Antibacterial Properties of Hemp Hurd Powder against E coli

Abstract

Hemp (Cannabis sativa L.) is an eco-friendly and multifunctional plant. Hemp hurd is a by-product of hemp plant during hemp fiber separation. Although hemp hurd is repeatedly announced owing antibacterial activity, it has never been systematically investigated and reported. In this study, the antibacterial activity of hemp hurd powder against Escherichia coli is investigated. This article reveals antibacterial activity of hemp hurd where hemp hurd powder inhibits the growth of E. coli. Meanwhile, the self-contamination (forming during retting process) inside hemp hurd has dramatic impact on the antibacterial performance. To achieve better antibacterial activity, hemp hurd was heat treated to eliminate self-contaminations. The impact of the particle sizes and heat treatment on the antibacterial effectiveness was evaluated.

Full text

Antibacterial Properties of Hemp Hurd Powder Against E. coli
Belas Ahmed Khan,
1
Jing Wang,
1
Philip Warner,
2
Hao Wang
1
1
Centre of Excellence in Engineered Fibre Composite, University of Southern Queensland, Toowoomba, Queensland 4350,
Australia
2
Ecofibre Industries Operations Pty Ltd, Maleny, Queensland 4552, Australia
Correspondence to: H. Wang (E-mail: hao.wang@usq.edu.au)
ABSTRACT: Hemp (Cannabis sativa L.) is an eco-friendly and multifunctional plant. Hemp hurd is a by-product of hemp plant during
hemp fiber separation. Although hemp hurd is repeatedly announced owing antibacterial activity, it has never been systematically
investigated and reported. In this study, the antibacterial activity of hemp hurd powder against Escherichia coli is investigated. This
article reveals antibacterial activity of hemp hurd where hemp hurd powder inhibits the growth of E. coli. Meanwhile, the self-
contamination (forming during retting process) inside hemp hurd has dramatic impact on the antibacterial performance. To achieve
better antibacterial activity, hemp hurd was heat treated to eliminate self-contaminations. The impact of the particle sizes and heat
treatment on the antibacterial effectiveness was evaluated. V
C2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015,132, 41588.
KEYWORDS: biomaterials; biomedical applications; morphology; properties and characterization; thermogravimetric analysis (TGA)
Received 26 May 2014; accepted 21 September 2014
DOI: 10.1002/app.41588
INTRODUCTION
Botanically, hemp is a member of the most advanced plant fam-
ily on earth. It is a dioecious woody, herbaceous annual plant
that uses the sun more efficiently than any other plants and it
can be grown in virtually any climate or soil condition. Hemp
has a long history of cultivation for a variety of applications
including textiles, medicine, recreational drugs, and food.
1,2
Hemp can be divided into fiber type (industrial hemp), inter-
mediate type, and drug type (known as marijuana), with tetra-
hydrocannabinol (THC) content ranging from <0.3%, 0.3–
1.0%, and 1–20%, respectively.
3,4
Industrial hemp is a kind of yearly harvested plant where hemp
fiber is the main product of its cultivation. Hemp hurd (also
called hemp core) is a by-product and agricultural waste of hemp
plant. It is a residue from the hemp stem after the bast fibers are
removed for textile. Hemp hurd is mostly used for animal bed-
ding because of its favorable properties: good absorbency, easy
handling, and rapid composting after use.
5
Another application
of hemp hurd is in construction sector. New utilizations of the
hemp hurd are under development,
6
however, a great amount of
hemp hurd (accounting for 70–80% of the hemp stem) is dis-
posed by combustion or landfilling, which results in resource
waste. To develop its utilizations, hemp hurd could be milled into
powder. For instance, hemp hurd powder is a good filler in
hemp-reinforced plastic composites.
7
Also, hemp hurd powder
can be incorporated in 3D printing filament material in the
emerging 3D printing technology.
8
Hemp hurd powder may also
be applied to produce activated carbon with high specific surface
areas, micro-porous structure, high adsorption capacity, and
degree of surface reactivity.
9
Recently, hemp as an antibacterial agent has been attracting
more and more attention.
10,11
Hemp fiber has been confirmed
with excellent antibacterial activity, which leads hemp fiber to
be a good material for functional textiles.
12
However, antibacte-
rial performance of hemp hurd has never been systematically
investigated, although it is repeatedly announced that hemp
hurd also has similar property.
13
The antibacterial properties in
hemp hurd may come from cannabinoids, alkanoids, other bio-
active compounds, or compounds of lignin.
11,14
In this study, hemp hurd was milled into powder with different
size. Then the antibacterial properties against Escherichia coli
were investigated. The impact of retting process and particle
size of hemp hurd powder on the antimicrobial performance
was studied. Due to huge amount of hemp hurd waste every
year, these observations may stimulate future inclusion of hemp
hurd in antibacterial food package, hence utilize agricultural
waste resource and reduce environmental pollution.
15
MATERIALS AND METHODS
Material
Three types of hemp hurd (retted, semi-retted, and non-retted)
were used in this study. Semi-retted and non-retted hemp hurd
was obtained from Ecofibre Industries Operations Pty (Aus-
tralia). Retted hemp hurd powder was received from Research
V
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Centre of China-Hemp Materials, Beijing, China. Retted hemp
hurd was obtained at about 20C for 8 days in dewing process.
Semi-retted hemp hurd was achieved at about at 20C for 3
days followed by mechanical separation. For the non-retted
hemp hurd, it was separated through mechanical decortication.
A cutter mill (Pulverisette 19 from Fritsch Gmbh, Germany) was
used for chopping hemp hurd chips into approximately 1 mm
snippets. Hemp hurd snippets were passed through a rotary mill
(Pulverisette 14 from Fritsch) to be milled repeatedly up to 55 times
to obtain desired particle size. Different size of sieve was applied to
separate the hemp hurd powder into different particle sizes.
Particle size distribution was measured using a Mastersizer 2000
(Malvern Instruments, UK) fitted with Hydro 2000S. The dis-
persion medium was deionized water.
16
Table I shows the parti-
cle size of the different retted samples with various time of
milling. All results were presented according to a volume-based
particle size distribution.
Antibacterial Test
The hemp hurd powder was tested for its antibacterial perform-
ance against E. coli (ATCC25922). The bacterial cultures were
maintained on nutrient agar slopes. They were grown in sterile
Tryptic Soy Broth and incubated at 37C for 18 h. Working
buffer solution (0.3 mMKH
2
PO
4
) was adjusted pH to 7.2 60.1
with a dilute solution of NaOH, and then capped, sterilized,
and stored at room temperature.
To prepare the working bacterial dilution, the culture was
diluted with the sterile buffer solution until the solution has an
absorbance of 0.28 60.02 at 475 nm (as measured spectropho-
tometrically), which corresponds to a concentration of 1.5–3.0
310
8
colony forming units per milliliter (CFU/mL). The Athe-
rton cyber series autoclave was used for sterilization and media
preparation at 121C for 20 min.
Antibacterial performance of the hemp hurd powder was inves-
tigated according to ASTM E2149-10, described as the follow-
ing: (1) 1.0 g of hemp hurd powder was placed into a 250-mL
flasks with 50 ml working bacterial dilution; (2) the flask was
shaken on an agitation shaker at a speed of 300 rpm at 25C
for 1 h 65 min; (3) 1 mL of the solution before or after shak-
ing was inoculated on a plate containing 15 mL tryptic soya
agars; (4) the inoculated plate were cultivated at 37C for 24 h;
and (5) active bacteria were counted and antibacterial effect was
calculated. Percent reduction of the organisms resulting from
treated sample directly compared to “inoculum only” sample
after specified contact time is calculated. Results are presented
in percent reduction by counting CFU/mL of bacteria.
Heat Treatment and Thermogravimetric Analysis
To study whether the antibacterial property in the hemp hurd
can be preserved after hemp hurd powder being processed at
different processing temperature, hemp hurd powder was heated
at 80, 120, 140, and 160C for 0.5–3 h in a dry oven. Hemp
hurd powder was kept open in a ceramic beaker to allow the
evaporation of moisture from the material.
Thermogravimetric analysis (TGA) was used to determine the
thermal stability of the hemp hurd powder. The constituents of
hemp hurd powder have different thermal reactivity and
decomposition at different temperatures. TGA analysis on hemp
hurd powder was carried out by a thermal gravimetric analyzer
(TGA Q500). In ramp method, the samples were heated from
10C to 400C at a rate of 20 C/min. In ramp and isothermal
method, the sample was heated up to 100C at a rate of 20 C/
min and held for 20 min, then it was heated up to 160Cata
rate of 20 C/min and held for another 20 min. Experiments
were carried out in a nitrogen medium (60 mL/min) and the
weight was recorded as a function of increasing temperature.
Morphologies
The morphologies of milled hemp hurd powder were studied
by scanning electron microscopy (SEM) images. The samples
were sputter gold-coated for 120 s and were examined in a
JEOL JCM 6000 SEM operated at 10 kV with use of the second-
ary electron signal.
Table I. Retting Status and Particle Size of Hemp Hurd
Retting status Mean particle size
Semi-retted 188.4 lm
85.4 lm
44.3 lm
21.2 lm
Non-retted 204.0 lm
99.8 lm
47.2 lm
20.5 lm
Retted 37 lm
19.1 lm
Figure 1. SEM images of hemp hurd powder: (a) retted; (b) semi-retted; (c) non-retted.
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RESULTS AND DISCUSSION
Morphologies
Figure 1 shows the SEM image (High-vac, 10 kV) of hemp hurd
powder. A number of thin hemp hurd slices and irregular-shaped
aggregation were clearly observed. In addition, some short fibers
still existed because of insufficient pulverization.
17
In general, it
shows an irregular rough surface with sharp edges and grooves.
The hemp hurd powder shows the porous structure, indicating
low bulk density and high water absorption capacity. The high
surface roughness of the hemp hurd powder ensures a good bond
with the polymer matrix, so that the hemp hurd powder will be
able to be applied to reinforced plastic and other composites.
Inhibition of Bacterial Growth
The antibacterial activity of hemp hurd powder is presented in
Figure 2. It is evident that the hemp hurd powder inhibited the
growth of E. coli as the plates of control and the hemp hurd
powder have shown different appearance in the bacterial lawn.
The controlled sample has the CFU 340; while the non-retted
hemp hurd powder CFU reduces 30% to 235; and the retted
hemp hurd powder CFU decreases 78% to 75. These results
demonstrate that retted hemp hurd is more effective for the
inhabitation of E. coli bacteria. However, in the petri dish, there
are still some other types of microorganisms remaining in both
retted and non-retted hemp hurd powder (although only E. coli
was expected). These contaminations are from hemp hurd pow-
der itself, and they might have occurred from the fields where
they grow up and existed during fiber processing (retted or
non-retted). It is also notable that retted hemp hurd embraced
more severe contamination than the non-retted hemp hurd.
The retting process is a controlled degradation of plant stems to
allow the fiber to be separated from the woody core (hurd).
Dew, water, enzymatic, mechanical, and chemical retting proc-
esses are common for hemp.
18,19
Therefore, the retting process
of hemp provides more opportunity for hemp hurd to be con-
taminated and promulgated.
Self-Contamination of Hemp Hurd
To remove self-contaminations, retted hemp hurd powder was
treated in the air oven at 180C for 20 min. Figure 3 shows the
antibacterial performance of retted hemp hurd powder before
and after heat treatment. It is clearly observed that self-
contaminations are eliminated after heat treatment. Compared
with the non-treated hemp hurd powder having around 78%
Figure 2. Inhibition of bacterial growth of hemp hurd powder: (a) control; (b) non-retted hemp hurd powder; (c) retted hemp hurd powder. [Color fig-
ure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 3. The antibacterial activity of retted hemp hurd: (a) before and (b) after heat treatment. [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]
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reduction in CFU, the heat treated hemp hurd powder had
more efficient antibacterial activity with 90% reduction in CFU.
Therefore, to achieve the best performance, the elimination of
self-contamination is certainly important. Inactivation of micro-
organisms (such as bacteria) can be achieved by chemical and/
or physical means, such as heat, chemical solutions, gases, and
radiation.
20–22
It has been confirmed that heat has detrimental
effects on living cells,
20
and the heat-based sterilization techni-
ques were developed and commercially used to dispel undesired
medium preservation.
23
To obtain appropriate and effective contamination elimination,
different heat treatment was investigated in this study. Hemp
hurd powder was kept in the oven at 80C for more than 3 h
until all the moisture was completely removed. Then, the pow-
der was spread on agar containing petri dishes and incubated at
35 62C for 24 h. Figure 4 shows the effect of low temperature
treatment to eliminate the self-contaminations, presenting that
Figure 4. Status of self-contaminations: (a) retted hemp hurd powder without heat treatment; (b) retted hemp hurd powder treated at 80C; (c) un-
retted hemp hurd powder without heat treatment; and (d) un-retted hemp hurd powder treated at 80C. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
Table II. Observation on Elimination of Self-Contaminations After Heat
Treatment
120C 140C 160C
30 min 333
60 min 33冑
90 min 3冑冑
120 min 3冑冑
150 min 3冑冑
180 min 冑冑冑
(3)5contamination remaining; (冑)5contamination completely eliminated. Figure 5. TGA curves of retted hemp hurd.
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heat treatment at 80C for 3 h was not sufficient to eliminate
the existing contaminations of hemp hurd powder. This is due
to the higher decomposing temperature of some contamina-
tions.
24
Dew, water, enzymatic, mechanical, and chemical retting
processes are common for hemp. Therefore, the retting process
of hemp provides more opportunity (along with the favorable
condition for bacterial growth) to hemp hurd to be contami-
nated and promulgated. It is necessary to remove those contam-
inations for further antibacterial application.
To investigate heat-based conventional sterilization methods,
both moist heat and dry heat were studied. In the case of moist
heat in an autoclave, a temperature of 120C at a pressure of 15
psi was applied for 20 min, while dry heat sterilization was pro-
ceeded at 170C for 1 h.
23
To study the impact of temperature
and holding time on the self-contamination elimination in hemp
hurd powder, a series of experiments were carried out, and the
status of self-contaminations was observed by simply spreading
out heat-treated hemp hurd powder on the agar containing petri
dish, followed by incubating at 35 62C for 24 h. Table II sum-
marizes the contamination status at different heat-curing condi-
tions, indicating that the higher the curing temperature, the
shorter the curing time to eliminate self-contaminations.
Thermal Degradation
TGA was applied to study the weight loss of hemp hurd powder
with the increase of temperature. The degradation process was
divided into four stages: moisture evaporation, hemicellulose
degradation, cellulose degradation, and lignin decomposition.
Moisture is present in the material in two forms: free water and
linked water. Free water is attached on the fiber surface and
evaporates at lower temperature (25–150C). The linked water
with the hydroxyl groups is bonded in hemicellulose and lignin,
and decomposes at higher temperatures.
25
After the removal of
free water, the degradation process begins in the cellulose, hemi-
cellulose, lignin constituents, and the associated linked
water.
26,27
TGA curves in Figure 5 suggest that at 160C, no
thermal degradation will occur to the hemp hurd powder itself.
Effect of Particle Size and Temperature
Retted (37 lm, 19.1 lm), semi-retted (188.4 lm, 85.4 lm, 44.3
lm, 21.2 lm), and non-retted (204 lm, 99.8 lm, 47.2 lm, 20.5
lm) hemp hurd powder of different particle sizes were investigated
for antibacterial activity. Heat treatment (160Cfor2h)wascar-
ried out to remove self-contaminations before testing. Particle size
did not show any effect on antibacterial activity, and 2-h contact
time was sufficient for both fine and coarse particle to present
Figure 6. Antibacterial activities of different types of hemp hurd powder (20 lm): (a) control; (b) retted hemp hurd powder; (c) semi-retted hemp hurd
powder; and (d) non-retted hemp hurd powder. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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maximum antibacterial activity. Figure 6 shows antibacterial result
of selected hemp hurd powder, where both retted and semi-retted
hemp hurd were heat-treated, and non-retted hemp hurd was
applied without further treatment due to non-contaminations. Fig-
ure 6(a) is the control without any hemp hurd powder, and Figure
6(b–d) contain hemp hurd powder. All the three types of hemp
hurd powder show similar antibacterial activity, which further con-
firmed that self-contaminations in hemp hurd are the main impact
factor on the antibacterial performance.
Potential Applications and Future Work
There is a great concern of contaminations by microorganisms
in a variety of areas, for example, medical devices, healthcare
products, water purification systems, hospitals, dental office
equipment, food packaging, food storage, and household sanita-
tion.
28
One possible way to address microbial contamination is
to develop materials with antimicrobial properties.
29
Polymers such as polyethylene, polyurethane, polytetrafluoroeth-
ylene, polyacetal, polymethylmethacrylate, polyethylene tereptha-
late, silicone rubber, polysulfone, polyetheretherketone,
poly(lactic acid), poly(glycolic acid), and so on, are used in vari-
ous biomedical fields.
30
Incorporating antibacterial hemp hurd
powder as filler in the polymer composites could provide not
only lighter weight but also protection against bacterial attach-
ment. Hemp hurd reinforced polymer composites could be an
excellent choice for prostheses. Similarly, hemp hurd powder can
be incorporated in food packaging composites, and be considered
as eco-friendly. Hemp hurd powder incorporated polymer com-
posites formed by injection molding can provide packaging mate-
rials with a wide range of shape and sizes for containing a range
of foods including meat, salads, and ready-made food products.
CONCLUSION
In this study, the antibacterial activity of hemp hurd powder
against E. coli was systematically studied by applying different
hemp hurd powder (retted, semi-retted, and non-retted) with dif-
ferent size. To apply the retted and semi-retted hemp hurd powder
as antibacterial materials, it is necessary to eliminate contamina-
tions that come from external surroundings (such as humid and
temperature) during retting process. The heat-treated hemp hurd
powder at 160C for 2 h showed efficient antibacterial activity up
to 90% reduction in CFU. It was also explored that the particle
size of hurd powder has no obvious impact on the antibacterial
performance. It is proposed that the hemp hurd powder has many
potential applications in biomedical, food packaging, polymer
composites, and other value added diversified products.
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