RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No.

62/168,638 filed May 29, 2016, the entirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

5,5'-dithiobis-(2-nitrobenzoic acid) ("DTNB") can react with volatile gas-phase thiols and produce the characteristic intensely yellow-colored dianion end product (structure shown in Fig. 1) without bulk aqueous solution.

A device for rapidly detecting and/or monitoring food spoilage is needed.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides a device comprising a solid pad on which DTNB has been immobilized and which is capable of detecting a volatile thiol in a gaseous state.

In certain embodiments, the present invention provides a device for food spoilage detection comprising an essentially clear first adhesive, to which is adhered a detector pad (medallion) by contact of the first adhesive with a face of the detector pad on which DTNB has been immobilized, to which detector pad, on its opposite face, is adhered a second adhesive which may be the same as or different from the first adhesive, to which second adhesive is adhered a porous backing.

In certain embodiments, the present invention provides a method for detecting food spoilage comprising placing a device as described above in proximity to a food within a container and monitoring the device for development of yellow or green color signal.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. Structure of 5,5'-dithiobis-(2-nitrobenzoic acid) ("DTNB").

Figure 2. Diffusion of volatile thiols on a pad.

Figure 3. Diffusion of volatile thiols in a closed environment.

Figure 4. Localized yellowing of DTNB-treated pads, presumably due to close- proximity out-gassing of volatile thiols from the rubber to the surface of the affected pads.

Figure 5. Possible combinations for active (treated/dried) and inactive (not treated) pad materials (and pad backing colors) Figures 6-8. initial experiments performed to demonstrate that dry reagent on pads might be adapted for food safety applications again looked only at the behavior of a model volatile thiol (2-mercaptoethanol) exposed to treated/dried pads.

Figures 9-17. Examples related to detection of spoilage of meats, fish, and poultry. Figures 18A-18D. Examples related to detection of spoilage of turkey.

Figures 19-21. Examples related to detection of spoilage of turkey.

Figure 22. Pad material treated with a detection solution comprising DTNB and inactive ingredients. DETAILED DESCRIPTION OF THE INVENTION

Diffusion (alone) of volatile thiols from one pad into nearby pad(s) can cause the reaction to proceed, without any physical connection between pads, as shown in the, in Fig. 2. A small aliquot (few μί) of allyl mercaptan (liquid) was applied to the DTNB-containing pad at the upper left (Fig. 2). Not only was there an immediate yellowing of that pad, but within about 20 seconds, nearby pads also became yellow colored (superimposed arrows suggesting the volatile thiol migration paths).

In a second example of the same phenomenon, an OraStrip® QuickCheck Canine test strip (ALT Bioscience, LLC, Lexington, KY) was placed in a container holding samples of methylmercaptan. The two screw-capped glass bottles containing the volatile thiol were stored in a clear plastic jar, and the test strip was taped to the inside of the outer container. Within a few hours of closing the system, the pad on the test strip became yellow (Fig. 3, arrow).

Finally, in a laboratory recreation of a 'real-world' situation, rubber bands were shown, after a few days of contact, to cause localized yellowing of DTNB -treated pads, presumably due to close-proximity out-gassing of volatile thiols from the rubber to the surface of the affected pads (Fig. 4).

A number of possible combinations for active (treated/dried) and inactive (not treated) pad materials (and pad backing colors) were conceived, based on our knowledge of how white and blue active pads reacted to thiols, as examples, offering scenarios for how each might appear, in the presence of little or no thiol, intermediate thiol levels, and at high thiol levels. A set of panels from that offering is shown in Figure 5.

Some of the initial experiments performed to demonstrate that such formats (dry reagent on pads to render them 'active', vs. no reagent as 'inactive') might be adapted for food safety applications again looked only at the behavior of a model volatile thiol (2- mercaptoethanol) exposed to treated/dried pads, albeit in a more controlled fashion. Briefly, a small clear plastic 'bell' was fitted in its top with a paper plug, and sealed with Parafilm. A small amount of the thiol was pipetted into the paper plug. Active/inactive pads were place on the plastic stage (into which the base of the bell fit). A count-up timer was started when the bell was fitted over the stage, and digital images were taken of the system (constant light and position) at various times. An example of this type of experiment is shown in Fig. 6. At the left are the starting pads aligned as parts of a square and/or circle; the pads below the " ^x " are not active, and those below the "^" are active. One clearly sees the active pads changing colors, from blue to greenish and from white to yellow, over the 10 minute time course of this series of images. A second, similar set of pads is shown in Fig. 7.

Such color developments tended to fade with time, once removed from the thiol- containing amospheres (see Fig. 8). The results indicate:

• For DTNB, a clear plastic cover slows pad color change.

• When more full exposed to the ambient atmosphere, thiol can cause rapid color change, even when the fluid containing it is a gas.

• Color change"reverts" to or toward pre-exposure levels, especially if exposure time to the thiol-containing atmosphere was relatively brief.

• One would expect rate of color development for such pads to be affected by ambient temperature, thiol concentration in the gas phase, detector concentration with in the pad, and factors affecting the effective acidity/alkalinity of the atmosphere.

EXAMPLE

Food safety/aging meat study

Examples related to detection of spoilage of meats, fish, and poultry (MFP) follow. First, three products were purchased at or near their store "sell by" dates. These were removed from their packages and placed into Zip Lock bags, and checked at various times using various test articles: contact tests such as OraStrip® (according to U.S. Pat. No.

8,815,152, incorporated herein by reference in its entirety) and OPA/DTNB tests (according to international (PCT) publication number WO/2014/193737, incorporated herein by reference in its entirety); a non-contact test including a pad thinner than an OraStrip® pad (this thinner pad is at times denoted a "Canine Retail" pad), on a white plastic backing (inserted into the Zip Lock bags with the samples). A slide presentation of such examples is presented infra. The L*a*b* values referred to below refer to the L*a*b* color space known in the color perception and detection arts. Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

Experiment 1

Sample items:

1. Beef cutlet with a sell by date of 8/15/15

2. Chicken gizzares with a use or freeze by date of 8/15/14

3. Fish filet with a sell by date of 8/13/14 Experiments were performed as indicated in Figures 9-17.

The results and conclusions were that there were not sufficient volumes of liquid samples from the packaging materials contacting the samples to carry out useful "rapid tests."

It was also difficult to interpret results wehe interfering substance, like hemoglobin were present. However, medallions (here using thiol pad of the Canine Retail type) placed ithin the packages showed promise as visual indicators of thiol in the ambient gas phase above the sample.

Experiment 2

In a second meat experiment, frozen turkey burger was thawed and allowed to sit with non-contact active/inactive pads, for many days, under refrigeration (2-8 ^° C). Visible color change (and evidence of malodor) was first noted on day 13. (Fig. 18A-18D)

Fig. 18A. 9/19 (day 10 - still no visible thiol detector changes, and no malodor detected)

Fig. 18B. 9/22 (day 13; first time visual changes were noted, as well as evidence of malodor)

Fig. 18C. 9/24 (day 15; visual change and malodor increasingly evident)

Fig. 18D. Results.

Experiment 3

An additional turkey burger experiment was done, this time monitoring pad L*a*b* values, and VSC levels above the sample (plastic tub, kept at ambient room temperature). Fig. 19.

In a refrigerated, multi-sample experiment, problems were noted with delayed color change of pads, when there was a close-fitting plastic film atop the pad, or when gas access was otherwise limited (in that instance, color changes occurred only along the edge of the pad with best exposure to the atmosphere containing the gaseous thiol). The graph for that 'delayed' thiol accumulation experiment is shown in Fig. 20.

Also, a multi-sample experiment was done, monitoring L*a*b* over time (again during refrigerated storage, but after rectifying the 'pad access' problem of the previous experiment). Here it was noted that there was not a perfect correspondence between the printed sell by date (dotted vertical lines in the graphs in Fig. 21) and the rises in b*, indicating accumulating thiol in the headspaces above the samples. A proposed article design meant to better allow unimpeded gas exchange is shown below, on the other side of the graphs.

Experiment 4

In a further example, a ransorb®' (pad) material from Essentra Porous Technol. (formerly Filtrona Fibertec) is treated with a detection solution comprising DTNB and inactive ingredients. Fig. 22. Such a pad material may be an off-white Nylon/polypropylene material in the form of a roll (width = 152 mm, length = 80 m, on 3" core; target thickness = 0.46 mm; target density = 0.286 g/cc). Such a roll may be contacted with a formulation solution having the following composition: Deionized Water (H20), -4.5-5 L; Sodium phosphate, dibasic, anhydrous, 22.50 g (21.30 - 23.70 g); Sodium phosphate, monobasic, dihydrate, 45.53 g (44.33 - 46.73 g); Glycerol, 37.80 g (36.6 - 39.00 g); myo-Inositol, 75 g (73.20 - 76.80 g); Beta-Cyclodextrin,15 g (14.64 - 15.36 g); Sorbitol, 75 g (73.20 - 76.80 g); Trehalose, 75 g (73.20 - 76.80 g); DTNB, 0.9 g (0.89 - 0.91 g). The pad material is dip- coated with the formulation solution, immobilizing the solutes on the pad, excess solvent being removed by air-drying, with or without gentle heating. All publications, patents and patent applications are incorporated herein by reference.

While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e. , meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. , "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein.

Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is

encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.