Over view on Bio Preservation-Fermentation-Bacteriocins-Mechanism-Classification-Future scope-Research focus

1. BIO-PRESERVATION OF
MEAT AND MEAT PRODUCTS
G.SUNDARESAN
MVM 15026
Credit seminar
Department of Livestock Products
Technology (Meat Science),
Madras Veterinary College,
Chennai – 600 007.

2. Introduction
 Meat is highly desirable, nutritious and rich in protein,
highly perishable.
 Ancient preservation- physical treatment and
application of preservatives.
(e.g) Heat treatment and
application of salt.
(Talukder et al. 2014)

3. Major concerns of meat preservation
Food Quality
Making food desirable to eat-
 Good taste
 Color
 Texture
(Nath et al., 2006)
Food Safety
 Infectious agents
 Toxic chemicals
 Foreign objects

4.  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
Existing Preservative Techniques

5. Bio preservation – an alternative
 Fermentation, bacteriocins, bacteriophages and bacteriophage-encoded
enzymes fall in this concept.
(Elsser et al.,2013)
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.
Definitions

6. Principles of bio preservation
Hindering the growth activity of the microorganisms.
e.g. pH
 Killing the microorganism.
e.g. Bacteriocins

7. Anti-microbial metabolites
Organic acids
Lactic acid
Acetic acid
Propionic acid
Bacteriocins
Class I
Class II
Class III
Molecular compounds
Hydrogen peroxide
Carbon dioxide
Diacetyl
Peptides

8. 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

9. VARIOUS METHODS OF BIO PRESERVATION
1. Fermentation
2. Bacteriocin application
3. Bacteriophages
4. Endolysins

10. Fermentation
 Fermentation is a metabolic
process that converts sugar to
acids, gases, or alcohol. It occurs
in yeast and bacteria.
Definition:
 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.

11. 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

12. 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).

13. General procedure for the preparation of fermented meat products
Fermentation in meat and meat products
Preparation of
meat emulsion
Shift it in
casing at -2.2
to 1.1° C
Addition of
starter culture
Fermentation-
lactic 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

14. 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.

15. Probiotics
"live micro-organisms which, when
administered in adequate amounts,
confer a health benefit on the host".
(WHO 2001) e.g. Lactobacillus plantarum
Definition
 Probiotics are poor survivors in meat – Challenging
environment .
 Micro-encapsulation may be an option for formulation
of fermented meat products with viable health-
promoting bacteria (Muthukumarasamy and Holley 2006).

16. Safety issues of fermented meat and meat products
I. Biogenic amines
Cadaverine
Putrescine
Tyramine
Toxicological symptom
Other symptom
1. Pseudo allergic reaction
2. Interaction with drugs
1. Gastric ulcers
2. Blood pressure problems
3. Nervous symptoms
II. Antibiotic resistance
1. Gene translocation
Suzzi and Gardini, 2003

17. 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).

18. Bacteriocins vs antibiotics
s.no Characteristic Antibiotics Bacteriocins
1 Synthesis
Secondary
metabolite
Ribosomal
2 Application Clinical Food
3 Activity
Varying
spectrum
Narrow spectrum
4 Host cell immunity No Yes
5 Mode of action
Cell membrane
or intracellular
targets
Mostly pore
formation, but in a
few cases possibly
cell wall
biosynthesis
6
Toxicity side effect
Yes None known

19. ADVANTAGES OF BACTERIOCINS
BACTERIOCINS
Reduce the
use of
chemical
preservatives
Better
preservation
Decrease the
risk of
disease
transmission
Extra
protection
It satisfy the
consumer
demand

20. FACTORS PROMOTING
USE
OF BACTERIOCINS AS
BIOPRESERVATIVES
Not altering
Nutritional
quality
Effective under
wide pH &
temperature range
Activity is not lost
Presence of other
preservatives Effective in low
concentrations
Broad spectrum of
activity from
food grade LAB
Preference over
traditional
preservative
Safe (FDA)
use of nisin for > 40 years
in several countries

21. Bio synthesis of Bacteriocins
Nisin A
Regulatory Gene
Cyclize &
Dehydrydrate
Pre
peptide
ABC
Transpoter
Cellular
space
Proteases
Active form
of Bacteriocin
Extra cellular
space
Lipoprotein &
Transporter
complex

22. Class I
Class II
Class III
Classification of Bacteriocins

23. 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 Class I b
Linear
Cationic and hydrophobic
peptides
Globular
No net charge
Cleveland et al., 2001
Nisin Mersacidin

24. Mechanism of action of Class I Bacteriocins

25. Class II bacteriocins or non Lantibiotics
1. Class IIa or pediocin-like bacteriocins
2. Class IIb or two-peptides bacteriocins
3. Class IIc
4. Class IId
 Cationic
 Hydrophobic
 Heat-stable
 Membrane active peptides
Klenhammer, 1993

26.  (e.g) Enterocin NKR-5-3C (Ent53C).
 Ent53C showed very strong microbial activity (in Nano
molar range) against Listeria spp.
Class IIa Bacteriocins (Pediocin like bacteriocins)
- anti-listerial bacteriocins
Mechanism of action
Enterocin

27. Class IIb bacteriocins (Two-peptide
bactetiocins)
 Killing activity of this
bacteriocin is based on the
synergistic activity of both the
peptide.
1. Type E (enhanced)
1. Type S (synergistic)
Action mechanism
(e.g.) Acidocin L

28. Class-IIc - Circular bacteriocins
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.
(e.g.) Lactococcin A

29. 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

30. 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

31. Production of Bacteriocins

32. 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
III. Use of previously fermented meat with
bacteriocin producing strains
(Thomas et al., 2000)
Bacteriocin producing strains
Purified bacteriocins
VI. Edible cellulosic films - New

33. 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 anti-
listerial activity, But they are not currently
approved.
 NISIN is the only approved Bacteriocin by
FAO/WHO 1969. (Devlieghere, et al., 2004)

34. 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)

35. 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)

36. 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.
Raw mince meat
(Pawar et al., 2000)

37. 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)

38. 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)

39. Examples of microorganisms that may be controlled by bio
preservation techniques

40. 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.
(Nes et al., 2007)
Commercially approved bacteriocin
Nisin – E234
(Nisaplin)

41. 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

42. 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.
 Bacteriophages will remain dormant unless their
specific target, L. monocytogenes is encountered.
(Kathy walker, 2006).
Phage (lytic)

43.  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.
ENDOLYSINS
(Jhamb and Spardha, 2014)

44. Endolysins mechanism of action
 Schematic representation
of the modular structure
 Mode of action of phage-
encoded - endolysins.

45. 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.

46. MERITS AND DEMERITS OF BIO PRESERVATION
Bio
Prn
Extended shelf-life of the
meat and meat product
Decrease the of food borne
pathogens transmission
Reduce use of chemical
preservatives
Resistant organisms are
controlled effectively
(e.g) L.monocytogenes
Minimal handling and
Natural way
Lack of research focus
Application deficit
Commercialization
Regulatory approval
MERITS DEMERITS

48. Current status
 Nowadays researchers are
focusing on the application of
bacteriocins in foods as part of
packaging films.
 The potential applications of
bacteriocins in the food and health
care sectors are evident.
(Cagri et al., 2004)

49. 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
(Billman Jacobe, 1996)
 Alternative food-grade organisms must be
Identified,
 Researchers are focusing on the application of
bacteriocins in foods as part of packaging films.
(Coma, 2008).

50. 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

51. THANK YOU