Credit seminar
BIO-PRESERVATION OF
MEAT AND MEAT PRODUCTS
Department of Livestock Products
Technology (Meat Science),
Madras Veterinary College,
Chennai – 600 007.
G.SUNDARESAN
MVM 15026
Introduction
 Meat is highly desirable, nutritious and rich in protein,
highly perishable.
 Ancient preservation- physical
application of preservatives.
treatment
and
(e.g) Heat treatment and
application of salt.
(Talukder et al. 2014)
Major concerns of meat preservation
Food Safety
 Infectious agents
 Toxic chemicals
 Foreign objects
Food Quality
Making food desirable to eat-
 Good taste
 Color
 Texture
(Nath et al., 2006)
Existing Preservative Techniques
 Drying, Curing, Smoking, Canning, Chilling, Freezing,
Direct microbial control
 The modern physical and chemical methods of food
preservation are more effective than the traditional methods,
with drawbacks.
 Chemical preservatives - Toxicity
 Change organoleptic and nutritional properties
 It made research interest on the
natural and effective
preservatives
Bio preservation – an alternative
Definitions
Meat preservation
Extending the shelf life of the food by
continuous fight against the spoilage organism.
Meat Bio-preservation
Extending the shelf life and food safety by natural
or controlled microbiota or their antimicrobial
components.
 Fermentation, bacteriocins, bacteriophages and bacteriophage-encoded
enzymes fall in this concept.
(Elsser et al.,2013)
Principles of bio preservation
Hindering the growth activity of the microorganisms.
e.g. pH
 Killing the microorganism.
e.g. Bacteriocins
Anti-microbial metabolites
Organic acids
Lactic acid
Acetic acid
Propionic acid
Molecular compounds
Hydrogen peroxide
Carbon dioxide
Diacetyl
Peptides
Bacteriocins
Class I
Class II
Class III
Mechanism of action of anti-microbial metabolites
 Organic acids:
By lowering pH.
 Oxidases
Hydrogen and Lactoperoxidase
• H2O2 producing reactions lower the
oxygen.
• Oxidize sulfhydryl groups of cell
proteins and membrane lipids.
 Diacetyl
• Interfering with the utilization
of amino acids
+
VARIOUS METHODS OF BIO PRESERVATION
3. Bacteriophages
1.
Fermentation
2.
Bacteriocin application 4. Endolysins
Fermentation
Definition:
 Fermentation is a metabolic
process that converts sugar to
acids, gases, or alcohol. It occurs
in yeast and bacteria.
 This process based on the growth of micro-organism in foods
it may be natural or added.
 Mainly comprised of lactic acid bacteria.
 Will produce the organic acid and other compounds.
 This compounds exhibits the preservative action.
Fermentation of meat by protective cultures
Selection of Cultures
• Generally Recognised As Safe (GRAS)
• Ability to produce antimicrobials in meats
• Limited sensory changes
• Limited acid production
• Weak protease activity
• Limited gas production
Starter culture for meat fermentation
 Meat starter cultures are preparations that contain active or
dormant microorganisms that develop the desired metabolic
activity in the meat (Hammes, Bantleon, and Min 1990).
Fermentation in meat and meat products
General procedure for the preparation of fermented meat products
Preparation of
meat emulsion
Shift it in
casing at -2.2
to 1.1° C
Addition of
starter culture
Fermentationlactic acid
Cooking or
drying
Upstream bioprocessing
 Inoculum development
 media development
 Enrichment
Downstream bioprocessing
 Separation of biomass
 Semi concentration
 Purification
 Laboratory and field trial
fermented
meat products
Effects of culture on the meat & meat products
Fermented sausages
Addition of curing agent and sugar and
fermentation condition preventing the growth of pathogens
in fermented sausages (Lucke, 1998 b).
Raw ham and Ready to eat
Injection of Psychotropic lactic acid bacteria even under the
aerobic condition observed approximately 2 log reduction of Listeria
monocytogenes during the Vacuum packaged conditions.
Semi processed raw meat
Strains of lactic acid bacteria that improves the self-life and freshness
of refrigerated semi-processed meat such as bacon and sausages.
Salted-Semi processed raw meat
Pediococcus starter grow and form the acids. Thus restricting the growth
of any clostridium botulinum and extend the shelf-life.
Probiotics
Definition
"live micro-organisms which, when
administered in adequate amounts,
confer a health benefit on the host".
(WHO 2001) e.g. Lactobacillus plantarum
 Probiotics are poor survivors in meat – Challenging
environment .
 Micro-encapsulation may be an option for formulation
of fermented meat products with viable healthpromoting bacteria (Muthukumarasamy and Holley 2006).
Safety issues of fermented meat and meat products
Toxicological symptom
I. Biogenic amines
Cadaverine
Putrescine
Tyramine
II. Antibiotic resistance
1. Gene translocation
1. Pseudo allergic reaction
2. Interaction with drugs
Other symptom
1. Gastric ulcers
2. Blood pressure problems
3. Nervous symptoms
Suzzi and Gardini, 2003
Bacteriocins
 Ribosomally synthesized extracellularly released
bioactive peptides or peptide complexes, having
bactericidal or bacteriostatic activity.
 Rapidly digested by proteases in human digestive
tract.
 This bacteriocins are first discovered in 1925 by
Andre Gratia
 Alternative to antibiotics and chemical
preservatives.
(Joerger et al., 2000).
Bacteriocins vs antibiotics
s.no Characteristic
Antibiotics Bacteriocins
1
Synthesis
Secondary
metabolite
2
Application
Clinical
Food
3
Activity
Varying
spectrum
Narrow spectrum
4
Host cell immunity
No
Yes
Cell membrane
or intracellular
targets
Mostly pore
formation, but in a
few cases possibly
cell wall
biosynthesis
Yes
None known
5
6
Mode of action
Toxicity side effect
Ribosomal
ADVANTAGES OF BACTERIOCINS
Extra
protection
Reduce the
use of
chemical
preservatives
Decrease the
risk of
disease
transmission
BACTERIOCINS
It satisfy the
consumer
demand
Better
preservation
Safe (FDA)
use of nisin for > 40 years
in several countries
Not altering
Nutritional
quality
Effective under
wide pH &
temperature range
Activity is not lost
Presence of other
preservatives
FACTORS PROMOTING
USE
OF BACTERIOCINS AS
BIOPRESERVATIVES
Effective in low
concentrations
Preference over
traditional
preservative
Broad spectrum of
activity from
food grade LAB
Bio synthesis of Bacteriocins
Regulatory Gene
Nisin A
Cyclize &
Dehydrydrate
Pre
peptide
ABC
Transpoter
Cellular
space
Proteases
Active form
of Bacteriocin
Lipoprotein &
Transporter
complex
Extra cellular
space
Classification of Bacteriocins
Class I
Class II
Class III
Class I Bacteriocins or Lantibiotics
• Small peptides (<5kD)
• More than 50 amino acids
• Made up of unusual amino acids (lanthionine, and
methyl-lanthionine)
Lantibiotics
Class I a
Nisin
 Linear
 Cationic and hydrophobic
peptides
Class I b
Mersacidin
 Globular
 No net charge
Cleveland et al., 2001
Mechanism of action of Class I Bacteriocins
Class II bacteriocins or non Lantibiotics




1.
2.
3.
4.
Cationic
Hydrophobic
Heat-stable
Membrane active peptides
Class IIa or pediocin-like bacteriocins
Class IIb or two-peptides bacteriocins
Class IIc
Class IId
Klenhammer, 1993
Class IIa Bacteriocins (Pediocin like bacteriocins)
- anti-listerial bacteriocins
 (e.g)
Enterocin NKR-5-3C (Ent53C).
 Ent53C showed very strong microbial activity (in Nano
molar range) against Listeria spp.
Mechanism of action
Enterocin
Class IIb bacteriocins (Two-peptide
bactetiocins)
 Killing activity of this
bacteriocin is based on the
synergistic activity of both the
peptide.
(e.g.) Acidocin L
1. Type E (enhanced)
1. Type S (synergistic)
Action mechanism
Class-IIc - Circular bacteriocins
(e.g.) Lactococcin A
Unique structural feature of a head-to-tail
cyclization of their backbones.
structural stability, higher thermal stress
resistance, and superior stability against
proteolytic digestion, compared to their
linear counterparts.
However, the biosynthetic mechanisms
currently remains unknown.
class IId – Non pediocin like bacteriocins Leaderless bacteriocins
 Diversity of primary structure.
(e.g.) Lacticin Q
Action mechanism
The highly cationic lacticin
Q molecules rapidly bind
with negatively charged
phospholipid bilayer
membrane.
After which, the
lacticin
Q
molecular
mass
translocate itself
from the outer to
inner membrane
as the pore closes
Class III Bacteriocins 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
Anti-microbial action
Production of Bacteriocins
Methods of bacteriocin application in
meat and meat products
I. Inoculation of meat with Lactic acid producing bacteria
- starter or protective culture
- Bacteriocins
II. Purified or semi purified
bacteriocins
Bacteriocin producing strains
III. Use of previously fermented meat with
bacteriocin producing strains
VI. Edible cellulosic films - New
(Thomas et al., 2000)
Purified bacteriocins
Application in meat products
Several bacteriocins from the bacteriocin
producing strains used as a food preservatives.
Still very limited.
Bacteriocins like Pediocin has very strong antilisterial activity, But they are not currently
approved.
 NISIN is the only approved Bacteriocin by
FAO/WHO
1969. (Devlieghere, et al., 2004)
Application in meat products
The most-studied bacteriocins in the
meat and meat products is  Nisin
 Enterocin AS_48
 Enterocin A and B
 Pediocin PA-I
(Saeed et al., 2014)
Use of Purified/ Semi purified bacteriocins
 To date, the only commercially produced
bacteriocins are:
 Nisin produced by Lactoccocus lactis ssp.
Lactis – Rogers and Whittier 1928.
 Pediocin PA-1, produced by Pediococcus
acidilactici (Not approved)
(Saeed et al., 2014)
Raw mince meat
The samples of raw mince meat were inoculated
with 103 CFU/g of Listeria monocytogenes and
stored at 40 c.
After 16 days, the control sample increased count
from 3log10 to 6.4log10 CFU/g.
Nisin – 2.4 log 10 CFU/g.
(Pawar et al., 2000)
Beef carcass
 Beef carcass were inoculated with approximately 4 log 10
CFU/cm2 of Brochothrix thermosphatca to evaluate the
effectiveness of nisin to sanitize the carcasses.
 Nisin spray treatment 1000 IU/ml reduced the population
at 0 day – 1.8 log 10 CFU/cm2
at 1 day - 3.6 log 10 CFU/cm2
( Deegan et al., 2006)
Bacteriocin producing culture addition:
 (Degnan et al., 1992) Demonstrated the possibility
of using bacteriocin producing culture LAB and
Pediococcus acidilactici (nisin and pediocin) to
control the L. monocytogenes growth in the all
vacuum packed Beef products
Combination with MAP
 (Fang and Lin, 1994) Was found to be more effective
when used in combination with modified
atmosphere packaging (100% co2, 80%
co2+20%o2)
Examples of microorganisms that may be controlled by bio
preservation techniques
Commercial status
 Subtilin, Cerein, Plantaricin, have been
isolated and characterized from different
bacteriocin producing strains.
 But they are yet to acquire a commercial
status.
 Nisin (Lactobacillus lactis) and Pediocin
(Pediococci acidilactici) are the only
commercially.
Commercially approved bacteriocin
Nisin – E234
(Nisaplin)
(Nes et al., 2007)
Bacteriophages
 Bacterial virus that infect the bacteria and reproduce by
hijacking their host biosynthetic pathway
 They are harmless to humans, animals and plants.
I. Classification
II. Mechanism of action
Bacteriophage application in meat
Application in meat industry
 FDA - Recently permit safe use of a bacteriophage
preparation as an anti-listerial agent in Ready To
Eat (RTE) meat and poultry products.
 Phage (lytic) preparation with 0.1ppm
concentration is sprayed directly on meat products
prior to packaging at the level of 1 milliliter
(ml)/500 cm2 surface area.
Phage (lytic)
 Bacteriophages will remain dormant unless their
specific target, L. monocytogenes is encountered.
(Kathy walker, 2006).
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 and Spardha, 2014)
Endolysins mechanism of action
 Schematic representation
of the modular structure
 Mode of action of phageencoded - endolysins.
Endolysin application
 Broad killing spectrum by cleaving peptidoglycan
linkage of bacterial membrane.
 It exhibits the antimicrobial activity at Nano gram
level .
 At the same time production cost is very high, because
use of genetically modified organism for the
production.
MERITS AND DEMERITS OF BIO PRESERVATION
MERITS
DEMERITS
Extended shelf-life of the
meat and meat product
Lack of research focus
Decrease the of food borne
pathogens transmission
Application deficit
Reduce use of chemical
preservatives
Resistant organisms
controlled effectively
(e.g) L.monocytogenes
Minimal handling and
Natural way
Bio
Prn
are
Commercialization
Regulatory approval
Current status
 The potential applications of
bacteriocins in the food and health
care sectors are evident.
 Nowadays
researchers
are
focusing on the application of
bacteriocins in foods as part of
packaging films.
(Cagri et al., 2004)
Research focus
 Commercial application and for production in large scale, both
genetic and fermentative protocols need to be optimized.
 E. coli has long been considered the
primary prokaryotic host for cloning
 Alternative food-grade organisms must be
Identified,
 Researchers are focusing on the application of
bacteriocins in foods as part of packaging films.
(Billman Jacobe, 1996)
(Coma, 2008).
Conclusion
Extend the shelf life and food safety of meat and meat
products by the use of natural or controlled microbiota and
antimicrobial compounds.
This preservation can be effectively used - hurdles.
Technology can be integrated as part of preservation
technology
THANK YOU