This document summarizes strategies for enhancing food safety using bacteriocins produced by lactic acid bacteria. It discusses bacteriocins' classification and detection methods. Bacteriocins like nisin have broad-spectrum antimicrobial activity and can be used as food preservatives. Methods for incorporating bacteriocins into foods include using purified preparations, fermenting foods with bacteriocin-producing cultures, and developing antimicrobial packaging. Emerging techniques like microencapsulation and hurdle technology could help control bacteriocin resistance and provide controlled release. Continued research on bacteriocins may lead to their increased use as natural preservatives to improve food safety.

1. Bacteriocins based strategy for
enhanced food safety
Diwas Pradhan
Dairy Microbiology Division
Oct., 2015
National Dairy Research
Institute
Karnal-132001

2.  Introduction to Food Safety
 Bacteriocins of Lactic Acid Bacteria
 Classification of bacteriocins
 Detection of bacteriocin activity
 Applications of bacteriocin in foods
Outline of the presentation

3. Food Safety Food Quality
Making food desirable to eat-
Good taste
Color
Texture
Making food safe to eat and free
from disease causing agents-
Infectious agents
Toxic chemicals
Foreign objects
Major Concerns of the Food Industry

4. Product Diversification
Upgraded Quality
Enhanced Shelf Life
Ready to Eat
Fresh- Tasting
Nutritious and Vitamin Rich
Minimally- Processed
Consumers’ Preference

5. Lactic acid
Propionic acid
Acetic acid
Antimicrobial
substances
LAB
produces
Organic acids
Low molecular
weight
compounds
Bacteriocins
Reuterin
Diacetyl & Acetaldehyde
H2O2
Fatty acids
Phenyl lactic acids
Nisin
Lacticin
Pediocin
Gassericin
Antimicrobial compounds produced by LAB
Today’s
area of
discussion

6. “Bacteriocins are ribosomally-synthesized peptides or proteins with
antimicrobial activity, produced by different groups of bacteria”
BACTERIOCINS
 Bacteriocins of lactic acid bacteria play a defining role in the preservation &
microbial safety of foods
 Bacteriocins may have broad or narrow spectrum of activity
 Bacteriocins with broad spectrum of activity are of great importance
in food safety while Bacteriocins with narrow spectrum of activity
may be used for specified use

7. Bacteriocins of lactic acid bacteria
Safe and efficacious use of nisin for >40 years in several countries
(GRAS status)
Effective under wide pH & temperature range
Activity is not lost in the presence of food additives and effective in
dairy foods during storage
Effective in low concentrations
Consumer resistance to traditional chemical preservatives and
concern over the safety of existing food preservatives such as sulfites
and nitrites
Do not alter acceptance quality of food and are safe for human
consumption
What makes LAB bacteriocins as promising agent for their use in biopreservation

8. Lantibiotics Unmodified
peptides
Large proteins Circular
peptides
Class I Class II
Class III Class IV
Heng et al., 2006
Classification of bacteriocins
So far many researchers have
classified the LAB bacteriocins
into many groups and hence led
to some controversies.
An appropriate classification of
bacteriocins has been given by
Hegg and Tagg in 2006
According to them, LAB
bacteriocins can be divided into
four classes

9. Circular peptides
characterized by a peptide
bond between the C- and
N-terminus
Large heat labile proteins
with modest prospects as
food biopreservatives
Post-translationally modified
peptides that contain
lanthionine amino acid
Heat stable non-modified
peptides and is the largest
class among Gram positive
bacteriocins
Class I
Class II
Class III
Class IV
Different classes of bacteriocins
Bacteriocins

10. Classification of bacteriocins
Ingolf F. Nes et al., 2015

11. Detection of Bacteriocin Activity
Agar well assay method
Indicator / sensitive strain or in soft agar is overlaid on TGE
hard agar plate and 6 mm well are cut with sterile borer.
100 µl of cell free culture supernatant (CFCS) is poured into the
wells and incubated at 37 ̊C for 24 h after diffusion of CFCS
Spot-on-lawn method
Indicator / sensitive strain or in soft agar is overlaid on TGE
hard agar plate
5µl of cell free culture supernatant (CFCS) is spotted onto the
overlaid surface and incubated at 37 ̊C for 24 h after diffusion
of CFCS

12. Measurement of Low concentrations of potassium ions
Bacteriocins usually act by permeabilizing
the cell membrane of indicator strain and
release of K+ ions
Low concentrations of potassium ions were
measured, so that the released potassium
ions from a bacteriocin-sensitive indicator
strain directly correlated to concentrations
of crude bacteriocin present in fermentation
broth injected into the cell.
Bactericidal action of bacteriocin
by potassium ion efflux and
increased ATP demand from K+
ATPase (adapted from Garneau et
al., 2002)

13. PCR methods can also be used to detect genes responsible for bacteriocin production
and regulation in bacterial cultures.
The DNA from bacteriocin positive strains can be subjected to a bacteriocin-specific
PCR array with primers representing known structural genes of bacteriocins from LAB.
PCR based methods

14. Contd…….
Optical density measurement
Optical density measurement in a microplate
system is also a convenient approach to check the
inhibition of target bacteria and determine the
inhibitory effects of bacteriocins.
ELISA technique has also been tested for the detection of
bacteriocins that mainly uses affinity-purified anti-nisin
immunoglobulin for the binding of nisin and anti-nisin peroxidise
linked with the substrate.
ELISA technique

15. 1
How to add bacteriocins in foods……………
Using a purified/semi-purified bacteriocin
preparation as an additive in food
Incorporation of an ingredient previously
fermented with a bacteriocin-producing strain
Use of a bacteriocin-producing culture to
replace the starter culture in fermented foods
to produce the bacteriocin in situ
Traditional methods for incorporation of bacteriocin preparations in foods

16. Prototypical lantibiotic, having been first marketed in England in 1953 and
has since been approved for use in over 50 countries.
Nisin has been assessed to be safe for food use by the Joint Food and
Agriculture Organization/World Heath Organization Expert Committee on
Food Additives in 1969.
In 1983, this bacteriocin was added to the European food additive list as
number E234 (indeed it is the only natural antibacterial to have been
approved for as a food preservative by the EU)
In 1988, it was approved by the US Food and Drug Agency (FDA) for use in
pasteurized, processed cheese spreads and is currently used in a wide
variety of foods across the world
1. Use of purified/ semi purified bacteriocins
Commercially available purified / semipurified Bacteriocins
Nisin Lactoccocus lactis ssp. lactis
Pediocin PA-1 Pediococcus acidilactici
Delves-Broughton et al., 1996

17. Use of nisin in the safety & biopreservation of dairy products
In pasteurized, processed cheese products to
prevent outgrowth of spores of Clostridium
tyrobutyricum
100-400 IU/g of nisin – for good quality cheese
Extends the shelf life of dairy desserts which cannot
be fully sterilized
Vegetables Target Organisms Bacteriocin
Fermented Vegetables L. monocytogenes Nisin
Non-fermented vegetables E. faecium Nisin-EDTA Treatment
Canned Vegetables Clostridium spp Nisin
Soy Milk Bacillus cereus Nisin EDTA-Na Acetate-
Citric Acid-K. sorbate

18. Use of Pedicin in the safety & biopreservation of dairy products
Pediocin AcH:
Active against both spoilage and pathogenic organisms
L. monocytogenes
Enterococcus faecalis
Staphylococcus aureus
Clostridium perfringens
Pediocin PA-1:
Inhibits Listeria in dairy products such as cottage cheese, ice cream, and
reconstituted dry milk

19. 2) Use of an ingredient previously fermented with a bacteriocin-producing strain
Pediocin 34 produced by Pediococcus pentosaceus 34
Spectrum of activity of pediocin 34
Gram positive Gram negative *
Staphylococcus aureus Escherichia coli
Listeria monocytogenes Pseudomonas spp.
Bacillus spp Salmonella spp.
Enterococcus spp. * In the presence of 20mM EDTA
Micrococcus spp.
Combination of Pediocin and Nisin has Synergistic Effect
Bacterial cells resistant to one bacteriocin can be sensitive to another
bacteriocin and the antibacterial spectrum of bacteriocins is effective by
using a combination of bacteriocins

20. Contd…..
MicroGARDTM
A product from DANISCO,
Denmark
Produced from skim milk
fermented by a strain of
Propionibacterium freudenreichii
ssp. shermanii
Used as biopreservative &
flavour enhancer
Approved by FDA (1990) and
granted GRAS status (1996
ALTA™ 2341
Quest International, US
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
LACTICIN 3147
fermentate
Lacticin 3147: Lc. lactis
DPC3147 fermentate
De-mineralized whey spray
dried to produce a
bioactive lacticin 3147
powder
Effective in inhibiting L.
monocytogenes Scott A
and Bacillus cereus in
natural yoghurt, cottage
cheese and soups

21. 3)Use of a bacteriocin-producing culture to replace the starter culture in fermented foods
to produce the bacteriocin in situ
The use of cultures to produce bacteriocins in situ
A more natural method of shelf-life extension and improving the safety of foods
BS-10®
Nisin producing L. lactis spp. Lactis, Chr. Hansen
BIOPROFIT™
L. rhamnosus LC705, BioGaia
BOVAMINE Meat CulturesTM
Texas Tech University
HOLDBAC™
L. plantarum, L. rhamnosus, L. sakei, L. paracasei and Propionibacterium
freundenreichii ssp. shermanii, DANISCO

22. Development of resistance against
antimicrobial
Natural degradation over time
Complex interaction with food matrix
Consumption of whole of the
antimicrobial in killing of target
microbes
(Balasubramanian et al., 2011)
Drawbacks of instant addition of antimicrobial
Antimicrobial becomes ineffective due to:-

23. Antimicrobial
packaging
(AMP)
Microencapsulation
Nanoencapsulation
Hurdle
technology
1 2
3 4
Contd…….
Newly emerged techniques for incorporation of bacteriocin preparations in
foods

24. 1) ANTIMICROBIAL PACKAGING (AMP)
“Anti Microbial Packaging (AMP) is the packaging system that is able to kill or inhibit
spoilage and pathogenic organism that are contaminating foods”
AMP
Bacteriocins of
LAB
Pre-existing
Packaging
concept
Antimicrobial packaging film prevents microbial
growth on food surface by direct contact of the
package with the surface of foods
The classical protective function of packaging is
supported by the antimicrobial action of relevant
bacteriocins

25. AMP
Nisin is a highly surface-active molecule that can bind to
different compounds, such as fatty acids of phospholipids
It a suitable feature for adsorption to solid surfaces and killing
bacterial cells that subsequently adhere
silica gel, gelatine, collagen casings, cast protein films; polymer film
coatings, calcium alginate, methyl cellulose films, corn zein, wheat
gluten films
Delivery systems used for immobilization of bacteriocins
Duraisamy et al., 2015

26. 2) Microencapsulation
“It is a technology whereby the target molecule is packaged in miniature, sealed capsules
to protect it from external factors and deliver in the targeted site under specific
conditions. ”
Minimizes bacteriocin resistance development and helps
to achieve controlled release of bacteriocins

27. Food-grade polymers like alginate,
chitosan, carboxy methyl cellulose
(CMC), carrageenan, gelatin and
pectin are mostly applied, using
various microencapsulation
technologies
2) Microencapsulation

28. 3) Nanoencapsulation
Bacteriocins are very small molecules to be efficiently retained in porous microcapsules
which may lead to leakage or inefficient delivery at the target site.
Hence many times primarily encapsulation of bacteriocins in nanosized liposomes is
popularly followed.
Nanoliposomes provide more
surface area
Improve the distribution in the
food system along with the
bioavailability of the
encapsulated bacteriocins
Added directly to the food
items
Malheiros et al. 2012

29. 4) Bacteriocins as part of Hurdle Technology
“ Use of hurdles of differing levels of intensity to bring
microbiological growth under control”

30. Bacteriocins as part of Hurdle Technology

31. Bioengineered Nisin
Deferred antagonism agar diffusion assay
highlighting the enhanced bioactivity of a
Nisin K22T producer, relative to a Nisin A
producer, against S. agalactiae AT CC13813
(colony size = 10 mm)
Cotter et al., 2012
The enhanced strain was found to produce a nisin that possessed a lysine to
threonine change at position 22 (K22T) of the nisin peptide,

32. Bioengineered Nisin
Cotter et al., 2012
Nisin T was enhanced against S. agalactiae, Streptococus mutans, Clostridium
difficile, several S. aureus strains, L. lactis and a variety of mycobacteria
Nisin V was enhanced against this same selection but differed by virtue of also
exhibiting enhanced activity against Listeria monocytogenes, Enterococcus faecium
and Bacillus cereus
Some other examples of bioengineered nisin prepared with food grade approach

33. MOVING TOWARDS BETTER TOMORROW
Cotter et al., 2012
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

34. CONCLUSIONS
Bacteriocins of LAB and bacteriocin-producing cultures are attractive options in food
safety & biopreservation
Further research into the synergistic reactions of these compounds and other natural
preservatives, in combination with advanced technologies could result in replacement
of chemical preservatives.
Application of bacteriocins could allow less severe processing treatments, while still
maintaining adequate microbiological safety and quality in foods

35. PROVIDING SAFE FOOD IS
NOW NOT A MATTER OF
CHOICE, IT IS A NECESSITY !!

36. Winter School, NDRI, 2015
THANK Y
U