Investigation of microbiological contamination in domestic refrigerators and an analysis of appropriate methods for reduction of contamination in ...
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Yvonne KAMPMANN1, Stefanie BRUCKNER1, Sandro KOHN2, Kerstin
KLOFT1 and Judith KREYENSCHMIDT1
1
Institute of Animal Science, Preventive Health Management Group, Katzenburgweg 7-9, 53115 Bonn; E-mail:
y.kampmannt@uni-bonn.de; Phone: +49 228 73 2053, Fax: +49 228 73 2617
2
Bosch und Siemens Hausgeräte GmbH, Robert-Bosch-Straße, D-89537 Giengen, Germany; E-
mail: sandro.kohn@bshg.com
Investigation of microbiological contamination in domestic
refrigerators and an analysis of appropriate methods for
reduction of contamination in private households
Abstract
This study provides original results concerning the microbial status of
domestic refrigerators and the testing of innovative antimicrobial
methods (ionization and silver coating) for their ability to reduce
airborne bacteria and bacterial count on refrigerator surfaces as well as
for their ability to decrease cross contamination.
1. INTRODUCTION
In food supply chains, from primary production to retail, rules and regulations
exist to ensure high quality and safe products. From the point of sale, end
consumers themselves have responsibility for the hygienic handling and storage of
food products. However, consumers are often not aware of the various factors
influencing the quality and safety of perishable products. In particular,
environmental factors like temperature and hygienic conditions have a huge
influence on the spoilage of food like fresh meat, fish and milk.
Studies in different European countries (France, Ireland, England, Greece,
Portugal and the Netherlands) as well as in the USA Canada and New Zealand,
have shown that many domestic refrigerators are operating above the
recommended refrigerator temperature of a maximum 5°C or 40°F (e.g. [1] - [3]).
This leads to faster spoilage of food.
Besides incorrect storage temperatures, cross-contamination is an area of great
concern in private households [4]. Bacteria can be transferred via hands,
contaminated dishcloths, and stored food to refrigerator surfaces and the
atmosphere [5]. Studies on the microbiological conditions found in domestic
refrigerators have shown a bacterial count on refrigerator surfaces between log 2,9
and log 7,1 CFU/cm2 on average. Furthermore, in more than 50% of investigatedrefrigerators, pathogen bacteria were found [3]. However, results vary greatly between countries. Furthermore, results concerning air contamination are missing. As a result, producers of domestic refrigerators are searching for innovative solutions to reduce microbiological contamination and to enhance food quality and safety. One possibility of reducing cross-contamination between surfaces and food is through treating the internal walls of refrigerators with an antimicrobial additive like silver ions. The antibacterial activity of silver ions is well documented. Both laboratory and clinical tests have demonstrated the effectiveness and safety of a range of silver-based antimicrobial additives (e.g. [6], [7]). The cellular effects of silver on several bacterial species were demonstrated by Feng et al. [8]. Silver ions react with electron donating groups, such as those containing sulphur, oxygen or phosphorus. This means target sites in bacteria are abundant, like proteins in the cell wall or cell membrane, enzymes and DNA [9]. Binding of silver ions to bacterial compounds results in protein inactivation, cell wall detachment, DNA condensation and will finally lead to cell destruction. The rate of bacterial inhibition is dependant on silver concentration and individual sensibility of microorganisms to silver ions. In general, gram-positive bacteria are less sensible to antibacterial compounds, since their peptidoglycan layer may protect them better from incoming silver ions [8], [10]. There are several factors that influence the antimicrobial action of silver. Amino acids, e.g. those with thiol groups like cysteine, bind to silver ions, thus lowering the amount of available silver for antimicrobial purposes. Temperature is another factor that influences the antibacterial action of silver. At low temperatures the release of silver from its carrier material decreases [11], [12]. Besides prevention of cross contamination between surfaces and food, prevention of cross contamination between air and food is an important factor in increasing food quality and safety. One possible approach to reduce air contamination is the fitting of refrigerators with ionisers. Ionization is a non-selective method affecting a wide spectrum of air pollutants (e.g. dust particles) and biological contaminants (e.g, microorganisms, pollen, and olfactory molecules) [13], [14]. This technology is already used in a range of industries, e.g. in dust-free rooms, in the food industry and medical techniques. Ionization is particularly important where both dust reduction and bactericidal properties are needed. [14], [15]. By air ionization, outer electrons are separated by single air molecules and attached to neutral molecules. Thus, positive and negative ions are built. Mostly, the following primary ions are formed: H+, H30+, O+, N+, Co4-, O-, OH-, H2O-, O2-, whereas superoxide (O2-) represents around 95% of negative charged ions and is more stable than the other primary negative charged ions [16], [17]. Cluster ions
accumulate on airborne pollutants, aerosols, and microorganisms in the air and give them a positive or negative charge. Ions are discharged and air contaminants are oxidised. Microorganisms are killed or inhibited in their growth [18], [19]. The production and the mode of action depend on many factors like humidity and the sensitivity of the bacteria [19], [20]. The methods described for reduction of microorganisms are already used in the food industry. However, studies about the application of these methods in the field of domestic refrigeration are rare. The aim of this study is the determination of the microbiological status of domestic refrigerators and to test two innovative antimicrobial methods (silver coating and ionization) for their ability to reduce both bacterial count on refrigerator surfaces and airborne bacteria. 2. MATERIAL AND METHODS The present study is divided into three stages. In the first stage, the microbiological status (surface and air contamination) in domestic refrigerators is quantified. In the second stage, silver is investigated for its ability to reduce surface contamination in refrigerators. In the last stage, ionization is tested for its ability to reduce air contamination in household refrigerators. Microbiological Status of the refrigerators To examine the microbiological flora on interior surfaces and the air contamination in refrigerators, refrigerators in private households in North Rhine Westphalia (a large German province) were checked. Bacterial count on inner surfaces Microbiological analyses of inner surfaces in 45 refrigerators were performed by using the stamping method according to DIN 10113-3. For determination of bacterial count, contact slides with differing agar for the following microorganisms were placed on different surfaces in the refrigerators (inner back wall, vegetable drawer and bottom shelf glass plate) and held against the surface for a few seconds (table 1):
Table 1. Contact Slices, incubation times and temperatures
Microorganisms Agar Incubation Incubation Company
temperature time (h)
(°C)
Total Viable Count Plate Count Agar 30°C 72h
Staphylococci Mannit-Salt- 37°C 36h
Phenolred-Agar
Staph. aureus Selective agar for 37°C 24h
Staph. aureus
indicator dye
Listeria spp. Agar Listeria 37°C 48h
according to Ottaviani
and Agosti (ALOA) Transia, Ober-
Lactobacilli Purpose Medium with 30°C 48h Mörlen, Germany
Tween
Enterobacteriaceae Violet Red Bile 30°C 24h
Glucose Agar
E. coli Selective agar for 37°C 24h
Entero-bacteriaceae
with β-Glucoronidase
Coliforms Violet Red Bile Agar 37°C 24 h
Salmonella spp. Rambach-Agar 37°C 24h
B. cereus Cereus Agar 37°C 24h Nissui
Pharmaceuticals,
Tokyo, Japan
Yeasts and Molds Potato-Dextrose-Agar 25°C 120 h Nissui
as well as Sabouraud- Pharmaceuticals,
Agar Tokyo, Japan
Evaluation and visual classification of the samples was done according to
specifications provided by Transia, medium manufacturers (Ober-Mörlen,
Germany). The bacterial count was expressed in 4 grades: clean (0 cfu/cm²),
reasonably clean (< 1 cfu/cm²), moderately contaminated (1-Testing of innovative methods to improve refrigerator hygiene
Two innovative methods to reduce bacterial counts on the surface and in the air of
refrigerators have been tested: embedding of silver into refrigerator interior
surfaces and ionization.
Silver
Investigation into the antimicrobial effect of silver incorporated in the inner liners
of refrigerators has been examined by laboratory tests according to JIS Z 2801 -
standard method for efficacy testing of plastics (Japanese Standards Association,
2000).
To test the antimicrobial activity of silver, sheets (50 x 50 mm) of High Impact
Polystyrene (HIPS) with a thin coating of silver containing HIPS were used
(AlphaSan®, Milliken, B). Uncoated test pieces without silver in the same size
were used as a reference. The described test-sheets and reference plates were
disinfected with ethanol impregnated bandages and dried in a sterile atmosphere.
24 plates containing silver and six reference sheets without silver were inoculated
with 0.4 ml of a 105 CFU/ml concentrated Staph. aureus suspension (DSM No.
346). In order to prevent evaporation and to standardize the contact area, the
inocula were covered loosely by sterile PE films (45 x 45 mm). The bacterial
concentrations on three untreated test pieces were determined immediately after
inoculation to determine the starting concentration. These sheets were placed in
sterile stomacher bags and doused with 10 ml of soybean-casein digest broth with
lecithin polysorbate (Roth, Karlsruhe, G) each. The other reference sheets and all
test sheets were incubated at 35°C (humidity 90%) for 24 hours and washed out
after incubation in a similar manner.
Viable counts were determined by plate counting using plate count agar (Roth,
Karlsruhe, G) of appropriate decimal dilutions, made in sterile phosphate buffered
saline (0.9%). Agar plates were incubated at 37°C for 48 hours before counting.
Antibacterial activity was calculated by subtracting the logarithmic value of viable
counts on coated material from untreated material after inoculation and incubation:
Tx , Re
log Reduction = log (2)
Tx , Pr
Tx,Re = Bacterial concentration on reference material, x hours after inoculation
Tx,Pr = Bacterial concentration on coated material, x hours after inoculation
For materials to account as antibacterial, the calculated value of antimicrobial
activity should be ≥ 2.0.In addition the examination method was adapted to refrigerating conditions. For
this reason, psychotropic microorganisms were applied and test temperatures were
lowered. Lact. delbrueckii, subspecies lactis (DSM Nr. 20072) and Ps. fluorescens
(DSM Nr. 304) were used as inoculums in a concentration of 105 CFU/ml each.
The incubation temperature was decreased to either 5°C. The incubation time was
extended up to 72 hours. The samples (for each trial and sampling point: 3 plates
containing silver and 6 untreated plates) were prepared, incubated, washed out and
plate counted in the way as described in JIS Z 2801.
Ionization
For testing ionization antimicrobial activity, two refrigerators, identical in
construction were used (volume 287 litres). A drawer with a holder for petri dishes
were installed to define an exact positioning and to allow the changing of petri
dishes at pre-determined time intervals. One refrigerator was equipped with an
electronically ionizer (Xi’an KongHong Information Technology Co., China). It
produces negative charged ions with a density of 5*106 Ions/cm3. The other
refrigerator served as reference. Both refrigerators were adjusted to 8°C.
4 ml aerosol from a Bacillus subtilis (DSM-Nr. 704) suspension at a concentration
of 106 CFU/ml was nebulised in both refrigerators. At defined time intervals (0.5
h, 1 h, 1.5 h, 2 h and 2.5 h), the airborne bacteria concentration was determined by
the use of the sedimentation method with caso agar for 30 minutes. Counting of
CFU followed after 72 h incubation at 30°C.
Reduction was calculated by the following formula:
Tx ,Re − Tx. Ion
Reduction = *100 (1)
Tx , Re
Tx.Ion = Ø airborne bacteria, x h after incubation in refrigerator with ionization
Tx,Re = Ø airborne bacteria, x h after incubation in refrigerator without ionization
3. RESULTS
Microbiological Status in the refrigerators
According to the total bacterial count on inner surfaces the vegetable drawer was
the most contaminated surface in household refrigerators (n=45). 25% percent of
the vegetable drawers showed a total bacterial count of more than 45 cfu/cm². On
all investigated surfaces the following microorganisms were found in descending
order: Staphylococci (62,6%), yeasts (62,3%), Bacillus cereus (47,9%),
Lactobacilli (32,8%), Listeria spp. (26,4%), Enterobacteriaceae (26,4%) and
Staphylococcus aureus (25,9%). Coliforms, Escherichia coli and Salmonella spp.
were not detected on any of the refrigerator surfaces investigated.The average airborne bacterial concentration in household refrigerators was 8.5 +/-
13.9 cfu/plate (n=90) and the maximum concentration was 62 cfu/plate. Moulds
were found in the air of more than 50% of the refrigerators investigated.
Testing of innovative methods to improve refrigerator hygiene
Silver
Laboratory tests on the antimicrobial activity of silver coated refrigerator surfaces
showed a reduction in average between 1.0 – 5.9 log10 units compared with
uncoated refrigerator surfaces, depending on bacterial strain, incubation time and
temperature.
The results, illustrated in Figure 1 for tests according to JIS Z 2801, indicate a
clear drop of S. aureus concentration on silver containing materials. Compared to
bacterial concentration on the reference materials after incubation, the coated
samples showed a reduction of 0.1 - 4.1 log10 units. Six of 24 tested samples
demonstrated a reduction of less than 1.0 log10 units. On the other hand, 14
samples showed a reduction of more than 2.0 log10 units. The median of reduction
was 2.7 log10 units.
Figure 1. Reduction of bacterial count on silver coated HIPS samples 24h after incubation with
S. aureus compared to reference plates without silver
Variation of bacterial strain, incubation temperature, and time showed that silver
coated plates had a bactericidal effect also at refrigerator normal temperatures.
However, at 5°C this effect became evident after a longer time period than at 35°C.
The reduction of Lact. delbrueckii is illustrated in table 2. At 35°C an antibacterial
decrease of 1.0 log10 units has been proven after 24 hours. At 5°C there was nodecline of bacterial concentration in the same time interval, but the bactericidal
effect (reduction of 1.4 log10 units) was achieved after 72 hours.
Table 2. Log10 - reduction of Lact. delbrueckii on silver containing HIPS materials at different
incubation temperatures and incubation times
Incubation- Incubation- Starting- Concentration after Concentration after Reduc-
temperature time (h) concen- incubation on incubation on silver tion
(°C) tration reference materials containing materials (lg CFU/
(CFU/ml) (lg CFU/ml) (lg CFU/ml) ml)
5
35 24 2.0*10 3.9 ± 0.15 2.9 ± 0.28 1.0
5 24 2.0*105 5.4 ± 0.15 5.6 ± 0.19 0*
5
5 72 2.0*10 4.9 ± 0.12 3.4 ± 0.14 1.4
*no significant difference [21]
At 35°C a drastic reduction of Ps. fluorescens by silver became apparent after only
24 hours (reduction of 5.1 log10 units), while at 5°C – as already shown for Lact.
delbrueckii – a similar reduction could only be achieved by longer storage periods
(up to a reduction of 5.4 log10 units, see table 3). Compared to the decline of Lact.
delbrueckii concentrations on silver coated surfaces, the reduction of Ps.
fluorescens is much higher.
Table 3. Log10 - reduction of Ps. fluorescens on silver containing HIPS materials at different
incubation temperatures and incubation times
Incubation- Incubation- Starting- Concentration after Concentration after Lg -
temperature time (h) concentratio incubation on incubation on silver reducti
(°C) n (CFU/ml) reference materials containing materials on
(lg CFU/ml) (lg CFU/ml)
35 24 7.9*105 6.1 ± 0.14 < 1.0 5.1
5
5 24 7.9*10 6.1 ± 0.06 5.9 ± 0.12 0.2*
5
5 72 7.9*10 6.4 ± 0.28 < 1.0 5.4
*no significant difference; [21]
Ionization
Tests with ionization showed that it is a possible method for reducing airborne
bacteria in domestic refrigerators.
Figure 2 shows the results of the development of air contaminants in refrigerators
with and without ionization.Figure 2. Airborne bacteria in a refrigerator with and without ionization at defined points in time It becomes evident, that after 0.5 h, TVC on average is 237 cfu/plate in a refrigerator with ionization and 300 cfu/plate in a reference refrigerator. This is equal to a moderately significant reduction of 21%. After 1h, the rate of reduction is highly significant at 58.2%. 4. CONCLUSION Investigations showed that domestic refrigerators in Germany are highly contaminated with a variety of spoilage and pathogenic bacteria. Especially on interior refrigerator surfaces, high bacterial counts of different microorganisms are found. Solutions to reduce refrigerator contamination are required. Laboratory tests with silver coated refrigerator inner surface indicate a strong antimicrobial activity of silver. This activity is dependent on temperature, time and the sensitivity of the bacterial strain. Tests with ionization showed, that in general it is possible to reduce air contamination in domestic refrigerators by this method. The combination of both methods can make an important contribution to the reduction of both surface and airborne contamination in household refrigerators. Further studies are necessary to investigate the inhibitory action of food proteins on the antimicrobial effect of silver and to analyse the long-term effects of ionization on food ingredients. 5. REFERENCES [1]. O’BRIEN, G.D. Domestic refrigerator air temperatures and the public’s awareness of refrigerator use. International Journal of Environmental Health Research 7, 1997,141-148. [2]. DANIELS, R.W. Home food safety. Food Technology 52, 1998, 54-56.
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