PROJECT RESULTS

Use of Lactoferrin and Lactoferricin to Inhibit Growth of Food Pathogens and Spoilage Microorganisms in Meat

Applicant: 

Dr. Richard A. Holley
Department of Food Science
University of Manitoba
Winnipeg, Manitoba  R3T 2N2  Canada

Table of Contents:

• Background and Objectives
• Procedure and Project Activities
• Results and Discussion
• Conclusions

ARDI Project:

#01-521

Total Approved:

$25,000

Date Approved:

February 7, 2003

Project Status:

Completed December, 2003

Background and Objectives:

The globalization of the world economy has resulted in meat and meat products being shipped over ever increasing distance to reach foreign markets.  The economic impact of foodborne pathogen outbreaks and less than desired shelf life of vacuum packaging and refrigerated products and consumer demand for all natural food products have necessitated the development of effective natural antimicrobial preservation systems for the meat industry.  Lactoferrin (LF) is the main iron-glycoprotein present in the milk of various mammals and it exerts an antimicrobial effect against a wide range of Gram-negative and Gram-positive bacteria, fungi, and parasites.  Although many studies have indicated that LF has the potential to be used as a natural antimicrobial preservative in the food industry, the success found in simple broth systems such as peptone or distilled water and buffered phosphate has not been seen in foods because antimicrobial activity of LF is reduced in the presence of divalent cations (calcium and magnesium).  In addition, LF activity was reported to be reduced at refrigeration temperatures.  Basic research on the effect of NaCl on the activity of LF had not been done and is needed to evaluate the usefulness of LF in cured meat systems.

Therefore, the objectives of this research were:

1. To optimize the ability of LF to inhibit foodborne pathogens and meat spoilage bacteria in broth systems similar to meat.  We chose to study the effect of growth media Lauria broth (LB) or All Purpose Tween broth (APT) which contain low and moderate cation concentrations, respectively, and examined effects of changes in incubation temperature and NaCl concentration on LF activity against Carnobacterium viridans (which causes spoilage of cured meat products) as well as the pathogens E.coli O157:H7 and Listeria monocytogenes.

2. To develop  a smart delivery system that could overcome the  interference with LF activity from cations in meat and meat products by using a microencapsulation system.  Microencapsulation may minimize the inactivation of LF and maximize opportunity for contact with pathogenic and spoilage bacteria at the sites where they most likely are to be found (the meat surface).  A temperature-induced release mechanism of LF from microscopic capsules containing LF and a food grade metal chelating agent (sodium bicarbonate, SB, sodium hexametaphosphate, SHMP, or sodium lactate, SL) at the surface of the meat or cured meat were used. 

3. To evaluate the effectiveness of encapsulated LF in extending shelf life and improving safety of fresh and cured meat products.  Packaging films used for processed meats (e.g. uncured turkey roll, bologna) were coated with microencapsulated LF (plus the chelator), packaged and stored at 4 and 10 °C ≤ 70 d.

Procedure and Project Activities:

In our initial work LF was found to have a lethal (bactericidal) effect against C.viridans at all incubation temperatures (4, 10 and 30 ēC) in both APT and LB broth.  LF effects were found to be dependent on the growth media since 32 mg/ml of LF killed C. viridans at 4 ēC in APT after 2 d and only 8 mg/ml was required in LB.  The increased resistance in APT can be explained by the higher concentrations of divalent cations (Ca⁺² and Mg ⁺²) in this broth.  On the other hand, LF effects became bacteriostatic (inhibitory but non lethal) at all incubation temperatures as the concentration of NaCl increased from 1.5 to 2.5% w/v.  Next, different chelating agents were used in an attempt to improve the activity of LF in the presence of 2.5% NaCl (similar to the concentration used in cured meat products).  SHMP (which is added to cured meat products to enhance emulsion stability) was able to improve the activity of LF in APT broth containing 2.5% NaCl but only at 30 ēC.  In contrast, no effect was observed in LB broth.  This can be explained by the ability of SHMP to chelate the higher levels of calcium and magnesium in APT and allow the destabilization of the cell membrane and improve LF performance.  This activity was temperature dependent.  It was unexpected that SL reduced the effect of LF against C.viridans in LB broth containing 0.5% NaCl.  When used up to 160mM SB also did not improve the activity of LF in both growth media containing 2.5%NaCl even though it is claimed that it can protect LF from divalent cations by enhancing its structural stability.

Results and Discussion:

By itself, LF had no activity against E.coli O157:H7 in either broth medium containing 0.5% NaCl.  In contrast, LF activity was enhanced against E.coli O157:H7 by the presence of 2.5% NaCl in LB medium at 37 ēC and in both LB and APT at 10 ēC.  The addition of SB, SL or SHMP resulted in synergistic interactions with LF in LB.  LF plus SB (160mM) produced a bactericidal effect with a higher lethality level in the presence of 2.5% NaCl, and at the lower incubation temperature only 40mM SB was needed to achieve the same result.  It was found that the reduced bactericidal activity of SL at the higher salt concentration and incubation at the lower temperature was restored by the addition of LF to the reaction mixture.   

Listeria monocytogenes was found to be resistant to LF under most test conditions.  The addition of SB (160 mM) to LF resulted in a bacteriostatic effect at both 37 ēC and 10 ēC in LB containing 2.5% NaCl.  However, after 5d at 10 ēC growth began, which reached 1 log₁₀ CFU/ml by10 d. SL and SHMP did not have either an additive or synergistic effect with LF. SL by itself was as bacteriostatic against this organism as was LF.

Conclusions:

Acknowledgement:

This project was made possible due to funding from the Governments of Manitoba and Canada through the Canada-Manitoba Agri-Food Research and Development Initiative (ARDI).

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