Biological methods for food preservation Flashcards
what is biopreservation?
is the use of microorganisms, their metabolic products, or both to preserve foods. However, this definition usually excludes fermentation
3 examples of biopreservation
- controlled acidification (where acid is produced by LAB in temperature-abused foods - extended period at 30C)
- use of bacteriocins
- use of bacteriophages
what is controlled acidification?
Organic acids can be added to foods, can be produced via fermentation, or LAB can produce lactic acid in situ and the controlled production of lactic acid can be an important form of biopreservation
what is microGARD?
natural, clean-label product designed to improve shelf-life protection
family of products that can be added to refrigerated food products:
- fermentable carbohydrate (usually lactose or dextrose)
- bacterial culture
=> combined to offer customizable protection for a variety of food products
why is microGARD useful in temperature abused products?
If the food is temperature abused the bacteria will grow and produce lactic acid, acetic acid, propionic acid, and bacteriocins to ensure the safety of the product despite the temperature abuse
what are the key benefits of MicroGARD? (3)
- Protect shelf life
- Maintain the organoleptic qualities of food
- Meet consumers’ demand for natural products
how was microGARD conceived?
was a way to use LAB to prevent the growth of C. botulinum in certain foods that could be temperature abused
=> The LAB do not grow under refrigeration conditions and will only grow if foods are temperature abused
what determined the effectiveness of in situ acidification?
depend on the products:
- pH
- buffering capacity
- target spoilage microorganisms
- concentration of fermentable carbohydrates
how are carcinogenic nitrosamines formed?
formed from nitrites in cured meats
what is the Wisconsin process?
When bacon without nitrites was inoculated with C. botulinum and incubated at 28C, 58% turned toxic
When bacon was prepared with less nitrite, 0.7% sucrose, and LAB starter cultures <2% became toxic
This is known as the Wisconsin process and was approved by the FDA for use in 1986
wha are bacteriocins?
antimicrobial peptides of bacterial origins that are lethal to some bacteria, but not the host that produced them. They are produced by virtually all bacterial species. Although, the bacteriocins produced by LAB are of particular interest to the food industry.
how to bacteriocins act to preserve foods?
are able to inhibit pathogens of serious concern, like L. monocytogenes, and are therefore of interest for food safety
bacteriocins are NOT …
antibiotics
bacteriocins can be divided into … major classes
4
what are the class 1 bacteriocins?
Contain unusual amino acids (dehydroalanine, dehydrobutyrine, lanthionine, and methyllanthione) produced by posttranslational modification. Dehydro- amino acids react with cysteine to form thioether lanthionine
bacteriocins containing lanthionine rings are commonly referred to as ,,,
lantibiotics
which class 1 lantibiotic bacteriocin is the best characterized LAB bacteriocin? how is it obtained?
Nisin - GRAS
obtained from the culturing of Lactobacillus lactis on natural substrates and is not chemically synthesized.
how does Nisin work?
used in processed cheese, meats, beverages, etc. during production to extend shelf life by suppressing Gram-positive spoilage and pathogenic bacteria.
- has anti-listeria properties
- sensitizes spores to heat (reduced thermal processing times)
- used in products where botulism is a concern
…. is also a lantibiotic, but is also an effective protease and can be used in food, laundry detergent, and contact lens cleaners. which MO produces it
Subtilin
Bacillus subtilis
what are the class 2 bacteriocins?
bacteriocins are small heat-stable proteins with a consensus leader sequence that signals to the producing cell that the protein must be exported
what are the 3 sub-classes of class 2 bacteriocins?
IIa: bacteriocins active against Listeria monocytogenes
IIb: bacteriocins that require two different peptides for activity
IIc: bacteriocins that require reduced cysteine for activity
what are the class 3 bacteriocins?
Are larger >30kDa heat-liable antimicrobial proteins
what are the class 4 bacteriocins?
Have lipid or carbohydrate moieties and the function of these non-protein portions are unknown
how do you discover bacteriocin?
most common method for bacteriocin discovery is to overlay a colony of the putative bacteriocin producer with an agar medium containing the bacterium being tested for sensitivity
Bacteriocin producers will form an inhibition zone with SHARP EDGES in the confluent growth
inhibition Zones produced by organic acids have … edges
fuzzy
how to identify inhibition Zones produced by bacteriophage
will not have a colony at the center
what verification test can be done for bacteriocin discovery?
A hole is poked in the center of the inhibition zone (where the colony is) and the hole is filled with a proteolytic enzyme. As this enzyme moves out it cleaves the bacteriocin and inactivates it, allowing the bacteria to grow where it once was inhibited
what is the mechanism of action of bacteriocin against vegetative cells?
All bacteriocins produced by LAB act by disrupting the integrity of the cytoplasmic membrane
The addition of bacteriocins to vegetative cells results in rapid and non-specific efflux of pre-accumulated ions, amino acids, and ATP molecules - dissipates chemical/electrical gradients
The cell is unable to regenerate the proton motive force, and ultimately increases in permeability render the cell usable to protect it’s cytoplasm from the environment, and this leads to cell inhibition and death
most bacteriocins have …. which allow for insertion into the membrane and permeabilization
amphiphilic cationic peptides
what are the two proposed models for bacteriocin insertion into membrane?
- The pore formation model, where bacteriocins bind, insert into the membrane, and oligomerize to form a pore
- The membrane solubilization model, where bacteriocins act as detergents resulting in lysis of the cell
what is the mechanism of action of bacteriocin against spores?
In spores nisin allows spores to germinate, and may even encourage germination => However, it inhibits the outgrowth of the preemergent spore (much less resistant to environmental stresses (i.e. heat) than the spore)
what are colicins?
proteins produced by, and are toxic for, some strains ofEscherichia coli.
What produces them?
They are produced by strains ofE. colicarrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity and release
how do colicins exert their lethal action?
- by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients
- They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system
- Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA.
what are the two classifications of colicins?
- enzymatic colicins
- pore-forming colicins
what are the different modes of action for enzymatic colicins?
- peptidoglycan synthesis block
- protein synthesis block by cleavage of tRNA
- Protein synthesis block by cleavage of 16S rRNA
- DNA degradation
what are the three wats that are approved methods to add bacteriocins to foods?
- purified bacteriocins can be added directly to the food product (nisin directly added to dairy products and other foods -> GRAS)
- Bacteriocinogenic cultures can be added to non-fermented food products, so that bacteriocins can be produced in situ
- Starter cultures that encode bacteriocins can be chosen for fermentation reactions (fermented meat/veg products where the indigenous communities are not inactivated by pasteurization)
Nisin is rarely used on it’s own, it’s generally part of a multiple-barrier inhibitory system which consists of:
modified-atmosphere (CO2) storage for delaying toxin production by C. botulinum in fresh fish
also added to ground pork stored in modified-atmosphere storage to delay or stop the growth of L. monocytogenes
… are not active against spores but are active agains vegetative L. monocytogenes cells
pediocins
what are pediocin cultures used for?
used as dried powder or culture liquid and applied to salad dressing, cream, cottage cheese, meat
what are the conditions which result in greater degree of inhibition by pediocin producing strain Pediococcus acidilactici?
The degree of inhibition is greater with decreasing temperatures, and is greater in anaerobic conditions than in aerobic conditions
what are the underlying mechanisms of bacteriocin resistance?
changes in membrane permeability
i.e. Membranes in nisin-resistant L. monocytogenes have more straight-chain fatty acids, and presumably this decrease in membrane fluidity hinders nisin insertion into the membrane
what are bacteriophages?
a natural component of food microbiota and are routinely consumed as part of our diet
what are bacteriophages used for?
control pathogens such as salmonella, E. coli, and listeria
what are some challenges associated with using bacteriophages in foods?
- Possible resistance
- Low numbers of bacteria in food (phage need 105 to 106 CFU of actively growing bacteria to be effective)
how is resistance to bacteriophages created?
generally created by changes in the bacteriophage receptor site. They can be proteins, LPS, or lipoproteins
how do you control bacteriophage resistance?
phage should be used in cocktails, containing several different strains and types
=> If a bacteria is resistant to one type, the other types should still be functional, and this strategy should curb the emergence of resistance
