4/18/2017

Biopreservation

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

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Food preservation
Food safety
Organoleptic quality
maintenance of food Reduction of the risk of
product throughout its pathogens in food product
shelf life 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

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Consumer’s Preference

Product Diversification
Upgraded Quality
Enhanced Shelf Life
Ready to Eat
Fresh
Fresh-- Tasting
Nutritious and Vitamin
Rich
Minimally
Minimally-- Processed

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

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Conventional Preservation Modern Preservation

Techniques Techniques

• Manipulation of the • High Hydrostatic

water activity Pressure (HHP)

• Lowering of pH • Pulsed Electrical Fields

(PEF)
• Heat treatment
• High pressure CO2
• Addition of chemical
treatment
preservatives and
• Ultraviolet light and
• Control of storage
temperature of foods • Radiation treatments
• Biopreservation

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

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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
Definition:
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

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Multiple Antimicrobial Effects of Lactic Acid Bacteria

• Acids
• Lactate, acetate, formate
• 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

• Sakacin ( bacteriocins produced by Lactobacillus sakei. They
are often clustered with the other lactic acid bacteriocins)
• ALCMix1 plantaricin Danisco
• Carnocin (Carnobacterium sp., inhibited lactic acid bacteria,
clostridia, enterococci, and some Staphylococcus aureus
strains)
• ALTA 2351 pediocin Quest
• Micocin carnocyclin Griffith Laboratories
• Piscicolin
• Carnobacteriocin

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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

Natural Food Safety in the DAIRY WORLD

Food Protection Solutions in Dairy

Applications
MicroGARD™ NATAMAX® NISAPLIN® HOLDBAC™
Fermentates Natural Antimicrobial Natural Antimicrobial Protective Cultures
Gram-neg.& Gram-pos. spoilage Yeasts & Moulds
Yeasts & Moulds Yeasts & Moulds Gram-positive Bacteria Listeria, Heterofermentative Lactobacilli

MicroGARD™ MicroGARD™ NATAMAX® NISAPLIN® HOLDBAC™ HOLDBAC™

100, 300, 400 200, 510 Standard, Salt , SF YM-B, YM-C Listeria, LC
Fermentation Metabolites CMP
Fermentation Metabolites CSP Natamycin Nisin Lactic bacteria & Propionibacteria Lactic bacteria

100 200 Salt & SF NISAPLIN® YM-B Listeria

Cottage Cheese Cottage Cheese Shredded cheese Cheese Yoghurt, Fresh Cheese Soft, blue, semi-hard cheeses
Yoghurt Yoghurt Cheese Coating Neutral Dairy Products Sour cream
Sour Cream Sour Cream Direct acidified MP Cottage Cheese etc.

300 510 NATAMAX® NISAPLIN® Milk Free YM-C LC

Flavoured Milks Dairy Spreads additionally All applications Yoghurt, Fresh Cheese Salt brine
Natural/processed Flavoured Milks Yoghurt, Sour Cream Sour Cream Semi-hard/White Cheeses
Cheese Cottage Cheese etc. Cottage cheese etc.

400
Dairy Spreads, Cheese Fillings

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HACCP: Hazard Analysis and Critical Control Points (Pendekatan rasional bahaya produk)
QMS: Quality Management System (Jaminan kesesuaian proses atau produk ie: ISO dll)
GMP: Good Manufacturing Practices (syarat minimum keamanan pangan)

HACCP-
HACCP-
Concept

QMS GMP

• Inhibition of food
pathogenic micro-
micro-
organisms
• growth control
• prevention of toxin-
formation
Food • reduction of pathogens
Micro-organisms
• final products
„Protective
• pre-stages and/or
Cultures““
Cultures
• or raw material
• Shelf life
extension based on
targeted inhibition of
specific spoilage micro-
organisms

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BIOPRESERVATION WITH FERMENTATION END

PRODUCTS OF LAB

LOW MOLECULAR
ORGANIC ACIDS WEIGHT COMPOUNDS
Lactic acid Reuterin
Diacetyl
Propionic acid
H2 O2
Acetic acid Fatty acids
Cyclic dipeptides
Phenyl lactic acids

BACTERIOCINS
(Antimicrobial Peptides)

Protection of food from spoilage

and pathogenic microorganisms by LAB

 Producing organic acids

 Hydrogen peroxide
 Diacetyl
 Antifungal compounds such as fatty
acids or phenullactic acid
 Bacteriocins

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Organic acids

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

Undissociated 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

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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

What makes them anti-microbial?

• Organic acids
• lactic acid
Anti-microbial metabolites • acetic acid
• propionic acid…

• Hydrogen peroxide

• Carbon dioxide

• Lacto peroxide

• Fatty acids

• Diacetyl

• Acetaldehyde

• Reuterin
• Other compounds of
low molecular mass

• Bacteriocins

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- more than anti-microbial metabolites!

anti-microbial
metabolites

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

= Inhibition of food pathogenic

and food spoilage micro-organisms

s - 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“

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Protective Cultures - categories

Starter Cultures STARTER fermentation

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

Multi-functional
Cultures

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

Protective Cultures - categories

Starter Cultures STARTER fermentation

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

Multi-functional
Cultures

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

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Protective Cultures - categories

Starter Cultures STARTER fermentation

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

Multi-functional
Cultures

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

Protective Cultures - categories

Starter Cultures STARTER fermentation

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

Multi-functional
Cultures

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

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Protective cultures - are safe

Protective cultures are:

generally recognised as safe (GRAS)

there is no indication of a health risk of

this bacterial group

• Lactic acid bacteria the use of lactic acid bacteria in biological

Lactobacillus sp. preservation might even contribute to the
Lactococcus sp. health benefits of a product *)
• Propionibacteria
*) Holzapfel et al., 1995, Int. J. Food Microbiol. 78: 119-131

Cultures - 4 steps from lab to customers

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

HOLDBAC™
HOLDBAC ™
in food & feed

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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 / Pack- End of

Preparation Storage
Ripening aging shelf
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

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Hydrogen Peroxide
 Strong oxidising effect on the bacterial cell wall

 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

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Bacteriocins

Bacteriocins are ribosomally synthesized single

polypeptides or posttranslationally 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,
facilitating genetic

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Bacteriocins of LAB
Main Characteristics Subcategory Examples
category
Class I Lantibiotics Type A Nisin
Type B Mersacidin
Class II Nonmodified 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
of 30 kDa B
Class IV Bacteriocins that form Leuconosin S
large complexes with other Lactococcin 27
macromolecules

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SOME USEFUL BACTERIOCINS FOR THE DAIRY

& FOOD INDUSTRY

Genus of Bacteria Bacteriocin Produced

Lactococcus lactis subsp. Nisin A,Z
lactis Pediocin PA-1, AcH
Pediococcus acidilactici
Pediococcus peantosaceus Pediocin 34
Leuconostoc spp. Leucocins
Lactabocillus sake Sakacin A
Lactabocillus plantarum Plantaricin
Lactabocillus helveticus Helveticin J
Carnabacterium 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
to traditional chemical
FACTORS PROMOTING preservatives and
USE concern over
Effective under OF BACTERIOCINS 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

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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:
are:
 Nisin produced by Lactoccocus lactis ssp
ssp.. lactis
 Pediocin PA-
PA-1, produced by Pediococcus acidilactici

۩ The use of purified bacteriocins have to be labeled

as additives and require regulatory approval.
approval.

Nisin is the only bacteriocin,

bacteriocin, approved for use as an
antimicrobial in food by the Joint FAO/WHO Expert
Committee on Food Additives

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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
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.

Nisin – E234
Nisaplin (Danisco)

GRAS status
1969 WHO and FDA
1983 EU
1988 US Food and Drug Agency

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•Used in dairy products, cheese, mayonnaise, canned

vegetables for shelf life extension.
•However, at present, only nisin and pediocin PA1/AcH
have found widespread use in food.
•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).

Nisin bactericidal mechanism

Nisin ihibits synthesis of peptidoglycan:
interaction between lipid I and lipid II
↓
pore forming
↓
Inhibition of cell wall synthesis

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•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.

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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.

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

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Nisin and type A lantibiotics kill bacteria by lipid II-targeted pore formation and
permeabilization of the membrane.

• 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 trans
membrane 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

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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
world wide interest

 In 1969 a joint FAO/WHO expert committee on food

additives recognised nisin as a safe and legal food additive

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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

Use of Nisin in the Biopreservation of Dairy Products

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

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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-
60-70 100-
100-150

Nisin in Stirred Yoghurt

• Addition of 50 IU nisin/g to yoghurt after

preparation gave an acceptable product with
increased shelf
shelf--life upto 10 days at
refrigeration temperature

• Prevents subsequent over-

over-acidification of the
yogurt.

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Effect of Nisin on the Shelf Life of Lassi

 The shelf life of lassi containing 100 to 200

IU nisin
nisin/ml
/ml increased by two folds and the
product was acceptable up to 24 hrs at room
temperature

 Lassi containing 50 IU nisin

nisin/ml
/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 1515--30 days as compared 7-15 days
shelf life of control at refrigeration temperature
(Khurana, 2006)

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Nisin: mechanism of activity

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

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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 cannot be used to hide poor manufacturing practice

 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

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
Gram-positive bacteria, including Listeria, Clostridium, Bacillus

Mode of action
Either a killing or growth inhibitory activity against vegetative
cells by targeting the cytoplasmic membrane, or prevention of the
outgrowth of heat-resistant spores.

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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

GUARDIAN™

 Solutions based on the synergy between nisin and rosemary

extract

 Kill Gram-positive bacteria and control their growth

 Rosemary extract also delays the oxidative rancidity of fats

and preserves the freshness of the foodstuff

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NovaGARD™ Combines 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 250 NovaGARD™ CB1

™ NR 100

Functionality Against Against Gram-positive Against Gram-positive

Gram-positive bacteria bacteria
bacteria Listeria inhibition Growth delay of
Growth Growth delay of Listeria, Clostridium,
inhibition of spoilage such as lactic Bacillus,
Bacillus bacteria heterofermentative
Growth delay Delay of oxidative lactic bacteria
of Listeria rancidity
Delay of
oxidative
rancidity
Application Culinary Heat-processed meat Deli salads (chicken,
products products such as tuna, seafood,
such as cooked sausages (hot coleslaw, egg),
pasteurised dogs, Frankfurters) and mashed potatoes,
soups & cooked hams meat broth soups,
sauces, low sauces, salad
pH sauces & dressings, RTE meals
marinades,
RTE foods

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Class II
On the basis of structure the class II
bacteriocins are subdivided into four
subclasses:
• Class IIa or pediocin-like bacteriocins
• Class IIb or two-peptides bacteriocins
• Class IIc
• Class IId

Class II
Class II bacteriocins are:
• 20-60 amino acids
• Post-translationally not modified
• Cationic
• Hydrophobic
• Heat-stable

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Class II bacteriocins

Class IIa Class IIb Class IIc Class IId

Bavaricin A - Lactobacillus sake Acidocin J1132 - L. acidophilus JCM Divergicin A - Cb divergens Plantaricin 1,25β - L.
MI401 1132 LV13 plantarum TMW1.25
Bavaricin MN - L. sake MN Lactacin F - L. johnsonii 11088 Lactococcin A - Lc. lactis
Carnobacteriocin B2 - Lacticin 3147 - Lc. lactis DPC3147 subsp. cremoris LMG
Carnobacterium pisciocola Lactobin A - L. amylovorus LMG P- 2130
LV17B 13139 Lactococcin 972 - Lc. lactis
Carnobacteriocin BM1 - Cb. Lactococcin G - Lc. Lactis IPLA972
piscicola LV17B LMG2081 Plantaricin A - L. plantarum
Curvacin A - L. curvatus LTH1174 Plantaricin EF - L. plantarum C11 C11
Divercin V41 - Cb. divergens V41 Plantaricin JK - L. plantarum C11
Enterocin A - Enterococcus faecium Plantaricin S - L. plantarum
CTC492/T136 LPCO10
Enterocin P - E. faecium P13 Thermophilin T - Streptococcus
Leucocin A/B-Talla - Leuconostoc thermophilus ACA-DC 0040
gelidum UAL187; Ln.
Carnosum Ta11a
Mesentericin Y105 - Ln.
mesenteroides Y105
Mundticin - E. mundtii AT06
Pediocin PA-1/AcH/SJ-1 -
Pediococcus parvulus
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. plantarum 423

PEDIOCINS
POTENTIAL BACTERIOCINS AS BIOPRESERVATIVES

Most pediocins are:
are:
Thermostable proteins
Active over a wide range of pH

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

Pediocin PA-
PA-1:
 Inhibits Listeria in dairy products such as cottage cheese,
ice cream, and reconstituted dry milk

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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
↓
by Decreasing
intracellular ATP
concentration leads to cell
death

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PA-1
Pediocin PA-1 also called pediocin AcH
ALTA 2431 (Quest)
• Broad inhibitory spectrum
• Most pediocin PA-1 producing bacteria are
pediococci
• Production of pediocin is assosiated with plasmid
pSRQ11
– Production by alternative host (dairy starter culture L. lactis
MM210 for cheddar cheese manufacture)

Class IIb
• Consist of two peptides
• For optimal activity both peptides are required
• Primary structure of peptides are very different
– could be subdivided into type E (enhanced) and
type S (synergistic) peptides

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Mechanism of action
• Formation of pores in cell wall

• The leakage of ions and/or a decrease in

intracellular ATP concentration

Mechanism of action
• Formation of pores in cell wall

• The leakage of ions and/or a decrease in

intracellular ATP concentration

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• 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.

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Class IIc
Bacteriocins are grouped on the basis of
their cyclic structure
N- and C-termini are covalently linked

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.

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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
• Function through the lysis of sensitive cells by
catalysing cell wall hydrolysis
• These proteins have a catalytic domain at the N-
terminus
• C-terminus probably represents the target recognition
site
• Do not always have specific immunity genes
• Might rely on modifications of the producer cell wall

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• Their mechanism of action is distinct from that of

bacteriocins as they function through the lysis of
sensitive cells by catalysing cell-wall hydrolysis .

• These proteins are also modular in structure and have a

catalytic domain atthe N-terminus that shows homology
to endopeptidases,and a C-terminus that probably
represents the target recognition site 62,63.

• 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 .

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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 control Specificity Application
of

Cheese, fresh dairy, bakery

Yeasts &
Natamax® Standard products, processed meat and
moulds
beverages

Shredded cheese, PVA cheese

Yeasts & No addition
Natamax® Salt coating
moulds of sugar
Dry sausages surface treatment

Yeasts & No addition

Natamax® G Beverages and wines
moulds of salt

Yeasts & Spraying, shredded and blocked

Natamax ® SF No carrier
moulds cheese, bakery products

Improved
Yeasts & Cheese surface treatment
Natamax® Gel surface
moulds Dry sausages surface treatment
adhesion

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MicroGARDTM
 MicroGARDTM (DANISCO, Denmark) is commercially
produced from grade A skim milk fermented by a strain of
Propionibacterium freudenreichii 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
• Processed meat products
• Chilled, pasteurised ready-to-eat meals
• Soups

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MG 300
MG 100 MG 200 MG CS1-50
MG CM1-50 MG 400

Gram- Gram- Gram-positive

Functionality negative negative bacteria
Gram-positive Gram-positive
: Growth bacteria bacteria Gram-negative
bacteria bacteria
control of Yeasts & Yeasts & bacteria
moulds moulds Yeasts & moulds

Cultured Cultured
Cultured Cultured grade A Cultured
Specificity grade A skim grade A skim
dextrose skim milk blend dextrose
milk milk

Grade A &
fermented
Grade A & milk, Cheeses, soups
Grade A & Cheeses, soups
fermented cheeses, dips & sauces,
fermented milk, & sauces,
milk, cheeses, & spreads, refrigerated
Application cheeses, salad refrigerated
salads, salad meats and
dressings, soups & meats and
dressings and dressings, processed
sauces and meats processed meats
soups soups & 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)

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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

LACTICIN 3147

Lacticin 3147 produced by Lc. Lc. lactis
DPC3147
DPC 3147 ferment reconstituted de
de--
mineralized whey (10%
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

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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
bio--preservation

 A more natural method of shelf- shelf-life

extension and improving the safety

Bacteriocin Based Bioprotective Cultures

• BS
BS--10
10®®
Nisin producing L. lactis spp
spp.. Lactis, Chris Hansen

• BIOPROFIT™
L. rhamnosus LC
LC705
705,, BioGaia

• BOVAMINE Meat CulturesTM

Texas Tech University

• HOLDBAC™
L. plantarum
plantarum,, L. rhamnosus
rhamnosus,, L. sakei
sakei,, L. paracasei and
Propionibacterium freundenreichii spp spp.. Shermanii,
DANISCO

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Basic Principle of Hurdle Technology

Hurdle technology in antimicrobial packaging system compared

to the conventional packaging system

BACTERIOCINS

Application of Bacteriocins as Part of Hurdle Technology

(Galvez et al., 2007)

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Sublethal Heat Injury and Bacteriocin

 Integrating bacteriocins of LAB with sub-lethal

stresses can be advantageous for greater control of
spoilage and pathogenic bacteria in food

 Heating of bacterial cultures at sub-lethal

temperatures (50-60oC/5-15 min) followed by treatment
with bacteriocin

Antimicrobial Packaging (AMP) Using

Bacteriocins

Incorporation of bacteriocins into packaging

films to control food spoilage and pathogenic
organisms

Antimicrobial packaging film prevents

microbial growth on food surface by direct
contact of the package with the surface of
foods

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Pre-Clinical Safety Evaluation of Bio-

preservative
• Acute toxicity Test
• Sub Chronic toxicity test
• Allergenicity Test
– The biochemical, hematological and allergenic
profiles normal.
– No abnormal changes in gross necropsy and
histopathology of major organs .
– No Genotoxicity potential

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Fig. 1. Influence of different factors on the efficacy of in situ

bacteriocin production for biopreservation

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

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