposed to Z-KMnO₄ as compared to an apple section from the original parent apple not exposed. Yeast/Mold growth rate was numerically equal in Z-KMnO₄ and non-exposed samples, respectively.

Discussion
Whole fruit or fruit juice may be the source of food-borne illnesses due to pre-harvest contamination or consumer-neglect from too long storage. Fresh apples and unpasteurized apple juice receive little to no antimicrobial treatment and, despite their health-promoting image, may transmit or harbor dangerous contaminants. Acid fruit juices below pH 4.6 were once deemed a minor health threat due to their high acidity. Furthermore, refrigeration temperatures (below 5ºC) were thought to resist pathogen growth, until the discovery that Listeria monocytogenes can grow in temperatures as low as 2ºC. Juice spoilage typically occurs as a reflection of the indigenous microflora, yeast, mold and/or lactic acid bacteria growth. Nonetheless, the emergence of hitherto unsuspected food pathogens with acid resistance combined with an increase in susceptible individuals, immunocompromised, chronically ill, the very young and very elderly, has dramatically changed this picture. Safety must always take precedent with strict limits on production, harvest, transportation, storage, manufacture, processing, labeling and distribution. These are incorporated into Good Agricultural Practices (GAPs) and Good Manufacturing Practices (GMPs) with Hazard Analysis and Critical Control Point (HACCP) procedures being applied throughout the food chain. These will be emphasized as appropriate. The National Food Processors Association (NFPA) has considered several options including current Good Manufacturing Practice (GMP) regulations. One of NFPA's officers wrote, "The only means of assuring that juice did not contain potentially pathogenic microorganisms was to include a microbial control step that has been scientifically proven to be effective in providing a level of protection equivalent to pasteurization in the process. Two percent of all juice products are not pasteurized or otherwise treated. Illness attributable to raw juice or juice includes imported frozen raw (unpasteurized) Mamey puree (13 cases, typhoid fever—Salmonella), raw apple juice in Canada (E. coli 0157:H7), raw orange juice in Australia (435 cases—Salmonella), raw orange juice from Arizona (300 cases—Salmonella muenchen in 20 states) and raw apple juice in Tulsa, Oklahoma (9 cases—E. coli 0157:H7). Only a microbial kill step applied to harvested raw fruit and/or juice itself can ensure that potentially pathogenic microorganisms are eliminated. Sorting and washing of fruit should be standard practice in all Good Manufacturing Practice operations for juice production but cannot be relied upon to ensure the complete removal of pathogenic microorganisms. While theoretically possible, achieving an appropriate level of protection from pathogenic microorganisms without applying some inactivating treatment to the juice seems technologically infeasible at this time. Processing methods that may provide an equivalent kill step include batch and continuous high-pressure processing systems, pulsed electric fields, ultraviolet light, electron beam treatment, irradiation, ultra filtration, or use of one or more of the preceding treatments in combination with an anti-microbial compound" (Matthys AW).

Potassium permanganate (KMnO₄) is a potent antimicrobial compound, which acts as an oxidizing agent directly reducing the contaminated environment of indigenous toxic substances and/or aerobic bacteria. KMnO₄ is so potent that it should not come in direct contact with humans or food nutrients. The packet-package of 7.5 grams Z-KMnO₄ permits the oxidizing, antimicrobial effect of the compound without transition or absorption into the adjacent foods stored within the same compartment. The chemical equation suggested by which ZPCC reduces ethylene gas and subsequently inhibits fruit ripening rate is through oxidization, namely as:



(Equation 1) 3CH₂ + 2 KMnO₄ + H₂O = 2MnO₂ + 3CH₃CHO + 2 KOH

(Equation 2) 3CH₃ CHO + 2 KMnO₄+ H₂O = 3CH₃ COOH + 2MnO₂ + 2KOH

(Equation 3) 3CH₃ COOH + 8KMnO KMnO₄ = 6CO₂ + 8KMnO₂ + 8KOH + 2H₂O

(Equation 4) Combining equations 1–3 = 3CH₂ CH₂ + 12KMnO₄ = 12MnO₂ + 12KOH + 6CO₂

Even if the reaction digresses or does not complete the carbon dioxide-producing equation, many of the intermediate products formed are irreversibly bound to the media or act as a reactant. Such is the case of the potassium hydroxide (KOH) formed in equations 1 and 2. The KOH will react with the acetic acid formed in equation 2 to produce the potassium acetate salt (KCOOCH₃) through a simple acid-base neutralization reaction shown as:

(Equation 5) = CH₃ COOH + KOH = KCOCH₃ + H₂O, or:

(Equations 1, 2, and 5) = 3CH₂CH₂ + 4KMnO₄ = 3KCOOCH₃ + 4MnO₂ + KOH =H₂O

Conclusion
One 7.5-gram packet of Zeolite-Coated Potassium Permanganate sachet stored in a plastic louvered container adjacent to an apple section for 28-days was observed to remarkably reduce ripening rate and aerobic bacteria growth by a factor of 1000, but failed to reduce the rate of yeast/mold spore growth. The data observed presents an advantageous method for reducing aerobic bacteria proliferation in stored produce, which is one marker of refrigerated spoilage, which otherwise may compromise health or increase the risk of food-borne illness. Z-KMnO₄-reduced produce ripening and subsequent aerobic bacteria contamination by this intervention should not be employed as a panacea for all indigenous microbes, such as yeast/mold spores.

Aerobic Bacteria Count In Two Isolated 25–28 gram Apple Sections
Samples (Post-Refrigeration 28-Days)

Is Ethylene Gas Control KMnO₄ Safe?
E.G.G. pellets oxidize the ethylene gas with nascent oxygen (nascent oxygen is a type of oxygen that oxidizes ethylene gas, molds, rots, and odors), converting the pellets into manganese dioxide, which is an organic fertilizer. Since February 2001, the Organic Material Review Institute for use with Organic food production and the material approve Ethylene Control E.G.G. for use and the FDA approves ink that is made in the plastic EGG-content sachets.

Correspondence:
Bill Misner Ph.D.
West 1140 Glass Avenue
Spokane, Washington 99205
509-327-5817
800-336-1977
drbill@e-caps.com

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Acknowledgements
I wish to acknowledge Biosan Laboratories and WayChem Incorporated for product donations to this research project:
Aerobic Bacteria and Yeast/Mold measures. Biosan Laboratories donated a SaniCheckAB & SaniCheckYM Test System For Counting Aerobic Bacteria and Yeast/Mold. Biosan Laboratories, Inc, 1950 Tobsal Court, Warren Michigan, 48091–1351.
Zeolite-Coated Potassium Permanganate (Z-KMnO₄) packets were donated by WayChem INC, P.O. Box 1450, 1101 Main Street, Evanston, Wyoming, 82931.
Disclosure: Neither competing interests nor remunerative relationship exist between the author and Biosan Laboratories or WayChem Inc, respectively.