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TRADITIONAL 
(CLASSICAL) 
BIOTECHNOLOGY 
Fermentation 
cellardoorfestival.com
recall: biotechnology 
•ancient 
•early history as related to food and 
shelter, including domestication 
! 
•traditional (classical) 
•built on ancient biotechnology 
•fermentation promoted food 
production and medicine 
! 
•modern 
•manipulates genetic information in 
organism 
•genetic engineering 
mitalee.wordpress.com
Grabbed from the PPT lectures of 
Professor/Dr. Arnold V. Hallare, 
(2013)
what to learn today… 
•overview of metabolism: 
aerobic and anaerobic 
respiration 
•Fermentation in Plants 
•Fermentation in 
Animals 
•Fermentation in 
Humans 
•traditional 
biotechnology: 
fermentation 
•virtual lab 
blog.leonardo.com
recall: metabolism 
academic.pgcc.edu
cellular respiration: a catabolic reaction 
•process of making ATP by breaking down 
organic compounds 
•exergonic 
•oxygen (O2) requiring 
•uses energy extracted from 
macromolecules (glucose) to produce 
energy (ATP) and water (H2O) 
enzymes 
6O2 + C6H12O6 6CO2 + 6H2O 
oxygen glucose carbon dioxide water 
ADP + Pi 
ENERGY transfer 
between enzymes, 
other molecules ATP
stages of aerobic respiration 
• glycolysis: 
cytosol 
• krebs cycle: 
mitochondrial 
matrix 
• ETC: inner 
mitochondrial 
membrane 
You may watch a video here about GLYCOLYSIS: http:// 
www.science.smith.edu/departments/Biology/Bio231/
anaerobic respiration (fermentation): 
if oxygen is absent 
Glucose Pyruvic 
acid 
cellardoorfestival.com 
classes.midlandstech.com
organic acids instead of atp 
www2.bc.cc.ca.us
prokaryotes vs eukaryotes 
www.hns.org.uk
anaerobic respiration in plants 
www.ipm.iastate.edu In response to flooding stress 
www.vce.bioninja.com.au
anaerobic respiration in animals 
www.fashioncentral.pk 
www.vce.bioninja.com.au 
slow twitch versus fast twitch 
muscles
slow twitch and aerobic respiration 
• example: dark leg meat of 
chicken 
• Specialised for slow, sustained 
contractions over a long period 
for endurance 
• contain lots of myoglobin which 
acts as a store of oxygen 
• Respire aerobically
slow twitch works best in: 
or if you wanna try duathlon 
when you try running the bdm ultramarathon 
(160km)
lactic acid in meat? 
• fast twitch 
• example: pectoral flight muscles 
(chicken breast) 
• for producing rapid, intense 
contractions of short duration for 
rapid movement 
• do not have myoglobin so Respire 
anaerobically 
• can accumulate lactic acid and 
leads to fatigue 
thoughtchalk.com
fast twitch works best in: 
Provide the muscle power for rapid, fast 
movement e.g. a cheetah's burst of speed to 
catch a gazelle, or the gazelles burst of 
speed to escape the cheetah 
or to power up usain bolt’s 
legs in sprints 
omarmcknight.com
fermentation in ruminants 
sci.waikato.ac.nz 
www.tankonyvtar.hu
fermentation in humans 
•farting 
•When carbon dioxide is 
used as an electron 
acceptor, the product is 
either methane or acetic 
acid 
•Methane produced in our 
gut is produced by this 
process 
www.ausforces.com
fermentation in microbes
fermentation : classical biotechnology 
•the use of microbes 
to enhance food 
flavor 
•the use of microbes 
to manufacture of 
beverages 
•the use of microbes 
to make the dough 
rise
products of fermentation: beer 
•An alcoholic beverage produced by the fermentation 
of sugar-rich extracts derived from cereal grains or 
other starchy materials 
•ancient biotechnology: beer brewing 
•Sumaria (4000 BC) 
•Sikaru 
•Egypt (3000 BC) 
•Zythum 
•India (2000 BC) 
•Sura 
•China (2000 BC) 
•Kiu 
www.nomad4ever.com
yeast in beer brewing 
•1680 Antonie van 
Leeuwenhoek Observed yeast 
in beer 
•1837 - Cagniard Latour 
decsribed that Microbe is 
responsible for alcoholic 
fermentation 
•1866 - Louis Pasteur stated 
that Yeast was responsible 
for alcoholic fermentation 
•1883 - Emil Christian Hansen 
Developed pure culture 
technique and Isolated pure 
cultures of brewing yeasts 
Weiss Ale 
Lager Lab 
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company 
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
microbes and beer brewing 
•malted barley 
Provides fermentable 
sugars, flavor, and 
color 
•hops Provides aroma 
and bitterness 
The Brewing Process 
Step Purpose 
Brewhouse 
Fermentation 
Lagering 
Starch Sugars 
Wort production 
Sugars Ethanol 
Flavor production 
Carbonation 
Flavor maturation 
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company 
The Brewing Process 
Malt Mill 
Mash Tun Cereal 
Cooker 
Lauter Tun 
Brew 
Kettle 
Hot Wort 
Receiver 
Wort 
Cooler 
Brink Fermentation 
Aeration 
Lagering 
Hops 
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
microbes and beer brewing 
Yeast Metabolism During Fermentation 
Sugars Oxygen 
Glucose 
Pyruvate 
TCA 
Cycle 
Amino Acids 
Energy 
CO2 
Ethanol 
Acetaldehyde 
Organic Acids 
Unsaturated Fatty Acids 
Sterols 
Amino Acids 
Esters 
Higher 
Alcohols 
VDK 
Sulfur 
Volatiles 
Membranes 
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
products of fermentation: cheese 
•cheese are ripened curds 
•milk is treated with lactic acid bacteria 
and rennin to coagulate proteins 
•curds + whey = milk 
•different microbes 
in the early and 
late stages of 
processing of 
cheese = different 
cheese 
characteristics 
idiva.com
types oTfy pcesh oef Cehseeese 
Acid Coagulated Fresh Cheese (lactic acid from 
bacteria) 
• no enzyme is used to finish the curd 
• Cottage and Cream Cheese 
Heat-Acid Precipitated Cheese (acid and heat 
precipitate/coagulate the protein and cause milk fat 
to curdle) 
• Add low amounts of acid to 75-100oC temp milk 
• High moisture and protein 
• Ricotta (Italy) Channa and Paneer (India) 
science of cooking
types oTfy pcesh oef Cehseeese 
Semi-hard Washed Cheese (washing cheese 
removes acid and lactose) 
• Acid and enzyme induced curdling 
• But removal of milk sugar and acid results in no 
fermentation results in a moist and less finished cheese 
• Gouda, colby, muenster, mozzarella … 
Hard Cheese (Low and High Temp) 
• Low moister makes a more dense hard cheese 
• Elevated temps and pressing drive off water 
• Cheddar, Romano, Parmesan, Swiss, 
science of cooking
swiss cheese and propionibacterium 
science of cooking 
Finishing Microbes 
Holy Cheese (cow)? – Propionibacteria: 
• Convert lactic acid to propionoic and acetic acid plus acetic 
acid and CO2. Also other flavors 
• Used to make Swiss Cheese 
• Need higher temps and time for bacteria to grow and produce 
• Growth requirements reflect 
origins of bacteria 
animal skin 
Lactic acid 
Propionoic acid 
+ 
Acetic acid Carbon Dioxide (g) 
Finishing up…
Blue Cheeses – Based on Origen 
fungi and blue cheese 
Roquefort - France Cambreles- Spain 
Stilton- England 
Danish Blue Cheese 
Gorgonzola- Italy 
science of cooking 
Penicillium roqueforti and P. camberti 
BLUE = MYCELIA/ or growth filaments
making the cheese 
Non Starter– 
ripening 
Starter – acid 
producing 
0 50 100 150 200 
science of cooking 
Bacteria Growth 
Time (Days)
product of biotechnology: breads 
•biotechnology’s first utilization of 
microbes = bread making 
•Around 4000 BC, Egyptians used the 
living organism yeast to make bread 
•Airborne wild yeast accidentally got 
their bread dough, causing it to rise 
www.acebakery.com
the sourdough bread 
•microbe one (AEROBIC): yeast 
•makes carbon dioxide and bread will rise 
•microbe two: anaerobic: lactic acid 
bacteria 
•make lactic acid and acetic acid that give 
rich complexity of flavors 
www.weekendbakery.com foodists.ca www.rootsimple.com
products of fermentation: wine 
•after bread comes wine: 3000 bc 
•converts sugars in grapes into alcohol 
www.cell.com
making your wine 
http://www.chinookwines.com 
1. harvest 
2. processing 
3. fermentation
making your wine 
http://www.chinookwines.com 
4. maturation 
6. bottling & corking 
5. fining and filtration
products of fermentation: yoghurt 
•FERMENTED MILK 
RESULTING TO A SEMI-SOLID 
CURD 
•LACTIC ACID BACTERIA = 
PROBIOTICS 
•AIDS IN DIGESTION 
•ACID PRODUCED DURING 
FERMENTATION CAUSES 
THE PROTEIN TO 
COAGULATE 
•Lactococcus lactis, 
Streptococcus 
thermophilus and 
Lactobacillus bulgaricus 
www.wombourneshopping.co.uk
how to make yoghurt 
Making Yogurt in 4 Simple Steps 
1. Start with Cow, Sheep, or Goat milk. 
2. Heat milk to 80 °C. Two purposes: 
• destroy existing bacteria 
• condition the proteins = begins the denaturing process 
(a whey protein molecule binds to a casein molecule which disrupts 
the casein bundles allowing them to make short branched micelle 
chains) 
3. Cool milk to 40 °C and innoculate with bacteria 
4. Incubate at 30 °C to 45 °C 
Casein before heat 
pre-treatment: 
Casein after heat 
pre-treatment: Casein after acid: 
www.bnc.asn.au
bacteria in yoghurt 
Milk Yogurt 
Casein protein micelles Bacteria produce acid 
(bundles) 
10-7 meters in diameter 
Fat 
globule 
Acid causes 
Casein bundles to 
fall apart into 
separate casein 
molecules. 
These rebind to 
each other in a 
network that traps 
water. 
= makes a gel

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Classical Biotechnology: FERMENTATION

  • 1. TRADITIONAL (CLASSICAL) BIOTECHNOLOGY Fermentation cellardoorfestival.com
  • 2. recall: biotechnology •ancient •early history as related to food and shelter, including domestication ! •traditional (classical) •built on ancient biotechnology •fermentation promoted food production and medicine ! •modern •manipulates genetic information in organism •genetic engineering mitalee.wordpress.com
  • 3. Grabbed from the PPT lectures of Professor/Dr. Arnold V. Hallare, (2013)
  • 4. what to learn today… •overview of metabolism: aerobic and anaerobic respiration •Fermentation in Plants •Fermentation in Animals •Fermentation in Humans •traditional biotechnology: fermentation •virtual lab blog.leonardo.com
  • 6. cellular respiration: a catabolic reaction •process of making ATP by breaking down organic compounds •exergonic •oxygen (O2) requiring •uses energy extracted from macromolecules (glucose) to produce energy (ATP) and water (H2O) enzymes 6O2 + C6H12O6 6CO2 + 6H2O oxygen glucose carbon dioxide water ADP + Pi ENERGY transfer between enzymes, other molecules ATP
  • 7. stages of aerobic respiration • glycolysis: cytosol • krebs cycle: mitochondrial matrix • ETC: inner mitochondrial membrane You may watch a video here about GLYCOLYSIS: http:// www.science.smith.edu/departments/Biology/Bio231/
  • 8. anaerobic respiration (fermentation): if oxygen is absent Glucose Pyruvic acid cellardoorfestival.com classes.midlandstech.com
  • 9. organic acids instead of atp www2.bc.cc.ca.us
  • 10. prokaryotes vs eukaryotes www.hns.org.uk
  • 11. anaerobic respiration in plants www.ipm.iastate.edu In response to flooding stress www.vce.bioninja.com.au
  • 12. anaerobic respiration in animals www.fashioncentral.pk www.vce.bioninja.com.au slow twitch versus fast twitch muscles
  • 13. slow twitch and aerobic respiration • example: dark leg meat of chicken • Specialised for slow, sustained contractions over a long period for endurance • contain lots of myoglobin which acts as a store of oxygen • Respire aerobically
  • 14. slow twitch works best in: or if you wanna try duathlon when you try running the bdm ultramarathon (160km)
  • 15. lactic acid in meat? • fast twitch • example: pectoral flight muscles (chicken breast) • for producing rapid, intense contractions of short duration for rapid movement • do not have myoglobin so Respire anaerobically • can accumulate lactic acid and leads to fatigue thoughtchalk.com
  • 16. fast twitch works best in: Provide the muscle power for rapid, fast movement e.g. a cheetah's burst of speed to catch a gazelle, or the gazelles burst of speed to escape the cheetah or to power up usain bolt’s legs in sprints omarmcknight.com
  • 17. fermentation in ruminants sci.waikato.ac.nz www.tankonyvtar.hu
  • 18. fermentation in humans •farting •When carbon dioxide is used as an electron acceptor, the product is either methane or acetic acid •Methane produced in our gut is produced by this process www.ausforces.com
  • 20. fermentation : classical biotechnology •the use of microbes to enhance food flavor •the use of microbes to manufacture of beverages •the use of microbes to make the dough rise
  • 21. products of fermentation: beer •An alcoholic beverage produced by the fermentation of sugar-rich extracts derived from cereal grains or other starchy materials •ancient biotechnology: beer brewing •Sumaria (4000 BC) •Sikaru •Egypt (3000 BC) •Zythum •India (2000 BC) •Sura •China (2000 BC) •Kiu www.nomad4ever.com
  • 22. yeast in beer brewing •1680 Antonie van Leeuwenhoek Observed yeast in beer •1837 - Cagniard Latour decsribed that Microbe is responsible for alcoholic fermentation •1866 - Louis Pasteur stated that Yeast was responsible for alcoholic fermentation •1883 - Emil Christian Hansen Developed pure culture technique and Isolated pure cultures of brewing yeasts Weiss Ale Lager Lab Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
  • 23. microbes and beer brewing •malted barley Provides fermentable sugars, flavor, and color •hops Provides aroma and bitterness The Brewing Process Step Purpose Brewhouse Fermentation Lagering Starch Sugars Wort production Sugars Ethanol Flavor production Carbonation Flavor maturation Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company The Brewing Process Malt Mill Mash Tun Cereal Cooker Lauter Tun Brew Kettle Hot Wort Receiver Wort Cooler Brink Fermentation Aeration Lagering Hops Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
  • 24. microbes and beer brewing Yeast Metabolism During Fermentation Sugars Oxygen Glucose Pyruvate TCA Cycle Amino Acids Energy CO2 Ethanol Acetaldehyde Organic Acids Unsaturated Fatty Acids Sterols Amino Acids Esters Higher Alcohols VDK Sulfur Volatiles Membranes Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
  • 25. products of fermentation: cheese •cheese are ripened curds •milk is treated with lactic acid bacteria and rennin to coagulate proteins •curds + whey = milk •different microbes in the early and late stages of processing of cheese = different cheese characteristics idiva.com
  • 26. types oTfy pcesh oef Cehseeese Acid Coagulated Fresh Cheese (lactic acid from bacteria) • no enzyme is used to finish the curd • Cottage and Cream Cheese Heat-Acid Precipitated Cheese (acid and heat precipitate/coagulate the protein and cause milk fat to curdle) • Add low amounts of acid to 75-100oC temp milk • High moisture and protein • Ricotta (Italy) Channa and Paneer (India) science of cooking
  • 27. types oTfy pcesh oef Cehseeese Semi-hard Washed Cheese (washing cheese removes acid and lactose) • Acid and enzyme induced curdling • But removal of milk sugar and acid results in no fermentation results in a moist and less finished cheese • Gouda, colby, muenster, mozzarella … Hard Cheese (Low and High Temp) • Low moister makes a more dense hard cheese • Elevated temps and pressing drive off water • Cheddar, Romano, Parmesan, Swiss, science of cooking
  • 28. swiss cheese and propionibacterium science of cooking Finishing Microbes Holy Cheese (cow)? – Propionibacteria: • Convert lactic acid to propionoic and acetic acid plus acetic acid and CO2. Also other flavors • Used to make Swiss Cheese • Need higher temps and time for bacteria to grow and produce • Growth requirements reflect origins of bacteria animal skin Lactic acid Propionoic acid + Acetic acid Carbon Dioxide (g) Finishing up…
  • 29. Blue Cheeses – Based on Origen fungi and blue cheese Roquefort - France Cambreles- Spain Stilton- England Danish Blue Cheese Gorgonzola- Italy science of cooking Penicillium roqueforti and P. camberti BLUE = MYCELIA/ or growth filaments
  • 30. making the cheese Non Starter– ripening Starter – acid producing 0 50 100 150 200 science of cooking Bacteria Growth Time (Days)
  • 31. product of biotechnology: breads •biotechnology’s first utilization of microbes = bread making •Around 4000 BC, Egyptians used the living organism yeast to make bread •Airborne wild yeast accidentally got their bread dough, causing it to rise www.acebakery.com
  • 32. the sourdough bread •microbe one (AEROBIC): yeast •makes carbon dioxide and bread will rise •microbe two: anaerobic: lactic acid bacteria •make lactic acid and acetic acid that give rich complexity of flavors www.weekendbakery.com foodists.ca www.rootsimple.com
  • 33. products of fermentation: wine •after bread comes wine: 3000 bc •converts sugars in grapes into alcohol www.cell.com
  • 34. making your wine http://www.chinookwines.com 1. harvest 2. processing 3. fermentation
  • 35. making your wine http://www.chinookwines.com 4. maturation 6. bottling & corking 5. fining and filtration
  • 36. products of fermentation: yoghurt •FERMENTED MILK RESULTING TO A SEMI-SOLID CURD •LACTIC ACID BACTERIA = PROBIOTICS •AIDS IN DIGESTION •ACID PRODUCED DURING FERMENTATION CAUSES THE PROTEIN TO COAGULATE •Lactococcus lactis, Streptococcus thermophilus and Lactobacillus bulgaricus www.wombourneshopping.co.uk
  • 37. how to make yoghurt Making Yogurt in 4 Simple Steps 1. Start with Cow, Sheep, or Goat milk. 2. Heat milk to 80 °C. Two purposes: • destroy existing bacteria • condition the proteins = begins the denaturing process (a whey protein molecule binds to a casein molecule which disrupts the casein bundles allowing them to make short branched micelle chains) 3. Cool milk to 40 °C and innoculate with bacteria 4. Incubate at 30 °C to 45 °C Casein before heat pre-treatment: Casein after heat pre-treatment: Casein after acid: www.bnc.asn.au
  • 38. bacteria in yoghurt Milk Yogurt Casein protein micelles Bacteria produce acid (bundles) 10-7 meters in diameter Fat globule Acid causes Casein bundles to fall apart into separate casein molecules. These rebind to each other in a network that traps water. = makes a gel