Media Preparation_Food Fermentation.pptx

Media Preparation_Food Fermentation

1. Fermentation Media

2. Introduction and Essentials of Media Composition Microorganisms used in lab- and industrial-scale fermentation are as diverse as the ingredients that can be used to support their growth. The challenge for fermentation scientists is to understand the requirements of the microorganism at hand and to design and develop the most suitable growth environment while minding such considerations as the product to be generated, the economics and suitability of the medium ingredients for scale-up, and the ultimate market of the product. Industrial microorganisms fall into four primary categories based on their mode of energy utilization: 1. Photoautotrophs: light as energy source, CO 2 as carbon (C) source; higher plants, most eukaryotic algae, some photosynthetic bacteria 2. Photoheterotrophs: light as energy source, organic C source; purple non-S photosynthetic bacteria, a few eukaryotic algae 3. Chemoautotrophs: chemical energy sources, CO 2 as C source (reduction of inorganic compounds as energy sources); only bacteria 4. Chemoheterotrophs: chemical energy sources, organic C source; most diverse— fungi, bacteria, some algae

3. Fermentation media can be crude or refined, complex or defined, and costly or inexpensive. No matter the process, the fundamental building blocks of biological molecules include C, nitrogen (N), oxygen (O), hydrogen (H), phosphorus (P), and sulfur (S). Chemotrophic microorganisms must obtain these elements, plus important cofactors like trace minerals, from their surrounding environment. Oxygen is largely accessed from the atmosphere or through supplementation of the growth medium with pure oxygen. The remaining elements (Table 2.1) are supplied by the growth medium. The exact chemical composition of cells varies from species to species and can be influenced by culture conditions.

4. The polysaccharide content of microalgae, for example, can vary widely depending on environmental conditions. Table 2.2 shows a typical chemical composition of Escherichia coli on a dry cell weight basis, with over 70% of the cell’s wet weight comprised of water. The key objective when developing fermentation media is to supply the microorganism with all required elements, in the proper balance and in accessible forms, for optimal growth, function, and production. Because of the wide array of sources and combinations, a rational approach to designing, developing, and optimizing fermentation media is important. The main inputs into fermentation media include sources of C and N, trace metals and cofactors, and other process-specific compounds such as antifoam and inducers. Optimizing medium formulations, with an eye on the final production scale and target market, is an additional step toward developing robust, scalable, and economical fermentation processes.

5. C Sources Microorganisms utilize C for both biosynthesis and energy generation. An adequate supply of C is vital for microbial growth and product formation. The choice of a suitable C source is influenced by various factors like the microorganism’s metabolism, biomass yield per unit of substrate, cost, availability of the source, and type of final product (e.g., whole cell, secondary metabolite, or native or recombinant protein). Defined C sources include simple sugars like glucose, fructose, mannose, galactose, sucrose, and xylose; polysaccharides such as starch; and lipids including oleates and glycerides. Peptones (digested proteins such as casein or soy), skimmed milk powder, molasses, corn steep liquor (CSL), and malt syrups are few of the complex C sources used in industry. The majority of the complex C sources also serve as a source of N and other nutrients essential for microbial growth. Less traditional sources like methane (CH 4 ), methanol (CH 3 OH), and CO 2 are also utilized in some types of microbial fermentations. Fats and oils can also serve as C sources, alone or in combination with defined or complex sources. For example, the medium used for lipase production by Candida rugosa contains both glucose and olive oil.

6. N Sources Nitrogen is a critical component in fermentation media, serving as a key constituent of nucleic acids, proteins, and co-enzymes such as vitamins. Some microorganisms, including most photosynthetic organisms and some bacteria and fungi, can access N from oxidized, inorganic sources such as NaNO 3 . Others, including industrial workhorses such as E. coli , require a reduced N source which can be supplied as ammonia salts or through complex sources including yeast extracts and peptones.

7. The Role of Water in Media With few exceptions, water comprises the vast majority of any growth medium. The mineral and salt content and pH of the water may influence the fermentation process. For food-grade fermentations, municipal water is routinely used. Some processes may require treatment of water by passing through sediment- and micro-filters and a UV chamber to eliminate physical and microbial contaminants present in the water. Reverse osmosis (RO) water is most frequently used in laboratory-scale fermentations. If a microorganism/process is sensitive to components present in the water, then deionized water may be used. Upon scale-up, the type of water can vary depending on the capabilities of the production facility. Some facilities may have large-scale RO systems, some may only filter municipal water across a large-bore membrane to remove particulates, and some may not treat city water in any way. In some low-margin processes like fuel ethanol fermentations, water may be recovered from DSP (e.g., distillation) and reused during fermentation.

8. Mineral Elements and Their Roles in Microbial Growth Ecologically, minerals and microorganisms interact intimately. For example, a lack of Fe in the animal gut could reduce the amount of propionate and butyrate produced by intestinal bacteria, whereas a high level of Fe could promote the development of pathogenic microflora. The presence of Mg on the other hand could improve the thermotolerance of probiotic bacteria L. rhamnosus GG , L. casei Zhang , and L. plantarum P-8 . Conversely, microorganisms play key geoactive roles in the biosphere, particularly in the areas of element bio-transformations and bio-geochemical cycling, metal and mineral transformations, decomposition, bio-weathering, and soil and sediment formation. Regarding the microorganisms themselves, certain minerals play important and indispensable roles in their metabolism and growth , and thus, these minerals must be available in the growth medium. For the preparation of fermentation media, it should be determined whether essential minerals are present in sufficient quantities, as is often the case with complex media, or must be individually added. Some trace minerals are frequently present in appropriate quantities in the water supply and as impurities in other media ingredients, reducing or eliminating the need for their specific addition.

9. Macro Minerals P, S, K, Ca, Mg, and Fe are the six main macro mineral elements required by microorganisms. P is important for energy transduction and is a component of phospholipids, proteins, and nucleic acids. S is a component of some amino acids (methionine and cysteine) and vitamins and also serves as a cofactor. K, Ca, and Mg exist in the cells as cations. K+ is needed for the activity of a number of enzymes and for ionic balance in yeast and fungi. Ca 2+ is a cofactor for enzymes such as proteases, is required for the heat resistance of bacterial endospores, and has a possible messenger role in yeast and fungi. Mg 2+ is needed as a cofactor for many enzymes, ATP complexes, and ribosome and cell membrane stabilization. Fe 2+ and Fe 3+ are components of cytochromes and cofactors for enzymes and electron-carrying proteins. Na+ and Cl− are major cations and anions in the cell and are thought to have a role in osmoregulation; however, despite their presence at high concentrations in many media, they are often excluded from the cell, being necessary only in micro-molar concentrations. Macro minerals are often supplied through complex ingredients. S is often supplied as SO 4 −2 together with another desired metal, e.g., ZnSO 4 .

10. P is commonly supplied in the form of PO 4 −3 and may serve a dual purpose along with pH buffering. K is often supplied as a phosphate salt or as KCl . Ca is often supplied as CaCl 2 and occasionally as CaSO 4 or CaCO 3 . Macro minerals may be required in higher concentrations when directly required for the synthesis of the desired fermentation product. For example, the synthesis of methionine by certain overproducing strains requires high concentrations of S in the medium. Bioavailability also needs to be considered for both macro and micro minerals that may be unavailable due to precipitation, chelation, or absorption. Precipitation of media components is the most common limitation of bioavailability, hampering the adequate supply of nutrients or interfering with the fermentation process and the monitoring devices. Precipitates can also affect DSP and purification operations.

11. Precipitation occurs when non-soluble complexes of divalent metal ammonium phosphates, magnesium phosphates, and other metal phosphates are formed. An approach to minimizing the formation of insoluble complexes is to sterilize problematic components separately. For example, a sterile solution of MgSO 4 is often added to PO 4 −3 -containing media post-sterilization to prevent the formation of NH 4 MgPO 4 .6H 2 O, which is highly insoluble. It is important to balance the relative concentration of different anions (PO 4 −3 , SO 4 −2 and Cl−) and cations (Na + , Ca2+, NH4+, and Mg2+) present in the medium. Precipitation may also form during the fermentation process due to the production of organic acids and CO 2 .

12. Micro Minerals Mn, Zn, Co, Mo, Ni, and Cu are often cofactors of enzymes, and they are functionally involved in the catalysis of reactions and maintenance of protein structure. These elements are generally required in the micro-molar range. Mn2+ helps many enzymes catalyze the transfer of PO 4 −3 groups. Zn 2+ is present at the active site of some enzymes. Co2+ is a component of vitamin B 12. Mo 2+ is required for N fixation. Ni 2+ is necessary for urease activity and is reportedly required for the growth of a bacterium ( Alcaligenes ( Hydrogenomonas ) eutrophus ), a cyanobacterium ( Oscillatoria ), and a green alga ( Chlorella vulgaris ), but its exact function is not clear. Cu is used as an electron carrier. As with macro minerals, the addition of micro minerals becomes more significant when they are required for the synthesis of certain fermentation products. For example, Zn is particularly important in alcohol fermentations, as it is a cofactor of alcohol dehydrogenase. Fermentation medium deficient in Zn may support growth, but the production of alcohol will be impaired. Furthermore, microorganisms from different environments may require special mineral elements that reflect the characteristics of their niches. For the growth of marine microbes, sea salt is frequently added to the media. Trace levels of toxic minerals (e.g., Ag, As, Ba, Cs, Cd, Hg, Li, Pb) which may be present in complex ingredients or even water may adversely affect growth of many microbes.

13. Growth Factors and Their Roles in Microbial Growth Growth factors are biologically active molecules such as amino acids, vitamins, purines, and pyrimidines. Amino acids are necessary for protein synthesis; purines and pyrimidines are the basis for DNA and RNA synthesis; and vitamins have diverse functions including as enzyme cofactors and precursors. Growth factors are classified as essential or accessory. Essential growth factors must be added to media for microorganisms incapable of synthesizing them; if they are absent, then growth cannot occur. Accessory growth factors are added to stimulate the rate and/or density of growth, despite the microorganisms possessing the synthesis capability. Some microorganisms can synthesize all growth factors such as nonsporiferous bacteria (i.e., Pseudomonas and Mycobacterium ) and many molds (i.e., Aspergillus and Penicillium ), and thus these microorganisms can grow on minimal media. However, the addition of even small amounts of growth factors can exert an effect on certain microorganisms and thus need to be taken into consideration in the design of fermentation media.

14. For the growth factors, several B group vitamins are described as essential, while others were stimulatory under certain growth conditions. A number of amino acids are described as essential (e.g., valine, leucine, isoleucine, methionine) or stimulatory for different strains of Lactococcus. Nucleotides are not essential; however, their addition to fermentation media stimulates growth. Acetic and lipoic acids can be essential or stimulatory under certain conditions, particularly in the absence of biotin. It is worth keeping in mind that a microorganism’s requirement for a growth factor can change depending on the C and N sources provided and on growth conditions such as aerobic or anaerobic environments.

15. The Role of Precursors in Fermentation Precursors are substances added prior to or during fermentation that are used in the synthesis of the fermentation product of interest. Precursors stimulate the synthesis of the product, increase the yield, or improve the quality of the product and may be provided in complex media or added as a pure compound. Manipulating microbial machinery through the addition of an alternative precursor is a frequently used technique that can give rise to alternative products. The production of β-lactam antibiotics has been optimized over many decades, with modern manufacturing delivering precursors continuously throughout fermentation. Natural penicillins are produced by Penicillum spp . in the absence of a side-chain precursor. However, a greater variety of penicillins (>100), and better control of specific penicillin, can be produced by adding a side-chain precursor. The most commonly manufactured penicillins , penicillin G and penicillin V are produced when phenylacetic acid and phenoxy acetic acid is added to the fermentation medium, respectively.

16. Metabolic Regulators and Their Roles in Fermentation Microbial regulation by metabolites is tightly controlled and critical to their efficient use of resources, preventing energy spent on futile production of unnecessary metabolites. These regulatory mechanisms can be manipulated or overcome through media design to enable high yield of desirable products. These metabolic regulators are classified as inhibitors, inducers, or enhancers. Metabolic inhibitors dampen or turn off a metabolic pathway and must be absent from the medium in order for the pathway to be activated. Inhibitors can be useful for turning off alternative pathways and allowing activity to be redirected to the pathway of interest. In addition, inhibitors can be used to avoid production of undesirable metabolites, such as those that impact on flavor of food and drink products. Inducers act to turn on the desired pathway and thus are essential for production of a compound under the control of the pathway. In certain scenarios, more than one inducer or structural analogue of the metabolite may be available, enabling selection of the most economical metabolite. Distinct from inducers, enhancers work to increase flux through a pathway as opposed to turning a pathway on.

17. Metabolic regulators are integral to industrial production of pectinases by bacteria and fungi. Pectinases hydrolyze pectin, a significant component of fruits, and are extensively used in the food industry, for example, in the clarification of fruit juice. Numerous publications describe complex media that induce pectinase synthesis due to the presence of pectinaceous substances, e.g., orange peel, wheat bran, and rice husk. In contrast, the use of some simple sugars including arabinose, glucose, and galactose, is reported to inhibit pectinase synthesis, likely through catabolite repression.

18. Antifoams Submerged fermentation of microorganisms generally starts in flasks, tubes, or microwell plates, where initial process development and strain selection can be accomplished in a relatively high-throughput manner. When growth is scaled to bioreactors, it is important to consider other factors that are absent or insignificant in smaller volumes. Not least of these considerations is foaming. Agitation, particularly in aerobic systems, can be high, with impeller tip speeds exceeding 3 m/s. In combination with aeration delivered through a sparger, considerable turbulence is present in a stirred-tank vessel. In conjunction with the nature of cell growth and many fermentation media, these physical conditions can lead to foaming. Foaming is challenging in several ways. Uncontrolled foaming during fermentation can lead to physical loss of microbial containment and product. It may also result in dangerous overpressure conditions if the exhaust path is blocked by foam fouling the exhaust filter. Foaming itself can cause the denaturation of proteins, which is particularly problematic if the product of interest is a secreted protein.

19. Why sterilization A fermentation product is produced by the culture of a certain organism, or animal cell line, in a nutrient medium. If a foreign microorganism invades the fermentation, then the following consequences may occur: 1. The medium would have to support the growth of both the production organism and the contaminant, resulting in a loss of productivity. 2. If the fermentation is a continuous one then the contaminant may “outgrow” the production organism and displace it from the fermentation. 3. The foreign organism may contaminate the final product, for example, single cell protein where the cells, separated from the broth, constitute the product. 4. The contaminant may produce compounds that make subsequent extraction of the final product difficult. 5. The contaminant may degrade the desired product; this is common in bacterial contamination of antibiotic fermentations where the contaminant would have to be resistant to the normal inhibitory effects of the antibiotic and degradation of the antibiotic is a common resistance mechanism, for example, the degradation of β-lactam antibiotics by β-lactamase-producing bacteria. 6. Contamination of a bacterial fermentation with phage could result in the lysis of the culture.

20. Avoidance of contamination may be achieved by: 1. Effective design and construction of the fermentation plant. 2. Using a pure inoculum to start the fermentation 3. Sterilizing the medium to be employed. 4. Sterilizing the fermenter vessel. 5. Sterilizing all materials to be added to the fermentation during the process, for example, air, nutrient feeds, antifoams, and pH titrants. 6. Maintaining aseptic conditions during the fermentation. 7. Putting in place detailed operating procedures for sterilization, aseptic maintenance, and staff training.

21. The control (inhibition) of microbial growth The control or prevention of microbial growth is necessary in many practical situations, particularly in health care, food processing and preparation, and in the preservation of materials. Control may be achieved using physical or chemical agents that either kill microorganisms or inhibit their further growth. Agents which kill cells are called ‘- cidal ’ agents, whereas ‘-static’ agents inhibit the growth of cells without killing them. Some agents affect groups of organisms; the term bactericidal, for example, refers to killing bacteria and bacteriostatic refers to inhibiting the growth of bacterial cells. Sterilization procedures completely destroy or eliminate all viable organisms, including spores, and may be performed using heat, radiation and chemicals, or by the physical removal of cells.

22. Microorganisms are not killed instantly on exposure to a lethal agent. The population decreases exponentially, by a constant fraction at constant intervals, and several factors influence the effectiveness of any antimicrobial treatment. These include: 1 population size: the larger the microbial population, the longer the time required to kill all the microorganisms present; 2 population composition: different microorganisms vary in their sensitivity to a specific lethal agent, and its effectiveness may be influenced by their age, morphology and physiological condition; 3 concentration of the antimicrobial agent or intensity of the treatment: higher concentrations or greater intensities are generally more efficient, but the relationship is rarely linear;

23. 4 period of exposure to the lethal agent: the longer the exposure, the greater the number of organisms killed; 5 temperature: normally a higher temperature increases the effectiveness of the agent; and 6 environmental conditions, such as pH, medium viscosity and the concentration of organic matter, can have major influences on the effectiveness of most antimicrobial agents.

24. Control of microbial growth by physical agents HEAT Whenever heat is used to control microbial growth both temperature and exposure time must be considered. The temperature required to kill a specific microorganism (the lethal temperature) varies quite widely. Also, the time needed to kill depends on several factors (described already). Heat is the most important and widely used means of sterilization and may be achieved through: 1 incineration, where burning at 500°C physically destroys the organisms and is particularly useful for some solid wastes; 2 moist heat, which is suitable for sterilizing most items, except heat-labile substances that would be denatured or destroyed. It is carried out using steam under pressure to achieve 121°C for 15min, and is extensively used in fermentation processes for the sterilization of vessels, connecting pipe work and culture media ; or 3 dry heat is less efficient than moist heat, requiring higher temperatures and much longer exposure times. It is performed in hot air ovens at 160°C for 2h, and can be used for glassware, metal objects and moisture-sensitive materials such as powders and oils.

25. Boiling of aqueous solutions or immersion of solid objects in boiling water at 100°C for 30min does not guarantee sterility. It kills most vegetative cells, but not all endospores. To kill endospores, intermittent boiling or tyndallization is required. This involves exposure of the material to elevated temperatures to kill the vegetative cells, followed by incubation at 37°C to allow any spores to germinate and form new vegetative cells. A second exposure to elevated temperatures kills the newly germinated vegetative cells. Heat sterilization is commonly employed in canning and bottling, and ultra-high temperature treatments (UHT) are used in some sterile packaging procedures. Pasteurization is a milder heat treatment, used to reduce the number of microorganisms in products or foods that are heat-sensitive and unable to withstand prolonged exposure to high temperatures. Batch pasteurization at 63∞C for 30min can be used for filled bottles; however, flash pasteurization at 72°C for 15s may be preferred for certain foods and beverages. For example, it is performed on milk and beer as it has fewer undesirable effects on quality or flavour . In the case of pasteurization of milk, the time and temperature used are targeted at killing milk-transmitted pathogens, notably staphylococci , streptococci , Brucella abortus , Mycobacterium bovis and Mycobacterium tuberculosis .

26. LOW TEMPERATURE These treatments involve refrigeration or freezing. Organisms are not usually killed, but the majority do not grow or grow very slowly at temperatures below 5 ° C. Perishable foods are stored at low temperatures to slow the rate of microbial growth and consequent spoilage. Often, it is psychrotrophs , rather than true psychrophiles, that are the cause of food spoilage in refrigerated foods. LOW WATER ACTIVITY This is used extensively to preserve foods, especially fruits, grains and some meat products. Methods involve removal of water from the product by heating or freeze-drying; alternatively, water activity may be reduced by the addition of solutes, usually salt or sugar.

27. IRRADIATION Microwave, UV, X-ray and gamma radiation can be used to destroy microorganisms. UV radiation is effective, but its use is limited to surface sterilization because it does not penetrate glass, dirt films, water and other substances. Ionizing radiation treatment mostly involves X-rays or g-rays. This is particularly effective due to its ability to penetrate materials. Irradiation is used commercially to sterilize items such as Petri dishes, and in some countries, spices, fruits and vegetables are irradiated to increase their storage life up to 500% by destroying spoilage microorganisms. The process is relatively expensive, and due to the nature of food materials and doses are given, not all organisms are killed. Irradiation may also destroy some vitamins. In some foods, the levels of vitamin C and E may be reduced by 5–10% and up to 25%, respectively. Such treatments are not suitable for all products; for example, when beer and some shampoos are irradiated, hydrogen sulphide may be generated. Irradiation of food has not been accepted worldwide. Currently, about 40 countries have passed legislation allowing its use for specified materials. However, it is now gaining greater acceptance, particularly with the increased incidence of food-borne diseases such as E. coli 0157:H7.