Lectures 11-14 Flashcards
1
Q
Mother dough
A
- 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
2
Q
Backstopping
A
take a piece of the mother dough from one batch and use it to start a new batch
3
Q
Sourdough type 1
A
- 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
4
Q
Sourdough type 2
A
- 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
5
Q
Type 1 vs Type 2
A
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
6
Q
Dough yield
A
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
7
Q
Sourdough type 3
A
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
8
Q
Main factors of Sourdough
A
- type of grain used
- age of mother dough
- DY
- co-presence of other organisms
- temp./season
- industrial vs. artisanal bakery
9
Q
LAB and Yeast Symbiosis
A
- 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
10
Q
Shelf-life stability
A
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
11
Q
Sourdough spoilage
A
- 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
12
Q
Kefir
A
- 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
13
Q
Kefiran
A
- unique kefir polysaccharide
- LAB symbiosis w/ saccharomyces cerevisiae: improves the quantity of kefiran made
14
Q
LAB present in Kefir
A
Primarily Lactobacillus, Lactococcus, Streptococcus, Leuconostoc (90%)
- act to preserve the milk -> acetic/lactic acid; flavour
15
Q
Yeast present in Kefir
A
mostly saccharomyces cerevisiae, kluyveromyces marxianus, kluyveromyces lactis, Candida kefir
- produces ethanol, CO2
16
Q
Role of kluyveromyces lactis
A
intracellularly produces B-galactosidase
- breakdown of lactose into glucose and galactose
17
Q
Role of LAB
A
- 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
18
Q
Kefir Production - Traditional/home
A
- 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
19
Q
Kefir Production - Industrial
A
- 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
20
Q
Cholesterol-lowering effects
A
- 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
21
Q
Milk
A
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
22
Q
Stabilizers for Yogurt
A
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
23
Q
What type of pasteurization does milk normally get?
A
High-temperature short time (HTST)
30 minutes, ~85 Celsius -> denatures whey proteins (alpha-lactalbumin and b-lactoglobulin)
- more protein unfolding = more water binding capacity
24
Q
Gel-Formation
A
“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
25
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
26
2 Starters in Yogurt
Streptococcus thermophilus (St) and Lactobacillus delbrueckii (Ld)
- thermotolerant bacteria
- both heterofermentative bacteria
- grown separately; different preferred growth conditions
27
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
28
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
29
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
30
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
31
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
32
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
33
Yogurt - Acetaldehyde
Primarily flavour
- light green apples/ tartness
- hydrolysis of threonine; decarboxylation of pyruvate; oxidation of acetyl-CoA
34
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
35
Syneresis
yellow/green H2O floating on top
- occur if too much acid
- week gel formation
- stabilizer -> bind up the extra water -> inc. gell strength
36
Yogurt stabilizers
- act to improve viscosity
- minimize syneresis (whey release)
- uniformity batch-batch
- must be functional at low pH
37
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
38
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
39
Coagulation in cheese production
liquid is removed from cheese through the coagulation of milk proteins
- casein micelles
40
Three Coagulation mechanisms in cheese production
1. Acid Coagulation
2. Acid/heat coagulation
3. Enzymatic coagulation
41
Acid Coagulation
1. 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,
42
Acid/heat coagulation
1. Cooking to 90 celsius
- denaturation of whey protein
2. 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
43
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
44
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
45
Three goals of the cheesemaker
1. expel the correct amount of whey
2. retain the correct amount of calcium phosphate
3. incorporate the correct amount of NaCl (preserve)
46
Cheese production steps
1. setting
2. cutting
3. cooking
4. draining/knitting/pressing
5. salting/brining
6 finishing/maturing/ripening
47
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
48
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
49
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
50
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
51
Step 5: salting/brining
- further removal of whey from, the curd
- provide an appropriate environment for ripening organisms
- prevents spoilage microorganism growth
52
Acidification
Fermentation of lactose to lactic acid by LAB
- naturally present; starter culture
Acid
- preserves cheese
- Inc., syneresis
- impacts enzymatic coagulation rate
53
The rate of acidification affects
- retention of colloidal calcium phosphate
- curd firmness
- gel syneresis
- pH at the start of ripening
54
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
55
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
56
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
57
Flavour development - three key components of cheese
1. Fermentation of lactose to lactic acid
2. Hydrolysis of lipids to fatty acids (lipases)
3. Breakdown of casein to peptides, amino acids,and ammonia
58
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
59
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
60
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
61
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
62
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
63
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.
64
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
65
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
66
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
67
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
68
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
69
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
70
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
71
Resolution of rigour
Proteolytic activity within muscle cells
disrupts actin/myosin associations
Muscles “relax”
72
Fish - Fermentation Routes
1. Fermentation via enzymes/autolysis
2. Microbial Fermentation
73
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
74
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
75
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
