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Antimicrobial Effects of Natural Tenderizing Enzymes on Different Strains of Escherichia coli O157:H7
and
Listeria
monocytogenes
on
Beef
LOGO
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Delete me & place your
Hanan Eshamah , Inyee Han , Hesham Naas , James Rieck and Paul Dawson
in this area.
1Food,
Nutrition and Packaging Sciences Department, Clemson University, Clemson, SC, 2Department of Food Hygiene, Faculty of Veterinary Medicine, University of Tripoli, Tripoli, Libya,
3Department of Mathematical Science, Clemson University, Clemson, SC
Abstract
Tenderization of beef meat is widely done for increasing consumer acceptance and enhancing shelf life.
This study was conducted on meat surface to determine the efficacy of proteolytic enzymes (papain and
actinidin), meat tenderizer enzymes, on the survivability of multiple strains of E. coli O157:H7 and L.
monocytogenes. Two overnight cultures of three strains of E. coli O157:H7 and L. monocytogenes were
separately suspended in 0.1% peptone water and were individually inoculated into beef meat surface
(ca. 106 CFU/ml). After 5 minute attachment time different enzyme concentrations were added. Treated
samples were then kept for 3h at 25°C and 35°C and 24h at 5°C. Actinidin concentration (700
mg/ml) tested at 25°C against E. coli O157:H7 and L. monocytogenes was the most effective
concentrations tested, reducing populations by 2.4 and 1.41 log CFU/ml after 3h, respectively. Papain
concentration of 10 mg/ml was the most effective concentrations tested at 25°C against E. coli
O157:H7 and L. monocytogenes reducing populations by 1.8 and 0.9 log CFU/ml after 3h, respectively.
If these proteolytic enzymes are combined with current antimicrobial treatments to form a hurdle effect,
higher pathogen reductions may be achieved. These findings suggest that, in addition to the potential for
improving the sensory attributes of beef meat, tenderization can enhance their safety and shelf life when
stored at suitable temperatures. The findings from this study suggest a promising approach in
developing antimicrobial systems for beef meat.
Keywords: proteolytic enzymes, actinidin, papain, meat tenderizing, Escherichia coli O157:H7, Listeria
monocytogenes, Meat surface
Introduction
Food borne diseases are a major cause of death in developed countries with estimation of 6.5– 33 million
illnesses and up to 900 deaths occurring each year from bacteria, viruses, parasites and fungi (Roberts,
2000). Escherichia coli O157:H7 and Listeria monocytogenes are pathogens that have received special
attention by federal agencies and food safety researchers due to their great economic impact when illnesses
occur. These pathogens are responsible for 3 billion dollars in economic loses each year (USDA, 2006).
Therefore, new alternatives are being studied to control these microorganisms. L. monocytogenes is widely
distributed in the nature. Some studies indicated that 1- 10 % of humans are intestinal carriers of L.
monocytogenes. Its association with meat and slaughter environment is well established (Benkerroum et al.,
2003). Consumption of raw and partially cooked contaminated meat can result Listeriosis, especially
among the immune-compromised populations, elderly and pregnant women (Shrinithivihahshini et al.,
2011).
Materials and Methods
Results
Inoculum preparation: Separate 16h cultures of L. monocytogenes and three strains of E. coli O157:H7 were grown at 37°C in Listeria
broth and tryptic soy broth (TSB) respectively. The cultures were centrifuged at 1107 X g for 15 min then the supernatant was decanted. The
bacteria pellet was resuspended in (0.1%) peptone water and then 1ml was transferred to 99 ml peptone water to reach a count of 5-6 log
CFU/ml.
Effects of papain on L. monocytogenes
Preparation of enzyme concentrations: Concentrations of papain used in this experiment were 0 mg/ml, 5 mg/ml, 8 mg/ml and 10 mg/ml
with E. coli O157:H7 and L. monocytogenes while concentrations of actinidin were 0 mg/ml, 175 mg/ml, 350 mg/ml and 700 mg/ml with E.
coli O157:H7 and L. monocytogenes.
Effects of papain on E. coli O157:H7
Meat sample preparation and inoculation: Chunk beef meat was purchased from a local store, transported to the laboratory under
refrigerated conditions (0–4°C) and stored in a chill condition at 4°C until use. The beef meat was cut using a sterile sharp knife and a
stainless steel square template into approximately 3x3 cm2. Meat samples were transferred into individual sterile plastic bags (WHIRL-PAK®,
Nasco, CA, USA). A 0.5 ml of the inoculum was pipetted on meat surface in the bags, giving a surface inoculum of 5-6 log CFU/ cm and
allowed to remain undisturbed for 5 min at room temperature to permit bacterial cell attachment before subjecting enzyme treatments. After
inoculation, 1 ml of each enzyme concentration was pipetted on the meat surface. The bags were then held at three different temperatures 5,
25, 35°C.
Enumeration of surviving bacteria and sampling time: Meat samples kept at 25°C and 35°C were sampled at 0, 1, and 3h while those at
5°C were sampled at 0, 6 , and 24 h. 20 ml peptone water solution was added to each bag and massaged for 1 minute then 0.1 ml was
removed and appropriate serial dilutions were surface plated on enrichment agar, Listeria agar (DIFCO Detroit, MI, ) for L. monocytogenes
and TSA (DIFCO, Detroit, MI ) for E. coli O157:H7, in duplicate.
The inoculated plates were incubated at 37°C for 48h for L. monocytogenes and 24h for E. coli O157:H7and dilution plates with 25-250
colonies were counted and populations were reported as a CFU/ml and log CFU/ml and statistically analyzed with SAS 9.2 (SAS institute,
Cary, NC). All experiments were repeated three times.
Determination of Enzymatic Activity and Protein Content: The enzyme activity of actinidin (KFPE OT1005X) and papain were measured
spectrophotometrically according to Kunitz with some modifications. The assay mixture (6 mL) contained 0.65% (w/v) of substrate, Casein
Solution; (Sigma, Sigma- Aldrich Pty Ltd.) dissolved in 50 mM Potassium Phosphate buffer, pH 7.5 at 37º C and different actinidin (KFEP
OT1005X) and papain dilutions. All reaction mixtures were incubated at 37 C for 10 minutes. The reaction was stopped by adding 5 ml of
110 mM Trichloroacetic Acid (TCA) and participated protein was removed by filtration through a 0.45 μm syringe filter. The absorbance of
filtrate was measured at 660 nm (Spectrophotometer 4001-000 GENESYS 20, 120V 50/60 Hz, US). Blank samples were performed by adding
the enzyme at the end of the incubation time, just before TCA addition and precipitation. One unit of the enzyme activity was defined as the
amount of enzyme which releases 1 µmol of tyrosine per min under the assay conditions. Specific activity was expressed as enzyme units per
mg protein. Protein content of actinidin (KFEP0T1005X) and papain were measured using the Modified Lowry protein assay method (Lowry
et al., 1951) with bovine serum albumin (Sigma) as the protein standard.
Escherichia coli O157:H7 is an emerging pathogen responsible for 73,000 illnesses and 61 deaths each year
in the United States (USDA-FSIS, 2002). Most of these illnesses are associated with eating undercooked,
contaminated ground beef.
Papain is important plant peptidase due to its powerful proteolytic activity, derived from the latex of unripe
papaya fruit (Carica papaya, Caricaceae). Papain is characterized by its ability to hydrolyze large proteins
into smaller peptides and amino acids. Many studies indicated that papain and other papaya extracts possess
antimicrobial activities against B. subtilis, Enterobacter cloacae, E. coli, Salmonella typhi, Staphylococcus
aureus, and Proteus vulgaris (Osato et al., 1993; Emeruwa, 1982).
Actinidin is another member of cysteine protease family present in kiwi fruit and it belongs to the same
class of enzymes as ficin, papain and bromelain. The important features of actinidin include a wide pH
range for catalytic activity and stability in high concentrations at moderate temperatures, but the enzyme is
susceptible to oxidation, a feature in common with other plant thiol proteases (Kaur, et al., 2010).
Antibacterial activity depends on many factors including pH, temperature and level of target microbial
population (Bloomfield, 1991). Nevertheless, the most important factor that affects the fate of
microorganisms in foods is the structure of the food matrix. Immobilized bacterial cells on solid surfaces
behave differently in terms of growth rate and survival; thus liquid laboratory media are not ideal for testing
real food conditions (Brocklehurst, et al., 1997; Robins, et al., 1994). It is important to test the antibacterial
activity of proteolytic enzymes used as a food preservative under actual food conditions.
Therefore, this study evaluated antibacterial activity of two proteolytic enzymes (actinidin and papain)
against pathogenic bacteria on meat surfaces in order to draw conclusions about their usefulness as food
preservatives beside their tenderizing effects. Two bacterial species were used, a mixed culture of E. coli
O157:H7 and L. monocytogenes, to determine the effect of these proteolytic enzymes on the bacterial
population (log CFU/ml) when held at different temperatures (5, 25 and 35°C).
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Papain concentration at 10 mg/ml at room temperature decreased the log CFU/ml of L. monocytogenes after 3 h by 0.9
while papain concentrations at 5 mg/ml and 8 mg/ml have no or little reduction (Fig.3). This effect of papain may
occur through inhibition of either the bacterial cell wall synthesis or protein synthesis (Osato et al. 1993).
There was no significant difference in log CFU/ml reduction of E. coli O157:H7 between papain concentrations tested
in this experiment 5, 8 and 10 mg/ml at room temperature. These reductions of the log CFU/ml were by 1.6, 1.7 and
1.8, respectively (Fig.4).
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Fig 1: Effect of Actinidin on L. monocytogenes at room temperature as a function of temperature* concentration *
time
Fig 2: Effect of Actinidin on E. coli O157:H7 at room temperature as a function of temperature* concentration
* time
Fig 3: Effect of Papain on L. monocytogenes at room temperature as a function of temperature* concentration *
time
Fig 4: Effect of Papain on E. coli O157:H7 at room temperature as a function of temperature* concentration *
time
Discussion
The inhibitory effect of actinidin and papain against foodborne pathogens used in this study was less effective on
meat than that when was added to 0.1% peptone water This is in agreement with the results of Shelef et al. (2006)
and Stecchini et al. (1993), who found that the potency of natural antimicrobial extracts decreases in complex food
systems.
Moreover, Hoa, et al. (1998) suggested that the differences in results could be due to the complexity of the food
system tested and/or the specific characteristic of the natural antibacterial used. Shelf, et al. (2006) also concluded
the antimicrobial activity of plant extracts increases by increasing its solubility in meat systems. Cutter, (2000);
Hsieh, et al., (2001) reported that the activity also increases under acidic conditions, high water contents, salt and
low fat contents of meat product. However, Robins and Wilson (1994) concluded that the growth of foodborne
pathogens in liquid culture provides a baseline for their behavior in complex structures.
Uhart, et al. (2006) have also observed the differences in the efficacy of natural antimicrobials when were studied in
vitro and when were added to a food matrix. They concluded that fat, oil droplets, and/or protein interaction are
responsible for this shortage of the activity. Furthermore, Farbood, et al. (1976) explained this shortage in three
reasons. First, the natural antimicrobials as spices may be absorbed or solubilized into the lipid fraction and then
react with fatty free radicals, leading to decrease its concentration in the aqueous phase and so its bactericidal effect.
Second, proteins may bind the active components of the natural antimicrobials thus, reducing their availability in the
aqueous phase. Third, the layer of fat surrounding the bacterial cell may protect the microorganisms from the
antimicrobial activity of spices. These three reasons may reduce the bactericidal strength of the natural
antimicrobials. However, regardless of the low antimicrobial reductions achieved, if these natural antimicrobials are
used as a part of a hurdle system, higher pathogen reductions may be attained.
References
Results
Actinidin
Effects of Actinidin on L. monocytogenes
Actinidin concentration at 700 mg/ml at room temperature decreased the log CFU/ml of L. monocytogenes after 3 h by 1.41 (Fig.1).
The relative effect on E. coli compared to L. monocytogenes agreed with Sparso & Moller (2002) who concluded that proteolytic
enzymes are more efficient against Gram-negative than Gram-positive bacteria.
Effects of Actinidin on E. coli O157:H7
Actinidin concentration at 700 mg/ml at room temperature decreased log CFU/ml of E. coli O157:H7 after 3 h by 2.4 (Fig.2). However,
there was not a difference in log CFU/ml reduction between the concentrations of 175 and 350 mg/ml. The exact mechanism by which
actinidin decreased bacterial growth of E. coli O157:H7 is not completely understood but it could be due to interaction with the proteins
in the outer cell membrane. These surface proteins may be degraded by actinidin causing a weakening of the cell wall resulting in cell
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