Patent Publication Number: US-2013251864-A1

Title: Method for Treating Shell Eggs with Gas

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is a continuation in part of and claims the benefit of priority to U.S. patent application Ser. No. 13/425,100, filed Mar. 20, 2012 and entitled “Apparatus for Treating Items with Gas”. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to a method for treating items, such as shell eggs, with a treatment gas to eliminate or render harmless bacterial contaminants. 
     BACKGROUND 
     Bacterial contamination of raw foodstuffs, such as poultry eggs, fresh fruits and vegetables, nuts and legumes presents a widespread health hazard to consumers. As many as 48 million Americans are sickened each year by contaminated food. The hazard is manifest by disease outbreaks costing billions in health care, lost wages, and lost business, not to mention fatalities. For example, even though only a very small percentage (estimated at 1 in 20,000) of raw poultry eggs are contaminated internally with  Salmonella  Enteritidis,  Salmonella  transmission through contaminated eggs results in approximately 700,000 cases of salmonellosis at a cost in excess of $1.1 billion annually. Other bacteria, such as  Escherichia coli  and  Listeria monocytogenes  account for similar suffering and costs. 
     Decontamination of foodstuffs is a challenge, as many known methods, while lethal to the bacteria, damage or otherwise render the foodstuffs inedible or undesirable to consumers. The wide range of foodstuffs, as exemplified by poultry eggs, fresh fruits and vegetables, as well as nuts and legumes, with their radically different physical characteristics, each has different requirements for treatments which will effectively eliminate contaminants while preserving the properties of taste, freshness, appearance and transportability which makes the foodstuffs desirable and wholesome. There is clearly a need for an apparatus which can be used to effectively treat different types of foodstuffs against various contaminants using a variety of methods while maintaining the quality and desirability of the foodstuffs to consumers. By virtue of its versatility, such an apparatus would also be useful for sterilizing items such as wound dressings, surgical instruments, aseptic containers or items required to be free of microbial contaminants. Such an apparatus may further be applied to deactivate hazardous toxins, particularly those produced by mold such as alflatoxin, as well as the elimination of pesticide residue. 
     SUMMARY 
     The invention concerns a method for treating shell eggs to reduce internal  Salmonella  Enteritidis concentration in the eggs. In an example embodiment, the method comprises: 
     (a) heating the eggs to an internal temperature of about 55-60° C. for about 2-25 minutes; 
     (b) subjecting the eggs to a pressure of about 60-81 kPa; 
     (c) subjecting the eggs to a treatment gas comprising about 8-12 wt. % ozone; 
     (d) maintaining the eggs in contact with the treatment gas for a period of time long enough so that the concentration of  Salmonella  Enteritidis in the eggs is reduced by an amount of at least log 5. 
     In a particular example, the eggs are heated to an internal temperature of about 55-57° C. for about 8-20 minutes. 
     In another example, the eggs are heated to an internal temperature of about 56-57° C. for about 8-15 minutes. 
     In another example, the eggs are subjected to a pressure of about 64-81 kPa (5-10 in Hg vac). 
     In another example, the eggs are subjected to a pressure of about 63-73 kPa (8-11 in Hg vac). 
     In another example, the eggs are subjected to a pressure of about 65-70 kPa (9-10 in Hg vac). 
     In another example, the treatment gas comprises bout 8-10 wt. % ozone. 
     In another example, the treatment gas is at a pressure between about 8-12 psig. 
     In another example, the eggs are maintained in contact with the treatment gas for less than 33 minutes. 
     In another example, the eggs are maintained in contact with the treatment gas for less than 30 minutes. 
     In another example, the eggs are maintained in contact with the treatment gas for less than 28 minutes. 
     In another example, the eggs are maintained in contact with the treatment gas for less than 26 minutes. 
     In another example, the eggs are maintained in contact with the treatment gas for about 25 minutes. 
     In another example, the eggs are maintained in contact with the treatment gas for about 20 minutes. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic diagram depicting an example apparatus for treating items with treatment gas. 
     
    
    
     DETAILED DESCRIPTION 
       FIG. 1  shows, in schematic form, an example apparatus  10  for treating items  12  with treatment gas. Treatment gas is defined herein to mean a single gas or a mixture of different gases. Apparatus  10  comprises a chamber  14  adapted to receive items  12 . Thus, chamber  14  has an opening  16  providing access to the chamber interior for loading and unloading items  12 , the opening being closable by a door  18 , shown in an open configuration in broken line. Racks  20  or other holding devices may be positioned within the chamber  14  as appropriate for the particular items being treated by the apparatus  10 . For some treatments, it is advantageous that the chamber  14  be substantially fluid tight, to allow treatment gas pressures above and below atmospheric to be maintained within the chamber. Furthermore, the shape of such a chamber  14  will be guided by well known engineering principles for pressure vessels, and may result in designs having a cylindrical shape with hemispherical ends. Other shapes are of course feasible and practical, such as flat sided chambers having a rectangular cross section to eliminate dead space within. The chamber material must of course be compatible with the treatment gas so that the chamber is not attacked and corroded by it. Stainless steel alloys, such as SS 316 or better are acceptable for many systems. Similarly, any gaskets or seals must also be compatible with the treatment gas. Rubber is generally avoided as it is susceptible to attack by ozone for example. Gasket materials such as silicone and polytetrafluoroethylene are used to advantage in the apparatus. It is found advantageous to include one or more sealed view ports  23  in the wall of chamber  14 . Viewports  23  could be equipped with a camera  25  to document changes in the items  12  as they occur during treatment. The view ports would also admit light into the chamber  14  to permit the photographic recording, for example, still photos, time lapse, or real time video. 
     Chamber  14  has a gas inlet  22 , and may have a separate gas outlet  24  which provides fluid communication between the chamber and the ambient  26  often through a gas destructing unit  88 . Destructing unit  88  may be a heater or a furnace which breaks down ozone or other heat-labile gases, a catalyst to catalyze the conversion of the gas to harmless product, a bed of gas-absorbent, or similar products. Gas inlet  22  is in fluid communication with a distribution duct  28  positioned within the chamber  14 . Distribution duct  28  extends throughout the chamber  14  and has a plurality of openings  30  for distributing treatment gas therein. The distribution duct  28  avoids stratification of treatment gas which enters chamber  14  through the gas inlet  22  and promotes a substantially homogeneous treatment gas mixture within the chamber. The homogeneous treatment gas mixture ensures that all of the items  12  within the chamber are exposed to the same concentration of treatment gas for effective treatment, regardless of their position within the chamber. 
     Apparatus  10  may also comprise one or more sources  32  of treatment gas, which may include, for example, an ozone generator  32   a,  a tank of carbon dioxide  32   b  and/or other devices or reservoirs capable of providing gas to the chamber  14 . An inlet duct  34  provides fluid communication between treatment gas source  32  and the gas inlet  22 . An inlet valve  36  may be positioned in the inlet duct  34  between the treatment gas source  32  and the gas inlet  22  to control the flow of treatment gas from the source to the chamber  14 . It may also be advantageous to use a bypass duct  38  to provide fluid communication between the gas inlet  22  and the ambient  26 . An exhaust valve  40  is positioned within the bypass duct  38  to control treatment gas flow through the bypass duct from the gas inlet  22  to the ambient  26 . The treatment gas may pass through the destructing unit  88  before being released to the ambient. A bypass valve  42  is positioned in fluid communication with the gas inlet  22 , the inlet duct  34  and the bypass duct  38 . The positioning of bypass valve  42  between the gas inlet  22  and both the inlet duct  34  and the bypass duct  38  allows treatment gas from the source  32  to flow either to the gas inlet  22  (and thereby into chamber  14  through distribution duct  28 ) or to the ambient  26  through the bypass duct  38 . Treatment gas flow from source  32  into chamber  14  is enabled by closing the exhaust valve  40  and opening inlet valve  36  and bypass valve  42 . Treatment gas from source  32  may be vented to the ambient  26  (again, through destructing unit  88  when necessary) by closing the bypass valve  42  and opening the inlet valve  36  and the exhaust valve  40 . Treatment gas venting to the ambient through the destructing unit  88  is useful when the treatment gas source  32  is a device, such as the ozone generator  32   a,  which may take time to achieve full gas flow rate. Bypass venting of the treatment gas allows full flow rate to be reached before admitting the treatment gas to the chamber  14 . 
     Chamber  14  is advantageously fitted with another exhaust valve  44 . Exhaust valve  44  is in fluid communication with the gas outlet  24  and is used to control the flow of treatment gas between the chamber  14  and the ambient  26  through the destructing unit  88 . A vacuum pump  46  may be used to evacuate chamber  14  as well as to draw treatment gas into the chamber from the source  32 . It is generally advantageous to use oil-less pumps to prevent explosions when highly-reactive gases are used. Vacuum pump  46  has an intake port  48  in fluid communication with chamber  14  and an exhaust port  50  in fluid communication with the ambient  26 . Treatment gas pressure within chamber  14  above atmospheric may be achieved and maintained by the action of treatment gas source  32  itself, or by a booster pump  52  in fluid communication with the inlet duct  34  between treatment gas source  32  and gas inlet  22 . Additionally, a gas reservoir  54  may be used in conjunction with the inlet duct  34  as an accumulator to provide a flow of treatment gas to the chamber at constant pressure and flow rate if desired. 
     To rapidly clear the chamber  14  of treatment gas after treatment, a purge pump  56  is used. Purge pump  56  has an intake port  58  in fluid communication with the ambient  26  and an exhaust port  60  in fluid communication with the chamber  14 . If ambient air is used as a purge gas, it may be advantageous to filter the air using a HEPA filter for example, so as not to introduce bacteria or other contaminates into the chamber  14 . Alternately, chamber  14  may be purged with an inert gas such as nitrogen from a pressurized purge tank  62 . 
     For most gas treatments, it is desirable to control the relative humidity within the chamber  14 . To that end, a liquid reservoir  64  may be provided within chamber  14 . Reservoir  64  may be, for example, an open container or recess in which water is held, the water evaporating and providing moisture to maintain a desired relative humidity favorable to the gas treatment. To facilitate humidification within chamber  14  it is advantageous to introduce at least a portion of the treatment gas through the water in the reservoir. This humidifies the treatment gas released into the chamber, which in turn, humidifies the chamber. In place of or in addition to the liquid reservoir  64 , an external liquid reservoir  66  may be employed. External liquid reservoir  66  may be, for example, a water tank, or the water service of the facility in which the apparatus  10  is located. Water or other liquid from the external reservoir  66  is injected into the chamber  14  using a nozzle  68  in fluid communication with both the chamber  14  and the external reservoir  66 . A control valve  70  positioned between the external reservoir  66  and the nozzle  68  may be used to control the flow of liquid to the chamber  14 . 
     It may also be advantageous to control the treatment gas temperature within chamber  14 . A heat exchanger  72 , operating between the chamber  14  and the ambient  26  may be used to transfer heat to or from the treatment gas and thereby control the temperature within chamber  14 . The treatment gas within chamber  14  may be heated or cooled using heat transfer surfaces  74 , such as coils through which a heated or chilled working fluid, such as water, propylene glycol or ethylene glycol, flows. Alternately, the heat surfaces could be the coils of a heat pump which uses the Joule-Thompson effect to heat or cool chamber  14 . Solid state heating and cooling devices, such as Pelletier devices are also feasible. It may be advantageous to employ a fan  76  within chamber  14  to augment heat transfer by forcing the treatment gas across the heat transfer surfaces  74 . Fan  76  would also promote circulation and mixing of the treatment gas within the chamber, preventing stratification and ensuring process uniformity, i.e., all items in the chamber are exposed to an effective concentration of treatment gas. Control of the temperature within chamber  14  may also be effected by providing a layer of insulation  77  surrounding the chamber to reduce heat transfer between the chamber and the ambient  26 . Additional temperature control may be afforded by a heating or cooling jacket  79  surrounding the chamber and though which a heating or cooling medium, such as water, glycol, or steam, is circulated. 
     It is advantageous to measure and monitor various operational parameters of the apparatus  10 . The operation parameters of interest include the treatment gas pressure, temperature and relative humidity within chamber  14 , as well as the concentration of treatment gases, such as ozone and carbon dioxide used within the chamber, and treatment time. To this end apparatus  10  is equipped with: a pressure transducer  78  for measuring gas pressure within chamber  14 ; a temperature transducer  80  for measuring the temperature within chamber  14 ; and a humidity sensor  82  for measuring relative humidity within chamber  14 . A treatment gas concentration monitor  84  is used to sample the gas from within chamber  14  and measure the concentration of its constituent gases. The monitor  84  may be used in an open loop configuration  86  to sample and measure small amounts of gas, exhausting the gas sample to the ambient (if environmentally acceptable) or to the destructing unit  88  which treats the treatment gas sample to render it harmless. Monitor  84  may also be used in a closed loop configuration  90 , which includes a control valve  92  controlling the flow of gas from the chamber  14  to the monitor  84  and a pump  94  for pumping the gas to the monitor. The monitor  84 , pump  94  and valve  92  are in fluid communication with one another and the chamber  14  through piping network  96  which permits gas samples to be drawn from the chamber  14 , conducted to the monitor  84  where treatment gas concentration is measured, and then the gas sample is returned to the chamber  14 . In an alternate embodiment, a probe may be inserted into chamber  14  to measure treatment gas concentration; this enables the operator to avoid the need to sample the treatment gas. 
     Apparatus  10  may be automated in its operation through the use of a controller  98 , which may comprise, for example, a programmable logic controller or other microprocessor based device. The pressure and temperature transducers  78  and  80 , the humidity sensor  82  as well as the treatment gas concentration monitor  84  each generate electrical signals indicative of the respective parameters which they measure and transmit these signals to the controller  98  over a communication network symbolized by dashed lines  100 . Lines  100  represent various types of communication means, for example, hard wired electrical conductors as well as wireless radio frequency communication. Resident software within controller  98  interprets the information contained in the signals generated by the transducers, sensors and monitors and uses this information in a feed-back loop to control the operation of the various components of the apparatus  10 , such as the various valves, pumps, fan, gas generators and heat exchanger which are also in communication with the controller over communication lines  100 . Either fixed orifice or adjustable orifice valves can be used for control of fluid flow coupled with pulsed or analog signals from the controller  98  to maintain less than maximum flow rates for the various valves that may be required during certain phases of the process. Not all of the communication lines are shown in  FIG. 1 , it being understood that controller  98  may be connected as necessary to any and/or all components as necessary for automated control. 
     System Operation 
     An example of apparatus operation for decontaminating poultry shell eggs is described below, considering that the apparatus  10  may be applied to other items, and that the particular parameters of operation will vary for different items as appropriate. 
     Eggs  102  are heated in a water bath (not shown) to a temperature of about 56-57° C. (as measured at the yolk) to denature the membrane attached to the inside surface of the egg shell. The eggs  102  are removed from the bath and positioned within chamber  14  while still wet. Door  18  is closed (shown in solid line), and vacuum pump  46  is used to draw a vacuum within chamber  14  that ranges from about 10 inches Hg vac to about 15 inches Hg vac. Application of vacuum allows sufficient water to be drawn out of the shells to prevent subsequent mold growth during product storage. Valve  106  is closed to isolate the vacuum pump  46  from the ambient. 
     In this example of apparatus operation the eggs  102  are to be decontaminated, both inside and outside their shell, by exposure to ozone. To that end, the treatment gas source  32  is the ozone generator  32   a  which is activated and begins to produce ozone. During the transient phase of ozone generator operation, the inlet valve  36  is open, the bypass valve  42  is closed and the exhaust valve  40  is open to permit the ozone generator  32  time to reach full ozone flow rate. Once this flow rate is achieved and the eggs have been subjected to vacuum, the exhaust valve  40  is closed, the bypass valve  42  is opened, thereby breaking the vacuum within chamber  14  by permitting ozone to flow into the chamber. Ozone flows through the bypass valve  42  and through the distribution duct  28  which distributes the ozone to all parts of the chamber  14 . The distribution duct promotes uniform ozone concentration throughout the chamber and thereby increases the effectiveness of the apparatus. 
     Ozone within the chamber  14  is maintained at 9-12 psig and a concentration of 8-12% by weight to ensure effective treatment of the eggs. Gas concentration monitor  84  samples the gas from chamber  14 , measures the ozone concentration, and signals the controller  98  over communication lines  100 , allowing the controller to increase or decrease the ozone concentration by control of the ozone generator  32  as required to maintain the desired concentration. Similarly, the pressure transducer  78  measures the gas pressure within chamber  14  and signals the controller, which increases or decreases the pressure as necessary to maintain the desired pressure. Booster pump  52  may be used in addition to the ozone generator  32  to maintain the desired gas pressure within chamber  14 . Temperature transducer  80  measures the temperature within the chamber  14  and signals the controller  98 , which activates the heat exchanger  72  to maintain the desired temperature. For egg decontamination using ozone, a temperature from about 15° C. to about 20° C. is desired, and generally the heat exchanger operates to cool the treatment gas within chamber  14  to maintain this temperature. Lower temperatures favor the stability of the ozone, which breaks down and becomes ineffective at higher temperatures. Fan  76  may also be operated as required to promote heat transfer and ensure proper circulation and mixing of the treatment gas for uniform gas concentration and temperature throughout the chamber. Uniform temperature and concentration ensure that all of the eggs are adequately exposed to an effective ozone bath. Water from the liquid reservoir  64  evaporates within the chamber  14  to maintain the desired relative humidity of 85-95%. The high relative humidity increases the antimicrobial effectiveness of the ozone. Should the humidity sensor  82  detect a decrease in the relative humidity its signals to the controller  98  will result in the controller injecting additional water into the chamber  14  from reservoir  66  through nozzle  68  via valve  72 . Under the desired conditions of ozone concentration, temperature, pressure and relative humidity prescribed above the eggs will be effectively sanitized after an exposure duration of 25-45 minutes. 
     After the eggs have been subjected to the ozone bath at the desired concentration of ozone within the desired temperature range, pressure range and humidity range for the desired amount of time, the ozone is vented properly and the eggs may be removed. Removing traces of ozone from the vessel may require flushing vessel contents with ambient air, several times. It is important that ozone level inside the vessel is lower to 0.1 ppm, or less, before the vessel is opened. Valves  92 ,  104  and  106  are closed to isolate, respectively, the gas concentration monitor  84  and the vacuum pump  46  from the chamber  14 . The inlet valve  36  is closed to isolate the ozone generator  32   a,  and the exhaust valves  40  and  44  are opened to permit treatment gas to escape from chamber  14 . The escaping treatment gas, having a high concentration of ozone, is conducted to gas destructing unit  88 , in this example a heater, which breaks down the ozone into oxygen and releases it to the ambient  26 . Valve  44  can be used to regulate flow of exhaust gases to maintain product quality. Once the pressure within chamber  14  reaches about atmospheric pressure the purge pump  56  is actuated to inject ambient air into the chamber. Chamber pressure is raised and maintained at approximately 3 psig as decrease in treatment gas concentration is measured by the monitor  84 . This gas purging step ensures that little, if any ozone remains within the chamber, allowing it to be safely opened for removal of the treated eggs. 
     Method of Decontaminating Eggs 
     The invention also encompasses a method of decontaminating eggs by treating them with ozone. At its core, the method comprises initially subjecting the eggs to gas pressure less than atmospheric, for example, at a vacuum pressure from about 1 inch Hg vac to about 29.9 inches Hg vac. The low pressure of 10-15 inches Hg vac is found to be advantageous. The vacuum pressure is then broken by subjecting the eggs to ozone. In this example method the eggs are subjected to ozone at a pressure from about 3 psig to about 15 psig, with a pressure of 9-12 psig being advantageous. The eggs are subjected to the ozone for a duration from about 5 minutes to about 60 minutes, with a duration of 25-45 minutes being advantageous. The concentration of ozone may be from about 1% by weight to about 14% by weight, with an ozone concentration of 8-12% by weight being advantageous. While subjected to the ozone the eggs are maintained in an environment at a relative humidity of at least 80%, with 80-100% relative humidity being acceptable and 85-95% relative humidity being advantageous. 
     Other steps may be added to the method. For example, it is advantageous to heat the eggs in a water bath to an internal temperature from about 55° C. to about 60° C. to denature the membranes under the shell. A temperature of 56-57° C. is found effective. This heating step using a water bath also serves to we the eggs, as it is advantageous to subject the eggs to the vacuum while wet. To ensure the effectiveness of the ozone as a decontaminant, it is advantageous to cool the eggs after the heating step. The eggs may be cooled to a temperature from about 5° C. to about 30° C., with a temperature of 15° C. to about 20° C. being effective. 
     The following examples illustrate use of the method disclosed herein for the decontamination of  Salmonella -inoculated shell eggs by heat-ozone combination and compares its effectiveness against other methods of treatment. 
     Shell eggs were inoculated with  Salmonella enterica  server Enteritidis to contain 10 7  colony forming units (cfu)/g of egg contents. Inoculated eggs were exposed to one of the following treatments: 
     (1) Heating in a circulating water bath and holding egg immersed at 57° C. for 20 minutes. 
     (2) Heating in the water bath and holding eggs immersed at 57° C. for 20 minutes, followed by a gaseous ozone treatment comprised of applying vacuum at 10 in Hg vac, vessel repressurization to 10 psig with a stream of ozone gas to achieve a concentration of 9% (weight basis), and maintaining the ozone concentration and pressure for 30 minutes. 
     (3) Heating in the water bath and holding eggs immersed at 57° C. for 25 minutes, followed by a gaseous ozone treatment comprised of applying vacuum at 10 in Hg vac, vessel repressurization to 10 psig with a stream of ozone gas to achieve a concentration of 9% (weight basis), and maintaining the ozone concentration and pressure for 40 minutes. 
     Surviving  Salmonella  populations were enumerated by plating egg contents, or their dilutions, onto a selective medium, xylose lysine deoxycholate (XLD) agar. Additionally, a  Salmonella  detection method (FDA Bacteriological Analytical Manual, BAM; http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm070149.htm) was carried out when survivors are expected to fall below the detection limit of the enumeration procedure. 
     Results 
     Heating only (treatment 1) produced 4-5 log inactivation of  Salmonella  in eggs but more than 50% of treated eggs were  Salmonella -positive. Mild heating followed by application of ozone (treatment 2) also decreased  Salmonella  populations by 4-5 log, with more than 50% of the eggs being  Salmonella -positive. The combined heat and ozone treatment (treatment 3) totally eliminated  Salmonella  populations in shell eggs since no survivors grew on the agar medium nor detected by the BAM protocol (e.g., 7-log reduction). 
     Additional testing has demonstrated that a reduction in  Salmonella  Enteritidis concentration in shell eggs by an amount of at least log 5 is possible by a treatment method which includes heating the eggs to an internal temperature of about 55-60° C. for about 10-25 minutes, and in which:
     (i) the eggs are subjected to a pressure about 60-81 kPa (5-10 in Hg vac), and   (ii) the ozone treatment step is carried out at a concentration of about 8-12 wt. % ozone for about 25 minutes.   

     It is to be noted that the stated temperatures are measured internal to the eggs, and are not the water bath temperature or temperature of other media heating the eggs. Consequently, the time durations during which the eggs are heated refer to the time which the internal temperature of the eggs spends at the stated temperature. Furthermore, pressures given in units of kPa are absolute pressures, and pressures given in terms of Hg vac are pressures below atmospheric, wherein atmospheric pressure is 29.9 in Hg. Pressures given in psig are gauge pressures, or pressures above atmospheric. 
     These parameters provide an example method for treating shell eggs to reduce internal  Salmonella  Enteritidis concentration in the eggs with less potential for adversely affecting the quality of the eggs. In various particular examples, the method comprises: 
     (a) heating the eggs to an internal temperature of about 55-60° C. for about 2-25 minutes, or, for example, to an internal temperature of about 55-57° C. for about 8-20 minutes, or, for example, to an internal temperature of about 56-57° C. for about 8-15 minutes; 
     (b) subjecting the eggs to a pressure of about 60-80 kPa (6-12 in Hg vac), or a pressure of about 64-81 kPa (5-10 in Hg vac) or a pressure of about 63-73 kPa (8-11 in Hg vac), or a pressure of about 65-70 kPa (9-10 in Hg vac); 
     (c) maintaining the eggs in contact with a treatment gas containing about 8-12 wt. % ozone, and advantageously, about 8-10 wt. % ozone, at a pressure of about 8-12 psig for a period of time long enough so that the concentration of  Salmonella  Enteritidis in the eggs, if any, is reduced by an amount of at least log 5, this period of time being about 33 minutes or less, or about 30 minutes or less, or about 28 minutes or less, or about 26 minutes or less. 
     Ozone treatment times of about 20-33 minutes, 22-30 minutes, 23-28 minutes and even 24-27 minutes have been shown to provide acceptable results consistent with the goals of the method.