RELATED APPLICATION

This is a patent of addition and claims priority to Indian Patent Application No. 520/DEL/2006 titled: “A REAGENT STRIP FOR RAPID DETECTION OF COLD SLAUGHTERED MEAT, A PROCESS OF PREPARING THE SAME, AND A METHOD OF DETECTION” filed by Director General, Defence Research and Development Organization on February 28, 2006 which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of determination of quality of raw meat to be used for human consumption and more particularly relates to a spot test strip for quick determination of microbial quality of raw chicken under field condition, process for preparing the same and method of use thereof.

BACKGROUND OF THE INVENTION

Meat is considered one among of the most highly perishable food commodity. Studies on meat spoilage have been concerned largely with processes occurring in red meat and poultry. The shelf-life of meat and meat products is the storage time until spoilage. The point of spoilage may be defined by a certain maximum acceptable bacterial level, or an unacceptable odour, flavour or appearance. The shelf-life depends on the numbers and types of microorganisms mainly result of activity of motile and non-motile bacteria, like Gram-negative, psychotropic and aerobic rods. Quality of raw meat in terms of microbial load plays an important role in consumption quality of final product. Therefor establishing the microbial quality of raw poultry meat as standard plate count (SPC) is very much important in accepting/rejecting a large consignment of transported meat. The SPC is most common and universally accepted index of microbial quality of foodstuffs (ISO, 1978; BAM 1992). It provides a standard of measuring composite microbial population in foods prior to the onset of sensory deterioration.

Conventional methods of SPC determination are very laborious and time consuming as it may take more than 24-48 hours. Moreover, keeping the meat at room temperature and waiting for the SPC results further accelerates the microbial deterioration and makes the meat unacceptable for cooking/consumption.

There are different methods/procedures available for rapid determination of microbial growth based on cell sizes, cell constituents, metabolic by-products and other immunofluorescence assays (Henk and Huis, 1997). Another method for determination of microbial load involves use of reducing dyes test, which depends on the ability of microorganisms to alter redox potential resulting in simple colour change reactions (Harrigan and McCance, 1976). The length of time taken to reduce (change) the colour depends on the load of the micro flora present in the sample, this can be understood as greater the number of microbes present, the shorter time required for the reduction. However, major disadvantages with the above technique are requirement of laboratory facilities, trained personnel, preparation of fresh dye solution, and inconvenience in transportation of the reagent solutions to field conditions.

Alfano (1993, US Patent No. 5,474,910) describes a method and device for detecting fluorescent biological molecules and/or microorganisms containing said fluorescent biological molecules within a given area or space. The method comprises illuminating the area of interest using a light of suitable wavelength to excite the fluorescent biological molecules and then measuring resultant fluorescent light from the illuminated area or space. Pahuski, et.al (1996, US Patent No. 5,587,286) illustrates methods and kits for detection of cells in food materials. The process involves removal of cells as cell pellets from liquid milk samples, or from cultures or extracts of other food materials or other materials of biological origin. The concentrated cells in the pellet can be analysed by various techniques to determine the relative cell count, such as by lysing of the cells followed by measurement of ATP. Pahuski, et.al (1997, US Patent No. 5,700,645) also describes about process and kits for separation, concentration and analysis of cells from food materials. Powers (1998, US Patent No. 5,760,406 and 1999, US Patent No. 5,968,766) developed method and apparatus for sensing the presence of microbes. In this method, the detection of aerobic microbes on non-living surfaces in which electromagnetic radiation having a wavelength greater than 350 nm is directed onto the surface to cause excitation of pyridine nucleotides present in microbial cells to emit radiation having a higher wavelength. That higher wavelength radiation and reflected radiation is then sensed as a measure of the amount of microbial cells on the surface. However, all known methods do not provide for instant determination of microbial quality of the food, more specifically of meat in the filed condition without requiring a laboratory and skilled personnel.

Hence, there is need for a ready-to-use convenient test kit for instantly determining the microbial quality of chicken/poultry meat in field condition.

SUMMARY OF THE INVENTION

In one aspect of the present invention, a reagent based test strip for rapid detection of microbial quality of a raw chicken meat sample, having chemically inert adsorbent strip having homogenously adsorbed resazurin, wherein level of adsorption of the resazurin is 19 – 21 µg/cm2 and has pH ranging within 6.8 – 8.2.

In another aspect of the present invention, a process for preparing reagent based test strip for rapid detection of microbial quality of a raw chicken meat sample, including the following steps of preparing resazurin solution of 0.1 – 0.15 % using distilled water, adjusting pH of the resazurin solution to range within 6.8 – 8.2, dipping one or more chemically inert adsorbent sheets in the pH adjusted resazurin solution, and drying the one or more adsorbent sheets having soaked in the resazurin solution, wherein the resazurin adsorbed over the one or more adsorbent sheets range from 19 – 21 µg/cm2.

In yet another aspect of the present invention, a process for detection of microbial quality of a raw chicken meat sample, including the following steps of soaking a reagent based test strip with clean water, pressing the wet reagent based test strip against the raw chicken meat sample for a duration of 20 – 30 seconds while keeping the reagent based test strip covered; and detecting the microbial quality of the raw chicken meat sample based on change in colour of the reagent based test strip within a pre-specified time frame.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for an easy and quick process of determination of microbial quality of raw chicken in field condition. To achieve this the present invention also provides for a reagent based test strip and process for preparing the same.
Raw/fresh meat (including mutton, goat, beef and buffalo meat) and poultry meat has got different microbial standards with respect to standard plate count (SPC) and coliforms as per the laid down legislation by European Union(EU) and some other individual nations at defined points in the production processes.

Table 1 indicates some of the references available for determining the upper limit/maximum microbial count for acceptance of raw meat and poultry, clearly showing differential microbial acceptance standards in each of the products. Therefore, use of microbial test strip used for assessing the meat freshness finds invalid application in poultry meat quality assessment since the dye reduction time for both conditions stays different.

Table 1: Acceptable upper limits for microbial load in meat and poultry meat. It is evident that tests prescribed for determination of microbial quality of meat will not be helpful in the same manner for raw chicken/poultry meat.

In one of the embodiments, the present invention provides for a reagent based test strip for rapid detection of microbial quality of a raw chicken meat sample. The test strip is a chemically inert adsorbent strip having homogenously adsorbed resazurin, wherein level of adsorption of the resazurin is 19 – 21 µg/cm2 and has pH ranging within 6.8 – 8.2.

The chemically inert adsorbent used for preparing the reagent based strip is selected from such as, but not limited to, medium ash WhatmanTM filter paper.

Dimension or size and shape of the test strip adsorbent is adjusted as per requirement of testing.

The test strip provided is suitable for carrying out the field testing of the raw poultry meat. The process of using the test strip is detailed later in the disclosure.

In another embodiment, the present invention also provides for a process for preparing reagent based test strip for rapid detection of microbial quality of a raw chicken meat sample, including the following steps:
preparing resazurin solution of 0.1 – 0.15 % using distilled water;
adjusting pH of the resazurin solution to range within 6.8 – 8.2;
dipping one or more chemically inert adsorbent sheets in the pH adjusted resazurin solution; and
drying the one or more adsorbent sheets having soaked in the resazurin solution, wherein the resazurin adsorbed over the one or more adsorbent sheets range from 19 – 21 µg/cm2.

In a further embodiment, the one or more chemically inert adsorbent sheets are cut to desired dimension before dipping in the pH adjusted resazurin solution.

The chemically inert adsorbent sheet(s) used are medium ash WhatmanTM filter paper.

The chemically inert adsorbent sheet(s) are dipped in the resazurin solution for a duration of 10 – 30 seconds. Subsequently, the chemically inert adsorbent sheet(s) are dried at a temperature of 40 - 45 oC for a duration of 1 – 2 hours. Later the dried chemically inert adsorbent sheet(s) are cut into strips of desired dimensions for the purpose of conveniently packing in a test kit.

In yet another embodiment, the present invention provides a process for detection of microbial quality of a raw chicken meat sample using the reagent based test strip, including the following steps:
soaking a reagent based test strip with clean water;
pressing the wet reagent based test strip against the raw chicken meat sample for a duration of 20 – 30 seconds while keeping the reagent based test strip covered; and
detecting the microbial quality of the raw chicken meat sample based on change in colour of the reagent based test strip within a pre-specified time frame.

After performing the steps above, the sample is considered to be fresh and fit for consumption when the reagent based test strip does not change its colour within the time frame of 20 minutes after being pressed against the sample. The sample is considered to be in incipient stage of spoilage when the reagent based test strip changes its colour from blue to pink within the time frame of 15 - 20 minutes after being pressed against the sample. The sample is considered to be spoiled when the reagent based test strip changes its colour from blue to pink within the time frame of 15 minutes after being pressed against the sample.

In one of the embodiments, the present invention provides for a microbial detection kit comprising the reagent based test strips sealed in total barrier packs, forceps for handling the test strips, and test manual with guidelines of using the test kit.

The present invention can be used by any layman to find out microbial quality of raw chicken meat on the spot without any training.

EXAMPLES

Example 1
Resazurin solution of 0.1 - 0.15% is prepared using distilled water. pH of the solution is adjusted in such a way that the final pH of the reagent strip would in the range of 6.8 to 8.2. Adsorbent, medium ash, qualitative Whatman No.1TM filter paper is used. Filter paper is cut into strips of 1cm x 2cm and 1cm x 3cm size. The strips are dipped in resazurin solution using a clean stainless steel forceps for 10-30 seconds. Resazurin on the paper should be in the range of 19-21 µg/cm2 when this range is altered on either side the reduction time would change accordingly. Strips are removed from the solution and drained thoroughly and dried on clean glass sheets at 40-45 oC for 1 - 2 hours. (Alternately, filter paper of convenient size may be dipped in the dye solution and cut into strips after drying). The size of the strip could be fixed as per convenience if the size is too small it could be inconvenient to handle and observed. If the size is too big, it would be wasted test material. Dried strips are stored in sealed total barrier packs.

Example 2
Resazurin solution of 0.1% is prepared using distilled water. Adsorbent, medium ash, qualitative Whatman No.1 filter paper is used. Filter paper is cut into strips of 1cm x 2cm and 1cm x 3cm size. The strips are dipped in resazurin solution using a clean stainless steel forceps for 10 - 30 sec. The resazurin on the paper is 21 µg/cm2. Strips are removed from the solution and drained thoroughly and dried an clean glass sheets at 40 - 45 oC for 1 - 2 hours. (Alternately, filter paper of convenient size may be dipped in the dye solution and cut into strips after drying). Dried strips are stored in sealed total barrier packs.

CLAIMS:
1. A reagent based test strip for rapid detection of microbial quality of a raw chicken meat sample, comprising:
a chemically inert adsorbent strip having homogenously adsorbed resazurin, wherein level of adsorption of the resazurin is 19 – 21 µg/cm2 and has pH ranging within 6.8 – 8.2.

2. The reagent based strip as claimed in claim 1, wherein the adsorbent is a medium ash WhatmanTM filter paper.

3. The reagent based strip as claimed in claim 1, wherein dimension of the test strip is adjusted as per testing requirement.

4. A process for preparing reagent based test strip for rapid detection of microbial quality of a raw chicken meat sample, comprising the following steps:
preparing resazurin solution of 0.1 – 0.15 % using distilled water;
adjusting pH of the resazurin solution to range within 6.8 – 8.2;
dipping one or more chemically inert adsorbent sheets in the pH adjusted resazurin solution; and
drying the one or more adsorbent sheets having soaked in the resazurin solution, wherein the resazurin adsorbed over the one or more adsorbent sheets range from 19 – 21 µg/cm2.

5. The process as claimed in claim 4, further comprising cutting the one or more chemically inert adsorbent sheets to desired dimension before dipping in the pH adjusted resazurin solution.

6. The process as claimed in claim 4, wherein the one or more chemically inert adsorbent sheet used are medium ash WhatmanTM filter paper.

7. The process as claimed in claim 4, wherein the one or more chemically inert adsorbent sheets are dipped in the resazurin solution for a duration of 10 – 30 seconds.

8. The process as claimed in claim 4, wherein the one or more chemically inert adsorbent sheets are dried at a temperature of 40 - 45 0C for a duration of 1 – 2 hours.

9. The process as claimed in claim 4, wherein the dried one or more chemically inert adsorbent sheets are cut into strips of desired dimensions.

10. A method for detection of microbial quality of a raw chicken meat sample, comprising the following steps:
soaking a reagent based test strip with clean water;
pressing the wet reagent based test strip against the raw chicken meat sample for a duration of 20 – 30 seconds while keeping the reagent based test strip covered; and
detecting the microbial quality of the raw chicken meat sample based on change in colour of the reagent based test strip within a pre-specified time frame.

11. The method as claimed in claim 10, wherein the sample is considered to be fresh and fit for consumption when the reagent based test strip does not change its colour within the time frame of 20 minutes after being pressed against the sample.

12. The method as claimed in claim 10, wherein the sample is considered to be in incipient stage of spoilage when the reagent based test strip changes its colour from blue to pink within the time frame of 15 - 20 minutes after being pressed against the sample.

13. The method as claimed in claim 10, wherein the sample is considered to be spoiled when the reagent based test strip changes its colour from blue to pink within the time frame of 15 minutes after being pressed against the sample.