Control of microbial growth in meat and

meat products: BIOPRESERVATION

Dr Ravi Kant Agrawal, MVSc, PhD

Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India
 Major Concerns of the Food Industry
Food Safety
Making food safe to eat and free from disease causing
agents-
•Infectious agents
•Toxic chemicals
•Foreign objects

Food Quality
Making food desirable to eat-
•Good taste
•Color
•Texture
Food Preservation
• Maintenance of organoleptic quality of food product
throughout its shelf life

Food safety
• Control of microbial load/Reduction of the risk of pathogens in
food product throughout its shelf life

 In spite of modern advances in technology, the preservation of

foods is still a debated issue, not only for developing countries
but also for the industrialized world
 Need for development of new technologies for better
processing, preservation, and storage of food
 Consumer’s Preferences

Product Diversification
Upgraded Quality
Enhanced Shelf Life
Ready to Eat
Fresh- Tasting
Nutritious and Vitamin Rich
Minimally-Processed
 Free from synthetic preservatives
 Challenges in Food Processing & Preservation
Challenges in processing
 Retaining the nutritional value, flavor, aroma, and
texture of foods
 Presenting them in near natural form with added
conveniences.

Challenges for the food preservation

 Diverse and demanding
 Need to be addressed on several fronts to derive
maximum market benefits
Conventional Preservation Modern Preservation
Techniques Techniques
 Manipulation of the  High Hydrostatic
water activity Pressure (HHP)
 Lowering of pH  Pulsed Electrical Fields
 Heat treatment (PEF)
 Control of storage  High pressure CO2
temperature of foods treatment
and  UV light and irradiation
 Addition of chemical  Biopreservation
preservatives
 Limitations

?
Thermal Treatment Radiation Treatments
Undesirable Organoleptic
Consumer Resistance
Changes

Chemical Preservatives High Pressure CO2

Safety Issues
Lack of Knowledge

HHP & PEF

Resistant Vegetative Bacteria, Cost
 Biopreservation
 Biopreservation refers to extended storage life and enhanced
safety of foods using the natural antimicrobial compounds that
are of plant, animal and microbial origin and have been used in
human food for long time, without any adverse effect on
human health
 Biopreservation reduces the amounts of chemical preservatives
as well as the intensity of heat treatments, both of which can
otherwise negatively affect the food quality
Biopreservation
Extended storage life and enhanced safety of
foods using the natural microflora and/or their
antibacterial products

Application in food:
1. use bacterial strains
2. add purified substance
3. add fermentation liquor or concentrate
 Multiple Antimicrobial Effects of Lactic Acid Bacteria
• Acids - Lactate, acetate, formate, propionate
• Diacetyl, acetaldehyde
• Reuterin = aldehyde
• Bacteriocins
 Antimicrobial peptides
 kill or inhibit growth of closely related bacteria
 bacteriocins of Gram-positive bacteria are seldom
active against Gram-negative bacteria
 Commercially Available Products
• Bactoferm F-Lc (pediocin Chr-Hansen)
• ALTA 2351 (pediocin-Quest)
• Sakacin
• ALCMix1 (plantaricin-Danisco)
• Carnocin
• Micocin (carnocyclin-Griffith Laboratories)
• Piscicolin
• Carnobacteriocin
 Selection of Cultures as Biopreservatives
 Ability to produce antimicrobials in meats
 Limited sensory changes
 Limited acid production
 Weak protease activity
 Limited gas production
 Absence of slime production
 • Inhibition of food
pathogenic micro-
organisms
• growth control
• prevention of toxin-
formation
Food • reduction of pathogens
 final products
Micro-organisms
Protective Cultures
 pre-stages and/or
 or raw material
• Shelf life
extension based on
targeted inhibition of
specific spoilage micro-
organisms
Biopreservation with fermentation end products of LAB
 Producing organic acids ORGANIC ACIDS
 Hydrogen peroxide Lactic acid
 Diacetyl Propionic acid
 Antifungal compounds such as fatty Acetic acid
acids or phenyl lactic acid LOW MOLECULAR
 Bacteriocins WEIGHT
COMPOUNDS
Reuterin
Diacetyl
H2 O2
Fatty acids
Phenyl lactic acids
Cyclic dipeptides

BACTERIOCINS
(Antimicrobial Peptides)
 What makes them anti-microbial?
 Organic acids
Anti-microbial metabolites  lactic acid
 acetic acid
 propionic acid…
 Hydrogen
peroxide
 Carbon dioxide
 Lacto peroxide
 Fatty acids
 Diacetyl
 Acetaldehyde
 Reuterin
 Other compounds
of low molecular
mass
 Bacteriocins
 Weak acids Have More powerful antimicrobial activity at low
pH than at neutral pH
 Acetic acid Is strongest inhibitor and has a wide range of
inhibitory activity, inhibiting Yeast, Molds and bacteria
 Un-dissociated molecule is the toxic form of a weak acid

Undissociated form of organic acid diffuses across the cell

membrane

Acid dissociates in the cytoplasm and releases the protons

Leads to acidification and dissipation of pH gradient over the

membrane causing the observed growth inhibition
Another Hypothesis
 Accumulation of anion is the cause of growth inhibition
 It reduces the rate of macromolecule synthesis and affects
transport across the cell membrane
- more than anti-microbial metabolites!

anti-microbial
metabolites

competitive Inhibition of
+ exclusion undesired
effects micro-organisms by
competing for
further, so nutrients, oxygen, etc.
+ far unknown e.g. quorum sensing
effects

= Inhibition of food pathogenic

and food spoilage micro-organisms
A natural way

Bio preservation
by protective cultures:

= Safer food

= Label friendly –
It´s a ‘culture’
and not a food preservative

= Positive image of
‘biopreservation’-
‘natural’
 Protective Cultures - categories

fermentation
Starter Cultures STARTER processes:
CULTURE Changes of
taste, flavour and
Adjunct-Cultures texture

Multi-functional
Cultures

Non-fermenting
Protective no influence on
PROTECTIVE
Cultures sensory
CULTURE
characteristics
 Protective Cultures - categories

Fermentation
Starter Cultures STARTER processes:
CULTURE Changes of
taste, flavour and
Adjunct-Cultures
texture

Multi-functional
Cultures

Non-fermenting
Protective No influence on
Cultures PROTECTIVE sensory
CULTURE characteristics
 Protective Cultures - categories

Fermentation
Starter Cultures STARTER processes:
CULTURE Changes of
taste, flavour
Adjunct-Cultures
and
texture
Multi-functional
Cultures

Non-fermenting
Protective No influence on
Cultures PROTECTIVE sensory
CULTURE characteristics
 Protective Cultures - categories

Fermentation
Starter Cultures STARTER processes:
CULTURE Changes of
taste, flavour and
Adjunct-Cultures
texture

Multi-functional
Cultures

Non-fermenting
Protective No influence on
Cultures PROTECTIVE sensory
CULTURE characteristics
 Protective cultures - are safe

Protective cultures are:

generally recognised as safe (GRAS)

there is no indication of a health risk of

• Lactic acid bacteria
Lactobacillus sp.
this bacterial group
Lactococcus sp.
Pediococcus sp. the use of lactic acid bacteria in
Carnobacterium sp.
Enterococcus sp
biological preservation might even
Leuconostoc sp. contribute to the health benefits of a
Weisella sp. product *)
Vagococcus sp.
*) Holzapfel et al., 1995, Int. J. Food Microbiol. 78: 119-131
• Propionibacteria
 Cultures - 4 steps from lab to customers

Tailor made
friendly label
• STEP 1: • STEP 2: • STEP 3: • STEP 4:
Isolation Application
HOLDBAC™
Process- Challenge
Selection Development tests
Application tests
applications
Up-Scaling
Screening in food model
Down-Stream-
systems
Processing in food & feed

HOLDBAC™
 Food Model Systems
To reproduce the food products from
production to the end of shelf life:
Continuous Monitoring:
• Behaviour of indicator strains
(growth – inhibition)
• Influence of the Food Protectants on
technological and organoleptic properties in
real food matrixes

Fermentation / End of
Preparation Pack- Storage
Ripening shelf
aging
life

Defined contamination with Microbiological Flora Analyses

L3* food pathogens
(e.g. Cl. botulinum, Physico-chemical Analyses
Campylobacter sp., E. coli H157,…)
Sensory Evaluation
Food spoilage microorganisms Technological Properties
 Hydrogen Peroxide
 Strong oxidising effect on the bacterial cell membrane
 Can oxidise sulfhydryl groups of cell proteins and
membrane lipids
 H2O2 producing reactions scavenge oxygen, thereby
creating anaerobic environment that is unfavourable
for some microorganisms
 Antimicrobial activity is enhanced by the presence of
Lactoperoxidase and thioccyanate

SCN- + H2O2 OSCN - +H2O

 Diacetyl
 Identified By Van Neil et al. (1929) as the aroma and flavor
component in butter
 Produced by species and strains of genera Lactobacillus,
Leuconostoc, Pediococcus and Streptococcus
 More active against Gram negative bacteria, Yeasts and molds
than against Gram Positive bacteria
 Reacts with the arginine binding protein of Gram negative
bacteria, thereby interfering with the utilization of arginine
 Bacteriocins
 Bacteriocins are ribosomally synthesized single polypeptides or
post-translationally modified ones that are usually inhibitory
only to closely related bacterial species
 Desirable properties that make Bacteriocins
suitable for food preservation
 They are generally recognized as safe substances
 They are not active and nontoxic to eukaryotic cells
 They become inactivated by digestive proteases, having little
influence on the gut microbiota
 They are usually pH and heat-tolerant
 They have a relatively broad antimicrobial spectrum against
many food-borne pathogenic and spoilage bacteria
 They show a bactericidal mode of action, usually acting on the
bacterial cytoplasmic membrane: no cross resistance with
antibiotics
 Their genetic determinants are usually plasmid-encoded
 Bacteriocins of LAB
Main Characteristics Subcategory Examples
category
Class I Lantibiotics Type A Nisin
Type B Mersacidin
Class II Non-modified heat-stable Subclass IIa Pediocin AcH
bacteriocins containing Enterocin FH99
peptides with molecular Pediocin 34
masses of 10 kDa Subclass IIb Lactacin F and
Lactococcin G
Class III Protein bacteriocins Helveticin J
with molecular masses Lactacins A and B
of 30 kDa
Class IV Bacteriocins that form large Leuconosin S
complexes with other Lactococcin 27
macromolecules
 Some useful bacteriocins for the dairy & food industry

Genus of Bacteria Bacteriocin Produced

Lactococcus lactis subsp. lactis Nisin A, Z
Pediococcus acidilactici Pediocin PA-1, AcH
Pediococcus peantosaceus Pediocin 34
Leuconostoc spp. Leucocins
Lactobacillus sake Sakacin A
Lactobacillus plantarum Plantaricin
Lactobacillus helveticus Helveticin J
Carnobacterium piscicola Carnocin/piscicolin
Do not alter Safe and efficacious
acceptance Economical use of nisin for > 40 years
quality of in several countries
food and are
safe for human
consumption
Consumer resistance
FACTORS PROMOTING to traditional chemical
preservatives and
USE OF BACTERIOCINS concern over
Effective under AS the safety of existing
wide pH & BIOPRESERVATIVES food preservatives
temperature range such as sulfites
and nitrites

Activity is not
lost in the Advent of novel
Presence of bacteriocins with
food additives broad spectrum of
and effective Effective in low activity from
in dairy Foods concentrations food grade LAB
during storage
 Bacteriocin Based Biopreservation Strategies
 Using a purified/ semi-purified bacteriocin preparation as an
additive in food
 By incorporating an ingredient previously fermented with a
bacteriocin-producing strain
 By using a bacteriocin-producing culture to replace all or part
of a starter culture in fermented foods to produce the
bacteriocin in situ.
 Use of Purified/ Semi purified Bacteriocins
To date, the only commercially produced
bacteriocins are:
 Nisin produced by Lactoccocus lactis ssp. lactis
 Pediocin PA-1, produced by Pediococcus acidilactici
The use of purified bacteriocins have to be labeled as
additives and require regulatory approval.

Nisin is the only bacteriocin, approved for use as an

antimicrobial in food by the Joint FAO/WHO Expert
Committee on Food Additives
 NISIN
Approved by FDA
Used in over 50 countries
Effective in a number of food systems
Wide range of inhibition spectrum
The level of nisin used depends on:
 Food composition
 Required shelf life
 Temperatures likely to be encountered
during storage
 Nisin
 Nisin was discovered in 1928 by Rogers and his workgroup
 Nisin has approved bactericidal effect against most LAB; S.
aureus; L. monocytogenes, Bacillus and Clostridium vegetative
forms.
 Belong to Class I, termed lantibiotics.
 Used in dairy products, cheese, mayonnaise, canned vegetables
for shelf life extension.
 The form of nisin used most widely in food is Nisaplin produced
by Danisco, which is a preparation that contains 2.5% nisin with
NaCl (77.5%) and non-fat dried milk (12% protein and 6%
carbohydrate).
• GRAS status from Food and Agriculture Organization-World
Health Organization approval in 1969
• European Union approval in 1983
• US Food and Drug Agency approval in 1988
 At present, only nisin and pediocin PA1/AcH have found
widespread use in food.
 Nisin bactericidal mechanism
Nisin ihibits synthesis of peptidoglycan:
interaction between lipid I and lipid II
(First uses lipid I and lipid II for docking to cell wall)
↓
pore forming
↓
Inhibition of cell wall synthesis
 They can bind to lipid II, the main transporter of peptidoglycan
subunits from the cytoplasm to the cell wall, therefore prevent
correct cell wall synthesis, leading to cell death.
 Furthermore, they can use lipid II as a docking molecule to
initiate a process of membrane insertion and pore formation
that leads to rapid cell death.
 Nisin: mechanism of activity
• First nisin reaches the plasma membrane where it bind to lipid
II with two of its amino terminal rings.
• This is then followed by pore formation, which involves a stable
transmembrane orientation of nisin.
• During or after assembly of four 1:1 (nisin: lipidII) complex, four
additional nisin molecules are recruited to form the pore
complex
Vegetative cells
 Nisin adsorbs to the cytoplasmic membrane where it forms
transient pores
 Low molecular weight compounds leak from the cell causing
loss of energy
 The pH gradient across the membrane becomes dissipated
 Collapse of the proton motive force (which drives ATP
synthesis- the cell’s energy)
 Heat-resistant endospores
 Nisin affects spores after germination, preventing their
outgrowth
 Does not normally kill bacterial spores
Bacteriocins: developing innate immunity for food Paul D. Cotter, Colin Hill & R. Paul
Ross Nature Reviews Microbiology 3, 777-788 (October 2005)
 Lactic acid bacteria (LAB) bacteriocins can be grouped on the
basis of structure, but also on the basis of mode of action.
 Some members of the class I (or lantibiotic) bacteriocins, such
as nisin, have been shown to have a dual mode of action.
 They can bind to lipid II, the main transporter of peptidoglycan
subunits from the cytoplasm to the cell wall, and therefore
prevent correct cell wall synthesis, leading to cell death.
 Furthermore, they can use lipid II as a docking molecule to
initiate a process of membrane insertion and pore formation
that leads to rapid cell death.
 A two-peptide lantibiotic, such as lacticin 3147, can have these
dual activities distributed across two peptides, whereas
mersacidin has only the lipid-II-binding activity, but does not
form pores.
 Large bacteriolytic proteins (here called bacteriolysins,
formerly class III bacteriocins), such as lysostaphin, can function
directly on the cell wall of Gram-positive targets, leading to
death and lysis of the target cell.
 Lantibiotics are produced
ribosomally as inactive
precursors that are
Nisin targeted by a leader
peptide to posttranslational
modification.

Precursor
 The realisation of Nisin as a food preservative
Nisin was used as a food preservative because:
 Nisin is non toxic
 The producer strain L. lactis is regarded as safe (food-grade)
 There is no apparent cross-resistance related to therapeutic
antibiotics
 It is degraded immediately during digestion
 It is heat stable at low pH

 The first commercial preparation of nisin (Nisaplin®) was made

in 1953 by Aplin & Barrett Ltd. (now Danisco).
 Originally intended only to control clostridial spoilage in
processed cheese, the application was so successful it lead to
worldwide interest.
 In 1969 a joint FAO-WHO expert committee on food additives
recognized nisin as a safe and legal food additive
 Use of Nisin in the Biopreservation of Dairy Products
 Nisin is used in pasteurized, processed cheese products to
prevent outgrowth of spores of Clostridium tyrobutyricum
 Used to extend the shelf life of dairy desserts which cannot be
fully sterilized
Preservation of Khoa with Nisin (100 IU/gm)
Temperature Shelf life
10 C up to 90 days
22 C up to 42 days
30 C up to 28 days

Preservation of Kheer with Nisin (200 IU/gm)

Temperature Shelf life (In days)
Control Sterilized Nisaplin added
37 C 2-3 3-4 8-10
4C 10 -15 60-70 100-150
 Nisin in Stirred Yoghurt
 Addition of 50 IU nisin/g to yoghurt after preparation
gave an acceptable product with increased shelf-life
upto 10 days at refrigeration temperature
 Prevents subsequent over-acidification of the yogurt.
 Effect of Nisin on the Shelf Life of Lassi
 The shelf life of lassi containing 100 to 200 IU nisin/ml
increased by two folds and the product was acceptable up to
24 hrs at room temperature
 Lassi containing 50 IU nisin/ml could be kept up to 8 to 10 days
at refrigeration temperature without much change in
acceptability (Kumar and Prasad, 1996).
• The combined use of Nisin (25 IU/ml) and GelodanTM increased
the shelf life of Srikhand to 10 days at 15 ± 1C (Sarkar et al.,
1996)
• Incorporation of 15 IU/gm Nisin into dahi retained all its
desirable characteristics up to 35 days at 15 C (Kumar et al.,
1998)
• The addition of 750 IU/ml of nisin extended the shelf life of
mango lassi to 15-30 days as compared 7-15 days shelf life of
control at refrigeration temperature (Khurana, 2006)
Important considerations to ensure good preservation

 Nisin works in a concentration dependent fashion

 Increasing bacterial cell or spore loads will require higher nisin
concentrations to achieve effective inhibition
 Nisin added to a food system will naturally and slowly degrade
during shelf life depending on the storage conditions and food
type
 For a continued inhibitory or sporostatic effect, there must be
sufficient nisin remaining within the food system at the end of
the required shelf life
 Nisin cannot be used to hide poor manufacturing practice
 Nisaplin® & Novasin™ antimicrobials
(Danisco)

 The active compound of Nisaplin® and Novasin™ products is

NISIN, a natural bacteriocin produced by fermentation of
Lactococcus lactis, a bacterial strain which occurs naturally in
milk

Effective against
 Effective against a broad range of Gram-positive bacteria,
including Listeria, Clostridium, Bacillus and lactic bacteria.

Mode of action
 Either a killing or growth inhibitory activity against vegetative
cells by targeting the cytoplasmic membrane and cell wall, or
prevention of the outgrowth of heat-resistant spores.
 Nisaplin® : composition
Appearance: Free flowing white powder

Average composition:
2.5% nisin
90% sodium chloride
4% protein
1.5% carbohydrate
2% moisture

Shelf life: 2 years at 4°C to 25°C

 GUARDIAN™ & NovaGARD™ antimicrobial systems
 The idea presiding over the development of GUARDIAN™ and
NovaGARD™ systems is the need for multifactorial food safety
and food preservation solutions combining and optimizing
known synergies between antimicrobials to control the growth
of microorganisms in foods, especially Gram-positive
pathogens.
 GUARDIAN™ NR systems are patented and complete natural
solutions based on the synergy between nisin and rosemary
extract - nisin activity is further enhanced by the synergistic
effect of a deodorised natural antioxidant such as rosemary
extract that increases the ability of GUARDIAN™ systems to kill
Gram-positive bacteria and control their growth.
 Rosemary extract delays also the oxidative rancidity of fats and
preserves the freshness of the foodstuff.
 NovaGARD™ systems are complete systems combining
antimicrobials and known chemicals such as organic acids and
their salts, which also increase their ability to kill Gram-positive
bacteria and control their growth.
 Main applications: soups, sauces, cooked sausages, salad
dressings, deli salads
 GUARDIAN™ GUARDIAN™ NR NovaGARD™ CB1
NR 100 250

Functionality Against Gram- Against Gram- Against Gram-

positive bacteria positive bacteria positive bacteria

Growth inhibition Listeria inhibition Growth delay of

of Bacillus Listeria,
Growth delay of Clostridium,
Growth delay of spoilage such as Bacillus,
Listeria lactic bacteria heterofermentative
lactic bacteria
Delay of oxidative Delay of oxidative
rancidity rancidity

Application Culinary products Heat-processed Deli salads (chicken,

such as meat products such tuna, seafood,
pasteurised as cooked coleslaw, egg),
soups & sauces, sausages (hot mashed potatoes,
low pH sauces & dogs, Frankfurters) meat broth soups,
marinades, RTE and cooked hams sauces, salad
foods dressings, RTE
meals
 Class II Bacteriocins
• Class II contains small heat-stable, non-modified peptides
• On the basis of structure the class II bacteriocins are subdivided
into four subclasses:
• Class IIa or pediocin-like bacteriocins: Class IIa includes
Pediocin-like Listeria active peptides with a conserved N-
terminal sequence Tyr–Gly–Asn–Gly–Val and two cysteines
forming a disulfide bridge in the N-terminal half of the peptide.
• Class IIb or two-peptides bacteriocins: Bacteriocins composed
of two different peptides comprise Class IIb. The two peptide
bacteriocins need both peptides to be fully active. The primary
amino acid sequences of the peptides are different.
• Class IIc: Cyclic
• Class IId: Miscellaneous
 Class II
Class II bacteriocins are:
• the most heterogenous and the biggest group of bacteriocins
• 20-60 amino acids
• Post-translationally not modified
• Cationic
• Hydrophobic
• Heat-stable
 Class II bacteriocins
Class IIa Class IIb Class IIc Class IId
 Bavaricin A - Lactobacillus Acidocin J1132 - L. Divergicin A - Cb Plantaricin 1,25β -
sake MI401 acidophilus JCM 1132 divergens LV13 L. plantarum
 Bavaricin MN - L. sake MN Lactacin F - L. johnsonii Lactococcin A - Lc. TMW1.25
 Carnobacteriocin B2 -
Carnobacterium pisciocola 11088 lactis subsp. cremoris
LV17B Lacticin 3147 - Lc. lactis LMG 2130
 Carnobacteriocin BM1 - Cb. DPC3147 Lactococcin 972 - Lc.
piscicola LV17B Lactobin A - L. lactis IPLA972
 Curvacin A - L. curvatus amylovorus LMG P-13139 Plantaricin A - L.
LTH1174 Lactococcin G - Lc. Lactis plantarum C11
 Divercin V41 - Cb. divergens
V41 LMG2081
 Enterocin A - Enterococcus Plantaricin EF - L.
faecium CTC492/T136 plantarum C11
 Enterocin P - E. faecium P13 Plantaricin JK - L.
 Leucocin A/B-Talla - plantarum C11
Leuconostoc gelidum Plantaricin S - L.
UAL187; Ln. Carnosum Ta11a plantarum LPCO10
 Mesentericin Y105 - Ln.
mesenteroides Y105 Thermophilin T -
 Mundticin - E. mundtii AT06 Streptococcus
 Pediocin PA-1/AcH/SJ-1 - thermophilus ACA-DC
Pediococcus parvulus 0040
ATO34/ATO77; Pediococcus
acidilactici H/SJ-1/PAC 1.0
 Piscicolin 126 - Cb. piscicola
JG126
 Sakacin 674 - L. sake LB764
 Sakacin A - L. sake LB 706
 Sakacin P - L. sake LB673
 Plantaricin C19 - L.
plantarum C19
 Plantaricin 423 - L.
 Pediocins
POTENTIAL BACTERIOCINS AS BIOPRESERVATIVES
 Most pediocins are:
Thermostable proteins
Active over a wide range of pH

 Pediocin AcH:
 Active against both spoilage and pathogenic organisms
 L. monocytogenes, Enterococcus faecalis, Staphylococcus
aureus, and Clostridium perfringens

 Pediocin PA-1:
 Inhibits Listeria in dairy products such as cottage cheese, ice
cream, and reconstituted dry milk
 Spectrum of Activity of Pediocin 34

Gram Positive Bacteria

Staphylococcus aureus
Listeria monocytogenes
Enterococcus spp. Gram Negative Bacteria*
Micrococcus spp. Escherichia coli
Bacillus spp. Pseudomonas spp.
Salmonella spp.
* In the presence of 20mM EDTA
Mechanism of action
Pediocin-like bacteriocins use
mannose permease for
docking to the cell wall
↓
Pore formation,
decreasing intracellular
ATP concentration leads to
cell death
 PA-1
• Most studied and well-known class IIa bacteriocin is pediocin PA-
1, which is at the market in Europe and USA under the name
ALTA2431 (Quest).
• Most pediocin PA-1 producing bacteria so far are isolated from
pediococci, organisms usually assosiated with vegetables and
meat products but poorly adapted for growing in milk and dairy
products.
• Therefore, the production of pediocin PA-1 by strains of dairy
origin is a higly desirable objective.
• Pediocin PA-1 also called pediocin AcH
• Broad inhibitory spectrum
• Production of pediocin is assosiated with plasmid pSRQ11
– Production by alternative host (dairy starter culture L. lactis
MM210 for cheddar cheese manufacture)
 ALTATM 2341
 ALTA™ 2341 (Quest International, US) is produced from
Pediococcus acidilactici fermentation and has to rely on the
inhibitory effects of pediocin PA-1/AcH
 Added to Mexican soft cheese to prevent Listeria
contamination
 Pediocin in the form of ALTA™ 2341 has been used in
combination with sodium diacetate (SD) and sodium lactate
(SL) as dipping solutions
 Class IIb
• Consist of two peptides.
• For optimal activity both peptides are required.
• In general, the class II peptides have an amphiphilic helical
structure, which allows them to insert into the membrane of
the target cell, leading to depolarisation and death.
• The two-peptide bacteriocins require the combined activity of
both peptides with a mechanism of action that again involves
the formation of pores in cell membrane/ dissipation of
membrane potential, the leakage of ions and/or a decrease in
intracellular ATP concentrations.
• These peptides display very low, if any,
bactericidal activity when tested individually.
• Primary structure of peptides are very
different
• Although members of this subgroup
are relatively heterogeneous, it has been
proposed that they could be subdivided
into type E (enhanced) and type S
(synergistic) peptides.
 LACTICIN 3147
 Lacticin 3147 produced by Lc. lactis DPC3147 ferment
reconstituted de-mineralized whey (10% solids), which was
pasteurized, concentrated and spray dried to produce a
bioactive lacticin 3147 powder.
 This powder was subsequently found to be effective in
inhibiting L. monocytogenes Scott A and Bacillus cereus in
natural yoghurt, cottage cheese and soups.
 Class IIc
 Bacteriocins are grouped on the basis of their cyclic structure.
 The class IIc (formerly class V) bacteriocins are grouped on the
basis that their N- and C-termini are covalently linked, resulting
in a cyclic structure.
• Although relatively few class IIc bacteriocins have been
identified, two subdivisions proposeD designated AS subclass
c(i) (comprising enterocin AS48 and the non-LAB circularin A)
and subclass c(ii) (comprising gassericin A, reutericin 6, the
non-LAB butyrivibriocin AR10 and, although a circular structure
has not yet been established, acidocin B) on the basis of
percentage amino-acid sequence identity.
• The class IIc bacteriocins gassericin A and reutericin 6 are the
only examples of non-lantibiotic LAB bacteriocins that contain
d-amino acids.
 Class IId
 Bacteriocins are usually combined in a ‘miscellaneous’ or ‘one-
peptide non pediocin linear’ group.
 Further subdivision on the basis of leader sequences is possible.
 Not well described yet
 Class III or bacteriolysins
• large > 15kDa
• heat-labile antimicrobial proteins
• have a domain-type structure
• different domains have different functions for translocation,
receptor binding, and bactericidal activity.
 Mode of action
 These proteins are also modular in structure and have a catalytic
domain at the N-terminus that shows homology to
endopeptidases, and a C-terminus that probably represents the
target recognition site.
 Their mechanism of action is distinct from that of bacteriocins as
they function through the lysis of sensitive cells by catalysing
cell-wall hydrolysis.
 Unlike the ‘true’ bacteriocins, they do not always have specific
immunity genes that accompany bacteriocin structural genes,
but might rely on modifications of the producer cell wall to
impart resistance.
 Natamax™ antimicrobials
 The active compound of Natamax® products is natamycin, a
natural antimycotic polyene macrolide produced by
fermentation of Streptomyces natalensis bacteria.

 Natamax® is one of the most effective natural technologies

against yeasts and moulds.

Main application areas

• Surface treatment of cheeses
• Surface treatment of dried and semi-dried cured meat products
• Yoghurt, sour cream
• Wines and fruit juices
• Bakery products
 Functionality:
Growth Specificity Application
control of

Yeasts & Cheese, fresh dairy, bakery

Natamax® moulds Standard products, processed meat
and beverages

No Shredded cheese, PVA

Natamax® Yeasts & addition of cheese coating
Salt moulds sugar Dry sausages surface
treatment

Yeasts & No
Natamax® G moulds addition of Beverages and wines
salt

Natamax ® Yeasts & Spraying, shredded and

SF moulds No carrier blocked cheese, bakery
products

Natamax® Yeasts & Improved Cheese surface treatment

Gel moulds surface Dry sausages surface
adhesion treatment
 MicroGARDTM
 MicroGARDTM (DANISCO, Denmark) is commercially produced
from grade A skim milk fermented by a strain of
Propionibacterium freundenreichii ssp. shermanii
 The mild, cultured flavor of MicroGARDTM can enhance and
protect the flavor of many food products
 The product was approved by FDA (1990) and granted GRAS
status (1996)

Main application areas:

• Bakery products and fillings
• Dairy products such as cheese
• Low pH dressings and sauces
• Chilled, pasteurized ready-to-eat meals
• Soups
• Processed meat products
 MG 300
MG 100 MG 200 MG CM1- MG 400 MG CS1-50
50

Gram- Gram- Gram-positive

Functionali negative negative Gram- bacteria Gram-positive
ty: Growth bacteria bacteria positive Gram-negative bacteria
control of Yeasts & Yeasts & bacteria bacteria
moulds moulds Yeasts & moulds
Cultured Cultured Cultured Cultured grade Cultured
Specificity grade A dextrose grade A A skim milk dextrose
skim milk skim milk blend

Grade A
&
Grade A & fermented
milk, Cheeses, Grade A & Cheeses,
fermented cheeses, soups & fermented milk, soups &
Application milk, dips & sauces, cheeses, salad sauces,
cheeses, spreads, refrigerated dressings, soups refrigerated
salads, salad meats and & sauces and meats and
dressings dressings, processed meats processed
and soups soups & meats meats
sauces and
bakery
 Shelf Life of Mango Lassi
 The use of MicroGARDTM 100 at the level of 1.5% was
quite effective in extending the shelf life of mango
lassi from 15 to 50 days at 4±1C

 The use of MicroGARDTM 100 at the level of 1.5% in low

calorie mango lassi was able to slow down the rate of
deteriorative changes and extended the shelf life from
12 days to 18 days at 4±1C
(Khurana, 2006)
USE OF A BACTERIOCIN-PRODUCING CULTURE TO REPLACE
ALL OR PART OF A STARTER CULTURE IN FERMENTED
FOODS TO PRODUCE THE BACTERIOCIN IN SITU
 The use of cultures to produce bacteriocins in situ as a means of
bio-preservation
 A more natural method of shelf-life extension and improving
the safety

Bacteriocin Based Bioprotective Cultures

BS-10® Nisin producing L. lactis spp. lactis, Chris Hansen
BIOPROFIT™ L. rhamnosus LC705, BioGaia
BOVAMINE Meat CulturesTM Texas Tech University
HOLDBAC™ L. plantarum, L. rhamnosus, L. sakei, L. paracasei and
Propionibacterium freundenreichii spp. Shermanii,
DANISCO
 MOVING TOWARDS BETTER TOMORROW……

• Identify new bacteriocins for application in foods

• Altering the specificity of existing bacteriocins
• Increasing the level of bacteriocin production
• Development of bacteriocin producing lactic starters
through gene transfer system
• Continued study of physical and chemical properties
 Other Antimicrobial agents
• Antimicrobials are agents that kill microorganisms or
inhibit their growth
• Biocontrol is the use of one or more organisms to
inhibit or control other organism
In Food industry
 Microbial Antagonism
 Competitive Exclusion Technology
 Antimicrobial Proteins
 Antimicrobial Peptides
 Antimicrobials of plant origin
 Antimicrobials of animal origin
 Bacteriophages
 B’dellovibrio
S. Waksman
 Microbial antagonism
 Microbial antagonism is the inhibition of undesired or
pathogenic microorganisms caused by competition for
nutrients, and by the production of antimicrobial metabolites.
 ‘Microbial Interference’ is also used which is a general
nonspecific inhibition or destruction of microorganism by
other members of the same habitat or environment.
 Lactic acid bacteria (LAB) have been used in food production as
an effective method for extending safe storage of foodstuffs
by simple fermentation
 Pure cultures of lactic acid bacteria (LAB): These organisms are
particularly suitable as antagonistic microorganisms in food
because of their ability to inhibit other foodborne bacteria by a
variety of means, including production of organic acids,
hydrogen peroxide and bacteriocins.
 Lactococcus, Streptococcus, Pediococcus, Leuconostoc,
Lactobacillus and Carnobacterium are the genera most
commonly used as starter cultures in the fermentation
processes of milk, meat and vegetable products (Stiles &
Hastings, 1991).
 Competitive exclusion technology
 Competitive exclusion (CE) as a technology, involves the
addition of a non-pathogenic bacterial culture to the
intestinal tract of food animals in order to reduce
colonization or decrease populations of pathogenic
bacteria in the gastrointestinal tract (Callaway et al.,
2004).
 The use of CE cultures has been studied extensively in
poultry to reduce Salmonella and Campylobacter carriage
(Chen & Stern, 2001; La Ragione & Woodward,
2003Wagner, 2006; Zhang, Ma, & Doyle, 2007a, 2007b).
 The use of CE cultures, including commercial products in
poultry has been reported to reduce the colonization of
poultry with Salmonella spp. by up to 70% or by 7–9 log10
cycles (Davies & Breslin, 2003; Hoszowski & Truszczynski,
1997; Schneitz & Hakkinen, 1998).
 Reductions of between 3–100% of Campylobacter spp.
colonization on poultry has also been reported (Schoeni &
Wong, 1994).
 The use of CE cultures in cattle and pigs to eliminate E. coli
O157:H7 and/or Salmonella from rumen and gastrointestinal
tract have also shown potential for further commercial
development (Brashears, Jaroni, & Trimble, 2003; Genovese
et al., 2003; Zhao et al., 2003).
 Antimicrobial proteins
 The production of one or more antimicrobial active metabolites
is part of the complex mechanisms by which a culture becomes
established in the presence of other competing organisms
(Holzapfel et al., 1995).
 These metabolites can include clinical or therapeutic low-
molecular weight antibiotics, lytic agents, toxins, bacteriolytic
enzymes and other metabolic products such as hydrogen
peroxide and diacetyl (Holzapfel et al., 1995; Kostrzynska &
Bachand, 2006).
Biocontrol of pathogenic bacteria through the food chain using microbial antagonistic
bacteria and/or their antimicrobial products. Antagonistic strains can be applied: (1) as
living cultures on livestock and fresh produce; (2) as protective cultures on ready-to-eat
food products; (3) as starter or protective cultures in fermented foods. They are
expected to grow and produce antimicrobial substances in situ, displacing unwanted
bacteria. Alternatively, food-grade preparations containing antimicrobials produced at
industrial scale by antagonistic strains can be applied as biopreservatives or as food
additives to inhibit transmission of food-borne and/or spoilage bacteria through the food
chain (1–4). Since the food microbiota may change considerably from farm to fork,
biocontrol strategies must be designed specifically for each type or category of food
product.
 Bacteriocins
 These are proteins that exert an antimicrobial action
against a range of microorganisms.
 Bacteriocins are ribosomally synthesized peptides whose
responsible genes are frequently associated with
transposons or on plasmids (Deegan, Cotter, Hill, & Ross,
2006).
 Their production can be related to the antagonism within a
certain ecological niche, as the producer strain, being itself
immune to its action, generally gains a competitive
advantage.
 Bacteriocins have a wide antibacterial spectrum with
feasible application in foods, such as meat and fish
products, fruits and vegetables, cereals and beverages
(Cleveland, Montville, Nes, & Chikindas, 2001).
 LAB-derived bacteriocins are generally recognized as safe
(GRAS) and are attractive to the food industry because of
their activity against key Gram-positive pathogens such as
Listeria monocytogenes or Staphylococcus aureus.
 The most-studied bacteriocins in meat and meat
products include nisin, enterocin AS-48, enterocins A
and B, sakacin, leucocin A, and especially pediocin PA-
l/AcH.
 These have shown promising results when used alone
or in combination with several physicochemical
treatments, modified atmosphere packaging, high
hydrostatic pressure, (HHP), heat, and chemical
preservatives, as an additional hurdle to control the
proliferation of L. monocytogenes and other pathogens
.
 Nisin
 Nisin is found to be more effective against Gram positive
bacteria such as Staphylococcus, Listeria and lactic acid
bacteria and also when used in combination with chemicals
such as EDTA.
 Reductions of 1.8-3.5 log in Gram positive bacteria have
been reported (Cutter and Siragusa 1995).
 Nisin is approved for use in the US in casings and on
cooked ready-to-eat (RTE) meat and poultry products.
 Several researchers have demonstrated the effectiveness
of nisin and/or nisin-producing strains against pathogenic
bacteria such as Clostridium botulinum in cheese and
against L. monocytogenes in cheeses such as Camembert,
Ricotta, and Manchego
 Pediocins
 Pediocin, produced by Pediococcus acidilactici, a
generally recognized as safe (GRAS) organism
 Commonly found and used in fermented sausage
production
 Pediocin AcH has been proven to be effective against
both spoilage and pathogenic organisms, including
Listeria monocytogenes, Enterococcus faecalis,
Staphylococcus aureus and Clostridium perfringens
 Reuterin
 Reuterin is a neutral, broad
spectrum antimicrobial
substance by Lactobacillus
reuteri.
 Antimicrobial activity of
reuterin against E. coli
O157:H7 and Listeria
monocytogenes on the
surface of cooked pork at 7°C
(El-Ziney et al. 1998).

Lactobacillus reutri
 Non-LAB bacteriocins
• Bacteriocins from non-LAB bacteria, such as variacin
(from Kocuria varians),
• Cerein 8A (from Bacillus cereus) or
• Colicins and microcins are also being investigated for
food biopreservation.
 Limitations
 Bacteriocins have narrow antibacterial activity.
 Large number of laboratory studies have been carried
out with different bacteriocins but still there is a long
way to go for industrial applications.
 Heterologous bacteriocin production and
development of large-scale production processes are
challenging.
 The impact of bacteriocin-producing strains on the
microbial ecology of the gastrointestinal tract and on
animal health is still not well understood.
 Zymosin
• Certain yeasts, such as strains of Saccharomyces
cerevisiae, produce several proteins (designated as
killer toxins or zymocins) that have limited
antimicrobial properties (Bakalinsky, 1992).
• Through genetic manipulation these can be altered to
have wider antimicrobial spectrum, especially against
fungi.
• These yeasts are normally present in fruits and
vegetables that are eaten raw, they are not considered
pathogenic, and thus can be used in place of
fungicides to enhance the preservation of fruits and
vegetables (Wilson 1991).
 Antimicrobial peptides
• Casecidin, obtained from milk casein by chymosin
digestion, exhibits activity against Staphylococcus aureus,
Sarcina, Bacillus subtilis, Diplococcus pneumoniae, and
Streptococcus pyogenes (Lahov and Regelson 1996).
• Isracidin is another casein-derived antimicrobial peptide. It
was tested against infection with Staphylococcus aureus
and Candida albicans in a mice (Lahov and Regelson 1996).
• Lactoferricins - several lactoferrin-derived antimicrobial
peptides named lactoferricin B have been isolated (Hoek,
Milne, Grieve, Dionysius, and Smith 1997; Shin et al. 1998).
• The hydrolyzate of lactoferrin exhibits broad antibacterial
activity against both Gram-positive and Gram-negative
bacteria.
 OTAP-92 - Hen egg ovotransferrin-derived antimicrobial
peptide that is active against S. aureus and E. coli has been
isolated (Ibrahim, Sugimoto, and Aoki 2000).
 Carnosine (β-alanyl-L-histidine) and Anserine (N-β-alanyl-1-
methyl-L-histidine) are endogenous antioxidative
dipeptides found in skeletal muscle (Lynch and Kerry 2000).
They are known to be the most abundant antioxidants in
meats.
 Microcins are small peptides that show inhibition of Gram-
negative bacteria.
 Microcin J25 (MccJ25) is active against Salmonella spp.,
Shigella spp., and Escherichia coli O157:H7.
 MccJ25 variant has been developed recently, which may be
used as a food preservative against the Gram-negative
pathogens (Pomares MF, Salomo´ n RA, Pavlova O,
Severinov K, Farı´as R, Vincent PA,2009).
 Some antimicrobial peptides of animal origin
 Pardaxin- Pardachiros maroratus (Red sea Moses sole),
Par. pavoninus
 Melittin- Bee venom
 Ceratotoxin- Ceratitis capita
 Histatins- Human saliva
 Trichorzin- Trichoderma (soil fungi)
 Cecropin- Humoral immune system of some insects i.e.
Hyalophora cecropia (giant silk moth)
 Magainins- Frog and other amphibians
 Defensins- Mammalian neutrophils
 Limitations
• Not much research

• Cytotoxicity

• Allergenicity

• Long way for industrialization

 Antimicrobials of Animal origin
 Enzymes and other polypeptides with antimicrobial
activity occur naturally in animals as well as plants.
 Lytic enzymes-
Lysozyme Its lysing activity has been demonstrated
against L. monocytogenes and C. botulinum (Hughey
and Johnson, 1987).
Glycosidases
Amidase
Lipases
Nielsen (1991) reported that lytic enzymes lysed
Campylobacter, E. coli, Salmonella, and V.
parahaemolyticus, indicating their potential use as
natural food antimicrobials to reduce the risk of
foodborne illness
Peroxidases and Oxidases
Lactoperoxidase
 Lactoperoxidase is the most abundant enzyme in bovine milk
and is also produced in salivary glands of mammals.
 In raw milk, the lactoperoxidase system (LPS) inhibits lactic
acid bacteria but is bactericidal to Gram-negative spoilage
psychrotrophs and Salmonella (Reiter and Harnulv, 1984; Leyer
and Johnson, 1993).
 The LPS is also active against Gram-positive pathogens such as
L. monocytogenes (Siragusa and Johnson, 1989), S. aureus
(Kamau et al., 1990), and B. cereus (Zajac et al., 1981).
Glucose oxidase
 Inhibition of growth of Salmonella infantis, S. aureus,
Clostridium perfringens, and B. cereus by glucose oxidase was
reported by Tiina and Sandholm (1989).
 Antimicrobials of plant origin
• There are numerous natural compounds in plants that
are known to prevent the growth of microorganisms
• Naturally occurring antimicrobial compounds are
present in plant leaves, stems, barks, roots, flowers,
and fruits
 Allium
• Garlic (Allium sativum), onion (A. cepa), and leek (A.
porrum) have allicin, a diallyl thiosulfinate (2-
propenyl-2-propenethiol sulfinate), as an antimicrobial
• Work on antimicrobial activity of garlic and other
Allium species has been done using foodborne
pathogenic bacteria, mycotoxigenic molds, and
spoilage microorganisms in general.
• Since sulfhydryl enzymes are common to all of these
microorganisms, the spectrum of activity of Allium
extracts is broad have effect against Staphylococcus
aureus and Bacillus species, Streptococcus
 Spices and herbs
 Cinnamic aldehyde (3-phenyl-2-propenal) has shown to be
the major antimicrobial compound in cinnamon.
 Also inhibits mold growth (Deans and Richie, 1987),
 Eugenol [2-methoxy-4-(2-propenyl)phenol], a major
constituent in clove oil and is present in considerable
amounts in the essential oil of allspice, possesses
antimicrobial activity.
 Vanillin (4-hydroxy-3-methoxybenzaldehyde), is a major
constituent in vanilla beans, which is structurally similar to
eugenol. Also antimycotic, Retard the growth of yeasts
(Boonchird and Flegel, 1982) and molds (Maruzzella and
Liguori, 1958). Vanillin is also inhibitory to L.
monocytogenes (Delaquis et al., 2005)
 Thymol [5-methyl-2-(1-methylethyl) phenol], present in the
essential oils of thyme, oregano, savory, sage, and several
other herbs, having a wide spectrum of antimicrobial
activity.
 Effective against V. parahaemolyticus, Shigella, C. botulinum,
S. aureus.
 Thyme extract has been reported to inhibit salmonellae, E.
coli O157:H7, Yersinia enterocolitica, Shigella flexneri, L.
monocytogenes, and S. aureus (Rota et al., 2004).
• Sage is inhibitory to V. parahaemolyticus (Shelef et al.,
1980), B. cereus, S. aureus, and S. typhimurium (Shelef et al.,
1984).
• Carvacrol, a major component of essential oil of oregano
and thyme, is lethal to S. cerevisiae (Knowles and Roller,
2000).
• Bhavanti Shankar and Sreenivasa Murthy (1979)
investigated the effect of turmeric on the growth of
intestinal and pathogenic bacteria and reported that the oil
fraction of turmeric was inhibitory toward numerous
• Allyl isothiocyanate, a nonphenolic compound naturally
occurring in plants belonging to the Crucifereae family,
has been successfully used to control or eliminate E.
coli O157:H7 in ground beef (Nadarajah et al., 2005).
• Growth of the mycotoxigenic molds such as, A. flavus,
A. parasiticus, Aspergillus versicolor, Aspergillus
ochraceus, Penicillium urticae, and Penicillium
roquefortii (Azzouz and Bullerman, 1982), as well as
food spoilage molds, yeasts, and bacteria is also
retarded or inhibited in the presence of many
commonly used spices and herbs.
• Phenolic compounds such as caffeic, chlorogenic, p-
coumaric, ferulic and quinic acids, are found in plant
parts used as spices (Beuchat and Golden, 1989).
• Hydroxycinnamic and cinnamic acids have been shown
to retard microbial invasion and delay rotting of fruits
and vegetables.
 Anthocyanin derivatives
 The antimicrobial properties of several compounds
responsible for color of plant tissues have been
demonstrated
 Pelargonidin, cyanidin, delphinidin, plonidin,
petunidin, and malvidin are among the most
important anthocyanins in terms of contributing to
the sensory quality of foods.
 Researchers have studied the mechanism of
antimicrobial activity of anthocyanins Carpenter et al.
(1967)
 Extracts and oils
• Recent research has focused on evaluating antimicrobial
activities of extracts of aromatic plants not necessarily
used as foods.
• Extracts of sumac (Nasar-Abbas and Halkman, 2004),
gardenia and cedar wood (Friedman et al., 2002), and
Lamiaceae species (Araujo et al., 2003), have been shown
to exhibit inhibitory activities against foodborne
pathogenic and spoilage microorganisms.
• Extracts of blueberries, crabapples, strawberries, red
wines, grape juice, apple juice, and tea have been studied
for their antiviral activity by Konowalchuk and Speirs
(1978). These inactivated poliovirus, coxsackievirus,
echovirus, reovirus and herpes simplex virus
• The primary inhibitors were thought to be tannins
• Tannic acid was antiviral against echovirus, poliovirus and
herpes simplex virus
 Phytoalexins
 Phytoalexins are low molecular weight compounds
produced by higher plants in response to microbial
infection and naturally occurring elicitors (Dixon et al.,
1983).
 Phytoalexins are known to occur in leaves, fruits,
seeds, roots, and tubers of a wide range of plants
 Glyceollin, coumestrol, and glycinol, are all
phytoalexins produced by soybeans, which inhibit
microbial membrane-associated processes (Weinstein
and Albersheim, 1983; Amin et al., 1988).
 Propolis
 Propolis is a mixture of resinous material collected by
honeybees from buds, blossoms, and leaves of plants,
pollen, and enzymes secreted by the bees.
 The antimicrobial activity of propolis has been
attributed to flavonoids and esters of phenolic acids
(Sforcin et al., 2000), as well as other components
(Kujumgiev et al., 1999).
 Bacteriophages
 Bacteriophages are viruses that infect and kill bacterial
cells by reproducing within the bacteria and disrupting the
host metabolic pathways, causing the bacterium to lyse.
 These specifically target bacterial cells and do not infect
mammalian cells, thus they are proposed as biocontrol
agents in human, animal, clinical and industrial
applications.
 The application of bacteriophage to control foodborne
pathogens such as Campylobacter, E. coli O157:H7, Listeria
and Salmonella in animal food products, and food
processing environments have been carried out
 Phages have been shown to reduce numbers of foodborne
pathogens such as L. monocytogenes on the surface
ripened cheeses as well as E. coli O157:H7 and Salmonellae
on fresh poultry.
 In 2006, the US Food and Drug Administration
approved a bacteriophage preparation to be applied
on Ready-To-Eat (RTE) meat and poultry products as
an antimicrobial agent against L. monocytogenes
(Federal Register, 2006).
 The bacteriophage preparation consists of six Listeria
specific bacteriophages, which were combined to
reduce the possibility of L. monocytogenes developing
resistance to the agent.
 Another commercially available product is Listex
TMP100, produced by EBI Food Safety.
 It is a bacteriophage used for the control of L.
monocytogenes in meat and cheese products. It is
recognized as GRAS by the FDA and has a wide host
range against Listeria strains (Carlton, Noordman,
Biswas, de Meester, & Loessner, 2005).
 Limitations
 Adsorption may be obstructed by the existence of
considerable numbers of non-host bacterial cells. This
type of environment may protect the bacterial cell from
bacteriophage infection.
 Host bacterial cells may also be protected by biofilms
present in a food environment
 Many meat products are distributed and stored at
refrigeration temperatures, conditions under which many
pathogens may not grow.
 Bacteriophages may be destroyed in food processing
environments as they are effectively cleaned and
sanitized.
 Bacteriophages also have the capability to transfer
unfavorable genes from one bacterium to another; these
could be virulence genes or antibiotic resistance genes.
 ENDOLYSINS
 Lysins, or endolysins , are hydrolytic enzymes produced
by bacteriophages.
 It cleave the host's cell wall during the final stage of the
lytic cycle.
 Endolysins - Degrading the peptidoglycan of Gram
positive bacteria when applied externally to the
bacterial cell, thereby acting as antibacterial agents.

(Jhamb
(Jhamband
andSpardha,
Spardha,2014)
2014)
 Endolysin
Endolysin application
application
 Broad killing spectrum by cleaving peptidoglycan linkage of
bacterial membrane.

 It exhibits the antimicrobial activity at Nanogram level .

 At the same time production cost is very high, because use of

genetically modified organism for the production.
 Bdellovibrios
 Bdellovibrio bacteriovorus is a tiny (0.2-0.5 um ×
0.5-2.5 um) Gram negative predatory bacteria
discovered by chance in 1962 by Stolp and Petzold.
 Parasitic bacteria B. bacteriovorus prey on a range
of Gram -ve pathogens and spoilage organisms.
 These organisms are present in soil and faecal
contents of many species, and can be isolated and
purified.
 Little work has been done on their applications to
foods.
 Bdellovibrio isolates have achieved 2.5-7.9 log
reductions in E. coli and Salmonella populations
during 7 hours in culture, and 3.0-3.6 log
reductions on stainless steel (Fratamico and Cooke
1996).
 Atterbury et al. 2011, Bdellovibrio-and-like-
organisms (BALOs) orally, to young chicks which
had experimentally colonized gut with S. enterica.
These predatory strains of BALOs could effectively
attack Salmonella, thereby reducing its number in
the cecum and significantly mitigating cecal
inflammation caused by the infection.
 Limitations
 BALOs don’t completely kill their prey.
 Their effects on the natural flora of different body cavities has
not been studied thoroughly and should be taken into
consideration, the use of BALOs in this case would need to be
monitored.
 These bacteria were found to be strict aerobes.
 BALOs is their inability to attack Gram negative bacteria with S-
layer on their surfaces.
 Their activity is affected by the physiological status of their
prey and by the presence of other bacteria.
 MERITS
MERITSAND
ANDDEMERITS
DEMERITSOF
OFBIO
BIOPRESERVATION
PRESERVATION
MERITS DEMERITS
Extended
Extended shelf-life
shelf-life of
of the
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Lackof
ofresearch
researchfocus
focus
meat
meatand
andmeat
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Application
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Decrease the
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pathogenstransmission
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Narrowactivity
activityof
ofspectrum
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Reduce
Reduceuse
useof
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Limited diffusion
diffusion inin solid
solid
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preservatives matrix
matrix

Resistant
Resistant organisms
organisms are
are Sometime
Sometimedifficult
difficultto
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apply
controlled
controlled effectively
effectively
e.g.
e.g.L.L.monocytogenes
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Commercialization

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