WO2011038168A1

WO2011038168A1 - Continous-flow bacterial disinfection of fruits, vegetables, fresh-cut produce and leafy greens using high-intensity ultrasound

Info

Publication number: WO2011038168A1
Application number: PCT/US2010/050091
Authority: WO
Inventors: Hao Feng; Arne Pearlstein; Bin Zhou
Original assignee: The Board Of Trustees Of The University Of Illinois
Priority date: 2009-09-24
Filing date: 2010-09-24
Publication date: 2011-03-31

Abstract

The invention provides methods and compositions for the continuous-flow, ultrasonic disinfection of produce.

Description

CONTINUOUS-FLOW BACTERIAL DISINFECTION OF FRUITS, VEGETABLES, FRESH-CUT PRODUCE AND LEAFY GREENS USING HIGH-INTENSITY

ULTRASOUND

CROSS-REFERENCE TO RELATED APPLICATION Pursuant to the provisions of 35 U.S.C. § 119(e), this application claims priority to

United States Provisional Application serial no. 61/245,382 filed September 24, 2009, which is incorporated in its entirely by reference herein.

BACKGROUND OF THE INVENTION

High-intensity ultrasound or power ultrasound has been widely used in industrial cleaning operations. Surface cleaning and decontamination by ultrasound is believed to be caused by very small, cavitation-generated water jets at a solid-liquid interface when a cavitating bubble collapses asymmetrically near the interface. Localized liquid velocities directed toward the solid surface of over 150 m/s can occur, which helps to remove particles from the surface. Surface shear caused by micro-streaming produced by stable cavitation bubbles can also help to clean the surface. Moreover, ultrasound can enhance interfacial mass transfer by renewing the boundary layer, as well as by acoustically-induced bulk flow. A recent study at Sandia National Laboratories reported ultrasound-enhanced chemical dissolution rates of patterned photoresists used in semiconductor and Lithography, Electroplating and Molding (LIGA) microdevice fabrication (Nilson & Griffiths, J. Electrochem. Soc. 149, G286 (2000)). Those authors proposed that acoustic streaming is responsible for the three- to four-fold increase in development rates of LIGA features in deep trench-like cavities having widths of a few micrometers or more. That report demonstrated the ability of power ultrasound to efficiently transport liquid into microtrenches and other microscopic topographic features.

The destruction of microorganisms by ultrasound is attributed to cavitation.

Cavitation can affect a biological system by virtue of a highly localized temperature/pressure rise, mechanical stress, and/or free radical production. See Riesz and Kondo, Free Radic. Biol. Med. 13, 247 (1992). These physicochemical mechanisms can lead to lethal damage through double-stranded DNA breaks, enzyme inactivation, and damage to liposomes and membranes.

Few and isolated studies have been conducted using batch operation units in recent years using ultrasound in combination with chemical sanitizers to reduce microbial populations on produce. Seymour et al. {Inter. J. Food Sci. Tech. 37, 547 (2002)) studied the application of power ultrasound to fresh produce decontamination in an ultrasound tank (batch operation) at frequencies of 25-70 kHz with seven vegetables, a fruit, and some herbs. They reported one log of additional reduction when ultrasound was combined with chlorinated water, compared to chlorine treatment without ultrasound. They reported, however, that the frequency of ultrasound treatment (25, 32-40, 62-70 kHz) had no significant effect on decontamination efficiency. Seymour concluded that "[w]ith the potentially high capital expenditure together with the expensive process of optimization and water treatment, it is unlikely that the fresh produce industry would be willing to take up this technology. Furthermore, the additional one log reduction achieved by applying ultrasound to a chlorinated water wash does not completely eliminate the risk of pathogens on fresh produce." See Seymour et al. abstract.

Scouten and Beuchat (J. Appl. Microbiol. 92, 668 (2002)) found that ultrasound in combination with 1% calcium hydroxide in a batch operation enhanced the decontamination efficacy on alfalfa seeds inoculated with S. enterica and E. coli 0157:H7. For apples, Huang et al. (J. Food Sci. 71, Ml 34 (2006)) reported up to one log additional reduction of S. enterica and E. coli 0157:H7 in an ultrasound and chlorine dioxide combined treatment, but found no obvious increase in log reduction for E. coli 0157:H7 inoculated on lettuce. Ajlouni et al. (J. Food Sci. 71, M62 (2006)) also found no effect of ultrasound on the inactivation of natural flora on Cos lettuce in a 20-min batch washing with any of four sanitizer solutions (0.02% peroxyacetic acid, 4 mg/L hydrogen peroxide, 2% acetic acid, 100 mg/L chlorinated water). The lack of effectiveness reported by Huang (2006) and Ajlouni (2006) might be attributable to the nonuniform ultrasound distribution in the ultrasonic cleaning baths due to standing wave formation, and to blockage of ultrasound propagation by produce leaves in the washing liquid.

Compositions and methods are needed in the art to improve ultrasound treatment of produce such that it efficiently and effectively kills microorganisms in a commercially high- throughput, continuous-flow manner.

BRIEF SUMMARY OF THE INVENTION

One embodiment of the invention provides an apparatus for continuous-flow disinfection of produce comprising a flume having an inlet and an outlet, wherein the inlet is configured to receive an aqueous suspension of produce, wherein the flume comprises: one or more agitators for mixing the aqueous suspension of produce in the flume; and two or more ultrasonic transducer blocks that provide ultrasound to the aqueous suspension of produce at two or more predetermined frequencies with controllable ultrasound power density and modulation of ultrasound frequency. The two or more ultrasonic transducer blocks can provide an improvement in acoustic field distribution by making the acoustic intensity more uniform than is possible with one transducer block or than with two or more transducer blocks operating at only one fixed frequency. The two or more ultrasonic transducer blocks can operate at two or more predetermined frequencies that differ from each other by at least 10 kHz. The agitators can be jets, paddles, mixers, baffles, flow diverters, floatation agents, surface active agents, or combinations thereof. The apparatus can comprise a controller for ultrasound power density, a controller for ultrasound frequency modulation, a controller for ultrasound frequency, or a combination thereof.

Another embodiment of the invention provides a method for continuous-flow disinfection of produce. The method comprises placing an aqueous suspension of produce in a flume having an inlet and an outlet, wherein the inlet is configured to receive the aqueous suspension of produce; wherein the flume comprises: one or more agitators for mixing the aqueous suspension of produce in the flume; and two or more ultrasonic transducer blocks that provide ultrasound to the aqueous suspension of produce at predetermined frequency, with controllable frequency modulation, and ultrasound power density; operating the two or more ultrasound transducer blocks at two or more central frequencies, wherein the two or more central frequencies can each be modulated about the two or more central frequencies such that the ultrasound produced by the ultrasound transducer blocks is provided to the aqueous suspension of produce in the flume; agitating the aqueous suspension of produce in the flume such that each piece of produce is subjected to ultrasound for approximately the same amount of time; and retrieving the produce from the aqueous suspension the outlet of the flume. The transducer blocks can comprise a controller for ultrasound power density, a controller for ultrasound frequency modulation, a controller for ultrasound frequency, or a combination thereof. In an alternative embodiment, the controllable ultrasound frequency modulation and/or controllable frequency are not present.

The two or more ultrasonic transducer blocks can provide a nearly uniform acoustic field to the aqueous suspension of produce. The ultrasonic transducer blocks can provide an acoustic field that is more uniform than an acoustic field that could be provided by one or more transducer blocks operating at a fixed frequency. The two or more ultrasonic transducer blocks can operate at two or more predetermined frequencies that differ from each other by at least 10 kHz. The two or more ultrasonic transducer blocks can operate at two or more predetermined frequencies of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 kHz; and at a modulation of frequency of about 0.5 to about 10 kHz; for about 15 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, to 5 minutes; and at an ultrasound power density in the flume of about 1, 2, 3, 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, 400 or more W/L. The agitators can be jets, paddles, mixers, baffles, flow diverters, floatation agents, surface active agents, or combinations thereof. The number of microorganisms on the produce can be reduced by greater than about 1 log, 1.5 logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 4.5 logs, or 5 logs. The aqueous suspension of produce can further comprise one or more disinfectants. The disinfectants can be chlorine, acidified sodium chlorite, peroxyacetic acid, acidic electrolyzed water, weak organic acids, cleaning agents, sodium hypochlorite, calcium hypochlorite, chlorine dioxide or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a lateral cross-section of a flume.

Figure 2 shows a lateral cross-section of a flume fitted with a channel.

Figure 3 A, 3B, and 3C show lateral cross-sections of flumes.

Figure 4 shows an aerial view of a flume.

Figure 5A-C shows the microbial count reduction on the surfaces of spinach leaves after individual spinach leaves were washed in a pilot-scale washer with and without ultrasound treatment, with a residence time of 60±20 seconds. (5A) Aerobic Plate Count; (5B) Yeast and Mold; and (5C) E. coli 87-23.

Figure 6A-C shows microbial count reduction on the surfaces of spinach leaves after one pound of spinach leaves was washed in a pilot-scale washer with and without ultrasound treatment, with a residence time of 60±30 seconds. (6A) Aerobic Plate Count; (6B)Yeast and Mold; and (6C) E. coli 87-23.

Figure 7A-E shows the effect of ultrasonication on the quality of spinach. (7 A)

Overall quality; (7B) Color; (7C) Sogginess; (7D) Off-odor; and (7E) Electro-Conductivity Rate.

Figure 8A-E shows the effect of ultrasonication on the quality of loose-leaf lettuce. (8A) Overall quality; (8B) Color; (8C) Sogginess; (8D) Off-odor; and (8E) Electro- Conductivity Rate.

Figure 9A-D shows the effect of ultrasonication on the quality of leaf portion of romaine lettuce. (9 A) Overall quality; (9B) Color; (9C) Sogginess; and (9D) Off-odor.

Figure 10A-D shows the effect of ultrasonication on the quality of rib portion of romaine lettuce. (10A) Overall quality; (10B) Color; (IOC) Sogginess; and (10D) Off-odor. Figure 11 shows the effect of ultrasonication on electro-conductivity rate of romaine lettuce.

Figure 12 shows the effect of ultrasound assisted washing on artificially contaminated baby carrots.

DETAILED DESCRIPTION OF THE INVENTION

Several well-publicized episodes (involving multiple deaths) in recent years have heightened awareness of the need to effectively disinfect produce before it is packaged and put into distribution. A key issue, particularly for delicate leafy produce, is to avoid degrading the appearance, taste, or other physicochemical properties of the produce during the disinfection process, and for those properties to remain undegraded for the anticipated shelf-life of the produce.

The instant invention provides equipment and methods to kill bacteria, yeast, fungi, viruses, and single cell organisms on produce, such as whole, sliced, diced, shredded, chopped or cut leafy produce (including delicate leaves such as spinach, cabbage, kale, lettuce, and other leafy vegetables) and whole, sliced, diced, shredded, chopped or cut fruits and vegetables (e.g., onions, peppers, tomatoes, apples, carrots, potatoes, grapes, oranges, soybeans, beets, seeds and grains) in a continuous-flow process consistent with commercial processing rates, without degradation of the produce. The processes use ultrasonic energy radiated into an aqueous suspension of whole or cut produce, such as leafy produce, vegetables, and fruits, to remove and/or kill bacteria (including potentially lethal E. coli) or other microorganisms, optionally in combination with low concentrations of chemical sanitizers (including e.g., ozone, chlorine, acidified sodium chlorite, chlorine dioxide, other chlorine compounds, hydrogen peroxide, the acidic portion of electrolyzed dilute NaCl solution ("acidic electrolyzed water"), peroxyacetic acid, or other weak organic acids). The methods of the invention provide for a continuous flow of produce and liquid moving through a flume, as opposed to batch processing where one batch of produce and liquid are treated in an apparatus, and then removed from the apparatus, followed by an identical series of steps for a second batch of produce and subsequent batches.

Flumes

The present invention provides compositions and methods for providing high- intensity ultrasound that kills or removes bacteria and other microorganisms in a continuous- flow system (systems where an aqueous suspension of produce is flowed through the system at a constant or variable flow rate) while maintaining acceptable levels of produce quality in a high-throughput, commercial-scale flume. Batch system experiments (i.e., closed containers with no mean flow) have demonstrated that high intensity ultrasound with suitable frequency content can lead to substantial augmentation of bacterial killing by chemical sanitizers, or enhanced removal of bacteria from produce surfaces. Batch systems, however, usually demonstrate higher bacterial killing rates than continuous flow systems, due to a higher acoustic energy density and longer residence time in the treatment chamber of a batch unit.

Produce, such as cut leafy produce, is traditionally washed and sanitized in a continuous-flow process in large stainless steel flumes. A typical flume size is about 3-18 m long (in the direction of mean flow), less than about 0.2-1.2 m deep, and about 0.4 to about 3 m wide. However, a flume can be about 10.0, 7.0, 5.0, 4.0, 3.5, 3, 2.5, 2.0, or 1.0 m (or any value between about 10.0 m and about 1.0 m) long; about 2.0, 1.5, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2 m (or any value between about 2.0 m and about 0.2 m deep); and about 4.0, 3.0, 3.5, 3.0, 2.5, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 m (or any value between about 4.0 and about 0.5 m) wide.

A flume can comprise a substantially planar bottom 160 with side walls 150, which extend upwardly from the planar bottom. See, e.g., Figures 1-3. The side walls can extend upward from the bottom at substantially 90 degrees {see Figure 3A) or can extend upward from the bottom at about greater than 90 degrees to about 140 degrees {see Figure 1, 110). Alternatively, the flume can be semi-circular, tubular, or cylindrical in shape {see Figure 3B, 3C) (or any other suitable shape) with or without an upper opening to allow access to the interior of the flume. An aqueous suspension of produce 120 is run through the flume.

Optionally, vertical channel walls 220 can be inserted longitudinally into the flume 200, with the space between adjacent walls constituting a channel 230 through which the aqueous suspension of produce passes 240. See Figure 2. Transducers 210 can be present, for example, within the vertical channel walls 220 or any other arrangement (see, e.g., Figure 1). The transducers can also be mounted at the bottom 160 and/or side wall of the flume 110.

A flume has an inlet 410 and outlet 420. See Figure 4. Produce, such as leafy greens in water or other liquid, enters through the inlet 410 and exits through the outlet 420. A flume can extend along a somewhat downwardly, generally horizontal direction, although other configurations are acceptable. A flume can be attached or coupled at the inlet or outlet to, e.g., a dip tank, a shaker, sprayers, a conveyor, an air knife, or other food processing equipment such that it forms a part of a food processing equipment system. Stone and/or insect traps can be present as part of the flume or attached to the flume to ensure that sand and other foreign matter settles and can be discharged. Wash water or liquid can be collected and reused, or recirculated in the treatment process.

The flow rate of water or other liquid and produce through a flume can be about 50, 75, 100, 200, 300, 400, 500, 600, 700, or 800 (or any value between about 50 and 800) gallons or more per minute. The capacity of a flume can be about 500, 1,000, 2,000, 5,000, 10,000, 15,000, 20,000 (or any value between 500 to about 20,000) or more pounds of produce per hour.

A flume and each of the components thereof comprise a suitable material that maintains proper sanitary properties and is sufficiently strong and durable, such as metal alloys (e.g., stainless steel) or suitable polymers, such as polyvinyl chloride.

The temperature of the produce and water or liquid suspension can be about 2, 4, 10, 15, 20, 25, 27, 30 (or any value between about 2 and about 30) degrees Celsius.

Ultrasonic Transducer Blocks

In one embodiment of the invention two or more ultrasonic transducer blocks (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) that provide high intensity ultrasound or power ultrasound are used in conjunction with a continuous-flow apparatus of the invention. Each ultrasonic transducer block is operated at a central frequency of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 kHz (or any value between about 5 and about 100 kHz). The two or more ultrasonic transducer blocks can be driven at quite different central frequencies (e.g., the central frequencies differ by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 kHz (or any value between about 5 and about 100 kHz)), each modulated by a maximum of about ± 10% around its central frequency (e.g., modulation of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kHz or any value between about 0.5 and about 10 kHz). For example, two transducer blocks can be present on or in a continuous-flow apparatus, with one transducer block operating at a central frequency of 20 kHz with a modulation of about 2 kHz and the second transducer block operating at a central frequency of about 40 kHz with a modulation of about 4 kHz.

Where there are more than two ultrasonic transducer blocks present, the transducers should be driven at least two different central frequencies. For example, where three transducer blocks are present, the first transducer block can be driven at a first central frequency, e.g., 20 kHz, and the second and third transducer blocks can be driven at a second central frequency, e.g., 40 kHz. Alternatively, all three (or more) transducer blocks can be driven at different central frequencies, e.g., 20 kHz, 40 kHz, and 75 kHz. Therefore, a plurality of transducer blocks can all be driven at different central frequencies or several transducer blocks in a plurality of transducer blocks can be driven at the same central frequency, while others are driven at a second central frequency, third central frequency, fourth central frequency, fifth central frequency, or more, as long as two or more central frequencies are employed.

In one embodiment of the invention the ultrasound frequency is controllable. That is, the operator can adjust and/or change the ultrasound frequency applied to flume using a controller associated with the transducer block or blocks. The ultrasound frequency controller can control one transducer block or a set of (two or more) transducer blocks. In another embodiment of the invention modulation of the ultrasound frequency is controllable. That is, the operator can adjust and/or change the modulation of the ultrasound frequency applied to flume using a controller associated with the transducer block or blocks. The modulation of ultrasound frequency controller can control one transducer block or a set of (two or more) transducer blocks.

The two or more ultrasonic transducer blocks are placed on or around the flume or channel walls to provide proper propagation of the ultrasonic waves from the ultrasonic transducer blocks through the water or liquid present in the flume. The intensity, frequency, location, and number of ultrasonic transducer blocks are matched to the dimensions of the flume.

Transducer blocks can be present at any position on exterior, interior or within the walls or the bottom surface of the flume or channel walls or may be at any position near the flume. Transducer blocks can be part of the flume, i.e., attached to the interior surface of the side walls 102, attached to the exterior surface of the side walls 103, present within the flume walls 101, attached to the interior surface of the flume bottom 108, attached to the exterior of the flume bottom 105, present within the flume bottom 106, near the flume side walls 104 or near the flume bottom 109, or combinations thereof. See Figure 1. The transducer blocks 107 can also be present within one or more housings 130 protruding into the volume of the flume at any depth. See Figure 1. Additionally, transducer blocks 210 can be imbedded in vertical channel walls 220 (or present on the walls) that are inserted longitudinally into the flume 200, with the space between adjacent walls constituting a channel 230 through which the aqueous suspension of produce 240 passes. See Figure 2. Ultrasonic transducer blocks can be present at two or more of any of the described locations and in any combination of described locations.

If attached to the interior or exterior of the two side walls of the flume or channel walls, or present within the two side walls of the flume or channel, each member of a pair of transducer blocks 441, 442 can be substantially opposing the other at the same position on each side wall 430 (e.g., at the same distance from the flume inlet and at the same height on each side wall). See Figure 4, 441, 442. Alternatively, pairs of transducer blocks can be present at staggered positions on each side wall. For example, a first transducer block 443 can be present on the exterior or interior of, or within, a first sidewall at a first certain distance from the inlet, and a second transducer block 444 can be present on the exterior or interior of, or within, a second sidewall at a different certain distance from the flume inlet. Optionally, a third transducer block can be present on the exterior or interior of, or within, a first or second sidewall at a third certain distance from the inlet. See e.g., Figure 4, 443, 444, 445. The first and second transducer blocks and optionally additional transducer blocks can be present at the same height on each of the sidewalls or may be present at different heights on each of the sidewalls. See e.g., Figure 1, placement of transducer blocks 101 and 102. A first or second (or additional) certain distance from the inlet can be, e.g., 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 7.0, 8.0, 9.0, 10.0 m (or any value between about 0.25 m and about 10 m) or more. A transducer block may be present at about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 m (or any value between about 0.2 and 2.0 m) from the bottom surface of the flume.

With the transducer blocks arranged as specified above, the ultrasound power density in the flume can be at about 1, 2, 3, 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, 400 (or at any value between about 10 and about 400) or more W/L. In one embodiment of the invention the ultrasound power density is controllable. That is, the operator can adjust and/or change the power density applied to flume using a controller associated with the transducer block or blocks. The ultrasound power density controller can control one transducer block or a set of (two or more) transducer blocks.

Equalizing Exposure to Ultrasound

Without adequate transverse mixing in planes perpendicular to the overall flow direction, some produce leaves or other produce elements will pass through the channel spending nearly their entire residence time very close to the channel or flume sidewalls, where the ultrasonic intensity is highest and the mean flow speed is lowest. For ultrasound levels required to achieve adequate bacterial (or other microorganism) kill on produce leaves or other produce elements that pass through the channel far from the channel or flume walls (near its vertical midplane), where the mean flow speed is higher, and residence time is shorter, this will lead to excessive ultrasound exposure near the walls, resulting in degradation of produce quality.

In one embodiment of the invention, each produce leaf or other element of produce in the continuous-flow apparatus has nearly equal exposure to ultrasound, that is, each produce leaf or other element is exposed to ultrasound for approximately the same amount of time. Appropriate choice of channel or flume width, and transducer placement in the flume or channel, coupled with transverse mixing of suspension of water or liquid the channel or flume will achieve nearly equal exposure of each piece of produce to the ultrasound. The use of jets 140, 250, such as zero-mass-flux synthetic jets (Nuventix, Inc.) or pressurized water jets along the flume can provide transverse mixing of liquid in the channel or flume. See Figures 1 and 2. Optionally, paddles or other mixers (including static mixers) can be used to agitate the suspension of produce. The bottom of the flume optionally can comprise one or more baffles or flow diverters that are spatially positioned along a bottom surface of the flume. The baffles or flow diverters can be used to agitate the produce leaves or other elements in the flow. Optionally, flotation agents or surface active agents, such as dioctyl sodium sulfosuccinate, can be added to the aqueous suspension of produce.

In one embodiment of the invention, the produce in the continuous-flow apparatus is subjected to ultrasound treatment for about 15 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes (or any value between 15 seconds and 5 minutes) or more.

Additional Disinfectants

The compositions and methods of the invention can function effectively in potable water to remove bacteria or other microorganisms from produce surfaces without the use of other disinfectants such as chemical processing aids or chemicals. Other disinfectants, however, can be used in conjunction with the compositions and methods of the invention.

Other disinfection methods can be combined with the methods of the invention to give augmented or synergistic microorganism killing results, e.g., chlorine (at, for example, 50, 100, 150, 200, 300, 400 (or any value between 50 and 400) mg/L)), acidified sodium chlorite (at, for example, 50, 100, 150, 200, 300, 400 (or any value between 50 and 400) mg/L), peroxyacetic acid (at, for example, 25, 50, 100, 150, 200, 300, 400 (or any value between 25 and 400) mg/L), acidic electrolyzed water (at, for example, 25, 50, 100, 150, 200, 300, 400 (or any value between 25 and 400) mg/L free chlorine), weak organic acids, cleaning agents, or sodium hypochlorite, calcium hypochlorite, and chlorine dioxide can be added to the aqueous suspension of produce. Methods of the invention can also be combined with other disinfection methods such as microfiltration, ultraviolet radiation, or ozonation to provide augmented or synergistic killing rates of microorganisms.

Reduction in Numbers of Bacteria

In one embodiment of the invention, the number of treated bacteria or microorganisms on the produce is reduced by greater than about 1 log, 1.5 logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 4.5 logs, or 5 logs (or any value between about 0.5 log and 5 logs) in the presence or absence of disinfectants. Advantageously, the methods of the invention effectively and efficiently reduce the numbers of microorganisms on produce without degradation of the appearance, taste, or other physiochemical properties of the produce over the whole shelf life of the produce.

All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference in their entirety. The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims. In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group. Examples

Example 1: Ultrasound- Assisted Spinach Washing with a Pilot-Scale Continuous-Flow Washer

A. Washing of individual spinach leaves in continuous-flow flume 1. Material and methods

Unwashed spinach was supplied by a California commercial grower. Ultrasound blocks used in the tests were designed to operate at 25 kHz, 40 kHz, and 75 kHz. One block at each frequency was embedded into each of two parallel walls, 12 inches apart, which formed a channel in a flume through which all produce samples passed. The blocks were arranged so that each was directly opposite the channel from a block of the same frequency.

The mean speed of aqueous disinfection solution in the channel was about 9 m/min. When chlorine was used, its total concentration was 50 mg/L.

Individual whole spinach leaves were placed, one-by-one, into a pilot-scale washer with the ultrasonic channel installed for sonication treatments. The residence time of each leaf flowing through the washer was recorded, and was in the range of 40 to 80 seconds. The microorganism used was E. coli 87-23, a nonpathogenic strain of E. coli 0157:H7. The E. coli cells were spot-inoculated on spinach leaves. The survival counts of E. coli after a treatment were analyzed by trypticase soy agar (TSA) with 50 ppm nalidixic acid, while those for natural microflora were monitored with 3M Petri films.

2. Results

Figures 5A-C show the microbial count reduction on individual spinach leaves after washing in a pilot-scale continuous-flow washer with and without ultrasound treatment, with a residence time for each leaf of 60±20 seconds. (5A) Aerobic Plate Count; (5B) Yeast and Mold; and (5C) E. coli 87-23.

B. Simultaneous washing of multiple spinach leaves

1. Material and methods

Unwashed spinach was supplied by a California commercial grower. Ultrasound blocks used in the tests were designed to operate at 25 kHz, 40 kHz, and 75 kHz. One block at each frequency was embedded into each of two parallel walls, 12 inches apart, which formed a channel in a flume through which all produce samples passed. The blocks were arranged so that each was directly opposite the channel from a block of the same frequency. The mean speed of aqueous disinfection solution in the channel was about 9 m/min. When chlorine treatment accompanied ultrasound, the total chlorine concentration was 50 mg/L. Water jets were used to provide mixing of the leaves in the flume during the treatment.

Multiple spinach leaves entered the washing facility nearly simultaneously at the upstream end. The minimum and maximum residence times for the leaves were obtained by recording when the first and last leaves, respectively, exited the washer. The residence time of each leaf flowing through the washer was recorded, and was in the range of 30 to 90 seconds. The microorganism used was E. coli 87-23, a nonpathogenic strain of E. coli 0157:H7. The E. coli cells were spot-inoculated on spinach leaves. The survival counts of E. coli after a treatment were analyzed by trypticase soy agar (TSA) with 50 ppm nalidixic acid, while those for natural microflora were monitored with 3M Petri films.

2. Results

Figure 6A-C shows microbial count reduction on spinach surfaces when one pound of leaves was washed in a pilot-scale continuous-flow washer with and without ultrasound treatment, with a residence time of 30-90 seconds. (6A) Aerobic Plate Count; (6B) Yeast and Mold; and (6C) E. coli 87-23.

Table 1. Summary of microbial count reduction.

¹ APC: Aerobic Plate Count.

Underscore indicates that the additional reduction is statistically significant (a As can be seen in Figs. 5-6 and Table 1, spinach leaves passing through the washer when the ultrasound was operating experienced a greater reduction in microbial count than those in washing tests without ultrasound. The increase was statistically significant in five of six cases, with the exception being the yeast and mold count for simultaneous washing of multiple spinach leaves. (Even in this case, the additional microbial reduction was 39.4%.) The ultrasonic enhancement of microbial reduction was greater for the leaf-by-leaf washing than in the multi-leaf experiments for each microbial test. For E. coli 87-23, the survival count with ultrasound and chlorine was one log below that for chlorine alone in the single- leaf tests. However, even for multi-leaf experiments, ultrasound reduced the E. coli 87-23 survival count by 72.8% (or 0.53 log) beyond what was achieved with chlorine alone.

Example 2

Effect of ultrasonication on the quality of leafy green produce

A. Material and Methods 1. Produce

Spinach, romaine lettuce, and loose-leaf lettuce were supplied by a California commercial grower.

2. Ultrasound equipment

Transducers driven by a variable-frequency ultrasonic generator attached to a stainless steel tube with inner diameter 1.5 inches were used in this study. The variable-frequency system, which has a central frequency of 28 kHz and random frequency sweeping and modulation in time domain and frequency domain can generate an acoustic intensity distribution in the tube with much less spatial variation than is possible at a single frequency. This is achieved by avoiding nonuniformities associated with standing wave formation in conventional ultrasonic treatment, which can cause localized damage to delicate vegetables at the antinodes of the ultrasonic field. A sample holder to secure cut produce samples in the tube was designed and fabricated.

3. Sample preparation

For romaine lettuce and loose-leaf lettuce, two outer layers were removed, and inner layers were sliced into 1.0x 1.0 inch squares, and treated. In spinach tests, only whole leaves were treated.

4. Ultrasound Treatment

100 g cut samples were placed into the sample holder, and the holder was placed into the tube. Pre-cooled tap water at 5°C was added to fill the tube. The variable-frequency ultrasound unit was turned on to start treatment. The treatment times were 0, 1, 2, 4, 8, and 16 min. After each treatment, water was drained from the tube, and the sample holder was removed. The treated samples were packaged into plastic bags made of a film having an oxygen transmission rate (OTR) of 380 cm³ per 100 cm² of surface area in 24 hrs. The samples were placed in a storage room at a temperature of 1°C. At days 0, 7, 14, and 21, samples were removed from storage and tested for quality.

5. Sensory evaluation

The effect of storage time and ultrasound treatment time on sensory perceptions (overall quality, color, sogginess, and off-odor) of lettuce was evaluated on days 0, 7, 14, and 21 by six untrained individuals. For each of the three vegetables samples with no treatment (the "controls") and samples treated by ultrasound were simultaneously presented to each panelist each week. The samples were placed on top of white paper plates which were randomly placed in trays, and. identified by three digits. For overall quality, a five-point hedonic scale (1-5) was used, with a score of 1 representing product strongly disliked, and a score of 5 representing product strongly liked. For color, the samples were rated on the same five -point scale, with 1 corresponding to no green and 5 corresponding to a fresh green appearance. Sogginess was rated as 1 (crisp) to 5 (soggy- watery). For off-odor, the samples were rated as 1 (none) and 5 (most severe). The minimum score for commercial consumer acceptance was set at 3 for color and overall quality. Maximum acceptable scores for sogginess and odor were 2. These limits of acceptability were based on our "acceptance test" profile. For example, for "overall quality" a rating of 3 out of a 1-5 scale was defined as "fair" and thus considered as the minimum score for salability to consumers.

6. Quality Evaluation (Electro-Conductivity Rate)

Five grams of each vegetable type sample (control and ultrasonically treated) were incubated at 23 °C in 100 ml glass bottles containing 50-70 ml deionized water. During incubation, samples were agitated using a shaker operating at a frequency of 100 cycles per minute. Electrical conductivity of the bath solution was measured at 1 min (CI) and 60 min (C60) of incubation using an Accumet Basic AB30 conductivity meter (Fisher Scientific). The samples were then autoclaved (121°C) for 25 min, and total conductivity (CT) of the bath solution was measured after cooling. Electrolyte leakage (E) was calculated from the following equation: E = 100 x (C60-C1)/CT.

B. Results

Figure 7A-E shows the effect of ultrasonication on the quality of spinach. (A) Overall quality; (B) Color; (C) Sogginess; (D) Off-odor; and (E) Electro-Conductivity Rate. Figure 8A-E shows the effect of ultrasonication on the quality of loose-leaf lettuce. (A) Overall quality; (B) Color; (C) Sogginess; (D) Off-odor; and (E) Electro-Conductivity Rate. Figure 9A-D shows the effect of ultrasonication on the quality of leaf portion of romaine lettuce. (A) Overall quality; (B) Color; (C) Sogginess; and (D) Off-odor. Figure 10A-D shows the effect of ultrasonication on the quality of rib portion of romaine lettuce. (A) Overall quality; (B) Color; (C) Sogginess; and (D) Off-odor. Figure 11 shows the effect of ultrasonication on electro-conductivity rate of romaine lettuce.

Summary for quality changes: The effects of ultrasound on produce quality immediately following sonication and during storage are shown in Figures 7-11. All produce samples (spinach and lettuce) showed minimal quality changes immediately following sonication, with no significant difference between the control and treated samples. During a 7-day storage at 0°C, most produce samples experience no noticeable quality changes compared to the control. At day 14, some treated samples showed reduced overall quality when observed with the naked eye, as reflected in lower overall quality scores, especially for the loose-leaf lettuce. Statistically, however, there was no significant difference in overall quality score between the samples treated for 1 min and the control, compared to the corresponding day-0 values. Each sample had an overall quality score greater than 3, and therefore in the range of consumer acceptance. In summary, with a 1 to 2 min ultrasonication in a variable frequency ultrasonication system carefully designed to provide a relatively uniform acoustic field, the quality of spinach and lettuce samples was adequate immediately after sonication, and for two weeks of storage thereafter.

Example 3

Removal of artificially contaminated E. coli 0157:H7 surrogate from baby carrot by ultrasound-assisted washing.

A. Material and methods:

1. Inoculum preparation: The E. coli 0157:H7, strain 87-23 (non-pathogenic), was transferred 3 times to tryptic soy broth (pH 7.3) by loop inoculation at successive 24-h intervals and incubated at 37°C. Bacterial cells were harvested, after 24 h of growth, by centrifugation (6000 x g) at 4°C for 10 min. The cell pellets from 200 mL culture were washed twice in peptone water (0.85% NaCl, 0.1% Bacto Peptone), and resuspended in 2000 mL of peptone water. The final concentration of E. coli 0157:H7 in the inoculum, determined by plating serial dilutions on TSA containing 50μg/mL nalidixic acid and incubating at 37°C for 24 h, was approximately 10⁸ CFU/mL.

2. Inoculation of baby carrot: Pre-washed and peeled baby carrots were supplied from a commercial source in California, kept at 4°C, and used within 3 days. 1000 grams of baby carrots were inoculated by dipping them into a beaker containing 2000 mL of the inoculum. The beaker was placed on a shaker at 60 rpm for 20 min. The carrots were air-dried for 20 min in a laminar flow biological hood before treatments.

3. Treatment: 100 grams of inoculated baby carrots were placed in the ultrasonic channel (25 kHz) in a pilot-scale washer containing 50 ppm chlorine prepared from CLOROX®, and treated for 1 min and 3 min with and without ultrasonication. Two ultrasonicator blocks that were each operated at 25 kHz were separately embedded into each of two parallel flume walls, 12 inches apart, which formed a channel in a flume through which baby carrots were treated. The blocks were arranged directly opposite from each other in the channel. Baby carrot samples were place in a nylon bag, and treated under manual agitation. The survival counts of E. coli after a treatment were analyzed by TSA enriched with 50 mg/L nalidixic acid.

4. Microbiological analysis: 25 grams of baby carrots were macerated in 75 mL 0.1% (w/v) sterile peptone water at 230 rpm for 2 min with a stomacher blender. The homogenate was filtered through sterile glass-wool, serially diluted in peptone water, plated (100 μΙ_, in triplicate) on trypticase soy agar (TSA) containing 50 μg/mL nalidixic acid, and incubated at 37 °C for 24 h before enumeration.

B. Results:

Figure 12 shows the reduction of E. coli 87-23 inoculated on baby carrots. The results (Figure 12) indicated that ultrasonication enhanced the removal of E. coli 0157:H7 from baby carrots by 1.24 log cycle and 0.65 log cycle when treated for 1 min and 3 min, respectively. Extending the ultrasonic treatment time did not result further reduce the E. coli population.

Claims

CLAIMS We claim:

1. An apparatus for continuous-flow disinfection of produce comprising a flume having an inlet and an outlet, wherein the inlet is configured to receive an aqueous suspension of produce, wherein the flume comprises:

(a) one or more agitators for mixing the aqueous suspension of produce in the flume; and

(b) two or more ultrasonic transducer blocks that provide ultrasound to the aqueous suspension of produce at two or more predetermined frequencies.

2. The apparatus of claim 1, further comprising a controller for ultrasound power density, a controller for ultrasound frequency modulation, a controller for ultrasound frequency, or a combination thereof.

3. The apparatus of claim 1, wherein the two or more ultrasonic transducer blocks each with a different frequency provide an acoustic field that is more uniform than an acoustic field provided by ultrasonic transducer blocks having a single frequency.

4. The apparatus of claim 1, wherein the two or more ultrasonic transducer blocks operate at two or more predetermined frequencies that differ from each other by at least 10 kHz.

5. The apparatus of claim 1, wherein the agitators are jets, paddles, mixers, baffles, flow diverters, floatation agents, surface active agents, or combinations thereof.

6. A method for continuous-flow disinfection of produce comprising:

(a) placing an aqueous suspension of produce in a flume having an inlet and an outlet, wherein the inlet is configured to receive the aqueous suspension of produce, wherein the flume comprises:

(i) one or more agitators for mixing the aqueous suspension of produce in the flume; and

(ii) two or more ultrasonic transducer blocks that provide ultrasound to the aqueous suspension of produce at predetermined frequency, with controllable ultrasound power density and ultrasound frequency modulation; (b) operating the two or more ultrasound transducer blocks at two or more central frequencies, wherein the two or more central frequencies can each be modulated about the two or more central frequencies such that the ultrasound produced by the ultrasound transducer blocks is provided to the aqueous suspension of produce in the flume;

(c) agitating the aqueous suspension of produce in the flume such that each piece of produce is subjected to ultrasound for approximately the same amount of time; and

(d) retrieving the produce from the aqueous suspension of produce at the outlet of the flume.

7. The method of claim 6, wherein the two or more ultrasonic transducer blocks provide to the aqueous suspension of produce an acoustic field that is more uniform than an acoustic field that is provided by transducer blocks operating at a fixed frequency.

8. The method of claim 6, wherein the two or more ultrasonic transducer blocks operate at two or more predetermined frequencies that differ from each other by at least 10 kHz.

9. The method of claim 6, wherein the two or more ultrasonic transducer blocks operate at two or more predetermined frequencies of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 kHz; at a modulation of frequency of about 0.5 to about 5 kHz; for about 15 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, to 5 minutes, and an ultrasound power density of about 1, 2, 3, 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, 400 or more W/L.

10. The method of claim 6, wherein the number of microorganisms on the produce is reduced by greater than about 1 log, 1.5 logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 4.5 logs, or 5 logs.

11. The method of claim 6, wherein the produce is suspended in an aqueous solution of one or more disinfectants.

12. The method of claim 11, wherein the disinfectants are chlorine, acidified sodium chlorite, peroxyacetic acid, acidic electrolyzed water, weak organic acids, cleaning agents, sodium hypochlorite, calcium hypochlorite, chlorine dioxide or combinations thereof.

13. The method of claim 6, wherein the agitators are jets, paddles, mixers, baffles, flow diverters, floatation agents, surface active agents, or combinations thereof.