Food Biotechnology Lesson 2: Complex Food Products PDF

Dr. Ilaria Benucci [email protected] Complex food products Dr. Ilaria Benucci [email protected] INDEX 1. Complex food products 2. Fermentations 2.1 Microbial Growth Curve...

Dr. Ilaria Benucci [email protected] Complex food products Dr. Ilaria Benucci [email protected] INDEX 1. Complex food products 2. Fermentations 2.1 Microbial Growth Curve 3. Alcoholic fermentation 3.1 Alcoholic fermentation in winemaking 3.2 Yeast metabolism during alcoholic fermentation 4. Homolactic fermentation 5. Heterolactic fermentation Dr. Ilaria Benucci [email protected] 1. Complex food products The complex products deriving from microbial biotechnology are defined as such because it is impossible to isolate the product, consisting of the transformed substrate and, in most cases, the microorganisms responsible for the modification. These products are obtained through processes developed starting from traditional technologies in the food sector, such as alcoholic beverages, dairy products and fermented vegetables. Dr. Ilaria Benucci [email protected] 1. Complex food products The production of alcoholic beverages has very ancient roots and is documented in all geographical areas of the planet. Fermentation has always been carried out starting from juices obtained from fruits, honey or pre-treated cereal starch. In some cases the products are used just after their production (e.g. beer), but more frequently after a relatively long period of conservation, in which the product undergoes significant changes in its organoleptic characteristics, like wine. The microorganisms involved in the production of alcoholic beverages are essentially yeast strains in pure selected culture or mixed culture of natural origin (lambic beers, so-called natural wines). The most represented genus is Saccharomyces, responsible for alcoholic fermentation. Dr. Ilaria Benucci [email protected] 2. Fermentations Fermentation processes represent, in the absence of atmospheric oxygen, an alternative to microbial respiration with lower energy yield, because the final products are not completely oxidized. The anaerobic catabolism of organic substances, mainly carbohydrates, by microbial enzymes. Homofermentative (formation of a single product); Heterofermentative (two or more final products). The microorganisms responsible for fermentation processes belong to: schizomycetes (bacteria): Lactobacillus, Clostridium, Nitrobacter, Acetobacter eumycetes (fungi): yeasts, molds Dr. Ilaria Benucci [email protected] 2. Fermentations Butyric ferm Alcoholic ferm Lactic ferm 2. Fermentations Lactic ferm Alcoholic ferm Propionic ferm Butyric ferm  Pyruvate ……… Pediococcus Lactobacillus Propioni- Saccharomyces Streptococcus Leuconostoc bacterium Enterobacter Clostridium Lactobacillus Homolactic Heterolactic Propionic Mixed-Acid Butyric Alcoholic ferm ferm ferm ferm ferm ferm Ethanol Ethanol Ethanol CO2 Lactic Lactic Propionic Formic, Butyric, acetic, lactic, acetic acid, + + acid acid, acid, succinic acid, isopropil CO2 CO2 ethanol acetic acid ethanol alcool, or acetic + + acetone, CO2, H2 butanol ac. CO2 + + CO2, H2 CO2 Late Wine Sparkling Beer Bread Yogurt Swiss Blowing in Kefir wine Cheese cheese Cheese 2.1 Microbial Growth Curve Yeast and bacteria during fermentation Dr. Ilaria Benucci [email protected] 3. Alcoholic fermentation Carbohydrate transformation for the production of wine, beer and bread. The substrates are glucose and fructose from grapes for wine, glucose resulting from the hydrolysis of starch from barley and wheat for beer and bread. Carried out by yeasts (Saccharomyces genus) that are resistant to low temperatures but sensitive to high temperatures (optimum 25 °C) Video: https://www.khanacademy.or g/science/high-school- biology/hs-energy-and- transport/hs-cellular- respiration/v/alcohol-or- ethanol-fermentation Dr. Ilaria Benucci [email protected] 3.1 Alcoholic fermentation in winemaking MICROBIAL ASPECTS In the grape must, just a few hours after pressing, anaerobic conditions prevent the development of microorganisms with oxidative metabolism, such as acetic acid bacteria and mold, favoring yeasts which prevail. In the early stages of fermentation, the group of apiculate yeasts (Kloeckera genus) dominates. When the concentration of ethyl alcohol is close to 5%, the apiculate yeasts stop their activity and Saccharomyces cerevisiae (elliptical) prevails completing the process. Kloeckera apiculata Saccharomyces cerevisiae Kloeckera apiculata, the indigenous yeast normally prevails in grape and must. It is easily recognized by the repeated budding on the two ends, the scars of which make the cell on a lemon shape Dr. Ilaria Benucci [email protected] 3.1 Alcoholic fermentation in winemaking MICROBIAL ASPECTS The spontaneous fermentation of grape must by indigenous/ "wild yeasts" yeasts is an oenological practice used only in some cellars. In order to avoid the development of "wild yeasts" (apiculates such as Kloeckera and Hanseniaspora responsible for sluggish and incomplete fermentations) in favor of elliptical yeasts of the Saccharomyces cerevisiae species, through guided alcoholic fermentations: Use of selected yeasts Scalar fermentations The use of sulfur dioxide Starter cultures of Saccharomyces cerevisiae are used, with inocula of 5-10×106/ml; elimination of apiculate yeasts to have a constant and complete fermentation Dr. Ilaria Benucci [email protected] 3.1 Alcoholic fermentation in winemaking STARTER CULTURES - Active Dry Yeast (ADY) The microbial biomasses that serve to govern a specific fermentation process are defined as starters. The main advantages of their use are : conduct fermentation more quickly; process control, avoiding the development of anomalous fermentations caused by unwanted and/or spoiling microorganisms; mitigation of problems relating to sluggish/incomplete fermentations; guarantee a final product with constant organoleptic characteristics. Dr. Ilaria Benucci [email protected] 3.1 Alcoholic fermentation in winemaking STARTER CULTURES - Active Dry Yeast (ADY) The recommended dose (20-25 g/hL) is rehydrated in a 10 times greater quantity of water (35-40°C) and subjected to gentle mixing for 15-20 min. After rehydration: the suspension is gradually added with must to bring the temperature closer to that of the tank to be fermented (max 25 min) the suspension is added to a quantity of must equal to 2% of the total mass and left for 24 hours for multiplication (musts that are more difficult to ferment); The dose of 20 g/hL of must provides 5x106 yeasts per ml; that is, approximately 5 times more than the indigenous microbial population; thus in 4 or 5 generations a sufficient population is produced. Dr. Ilaria Benucci [email protected] 3.1 Alcoholic fermentation in winemaking YEAST INOCULUM PREPARATION - Active Dry Yeast (ADY) inoculate the musts as soon as possible to facilitate the dominance of the selected strain inoculate the must when the temperature is above 12 °C use of a clean container; first addition of water at 37 °C; reidratation - 15/20 min slowly add the active dry yeast; The addition of must during the rehydration phase is NOT recommended (it promotes acclimatization and activation of indigenous flora) Dr. Ilaria Benucci [email protected] 3.1 Alcoholic fermentation in winemaking YEAST REQUIREMENTS DURING THE DIFFERENT GROWTH PHASES Survival factors 108 Oxygen Total cells/mL 107 106 Ammonium (NH 4) and aminic nitrogen(200-250 mg/L) 1 3 11 Days Dr. Ilaria Benucci [email protected] 3.1 Alcoholic fermentation in winemaking YEAST REQUIREMENTS DURING THE DIFFERENT GROWTH PHASES Log phase Macro and micronutrients Stationary phase Survival factors ▪ Oxygen ▪ Fatty acids ▪ Sterols ▪ Survival nutritional factors (Vitamins)  3.1 Alcoholic fermentation in winemaking SURVIVAL NUTRITIONAL FACTORS Macronutrients (C, N, P): raw material for building cells. N possible limiting factor. Micronutrients: act at low concentrations on cellular multiplication and activity. Essential constituents of coenzymes, they are able to facilitate biochemical reactions. Minerals (Mg, Ca, Mn, K, Zn, Fe, Cu). Growth factors - vitamins (biotin is the only essential, the others have a stimulating effect).  3.1 Alcoholic fermentation in winemaking SURVIVAL NUTRITIONAL FACTORS Solid sediments (fine lees) constituting the pruina and cuticular wax of grapes, e.g. oleanolic acid, in particular if associated with oleic acid and long-chain unsaturated fatty acids (C18:1, C18:2) and deriving from yeast cell membranes. Sterols their presence is necessary to ensure the permeability of the membranes. In the presence of oxygen the cells are able to synthesize them. In anaerobic conditions ergosterol, other sterols and long-chain fatty acids act as oxygen substitutes for the cell. The lipidic supplementation provided to yeast by the solid sediments (fine lees) is the main factor controlling the production of acetic acid in white and rosé winemaking (favor the penetration of amino acids into the cell, limiting the production of acetic acid). Inactivated yeast 3.2 Yeast metabolism during alcoholic fermentation 3.2 Yeast metabolism during alcoholic fermentation Dr. Ilaria Benucci [email protected] 4. Homolactic fermentation Microbiological transformation of glucose to lactic acid which is formed by the reduction of pyruvic acid. The responsible microorganisms are homofermentative bacteria called Gram+ acidogenic bacteria (Lactobacillus and Streptococcus genus). They normally produce L(+)lactic acid. It is a fermentation used in the preparation of yogurt, in the ripening of cheeses and in the Regenerated NAD+ preservation of vegetables (sauerkraut, cucumbers, pickled olives). Video: https://www.khanacademy.org/science/high- school-biology/hs-energy-and-transport/hs- cellular-respiration/v/lactic-acid-fermentation Dr. Ilaria Benucci [email protected] 5. Heterolactic fermentation Heterolactic fermentation is the conversion of one glucose molecule into the lactic acid molecule, carbon dioxide, and ethanol. This process involves heterofermentative bacteria that are able to produce less lactate and less amount ATP comparatively, but they can produce several other products, including ethanol and carbon dioxide. The chemical reaction for this process is as follows: Glucose + ADP + 2Pi → lactate + ethanol + CO2 + ATP Some examples of heterofermentative bacteria include Leuconostoc mesenteroides, Lactobacillus bifermentous, and Leconostoc lactis.