BIO-PRESERVATION OF MEAT AND MEAT PRODUCTS

G.SUNDARESANMVM 15026

Credit seminar

Department of Livestock Products Technology (Meat Science), Madras Veterinary College, Chennai – 600 007.

IntroductionMeat 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)

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

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

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

Principles of bio preservation

Hindering the growth activity of the microorganisms.

e.g. pH

Killing the microorganism.

e.g. Bacteriocins

Anti-microbial metabolites

Organic acidsLactic acid Acetic acidPropionic acid

Bacteriocins Class I Class II Class III

Molecular compounds Hydrogen peroxide Carbon dioxide Diacetyl Peptides

Mechanism of action of anti-microbial metabolites

Organic acids:

By lowering pH.

OxidasesHydrogen 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

1. Fermentation

2. Bacteriocin application

3. Bacteriophages

4. Endolysins

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.

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).

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

Effects of culture on the meat & meat products

Fermented sausagesAddition of curing agent and sugar and

fermentation condition preventing the growth of pathogens in fermented sausages (Lucke, 1998 b).

Raw ham and Ready to eatInjection 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 meatStrains 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 meatPediococcus starter grow and form the acids. Thus restricting the growth

of any clostridium botulinum and extend the shelf-life.

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).

Safety issues of fermented meat and meat products

I. Biogenic amines CadaverinePutrescineTyramine

Toxicological symptom

Other symptom

1. Pseudo allergic reaction2. Interaction with drugs

1. Gastric ulcers2. Blood pressure problems3. Nervous symptoms

II. Antibiotic resistance

1. Gene translocation

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 Ribosomal

2 Application Clinical Food

3 Activity Varying spectrum Narrow spectrum

4 Host cell immunity No Yes

5 Mode of actionCell membrane or intracellular targets

Mostly pore formation, but in a few cases possibly cell wall biosynthesis

6Toxicity side effect 

Yes None known

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

FACTORS PROMOTING USE

OF BACTERIOCINS ASBIOPRESERVATIVES

Not alteringNutritionalquality

Effective under wide pH &

temperature range

Activity is not lostPresence 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

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

Class IClass IIClass III

Classification of Bacteriocins

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

Mechanism of action of Class I Bacteriocins

Class II bacteriocins or non Lantibiotics

1. Class IIa or pediocin-like bacteriocins2. Class IIb or two-peptides bacteriocins3. Class IIc4. Class IId

Cationic Hydrophobic Heat-stable Membrane active peptides

Klenhammer, 1993

(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

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

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

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

III. Use of previously fermented meat with bacteriocin producing strains

(Thomas et al., 2000)

Bacteriocin producing strains

Purified bacteriocins

VI. Edible cellulosic films - New

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)

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)

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)

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.

(Nes et al., 2007)

Commercially approved bacteriocinNisin – E234 (Nisaplin)

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.

Bacteriophages will remain dormant unless their specific target, L. monocytogenes is encountered.

(Kathy walker, 2006).

Phage (lytic)

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)

Endolysins mechanism of action

Schematic representation of the modular structure

Mode of action of phage-encoded - 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

s.no MERITS DEMERITS

1 Narrow activity of spectrum

2 Limited diffusion in solid matrix

3 Sometime difficult to apply

4 Prevent the colorectal cancer Consumers are reluctant to use of meat preserved using micro-organisms

5 R  

6  

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

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)

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).

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