Article pp.327-334 du Vol.28 n°4-5 (2008)

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FOCUS : JSMTV

Food biopreservation strategies

P. Garry

¹

*, S. Christieans

²

, P. Cartier

³

RÉSUMÉ

Les procédés de biopréservation alimentaire

La biopréservation est une méthode « naturelle » de conservation des aliments, destinée à contrôler la croissance des germes pathogènes et/ou bactéries d’alté- ration tout en préservant les qualités sensorielles et nutritionnelles. Il existe diffé- rentes techniques de biopréservation, comme l’utilisation de bactéries lactiques ou de métabolites bactériens (acide lactique, peroxyde d’hydrogène ou encore des bactériocines...). Des systèmes enzymatiques naturels tels que le système lactoperoxydase peuvent également être utilisés en biopréservation. Des études récentes ont par ailleurs montré l’intérêt de l’utilisation de bactériophages pour la maîtrise de pathogènes tels que Listeria monoytogenes.

Le recours à la biopréservation peut également permettre de réduire l’intensité de certains traitements thermiques appliqués aux denrées. Enfin, les différentes voies de biopréservation peuvent être associées pour agir en synergie (théorie des barrières).

Mots clés

Biopréservation, bactéries lactiques, bactériocines, bactéries pathogènes, flore d'altération.

SUMMARY

Food biopreservation is a method of conservation, destinated to control the growth of pathogenic and/or spoilage bacteria in food and also to preserve sensory and nutritional qualities of food. There are different ways of biopreservation:

microorganisms as lactic acid bacteria, bacterial metabolites (lactic acid, hydrogen peroxide and bacteriocins...), natural enzyme systems such as lactoperoxidase, molecules derived from plants such as essential oils and bacteriophages.

The biopreservation can also reduce the intensity of heat treatments or to make the products ready to eat safe. It is also possible to associate several of these techniques of biopreservation which will act in synergy (hurdles theory).

Keywords

biopreservation, lactic acid bacteria, bacteriocin, pathogenic bacteria, spoilage bacteria.

1. IFIP Ð Institut du porc Ð 7, avenue du GŽnŽral-de-Gaulle Ð 94700 Maisons-Alfort Ð France.

2. ADIV Ð ZAC Parc Industriel des Gravanches Ð 10, rue Jacqueline-Auriol Ð 63039 Clermont-Ferrand Ð France.

3. Institut de lÕŽlevage Ð Route dÕƒpinay Ð 14310 Villers-Bocage Ð France.

* Correspondence: pascal.garry@iÞp.asso.fr

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1 – INTRODUCTION

Biopreservation is a method of preserving food using natural molecules or microorganisms. In food industry, the biopreservation can be used to minimize the addi- tion of chemical additives such nitrite sodium chloride… Since 1990, many scientific papers describe different strategies for food preservation.

The biopreservation, is an emergent technology of conservation, designated to control the growth of pathogenic and/or spoilage bacteria and also to preserve sensory and nutritional qualities of food.

There are different ways of biopreservation. So it is possible to use:

– Microorganisms as lactic acid bacteria (LAB). These flora play an essential role in the majority of food fermentations, and a wide variety of strains are routinely used as starter cultures in dairy, meat, vegetable and bakery products.

– Bacterial metabolites such as lactic acid, hydrogen peroxide and bacteriocins.

The bacteriocins form a heterogeneous group of anti-bacterial proteins that vary in spectrum of activity, mode of action, molecular weight, genetic origin and biochemical properties. Only the nisin is currently authorized.

– Natural enzyme systems such as lactoperoxidase.

– Molecules derived from plants such as essential oils.

– Bacteriophages.

2 – LACTIC ACID BACTERIA

Lactic acid bacteria are commonly found in meat and it’s a predominant flora in vacuum- or modified-atmosphere packaging for meat and meat products. Some lactic acid bacteria species can spoil the meat product (gas production, tastes and sour smells, acidification…), while others species do not affect the sensory quality of the product even though they are present in high numbers. This last group can be selected and studied as bioprotective culture to enhance the microbial safety of meat.

The selected bacteria for biopreservation must satisfy the four following criteria:

they must be isolated preferably from a similar product, grow at low temperature, not modify the sensory quality of the product and produce antagonistic molecules active against undesirable microflora. Among the species able to be used as bioprotective cultures, three are predominant in meat and meat products: Lactococcus, Lactobacillus and Pediococcus.

The antimicrobial activity of lactic acid bacteria is mainly due to their properties of:

– Production of organic acids such as lactic acid and acetic acid;

– Production of bacteriocins such as nisin, pediocin…;

– Competition for nutrients;

– Carbon dioxid production (CO₂);

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– Hydrogen peroxide production (H₂O₂);

– pH reduction.

Many tests were carried out in meat products (cooked or raw meat). Two exam- ple are illustrated in the two figures bellow.

0 1 2 3 4 5 6 7 8 9

0 5 10 15 20 25 30 35

Time (d)

Log (CFU/g)

Figure 1

Impact of Lactobacillus sakei (LAB) on the growth at 7°C of Listeria monocytogenes (Lm) in cook meat product. ( ) Growth of Lm in presence of LAB, ( ) Growth of Lm

in absence of LAB, ( ) growth of LAB according to Vermeiren et al, 2006.

(CFU: Colony Forming Unit; d: day).

- 1 - 0,5 0 0,5 1 1,5 2

0 1 2 3 4 5 6 7 8

Time (d)

log (CFU/g)

Figure 2

Impact of Lactobacillus sakei (LAB) on the growth of Listeria monocytogenes at 4°C in raw meat. Growth of Lm in absence of LAB ( ), in presence of LAB inoculated

at 10⁴ CFU/g ( ), in presence of LAB inoculated at 10⁸ CFU/g ( ) (ADIV TrueFood program, 2008). (CFU: Colony Forming Unit; d: day).

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To be able to use these bacteria as bioprotective cultures, it is necessary to show their innocuousness for consumers. For these cultures, the European authori- ties defined the QPS concept (Qualified Presumption of Safety). This status is the equivalent of GRAS (Generally Recognised As Safe) status in the United States with some European specificities.

Micro-organisms which cannot follow the “QPS approach” have to be subjected to a complete evaluation to ensure their safety.

The QPS status determine according to the taxonomic group of the strain, knowledge about the taxonomic group in term of safety, existence of pathogens in the taxonomic group and the final use of the product.

3 – POSITIVE BIOFILMS

The lactic bacteria can be used to colonize surfaces in contact with food and to prevent the pathogenic flora implementation. This approach called “positive biofilms” is known for ten years (Leriche et al. 2000; Briandet 1999).

The French project « UNIR » proved that a positive biofilm was able to reduce until 95% of the Listeria monocytogenes contamination on inert surfaces (Briandet, 1999). As reported by Moreau (2008) positive biofilms composed of Lactococcus lactis (starter cultures classically used in milk industry), strongly inhibit the Listeria monocytogenes adhesion. Also Leriche and Carpentier (2000) showed that a Staphylococcus sciuri biofilm reduced the Listeria monocytogenes adhesion on stainless surfaces (figure 3).

However, before using “positive biofilm” in food industry, it is necessary to prove a good colonization of the selected strains.

2 2,5 3 3,5 4 4,5 5 5,5 6 6,5 7

(a) (b)

Log (CFU/cm²)

Figure 3

Percentage of Listeria monocytogenes adhering to stainless stell in a single culture (a) or in a presence of a biofilm of Staphylococcus sciuri (b) Adherent cells of L.

monocytogenes were harvested in exponential ( ) or stationary growth phase ( ).

Bars represent 95% confidence intervals (one experiment performed in duplicate) according to Leriche and Carpentier 2000. (CFU: Colony Forming Unit).

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4 – BACTERIOCIN

Bacteriocins are bacterial peptides having a bactericidal activity. They are produced by various lactic acid bacteria including Lactococci, Lactobacilli and Pedio- cocci. They can be used to control pathogenic or/and spoilage bacteria according to their spectrum.

According Galvez et al. (2007), using bacteriocin does not lead to cross-resist- ance to antibiotics. The bacteriocins are generally recognized as safe substances, because they are specific for prokaryotic cells, not active and nontoxic on eukaryo- tic cells, and become inactivated by digestive proteases (Galvez et al., 2007).

The bacteriocins are obtained by producing more or less purified strains in fer- menters. They can be then incorporated in the product as additive. The use of bacteriocin for the biopreservation can also be done by a direct inoculation of bacteria which produce the bacteriocin into the food. In this case the synthesis and the quantity of bacteriocin present in the product as well as the desired effects are difficult to control.

The nisine produced by Lactococcus lactis is the greatest studied. This peptide obtained the GRAS status (Generally Recognized As Safe) in the United States. In European Union, by the directive 95/2/CE relating on food additives other than col- ours and sweeteners, the nisine (E234) is authorized as preservative in cheeses and cheese spreads, in puddings, and in some other dairy products.

5 – PHAGES

Bacteriophages are viruses that colonize the bacteria. Each phage is specific of bacterial specie particularly the specific phage of Listeria monocytogenes. When a virus infects a cell, the phage uses the cellular machinery of the bacterium for its own multiplication and thus leads to the bacteria lyses. These two properties (spe- cificity of the host and lysis of the cell) are used to eliminate specifically undesirable bacteria in food matrices.

For instance, Bigwood et al. (2008) shows the system effectiveness on Salmo- nella and Campylobacter in meat products preserved at 5°C (figure 4).

Figure 4

Evolution of Salmonella (A) and Campylobacter (B) in fresh meat beef in presence ( ) or absence ( ) of phages (according to Bigwood et al. 2008).

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The FDA and the American department of agriculture authorized the use of the LISTEX^TM P100, a product containing natural bacteriophages against Listeria in food.

This product is considered as a GRAS substance. In France the requests for use of this product are under investigation.

6 – PLANT EXTRACTS

Antimicrobials like thymol, carvacrol and eugenol respectively extracted from thyme and Clove showed their interest for the conservation of the food (Nazer et al., 2005, Guillier et al., 2007). Principal antimicrobial compounds are presented in table 1.

Some of these compounds have also been tested for the surface disinfection (Dubois-Brissonnet et al., 2008). These authors show that these molecules used between 5 to 10 mM would reduce the surface microbial population of 5 decimal in 10 min.

Table 1

Principal antimicrobial compounds present in spices according to Fine and Gervais (2007).

7 – LACTOPEROXIDASE

The lactoperoxidase is the most abundant enzyme in milk. In the presence of thiocyanate ion (SNC-) and hydrogen peroxide (H₂O₂₎), this enzyme can produce hypothiocyanate ion (OSNC-) which oxidative function acts as a good antimicrobial.

In biopreservation, this enzyme is often associated with glucose oxidase to provide hydrogen peroxide to lactoperoxidase (figure 5).

Spice Antimicrobic compounds

Garlic Allicin

Mustard Allyl Isothiocyanate

Anise Anethole

Paprika Capsicidine

Chilli, Paprika Capsaicin

Rosemary Carnosol

Oregano, thyme Carvacrol

Cinnamon Cinnamic aldehyde

Cinnamon, cumin Cuminaldehyde

Clove Eugenol

Ginger, thyme Geraniol

Mint Compounds derived from p-menthan

Oregano, thyme Thymol

Rosemary Ursolic acid

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Glucose + O₂ + H₂O

→

glucono-1,4-lactone + H₂O₂ (1) SCN- + H₂O₂

→

^{OSCN- + H}²^{O (2)}

Figure 5

Actions of Glucose Oxidase (1) and Lactoperoxidase (2).

If the lactoperoxidase effectiveness could be shown on various foods like dairy products or salmon, to date, only one application on salads received a favorable opinion of AFSSA (French Agency for Food Safety).

8 – CONCLUSION

The use of biopreservation techniques seems to be a good alternative to “chemical” molecules and is a way to answer consumer’s expectations for “more natural”

products. The biopreservation can also reduce the intensity of heat treatments and make the products ready to eat safe. A professional who intends to use a biopreservation method to develop new products or to make safe sensitive products should check the effectiveness and the impact on organoleptic qualities by challenge-tests or pilot manufacturing. It is also possible to associate several of these techniques of biopreservation which will act in synergy (hurdles theory). Also tests were carried out to test the joint action of nisine and phages against Listeria monocytogenes in beef (Dykes and Moorhead, 2002).

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