Bacteriocins based strategy for

enhanced food safety

Diwas Pradhan

Dairy Microbiology Division

Oct., 2015

National Dairy Research Institute

Karnal-132001

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

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

Product Diversification

Upgraded Quality

Enhanced Shelf Life

Ready to Eat

Fresh- Tasting

Nutritious and Vitamin Rich

Minimally- Processed

Consumers’ Preference

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

“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

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

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

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

Classification of bacteriocins

Ingolf F. Nes et al., 2015

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

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)

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

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

1

How to add bacteriocins in foods……………

Using a purified/semi-purified bacteriocinpreparation as an additive in food

Incorporation of an ingredient previouslyfermented with a bacteriocin-producing strain

Use of a bacteriocin-producing culture toreplace the starter culture in fermented foodsto produce the bacteriocin in situ

Traditional methods for incorporation of bacteriocin preparations in foods

Prototypical lantibiotic, having been first marketed in England in 1953 andhas since been approved for use in over 50 countries.

Nisin has been assessed to be safe for food use by the Joint Food andAgriculture Organization/World Heath Organization Expert Committee onFood Additives in 1969.

In 1983, this bacteriocin was added to the European food additive list asnumber E234 (indeed it is the only natural antibacterial to have beenapproved for as a food preservative by the EU)

In 1988, it was approved by the US Food and Drug Agency (FDA) for use inpasteurized, processed cheese spreads and is currently used in a widevariety 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

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

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

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

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

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 Propionibacteriumfreundenreichii ssp. shermanii, DANISCO

Development of resistance against antimicrobial

Natural degradation over time

Complex interaction with food matrixConsumption 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:-

Antimicrobial packaging

(AMP)

Microencapsulation

NanoencapsulationHurdle

technology

1 2

3 4

Contd…….

Newly emerged techniques for incorporation of bacteriocin preparations in

foods

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”

AMPBacteriocins of

LAB

Pre-existingPackaging 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

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

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

Food-grade polymers like alginate,

chitosan, carboxy methyl cellulose

(CMC), carrageenan, gelatin and

pectin are mostly applied, using

various microencapsulation

technologies

2) Microencapsulation

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 moresurface areaImprove the distribution in thefood system along with thebioavailability of theencapsulated bacteriocinsAdded directly to the fooditems

Malheiros et al. 2012

4) Bacteriocins as part of Hurdle Technology

“ Use of hurdles of differing levels of intensity to bringmicrobiological growth under control”

Bacteriocins as part of Hurdle Technology

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,

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

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

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

PROVIDING SAFE FOOD IS

NOW NOT A MATTER OF

CHOICE, IT IS A NECESSITY !!

Winter School, NDRI, 2015

THANK Y U