Title of Invention

A NOVEL PROCESS OF SPORULATION, ACTIVATION AND GERMINATION IN THERMOPHILIC BACTERIA FOR RAPID DETECTION OF ANTIBIOTIC RESIDUES IN MILK

Abstract

This invention relates to a novel analytical process of detection of antibiotic residues in milk system involving sporulation upto 80-90% in thermophillic strains B. stearothermophilus seeded in selective sporulation medium containing Beef extract 0.15-0.30% preferably 0.3%; peptone 0.25-0.5% preferably 0.5%; tryptone .09-0.17% preferably 0.17%; sodium chloride 0.025-0.5% preferably 0.05%; agar 1.5+0.2% preferably 1.5%; brombcresol purple .004-.008% preferably .006% as color indicator, followed by activation of dormant spores of B. stearothermophilus in BPTS medium at 55±5°C for 12+3 hours preferably at 55°C for 14 hours incubation, wherein the activated spores are forced for germination and out growth using 0.5+0.05mg D-dextrose preferably 0.5 mg; 5.0+0.5mg skimmed milk powder preferably 5.0 mg; 2.0±0.2mg whey powder preferably 2.0 mg at incubation temp. 64±2°C preferably at 64°C as germinant, wherein change of colour of the medium from purple to yellow within 2-3 hours is taken as criteria of dormant spores into vegetative cells and finally mixing germinant mixture 55+5.0 mg preferably 60 mg in milk and heated to 95 to 105°C preferably 100°C for 2-3 minutes and adding .01-.02 ml preferably .015 ml of milk in BPTS medium and incubating at 64±2°C preferably 64°C for 2.30-3 hours and observing the change of color of the medium.

Full Text

Field of Invention; This invention relates to an analytical process for detection of antibiotic residues in milk using dormant bacterial spores as biosensor. Background of Invention; The EU reference method for the determination of antibiotic residues in raw and heat-treated milk is based on microbial inhibition principle using B. stearothermophilus var. calidolactis ATCC 10149 as test organisms because of its relatively high sensitivity to inhibitory substances (IDF, 1991). However, the IDF method is quite complex and lengthy to carry out as it involves the continual growth of large quantities of B. stearothermophilus spores. Therefore, due to technical difficulties in carrying out the reference method, microbial inhibitor test kit assays based on the IDF method, are currently in use for the determination of antibiotic residues in milk. The detail list of such microbial inhibition assays for the detection of antibiotics in milk is as follow: (Table Removed) Objects of the Invention; An object of this invention is to propose a process for detection of antibiotic residues in milk using dormant bacterial spores as biosensor. Another object of this invention is to propose a process for detection of antibiotic residues in milk using dormant bacterial spores as biosensor where heat resistance and non metabolic state of spores are used. Still another object of this invention is to develop an analytical process of transformation of dormant spore of Bacillus stearothermophilus into active vegetative cell through activation, germination and outgrowth. Further object of this invention is to propose a process for detection of antibiotic residues in milk using dormant bacterial spores as biosensor where antibiotic residues are tracked within 2-3 hour during different stages of milk production and processing. At the outset of the description that follows, it is to be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only an exemplary embodiment and is not intended to be taken restrictively to imply any limitation on the scope of the present invention. Detailed Description of the Invention; This invention relates to application of dormant bacterial spores as biosensor. The bacterial spores have unique ability to sense environmental changes in response to specific "germinant" and transform rapidly into growing vegetative cells. The spores are heat resistant and can remain in non metabolic state for many years. This characteristic can effectively be used as a biosensor for tracking these residues in milk and milk products. In the present invention, an analytical process of transformation of dormant spore of Bacillus stearothermophilus into active vegetative cell through activation, germination and outgrowth has been developed. This analytical process can track major groups of antibiotic residues in milk within 2.30-3.0 hours at MRL/or above levels recommended by codex. Development of new sporulation medium; Sporulation up to 80-90% in thermophilic strains [B. stearothermophilus) seeded in selective sporulation medium containing different ingredients i.e. beef extract, peptone, tryptone, sodium chloride (BPTS), agar and bromo cresol purple (BCP) as color indicator has been established after incubation at 55C. Effective sporulation using similar medium was also achieved using mesophilic strains of Bacillus spp. Simultaneous process of sporulation and their heat activation; The BPTS medium containing cell pellets/spores of B.stearothermophilus was incubated at 55C for overnight period. It was established under phase contrast microscope that during sporulation and activation, sporulating cells undergo a chain reaction with visibility of spores as light structure outside the vegetative cells (sheet no. 1, plate-I). The chain behavior was intact till germinant were not added in the medium (sheet no. 1, plate-II). The activated spores were stable up to 12 months period without any impact on their stability, kit performance, detection time etc, when kept under refrigeration temperature. Development of new Germinant mixtures; The process of Germination and outgrowth of activated spores using mixture of different germinant is established. Germination of activated spores required the presence of a chemical trigger, a germinant. The effective combination of three different germinant i.e. dextrose, skimmed milk powder and whey powder was established with consistent conversation of activated spores into vegetative cells of B. stearothermophilus. The germinant mixture was found effective when added to their specific concentrations. Broad spectrum detection of antibiotic residues based on inhibition principle; Germination and Outgrowth of activated spores into vegetative cells was established within 2.30-3.0 hrs of addition of germinant 85 milk in BPTS medium when incubated at 64C. The process details as depicted in sheet no. 2 are as follow: BPTS medium" seeded with spores of B. stearothermophilus and color indicator was inoculated with .01-.015 ml of reconstituted milk without antibiotics (control)/or with antibiotic milk containing germinant mixtures (55+5.0 mg in 1.5±lml of milk heated to 95-105°C for 2-3 minutes) 8B incubated at 64C for 2.30-3.0 hrs periods (sheet no. -2 Fig. I). The change in color of the medium from purple to yellow was used as a criterion for semi quantitative detection of antibiotic residues in milk (sheet no-2. Fig II). The detailed interpretation of test results are as follows: Sheet 2 Fig: I Development of yellow color after incubation (2-3 hrs) at 54C: The color change of the medium from purple to yellow is due to release of calcium dipicolonic acid in the medium which indicates the conversion of activated spores into vegetative cells (antibiotic-ve) Sheet2 Fig.II No change in color after incubation (2-3hrs) at 64C: antibiotic + ve at MRL level The culture was grown in 100 ml of pre-sporulation medium, B. stearothermophilus broth (BST) and incubated at 50-64°C, preferably 55°C for 72+6 hours, preferably 72 hours. The cell suspension containing spores were centrifuged at 8000-12000 rpm for 10+2 minutes, preferably at 10,000 rpm for 10 min., washed and recentrifuged. The pellet containing spores were suspended in sterile saline and preserved under refrigeration condition. Cells pellet containing spores were seeded @ 12.5±2.5% in BPTS medium containing Beef extract (0.15-0.30%), peptone (0.25-0.5%), tryptone (0.09-0.17%) sodium Chloride (0.025-0.5%), agar (1.5+2%), bromo cresol purple (.004-.008%) as color indicator preferably in BPTS medium containing 0.3% Beef extract, 0.5% peptone, 0.17% tryptone, 0.05% sodium chloride, 1.5% agar and .006% Bromo cresol purple as colour indicator. Cell pellet containing spores seeded in BPTS medium were activated at 55±5°C for 12+3 hrs preferably at 55°C for 14 hrs incubation. The activated spores were forced for germination and out growth using D-dextrose (0.5+0.05 mg), skimmed milk powder (5.0+0,5 mg), whey powder (2.0+0.2 mg) incubation temp (64±2°C), preferably with D-dextrose 0.5mg, skimmed milk powder 5.0 mg., whey powder 2.0 mg as germinant. The change of colour of the medium from purple to yellow within 2-3 hours when incubated at 64°C was taken as criterion for transformation of dormant spores into vegetative cells. Antibiotic residues in milk are detected using developed analytical process. Mixing of germinant mixture (55+5.0 mg) preferably 60mg in milk and heat treated to 95-105°C preferably 100°C for 2-3 minutes. Then added accurately .01-.02 ml of milk preferably .015 ml in BPTS medium and incubated at 64±2°C preferably 64°C for 2.30-3.0 hours and the change in the colour of the medium is observed. Detection of antibiotic groups at MRL/ or above levels as per EU/codex limits; The present invention is working in broad spectrum detection of antibiotic residues at their respective MRL/ or above levels as specified by the EU/codex as per the details given below: (Table Removed) The analytical process is calibrated with AOAC approved Charm 6602 system for detection of antibiotic residues in Milk. Insensitivity towards inhibitors other than antibiotics present in milk: The present invention is superior over the existing technologies in terms of its response toward the presence of other inhibitors like detergents, sanitizers, preservatives etc in milk. Broad application areas; The process can be used successfully for different types of raw milk (cow/buffalo/mixed), heat treated fluid milk (skimmed, toned/double toned/standardized/full cream) 85 dried products (SMP/partially skimmed milk powder/full cream powder, baby foods etc) Tracking spoilage & pathogenic organisms in dairy food chain; The develop analytical process of transformation of dormant spores into vegetative cells can also be useful in targeting microbial contaminants like spoilage and pathogenic organisms in raw and processed foods using suitable germinogenic substrates. It is to be noted that the formulation of the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant formulations are intended to be within the scope of the present invention which is further set forth under the following claims:- We claim; 1) A novel analytical process of detection of antibiotic residues in milk system involving sporulation upto 80-90% in thermophillic strains B. stearothermophilus seeded in selective sporulation medium containing Beef extract 0.15-0.30% preferably 0.3%; peptone 0.25-0.5% preferably 0.5%; tryptone .09-0.17% preferably 0.17%; sodium chloride 0.025-0.5% preferably 0.05%; agar 1.5+0.2% preferably 1.5%; bromocresol purple .004-.008% preferably .006% as color indicator, followed by activation of dormant spores of B. stearothermophilus in BPTS medium at 55+5°C for 12+3 hours preferably at 55°C for 14 hours incubation, wherein the activated spores are forced for germination and out growth using 0.5+0.05mg D-dextrose preferably 0.5 mg; 5.0+0.5mg skimmed milk powder preferably 5.0 mg; 2.0±0.2mg whey powder preferably 2.0 mg at incubation temp. 64±2°C preferably at 64°C as germinant, wherein change of colour of the medium from purple to yellow within 2-3 hours is taken as criteria of dormant spores into vegetative cells and finally mixing germinant mixture 55±5.0 mg preferably 60 mg in milk and heated to 95 to 105°C preferably 100°C for 2-3 minutes and adding .01-.02 ml preferably .015 ml of milk in BPTS medium and incubating at 64±2°C preferably 64°C for 2.30-3 hours and observing the change of color of the medium. 2) The process as claimed in claim 1, wherein during sporulation and activation, sporulating cells undergo a chain reaction with visibility of spores as light structure outside the vegetative cells. 3) The analytical process as claimed in claim 1, wherein germination of activated spores is achieved in the presence of a chemical trigger, a germinant wherein the germinant mixture is found to be active at their specific concentration. 4) The process as claimed in claim 1, wherein the colour change of the medium from purple to yellow due to release of calcium dipicolonic acid in the medium indicates the conversion of activated spores into vegetative cells in case of antibiotic negative sample and no change in colour in case of antibiotic positive samples. 5) The analytical detection process as claimed in claim 1, wherein the process in broad spectrum detection of antibiotic residues at their respective MRL/or above levels as specified by EU/codex.

Documents:

1479-del-2006-Abstract (16-03-2007).pdf

1479-del-2006-Abstract-(07-08-2014).pdf

1479-del-2006-Assignment (19-10-2006).pdf

1479-del-2006-assignment.pdf

1479-del-2006-Claims (16-03-2007).pdf

1479-del-2006-Claims (19-11-2012).pdf

1479-del-2006-Claims-(07-08-2014).pdf

1479-del-2006-Correspondence Others-(07-08-2014).pdf

1479-del-2006-Correspondence Others-(17-10-2014).pdf

1479-del-2006-Correspondence-others (03-09-2010).pdf

1479-del-2006-Correspondence-others (03-11-2008).pdf

1479-del-2006-Correspondence-others (19-11-2012).pdf

1479-del-2006-correspondence-others.pdf

1479-del-2006-Description (Complete) (16-03-2007).pdf

1479-del-2006-description (provisional).pdf

1479-del-2006-drawings.pdf

1479-del-2006-form-1.pdf

1479-del-2006-Form-2 (16-03-2007).pdf

1479-del-2006-Form-2-(07-08-2014).pdf

1479-del-2006-form-2.pdf

1479-del-2006-form-26.pdf

1479-del-2006-form-5.pdf

1479-del-2006-form-6.pdf

1479-del-2006-gpa.pdf