Fermentation (CHM226) PDF

# Fermentation Fermentation is a metabolic process that converts sugar to acids, gases or alcohol. It occurs in: * yeast and bacteria * oxygen-starved muscle cells, as in the case of lactic acid fermentation Fermentation is also used more broadly to refer to the bulk growth of microorganisms on...

# Fermentation Fermentation is a metabolic process that converts sugar to acids, gases or alcohol. It occurs in: * yeast and bacteria * oxygen-starved muscle cells, as in the case of lactic acid fermentation Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth medium, often with the goal of producing a specific chemical product like enzyme, vaccines, antibiotics, food product/additive etc. French microbiologist Louis Pasteur is often remembered for his insights into fermentation and its microbial causes. The science of fermentation is known as **zymology.** ## Fermentation Process Fermentation takes place in the lack of oxygen (when the electron transport chain is unusable) and becomes the cell's primary means of ATP (energy) production. It turns NADH and pyruvate produced in the glycolysis step into NAD⁺ and various small molecules depending on the type of fermentation. * **In the presence of O2, NADH and pyruvate are used to generate ATP in respiration.** This is called oxidative phosphorylation, and it generates much more ATP than glycolysis alone. For that reason, cells generally benefit from avoiding fermentation when oxygen is available. * **The exception being obligate anaerobes which cannot tolerate oxygen.** ## Glycolysis The first step, glycolysis, is common to all fermentation pathways: $C_6H_{12}O_6 + 2 NAD^+ + 2 ADP + 2 P_i → 2 CH_3COCOO¯ + 2 NADH + 2 ATP + 2 H_2O + 2H^+$ Where: * Pyruvate is $CH_3COCOO^-$. * $P_i$ is inorganic phosphate. * Two ADP molecules and two $P_i$ are converted to two ATP and two water molecules via substrate-level phosphorylation. * Two molecules of $NAD^+$ are also reduced to NADH. In oxidative phosphorylation the energy for ATP formation is derived from an electrochemical proton gradient generated across the inner mitochondrial membrane (or, in the case of bacteria, the plasma membrane) via the electron transport chain. Glycolysis has substrate-level phosphorylation (ATP generated directly at the point of reaction). ## Humans and Fermentation Humans have used fermentation to produce food and beverages since the Neolithic age. * For example, fermentation is used for preservation in a process that produces lactic acid as found in such sour foods as pickled cucumbers, kimchi and yogurt. * Fermented foods are also used for producing alcoholic beverages such as wine and beer. * Fermentation can even occur within the stomachs of animals, such as humans. ## Definitions of Fermentation There are a number of definitions of fermentation that range from informal, general usage to more scientific definitions. 1: Preservation methods for food via microorganisms (general use). 2: Any process that produces alcoholic beverages or acidic dairy products (general use). 3: Any large-scale microbial process occurring with or without air (common definition used in industry). 4: Any energy-releasing metabolic process that takes place only under anaerobic conditions (becoming more scientific). 5: Any metabolic process that releases energy from a sugar or other organic molecules, does not require oxygen or an electron transport system, and uses an organic molecule as the final electron acceptor (most scientific). ## Examples of Fermentation Fermentation does not necessarily have to be carried out in an anaerobic environment. For example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to aerobic respiration, as long as sugars are readily available for consumption (a phenomenon known as the Crabtree effect). * **The antibiotic activity of hops also inhibits aerobic metabolism in yeast.** * Fermentation reacts NADH with an endogenous, organic electron acceptor. Usually this is pyruvate formed from the sugar during the glycolysis step. * During fermentation, pyruvate is metabolized to various compounds through several processes. ### Ethanol fermentation **aka alcoholic fermentation** Ethanol fermentation is the production of ethanol and carbon dioxide. ### Lactic acid fermentation Lactic acid fermentation refers to two means of producing lactic acid: * **Homolactic fermentation** is the production of lactic acid exclusively. * **Heterolactic fermentation** is the production of lactic acid as well as other acids and alcohols. ## Substrate of Fermentation Sugars are the most common substrate of fermentation, and typical examples of fermentation products are ethanol, lactic acid, carbon dioxide, and hydrogen gas ($H_2$). However, more exotic compounds can be produced by fermentation, such as butyric acid and acetone. * Yeast carries out fermentation in the production of ethanol in beers, wines, and other alcoholic drinks, along with the production of large quantities of carbon dioxide. * Fermentation occurs in mammalian muscle during periods of intense exercise where oxygen supply becomes limited, resulting in the creation of lactic acid. ## Ethanol Fermentation Chemical Reaction The chemical equation below shows the alcoholic fermentation of glucose, whose chemical formula is $C_6H_{12}O_6$. One glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules: $C_6H_{12}O_6→2 C_2H_5OH + 2 CO_2$ Where: * $C_2H_5OH$ is the chemical formula for ethanol Before fermentation takes place, one glucose molecule is broken down into two pyruvate molecules. This is known as glycolysis. ## Lactic Acid Fermentation **Homolactic fermentation** (producing only lactic acid) is the simplest type of fermentation. The pyruvate from glycolysis undergoes a simple redox reaction, forming lactic acid. It is unique because it is one of the only respiration processes to not produce a gas as a byproduct. Overall, one molecule of glucose (or any six-carbon sugar) is converted to two molecules of lactic acid: $C_6H_{12}O_6→2CH_3CHOHCOOH$ It occurs in the muscles of animals when they need energy faster than the blood can supply oxygen. It also occurs in some kinds of bacteria (such as Lactobacilli) and some fungi. * This type of bacteria that converts lactose into lactic acid in yogurt, giving it its sour taste. * These lactic acid bacteria can carry out either homolactic fermentation, where the end-product is mostly lactic acid, or ### Heterolactic Fermentation **Heterolactic fermentation** is where some lactate is further metabolized and results in ethanol and carbon dioxide (via the phosphoketolase pathway), acetate, or other metabolic products, e.g.: $C_6H_{12}O_6 CH_3CHOHCOOH+C_2H_5OH+CO_2$ If lactose is fermented (as in yogurts and cheeses) it is first converted into glucose and galactose (both six-carbon sugars with the same atomic formula): $C_12H_{22}0_{11} + H_2O → 2 C_6H_{12}O_6$ Heterolactic fermentation is in a sense intermediate between lactic acid fermentation, and other types, e.g. alcoholic fermentation. The reasons to go further and convert lactic acid into anything else are: * **The acidity of lactic acid impedes biological processes:**This can be beneficial to the fermenting organism as it drives out competitors who are unadapted to the acidity; as a result the food will have a longer shelf-life (part of the reason foods are purposely fermented in the first place). * However, beyond a certain point, the acidity starts affecting the organism that produces it. * **The high concentration of lactic acid (the final product of fermentation) drives the equilibrium backwards (Le Chatelier's principle), decreasing the rate at which fermentation can occur, and slowing down growth** * **Ethanol, that lactic acid can be easily converted to, is volatile and will readily escape, allowing the reaction to proceed easily.** $CO_2$ is also produced, however it's only weakly acidic, and even more volatile than ethanol. * **Acetic acid (another conversion product) is acidic, and not as volatile as ethanol; however, in the presence of limited oxygen, its creation from lactic acid releases a lot of additional energy** * It is a lighter molecule than lactic acid, that forms fewer hydrogen bonds with its surroundings (due to having fewer groups that can form such bonds), and thus more volatile and will also allow the reaction to move forward more quickly. * **If propionic acid, butyric acid and longer monocarboxylic acids are produced (see mixed acid fermentation), the amount of acidity produced per glucose consumed will decrease, as with ethanol, allowing faster growth.** ## Aerobic Respiration In aerobic respiration, the pyruvate produced by glycolysis is oxidized completely, generating additional ATP and NADH in the citric acid cycle and by oxidative phosphorylation. * However, this can occur only in the presence of oxygen. * Oxygen is toxic to organisms that are obligate anaerobes, and is not required by facultative anaerobic organisms. * In the absence of oxygen, one of the fermentation pathways occurs in order to regenerate NAD+; lactic acid fermentation is one of these pathways. ## Hydrogen Gas Production in Fermentation Hydrogen gas is produced in many types of fermentation (mixed acid fermentation, butyric acid fermentation, caproate fermentation, butanol fermentation, glyoxylate fermentation), as a way to regenerate NAD⁺ from NADH. Electrons are transferred to ferredoxin, which in turn is oxidized by hydrogenase, producing $H_2$. * Hydrogen gas is a substrate for methanogens and sulfate reducers, which keep the concentration of hydrogen low and favor the production of such an energy-rich compound. * But hydrogen gas at a fairly high concentration can nevertheless be formed, as in flatus. **As an example of mixed acid fermentation, bacteria such as **Clostridium pasteurianum** ferment glucose producing butyrate, acetate, carbon dioxide and hydrogen gas.** The reaction leading to acetate is: $C_6H_{12}O_6 + 4 H_2O → 2 CH_3COO¯ + 2 HCO_3¯ + 4 H^+ + 4 H_2$ Glucose could theoretically be converted into just $CO_2$ and $H_2$, but the global reaction releases little energy. ## Methane Gas Production in Fermentation Acetic acid can also undergo a dismutation reaction to produce methane and carbon dioxide: $CH_3COO¯ + H^+ → CH_4 + CO_2 ∆G° = -36 kJ/reaction$ This disproportionation reaction is catalysed by methanogen archaea in their fermentative metabolism. One electron is transferred from the carbonyl function (e¯ donor) of the carboxylic group to the methyl group (e¯ acceptor) of acetic acid to respectively produce $CO_2$ and methane gas. ## History of Fermentation The use of fermentation, particularly for beverages, has existed since the Neolithic and has been documented dating from: * 7000–6600 BCE in Jiahu, China * 6000 BCE in Georgia * 3150 BCE in ancient Egypt * 3000 BCE in Babylon * 2000 BCE in pre-Hispanic Mexico * 1500 BC in Sudan Fermented foods have a religious significance in Judaism and Christianity. The Baltic god Rugutis was worshiped as the agent of fermentation. ### Louis Pasteur Louis Pasteur (1822–1895) showed that fermentation is initiated by living organisms in a series of investigations. * In 1857, Pasteur showed that lactic acid fermentation is caused by living organisms. * In 1860, he demonstrated that bacteria cause souring in milk, a process formerly thought to be merely a chemical change, and his work in identifying the role of microorganisms in food spoilage led to the process of pasteurization. In 1877, working to improve the French brewing industry, Pasteur published his famous paper on fermentation, "Etudes sur la Bière", which was translated into English in 1879 as "Studies on fermentation". * He defined fermentation (incorrectly) as "Life without air", * He correctly showed that specific types of microorganisms cause specific types of fermentations and specific end- products ### Eduard Buechner Although showing fermentation to be the result of the action of living microorganisms was a breakthrough, it did not explain the basic nature of the fermentation process, or prove that it is caused by the microorganisms that appear to be always present. * Many scientists, including Pasteur, had unsuccessfully attempted to extract the fermentation enzyme from yeast. * Success came in 1897 when the German chemist Eduard Buechner ground up yeast, extracted a juice from them, then found to his amazement that this "dead" liquid would ferment a sugar solution, forming carbon dioxide and alcohol much like living yeasts. * Buechner's results are considered to mark the birth of biochemistry. * The "unorganized ferments" behaved just like the organized ones. * From that time on, the term enzyme came to be applied to all ferments. It was then understood that fermentation is caused by enzymes that are produced by microorganisms. * In 1907, Buechner won the Nobel Prize in chemistry for his work. ## Advances in Fermentation Advances in microbiology and fermentation technology have continued steadily up until the present. For example, in the late 1970s, it was discovered that microorganisms could be mutated with physical and chemical treatments to be higher-yielding, faster-growing, tolerant of less oxygen, and able to use a more concentrated medium. * Strain selection and hybridization developed as well, affecting most modern food fermentations. * Other approach to advancing the fermentation industry has been done by companies such as BioTork, a biotechnology company that naturally evolves microorganisms to improve fermentation processes. * This approach differs from the more popular genetic modification, which has become the current industry standard. ## Industrial Fermentation **Industrial fermentation is the intentional use of fermentation by microorganisms such as bacteria and fungi to make products useful to humans.** Fermented products have applications as food as well as in general industry. * Some commodity chemicals, such as acetic acid, citric acid, and ethanol are made by fermentation. * The rate of fermentation depends on the concentration of microorganisms, cells, cellular components, and enzymes as well as temperature, pH and for aerobic fermentation oxygen. Product recovery frequently involves the concentration of the dilute solution. * Nearly all commercially produced enzymes, such as lipase, invertase and rennet, are made by fermentation with genetically modified microbes. * In some cases, production of biomass itself is the objective, as in the case of baker's yeast and lactic acid bacteria starter cultures for cheese making. In general, fermentations can be divided into four types: * **Production of biomass (viable cellular material)** * **Production of extracellular metabolites (chemical compounds)** * **Production of intracellular components (enzymes and other proteins)** * **Transformation of substrate (in which the transformed substrate is itself the product)** These types are not necessarily disjoint from each other, but provide a framework for understanding the differences in approach. * The organisms used may be bacteria, yeasts, molds, animal cells, or plant cells. * Special considerations are required for the specific organisms used in the fermentation, such as the dissolved oxygen level, nutrient levels, and temperature. ## General Overview of Industrial Fermentation * In most industrial fermentations, the organisms are submerged in a liquid medium; * In others, such as the fermentation of cocoa beans, coffee cherries, and miso, fermentation takes place on the moist surface of the medium. There are also industrial considerations related to the fermentation process. * For instance, to avoid biological process contamination, the fermentation medium, air, and equipment are sterilized. * Foam control can be achieved by either mechanical foam destruction or chemical anti-foaming agents. * Several other factors must be measured and controlled such as pressure, temperature, agitator shaft power, and viscosity. An important element for industrial fermentations is scale up. * This is the conversion of a laboratory procedure to an industrial process. * It is well established in the field of industrial microbiology that what works well at the laboratory scale may work poorly or not at all when first attempted at large scale. * It is generally not possible to take fermentation conditions that have worked in the laboratory and blindly apply them to industrial-scale equipment. Although many parameters have been tested for use as scale up criteria, there is no general formula because of the variation in fermentation processes. * The most important methods are: * the maintenance of constant power consumption per unit of broth and * the maintenance of constant volumetric transfer rate. ## Phases of Microbial Growth When a particular organism is introduced into a selected growth medium, the medium is inoculated with the particular organism. Growth of the inoculum does not occur immediately, but takes a little while. This is the period of adaptation, called the lag phase. Following the lag phase, the rate of growth of the organism steadily increases, for a certain period—this period is the log or exponential phase. After a certain time of exponential phase, the rate of growth slows down, due to the continuously falling concentrations of nutrients and/or continuously increasing (accumulating) concentrations of toxic substances. This phase, where the increase of the rate of growth is checked, is the deceleration phase. After the deceleration phase, growth ceases and the culture enters a stationary phase or a steady state. * The biomass remains constant, except when certain accumulated chemicals in the culture lyse the cells (chemolysis) * Unless other microorganisms contaminate the culture, the chemical constitution remains unchanged. * If all of the nutrients in the medium are consumed, or if the concentration of toxins is too great, the cells may become scenescent and begin to die off. * The total amount of biomass may not decrease, but the number of viable organisms will decrease. ## Fermentation Medium The microbes used for fermentation grow in (or on) specially designed growth medium which supplies the nutrients required by the organisms. * A variety of media exists, but invariably contains a carbon source, a nitrogen source, water, salts, and micronutrients. * In the production of wine, the medium is grape must. * In the production of bio-ethanol, the medium may consist mostly of whatever inexpensive carbon source is available. ### Carbon Sources Carbon sources are typically sugars or other carbohydrates, although in the case of substrate transformations (such as the production of vinegar) the carbon source may be an alcohol or something else altogether. * For large scale fermentations, such as those used for the production of ethanol, inexpensive sources of carbohydrates, such as molasses, corn steep liquor, sugar cane juice, or sugar beet juice are used to minimize costs. * More sensitive fermentations may instead use purified glucose, sucrose, glycerol or other sugars, which reduces variation and helps ensure the purity of the final product. * Organisms meant to produce enzymes such as beta galactosidase, invertase or other amylases may be fed starch to select for organisms that express the enzymes in large quantity. ### Nitrogen Sources Fixed nitrogen sources are required for most organisms to synthesize proteins, nucleic acids and other cellular components. Depending on the enzyme capabilities of the organism, nitrogen may be provided as: * bulk protein, such as soy meal * pre-digested polypeptides, such as peptone or tryptone * ammonia or nitrate salts Cost is also an important factor in the choice of a nitrogen source. Phosphorus is needed for: * production of phospholipids in cellular membranes * production of nucleic acids The amount of phosphate which must be added depends upon the composition of the broth and the needs of the organism, as well as the objective of the fermentation. * For instance, some cultures will not produce secondary metabolites in the presence of phosphate. ### Growth Factors and Trace Nutrients Growth factors and trace nutrients are included in the fermentation broth for organisms incapable of producing all of the vitamins they require. * Yeast extract is a common source of micronutrients and vitamins for fermentation media. * Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and cobalt are typically present in unrefined carbon and nitrogen sources, but may have to be added when purified carbon and nitrogen sources are used. ### Foam Control Fermentations which produce large amounts of gas (or which require the addition of gas) will tend to form a layer of foam, since fermentation broth typically contains a variety of foam-reinforcing proteins, peptides or starches. * To prevent this foam from occurring or accumulating, antifoaming agents may be added. * Mineral buffering salts, such as carbonates and phosphates, may be used to stabilize pH near optimum. * When metal ions are present in high concentrations, use of a chelating agent may be necessary. ## Production of Biomass Microbial cells or biomass is sometimes the intended product of fermentation. Examples include: * single cell protein, * baker's yeast, * Lactobacillus, * E. coli, * and others In the case of single-cell protein, algae are grown in large open ponds which allow photosynthesis to occur. If the biomass is to be used for inoculation of other fermentations, care must be taken to prevent mutations from occurring. ## Production of Extracellular Metabolites Microbial metabolites can be divided into two groups: 1. **Those produced during the growth phase of the organism, called primary metabolites** 2. **Those produced during the stationary phase, called secondary metabolites.** **Examples of primary metabolites:** * ethanol * citric acid * glutamic acid * lysine * vitamins * polysaccharides **Examples of secondary metabolites:** * penicillin * cyclosporin A * gibberellin * lovastatin ## Primary Metabolites Primary metabolites are compounds made during the ordinary metabolism of the organism during the growth phase. * **A common example is ethanol or lactic acid, produced during glycolysis.** * **Citric acid is produced by some strains of Aspergillus niger as part of the citric acid cycle to acidify their environment and prevent competitors from taking over.** * **Glutamate is produced by some Micrococcus species, and some Corynebacterium species produce lysine, threonine, tryptophan and other amino acids.** All of these compounds are produced during the normal "business" of the cell and released into the environment. There is therefore no need to rupture the cells for product recovery. ## Secondary Metabolites Secondary metabolites are compounds made in the stationary phase; penicillin, for instance, prevents the growth of bacteria which could compete with Penicillium molds for resources. * Some bacteria, such as Lactobacillus species, are able to produce bacteriocins which prevent the growth of bacterial competitors as well. * These compounds are of obvious value to humans wishing to prevent the growth of bacteria, either as antibiotics or as antiseptics (such as gramicidin S). * Fungicides, such as griseofulvin are also produced as secondary metabolites. Typically secondary metabolites are not produced in the presence of glucose or other carbon sources which would encourage growth, and like primary metabolites are released into the surrounding medium without rupture of the cell membrane. ## Production of Intracellular Components Of primary interest among the intracellular components are microbial enzymes: * catalase * amylase * protease * pectinase * glucose isomerase * cellulase * hemicellulase * lipase * lactase * streptokinase * and many others. Recombinant proteins, such as: * insulin * hepatitis B vaccine * interferon * granulocyte colony-stimulating factor * streptokinase * and others are also made this way. The largest difference between this process and the others is that the cells must be ruptured (lysed) at the end of fermentation, and the environment must be manipulated to maximize the amount of the product. * Furthermore, the product (typically a protein) must be separated from all of the other cellular proteins in the lysate to be purified. ## Transformation of Substrate Substrate transformation involves the transformation of a specific compound into another, such as in the case of phenylacetylcarbinol, and steroid biotransformation, or the transformation of a raw material into a finished product, in the case of food fermentations and sewage treatment. ## Food Fermentation Ancient fermented food processes, such as making bread, wine, cheese, curds, idli, dosa, etc., can be dated to more than seven thousand years ago. *They were developed long before man had any knowledge of the existence of the microorganisms involved.* Some foods such as Marmite are the byproduct of the fermentation process, in this case in the production of beer. ## Ethanol Fuel Fermentation is the main source of ethanol in the production of Ethanol fuel. Common crops such as sugar cane, potato, cassava and corn are fermented by yeast to produce ethanol which is further processed to become fuel. ## Sewage Treatment In the process of sewage treatment, sewage is digested by enzymes secreted by bacteria. Solid organic matters are broken down into harmless, soluble substances and carbon dioxide. Liquids that result are disinfected to remove pathogens before being discharged into rivers or the sea or can be used as liquid fertilizers. *Digested solids, known also as sludge, is dried and used as fertilizer.* Gaseous byproducts such as methane can be utilized as **biogas** to fuel electrical generators. One advantage of bacterial digestion is that it reduces the bulk and odor of sewage, thus reducing space needed for dumping. The main disadvantage of bacterial digestion in sewage disposal is that it is a very slow process. ## Agricultural Feed A wide variety of agroindustrial waste products can be fermented to use as food for animals, especially ruminants. Fungi have been employed to break down cellulosic wastes to increase protein content and improve in vitro digestibility. ## Bioassay Bioassay (commonly used shorthand for biological assay or assessment), or biological standardization is a type of scientific experiment. * A bioassay involves the use of live animal or plant (in vivo) or tissue or cell (in vitro) to determine the biological activity of a substance, such as a hormone or drug. * Bioassays are typically conducted to measure the effects of a substance on a living organism and are essential in the development of new drugs and in monitoring environmental pollutants. * Both are procedures by which the potency or the nature of a substance is estimated by studying its effects on living matter. * A bioassay can also be used to determine the concentration of a particular constitution of a mixture that may cause harmful effects on organisms or the environment. ## Use of Bioassays Bioassays are procedures that can determine the concentration or purity or biological activity of a substance such as vitamin, hormone or plant growth factor by measuring the effect on an organism, tissue, cells, enzyme or receptor. Bioassays may be: * qualitative * quantitative ### Qualitative Bioassays Qualitative bioassays are used for assessing the physical effects of a substance that may not be quantified, such as seeds fail to germinate or develop abnormally deformity. **An example of a qualitative bioassay includes Arnold Adolph Berthold's famous experiment on castrated chickens. This analysis found that by removing the testicles of a chicken, it would not develop into a rooster because the endocrine signals necessary for this process were not available.** ### Quantitative Bioassays Quantitative bioassays involve estimation of the dose-response curve, how the response changes with increasing dose. That dose-response relation allows estimation of the dose or concentration of a substance associated with a specific biological response, such as the LC50 (concentration killing 50% of the exposed organisms). Quantitative bioassays are typically analyzed using the methods of biostatistics. ## Purpose of Bioassays 1. Measurement of the pharmacological activity of new or chemically undefined substances 2. Investigation of the function of endogenous mediators 3. Determination of the side-effect profile, including the degree of drug toxicity 4. Measurement of the concentration of known substances (alternatives to the use of whole animals have made this use obsolete) 5. Assessing the amount of pollutants being released by a particular source, such as wastewater or urban runoff. 6. Determining the specificity of certain enzymes to certain substrates. ## Types of Bioassays Bioassays are of two types: ### Quantal Bioassays A quantal assay involves an "all or none response". ### Graded Bioassays Graded assays are based on the observation that there is a proportionate increase in the observed response following an increase in the concentration or dose. The parameters employed in such bioassays are based on the nature of the effect the substance is expected to produce. * For example: contraction of smooth muscle preparation for assaying histamine or the study of blood pressure response in case of adrenaline. A graded bioassay can be performed by employing any of the below-mentioned techniques. The choice of procedure depends on: 1. The precision of the assay required 2. The quantity of the sample substance available 3. The availability of the experimental animals. ## Bioassay Techniques 1. Matching Bioassay 2. Interpolation Method 3. Bracketing Method 4. Multiple Point Bioassay (i.e.-Three-point, Four-point and Six Point Bioassay) 5. Divided bioassay ### Matching Bioassay Matching Bioassay: It is the simplest type of the bioassay. In this type of bioassay, response of the test substance taken first and the observed response is tried to match with the standard response. Several responses of the standard drug are recorded till a close matching point to that of the test substance is observed. A corresponding concentration is thus calculated. This assay is applied when the sample size is too small. Since the assay does not involve the recording of concentration response curve, the sensitivity of the preparation is not taken into consideration. Therefore, precision and reliability is not very good. ### Interpolation Bioassay Interpolation bioassay: Bioassays are conducted by determining the amount of preparation of unknown potency required to produce a definite effect on suitable test animals or organs or tissue under standard conditions. This effect is compared with that of a standard. Thus the amount of the test substance required to produce the same biological effect as a given quantity the unit of a standard preparation is compared and the potency of the unknown is expressed as a % of that of the standard by employing a simple formula. Many times, a reliable result cannot be obtained using this calculation. Therefore it may be necessary to adopt more precise methods of calculating potency based upon observations of relative, but not necessarily equal effects, likewise, statistical methods may also be employed. The data (obtained from either of assay techniques used) on which bioassay are based may be classified as quantal or graded response. Both these depend ultimately on plotting or making assumption concerning the form of DRC. ## Environmental Bioassays Environmental bioassays are generally a broad-range survey of toxicity. * A toxicity identification evaluation is conducted to determine what the relevant toxicants are. * Although bioassays are beneficial in determining the biological activity within an organism, they can often be time-consuming and laborious. * Organism-specific factors may result in data that are not applicable to others in that species. For these reasons, other biological techniques are often employed, including radio-immunoassays. ## Quality Control Quality control, or QC for short, is a process by which entities review the quality of all factors involved in production. ISO 9000 defines quality control as "A part of quality management focused on fulfilling quality requirements". This approach places an emphasis on three aspects: 1. Elements such as controls, job management, defined and well managed processes, performance and integrity criteria, and identification of records 2. Competence, such as knowledge, skills, experience, and qualifications 3. Soft elements, such as personnel, integrity, confidence, organizational culture, motivation, team spirit, and quality relationships. Controls include product inspection, where every product is examined visually, and often using a stereo microscope for fine detail before the product is sold into the external market. Inspectors will be provided with lists and descriptions of unacceptable product defects such as cracks or surface blemishes for example. The quality of the outputs is at risk if any of these three aspects is deficient in any way. Quality control emphasizes testing of products to uncover defects and reporting to management who make the decision to allow or deny product release, whereas quality assurance attempts to improve and stabilize production (and associated processes) to avoid, or at least minimize, issues which led to the defect(s) in the first place. For contract work, particularly work awarded by government agencies, quality control issues are among the top reasons for not renewing a contract. ## Shelf Life of Products Shelf life is the length of time that a commodity may be stored without becoming unfit for use, consumption, or sale. * In other words, it might refer to whether a commodity should no longer be on a pantry shelf (unfit for use), or just no longer on a supermarket shelf (unfit for sale, but not yet unfit for use). * It applies to cosmetics, foods and beverages, medical devices, medicines, explosives, pharmaceutical drugs, chemicals, car tires, batteries, and many other perishable items. * In some regions, an advisory best before, mandatory use by, or freshness date is required on packaged perishable foods. ## Background Shelf life is the recommended maximum time for which products or fresh (harvested) produce can be stored, during which the defined quality of a specified proportion of the goods remains acceptable under expected (or specified) conditions of distribution, storage and display. Most expiration dates are used as guidelines based on normal and expected handling and exposure to temperature. * Use prior to the expiration date does not guarantee the safety of a food or drug, and a product is not necessarily dangerous or ineffective after the expiration date. According to the USDA: "Canned foods are safe indefinitely as long as they are not exposed to freezing temperatures, or temperatures above 90 °F (32.2° C). If the cans look ok, they are safe to use. Discard cans that are dented, rusted, or swollen. High-acid canned foods (tomatoes, fruits) will keep their best quality for 12 to 18 months; low-acid canned foods (meats, vegetables) for 2 to 5 years 80 °F (27 °C)." ## "Sell By" Date "Sell by date" is a less ambiguous term for what is often referred to as an "expiration date". Most food is still edible after the expiration date. * A product that has passed its shelf life might still be safe, but quality is no longer guaranteed. * In most food stores, waste is minimized by using stock rotation, which involves moving products with the earliest sell by date from the warehouse to the sales area, and then to the front of the shelf, so that most shoppers will pick them up first and thus they are likely to be sold before the end of their shelf life. * This is important, as consumers enjoy fresher goods, and furthermore some stores can be fined for selling out of date products; most if not all would have to mark such products down as wasted, resulting in a financial loss. ## Factors that Influence Shelf Life Shelf life depends on the degradation mechanism of the specific product. Most can be influenced by several factors: * exposure to light * heat * moisture * transmission of gases * mechanical stress and * contamination by things such as micro-organisms. Product quality is often mathematically modelled around a parameter (concentration of a chemical compound, a microbiological index, or moisture content). ## Food Spoilage For some foods, health issues are important in determining shelf life. Bacterial contaminants are ubiquitous, and foods left unused too long will often be contaminated by substantial amounts of bacterial colonies and become dangerous to eat, leading to food poisoning. However, shelf life alone is not an accurate indicator of how long the food can safely be stored. * For example, pasteurized milk can remain fresh for five days after its sell-by date if it is refrigerated properly. * In contrast, if milk already has harmful bacteria, the use-by dates become irrelevant. The expiration date of pharmaceuticals specifies the date the manufacturer guarantees the full potency and safety of a drug. * Most medications continue to be effective and safe for a time after the expiration date. * A rare exception is a case of renal tubular acidosis purportedly caused by expired tetracycline. A study conducted by the U.S. Food and Drug Administration covered over 100 drugs, prescription and over-the-counter. The study showed that about 90% of them were safe and effective as long as 15 years past their expiration dates. Joel Davis, a former FDA expiration-date compliance chief, said that with a handful of exceptions - notably nitroglycerin, insulin and some liquid antibiotics - most expired drugs are probably effective. ## Shelf Life and Drug Manufacturers Shelf life is not significantly studied during drug development, and drug manufacturers have economic and liability incentives to specify shorter shelf lives so that consumers are encouraged to discard and repurchase products. One major exception is the Shelf Life Extension Program (SLEP) of the U.S. Department of Defense (DoD), which commissioned a major study of drug efficacy from the FDA starting in the mid-1980s. * One criticism is that the U.S. Food and Drug Administration (FDA) refused to issue guidelines based on SLEP research for normal marketing of pharmaceuticals even though the FDA performed the study. * The SLEP and FDA signed a memorandum that scientific data could not be shared with the public, public health departments, other government agencies, and drug manufacturers. * State and local programs are not permitted to participate. * The failure to share data has caused foreign governments to refuse donations of expired medications. One exception occurred during the 2010 Swine Flu Epidemic when the FDA authorized expired Tamiflu based on SLEP Data. The SLEP discovered