Fermentation and fermented products FOOD304 – Microbial Biotechnology Fermentation and fermented products Dr Stephen On Associate Professor in Food Microbiology Adapted from materials prepared by: Dr Malik Altaf Hussain Senior Lecturer in Food Microbiology

Previously in Bios110… All living things require energy for core functions Energy is generated by the catabolism of substrates Autotrophs – can fix and use carbon for growth Photoautotroph – use light as energy source to use Lithoautotroph – use inorganic chemicals as energy source Heterotrophs – must acquire carbon for use (but cannot fix CO2) Photoheterotroph – energy from light to use external carbon for growth Chemoorganoheterotroph - energy from chemically changing external carbon for growth 95% of known life forms are chemoorganoheterotrophs (aka organotrophs)

For organotrophs Glucose is a key substrate Glycolysis is a major pathway for catabolising glucose to generate energy (ATP) Glycolysis results in the production of pyruvate and ATP

Glycolysis at-a-glance Note – this is a critical step, generating 2 triose sugar molecules. Each sugar is further processed resulting in a net energy gain Glycolysis does not require molecular oxygen and is a pathway common to aerobes and anaerobes Thomas Shafee. Creative commons licence 4.0. https://en.wikipedia.org/wiki/File:Glycolysis_metabolic_pathway_3_annotated.svg

How significant is Glycolysis? https://upload.wikimedia.org/wikipedia/commons/thumb/7/70/Phylogenetic_tree.svg/450px-Phylogenetic_tree.svg.png. Public domain. Glycolysis is an almost universal metabolic process known to occur (with variations) in many types of cells in nearly all organisms. The wide conservation includes the most phylogenetically deep rooted organisms and thus glycolysis is considered to be one of the most ancient metabolic pathways. Glycolysis, through anaerobic respiration, is the main energy source in many prokaryotes, eukaryotic cells that do not have mitochondria – and is the start of the energy production process in organisms that do have mitochondria

Where does Glycolysis occur? In eukaryotes and prokaryotes, glycolysis takes place within the cytosol of the cell. In plant cells some of the glycolytic reactions are also found in the chloroplasts. https://commons.wikimedia.org/wiki/File:Average_prokaryote_cell-_en.svg. By Mariana Ruiz Villarreal, LadyofHats [Public domain], via Wikimedia Commons Plant cell. By LadyofHats [Public domain], via Wikimedia Commons https://commons.wikimedia.org/wiki/File:Plant_cell_structure-en.svg

Glycolysis: fermentation’s first step The end product of glycolysis = pyruvate In anaerobic conditions, the further conversion of pyruvate is the organisms main source of energy The end-product is organism-dependant: Lactic acid Ethanol

Fermentation: the last resort? Fermentation does not release as much energy as aerobic respiration (2 vs. 38 ATP molecules!) and is a “last resort” for most facultative anaerobes Fermentation’s main energy generating contribution is to regenerate NAD+ that can then be used in glycolysis However in the presence of excess sugars (C-source), yeasts will prefer to use fermentation to generate energy (the Crabtree effect) Ethanol can also be used as an energy source direct

Alcohol fermentation One glucose molecule breaks down into two pyruvates via glycolysis (1). The energy from these exothermic reactions is used to bind inorganic phosphates to ADP and convert NAD+ to NADH. The two pyruvates are then broken down into two Acetaldehyde and give off two CO2 as a waste product (2). The two Acetaldehydes are then reduced to two ethanol, and NADH is oxidized back into NAD+ (3). By Davidcarmack (Own work) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

Alcohol Fermentation Only 2 ATP End products: Alcoholic Beverages (Wine, Beer) Bread dough to rise Saccharomyces species used to produce food and beverages include: Saccharomyces pastorianus - For beer and lager making Saccharomyces bayanus - For cider and wine making Saccharomyces cerevisiae - Bread, beer, sake and wine making

Wild vs. Commercial Fermentation “Wild” fermentations use yeast naturally present in/on the food being fermented to produce the food. Tend to take longer and flavour development is less consistent Commercially available yeast strains are generally more dependable, more robust Less complexity and less reflective of the region’s ecosphere

Lactic acid fermentation By Sjantoni (Own work) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

Lactic Acid Fermentation Only 2 ATP End Product - Lactic Acid Food Spoilage Food Production Yogurt - Milk Pickles - Cucumbers Sauerkraut - Cabbage 2 Genera: Streptococcus Lactobacillus

Malolactic fermentation - Is not strictly speaking fermentation. It is a decarboxylation reaction that converts malic acid (naturally present in grape must) to lactic acid. It is used in winemaking (often in a secondary process) to remove the acidic taste imparted by lactic acid, and to improve the mouthfeel of the wine. MLF is widely used in red wine production. The “buttery” texture of chardonnay occurs as a result of the diacetyl by-product. MLF is undertaken by bacterial species, notably Lactic Acid Bacteria (LAB) such as Oenococcus oeni, and Lactobacillus or Pediococcus species. By Yikrazuul (Own work; PMID 16826375) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

Fermentation, Food culture and food preservation It was most likely the beneficial influence of fermentation on food preservation (more than any other factor) that initially cemented fermentation into food culture. Food can be preserved by fermentation, since fermentation through the production of (mostly) acids or alcohol can make conditions unsuitable for undesirable or pathogenic microorganisms. The effect of alcohol on human behaviour has only served to further secure the importance of fermented products to mankind!

Fermentation and mankind’s history Evidence for bread making in Italy, Russia and the Czech Republic 30,000 years ago! Leavened bread would have been produced naturally; historians cite the intentional production of leavened bread started around 6000 years ago. Historians believe yogurt originated in Turkey / Central Asia 10,000 years ago Jugs discovered in this period have suggested brewing of alcoholic beverages begun at this time also Pottery from North China 8-9000 years old evidence of fermented drink from rice, honey and fruit Pottery from Romania, Switzerland and Hungary 8000 years ago indicate traces of yogurt, cheese, sour cream Jars of wine from Iran 7400 years old Brewing in Egypt – Heirakonpolis’ ruins contain the remains of the world’s oldest brewery - 5400 years Pickling in India of cucumbers - 4000 years ago

Purpose of Fermentation Preserve food particularly in warm climates Produce new food products

Two Major and One Minor Type of Fermentation Lactic acid fermentation (homolactic - produces lactate only) Performed by: Lactococci, Leuconostoc, Lactobacilli, Streptococci, Bifidobacterium Lack enzymes to perform the TCA cycle. Often use lactose as the input sugar (found in milk) Alcoholic fermentation (produce ethanol) Saccharomyces spp. Also: heterolactic fermentation (lactate and other products) Clostridium, E. coli etc

Saccharomycetes Clostridium E. coli Propionebacterium Enterobacter Streptococcus Lactobacillus Diagram by M. Hussain, Lincoln University

Fermentation: what’s in a word? Classic fermentation involves conversion of sugars to acids and/or alcohols, usually in anaerobic conditions In an industrial context, the term has also been used to describe the bulk growth of microorganisms in a liquid growth medium These microbiological processes can occur aerobically as well as anaerobically.

Industrial Fermentation Products Category Examples Uses Food Beer, wine, vinegar, coffee, tea, pickles cheese, salami, sauerkraut, yoghurt, soy sauce Preservation of food by the use of acids and alcohols Feed Silage Preservation of feed by organic acids Cell mass Yeast, bacteria Starter cultures and animal feeds Organic Solvents Ethanol, Glycerol, acetone etc Cosmetics, pharmaceuticals, manufacturing etc Organic Acids Lactic, citric , acetic, formic etc Food additive, manufacturing etc Amino Acids L- Lysine, L-Tryptophane, L-Phenalaline, Glutamic acid Food and feed additive Antibiotics Penicillin, streptomycin, tetramycin etc Human and animal health Vitamins B12, Riboflaflaven Food and feed supplements Enzymes Amylase, cellulase, protease, lipase etc Food processing, tanning, detergents etc Bio-Polymers Dextran, polyhydroxybuyarate etc Food additive, packaging etc Pharmaceuticals Insulin, interferon , growth hormones etc Human medicines Environmental Waste and waste water treatment Public hygiene Energy Ethanol, methane Fuel additive and heating

Industrial Fermentation products can be classed as: Primary metabolites Produced in parallel with cell growth E.g. Ethanol, lactic acid Secondary metabolites Not directly related to the growth of the cell E.g. Amino acids, proteins etc

Why use fermentation in food? Preservation of food through lactic acid, alcohol, acetic acid, and alkaline fermentations. Enrichment of the food through development of a diversity of flavours, aromas, and textures. Enhancement of social interaction and relaxation Enrichment of food nutrition through production of proteins, amino acids, fatty acids, and vitamins. Detoxification during food-fermentation (e.g. Cassava) Health benefits (eg probiotic yogurt)

Acid and alcohol production Production of bacteriocins Benefits of fermentation Raw material Fermented food Benefit Preservation Milk (Most materials) Yoghurt, cheese Enhancement of safety Vinegar Beer Wine Salami Gari, polviho azedo Soy sauce Acid production Acid and alcohol production Production of bacteriocins Removal of toxic components Fruit Barley Grapes Meat Cassava Soybean Enhancement of nutritional value Bread Kimchi, sauerkraut Nata de coco Bifidus milk, Yakult, Acidophilus yoghurt Improved digestibility Retention of micronutrients Increased fibre content Synthesis of probiotic compounds Wheat Leafy veges. Coconut Milk Improvement of flavour Coffee beans Grapes Coffee Wine