Sterilization is a process of eliminating (killing) transmissible microbial agents like fungi, bacteria, virus, spores and enzymes in biological cultures and processed formulations. Sterilization can be achieved by combination of heat, chemicals, irradiation, high pressure, and filtration.
Complete sterilization of biological and pharmaceutical grade material, as well as production containers and equipment is highly desirable for most commercial applications because it prevents proliferation of microbes in required product. Typical sterilization of production equipments is achieved with the help of super-heated steam at 121°C for few minutes, as determined by experimental data.
Sterilization aims to reduce viable micro-organisms and spores to about 1/1000 per ml of their original value, which directly implicates that there is possibility of contamination of one batch in 1000 batches. Clean-in-place (CIP) and Sterilization-in-place (SIP) is commonly used for cleaning and sterilization of production equipments, pipe-work, valves and filters in continuous production line.
CIP & SIP for batch processes can be controlled by dismantling production equipment and parts to achieve results as per desired standards. Superheated steam destroys most spores and bacteria but also made to react with chemical disinfectants and cooled prior to regeneration of steam for next sterilization process or use of water in waste-treatment or recirculation in heat-exchangers.
Culture media and raw ingredients derived from biological source are most prone to bacterial and fungal growth. These are highly potent and possibly alter the quality of ingredient or even chemical nature of the ingredient. For Example, dairy products are common ingredients used in biological formulations. These contain organically rich biomolecules like lactose sugar, phospholipids (fats), proteins, lactic acid, vitamin B, vitamin C, calcium carbonate, magnesium carbonate and other trace minerals.
Many harmful micro-organisms, yeasts, molds and pathogenic bacteria such as Salmonella, Yersinia, Campylobacter, Staphylococcus, Escherichia, Listeria, Mycobacterium and Coxiella species proliferates dairy products. These microbes are responsible for a number of diseases in animals and humans such as brucelliosis, diphtheria, scarlet fever, Q fever and tuberculosis. Similarly, sap, resins, vitamins and minerals derived from biological sources of aromatic plants, herbs, vegetative parts of plants and fruits contain a variety of micro-organism that alter the chemical and biological nature of principle ingredient. Therefore, it is essential to sterilize culture media and raw materials prior to use as active pharmaceutical ingredient (API) or use as binders and additional agents in making drug formulations.
Culture media can be sterilized using UV radiations, IR radiations, combination of heat and chemical disinfectants, and through use of chemically activated filters (pre-filters and post-filters). Excessive heat can alter the biological nature of enzymes and render the ingredient useless for desired applications. Chemical disinfectants must be optimized for desired use as they might react with reactive group of ingredient. Air sterilization and air purification for production processes that utilize N2, O2, CO2 is equally essential as there is a possibility of entry of spores or toxins from unpurified air. Air filters and chemical activators at entry of air are deployed for this purpose. Similarly, purified water, either distilled water or nanofiltration technique is used for production of drug formulations.
Pasteurization is the process heating of beverages such as milk, wine, fruit-juice to destroy/inactivate living bacteria and enhance the flavour, bouquet, and shelf-life for preservation under optimal conditions. Pasteurization is achieved by heating material for about 30 minutes at 68°C (154.4°F). This heat is intended to destroy spores but prevent alteration of natural product by heat. Liquid is immediately cooled to 10°C (50°F) or lower to prevent spores from developing.
Pasteurization is the reason for milk's extended shelf life. High-temperature short-time pasteurization (HTST) of milk typically has refrigerated shelf life of two to three weeks, whereas ultra heat treatment pasteurization (UHT) combined with sterile handling and container technology can preserve milk for more than nine weeks. High boiling temperatures for natural material like fruit juice or milk is avoided because it generally results in casein aggregation, micelle formation and curdling. High temperatures also result in slightly altering the taste of compound and vapour loss into the environment. HTST or flash pasteurization forces milk between preheated metal places or pipes to 72 °C (161 °F) for 15 seconds.
UHT processing holds the milk at a temperature of 140 °C (284 °F) for four seconds. During UHT processing milk is more sterilization than pasteurization. This process allows milk or juice to be stored several months without refrigeration. The process is achieved by spraying the milk or juice through a nozzle into a chamber that is filled with high-temperature steam under pressure. After the temperature reaches 140 °C, the fluid is cooled instantly in a vacuum chamber, and packed in a pre-sterilized airtight container. The efficiency of HTST pasteurization standard is designed to achieve five-log reduction, killing 99.999% of the number of viable micro-organisms in milk, considered adequate for destroying almost yeasts, molds and spoilage bacteria. Heat pasteurization may result in loss of some vitamins and minerals but the process is weighed in terms of prevention from diseases and prolonged storage rather loss of trace nutrients.
History and invention
French chemist and microbiologist Louis Pasteur (December 27, 1822 – September 28, 1895) is renowned for his discovery of principles in microbial fermentation and pasteurization. He pioneered remarkable breakthroughs in vaccination for diseases like rabies and anthrax that were widespread during his age. In 1864, Pasteur discovered that beer and wine (ale) turned sour on ageing or prolonged storage that was later demonstrated as result of microbial growth or enzyme activity which caused wines to turn sour. To remedy the frequent acidity of the local wines, he observed experimentally that heating young wine to only about 50–60 °C (122–140 °F) for a brief time killed microbes and that the wine could subsequently be aged (stored) without sacrificing the final quality or taste of wine. He noted that milk is an excellent source of bacteria and storing the milk at normal temperature results in proliferation of bacteria that spoils the quality of milk. He became successful in eliminating the growth of pathogenic microbes from milk by extending the same approach used for wines.
In honour of Pasteur’s work (invention), the process became known as "Pasteurisation". Pasteurization was originally used as a way of preventing wine and beer from souring, and it would not be many years before milk was pasteurized. Today the process of pasteurization is used widely in the dairy and food industries for microbial control and preservation of the food we consume. Pasteurization does not result in complete elimination of pathogenic microbes but lowers the total microbial content in beverages, which enables storage of beverages for extended period of time without compromising on nutritional quality or taste.
Prior to work of Pasteur, a traditional form of pasteurization was practised in England by scalding and straining milk cream to increase the quality of butter. Similar process was introduced to Boston, USA in 1773, although not widely until late 18th century. In 1768, an Italian priest and scientist Lazzaro Spallanzani observed experimentally that heat killed bacteria such that they do not re-appear if the product is hermetically sealed.
In 1795, a Parisian chef and confectioner named Nicolas Appert studied ways to preserve foodstuffs and succeeded in preserving soups, vegetables, juices, dairy products, and fruit syrups by placing food in glass jars and sealing them with cork and sealing wax and finally putting them in boiling water. After 14 – 15 years of research in food preservation, Appert submitted his invention and won the noble prize from French government in January 1810. Appert published the art of preserving animal and vegetable substances (L'Art de conserver les substances animales et vegetables).
Conclusion
F&B processing, drug processing and medicine is both an art and science practised since pre-historic times to modify or preserve variety of organic raw ingredients by cooking, salting, dehydrating, acidification, drying, cold storage, pickling (fermentation) and many other similar processes. These traditional methods serve to protect both the nutritional composition of food and reduce spoilage from pathogens. Commercial scale storage and preservation is practised in a variety of ways that does not cause deterioration of food and also increases the shelf-life (time-period through which food can be stored without adversely affecting its nutritional composition, taste, odour and appearance). Pasteurization process demonstrates that number of viable organisms could be reduced internally (up to efficiency of 99.9% i.e. one in 1000 bacteria) before preserving the food stuff in a sealed container. Additionally, cold storage or refrigeration can minimize the activity of internal enzymes and further restrict growth of micro-organism, which allows extending age of the product.
(The author is MD of VMG Biotech Consultants, New Delhi)