Food and industrial microbiology
BIOS 303: Lecture 22
Lecture 22: food & industrial microbiology
§ Food spoilage and preservation
§ Foods made by microbes
§ Especially dairy, chocolate, and alcoholic beverages!
§ Basics of industrial microbiology
§ Products of industrial microbiology
§ Antibiotics, vitamins, and enzymes
§ Biofuels and biocontrol
§ Microbe-made mammalian products, transgenic
plants and animals, and metagenomics/genome
mining
Food spoilage
§ Any change in the appearance, smell, or taste of a food
product that makes it unacceptable to the consumer
§ May or may not be safe to eat,
but unpalatable = will not be
purchased or readily consumed
§ Loss of $$$ in food industry =
higher prices!
Kulmalukko
Categories of food spoilage
§ Vary in water activity (aw) = water available for metabolic
use
1. Perishable: spoils fairly quickly; fresh food items (e.g.,
meats, fish, eggs, milk, fruits and veggies); high aw
2. Semiperishable: slower than #1 (e.g., nuts and potatoes)
3. Stable/non-perishable: flour, sugar, rice, dry beans, etc.
§ Microbial growth rate/lag phase length depend on:
§ Size of inoculum, temp, nutrients available, aw,
preservation methods, etc.
Common fresh food spoilage microbes
§ Fruits and vegetables: Erwinia, Pseudomonas,
Corynebacterium sp. mostly veggies
§ Fungi: Rhizopus, Aspergillus,
Penicillium may affect both
§ Meat/poultry/eggs: Acinetobacter,
Pseudomonas, Salmonella,
Escherichia, Listeria, Campylobacter, fungi, etc.
§ Milk: Pseudomonas, lactic acid bacteria (Lactobacillus),
Streptococcus, etc.
§ High-sugar foods: Clostridium, Bacillus, fungi like
Saccharomyces, Penicillium, etc.
See text table
Food preservation
§ Cold/refrigeration (but psychrophiles can still grow!)
§ Pickling: weak acids (e.g., vinegar) to
lower pH
§ Drying/dehydration: Decreases aw
§ High salt and sugar do this
§ Drying: lyophilization (freeze-dry)
§ Spray drying: rapid drying with heat;
powdered milk, concentrated liquids
Spray dryer
§ Heating: (ultra)pasteurization (= flash-heat
and cool) helps preserve food quality
Food preservation
§ Canning: seal food in and heat it; key = temp and time
§ Depends on food, density, acidity, container size, etc.
§ Can use steam + pressure
§ Cans are anoxic = never aerobes
§ If not properly sterilized, anaerobes (e.g., Clostridium)
can grow -> produce exotoxins
§ Gas = bulging cans bad!
Food preservation
§ High pressure: ≤ 100,000 psi
§ E.g., salsas, juices, and ready-to-eat meats
§ Chemical: > 3,000 food additives
§ E.g., sodium or calcium propionate, sodium benzoate,
sorbic acid, ethylene oxide, sodium nitrite, and
formaldehyde!
§ Irradiation: UV, gamma, beta, etc. rays
§ Fresh meat and flour
Most importantly…
§ Without microbes, there would be no…
§ Fermentation: anaerobic catabolism of organic C by
yeasts (e.g., Saccharomyces cerevisiae) and bacteria
into alcohol, acid, and gas H. Thompson
Microbes also help make…
§ Often acidic products = also prevent spoilage!
See text table for a bunch! Shant, H. Thompson
Other fermented stuff
§ Meats like sausages; veggies like cabbage
(sauerkraut), olives, pickles, other pickled stuff
§ Soy sauce: fermentation of soybeans and wheat by the
fungus Aspergillus
§ Vinegar is produced by Acetobacter, Gluconobacter
Microbes + Wisconsin = cheese!
§ By 1915, Wisconsin was the
leading U.S. producer of
dairy
§ Still the leading producer of
cheese
§ 1990s: Wisconsin lost the
top milk production crown to
California…
§ …but 90% of our milk goes
to make cheese!
H. Thompson, K. Weller (USDA), WI Milk Marketing Board
Microbes + Wisconsin = cheese!
§ 90% of that milk goes to make a LOT of cheese varieties…
WI Milk Marketing Board
It’s a great time to be in the cheese biz
§ Rennet: complex set of enzymes from rumen; separate
into solid curds and liquid whey
§ Chymosin: protease that curdles milk casein
WI Milk Marketing Board
The state microbe: Lactococcus lactis
§ 2010: Wisconsin state legislators
tried to adopt our official state
microbe: Lactococcus lactis
§ Not so frivolous when you look at
the varieties (and economy):
§ Cheddar, Colby, Monterrey jack,
Camembert, Brie, Roquefort
§ Cottage, cream cheese, sour
cream, and buttermilk!
L. lac's
J. Heintz, K. Todar (UW-Madison)
Chocolate is fermented!
§ Ripe cocoa tree pods are harvested and immediately
slashed
§ Begins fermentation process on the seeds and pulp
§ Placed in a large “sweat box” or in the ground and
covered with banana leaves
Chocolate fermentation
1. Yeasts break down the pectin
around seeds, then ferment the
sugars into ethanol and gas (CO2)
§ Stopped by increasing temps and
ethanol production
2. Lactic acid bacteria then produce
lactic acid = lower the pH
H. Thompson
Chocolate fermentation
3. The acid helps other bacteria grow
(e.g., acetic acid bacteria like
Acetobacter) -> ferment and make
acetic acid
§ Acetic acid is essential to making
chocolate ->
§ It not only kills the seed sprout, but
adds to the delicious flavors!
H. Thompson
Chocolate fermentation
§ Lasts about 5-7 days
§ Stop too soon = bitter
§ Stop too late = microbes
produce bad tastes
§ End product: fermented seeds =
chocolate beans
§ Beans are sun- or oven-dried, then
roasted -> kills most microbes
H. Thompson
Microbes and bread
§ Ancient Egyptians used yeast to leaven bread
§ A bakery from 2575 B.C. found at the
Giza pyramid site
§ Oxic conditions: yeast make CO2 but
minimal alcohol
§ Amylases in moistened dough release
maltose and glucose from starch
§ S. cerevisiae maltase/other enzymes break down bread
sugars -> ferment to CO2 = makes dough rise!
§ Fermentation products contribute to flavor
Wine production
§ Enology: the study of wine production
§ First crush grapes and separate the
juice = must
§ Red wine: leave red grape skin w/ must
to release pigments
§ White wine: no skin or white grapes
§ Fruit juices can be naturally fermented w/ wild yeasts, but
results can be inconsistent…
§ So, Saccharomyces ellipsoideus is often added
Wine fermentation
§ Primary fermentation for ~3-5 days at ~20-28°C
§ Ferments to ~10-14% ethanol; higher kills the yeast
§ Then, aged: clearing and flavor development
§ Red wine > white
§ Malolactic fermentation: secondary; important part of
red wine (and some white) production
§ B/C grapes have too much malic acid = sharp/bitter
§ Done by lactobacilli
§ Ferment malic acid -> lactic acid and CO2
§ Other reactions also change composition
Summary of red and white wine production
Brewing beer
§ Malt: natural enzymes digest starch of germinated
barley seeds/grains -> glucose
§ = Food for yeast
§ Mash: malt, etc. is cooked in water and
steeps in large tub
§ Goal: obtain fermentable liquid
§ Malt enzymes keep breaking down complex
nutrients to maltose/glucose/proteins/amino acids/etc.
§ Wort: aqueous part separated by filtering (lauter tun)
§ Hops (dried female flowers of the hops plant) are added
§ Hops inhibit spoilage microbes and add flavor
Brewing beer: fermentation
§ Wort boiled for hours, then filtered
§ Heat inactivates the
hydrolytic enzymes
§ Yeast then added
§ Ferment sugars to ethanol
and CO2
§ Top yeasts = ales =
Saccharomyces cerevisiae
§ Bottom yeasts = lagers = S. carlsbergensis
§ Top yeasts ferment at warmer temps = faster
§ 5-7 days at 14-23°C, vs. 8-14 d at 6-12°C for bottom
Photo courtesy G. Richards, from the delicious Anchor Brewery in San Francisco
Brewing beer: sanitation
§ Beer production: pathogens almost never survive
§ (Contamination can occur)
§ Prior to water sanitation, beer was actually “healthier”
than water = less likely to give you gastroenteritis!
Distilled alcoholic beverages
§ Distillates = products of heating fermented liquids to
volatilize alcohol (and other parts)
§ Distillation = condense and collect distillate
§ Consequence: jacks up the
liquor concentration!
§ Distilling any alcoholic liquid gives a distinct product:
§ Distilled malt brews -> whiskey; wine -> brandy
§ Fermented molasses -> rum; grain/potatoes -> vodka;
grain + juniper berries -> gin
§ Have good and bad volatiles; aging gets rid of bad
Industrial microbiology
§ Commercial products produced on a large scale by
microbes
§ Distinct from biotechnology = using
microbes to produce commercial
products (often via genetic engineering)
§ May use in industrial, but…
§ Scale: getting microbes to overproduce
stuff on a large scale as efficiently as
possible Industrial-scale
§ = Optimize growth rate fermenters
(each 240 m3)
Examples of industrial microbial products
§ Antibiotics (penicillin, tetracycline)
§ Enzymes (laundry proteases and lipases)
§ Food additives (vitamins, amino acids)
§ Chemicals (biofuels, biocontrol, citric acid)
§ Alcoholic beverages (see previous!)
Microbial production = efficient metabolism
§ Primary metabolite: forms during
log phase
§ E.g., ethanol
§ Often need to grow/survive (e.g.,
E get from fermenting to EtOH)
§ Secondary metabolite: near end of
growth = stationary phase
§ E.g., antibiotics
§ Often more complex = important!
§ Often don’t need for growth =
trickier for optimal production
Fermentors and scale-up
§ Fermentation: in industry, any large-scale microbial
process (whether or not true fermentation)
§ Fermentor: the vessel fermentation takes place in
§ 1,000 L (enzymes, molecular biology products) to
500,000 L (amino acids, crappy beer)
§ Scale-up: transfer from small-scale lab process to large-
scale industrial/commercial equipment
§ Rarely behave same on large scale
§ E.g., lotta cells = high O2 demand;
often limiting
§ In lab, easily tweak pH, temp, nutrients
§ Then, pilot (~300-3,000 L) before large
scale
Antibiotic isolation and production
§ Isolate and screen for new; now, computer modeling of
drug-target interactions
§ Past: more via screening (still in academics)
§ E.g., cross-streak method: grow candidate antibiotic
guy, then streak other species and see if inhibited:
ß Isolate,
then screen à
§ Produce and purify: isolate high-yield strains (e.g.,
mutagenesis)
Vitamin, enzyme, etc. production
§ Vitamins: total sales second only to antibiotics in the
pharmaceutical industry
§ Very complex = best to have
microbes do the work! à
§ Other nutrients: amino acids, etc.
§ Exoenzymes: secreted microbial
enzymes
§ Food/health, laundry, and textile industries
§ E.g., proteases (laundry detergents); amylase digests
starch (bread)
See text tables for more e.g.’s
Biofuels
§ Fuels derived by microbes from recent organic matter
Ethanol:
§ 60 billion liters of ethanol per year!
§ Mostly Saccharomyces et al.
fermenting glucose from cornstarch
§ Burning releases CO2, so why better Methane:
for the environment than fossil fuels?
Biofuel use and production
§ Gas often ~10% EtOH; used in almost all gasoline
engines
§ Higher EtOH (85%) only usable by modified engines
§ DOE goal: substitute 30% of gas w/ biofuel by 2030
§ Bioethanol production from corn is not ideal:
§ Less energy per weight vs. other fuels
§ Currently: 25% more energy to produce 1 L EtOH
than the EtOH itself has!
§ Absorbs water = can’t transport via current pipelines
§ Still, easily replenishable C + good enviro. = why use!
Alternate biofuel sources
§ Other plant sources (e.g., switchgrass): grow fast, easy
to harvest/degrade cellulose -> glucose
§ Harvest CH4 from agriculture,
wastewater plants!
§ H2: 3X more potential energy per weight vs. EtOH
§ Working at making efficient, but…
§ No liquid form yet = storage and distribution probs
§ Can’t be mixed with current fuel or used by cars
§ Use as fuel cells likely better
§ Algae: some produce long-chain hydrocarbons ~crude
oil; scale-up is current challenge
Microbes: insect pest control in agriculture
§ Insects pests cause billions of dollars of damage in
agriculture and forestry (spongy moth, corn borers, etc.)
§ Biocontrol: bacteria like Bacillus thuringiensis produce
toxins used to kill pests = natural alternative to pesticides
§ Bioinsecticides, biopesticides, etc.
Bacillus thuringiensis Bt toxin
§ Bt insecticide used commercially in U.S. since 1958
§ Sprayed on plants to limit
destruction by insects
§ Toxin genes can be introduced into
genetically-modified plants
a) Normal leaves eaten;
b) Leaves expressing Bt toxin
Transgenic plants and animals
§ Transgene: gene from one org. inserted into another
§ Genetically modified organism: any org. genetically
engineered, but often used to refer to those with transgene
§ One gene with well-defined function = fairly safe!
§ E.g., plants expressing Bt toxin in their chloroplasts
§ E.g., immune-deficient mice in medical research
§ E.g., improve livestock (e.g., metabolize vitamin)
Verifying pig gene
expression system with GFP
Expressing mammalian genes in bacteria
§ = biotechnology and genetic engineering/cloning
§ Examples used in therapy:
§ Insulin (diabetes)
§ Nerve growth factor (stroke, degenerative neurological
disorders)
§ Relaxin (facilitates childbirth)
§ Somatotropin (certain growth abnormalities)
§ Urokinase (blood clotting)
§ Erythropoietin (certain types of anemia)
§ α-interferon (antiviral, antitumor)
§ Human DNase I (cystic fibrosis)
Genome mining and pathway engineering
§ Genome mining: isolate novel, potentially useful genes
from the environment without culturing
§ E.g., metagenomics of
environmental sample
§ Clone genes into plasmids ->
express (e.g., in E. coli) ->
screen properties of interest
§ Test for, e.g., novel antibiotic
activity
§ Pathway engineering: modify
metabolic pathways by adding/
changing genes = microbe makes a new product!
