Begin Fermentation
“Wild Yeasts” Dominate Grape
Skins
• Kloeckera and Hanseniospora (50-75%),
and at lesser levels, Candida, Cryptococcus,
Rhodotorula, Pichia, Kluyveromyces and
Hansenula
Fermentation Yeast
Saccharomyces cerevisiae
Number of S. cerevisiae yeast on a
grape: 1-10 Cells
= 40 thousand cells per gallon of juice
Number of S. cerevisiae yeast in a
gallon of fermenting wine:
2 x 10 12 or 2 Trillion Cells
Alcoholic Fermentation
• Grape Sugars Ethanol + Carbon Dioxide + Heat
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Yeast tolerance limits:
– Ethanol up to about 16%
– Pressurized up to about 120 psi
– Heat above 110 F
Purpose of Inoculation
• Domination by the “good guys”
Starter Yeast Cultures
ferment 25-30% sugar to dryness
tolerate 50-100 mg/L free SO2
produce minimum amounts of acetic
acid, hydrogen sulfide or sulfur dioxide
tolerate cool (50º F) to very warm (100ºF) fermentation
temperatures
• tolerate low pH musts (<3.0)
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Happy Yeast
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Replication
– Oxygen
– Nitrogen
– Sugar
Fermentation
– Temperature 75F
– Sugar
Yeast Food
• Nitrogen deficiencies in juice
– diammonium phosphate (DAP) &/or Yeastex or
Fermaid
• proprietary formulations
– Add up to 2 grams/gallon at inoculation
– Helps avoid stuck fermentations and hydrogen
sulfide formation
Starter Culture Preparation
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1 gram yeast per gallon juice
Warm 10 ml of water per 1 gram yeast to 104 F
Sprinkle yeast on top
Hold at 100-102F for 15-30 minutes
Add to juice, temperature compensated, and
stir in well
Typical profile of yeast growth versus sugar utilization
Monitor Fermentation
• atch for signs
• Reinoculate if no activity after 48 hours
• Take hydrometer readings
– Determine when to press red wines
–Watch for stuck fermentations
You’’re a Winner!!
Indy International Wine Competition
Industrial fermentation
Production with microbes
• Industrial microbiology
– The use of microbes in the production
– Start from an ancient time
– Large scale production –
• Early 20th century – beverage industry, vinegar,
baking yeast, citric and lactic acid
• Late 20th century – development of
biotechnology from the antibiotic industrial
Production with microbes
• Fermentation industry
– Microbes to produce ‘useful’ product
– Usually a naturally produce substances for
growth, and maintenance.
– Metabolites
• Primary metabolite – produce during growth
• Secondary metabolite – produce after growth
stage
Secondary metabolite
Primary metabolite
Products
I. Foods and foods related
– Fermented meat, Cheeses and milk products,
mushrooms, baker’s yeast, coffee, pickles, single
cell-proteins, vinegar, amino acids, vitamins,
alcoholic beverages
Products
II. Organic acids
– Citric acids, Itaconic acid
III. Enzymes and microbial transformation
– Commercial enzyme, sterol conversion
IV. Engineered microbes
– Insulin, human growth factor
Production with microbes
• Steps in Industrial fermentation
– Isolation of microbes that produce your
interested product
– Screen for the best producing strain: naturally
or mutation.
– Optimize production condition (growth –
basically) → in lab
– Scale up from lab scale (up to 10 L) to industrail
scale (>10,000 L) (raw material?)
Food and food related products
• Whole cells products
– Including – mushrooms and Single-cell protein
(SCP)
• Mushrooms – fruiting body of mold!
– Highly consumed every year – high nutrition.
– Grow on decaying organic matter in soil or wood.
• Stalk and cap = compact mycelium
• Gills = site of reproductive spores
– E.g. Agaricus bisporus (botton mushroom),
Lentinus edulus (Shitake mushroom)
Food and food related products
• Single-cell protein – yeast, and cyanobacteria
– Alternative food sources – mostly used now as
supplementary diet e.g. yeast is a source for B
Vitamins.
– Organisms: yeast, Spirulina
– Growing biomass and freeze dry
– Raw material from other industrial, farming
Food and food related products
• Baker’s yeast
– Same process as in single cell protein
Food and food related products
• Cheese production
– Can produce from any milk.
– 4 phases: coagulation, separation, shaping
and ripening
• Coagulation: rennin enzyme or acids precipitate
casein
– From calves’s stomachs, engineered bacteria
– Acid production by lactic acid bacteria
– Curd entrap bacteria, fat globules and other
material.
• Cheese production
– Separation & Shaping
• Additives (salt and herbs) may be added after seperation
• or shaping: effect property of cheese product
– Ripening – add flavor and look
– Natural or inoculation of microbes e.g. Penicillium
roquefortii (spores are usually added into blue
cheese), and Penicillium camemberti is added to
the surface of a cheese known as Camenbert
Blue cheese
Brie
Gorgonzola
Swiss
Food and food related products
• Yogurt and other fermented milk products
– Yogurt, kefir, buttermilk, sourcream
– Yogurt usually use mixture of 2 cultures:
• Streptococcus thermophilus: ini. acid production
• Lactobacillus bulgaricus
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0.5 - 1.0% lactic acid
Fermented at 28-32 °C
Taste due to acetaldehyde, diacetyl, and acetoin
Product need to contains live cultures
Some Foods Produced from Fermented Milk.
Fermented
Microorganisms
Product
Description
Sour cream
Streptococcus sp.
Leuconostoc sp.
Cream is inoculated and leave
to develop acidity
Cultured
buttermilk
Streptococcus sp.
Leuconostoc sp.
Made with skimmed or partly
skimmed pasteurized milk.
Acidophilus
milk
Lactobacillus
acidophilus
This milk product is used for its
medicinal therapeutic value.
Kefir
Streptococcus lactis,
Lactobacillus
bulgaricus, yeasts
A mixed lactic acid and
alcoholic fermentation.
Industrial production
of yogurt
Kefir
Buttermilk
Food and food related products
• Alcoholic beverages
– Accidental discovered from ancient human
– Saccharomyces cerevisiae
– Raw materials are regional….variety of
products
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Wine – grapes, fruits
Beer – cereal grain – barley, sorgum, wheat
Other – rice wine (rice), teguila (agave cactus),
vodka (corn), mead (honey).
Food and food related products
• Fermented meat
– Use to preserve food E.g. salami, summer
sausage, cured ham
– Thailand – naam (fermented pork), fermented
sausage, fermented fish, fish sauce…etc
– Main organism – Pediococcus serevisiae,
Lactobacillus plantarum, other lactic acid bacteria
– Production of lactic acid to change pH of food
– Production of probiotics in some strains
Food and food related products
• Fermented fruits and vegetables
– Found around the world
– With bacteria, yeast and mold
– Depends mainly on native microbes
associate with the plants.
– Most included mixed culture
Fermentation
– Conditioning by human to favor growth
of beneficial microbes - salting
Products
Organisms
Sauerkraut
Lactobacillus mesenteroids, Lactobacillus
plantarum
Pickles
Pediococcus cerevisiae, Lactobacillus
plantarum
Soy sauce
Rhizopus oligosporus, Rhizopus oryzae,
Lactobacillus delbrueckii, Saccharomyces
rouxii
Coffee
Leuconostoc mesenteroides,
Saccharomyces marscianus,
Flavobacterium spp., Fusarium spp.
Cocoa products
Leuconostoc mesenteroides, Candida sp.
Food and food related products
• Flavoring agents: amino acids and nucleotides
– Monosodium glutamate, inosinic acid – flavor
enhancers
– Originally use seaweed, fish
– Produce by enzymatic hydrolysis of yeast RNA
(Candida utilis)
– Now direct fermentation = monosodium glutamate
and 5’-IMP (inosine monophosphate)
– Brevibacterium ammoniagenes produce inosine and
then chemically phosphorylated
Non-food products
• Enzyme production
– Produce in large scale around the world
– Major producer – Novozyme (44%), Genencor
Intl, USA (18%)
Enzyme
Organisms
Use
Bacterial proteases
Bacillus, Streptomyces
Detergents
Asparaginase
Escherichia,
Serratia
Antitumor agent
Glucoamylase
Aspergillus
Fructose syrup production
Bacterial amylases
Bacillus
Starch liquefaction, brewing,
baking, feed, detergents
Glucose isomerase
Bacillus, Streptomyces
Sweeteners
Rennin
Alcaligenes,
Aspergillus,
Candida
Cheese manufacture
Pectinase
Aspergillus
Fruit juice clarification
Lipases
Micrococcus
Cheese production
Penicillin acylase
Escherichia
Semisynthetic penicillins
Non-food product
1. Vitamins
2. Antibiotics
3. Alkaloids
4. Steroids
5. Non-Steroid Hormones/cell regulators (cytokines):
1. Epidermal growth factor
2. Proinsulin
3. Insulin
4. Human growth hormone
5. Somatostatin
6. Interferons
7. Platelet-derived growth factor
8. Fibroblast growth factor
9. Tumor Necrosis Factor
10. Other cytokines are coming on line all the time
Non-food product
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Blood coagulating factor XIII
The restriction enzymes
Other enzymes (e.g. ligase, DNA polymerases etc.)
Solvents
How do the microbes produce these products?
• Genetically modified microorganisms
• Clone genes that encode protein of interest into
vectors and express this genes in microbes used
in production.
• Able to produce high amount of compounds than
in original organisms with ease of manipulation.
• Microbes able to utilize waste from other process
as a raw material for fermentation.
• Antibiotics
– Penicillin by Penicillium chrysogenum
– Now most of the antibiotics were discover from soil
microorganisms especially in Streptomyces spp.
– Engineered genes into production strains.
– Modification of existing antibiotics.
Streptomycin from Streptomyces griseus
• Alkaloids
– Nitrogen containing organic compounds that
derived from plants
– Have medicinal purposes:
• Atropine – dilates pupils of the eyes (some muscle
disease)
• Morphine and codeine – relief of pain
• Cocaine – local anesthetic
• Quinine, caffeine, nicotine, strychnine, serotonin , and
LSD
serotonin
• Steroids
– Types of products
include cortisone
and reproductive
hormones
• Human proteins
• Biotech Drugs
– Mainly recombinants proteins and monoclonal
antibodies.
– Highly active fields of biotech
– Lots of new products each year
• For list of product approve in US – www.bio.org
– Timeline in drug development