Biological Methods of Preservation

Question 1

Define Biopreservation:

Give some examples:

Answer 1

Use of Microorganisms, their metabolic products, or both to preserve food (excluding fermentation)

• controlled acidification
• bacteriocins
• bacteriophages

Question 2

What is controlled acidification?

Answer 2

acidification of food for preservation:

done by direct addition of organic acid, fermentation, or use LAB to produce in situ

Question 3

What is MicroGard?

Answer 3

form of controlled acidification:
contains fermentable carb + customized culture (depending on product)
acts as safeguard: if food temperature abused -> MicroGard microbes will grow and make acid, prevent pathogens

Question 4

What are the main benefits of MicroGard?

Answer 4

1. protect shelf life/safety
2. maintain organoleptic qualities
3. “natural” product

Question 5

What is the Winsconsin process, and what are the advantages?

Answer 5

add less nitrite, 0.7% sucrose, LAB: to prevent C. botulinum in bacon

less nitrite needed (less potential carcinogens)

Question 6

What are bacteriocins?

Bacteriocins produced by ____ are of particular interest.

Answer 6

antimicrobial peptides produced by bacteria that are lethal to others, but not the producer (or humans)

LAB

Question 7

What are the different categories of bacteriocins?

Answer 7

Class I, Class II, Class III, Class IV

Question 8

What are the Class I bacteriocins and how are they made?

Answer 8

unusual AA; made by posttranslational modification

ex: dehydroalanine, dehydrobutyrine, lanthionine, methyllanthione

Question 9

What is a specific subcategory of Class I bacteriocins? How is it produced?

Answer 9

Lantibiotics (containing lanthionine ring)

rxn of dehydro-amino acids with cysteine -> thioether lanthionine

Question 10

What is the best characterized LAB bacteriocin? What category does it belong in, and how is it produced industrially?

Answer 10

NISIN
class I bacteriocin

made by culturing L. lactis (not synthesized chemically) -> for cheese/meat/beverages

Question 11

True/False: Nisin is produced by lactic acid bacteria

Answer 11

True

Question 12

What is an example of a lantibiotic not made by LAB? What is it used for?

Answer 12

subtilin (made by Bacillus subtilis)

antibacterial action; also protease (food, laundry detergent, lens cleaners)

Question 13

What are 2 examples of lantibiotics?

Answer 13

subtilin, nisin

Question 14

What are class II bacteriocins?

Answer 14

small heat stable proteins

with consensus leader sequence that signals to cell to export protein

Question 15

What are the subclasses of Class II bacteriocins?

Answer 15

IIa: active against L. monocytogenes
IIb: requires 2 different peptides for activity
IIc: requires reduced cysteine for activity

Question 16

What are class III bacteriocins?

Answer 16

> 30kDa (large) heat-liable proteins

Question 17

What are class IV bacteriocins?

Answer 17

with lipid or carb components (unknown function)

Question 18

What method is used to discover new bacteriocins?

Answer 18

Place colony of bacteriocin producer onto agar inoculated with lawn of other bacterium -> incubate, observe for ZONE OF INHIBITION (circle of no growth)

Question 19

True/False: a zone of inhibition indicates there is definitely bacteriocin production

Answer 19

False: could be other inhibitory substance (organic acid, H peroxide, phage contamination)

Question 20

How can you differentiate between the potential causes of a zone of inhibition?

Answer 20

bacteriocin: sharp edges, colony in center
organic acid: fuzzy edges
Phage: no colony n center

Perform test: remove colony, apply protease -> if it was bacteriocin, then the bacteria should now be able to grow over

Question 21

What is the general mechanism of all LAB bacteriocins?

Answer 21

disrupt integrity of cytoplasmic membrane

1. bind and create pore (hole)
   or
2. membrane solubilization

Question 22

Bacteriocins are most effects against cells in what state?

Answer 22

vegetative

Question 23

What is the effect of bacteriocins on vegetative cells?

Answer 23

disrupt membrane -> ions, AA, ATP will spill out -> ruins chemical/electrical gradient -> cannot generate energy, cannot protect from outer environment -> inhibition or death

Question 24

What property of bacteriocins allows for its effects against the membrane?

Answer 24

amphiphilic cationic peptides -> can insert into membrane

Question 25

True/False: Nisin will encourage germination of spores, making it risky to use as a bacteriocin

Answer 25

False: it DOES encourage germination, BUT: will attack pre-emergent spore (before starts growing) (germinated spore is more vulnerable than dormant)

Question 26

What are Colicins?

Answer 26

bacteriocins made by some strains of E. coli against other strains

Question 27

What do E. coli strains need to be able to produce colicin?

Answer 27

colicinogenic plasmic | genetic info for synthesis, immunity, and release

Question 28

Colicin expression is under the ____ regulatory system

Answer 28

SOS

Question 29

Describe the mechanism of action for colicin:

Answer 29

binds to specific receptors (outer membrane proteins for nutrient uptake) -> enter, transported by Tol or TonB -> disrupt inner membrane or attack DNA, rRNA, or tRNA

Question 30

True/False: colicin must enter the bacterial cell in order to be lethal

Answer 30

true (attacks inner mechanisms)

Question 31

The 2 classes of colicins:

Answer 31

enzymatic pore-forming

Question 32

What are the lethal modes of action of enzymatic colicins?

Answer 32

1. block synth of peptidoglycans 2. cleave tRNA -> stop protein synth 3. cleave 16S rRNA -> stop protein synth 4. DNA degradation

Question 33

What are the approved ways to add bacteriocins to food?

Answer 33

1. add purified bacteriocin directly 2. add producing culture (make in situ) 3. choose bacteriocin making culture to include for fermentation

Question 34

Give an example of a bacteriocin that is added to food: | What are its main purposes?

Answer 34

Nisin: for milk, cheese, dairy, canned food, smoked fish, mayo, baby food (GRAS) anti-listerial agent; sensitize spores to heat (prevent botulism, lessen thermal treatment time needed)

Question 35

Is Nisin used alone or in conjunction with other treatments?

Answer 35

Together - part of a multiple barrier inhibitory system ex: with mod. atmosphere storage (fresh fish, ground pork)

Question 36

What is a bacteriocin that is produced in situ in food? What does it protect against?

Answer 36

Pediocins (in wieners, dressings, cottage cheese, meat, RTE salad) effective against vegetative L. monocytogenes (not good for spores)

Question 37

The protective effect of pediocin is more effective with (higher/lower) temperatures, and better in (aerobic/anaerobic) conditions

Answer 37

lower | anaerobic

Question 38

Why is a bacteriocin producing strain beneficial for the making of fermented foods? Give an example

Answer 38

helps inactivate existing bacteria -> allow for culture to grow, better chance of success ex: adding pediocin producers to sausage -> reduces L. monocytogenes

Question 39

how does bacteriocin producers affect shelf life in fermented foods? give an example:

Answer 39

Increase shelf life | ex: add nisin producing LAB to cheese -> shelf life increased from 14 to 87 days

Question 40

True/False: unlike antibiotics, bacterial resistance is not a concern with bacteriocins.

Answer 40

False: resistant strains exist and can develop!

Question 41

What are some developed resistance mechanisms to bacteriocins that have been observed? How does specificity vary?

Answer 41

1. destruction of bacteriocin (specific or general) 2. modification (specific) 3. Altered receptors (specific) 4. Membrane composition (general)

Question 42

Can bacteriocin resistance be overcome by applying a combo of different bacteriocins? Why or why not?

Answer 42

Yes, if it is a specific resistance mechanism No, if it is a general mechanism (need a different functioning bacteriocin)

Question 43

Is cross-resistance with antibiotics an issue for bacteriocin resistance?

Answer 43

Usually no; | Multidrug resistant bacteria can still be sensitive to bacteriocin

Question 44

What is a resistance mechanism observed in L. monocytogenes against nisin?

Answer 44

modified membrane composition - more straight chain FA (less fluidity) -> nisin can't insert as easily

Question 45

What is a specific defense mechanism against dehydroreductase bacteriocins?

Answer 45

Modification

Question 46

What is important in the food industry to combat bacteriocin resistance?

Answer 46

Apply multiple hurdle technology (use many diff steps/methods to kill bacteria) Bacteriocin resistant cells can be sensitive to other factors (heat, cold, etc)

Question 47

True/False; we normally consume bacteriophages

Answer 47

True (part of natural food microbiota)

Question 48

What are the main challenges with using phages for microbial control in food?

Answer 48

1. lack of consumer acceptance 2. possible resistance 3. low numbers of bacteria in food (phage cannot effectively grow) 4. targets specific species/strain

Question 49

Phages need _____ actively growing bacteria to be effective

Answer 49

10^5 - 10^6

Question 50

There are commercial phages used to control growth of ___, ___ and ____.

Answer 50

Salmonella, E. coli, Listeria

Question 51

How is bacteriophage resistance created? How can we overcome this?

Answer 51

changes in bacteriophage receptor site (proteins, LPS, lipoproteins on surface) use mix of different phages (if one is useless, other still are functional)