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Background and Objectives:
Increased consumer demand for
all natural food products has put pressure on industry and regulatory
agencies to closely examine the potential for use of natural
antimicrobials that prevent or control the growth of foodborne pathogens
and spoilage microorganisms.
Considerable interest has arisen
in the use of lactoferrin (LF) for the surface decontamination of beef
carcasses and subsequently its possible use as a natural food
preservative. Lactoferrin 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. In addition, LF has antioxidant, antiviral,
anti-inflammatory, immune-modulating, anti-cancer effects and can
promote the growth of probiotic bacteria like Bifidobacterium.
Although many studies have
indicated that LF has the potential to be used as a natural
antimicrobial preservative in the food industry, the success in simple
broth systems such as peptone or distilled water and buffered phosphate
has not been seen in foods because the antimicrobial activity of LF is
reduced in the presence of divalent cations (calcium and magnesium) at
concentrations between 1-5 mM.
The overall objective of the
presently reported study was to overcome the inhibitory effects of
cations on the antibacterial activity of LF by using a smart delivery
system in food for LF by its microencapsulation. Two types of
microcapsules were developed, each based on emulsion technology and
these were tested first in broth and then in cured meats against a
spoilage bacterium (Carnobacterium viridans) and a mixture of
pathogens (2-5 strains of E.coli 0157:H7). Tests were conducted
in vacuum packaged bologna against the spoilage organisms and in dry
fermented sausages against E.coli 0157:H7. Work was undertaken
to improve the shelf-life of the inoculated bologna and to improve the
safety of dry fermented sausages (known to be periodically contaminated
with the E.coli pathogen) using microencapsulated LF.
Procedure and Project
Activities:
A primary emulsion was made by
adding LF with chelators (to complex cations) to a mixture of corn oil
and butter fat designed to melt and release LF when temperatures
exceeded 5.5ºC (temperature abuse). This water-in-oil (w/o) emulsion
effectively retained LF until temperature abuse occurred. These
microcapsules were paste-like in consistency and difficult to distribute
as an ingredient in a mixed ingredient food, but were easily
incorporated in an antimicrobial film made from whey protein isolate.
This was done and the antimicrobial effects of the LF containing film
were examined on inoculated bologna following vacuum packaging. The
test organism was Carnobacterium viridans and treated meat was
stored at 4 and 10°C for 28 days.
A secondary or multiple emulsion
of LF was made by re-emulsifying the primary w/o emulsion in a solution
of whey protein isolate to yield a water-in-oil-in-water or w/o/w
emulsion which was then freeze-dried to form a powder. The action of
freezing destroyed the temperature sensitive release of LF, however,
this was less important than maintenance of LF antimicrobial activity.
The purpose here was to use the powder as an ingredient in dry sausages
to reduce viability of E.coli 0157:H7 in these uncooked,
fermented products. Processing of the sausages involves exposure to
fermentation temperatures of 24-26°C for 3 days with drying at 13°C for
28 days. Products are shelf stable at room temperature for
≤ 6 months.
There is a regulatory requirement that E.coli 0157:H7 must be
absent following processing, and the criterion used is a 5 log colony
forming unit/g (cfu/g) reduction in the number of E.coli 0157:H7
present below their initial numbers.
Results and Discussion:
In broth studies it was found
that Carnobacterium viridans was sensitive to LF but salt (NaCl)
at 2.5% reduced LF effectiveness. In contrast, NaCl increased the
antimicrobial activity of LF against E.coli 0157:H7. When LF was
incorporated into an antimicrobial whey protein isolate film and applied
to the bologna surface, growth of C.viridans was delayed at both
4 and 10°C and microencapsulated LF had greater antimicrobial activity
than when unencapsulated. Bologna shelf-life was extended by LF
addition.
LF was not found to have
significant inhibitory activity against meat starter cultures and
therefore was suitable to use in fermented sausages.
Not all of the E.coli
0157:H7 strains tested were sensitive to LF, and inhibition involved
both injury and lethal effects. From among the 5 tested strains, two of
the more sensitive strains were selected for testing during dry
fermented sausage manufacture. When unencapsulated LF and LF
microencapsulated in primary and multiple emulsions were tested it was
found that the two strain mixture of E.coli 0157:H7 was reduced
≤ 4
log cfu/g by the antimicrobials. Unencapsulated LF was slightly more
effective than when microencapsulated, however, the amount of LF used in
treatments containing microcapsules was between 2-3 times lower than in
unencapsulated treatments because of technical limitations associated
with formulation. In some treatments there were as many as 25% of cells
injured and capable of recovery. Unfortunately, whether encapsulated or
not, LF was not able to achieve the required 5 log cfu/g reduction in
pathogen viability, even though reductions were substantial.
Glanbia Nutritionals (Munroe,
WI) contributed $10,000 (US) and $2,500 in kind (in the form of LF)
toward completion of this project. Graduate student Al-Nabulsi was
supported by a scholarship ($25,000 US) from Jordan University.
Research papers published or
submitted and presentations made at scientific meetings as a result of
work conducted during this project are listed below.
Conclusion:
During this project, work
undertaken examined the development of an antimicrobial mixture
containing the milk protein lactoferrin (LS) plus agents (chelators)
capable of enhancing its activity in foods (bologna, dry fermented
sausages) against both spoilage (Carnobacterium viridans) and
pathogenic (E.coli 0157:H7) bacteria. LF plus chelators were
microencapsulated and applied in a film to bologna surfaces, and
capsules were freeze dried to form a powder used as an antimicrobial
ingredient in sausages. The antimicrobial film was successfully used to
extend the shelf-life of refrigerator-stored bologna and significantly
reduced E.coli 0157:H7 viability in sausages. Although lethal to
C.viridans, LF caused injury in about 25% of sensitive E.coli
0157:H7 cells, but not all strains of E.coli 0157:H7 were
sensitive. While it is unlikely that lactoferrin itself will find
application in dry fermented sausages to control E.coli 0157:H7,
a LF derivative (hydrolysate) lactoferrin (Lfcin), known to be 25 times
more potently lethal has potential for this application and is the
subject of a research proposal submitted to the Natural Sciences and
Engineering Research Council (Canada) for evaluation/funding. The
microencapsulation procedure used is novel and the technology may be
useful for other natural antimicrobials where agent stability and
maintenance of critical concentrations are problematic. It is expected
that these results will lay the foundation for additional work where
natural antimicrobials can be used with or instead of conventional
preservatives to improve the shelf-life and safety of perishable foods.
Acknowledgements:
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),
Glanbia
Nutritionals (Munroe, WI), the
Natural Sciences and Engineering Research Council (Canada) and Jordan
University (Amman, Jordan).
Publications:
Al-Nabulsi, A. A. (2006). Use of
the milk protein lactoferrin as a natural antimicrobial in meat
products. PhD Thesis, University of Manitoba.
Al-Nabulsi, A. A., Han, J. H.,
Liu, Z., Rodrigues-Vieira, E. and Holley, R. A. (2006). Effect of
microencapsulated bovine lactoferrin incorporated in whey protein
isolate packaging film against Carnobacterium viridans in
bologna. J. Food Sci. (Submitted on Feb. 22, 2006).
Al-Nabulsi, A. A. and Holley, R.
A. (2006). Activity of bovine lactoferrin against Escherichia coli
0157:H7 strains and meat starter cultures in broth and during dry
sausage manufacture following its microencapsulation. Int. J. Food
Microbiol (Submitted on Jan. 16, 2006).
Presentations:
Al-Nabulsi, A. A. and Holley, R.
A. (2006). Activity of bovine lactoferrin against Escherichia coli
0157:H7 strains and meat starter cultures in broth and during dry
sausage manufacture following its microencapsulation. 93rd
Annual Meeting of IAFP. Aug. 13-16, Calgary, AB.
Al-Nabulsi, A. A. Han, J. H.,
Rodrigues-Vieira, E. and Holley, R. A. (2006). Effect of
microencapsulated bovine lactoferrin against Carnobacterium viridans
in bologna. Annual Meeting of IFT. Jun. 24-28, Orlando, FL.
Al-Nabulsi, A. A. and Holley, R.
A. (2005). Effect of sodium hexametaphosphate against Carnobacterium
viridans. 92nd Annual Meeting of IAFP. Aug. 14-17,
Baltimore, MD.
Al-Nabulsi, A. A. (2005). Use of
lactoferrin to inhibit the growth of foodborne pathogens and spoilage
bacteria in meat and meat products. Annual meeting of CMSA. (Oral). Feb.
2002, Gatineau, QC.
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