Lectures 11-14 Flashcards
Mother dough
- continuously maintained
- contains the microbial inoculum for subsequent doughs
- made primarily of water and flour
- rich in fermentable CHO (maltose)
- low pH allows LAB to flourish
- endophytes
Backstopping
take a piece of the mother dough from one batch and use it to start a new batch
Sourdough type 1
- spontaneous and relies on the naturally present microflora
- mixing flour + water, adding some of the previous batch’s mother dough
- Saccharomyces cerevisiae NOT added
- LAB/yeast are ‘fed’ daily w/ freshwater/flour
- fermented at ambient temperature for 6-24hrs
ph 3.8-4.5
-rise due to CO2 production
Sourdough type 2
- adding a starter culture of LAB to flour-water mixture -> acid tolerant Lactobacilli used
- strain chosen
a) fast acid producers
b) produce desirable flavour compounds - Saccharomyces cerevisiae added as a leavening agent -> rise
- temp >30 Celsius for 1 to 3 days; no extra feeding
- pH < 3.5
- pumpable liquid form and produced in bioreactors/tanks
- liquid culture sold commercially to bakeries
Type 1 vs Type 2
type 1:
- pH 3.8 - 4.5 ‘
- thick dough (low DY)
- temp: 20 - 30
Type 2:
- pH < 3.5
- thin dough (high DY)
- temp: > 30
Dough yield
Formula: (flour mass + water mass) * 100/ flour mass
high DY -> more water and thinner dough
- faster acidification
- better diffusion of produced organic acids/secondary metabolites
- better access to substrates
- high temperature and water content -> enhances acid production
Sourdough type 3
type 2 dried
- microflora inactivated; heat-resistant LAB is used
- Saccharomyces cerevisiae added
- adding flavour/texture to the product
- dried using Drum drier and miller down after
- stream heat; minimal heat damage; no/minimal caramelization/Mailard rxn; > 113 -> acetic acid
Main factors of Sourdough
- type of grain used
- age of mother dough
- DY
- co-presence of other organisms
- temp./season
- industrial vs. artisanal bakery
LAB and Yeast Symbiosis
- LAB -> high adaptable CHO metabolism (sugars) and acidification creates a low pH environment (proteases -> free amino acids for their growth)
- Yeast -> flavour, leavening, breakdown of phytic acid = more mineral availability)
- relationship between F. sanfransicisensis (process maltose) and k. humilis (can’t process maltose; acid-tolerant)
- F. sanfransicisensis imports and processes maltose into glucose and glucose-6-phosphate vis isomerizes
- glucose is released into the extracellular medium for use by K. humilis
- K.humilis supplies F. sanfransicisensis with vitamins and minerals
Shelf-life stability
Retrogradation -> crust becomes leathery, flavour diminished
mould contamination and development of rope caused by bacillus spp.
- unpleasant odours
- discoloured, sticky bread crumbs
addition of sourdough to bread dough -> slow stale process, prevent ropiness and prolong mould period
- due to the acidification of acetic acid
Sourdough spoilage
- prone to mould growth
- baking -> good kill step
- cooling bread before bagging to remove moisture
- use preservatives -> calcium phosphate, potassium sorbate, calcium lactate -> inhibits growth of mould
Kefir
- insoluble macroscopic particles
- protein mostly casein
- CHO mostly LAB exopolysaccharides
- unique kefir polysaccharide
- 1:1 glucose to galactose
- LAB + AAB + yeast + maybe some fungi
- hetero- and homo- LAB
- assimilating
Kefiran
- unique kefir polysaccharide
- LAB symbiosis w/ saccharomyces cerevisiae: improves the quantity of kefiran made
LAB present in Kefir
Primarily Lactobacillus, Lactococcus, Streptococcus, Leuconostoc (90%)
- act to preserve the milk -> acetic/lactic acid; flavour
Yeast present in Kefir
mostly saccharomyces cerevisiae, kluyveromyces marxianus, kluyveromyces lactis, Candida kefir
- produces ethanol, CO2
Role of kluyveromyces lactis
intracellularly produces B-galactosidase
- breakdown of lactose into glucose and galactose
Role of LAB
- intracellular produces B-galactosidase
- glucose -> homofermentative pathway
- galactose -> Leloir pathways - Lactose phosphorylated during transport and split by 6-phospho-B-galactosidase
- glucose -> homofermentative pathway
- galactose-6-phosphate -> Tagatose-6-pathways
Kefir Production - Traditional/home
- initially aerobic, but O2 is consumed and becomes anaerobic
- LAB -> combination of homo- and heterofermentative
- lactic acid, acetic acid, diacetyl, acetaldehyde, CO2
- yeast -> ethanol production
- recover grains afterwards for re-use
Kefir Production - Industrial
- milk content standardized
- pasteurized -> rid of undesired microflora
Starter culture - lactobacillus kefir, lactobillus kefiranfaciens
- no yeast
- anaerobic
after 24hr add flavour, etc. and packages - final product -> 0.8% to 1.% lactic acid
- tart flavour, smooth, viscous body
Cholesterol-lowering effects
- kefir grains reduce CH levels in milk
K. marianus -> ability to assimilate CH; put it in the grain
L. plantarum -> inhibits host CH uptake -> bile salts hydrolase that cuts up bile salts and prevents lipid uptake
Milk
initially pH: 6.0-6.5
- composition standardized
skim milk; extra milk solids added to facilitate texture
- total non-fats milk solids: 12 -15%
- enhances water binding, prevents syneresis
Stabilizers for Yogurt
high water availability (~0.97-0.99)
- spoilage is not an issue
improves viscosity/body; minimizes syneresis; uniformity batch -batch; function at low pH
examples: gelatin, pectins, starches, whey proteins, locust/carob, ultrafiltered milk
What type of pasteurization does milk normally get?
High-temperature short time (HTST)
30 minutes, ~85 Celsius -> denatures whey proteins (alpha-lactalbumin and b-lactoglobulin)
- more protein unfolding = more water binding capacity
Gel-Formation
“acid-induced milk gel”
Pre-starter:
- whey proteins denatured w/ heat -> these interact w/ k-caseins via hydrophobic interactions and cross-link w/ k-caseins through disulphide bonds
Post-starter:
- acidification (protons) leads to charge neutralization facilitating more casein-casein interactions
Calcium phosphates leached out of micelle
- normal complexes w/ phosphoserine residues in casein - further destabilize micelles
- isoelectric point (pH ~ 4.6) gelation of caseins occurs
Key properties of Yogurt Starter
- Freeze well
- rehydrates/wakes up and grows well
- make acid quickly - drop pH to required to target in 4-6 hours
- resistant to bacteriophages
- able to create the ‘right’ viscosity/body
- no acid production at low temp
- mild flavours/no off flavours
2 Starters in Yogurt
Streptococcus thermophilus (St) and Lactobacillus delbrueckii (Ld)
- thermotolerant bacteria
- both heterofermentative bacteria
- grown separately; different preferred growth conditions
Streptococcus thermophilus (St)
- grows first, lowers pH of milk for Ld
- anaerobic, but aerotolerant
- more acid-sensitive; inhibited sooner
Proteolytic system:
- casein is degraded by cell envelope-associated proteases (CEPS) -> in St called Prts
- serine protease
Lactobacillus delbrueckii
hydrolases caseins peptides via PrtB -> peptides transported via various transporters for different size fragments
- hydrolyzed by peptidases to amino acids -> feed St. higher acid tolerance
Metabolism
Both microorganisms express cytosolic B-galactosidase
- glucose -> homofermentative pathways
- galactose -> exports galactose to pump in lactose via lactose permeases (Lacs)
- energetically favourable to use galactose and lactose permease to transport lactose
Protocooperation
shift pH of milk closer to optimal for Ld
CO2, formic acid, folic acid (St. -> Ld.) -> cofactor/precursors in purine biosynthesis
Purines (Ld -> St) -> St is capable of making them but in co-culture, all turned off
LCFA (St. -> Ld) -> growth for Ld; lack the genes encoding enzymes capable of making LCFA; synthetase, desaturase, dehydrase
Where is the CO2 coming from?
Urease (St. -> Ld) -> enzymes, breaks urea into ammonia and Co2
ammonia
- CO2 -> Ld -> purines
- controls acidity in the media; acting as a protein sink
Exopolysaccharides (EPS)
modulate texture using high or low levels
potential issues -> excessive
- ropiness/stringiness
- undesirable; mask flavours; ‘slick’ mouthfeel
Controlled
- dec. temp = more EPS production
EPS -> homo- or heteropolymers; starts out as glucose-6-phosphate
Yogurt - Acetaldehyde
Primarily flavour
- light green apples/ tartness
- hydrolysis of threonine; decarboxylation of pyruvate; oxidation of acetyl-CoA
Yogurt Defect - overproduction of acid
pH < 4.0 and acid > 2.0% acid
- too high temp; cooling rate too low; acid production during storage
- control -> LAB strains sensitive to low pH or high temperature; incubate at 37 celsius
Syneresis
yellow/green H2O floating on top
- occur if too much acid
- week gel formation
- stabilizer -> bind up the extra water -> inc. gell strength
Yogurt stabilizers
- act to improve viscosity
- minimize syneresis (whey release)
- uniformity batch-batch
- must be functional at low pH
Villi
“capsular” EPS produces -> stays attached to the cell wall
- musty flavour -> yeast (Geotrichrim candidum)
- yeast and LAB present
- velvet like surface; aerobic; catabolizes lactic acid (pH ~4.4) ; release protease that breakdown amino acids and release ammonia
- secretes lipases
- not homogenized; mesophilic cultures
What is cheese?
cheese is the fresh or matured product obtained by the drainage after the coagulation of milk, cream, skimmed or partly skimmed milk, buttermilk or a combination thereof
Coagulation in cheese production
liquid is removed from cheese through the coagulation of milk proteins
- casein micelles
Three Coagulation mechanisms in cheese production
- Acid Coagulation
- Acid/heat coagulation
- Enzymatic coagulation
Acid Coagulation
- drop pH to 4.6: isoelectric point of casein
- addition of aid or lactic acid by LAB
- neutralizes charges on casein micelle
fresh cheese - 70-80% moisture
can be strained or pressed to remove whey and reduce moisture
ex. cottage cheese, cream cheese, quark,
Acid/heat coagulation
- Cooking to 90 celsius
- denaturation of whey protein - Drop pH to ~5.3
- addition of acid
fresh cheese with 50-80% moisture
can be pressed to remove extra moisture
e. ricotta cheese, paneer
Enzymatic Coagulation
Enzymes induce curd formation
Rennet: a mixture of enzymes found in the stomach of ruminant animals (main enzyme: chymosin, an aspartic protease)
Fungal proteases
Plant-derived proteases
Function: cleavage of k-casein
Enzymatic Coagulation - 2 phases
Primary phase
- cleavage of k-casein on the exterior of casein micelles = reduction in negative charge
- micelles being to aggregate
Secondary phase
- residual negative charge in casein micelles reduced by free Ca2+; further aggregation occurs
Three goals of the cheesemaker
- expel the correct amount of whey
- retain the correct amount of calcium phosphate
- incorporate the correct amount of NaCl (preserve)
Cheese production steps
- setting
- cutting
- cooking
- draining/knitting/pressing
- salting/brining
6 finishing/maturing/ripening
Step 1: Setting
Rennet/chymosin is added to the milk
- chymosin-acid protease; most active at pH 5.5
- the rate of acidification important - rapid causes a loss of calcium phosphate - crumbly cheese
Culture may be added or slightly before rennet to allow ph drop
a) coagulation
- length of renneting affects curd firmness
b) acidification by LAB
- final pH 4.6-5.1
- the rate of pacification affects calcium phosphate retention
- pH affects curd firmness -> low pH = firmer curd
Step 2: Cutting
curd is cut to initiate syneresis
surface area to volume ratio
- to inc. water removal, cut the curd into smaller pieces
smaller surd (grain sized) used for hard cheese, larger curds for softer cheese
Step 3: Cooking
Curds and whey are cooked and stirred to help remove moisture
higher temp/longer time = drier curd
affect mineral retention, buffer capacity, lactic acid production
Step 4: Draining/knitting/pressing
- curd is separated from the whey
- curd is allowed to fuse
- added pressure is often used to facilitate moisture removal and knitting
Step 5: salting/brining
- further removal of whey from, the curd
- provide an appropriate environment for ripening organisms
- prevents spoilage microorganism growth
Acidification
Fermentation of lactose to lactic acid by LAB
- naturally present; starter culture
Acid
- preserves cheese
- Inc., syneresis
- impacts enzymatic coagulation rate
The rate of acidification affects
- retention of colloidal calcium phosphate
- curd firmness
- gel syneresis
- pH at the start of ripening
Starter cultures microorganisms
Harder cheeses are made with thermophilic cultures
- more syneresis/moisture removal at higher temperatures
Mesophiles (< 39°C):
Lactococcus lactis subsp. lactis
Lactococcus lactis subsp. cremoris
Thermophiles (> 39°C):
Lactobacillus delbrueckii subsp. bulgaricus
Lactobacillus delbrueckii subsp. lactis
Lactobacillus helveticus
Streptococcus thermophilus
Lactococcus lactis
- Gram +ve cocci
- Mesophilic (20-
30°C) - Homofermentative
- Acid production inhibited above 39°C
- Cell death above 45°C
- membrane transport consumes ATP
- 1 lactose = 4 lactic acid
- galactose: TGP pathway
Lactobacillus delbrueckii/helveticus
- Gram +ve rods
- Thermophilic (40°C - 44°C)
- Homofermentative
- Used in high-temperature cooked
cheese (< 65°C) - Protocooperative with Streptococcus
thermophilus - antiporter transport of LacA Galactose is lost
- 1 lactose = 2 lactic acid + 1 galactose
Flavour development - three key components of cheese
- Fermentation of lactose to lactic acid
- Hydrolysis of lipids to fatty acids (lipases)
- Breakdown of casein to peptides, amino acids,and ammonia
Bloomy-rind cheese
Ex. Brie, Camembert
Penicillium camemberti can be added as freeze-dried spores
Geotrichum candidum can be added directly to the milk or to the salt brine or sprayed on the surface
Pre-ripening conditions:
- rapid acidification w/ mesophilic cultures
- low pre-aging pH: 4.6-4.7
- no cooking or pressing
- moisture >50%
- dry salt
Yeasts grow first, consume lactate and raise pH
- Geotrichum candidum
- Kluyveromyces lactis
- Saccharomyces cerevisiae
- Debaromyces hansenii
Molds (Penicillium) follow later
Geotrichum candidum
- proteinase activity releases peptides that stimulate the growth of Penicillium camemberti
Penicillium camemberti
- appears after 6-7 days
- lactic acid metabiluzes, raising pH > 7.0
- allows entry of flavour-producing bacteria
Why is bloomy-rind cheese softer near the exterior? A zonal cheesy gradient
- pH inc. on the cheese surface and a pH gradient forms
- CaPO4 precipitates at the rind as pH inc. and forms a gradient (high at the rind and low at the centre
- softening from the inc. pH, precipitation of CaPO4 at the rind results in a zonal pattern of ripening
Bloomy-rind cheese flavour
- A synergistic effect between G. candidum
and P. camemberti prevents bitterness
from developing in the cheese - P. camembert releases proteinases that
create large, bitter peptides - G. candidum and P. camemberti produce
aminopeptidases and carboxypeptidases
that break down bitter peptides
Washed-rind cheese
Ex. Limburger, Talgeggio, Muenster
Pre-ripening conditions:
- similar to bloomy rind, except:
- slower acidification
- higher starting pH of 5.0 to 5.4
Yeasts (G. candidum) raise pH to ~6.0 within a few days of ripening, opening the way for
coryneform growth
Cheese is washed regularly with salt solution: which stops mold growth
Brevibacterium linens
- yeast
- appears when surface pH rises above ~6
- extensive proteolytic activity
- volatile sulphur compounds and ammonia development = smelly cheese
- gives the cheese a red-orange colour
Interior Ripening - Cheese
- The interior of most cheeses are
anaerobic, inhibiting the growth of
cheese yeasts - Flavour development is primarily
the result of starter LAB (SLAB)
and non-starter LAB (NSLAB) - Proteolytic cascades convert the
casein to peptides, amino acids, and
finally to flavour compounds - This is the dominant ripening method
of common hard cheeses: Cheddar,
Gouda, etc.
Alpine (swiss) cheese
examples: Emmental
- S. thermophilus and L. helveticus work protocooperatively to ferment cheese after cooking
- S. thermophilus metabolizes lactose and excretes galactose back into the cheese
- L.helveticus imports galactose and uses it (Leloir pathway)
- Final pH = 5.1 - 5.3
- Brine in 20% salt and then ripen
Alpine (swiss) cheese - Pre-ripening conditions
- delayed acidification
- cooked up to 50 Celsius
- curd pressed
- low salt
- Warm temperature ripening (20°C - 24°C)
encourages the growth of Propionibacteria:
lactate -> propionic acid + acetic acid + CO2 - CO2 is trapped inside, forming the ‘eye’ associated with alpine cheeses
Propionibacterium freundenreichii
- Gram +ve rods, anaerobic to
aerotolerant - Use lactate as a substrate
- Sensitive to NaCl
- Optimal growth temperature 25°C
- Tolerant to 55 °C
- 3 Lactate = 2 propionate + acetate +
CO2 + ATP - Lactate -> pyruvate is oxidized to acetate or pyruvate can be reduced to propionate through the wood-werkmen pathway
Blue-veined cheese
Pre-ripening conditions:
- low pH of 4.5 - 6.0
- no cooking or pressing
- moisture 38-50%
- high salt
The interior aerobic environment required for Penicillium roqueforti
- No pressing: keeps space between curds
- Citrate heterofermentation by Leuconostoc cremoris and Lactococcus lactis
subsp. lactis bv. Diacetylactis produces CO2, fracturing the curds
Penicillium roqueforti
- Aerobic mould grows through cheese wherever oxygen is available
- Carries out extensive lipolysis and proteolysis relative to other hard cheeses, leading to strong flavours
- Converts free lipids to methyl ketones (fruity, floral, musty, spice, “blue cheese”)
- Flavour intensity correlates with methyl ketone concentration
Fish Spoilage
Relatively fast
- high water content in general
- historically, no access to refrigeration -> faster microbial spoilage
seawater harbour a large number of spoilage bacteria (psychrotophs)
fermentation allows for extending the edibility of the product in some form
Fish - Post mortem
4 main steps following death:
Rigour mortis -> resolution of rigour -> autolysis -> microbial spoilage
All contribute to spoilage or if controlled via external factors, fermentation
- temperature, fish size, species
Resolution of rigour
Proteolytic activity within muscle cells
disrupts actin/myosin associations
Muscles “relax”
Fish - Fermentation Routes
- Fermentation via enzymes/autolysis
- Microbial Fermentation
Fish - Alkaline Fermentation
The end product is ammonia
extensive autolysis occurs (post-mortem step 3) and salt represses unwanted microorganisms
Fish sauce pH < 7.0
- ammonia is a hallmark indicator that alkaline fermentation has occurred
pH range of the main classes of proteases
- aspartic proteases, cysteine proteases, serine proteases
main proteases in alkaline fermentation trypsin-like serine proteases
Endopetidases and exopeptidases
Microbial Fermentation
Natural seas salt
Lactic acid could be generated by LAB even without CHO present
Common genera:
- Bacillus, staphylococcus
LAB: Tetragenococcus
- generally contributes to the breakdown of fish
Post-Fermentation
Sauce allowed to settle
- The liquid removed is 1st grade
brine can be added to the solids
- 2nd grade but not as flavourful
solids can be ground and made into a paste
Airing out the sauce
- allows for the dissipation of “fishy” odours
- usually done out in the open with a cover
