1. 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

2. 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)

3. 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

4. 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

5. How significant is Glycolysis?
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

6. 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.
By Mariana Ruiz Villarreal, LadyofHats [Public domain], via Wikimedia Commons
Plant cell. By LadyofHats [Public domain], via Wikimedia Commons

7. 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

8. 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

9. 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 ( via Wikimedia Commons

10. 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

11. 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

12. Lactic acid fermentation
By Sjantoni (Own work) [CC BY-SA 3.0 ( via Wikimedia Commons

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

14. 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 ) [CC BY-SA 3.0 ( via Wikimedia Commons

15. 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!

16. 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 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 years
Pickling in India of cucumbers years ago

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

18. 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

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

20. 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.

21. 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

22. 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

23. 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)

24. 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