FE 462 BIOCHEMICAL ENGINEERING Introduction-A Little Microbiology Applications of Microbiology to Foods Ancient Egyptians used fermentation to produce beer and convert grape juice to wine. They also practiced the aerobic conversion of the alcohol in wine to the acetic acid of vinegar, and the leavening of bread. The present practices of using, for example, pectinases for enhanced release of fruit juices from tissue and amylases for the enzymatic modification of starches, are examples involving the indirect application of microorganisms to foods and food components. Applications of Microbiology to Foods The production of xanthan gum by the plant pathogenic bacterium Xanthomonas campestris for use as a viscosity agent in beverages and semisolid food products is an example of the use of an originally undesirable organism for the production of a desirable food and beverage additive. Applications of Microbiology to Foods The use of the mold Aspergillus niger to produce high yields of citric acid as a food and beverage acidulant was established in the 1920s and is a classic example of an initial surface culture process that was eventually converted to a submerged aerated process with the use of mutants. The Nature of Microorganisms Microorganisms can be found almost anywhere in the taxonomic organization of life on the planet. Bacteria and archaea are almost always microscopic, while a number of eukaryotes are also microscopic, including most protists, some fungi, as well as some animals and plants. Viruses are generally regarded as not living and therefore are not microbes, although the field of microbiology also encompasses the study of viruses. The Nature of Microorganisms All microorganisms are allocated to a specific group with respect to growth temperature. Obligate psychrophiles are defined as those organisms capable of growth at or near 0°C but not at 20°C. Such organisms usually have a maximum growth temperature of 15–17°C. Psychrotrophic organisms are capable of growth at or near 0°C but exhibit optimum growth at approximately 25°C and are frequently unable to grow at 30°C. Mesophiles exhibit growth from 20–45°C with an optimum growth temperature usually in the range of 30–35°C. Thermophiles exhibit growth in the range of 45–65°C. Hyperthermophiles are organisms from oceanic thermal vents and hot springs that are restricted to growth temperatures from 70–120°C. Hyperthermophiles have not yet been isolated from foods. Nutritional Requirements All biological systems, from microorganisms to man, share a set of nutritional requirements, which are:
1. Sources of energy
a. Phototrophs organisms which are capable of employing radiant energy.
b. chemotrophs organisms which obtain the energy for their activities and self- synthesis from chemical reactions that can occur in the dark.
2. Sources of carbon
a. autotrophs organisms which can thrive on an entirely inorganic diet, using CO2 or carbonates as a sole source of carbon.
b. heterotrophs organisms which cannot use CO2 as a sole source of carbon but require, in addition to minerals, one or more organic substances, such as glucose or amino acids, as sources of carbon.
3. Sources of nitrogen:
atmospheric nitrogen, inorganic nitrogen compounds, or other derived nitrogen.
4. Sources of sulfur and phosphorus:
elementary sulfur, inorganic sulfur, or organic sulfur.
5. Sources of metallic elements:
sodium, potassium, calcium, magnesium, manganese, iron, zinc, copper, and cobalt.
6. Sources of vitamins.
Physical Conditions After determining the proper nutrients for the cultivation of bacteria, it is necessary to determine the physical environment in which the organisms will grow best. Three major physical factors to be taken into consideration are temperature, the gaseous environment, and pH. Since microbial activity and growth are manifestations of enzymatic action, and since the rates of enzyme reactions increase with increasing temperatures, the rate of microbial growth is temperature dependent. Depending on the temperature range over which they grow, bacteria are called psychrophiles, mesophiles, or thermophiles. Physical Conditions The principal gases in the cultivation of bacteria are oxygen and carbon dioxide. There are four types of bacteria, according to their response to oxygen: 1. Aerobic bacteria grow in the presence of free oxygen. 2. Anaerobic bacteria grow in the absence of free oxygen. 3. Facultatively anaerobic bacteria grow in either the absence or the presence of free oxygen. 4. Microaerophilic bacteria grow in the presence of minute quantities of free oxygen. For most bacteria the optimum pH for growth lies between 6.5 and 7.5. Although a few bacteria can grow at the extremes of the pH range, for most species the minimum and maximum limits fall somewhere between pH 4 and pH9. Culture Media The growth of microbial population in artificial environments is called cultivation. A culture that contains only one kind of microorganism is a pure culture. A mixed culture is one that contains more than one kind of microorganism. The necessary steps for cultivating microorganisms are: 1. Preparing a culture medium in which a microorganism can grow best. 2. Sterilizing in order to eliminate all living organisms in the vessel. 3. Inoculating the microorganism in the prepared medium. Sterilization After a suitable culture medium is selected for the cultivation of a specific microorganism, it is poured into a culture vessel. If you use test tubes or flasks as your culture vessel, the ends of test tubes or flasks should be covered with a suitable closure to allow for the exchange of gases with the atmosphere, yet to keep foreign organisms out of the media. Various types of closures are used in the modern laboratory including cotton plugs, plastic foam, screw caps, metal caps, and aluminum foil. Sterilization The medium is then sterilized to eliminate all living organisms in the vessel,-The most common method of sterilization is by moist heat (steam under pressure) in an autoclave. Generally, the autoclave is operated at approximately 15 psi at 121°C. The time of sterilization depends on the nature of the material, the type of container, and the volume. For example, test tubes of liquid media can be sterilized in 15 to 20 minutes at 121°C. Inoculation Inoculation is the seeding of a culture vessel with the microbial material (inoculum). The inoculum is introduced with a metal wire or loop which is rapidly sterilized just before its use by heating it in a flame. Transfers of liquid culture are often made by using a sterilized pipette. The inoculation is usually done in a laminar flow hood to minimize the risk of contamination. It is important to know proper pipetting techniques for inoculating or sampling during cultivation. CELL GROWTH MEASUREMENT In any biological system, growth can be defined as the orderly increase of all chemical components. To follow the course of growth, it is necessary to make quantitative measurements. Cell growth can be determined by measuring cell number, cell mass, or cell activity. Measurement of Cell Number Microscopic Counts: The number of cells in a population can be measured under a microscope by counting cells placed in special counting chambers. CELL GROWTH MEASUREMENT Viable Plate Count: A viable cell is defined as one that is able to divide and form a colony. CELL GROWTH MEASUREMENT Measurement of Cell Mass Cell Dry Weight: Cell dry weight can be measured directly by taking an aliquot of cell suspension and centrifuging it. After the supernatant is discarded, the cells are thoroughly washed with distilled water to eliminate all soluble matter. The suspension is recentrifuged and the settled cells are dried in an oven and weighed. Turbidity: The cell mass can be measured optically by determining the amount of light scattered by a suspension of cells. The technique is based on the fact that small particles scatter light proportionally, within certain limits, to their concentration. CELL IMMOBILIZATION As in the case of enzymes, whole cells can be immobilized for several advantages over traditional cultivation techniques. By immobilizing the cells, process design can be simplified since cells attached to large particles or on surfaces are easily separated from product stream. This ensures continuous fermenter operation without the danger of cell washout. Immobilization can also provide conditions conducive to cell differentiation and cell-to-cell communication, thereby encouraging production of high yields of secondary metabolites. Immobilization can protect cells and thereby decrease problems related to shear forces.