An official website of the United States government.
The overall goal of this SBIR project is to develop branding inks containing biologically labile derivatives of FD&C dyes (i.e., "pro-dyes") that are not detected by the VerifEYE Carcass Inspection System and are adherent to beef carcasses throughout the multiple steps involved in meat processing. Success in meeting this goal will advance the capability to monitor and maintain meat quality in two significant ways: (1) It will eliminate the current shortcomings of the VerifEYE system in detecting contamination on beef carcasses. (2) It will provide a practical method for expansion of applications of the VerifEYE system to contamination detection during processing of non-plant eating animals (e.g., poultry).
<P>
The aim of the Phase I SBIR research is to provide prototype branding inks incorporating pro-dyes that: (1) will not be detected as "false positives" by the VerifEYE system; (2) will be adherent to beef carcasses throughout processing, including multiple water spraying cycles; (3) are compatible with commonly used ink formulations; and (4) are converted after ingestion by humans to the parent FD&C dye and non-toxic by-products.
<P>
In Phase I, two FD&C dyes, a red dye and a blue dye, will be converted to ester or amide pro-dyes that exhibit the four properties cited above. Acceptable pro-dyes will be formulated into prototype branding inks and screened for utility during simulated meat processing. The pro-dye approach we will use is analogous to the pro-drug approach that has been employed by the pharmaceutical industry for many decades to convert drugs that have poor performance characteristics to pro-drugs that are metabolized back to the parent drug. <P>
The benefits of our pro-dye approach include: (1) continued use of USDA-approved dyes, eliminating the potential cost associated with new compound development and toxicological studies associated with new chemical entities; (2) the use of components in the pro-dye that are inexpensive, non-toxic, and possess an extensive history of safe use by humans; (3) improvements in the solubility of low-solubility dyes, such as Blue #2, thereby broadening potential applications in the food industry; (4) the potential for altering adhesion properties of currently used dyes, such as Blue #1, thereby broadening potential applications in meat processing; and (5) expanded use of the VerifEYE system, helping to make the U.S. and world food supply safer for consumers.
NON-TECHNICAL SUMMARY: Bacterial contamination in the American beef supply continues to be a serious concern for the meat processing industry. As part of a broad program of improvements in processing procedures and sanitary methods, meat processors in the U.S. and abroad have initiated use of the VerifEYET Carcass Inspection System (eMerge Interactive, Sebastian, FL). The VerifEYE system automatically inspects each beef carcass, identifying areas contaminated by organic material which may harbor dangerous pathogens such as E. coli O157:H7, Salmonella, or Listeria. Unfortunately, the system also detects the purple inks currently used by the USDA to brand carcasses and reports responses to the inks as "false positives" of fecal and/or intestinal contamination. Experiments in which other FD&C dyes were substituted for the interfering dyes in the purple inks have repeatedly failed, and inks containing dyes that are not approved for use in foods cannot be used without FDA- and USDA-approval. This Phase 1 SBIR project is designed to develop prototypes of branding inks containing novel, biologically labile derivatives of FD&C dyes (i.e., "pro-dyes") that are not detected by the VerifEYE system and are adherent to beef carcasses throughout the multiple steps involved in meat processing. In the future, related applications of this new pro-dye technology may expand use of the contamination detection system to other food safety areas. The improved quality and safety that are likely to result from use of these unique pro-dye products will ultimately benefit the food processing industry and the public.
<P>
APPROACH: During Phase 1 of this SBIR project, BioLink will complete five tasks. Task 1 consists of synthesis of pro-dyes that are esters or amides of Red #3 and Blue #1. Synthesis will require activation of the carboxylic or sulfonic acid moiety on the dye and reaction with an alcohol or amine to provide an ester or amide, respectively. Esters will be synthesized by activation of a carboxylic or sulfonic acid moiety through conversion to an acid halide, followed by reaction with an alcohol or phenol in the presence of a tertiary amine, such as triethyl- or tributylamine. Amides will be synthesized by activation of a carboxylic or sulfonic acid moiety through conversion to an acid halide or activated ester, followed by reaction with an amine. Specifically, the parent Red #3 dye will be converted to a Pro-Dye which is an ester or amide of Red #3. Likewise, the parent Blue #1 will be converted to a Pro-Dye which is an ester or amide of Blue #1. In each case, the ester or amide portion of the Pro-Dye will be selected to be (1) a GRAS substance or Inactive Ingredient. In the case of Red #3, the ester or amide will also quench fluorescence of Red #3, while for Blue #1, the ester or amide will promote adherence to fat or meat. The facilities and equipment required for this task are available at BioLink. Task 2 will consist of spectrophotometric measurement of the fluorescence response of each pro-dye. Excitation at 340-440 nm and fluorescence at 440-750 nm will be monitored using a Waters Model 470 scanning fluorescence detector. If fluorescence of the pro-dye to Red #3 is abated or quenched in the 660-680 nm range used by the VerifEYE system, or if the pro-dye to Blue #1 shows increased adherence, the pro-dye will be further tested in two ways: (a) it will be formulated in an experimental ink and its adherence to carcass components during pseudo-processing steps will be evaluated (Tasks 3 and 4); and (b) its conversion to the parent dye will be assessed (Task 5). Prototype ink formulations typically contain components such as water, alcohols, propylene glycol, Methocel, and citric acid. Prototype inks will be stamped on meat, fat, and carcass parts to evaluate adherence to the carcass. In addition, the stamp will be subjected to each of the typical carcass cleaning steps, as well as the hot and cold water washes that are used in meat processing. If the prototype ink remains clearly visible on the carcass after these tests and does not interfere with contamination detection, three of the Critical Performance Objectives of the ink will have been met. Conversion of the pro-dye to its parent FD&C dye will be tested using Simulated Gastric Fluid with enzymes (USP Test Solution) and Simulated Intestinal Fluid with enzymes (USP Test Solution). Simulated Gastric Fluid is a solution of sodium chloride and pepsin in dilute hydrochloric acid that mimics the properties of human stomach acid. Simulated Intestinal Fluid is a solution of pancreatic enzymes in phosphate buffer, pH 7.5, which mimics the properties of enzymes and fluids in the human intestine. The rate of hydrolysis of the pro-dye to its parent FD&C dye will be monitored using HPLC (BioLink equipment).
<P>
PROGRESS: 2005/05 TO 2005/10 <BR>
In its USDA-supported SBIR Phase I research, BioLink has prepared ten novel pro-dyes and innovative prototypes of branding inks that do not interfere with contamination detection by the VerifEYE Carcass Inspection System. Pro-dyes are innovative derivatives of FDA-approved colors that after being ingested, break down to the original FDA-approved color. BioLink completed three Phase I tasks: (1) Pro-dyes were synthesized from FD&C Red No. 3, Red No. 40, and Blue No. 2. To date, attempts to prepare pro-dyes from FD&C Blue No. 1 have failed. Two pro-dyes are novel derivatives of FD&C colors that have been PEGylated (i.e., covalently bonded to poly(ethylene glycol) (PEG) to change their solubility in water). (2) The physicochemical and spectroscopic properties of each pro-dye were compared to those of the precursor FD&C dye. (3) The dyes were incorporated into prototype branding inks, and the color and short-term stability of the inks were monitored. In addition, the research and its objectives were presented at a joint meeting with representatives of the Office of Food Additive Safety of the U.S. Food & Drug Administration and the U.S. Department of Agriculture. Taken together, the experimental data indicate that each pro-dye offers the combined advantages of (a) the color of the parent dye, (b) improved solubility in branding ink formulations, and (c) no interference with contamination detection by the VerifEYE system during beef processing. In addition, the pro-dye approach holds promise for eliminating FD&C Red No. 3, a known carcinogen, from the American diet. The improved quality and safety of the food products that result will benefit the consumer, and the food processing industry, and the public.
<BR>
<BR>
IMPACT: 2005/05 TO 2005/10 <BR>
U.S. meat processors have recently implemented the VerifEYE (TM) Carcass Inspection System (eMerge Interactive, Sebastian, FL), a system that automatically inspects each beef carcass and identifies contaminated areas that may harbor dangerous pathogens such as E. coli O157:H7. Implementation of the VerifEYE system has already demonstrated that microbiological contamination detection and removal from the store-ready product can be achieved, provided that interferences (i.e. "false positives") from currently used branding inks can be eliminated. Solving this interference problem is critical to public safety, and the barrier to reliable contamination detection must, therefore, be eliminated. In its USDA-supported SBIR research, BioLink has provided a solution: "pro-dyes" that do not interfere with contamination detection by the VerifEYE system. Pro-dyes are innovative derivatives of FDA-approved colors that after being ingested, break down to the original FDA-approved color. Each pro-dye offers the combined advantages of (a) the color of the parent dye, (b) improved solubility in branding ink, and (c) no interference with contamination detection during beef processing. In addition, the pro-dye approach holds promise for eliminating FD&C Red No. 3, a known carcinogen, from the American diet. The improved quality and safety of the food products that result will benefit the consumer, and the food processing industry, and the public.