The anti-microbial effect of food wrap containing beeswax products

Abstract

Food preservation is a vital issue on a global scale. It reflects both the need to prevent food spoilage as well as the necessity for reduction in the occurrence of food-borne pathogens. Progress of technology allowed for the development of various techniques that inhibit the growth or activity of food contaminating microbes. In the last few years we saw creation of various types of antimicrobial food packaging systems. They contain active antimicrobial agents immobilized on a material that acts as a food wrap. In this paper we have tested the efficiency of Abeego, a commercially available food wrap, against representatives of different groups of microbes. The Abeego wrap contains natural products such as waxes and oils, which can display antimicrobial activity. We analyzed the ability of the wrap and its components to affect the growth and replication of various groups of microorganisms. We have discovered that the Abeego wrap was capable of significantly inhibiting the viable cell count of bacterial species - both gram positive and gram negative. However no noticeable antifungal or antiviral activity has been observed. This implies that Abeego wrap can act as an efficient mean of inhibiting the spread of food-borne bacterial pathogens, as well as contribute to preventing food spoilage.

Full text

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THE ANTI-MICROBIAL EFFECT OF FOOD WRAP CONTAINING BEESWAX PRODUCTS
Crystal T. Pinto1, Jarosław A. Pankowski* 2, Francis E. Nano1
Address(es):
1University of Victoria, Department of Biochemistry and Microbiology, 3800 Finnerty Road, Victoria BC V6P 4W8, Canada.
2University of Nebraska-Lincoln, Department of Chemistry, 1400 R Street, Lincoln, NE 68588, USA.
*Corresponding author: jaroslaw.pankowski@unl.edu
ABSTRACT
Keywords: food preservation; antimicrobial; food wrap; Salmonella
INTRODUCTION
The spoilage and contamination of groceries caused by microbes is a serious
concern for many branches in the food industry. It results in food products being
unsuitable for consumption and can account for significant financial losses. It is
estimated that approximately 25% of world’s food supply is lost due to the
activity of microorganisms (Singhet al. 2016). Therefore preventing product
spoilage in food industry is one of the most vital matters. Diseases caused by
food-borne pathogens are another major issue for food distribution. The problem
of food preservation has been known since the beginning of civilization and as a
result multiple techniques of conserving food have been developed. This includes
methods such as drying, salting and pickling. In many cases, especially in
modern food preservation, use of antimicrobial agents plays a significant role.
Especially the chemical compounds that stop the growth and activity of microbes
allow for food to remain consumable for much longer. New possibilities in the
field of food preservation have become available thanks to the use of
antimicrobial food packaging (Malhotra et al. 2015). This type of packaging
material incorporates elements that inhibit the growth of pathogenic bacteria and
fungi. In some cases the active agent can be a natural product.
Abeego is a consumer food wrap material that consists of a coated fabric. The
main constituent of Abeego is beeswax which has been deemed “generally
regarded as safe” (GRAS) (“Select Committee on GRAS Substances (SCOGS)
Opinion: Beeswax (yellow or white),” 1975) by the U.S. Food and Drug
Administration. Lesser components include tree resin and oils. One of the
potential advantages of using beeswax as part of a food wrap material is its anti-
microbial properties (Antúnez et al., 2008; de Andrade Ferreira et al., 2007;
Orsi et al., 2005; Wilsonet al. 2015; Zhang et al. 2013). The anti-microbial
effects of beeswax are attributed to propolis (Marcucci et al., 2001), a sticky
material used by bees to seal holes and cracks in beehives. The major constituents
of propolis are resins derived from the plants that the bees visit while collecting
pollen. The variation in flora in different geographical locations will affect the
constituents of propolis (Wilson et al. 2013; 2015) and, in turn, this will affect
the potency of the anti-microbial effect of each lot of beeswax. Thus, it is
important for manufacturers of food service products using beeswax to have
evidence that each lot of beeswax has a minimal level of anti-microbial activity.
Presumably, much of the anti-bacterial activity of Abeego wrap comes from the
propolis fraction of the beeswax. However there could be contributions from the
resin, oil and fabric components as well.
The aim of this project was to investigate the anti-microbial activity of the
Abeego wrap and its components. The anti-bacterial activity of Abeego is
predominently attributed to the propolis fraction of the beeswax, however there is
a possibility of resin, oil and fabric components to play an important role too.
Therefore we decided to investigate the effect of the Abeego product and its
individual components on different groups of microbes. Since food-borne
pathogens can be found among bacteria, fungi and viruses, we have investigated
representatives of each group. The individual components of Abeego wrap
analysed during this project have been taken from the lot used to make the final
product.
MATERIAL AND METHODS
Preparation of the Abeego wrap and its components
The Whatman™ Filter Paper Circles (12.7 mm) were used as carriers for the oil
and wax. Fragments of Abeego wrap and fabric were cut out to have the same
shape and size as paper circles. Papers and wrap were sterilized in the autoclave.
Oil was prepared by leaving it in boiling water for 1 hour. The resins were
sterilized with 70% ethanol. The sterile paper circles were dipped in oil or molten
wax. For standardization purposes pieces of resins were selected so that their
weight would be within 10% of each other.
Strains and growth conditions
Bacterial strains of Bacillus cereus, Escherichia coli W3110, Pseudomonas
aeruginosa, Salmonella enteritidis and Staphylococcus aureus were used for the
anti-bacterial activity experiments. For positive control discs soaked in
Food preservation is a vital issue on a global scale. It reflects both the need to prevent food spoilage as well as the necessity for
reduction in the occurrence of food-borne pathogens. Progress of technology allowed for the development of various techniques that
inhibit the growth or activity of food contaminating microbes. In the last few years we saw creation of various types of antimicrobial
food packaging systems. They contain active antimicrobial agents immobilized on a material that acts as a food wrap. In this paper we
have tested the efficiency of Abeego, a commercially available food wrap, against representatives of different groups of microbes. The
Abeego wrap contains natural products such as waxes and oils, which can display antimicrobial activity. We analyzed the ability of the
wrap and its components to affect the growth and replication of various groups of microorganisms. We have discovered that the Abeego
wrap was capable of significantly inhibiting the viable cell count of bacterial species – both gram positive and gram negative. However
no noticeable antifungal or antiviral activity has been observed. This implies that Abeego wrap can act as an efficient mean of inhibiting
the spread of food-borne bacterial pathogens, as well as contribute to preventing food spoilage.
ARTICLE INFO
Received 14. 11. 2016
Revised 5. 8. 2017
Accepted 12. 9. 2017
Published 1. 10. 2017
Regular article
doi: 10.15414/jmbfs.2017.7.2.145-148

J Microbiol Biotech Food Sci / Pinto et al. 2017 : 7 (2) 145-148
146
kanamycin (25 µg/ml or 50µg/ml) were used. Two strains of Saccharomyces
cerevisiae – CRY1 and MYA3666 (VL6-48) – were used as representatives of
yeasts. Bacteriophages M13 (Messing, 1993) and P1vir (Luria et al. 1960) were
used for the viral assays. The M13 phage was amplified by infecting E. coli
JM109 strain, while the P1vir was amplified by infecting E. coli DH10B.
Bacterial broth growth was done in Luria-Bertani (LB) medium (Bertani, 1951).
Yeast broth growth was done in the yeast extract-peptone-dextrose (YPED)
medium supplemented with adenine (YPAD) (Murthy et al. 1975). For solid
media agar was added to concentration of 1.5%.
Zone of inhibition assay
The strains were grown overnight in LB and undiluted samples were spread on
LB plates using sterile cotton swabs. Discs of Abeego wrap, fabric, oil, resin and
beeswax were prepared and sterilely placed on the plates with the above cultures.
The Abeego fabric was used as a negative control (as it contains no beeswax) and
discs soaked in kanamycin were used as positive control. The plates were
incubated at 37⁰C for 18-20 hours until the bacterial lawn of growth was
observed. The zones of inhibition were analyzed by measuring their radius i.e.
the distance from the center of zone to its border.
Bacteria survival assay
For S. enteritidis assay the Abeego discs were left in 1ml LB medium in a
microtiter plate for 18 hours. Then 100µl of S. enteritidis inoculum (~105 cfu/ml)
was added into the wells. The cultures were incubated with shaking at 30°C for
24 hours. After that time the cells in each well were enumerated on dilution
plates. This experiment was performed in quadruplicates. For S. aureus assay the
Abeego discs were left in 1ml LB medium in a microtiter plate for 72 hours.
Then 100µl of S. aureus inoculum (~106 cfu/ml) was added into the wells. The
cultures were incubated with shaking at the room temperature for 40 hours. After
that time the cells in each well were enumerated on dilution plates. This
experiment was performed in pentaplicates. For both experiments the data was
analyzed using unpaired T-test.
Yeast survival assay
For the yeast experiments the Abeego discs were left in YPAD medium in a
microtiter plate for 24 hours. Then inoculum from an overnight S. cerevisiae
culture (~10-6 cfu/ml) was used to start the fresh cultures. The cultures with and
without the Abeego wrap were grown at a room temperature for 48 hours. After
that time the cells in each well were enumerated on dilution plates. These
analyses were performed in triplicates. For this experiment the data was analyzed
using unpaired T-test.
Phage activity assay
For the M13 experiment ~103 phage particles were suspended in LB medium in a
microtiter plate. The phages were incubated with and without sterile Abeego
wrap, at a room temperature with shaking. After 50 hours, plaque assay was
carried out by inoculating the phage with the JM109 strain into LB soft agar and
creating an overlay. For the P1/vir experiment ~106 phage particles were
suspended in LB medium in a microtiter plate. The phages were incubated with
and without sterile Abeego wrap, at a room temperature with shaking. After 24
hours, plaque assay was carried out by inoculating the phage with the DH10B
strain into LB soft agar and creating an overlay.
RESULTS AND DISCUSSION
Growth inhibition assay on agar plates
To test the spectrum of anti-bacterial activity of the commercial products, a zone
of inhibition assay was carried out against the bacteria that are some of the most
common food-borne pathogens and environmental contaminants. The zone of
inhibition assay was utilized as it allows for a quick determination of an
inhibitory effect of a given substance. The impact of Abeego wrap and its
components was compared to a positive control in form of antibiotic discs. After
the incubation no zone of inhibition was observed with Abeego fabric, beeswax,
oil and resin against any of the analysed bacteria. At the same time the positive
control generated visible zone of inhibition (Fig 1). The likely explanation for
this lack of effect from the tested products is the limited diffusion into the
surrounding medium. Therefore in order to properly asses the activity we had to
utilize an alternative method.
Figure 1 Zone of inhibition assay to test the antibacterial activity of Abeego
Fabric and its constituent parts. A) Zone of inhibition assay for Abeego wrap
and fabric against B. cereus B) Zone of inhibition assay for Abeego oil and wax
against B. cereus C) Zone of inhibition assay for Abeego resin against B. cereus
D) Zone of inhibition assay for Abeego oil and wax against P. aeruginosa E)
Zone of inhibition assay for Abeego oil and wax against P. aeruginosa F) Zone
of inhibition assay for Abeego oil and wax against S. aureus G) Zone of
inhibition assay for Abeego resin against S. aureus H) Zone of inhibition assay
for Abeego wrap and fabric against E. coli
Anti-bacterial activity of Abeego wrap
We decided that the best way to detect the anti-microbial activity of the Abbego
wrap was to incubate it with bacterial cells in the liquid phase. To represent the
diverse pathogens we have selected both a gram negative bacterium - Salmonella
enteritidis and gram positive bacterium -Staphylococcus aureus. S. enteritidis is
an organism that is commonly associated with gastrointestinal infections (Feasey
et al. 2012). On the other hand S. aureus is a commensal inhabiting skin of
people and animals. It can however act as an opportunistic pathogen. It can also
become a food-borne pathogen when transferred onto dairy products (Fooladi et
al. 2010). During the test bacterial cells of both strains were exposed to Abeego

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147
wrap and its individual components. We discovered that incubation with the wrap
has caused a significantly decline in the number of cells of both S. enteritidis and
S. aureus (Fig 2). At the same time some of the individual components of the
Abeego wrap also had an impact on the number of observed colony forming units
(cfu). However there is no consistency in that manner between both tested
organisms. This could mean that each compound has a major effect on a different
type of bacterial cell. Therefore the effect of wrap could be attributed to a
combined impact of all its components. Overall this confirms the previous
suspicion of the anti-bacterial activity of the tested material.
Anti-yeast activity of Abeego wrap
While the Abeego product shows an effect on bacterial cells it has to be further
investigated if it can inhibit the survival of eukaryotic pathogens. For this
purpose we have used two strains of Saccharomyces cerevisiae – CRY1 and
MYA 3666. S. cerevisiae is the most commonly used representative of Eukarya.
It serves as a good approximation of fungal food-borne pathogens like
Aspergillus flavus (Kim et al. 2004). The yeast cells were treated in a similar
manner as S. enteritidis and S. aureus in the survival assay. The analysis of the
number of cfu revealed a small decrease after treatment of CRY1 cells with the
Abeego product. However the observed results were not considered to be
significant (Fig. 3). This strongly suggests that the tested wrap has no impact on
the fitness of eukaryotic cells. It is likely that the composition of yeast membrane
and cell wall gives resistance to the effects that were observed in bacteria.
Therefore similar reaction might be expected from food-borne fungal
microorganisms.
Activity on viral capsid
The last relevant group of food-borne pathogen consists of viruses. The common
examples could be noroviruses, rotaviruses as well as hepatitis A and E viruses
(Atreya, 2004; Carter, 2005). All of these pathogens possess non-enveloped
capsids. On the other hand there are much fewer enveloped viruses that can cause
food-borne infections i.e. astroviruses have enveloped viroids and are associated
with gastroenteritis (Fleet et al. 2000). Additionally coronaviruses can cause
gastrointestinal symptoms, however these viruses are mostly associated with
pneumonic infections (Jevšnik et al., 2013). To investigate the effect of Abeego
wrap on viral capsids we tested representatives of non-enveloped viruses. For
quick and easy assay we decided to analyse the phage particles. These viruses can
be replicated in short time and assayed in a reproducible manner. As viral
particles they can act as an approximation of the eukaryotic viruses. The
commonly used bacteriophages M13 and P1 were chosen for this experiment.
The phage particles were tested using similar method as the one used for bacterial
and yeast survival assay. Each phage was then enumerated on two-layer plates by
infecting E. coli cells. Results show decrease in the overall number of active
phage particles (Fig 4). However these changes are not significant enough to
support conclusion that the Abeego wrap can inactivate viral particles. A possible
explanation for this result would be that the effect of beeswax components is
primarily targeting the membrane and therefore has no strong impact on the
protein structure of viroid.
Figure 2 Anti-bacterial activity of Abeego product. The Abeego wrap and its components (wax, resins, oil and fabric) were tested for their anti-microbial effect on
cells of Salmonella enteridis and Staphylococcus aureus. Pure LB medium and antibiotic were used as negative and positive control, respectively. Significant decrease
in cell number has been observed for S. enteridis when comparing LB medium with wrap (P=0.002) or resins (P=0.043). Other individual elements of Abeego wrap
(wax, oil and fabric) had no significant impact on cell number. Significant decrease in cell number has been observed for S. aureus when comparing LB medium with
wax (P=0.02), oil (P=0.04) or wrap (P=0.02). The exposure to antibiotic reduced number of cells to zero in both cases. The significance level of 0.05>P>0.01 is
represented by * symbol on the graph. The significance level of P<0.01 is represented by *** symbol on the graph. Bars represent standard deviation.
Figure 3 Effect of Abeego product on yeast cells. The Abeego wrap was tested for its anti-microbial effect on cells of Saccharomyces cerevisiae strains CRY1 and
MYA 3666. The yeast cells grown in regular YPAD medium were compared to the ones grown in the presence of the wrap. After enumerating cells in both populations
no significant differences were observed (P>0.05, not significant indicated as ns). Bars represent standard deviation.

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Figure 4 Effect of Abeego product on activity of phage particles. The Abeego wrap was tested for its effect on suspended particles of P1vir phage and M13 phage.
The phages exposed to the wrap and the negative control were enumerated on two-layer plates with appropriate strains of E.coli. No significant differences were
observed between the two populations (P>0.05, not significant indicated as ns). Bars represent standard deviation.
CONCLUSION
The presented results show that Abeego wrap possesses anti-bacterial activity.
This effect can be observed when the microorganisms are exposed to the surface
of the wrap, since the active substance cannot diffuse into the environment. The
tested product displayed activity against both gram negative and gram positive
cells. At the same time this effect does not extend to yeast cells and viral capsids.
Acknowledgments: The work was supported by grant from the Natural Sciences
and Engineering Council of Canada, EGP 477804-14. We woud also like to thank
Toni Desrosiers for providing resources necessary for this project.
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