Food Biotechnology Lecture Notes PDF

Biotechnological Applications In Food Industry Dr. Eman Owis Lecturer Of Microbial Biotechnology – Mansoura Uni P h. D. G ö t t i n g e n U n i - G e r m a n y [email protected]  Food b...

Biotechnological Applications In Food Industry Dr. Eman Owis Lecturer Of Microbial Biotechnology – Mansoura Uni P h. D. G ö t t i n g e n U n i - G e r m a n y [email protected]  Food biotechnology Nanotechnology in Microorganisms Fermentation Genetically Modified Enzymes in food Introduction agriculture and food associated with food Biotechnology Food industry industry & Bioethics Bacteria, yeast and Applications of Definition Introduction Genetic Engineering Introduction molds nanotechnology Production of food Ethical aspects of food Branches of Factors influencing Fermenter DNA, RNA, Peotein enzymes from and agricultural biotechnology microbial activity microorganisms biotechnology Benefits of Importance of Molecular Biology Different enzymes in Types of bacteria in the food biotechnology industry fermentation techniques food industry Food biotechnology Importance of yeasts safety and regulations in foods Different techniques associated with food biotechnology  Outputs of this Lecture The Process of Fermentation (Principles & Bases). Principles of fermenters or bioreactors. Principles of down streaming. General concept of fermentation: -The Old Concept: It is a vital anaerobic process in the presence of yeast until organic matter is converted into simpler and more important materials. - The Modern Concept: Is the use of microorganisms or a product of the microorganism (enzymes) to convert a complex organic compound medium into a simpler material of interest to the human race or to create a complex substance used to improve or increase the quality of life.  Object Object Fermentation Products Fermentation Products Applications Applications  Primary products Secondary According to the object products Enzymes Applications Medical Chemicals Agriculture Fuel Food Environment Proteins Inorganic Green Manure Biofuel Dairy products Solid Waste recycling Alternate The antibiotics Organic Fodder Beverages Waste recycling Vitamins Sewage Treatment Organic acids Toxins An overview of a typical industrial fermentation process Difference between FERMENTER AND BIOREACTOR Parameter Fermenter/ Microbial Bioreactor Mammalian Cell Bioreactor Growth and maintenance of Growth and maintenance of Use for bacterial and fungal cell. Mammalian cell. Size Large Small Growth Rate Few days to week Several weeks pH Addition of acids and bases Addition of gases (CO2 Cylinder) rpm 800 or more 150 or less Antifoam agents, Sensor, Foam Not common Peristaltic pump Condition Aerobic and Anaerobic Aerobic only Basic design of fermenter Parts of a fermentor 1. Impeller (agitator): To stir the media continuously and hence prevent cells from settling down and distributing oxygen throughout the medium. 2. Sparger (aerator): Introduce sterile oxygen to the media in case of aerobic fermentation process 3. Baffles (vortex breaker): Disrupt vortex and provide better mixing 4. Inlet Air filter: Filter air before it enters the fermenter 5. Exhaust Air filter: Trap and prevent contaminants from escaping 6. Rota meter: Measure the flow rate of Air or liquid 7. Pressure gauge: Measure pressure inside the fermenter 8. Temperature probe: Measure and monitor change in temperature of the medium during the process 9. Cooling jacket: To maintain the temperature of the medium throughout the process 10. pH probe: Measure and monitor the pH of the medium 11. Dissolved oxygen probe: Measure dissolved oxygen in the fermenter 12. Level probe: Measure the level of the medium 13. Foam probe: Detect the presence of the foam 14. Sampling point: To obtain samples during the process 15. Valves: Regulates and controls the flow of liquids and gases Types of Fermentation Process Hence, the fermenters can be divided according to the method of fermentation (reactor nature) 1- Batch reactors. The simplest type. Reactor is filled with medium and the fermentation is allowed. Fermentation has finished, and contents are emptied for downstream processing. The reactor is then cleaned, re-filled, and re-inoculated and the fermentation process starts again. 2- Continuous reactors. The fresh media is continuously added and bioreactor fluid is continuously removed. As a result, cells continuously receive fresh medium and products and waste products and cells are continuously removed for processing. The reactor can thus be operated for long periods of time without having to be shut down. Many times more productive than batch reactors. The growth rate of the organism in the reactor can be more easily controlled and optimized. Cells can also be immobilized in continuous reactors, to prevent their removal (semi-continuous). 3- Fed batch reactor: Most common type of reactor used in industry. Fresh media is continuous or sometimes periodically added. The fermentation triangle in which the bio-reactor, the biocatalyst, and purification represent the three sides of the triangle, and the surface of the triangle occupied by the raw material (Media) where the integration of the four previous inputs is necessary to complete the fermentation process itself. Raw material Down stream Factors that Influence Growth Phile vs. Tolerance – “Growth” is generally used to refer to the acquisition of biomass leading to cell division, or reproduction – Many microbes can survive under conditions in which they cannot grow – The suffix “-phile” is often used to describe conditions permitting growth, whereas the term “tolerant” describes conditions in which the organisms survive, but don’t necessarily grow – For example, a “thermophilic bacterium” grows under conditions of elevated temperature, while a “thermotolerant bacterium” survives elevated temperature, but grows at a lower temperature  Obligate (strict) vs. facultative – “Obligate” (or “strict”) means that a given condition is required for growth – “Facultative” means that the organism can grow under the condition, but doesn’t require it – The term “facultative” is often applied to sub-optimal conditions – For example, an obligate thermophile requires elevated temperatures for growth, while a facultative thermophile may grow in either elevated temperatures or lower temperatures The Requirements for Growth Physical requirements – Temperature – pH – Light – Radiation – Water – Osmotic pressure Chemical requirements – Carbon – Nitrogen, sulfur, and phosphorous – Trace elements – Oxygen – Organic growth factor Temperature Optima – Microbial cells cannot control their temperature, so they assume the ambient temperature of their natural habitat – The range of temperatures for the growth of a given microbial species can be defined by three cardinal thermal points: a minimum, optimum, and maximum growth temperature Optimum growth temperature is usually near the top of the growth range Death above the maximum temp. comes from enzyme inactivation Mesophiles most common group of organisms 40ºF (5°C) slows or stops the growth of most microbes e.g. Growth temperature (cardinal ) The minimum growth temperature is the lowest temperature at which a species will grow The optimum growth temperature is the temperature at which it grows best The maximum growth temperature is the highest temperature at which growth is possible. Effects of Temperature on Growth Typical Growth Rates and Temperature Oxygen Requirements Oxygen is essential for obligate aerobes (final electron acceptor in ETC). Oxygen is deadly for obligate anaerobes. How can this be true? – Neither gaseous O2 nor oxygen covalently bound in compounds is poisonous …they are nonreactive. – The forms of oxygen that are toxic are highly reactive oxidizing agents. – They do damage to cells by oxidizing compounds such as proteins and lipids. Oxygen Requirements Culture Medium ❖ Obligate Anaerobes Forms an oxygen Killed by oxygen gradient More Oxygen on top Clostridium spp. ❖ Obligate Aerobes ❖ Aerotolerant Anaerobes Aerobic only (O2) Growth not affected by Oxygen Mycobacterium tuberculosis Enterococcus faecalis ❖ Facultative Anaerobes ❖ Microaerophiles Aerobic Primarily aerobic Alternatively anaerobic Killed by high (20%) O2 concentration Escherichia coli Campylobacter spp. Classification of Organisms Based on Oxygen Requirements Aerobes – undergo aerobic respiration. Anaerobes – do not use aerobic metabolism. Facultative anaerobes – can maintain life via fermentation or anaerobic respiration or by aerobic respiration. Aerotolerant anaerobes – do not use aerobic metabolism but have some enzymes that detoxify oxygen’s poisonous forms. Microaerophiles – aerobes that require oxygen levels from 2-10% and have a limited ability to detoxify hydrogen peroxide and superoxide radicals.  The Effect of Oxygen (O2) on Growth Grows best in oxygen, but can Only grows without Grows with or Grows in low conc. Needs oxygen grow without oxygen without oxygen of oxygen Chemical requirements Microbial Nutrition (Media) A. Nutrient Requirements B.Culture Media Microbiological Media Preparations devised to support the growth (reproduction) of microorganisms Can be liquid or solid Chemically defined vs. complex media – Chemically defined media The exact chemical composition is known – Complex media Exact chemical composition is not known Often consist of plant or animal extracts, such as soybean meal, milk protein, etc. Include most routine laboratory media, The Common Nutrient Requirements Water Macroelements (macronutrients) required in relatively large amounts – C, N – O, H, S, P, K, Ca, Mg, and Fe Micronutrients (trace elements) often supplied in water or in media components – Mn, Zn, Co, Mo, Ni, and Cu – required in trace amounts – Growth Factors (Special requirements) Amino acids are needed for protein synthesis, purines and pyrimidines for nucleic acid synthesis. Vitamins are small organic molecules that usually make up all or part enzyme cofactors, and only very small amounts are required for growth. Growth – “Growth” is generally used to refer to the acquisition of biomass ) irreversible( leading to cell division, or reproduction – There are two important factors for determining the living state of the organism: * Generation time: Time is needed to multiply the number of cells. * Multiplication time: Time to multiply mass (weight). – Many microbes can survive under conditions in which they cannot grow  Growth in Batch Culture (A) For Unicellular organisms A “batch culture” is a closed system in broth medium in which no additional nutrient is added after inoculation of the broth. Typically, a batch culture passes through four distinct stages: Lag stage Logarithmic stage Stationary stage Death stage Standard Growth Curve e.g. Bacterial Growth Bacterial division occurs according to a logarithmic progression (two cells, four cells, eight cells, and so on). Generation or Doubling Time Time required for a cell to divide or a population to double in number. Highly variable, 20 minutes to 24 hours. ❑ Escherichia coli 20 minutes ❑ Mycobacterium tuberculosis 12 hours Escherichia coli’s generation time 32 16 8 4 2 1 0 20 40 60 80 100 120 140 Minutes Logarithmic growth  Bacterial Growth Curve Number of bacteria Lag phase Time During the lag phase, there is little or no change in the number of cells, but metabolic activity is high.  Bacterial Growth Curve Number of bacteria Log Phase Time During the log phase, the bacteria multiply at the fastest rate possible under the conditions provided.  Bacterial Growth Curve Number of bacteria Stationary Phase Time During the stationary phase, there is an equilibrium between cell division and death.  Bacterial Growth Curve Number of bacteria Death Phase Time During the death phase, the number of deaths exceeds the number of new cells formed. Phases of Growth 1. Lag Phase Considered as the adjustment period when the organism adapts to new surroundings cells are very active metabolically No Microbial growth Synthesize enzymes to adapt to the environment Recovery from stress or injury This period may be extended in unfavorable environments. In extreme cases, the lag phase can last for weeks Factors Influencing the Lag Phase Chemical composition of the fermentation media influences the length of the lag phase. Longer lag phase is observed if the inoculum is transferred into a fresh medium of different carbon source. Age of the inoculum. If the inoculum is in exponential growth phase, it will exhibit shorter lag in the fresh medium. Concentration of the inoculum. Viability and morphology of the inoculum. 2. Log Phase (Exponential) Rapid cell growth (exponential growth) Population doubles every generation Microbes are sensitive to adverse conditions – antibiotics – anti-microbial agents Growth is stable Growth rate is constant for a given bacteria under specified conditions Catabolic processes generate energy Anabolic processes build cell structures 3. Stationary Phase Cells begin to encounter environmental stress Over time, essential nutrients become depleted or waste products build up to toxic levels so that logarithmic phase ceases and results in stationary phase No net growth in stationary phase (cell ‘replacing’ but number not increasing (Death rate = rate of reproduction) Cell functions such as energy metabolism may continue The specific growth rate of the microorganism continues decelerating until the substrate is completely depleted. Microorganisms are still metabolically active, metabolizing intracellular storage compounds, utilizing nutrients released from lysed cells and in certain cases produce secondary metabolites. 4. Death Phase Due to limiting factors in the environment Death exceeds division (Death rate > rate of reproduction) Viable cell count decreases Under certain circumstances cell death is accompanied by cell lysis (B) Growth in Filamentous organisms Growth on solid media Lag stage Rapid growth stage Retarded growth stage (B) Growth in Filamentous organisms Growth on Liquid media Lag stage accelerated stage Stationary stage Death stage