Title of Invention

"A BIOLOGICAL INDICATOR TO ASSESS STEAM STERILIZATION OF FOODS"

Abstract

A biological indicator to assess steam sterilization of foods comprising of atleast two packs made of flexible packaging materials, wherein outer pack is made out of flexible multilayer packaging material characterized by containing spore suspension of Bacillus Stearothermophilus and having thermal resistance of 250°C and inner pack is made out of flexible single layer packing material containing dextrose tryptone broth and having thermal resistance of just below 105°C.

Full Text

This invention relates to a biological indicator to assess steam sterilization of foods. PRIOR ART Thermal processing of foods is one of the most successful and acceptable ways of food preservation. Designing of heat treatment is very important and considered as a critical control point to ensure the shelf life and microbial safety particularly with respect to Cl. botulinum 12D reduction in thermal processing of ready-to-eat foods. Many methods for thermal process design and calculation have been developed based on three types of approaches physical-mathematical procedure, chemical markers and bio-indicator. Despite, the large amount of work that has been done on physical-mathematical methods of evaluation, it still remains mainly empirical and knowledge in several cases is limited (Nikolas et al., Crit. Rev Food Sci & Nutr. 1997,37(5)pp.411-441). Intrinsic chemical markers have been used for thermal process evaluation such as loss in thiamin, vitamin C and degradation of food colour pigments such as anthocyanic (Kim et al., Food Tech. 1993, 47(1) pp,91-99) and other chemical marker compounds like maillard reaction products (MRPs) formed at sterilization temperature in the food have also been reported (Wnorowski et al. J Food Sci. 2002 67(6) 2149-2152), however the quantification and correlation with process lethality has not bee established completely. Inoculum pack study of microorganisms/spores as bio indicator for process verification has also been reported (Sastry et al., J Food Sci. 1988, 53 (5) pp. 1528-1530), however, analysis of microorganism usually takes two to ten days, which is too much of a time lag for evaluating process conditions. In one commonly used method of testing sterilizers, a piece of filter paper having standard biological spores of a strain (spore strip or spore dot) sufficiently resistant to the sterilization medium is exposed to the sterilization process being tested. Then the "sterilized" strip is exposed to a large amount of fluid nutrient. Sterilization of the standard spore strain ensures sterilization of corresponding bacterial cells in the chamber load. Survival of the spores subsequent to the sterilization process is typically determined by mixing a test solution consisting of nutrient growth medium containing a pH indicator with the spores and incubating the culture for growth. Due to acid production by microorganism the pH indicator in the solution changes color. It there are now viable spores following sterilization, the pH of the test solution remains essentially unchanged. While survival of the standard spore strain indicates unsatisfactory sterilization of the load. The immediate and massive outflow of the nutrient containing fluid from the ampoule to conduct the procedure described above has been described in U.S. Pat. Nos. 3, 440, 144, 3, 661, 717 and 4, 304, 869. While the methods and apparatus described therein are effective to provide the desired immediate outflow of the contents of the ampoule. U.S. Pat. No. 4732850 describes a biological indicator, made of a vial which contains a spore dot and a nutrient solution in a frangible ampoule. The nutrient solution typically contains a pH indicator to determine whether all active spores on the strip were killed by the sterilization process which takes place prior to breaking the ampoule to release the nutrient solution when the cap of the vial is fully pressed on. Some of the methods described therein are associated with the risk of injury to the user as well as the possibility of external contamination. Also, such methods do not provide a simple apparatus for effectively mixing the nutrient fluid. One of the first unitary or self-contained biological indicators has been described in U.S. Pat. No. 3, 346, 464. This patent describes a sterilization indicator in the form of a semi-permeable envelope, which is capable of transmitting water and a gaseous sterilizing media without allowing the passage of microorganisms. The envelope contains both test microorganisms and a dehydrated nutrient media. After exposure to a sterilization cycle, the envelope is immersed in warm water. The water permeates the envelope and re-hydrates the nutrient media allowing for contact with the test microorganism and growth of any remaining viable microorganism. This structure, however, suffered from a number of design defects and did not achieve wide success. For example, the performance of this device was dependent upon the characteristics of the water used in warm water incubation and permeability of envelope. The improvements to unitary or self-contained biological indicators have been illustrated by U.S. Pat, Nos. 3, 661, 717 and 4, 291, 122. Both of these patents, however, explain devices which suffer performance nomalies over a range of sterilization temperatures from 270°F.-285°F. (Reich, R. R. and Fitzpatrick, B. G. Journal of Hospital Supply, Processing and Distribution, 1985, 3(3) pp. 60-63). These performance anomalies are due to thermal lag caused by their design and construction (Joslyn L. Sterilization by Heat, in Disinfection, Sterilization and Preservation. Lea Febiger, Phila, Pa. 1983 pp. 33-34). Common to both the U.S. Pat. Nos. 3, 661, 717 and 4, 291, 122 is that they provide for an outer compartment or vial containing a pressure open able inner compartment (i.e., the media ampoule) with the microorganisms or spores on their respective carriers located between and adjacent to the two compartments. It is this disposition of the spores between the two compartments and their close juxtaposition with the ampoule of media, which retards the penetration of the sterilant, augmenting the effect of thermal lag thereby contributing to the performance anomalies of these indicators. Investigations have shown and inferences from the literature (Perkins et al., Applied and Environmental Microbiology Aug., 1981, Vol. 42, No. 2, (pp. 383-384) indicate that the sterilization times required at temperatures at or above 270°F. for conventional self-contained biological indicator designs are excessive in comparison to conventional biological indicator designs consisting of spores inoculated onto a paper chromatography grade carrier (or other suitable carrier) and enclosed in a glassine or other suitable material envelope. This can result in false positive readings and lead to the reprocessing of an otherwise sterile load and unnecessary equipment downtime. False negative readings can also occur at lower sterilization temperatures (leading to release as sterile, of an under processed load whose contents may in fact not be sterile. OBJECTS OF THE INVENTION The primary object of the present invention is to propose a biological indicator to assess steam sterilization of foods which overcomes disadvantages such as spore inoculation on the paper strip and dehydration, rupture of a sealed frangible container, such as ampoule, rigid tubular vial, in which the frangible ampoule is retained and breaking the ampoule to release the nutrient. Another object of this invention is to propose a biological indicator to assess steam sterilization of foods which is glass free flexible biological indicator system. Further object of this invention is to propose a biological indicator to assess steam sterilization of foods which is able to assess microbial sterility of a thermal sterilizer and packaged products upon completion of a thermal sterilizing cycle. Still another object of this invention is to propose a biological indicator to assess steam sterilization of foods which is simple and cost effective. Yet another object of this invention s to propose a biological indicator- to assess steam sterilization of foods which is based on chemometry for steam sterilization. STATEMENT OF INVENTION According to this invention there is provided a biological indicator to assess steam sterilization of foods comprising of atleast two packs made of flexible packaging materials, wherein outer pack is made out of flexible multilayer packaging material characterized by containing spore suspension of Bacillus Stearothermophilus and having thermal resistance of 250°C and inner pack is made out of flexible single layer packing material containing dextrose tryptone broth and having thermal resistance of just below 105°C. DETAILED DESCRIPTION OF THE INVENTION The indicator of the present invention is pack-in-pack type, comprising of atleast two packs made of flexible packaging materials. The outer pack, also known as biological indicator sachet (henceforth mentioned as Tack A') is made out of flexible multilayer packaging material comprising of approximately 12µm Polyethylene terephthalate/9 µm Aluminium foil/15 µm nylon /70 µm Cast Polypropylene (PET/Al.foil/Nyl/C.PP) of total thickness of 90-120 µm. The thermal resistance of the materials is above 250°C. To see the colour change, two transparent windows are provided at one side of this pack. The inner pack, also known as 'culture media pack' (henceforth mentioned as Tack B') is made out of flexible single layer polyolefin packaging material of 40-60 µm thickness. The thermal resistance of the material is just below 105°C. It is initially filled with approximately 0.5 ml of triple strength dextrose tryptone broth containing bromo-cresol purple and then heat-sealed. The heat sealed 'Pack B' is placed in prefabricated 'Pack A' whose 3 sides are already heat-sealed. Subsequently it is filled with approximately 1 ml of spore suspension with the count of 6.3 X 105 spores of Bacillus stearothertnophilus per ml in which count can be varied as per the Fo value requirements). Finally, top of pack A is heat-sealed. When the indicator of the present invention is subjected to moist heat treatment, due to the increase in the temperature to about 105°C, 'Pack B' undergoes disintegration and simultaneously allows the culture media to mix with spore suspension (kept in 'Pack A'). As the temperature reaches 110°C, the colour of the aliquot (culture media + spore) changes from colourless to purple. As the temperature increases progressively, the intensity of the purple colour of aliquot remains constant. This gives a presumptive indication of the adequacy of thermal processing. In order to confirm adequacy of thermal processing, the system is incubated at 56±2°C for 24-48 h. If the process is inadequate (the required temperature and time is not received) due to the survival of Bacillus stearothermophilus spores, the purple colour of the aliquot changes to yellow. However, under adequate processing conditions, the purple colour of the aliquot is retained. Methodology: Spore Production and Thermal Resistance: Spore production: Bacillus stearothermophilus ATCC 7953 was obtained from Microbial Type Culture Collection (MTCC) Chandigarh, India. The spore production and heat resistance studies were carried out as described by Fernanzez et. Al., J Food Prot. 1994, 57 (1) pp.27-41, The spore population per ml was 6.3 X 105 and D value at 121°C was 2.1 minutes. In actual thermal processing, the determined killing time of above organism at 121°C is 10 minutes. However, this varies depending on the D value of microorganisms (Pflug. I. J., J Food Prot. 1987, 50 (7) pp. 608-615). Procedure to use Biological Indicator The 'Pack A' and 'Pack B' were made and filled with spores and culture media as described hereinabove. Biological indicator sachet was kept at geometric center of the retort as well as inside the food packaged in can/retortable pouches placed at different locations inside the retort. Thermal process is carried out as per standard procedure for commercial sterility. After the completion of sterilization cycle, colour change from colour less to purple is checked. This colour change may presumptively indicate the adequacy of thermal processing. In order to obtain confirmatory result, the whole biological indicator system is incubated at 56±2°C for 24 - 48 h. The presence of curvival Bacillus stearothermophilus spores changes the colour of culture media from purple to yellow indicating that the process is incomplete and hence the product is under-processed. Thus, the self-contained biological indicator system consists of bacterial spore suspended in pre-sterilized distilled water and broth culture medium packed separately in flexible plastic packaging materials. The liquid culture media contains a pH indicator to detect the surviving spores after incubation followed by heat sterilization. Since it is a glass free, plastic based flexible sachet system it can be placed at any place in retort .and/or packaged product. This biological sterilization indicator packed in heat-sealed plastic packaging sachet is capable of withstanding the thermal .exposure/high temperature of the sterilizer. After completion of the sterilization process, the system can be easily removed from the sterilizer and examined for its effectiveness. Easy interpretation is quit possible based on visible colour change from colour less to purple which ensures the adequacy of steam temperature in the retort presumptively and subsequently confirming the result through change in colour of media from purple to yellow due to change in pH after 24-48 h incubation at 56±2°C, attributed to the presence of Bacillus stearothermophilus spores. This indicator can also be calibrated to a predetermined sterilization value (Fo-value) based on destruction/inactivation of test organisms in logarithmic scale. This provides simple tool to analyze and verify the adequacy of heat distribution in a loaded sterilizer as well as microbial sterility of the packaged products after exposure to heat at a. pre-determined sterilization temperature and time combination after 48 hrs of incubation at 56±2°C. Features and Advantages of present invention 1. It is self-contained, glass free, flexible sachet-packaging system applicable to both retort and food products packed in metal cans and/or flexible packaging materials. 2. The suspended spores in liquid medium are distributed uniformly and facilitate moist heat sterilization. 3. It ensures the steam temperature of the retort at 100°C-110°C presumptively. 4. It.confirms the commercial sterilization of the food products after incubation at 56±2°C for 24-48 h. 5. It ensures the adequacy of heat distribution in the retort as well as ensures microbial safety of products. 6. It can be calibrated to any specified sterilizing value (Fo-value) based on inactivation of test organisms. 7. It contains a pH indicator, which confirms the survival of spores after incubation by prominent visible colour change. >, 8. No further procedure such as rehydration and mixing of culture media are required after sterilization except incubation. 9. It is suitable for moist heat sterilization. 10. It is storable at any storage condition before subjecting to heat treatment. 11. It is user friendly and cost effective as it is devoid of impregnation/lyophilization of spore procedure. 12. It consists of cost effective and indigenously available packaging materials. 13. The results obtained by using this system are consistent and reproducible. 14. It can be easily adopted in commercial production of thermally processed foods. It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which is further set forth under the following claims:- WE CLAIM: 1. A biological indicator to assess steam sterilization of foods comprising of atleast two packs made of flexible packaging materials, wherein outer pack is made out of flexible multilayer packaging material characterized by containing spore suspension of Bacillus Stearothermophilus and having thermal resistance of 250°C and inner pack is made out of flexible single layer packing material containing dextrose tryptone broth and having thermal resistance of just below 105°C. 2. A biological indicator as claimed in claim 1, wherein the outer pack comprising of approximately 12µm of polyethylene terephthalate/9 µm of Aluminium foil/15 µm of Nylon/70 µm of cast polypropylene having total thickness of 90-120 µm. 3. A biological indicator as claimed in claim 1, wherein the outer pack is provided with transparent window(s) to see the colour change. 4. A biological indicator as claimed in any of the preceding claims, wherein the inner pack is made out of polyolefin of 40-60 µm thickness. 5. A biological indicator as claimed in claim 1, wherein the dextrose tryptone broth containing bromo-cresol purple is having triple strength.

Documents:

428-DEL-2007-Abstract-(05-12-2011).pdf

428-del-2007-abstract.pdf

428-DEL-2007-Assignment.pdf

428-DEL-2007-Claims-(05-12-2011).pdf

428-del-2007-claims.pdf

428-DEL-2007-Correspondence Others-(05-12-2011).pdf

428-del-2007-Correspondence Others-(18-05-2012).pdf

428-del-2007-Correspondence-others-(23-09-2013).pdf

428-DEL-2007-Correspondence-Others.pdf

428-DEL-2007-Description (Complete)-(05-12-2011).pdf

428-del-2007-description (complete).pdf

428-DEL-2007-Form-1.pdf

428-del-2007-form-2.pdf

428-DEL-2007-Form-5-(05-12-2011).pdf

428-DEL-2007-Petition-137-(05-12-2011).pdf