Quiz 2 Content Flashcards
Which foods are microbiological contamination a universal concern?
- Meat, poultry, and fish are highly susceptible
- Foodborne pathogens: E. coli O157; Salmonella (non-typhoidal); Listeria monocytogenes
Describe Listeria monocytogenes.
- Rod shaped, Gram positive
- Facultative anaerobe - can survive without oxygen
- Grows from 0-45C with optimum temp between 30-37C
- Common in many environments
- Not completely inhibited by high salt concentrations or the presence of nitrite
Describe the prevalence of Listeria monocytogenes in processing plants.
Describe the persistence of Listeria monocytogenes in food processing plants.
Months to years!
Describe the bacterial stress response.
- Foodborne pathogens can sense changes in their surroundings and respond by altering gene expression
- A protective response may follow that increases tolerance to one or more stresses - aids in the survival in food products and in the food processing environment
- Alter the virulence properties of pathogens and can contribute to survival in vivo during infection
RpoS/SigS bacterial stress response occurs in […]
Rpos/SigS bacterial stress response occurs in Gram-negative bacteria.
RpoB/SigB bacterial stress response occurs in […]
RpoB/SigB bacterial stress response occurs in Gram-positive bacteria.
What type of stress response occurs in Gram-positive bacteria?
RpoB/SigB
What type of stress response occurs in Gram-negative bacteria?
RpoS/SigS
Give three examples of the bacterial stress response.
- E. coli O157 can survive at pH < 2.5 as it has acquired acid resistance
- Listeria monocytogenes can grow in cold environments
- Salmonella can survive in dry foods
Why do we need food antimicrobials? [3]
Because foodborne pathogens are (1) prevalent, (2) persistent, and (3) stress resistant.
Give three examples of the use of antimicrobials to reduce foodborne pathogens and spoilage organisms.
- Use of bacteriocins to reduce Campylobacter in poultry
- Use of nisin on processed cheese to prevent growth of Clostridial spores and toxin production
- Use of green tea extracts to control spoilage caused by yeasts and mould
What types of antimicrobials are used in foods?
- Natural (spices, oils)
- Artificial (organic acids)
- Traditional (organic acids, esters, salts)
- Emerging (bacteriocins, bacteriophages)
- Bacteriostatic and bacteriocidal agents
What is the difference between bacteriostatic and bacteriocidal agents?
Bacteriostatic: inhibit growth
Bacteriocidal: kill bacteria
Give examples of bacteriostatic and bacteriocidal agents.
Bacteriostatic: organic acids like lactate and diacetate
Bacteriocidal: antimicrobial peptides (e.g., nisin)
Note that sometimes antimicrobials may work against one another as well, which is why we need to study the effects of antimicrobials in combination.
How do the bacteriostatic agents work?
- Organic acids (lactate; diacetate)
- Reduce proton motive force, which is required to drive ATP synthesis, by decreasing intracellular pH
What are the most widely used compounds for control of L. monocytogenes in foods?
The organic acids (bacteriostatic agents): sodium lactate and sodium diacetate.
Note, these are widely used because they not only work well, but also because organic acids are affordable (e.g., cost effective for industry).
How do bactericidal agents work? [7]
By causing disruption to:
1. The cell wall,
2. cellular membrane,
3. protein synthesis,
4. nucleic acid synthesis,
5. DNA repair,
6. other metabolic pathways, or
7. producing toxins or free radicals.
What is nisin?
- A bacteriocidal agent (a Class I antimicrobial peptide)
- Naturally produced by Lactococcus lactis
How is nisin used in preventing food borne illness & food spoilage?
- Bacteriocidal agent
- Binds to lipid II in the cell membrane and forms pores that dissipate the proton motive force and make the bacterial cells leaky
- Effective against Gram-positive (e.g., Listeria monocytogenes), but not Gram-negative (e.g., E. coli, Salmonella), yeast or fungi
- Generally recognized as safe
Why isn’t nisin effective against Gram-negative bacteria?
- The outer membrane of Gram-negative bacteria effectively excludes nisin from making contact with the cell wall and interacting with the cytoplasmic membrane.
- However, in combination with chelating agents, such as EDTA, nisin can be effective against Gram-negative bacteria too
Foodborne pathogens cannot become resistant to nisin.
True or False?
False - they can become resistant.
Foodborne pathogens can become resistant to nisin.
True or False?
True.
What are the chemical properties of lauric arginate?
- Derived from lauric acid, L-arginine, and ethanol
- Active over a wide pH range (3 - 7) - so not effective in alkaline environments
This is a chemically synthesized product.
What is the antimicrobial spectrum of lauric arginate? [6]
- E. coli
- S. typhimurium
- S. aureus
- L. monocytogenes
- Lactobacillus
- Yeasts and moulds
Notice this range is much wider than for the antimicrobial nisin, which is not effective against Gram-negative bacteria, yeasts or moulds when used in isolation.
What are food applications of lauric arginate? [4]
- Cheeses
- Beverages
- Processed fruits & veggies
- Meats & fish
What is the proposed mechanism of action of lauric arginate?
Disruption/instability of cytoplasmic membrane and alter metabolic processes without causing cellular lysis.
Lauric arginate is considered bacteriocidal, and is GRAS.
What are the origins of natural antimicrobials?
- Animal: lysozyme (eggs); lactoferrin (milk); chitosan (shellfish)
- Microbial: nisin (Lactococcus lactis); natamycin (Streptomyces natalensis)
- Plants: essential oils; spices
What is natamycin?
- Produced by Streptomyces natalensis
- Broad-spectrum antifungal agent
Describe the synergy of antimicrobials.
Some combinations are more effective together than are either of the antimicrobials used alone (recall nisin + sodium lactate used to inhibit L. monocytogenes)
What makes a good antimicrobial to be used in the food industry? [6]
- Generally recognized as safe
- Able to efficiently inhibit the growth of target microorganisms within the given shelf-life
- Have minimum impact on sensory odour and colour characteristics
- Easy to apply
- Stable at various conditions (pH, temperature)
- Cost-effective
How are antimicrobials applied? [3]
- Applied to products as solutions (e.g., add to the brine)
- Edible films (e.g., edible Chitosan films containing lactoferrin significantly decreased the growth of E. coli O157:H7. Brown, 2008)
- Applied to the packaging material
What challenges are associated with antimicrobials, from the producer’s perspective? [3]
- May introduce sensory colour and odour characteristics defects
- Adds extra steps in food production
- Extra cost
Describe factors that affect antimicrobial activity. [3]
- The complexity and/or biochemical composition of the foods themselves (e.g., physical properties like the CHO, FAT, and PRO content as well as moisture content and water activity; food substances like native microbes; other preservatives)
- Food packaging materials and packaging conditions (e.g., gas composition; water vapour transmission rate; light access etc.)
- The metabolic state and growth phase of the microbial cells (pathogens in stationary phase can be more resistant than pathogens in the log phase that are rapidly growing)
What are the antimicrobials used here?
- Honey
- Sodium lactate is an organic acid (this is bacteriostatic)
- Sodium nitrite
- Salt
- Spices
- Brown sugar
- Sodium phosphate (preservative; mostly there to thicken and stabilize)
What are the antimicrobials used here?
- Likely applied to the packaging since nothing is listed in the ingredients; when applied to the packaging, it is not necessary to list it on the label (e.g., lauric arginate)
- Salt
What are the antimicrobials used here?
- Natural green tea extract
- Nisin
- Salt
- Sugar
- Celery extract (= nitrite!)
GRAS stands for:
generally recognized as safe
[…] works against Gram-positive, but not effective against Gram-negative, mould or fungi.
Nisin works against Gram-positive, but not effective against Gram-negative, mould or fungi.
Lactoferrin is a natural antimicrobial coming from […]
Lactoferrin is a natural antimicrobial coming from milk
What is biocontrol?
‘Taming’ the good microbes to (better) control the bad ones
List 6 methods of biocontrol.
- Fermentation
- Probiotics and prebiotics
- Competitive exclusion
- Bacteriophages
- Bacteriocins
- Quorum sensing inhibitors (quorum quenching)
What is the antimicrobial spectrum of nisin?
Works against Gram-positive, not generally against Gram-negative, yeast, and fungi
What is the method of action of nisin as an antimicrobial?
It binds to lipid II in the cell membrane and forms pores that dissipate the proton motive force.
What are bacteriocins?
- Ribosomally synthesized antimicrobial peptides produced by bacteria
- They can kill or inhibit bacterial strains closely-related or non-related to producer bacteria
- They will not harm the bacteria themselves by specific immunity proteins
What are Class I bacteriocins?
- Post-translationally modified (PTM) bacteriocins with less than 28 amino acids
- Small membrane-active peptides (<5 kDa)
- Example: nisin
What is a post-translational modification of a peptide?
- Covalent processing events that change the properties of the protein by proteolytic cleavage and adding a modifying group
What are Class II bacteriocins? [4]
- 30 - 60 amino acids (<10 kDa)
- Not PTM
- Always exhibit unique properties of heat tolerance
- Unmodified and positive charge
What are Class III bacteriocins?
- Large molecular weight (> 30 kDa)
- Heat unstable proteins
Describe pediocins.
- A bacteriocin
- Produced by Pediococcus spp.
- Class II, heat stable
- Particularly effective against Listeria monocytogenes
- GRAS
Describe enterocins.
- A bacteriocin
- Produced by Enterococci
- Class II, heat stable
- GRAS
Describe lacticins.
- A bacteriocin
- Produced by Lactococcus lactis
- Not GRAS, but L. lactis is GRAS
Add the bacteria directly to food, not the bacteriocin. These are Class I (not specified in slides, but determined via Google)
Give an example of a class I bacteriocin.
Nisin
Give two examples of class II bacteriocins.
- Enterocins
- Pediocins
[…] is the only bacteriocin licensed as a biopreservative.
Nisin is the only bacteriocin licensed as a biopreservative.
Compare and contrast these two commercially available products:
* DelvoNis
* Nisaplin
- How were these products made?
- In which countries are these products approved?
- What pathogens are they targeting?
- What foods can these products be applied to?
- Nisin is produced through fermentation process using strains of L. lactis which produce nisin as a byproduct, which is purified and concentrated.
- Nisaplin is approved in the US, UK and EU. DelvoNis is approved in EU.
- They both target Gram-positive bacteria [e.g., Bacillus cereus (a spore former) and Listeria monocytogenes (a non-spore former)]
- DelvoNis is for use in cheeses, dairy desserts, meats, beverages, and baking products. Nisaplin is for use in meats, poultry, seafood and alternatives.
Differentiate between bacteriocins and antibiotics.
Bacteriocin: restrict their activity to strains of species related to the producing species (particularly to strains of the same species)
Antibiotic: has a wider activity spectrum; even if their activity is restricted this does not show any preferential effect on closely related strains
Describe competition with native microbiota.
i.e., competitive exclusion
- Production of antimicrobial compounds (e.g., bacteriocins)
- Acid production
- Competing microbes
- Interactions of unidentified antimicrobial substances
These are two different products. Which one is the better choice?
- Salt, vinegar, lactic acid, and sugar are all antimicrobials, however the product on the left has been associated with L. monocytogenes outbreaks.
- Choosing the product on the right would be smarter because the company has not been associated with Gram-positive bacteria.
What is a ‘food processing aid’?
- A substance that is used for a technical effect during food processing or manufacture but, unlike food additives, its use does not affect the intrinsic characteristics of the food and it results in no or negligible residues of the substance or its by-products in or on the finished food. (e.g., bacteriophage)
What is a bacteriophage?
- “Bacteria eater”
- These are viruses that infect and replicate only in bacterial cells.
- Used as a food prodcessing aid
Describe antibiotic resistance.
- This is a naturally occurring phenomenom
- Bacteria adapt to the drugs that are designed to kill them and change to ensure their survival
- Form ‘superbugs’
Describe ‘superbugs in the media’
What are phages?
- Predators of bacteria
- Most abundant replicating biological entity on Earth
- Tiny (0.2-0.4 microns (5-10x smaller than bacteria)
- Cause lysis (killing) of hosts (specific to bacterial hosts; harmless to humans, animals, and plants)
Desribe the anotomy of a tailed bacteriophage of the order Caudovirales
- The capsid contains the genetic material
How do phages infect their bacterial hosts?
- They use the tail and associated receptor-binding proteins to interact with the host and to create a channel through which the DNA enters the cell. The baseplate initiates infection when the tail fibers bind to a host cell.
Describe the phage life cycle.
-
Lytic cycle
- Host recognition
- Adsorption
- Penetration and DNA injection
- Phage DNA replication
- Synthesis of new phage
- Phage assembly
- Host cell lysis and phage release
-
Lysogenic cycle
- Host recognition
- Adsorption
- Penetration and DNA injection
- Insertion of prophage into host genome
- Cell division
- Release of prophage due to environmental signals
- Phage DNA replication
- Synthesis of new phage
- Phage assembly
- Host cell lysis and phage release
Compare the lytic and lysogenic phage life cycles.
Lytic Life Cycle:
In the lytic cycle, the phage infects the host bacterium and immediately takes control of the host’s cellular machinery. The phage replicates its DNA and produces numerous copies of itself (virions) within the host. The host cell is eventually lysed (ruptured), releasing the newly formed phage particles, which can go on to infect other bacteria. This cycle results in the rapid destruction of the host cell.
Lysogenic Life Cycle:
In the lysogenic cycle, the phage integrates its DNA into the host bacterium’s genome. The integrated phage DNA is called a prophage and becomes a part of the host’s genetic material. The host bacterium continues to replicate normally, including the prophage in its genome. Under certain conditions (e.g., stress or environmental triggers), the prophage may exit the host genome and initiate the lytic cycle, leading to the destruction of the host cell.
Alternatively, the lysogenic cycle can persist without harming the host for generations.
What are the desirable attributes of bacteriophages as food antimicrobials? [5]
- ‘Green’ technology (organic; clean label; non-GM; Kosher)
- Kill ONLY the specific target bacteria
- Safe for human consumption
- Self-replicating and self-limiting
- Ubiquitously distributed in nature
Compare and contrast these two products:
* SalmoFresh
* PhageGuardS
- What bacteriophages were used in these products?
- In which countries are these products approved?
- What salmonella strains are they targeting?
- What foods surfaces can these products be applied to?
- SalmoFresh uses a blend of six lytic phages. Phage Guard S uses
- SalmoFresh is approved in the US. Phage Guard S is approved in the US.
- SalmoFresh targets Salmonella enterica and Phage Guard S targets Salmonella
- SalmoFresh can be used for direct food applications, and in some cases it can be applied on surfaces involved in food processing. Phage Guard S
Nisin is a Class […] bacteriocin that has received GRAS status.
Nisin is a Class I bacteriocin that has received GRAS status.
Lacticins are produced by […] and are not considered as GRAS.
Lacticins are produced by Lactococcus lactis and are not considered as GRAS.
