FST 114: Midterm 2

What is the metabolism for Leuconostoc?

facultative hetero fermentative

What is the metabolism for Lactococcus?

obligate homo fermentative

What is the metabolism for propionibacterium?

hetero fermentative

What is the metabolism for streptococcus?

obligate homo fermentative

What is the metabolism of cheddar cheese?

obligate homo fermentative

What are the two main proteins in cultured dairy products?

casein and whey

What are caseins?

majority of the milk proteins, forms micelles, colloidal solution

What are micelles? What are its features?

amphipathic molecules, - charged

What are the subgroups of casein?

alphaS1, alphaS2, beta, and kappa

What is the feature of kappa casein?

glycosylated by sialic acid, forms the outer layer of micelle

What does "glycosylated" mean?

carbohydrate or sugar chain attached

Why is milk considered a colloidal solution?

casein protein is dispersed in water as micelles

What is whey?

20% of milk proteins, very soluble

What is the normal milk pH? How can this pH be dropped?

6.5 to 6.7, addition of acid or growth of acid producing bacteria

What is the isoelectric point (of casein)?

solubility of casein is at its lowest point

Why is reaching the isoelectric point of casein important?

casein loses its negative surface charge, causes micelle to aggregate and precipitate

What is the first step of precipitation of acid (in milk)?

Ca phosphate will dissolve, formation of ionized Ca, more rigidity from strong ionic bonds

What is the second step of precipitation of acid (of milk)?

pH of solution approached the isoelectric point of casein

What is the feature of LAB metabolism?

auxotrophic, require exogenous branched AA

What AA to LAB metabolism require?

glutamate, histidine, methionine

What do starter bacteria must have to hydrolyze milk proteins to liberate essential AA?

proteolytic system, proteolysis to obtain AA

What is the test used for lactose utilization and for proteolysis?

litmus milk test

What cleaves casein into oligopeptides? What are its features?

protease, anchored to the cell wall

What do proteases cleave?

casein

What and where do the oligopeptides get transported to?

cytoplasm, oligopeptide transport system

What transports the Di/Tri peptides?

di/tri peptide transport system

What are the two key enzymes found in the proteolytic system in LAB? Where are they found?

protease outside of the cell , peptidase in the cytoplasm

What does peptidase cleave?

oligopeptides into di/tri peptides

What cleaves Di/Tri peptides into AA?

di/tri peptidase

How many transport systems are found in the proteolytic systems in LAB? Where does it go?

three, outside of the cell to the cytoplasm

What is the major enzyme found in the Lactoccal Proteolytic System?

PrtP

What is the PrtP? What is its purpose?

extracellular serine proteinase

Why is PrtP important to LAB?

cannot grow in milk because it lacks AA precursors

Where is PrtP found?

outside of the cell, attached to the peptidoglycan

What is PrtP activated by? What is the purpose of this?

PrtM cleaving the pro sequence, folding helper and maturation protein, ensure proper activation

If there is a mutation with PrtM, what occurs?

PrtP cannot fold properly and remain inactive, casein can't be cleaved into peptides

What are the two parts of PtrP?

pre-pro sequence

What is the purpose of the pre-sequence?

signal peptides that directs protein through the sec secretion pathway

What is the purpose of the pro-sequence?

keeps enzyme inactive until PtrM folds PtrP, localized outside of the cell

What is the first step of Lactococcal Proteolytic System?

PrtP gene is transcribed, translated into pre-pro protein

What is the second step of Lactococcal Proteolytic System?

pre sequence directs PrtP to the sec secretion system, pre sequences cleaved

What happens to the protein during the second step?

anchored to the cell wall, remains inactive

What is the third step of Lactococcal Proteolytic System?

pro proteins folds PtrP, inhibiting catalytic activity

What is the fourth step of Lactococcal Proteolytic System?

PrtM cleaves the pro sequence, activating PrtP

What does it mean when the enzyme is activated (in the Lactoccal Proteolytic System)?

maturation into functional serine protease

What is the fifth step of Lactococcal Proteolytic System?

C terminal anchoring domain of PrtP interacts with the cell wall to allow casein interaction

What is the last step of Lactococcal Proteolytic System?

active PtrP cleaves milk/beta casein into oligopeptides

What are the two types of PrtP?

PI and PII proteinase

What is the purpose of the PI proteinase?

hydrolyzes the C terminal of beta casein, produces bitter peptides

What does PI proteinase generate?

8-17 mer peptides that can be transported into the LAB cell via the oligopeptide transport system

What is the purpose of PIII proteinase?

acts efficiently on diverse set of casein

What are the membrane transport proteins for peptides generated by? Where?

outside of the cell by PrtP

What cleaves from the first AA from the N terminal of the protein?

3 aminopeptidase, Pep N, A, C

What releases AA from tripeptides?

tripeptidase, Pep T and 53

What releases AA from dipeptides>?

dipeptidase, Pep V and D

What does Lc. lactis subsp. cremoris use to hydrolyze internal peptides?

endopeptidase, Pep O and F, not in intact casein

What are critical for LAB's efficient growth on milk? Why are proteases not enough?

internal peptidases, proteases doesn't release free AA, only fragments

Why are internal peptidase important for cheese ripening?

dying starter culture release intracellular peptides, need to cleave into AA as precursors for flavor compounds

What are the components for the proteolytic system of Lactococcus?

cell wall associated proteases, transport systems, intracellular peptidase

What is the global transcriptional repressor in the proteolytic system?

codY

What is the purpose of codY repressor?

down regulates genes when nutrients are plentiful

When is the codY repressor active? What does that mean?

bound to isoleucine, leucine, and valine, repressing expression of the whole proteolytic system

When is the codY repressor inactivarted?

scarcity of nutrient like milk, cannot bind to DNA, increase breakdown of proteins for AA

What are the two starter cultures of yogurt? What are their features?

S. thermophilus, Lactobacillus bulgarius or helveticus, strictly homo fermentative

Who invented the term dysbiosis?

Mechnikov

What did Mechnikov do?

proposed fermented milk contains factors that prevent putrefaction

What is the goal of LAB fermentation in yogurt?

produce lactic acid, drop the pH to the isoelectric point of casein

What is the defect in production of yogurt?

syneresis, watery layer of yogurt

What are the shared features of S. thermophilus and L. bulgaricus/helevticus?

homofermenters, transport lactose via symport, use beta-galactosidase, export galactose back into media

Explain the features of the homo fermentation of S. thermophilus and L. bulgaricus/helevticus.

lactic acid production only, critical for acidification, pH drop to 4.6, and casein coagulation

Explain the features of the transport of lactose via symport of S. thermophilus and L. bulgaricus/helevticus.

transported into the cell with H+, maintains PMF, uses leloir pathway to metabolize galactose

Explain the features of the export of galactose of S. thermophilus and L. bulgaricus/helevticus.

don't metabolize galactose, results in galactose accumulation in final product

Explain the feature of beta-galactosidase of S. thermophilus and L. bulgaricus/helevticus.

LacZ, uses it to cleave lactose into glucose and galactose

What stops fermentation with S. thermophilus and L. bulgaricus/helevticus?

enough sugar present that causes pH drop to be toxic to them

Why is the galactose build up not a problem with yogurt?

meant to be consumed fast

Why would the galactose build up be problematic to cheese production?

can be fermented by other microbes, influence flavor

Why is there a proto-cooperation among yogurt starters?

lactobacillus bulgarius creates low pH environment for s. thermophilus

What does S. thermophilus create during the metabolism of lactose? How does this help L. Bulgarius?

CO2, formic and lactic acid, promotes folic acid and FA biosynthesis for L. bulgarius growth

What are the factors that help flavor development in yogurt?

non-volatile acids, volatile acids, carbonyl compounds

What are non-volatile acids? What are some examples?

do not evaporate easily, stay dissolved in yogurt, lactic and pyruvic acid

What are volatile acid? What are some examples?

evaporate easily, not high in concentration, acetic acid

What are the carbonyl compounds found in yogurt?

acetaldehyde, acetoin, diacetyl

What is the production of diacetyl responsible for?

butter flavor

What are the main enzymes found in the diacetyl pathway?

citrate lyase, oxaloacetate, dehydrogenase

What are the transporters found in the diacetyl pathway?

citrate permease

How many carbons does citrate have?

6C

How many carbons do oxaloacetate have?

4C

How many carbons do pyruvate have?

3C

How many carbons do acetate have?

2C

How many carbons do acetoin have?

4C

How many carbons do alpha-acetolactate have?

5C

What is the first step of the diacetyl pathway?

citrate is transported into the cell via citrate permease

What is the second step of the diacetyl pathway?

Intracellular citrate is converted into oxaloacetate and acetate via citrate lyase

What is the third step of the diacetyl pathway?

oxaloacetate is converted to pyruvate and CO2 via the oxaloacetate decarboxylase

What is the fourth step of the diacetyl pathway?

pyrvate is converted into diacetyl ONLY in the presence of oxygen

If there is no oxygen, what happens to diacetyl?

converted to 2,3 butanediol, diacetyl concentration decreases

What are the key enzymes in the butanediol fermentation pathway?

ALS, ADC, BDH

What is ALS?

alpha acetolactate synthase

What is BDH?

butanediol dehydrogenase

What is ADC?

acetolactate decarboxylase