Investigation and Spectroscopic Analysis of the Effectiveness of Biocidal Product Added Erasers Against Escherichia Coli and Staphylococcus Aureus Bacteria
1. Abstract
Today, antibacterial materials have many application areas. There is an intense need to destroy and prevent the spread of microorganisms in many different fields such as medical instruments, dental fillings, and the food industry.
Especially heavy metal ions such as silver (Ag), zinc (Zn), copper (Cu), mercury (Hg), titanium (Ti) have high antibacterial activity. These ions deactivate the bacterial enzymes,
prevent the bacteria from performing enzyme reactions and shorten their lifespan [1].
Although antibacterial products have always attracted attention by consumers, they have become even more important especially after the Covid 19 epidemic.
In this study, it is aimed to gain antibacterial properties in order to prevent the formation of bacteria on the erasers, which can be carried from hand to hand and have a high risk of spreading bacteria, in many areas such as schools, offices, houses and besides, it is aimed not to pose any risk in terms of
chemicals.
2. Introduction
Although microorganisms such as bacteria and fungi cannot be seen with the naked eye, they live in all environments, and harmful bacteria that are pathogenic may also be present among them.
Bacteria can reproduce even in normal humidity and temperature conditions, they can easily create a habitat on commonly used objects, including plastics, and cause negative effects on hygiene, health and esthetics. They can cause surface deterioration and cracks, discoloration, slime formations and unpleasant odors on the objects on which they multiply.
In addition, they can create an environment and conditions that will cause infectious diseases. Antimicrobial compounds are widely used in various industrial processes such as drinking water, waste management, daily use cosmetics, furniture and bacterial drugs.
Antimicrobial compounds are commonly used to control the growth of pathogenic microorganisms or to remove them from inanimate objects, surfaces or intact skin [2].
Products with antimicrobial surfaces are highly desirable. These surfaces preferably exhibit an antibacterial effect against both Gram positive bacteria (eg Staphylococcus aureus) and Gram negative bacteria (eg E. coli) [3]. Antimicrobial products offer broad protection: inhibit the growth of bacteria, fungi and viruses.
These products are integrated into various plastic items and provide antibacterial protection, especially on the surface of the item. Eraser is one of the tools (write, design, modify) applied and widely used for educational purposes. The use of the eraser is not limited to just erasing, it can be applied to create highlights, textures and even deep visual effects People (for example, students, teachers, and other professionals) are in frequent contact with such products.
Therefore, their contents are subject to some chemical limitations. The presence of antibacterial properties in such widely used tools plays a critical role in humans, especially those with sensitive health conditions.
As Adel, we have added antibacterial functionality to our newly developed erasers by using antimicrobial technology with Avient masterbatch, which is in the list of ‘Ministry of Health Biocidal Inventory Registered Products’.
3. Material and Method
3.1. Performing the Antibacterial Test
3.1.1. Preparation of Test Inoculum:
From the culture incubated for 16-24 hours in PCA, the density is adjusted to 6x108 cfu/ml with a sterile loop into 1/500 NB. Ensure that the bacteria are completely dispersed. It is diluted again with 1/500 NB to adjust the bacterial concentration.
Bacteria concentration should between 2.5x105 and 10x105 kob/ml. This prepared suspension is used in the test inoculum.
3.1.2. Preparation of Samples
6 untreated and 3 treated test specimens are used. Treated or untreated test specimens cut 50x50 mm ±2 straight. Samples should not be thicker than 10 mm. The surface area should be between 400 mm2 and 1600 mm2.
40×40 mm sterile film is cut for each sample. While the samples are being prepared, contamination of microorganisms and organic waste should be avoided. Surface cleaning is done using 70% ethanol.
3.1.3. Inoculate of Samples
3 treated and 6 untreated samples are placed in sterile petri dishes separately. The surface to be tested must be on the upper side. 0.4 ml of the prepared test inoculum is added to each sample with a pipette. 40mmx40mm sterile film is placed on the inoculum and pressed gently.
After the sample is covered with film, the petri dish is closed. 3 treated and 3 untreated samples are placed in the incubator (These samples are considered to be 24 hours of
contact). Incubate at 35±1ºC for 24±1 hours. The relative humidity in the incubator should not be less than 90%.
The other 3 untreated samples are considered as 0 hour contact time. After inoculation immediately washing is performed. After the sample is covered with sterile film, there should be no air bubbles or air on the inoculum.
3.1.4. Recovery of Bacteria from the Sample Immediately after inoculation
Immediately after the inoculum, 10 ml of SCDLP solution (Soyabean casein solution containing lecithin and polyoxyethylene sorbitan monooleate) is added directly onto the remaining 3 untreated samples and mixed gently by closing the petri dish. It is ensured that the sample is completely wetted with the neutralization solution.
With the help of a pipette, the solution is taken from the petri dish at least 4 times and pipetted onto the sample again. If less than 10 ml is required depending on the shape and size of the sample, the volume used is recorded at the report.
3.1.5. The Method of Finding Bacterial Counts with the Cast Plate Method
After washing, 1 ml of the suspension is taken and into 9 ml of phosphate buffer (1/10 dilution) with pipette. 1 ml of this prepared solution is taken again and pipetted
into 9 ml of phosphate buffer. In this way, a 1/100 diluted solution is prepared.
1 ml of each dilution is placed in 2 separate petri dishes. 15-20 ml of bacterial suspension
from PCA (Plate count agar) cooled to 46-48ºC is poured over. Mix it gently and let it cool at room temperature. After the agar solidifies, it is incubated at 35±1°C for 40-48
hours.
After incubation, bacteria grown in serially diluted petri dishes are counted. Between 30 and 300 colonies are observed. If the number of growing colonies is <30, 1 ml more of this solution is planted on agar. If there is no growth on the agar, it is reported as <1.
3.1.6. Finding the Bacteria Count
The number of bacteria that reproduce for each sample is calculated using the below formula.
N= (100×C×D×V) / A
N : Number of bacteria
C : Average number of colonies in a double petri dish
D : Dilution factor
V : Volume of SCDLP solution used (ml)
A : Surface area of the film (mm²)
If no bacterial colonies have grown on the agar, write <V
as up the volume of SCDLP solution added to the sample.
If V= 10ml SCDLP, the result is <10.
3.1.7. Checking the Validity of the Test
In order for the test to be valid, the following 3 conditions must be met.
a) (Lmax - Lmin) / (Lmean) ≤ 0,2 After inoculation, the log values of the bacterial counts in the untreated sample are taken.
b) The average number of bacteria in the untreated sample immediately after inoculation should be between 6,2x103 cells/cm2 and 2,5x104 cells/cm2.
c) The number of bacteria obtained after 24 hours of incubation of untreated samples should not be less than 6,2x101 cells/cm2 [4].
Lmax: Logarithm of the maximum number of cfu on the parallels of the untreated sample
Lmin: Logarithm of the minimum number of cfu on the parallels of the untreated sample
Lmean: Logarithm of the mean number of cfu of untreated sample parallels
3.1.8. Calculation of Antibacterial Activity
R= (Ut – U0) – (At – U0) = Ut – At
R: Antibacterial activity
U0: Logarithmic value of the untreated sample immediately after inoculation
Ut: Logarithmic value of the untreated sample immediately after 24 hours of incubation
At: Logarithmic value of the treated sample after 24 hours of incubation
Results to be considered effective R value should be ≥ 2 for [5].
3.2. Physical and Spectroscopic of Characterizations of Erasers
3.2.1. Mechanical Properties
Density, hardness, tensile strength at break, elongation at break, and modulus values at 100% of the test samples with and without antibacterial agent were examined. Density value is made according to ASTM D792, hardness value is ASTM D2240, other mechanical values are made according to ASTM D412 standards.
3.2.2. Chemical Analysis with Spectroscopic Methods
3.2.2.1. Determination of Polyaromatic Hydrocarbones (PAH)
20 ml of Toluene and internal standard mixture is added on 500 mg eraser sample and extracted in an ultrasonic bath at 600C for 1 hour. After 1 hour, the solution cooled to room temperature was taken into a 1 ml vial and the determination of Polyaromatic Hydrocarbons was made by AfPS GS 2019:01 PAH method in GC-MS device [6].
3.2.2.2. Determination of Phthalates
5 ml of Tetrahydrofuran (THF) solution was added to 50 mg of the eraser sample and left in a shaker mixer for 30 minutes to dissolve. After 30 minutes, 10 ml of Acetonitrile or Hexane was added to each 5 ml of THF and shaken for 5 minutes. After the shaking was finished, the solution was filtered through a 45 μm membrane filter and taken into a vial and the phthalates were determined by the CPSC-CH-C1001-09.4 method in the GC-MS device [7].
3.2.2.3. Determination of Cadmium
0.5 gram eraser sample was weighed into the Kijeldal tube. The tube was placed on the heater and 10 ml of sulfuric acid was added into it. Heated to high temperature to remove organic compounds, after the formation of white smoke, heating was continued for another 15 minutes and then the tube was removed from the heater and cooled for 10 minutes.
With the help of a funnel, 5 ml of hydrogen peroxide solution was slowly added into
the tube and heated to high temperature, then cooled to room temperature and again 5 ml of hydrogen peroxide was added and heated to high temperature again.
This hydrogen peroxide addition was done 4 times with 5 ml added. The solution cooled to room temperature was taken into a 100 ml flask and completed with distilled water. 1 ml sample from the solution was taken into a vial with a membrane filter, and Cadmium was determined in the ICP-MS device with the BS EN 1122:2001 method [8].
3.2.2.4. Determination of Lead
150 mg of the eraser sample was weighed into the microwave tube. 5 ml of concentrated nitric acid was added to the tube at room temperature. The tube was closed and kept in the microwave device at 2000C for 20 minutes.
The sample was allowed to cool for 5 minutes before being removed from the microwave. The microwave container was opened under the fume hood and the solution in it was taken into a 50 ml balloon jug and made up to 50 ml with distilled water. By taking 1 ml vial from the solution completed to 50 ml and Lead was determined by CPSC-CH-E1002 method in ICP-MS device [9].
4. Results
As a result of the studies, the Antibacterial Activity (R) value against E.Coli and S.Aureus microorganisms of the eraser sample tested with the ISO 22196:2011 standard was found to be greater than 2, and the Antibacterial Activity value in percentage was 99.99%.
Antibacterial activities of our product have been proven against infectious bacteria that are harmful to humans, such as E. Coli and S. Aureus. The following results were obtained in the antibacterial test.
Control of standard characteristics such as mechanical property is important in the materials industry to ensure product quality. Therefore, an additive agent should allow
the performance of the polymeric composition to improve without compromising its original character. [5]. The mechanical test results of the standard eraser without antibacterial agent and the eraser with antibacterial agent were found in the table below.
Table 3 shows the mechanical test results of standard eraser samples with and without antibacterial agent additives. According to the data, it was observed that the density values of both samples are the same.
While the tensile strength at break and the elastic modulus at 100% were very close to each other, it was observed that the antibacterial agent increased the hardness of the finished product by around 4 units. With the increase in hardness, a decrease in the elongation value of the material containing antibacterial agent was observed.
This increase in the hardness value and the decrease in the elongation at break are within the desired limit values of the standard eraser sample. As a result of the spectroscopic analysis of the eraser sample; phthalate species were checked with GC-MS device and none of the following (Table 4) phthalate species were detected.
According to the Turkish KKDIK (Registration, Evaluation, Authorization and Restriction
of Chemicals) regulation, it is forbidden to use these phthalates in plastic materials at concentrations higher than 0.1% by weight. [10]. As a result of the analysis of our eraser, it has been seen that it is in accordance with the values of the relevant regulation.
PAH species of the eraser sample were checked with GC-MS device and none of the following (Table 5) PAH species were detected. According to the Turkish KKDIK regulation, some of the following PAH types(*) cannot be found in plastic or rubber materials that may come into contact with human skin in amounts greater than 0.5 mg/kg. [10].
As a result of the analysis of our eraser, it has been seen that it is in accordance with the values of the relevant regulation. Lead and Cadmium content of the eraser sample was checked with the ICP-MS device, and Lead (Table 6) and Cadmium (Table 7) compounds were not detected.
If the lead concentration is equal to or higher than 0.05% by weight in the articles and in the normal and anticipated uses of the article parts, it is not used in articles that are
offered to the public and that children can put in their mouths and cannot be placed on the market.
It is stated that mixtures and articles produced from plastic materials cannot be placed on the market if they contain cadmium in a concentration equal to or greater than 0.01% by weight of the plastic material. [10]. As a result of the analyzes of our eraser, it has been seen that the Lead and Cadmium values are in accordance with the limit values of the relevant regulation.
5. Conclusion and Comments
In this study, we have progressed in our eraser production with a thermoplastic elastomer-containing polymer and Avient antimicrobial solution. Antimicrobial actives act within the polymer matrix and spread over the surface of the materials, providing a protective shield with antimicrobial activity.
Organic antimicrobials are effective at low concentrations and offer a ideal cost/performance ratio in polymers processed at temperatures not exceeding 250°C. Antibacterial effect tests are given in the tables above (Tables 1 and 2). Escherichia coli and
Staphylococcus aureus bacteria decreased by 5.63 and 4.35 log, respectively, after 24 hours in tested samples according to ISO 22196:2011 standards.
This shows that the antibacterial activity R is greater than 2 and is 99.99 percent. These erasers, which show high antibacterial activity, have become a product that consumers can use safely for a long time. According to the study, the hardness value of the eraser, which was 61 Shore A, increased to 65 Shore A with the addition of antibacterial agent.
This value, which is within the standards, is suitable for use. As a result of all the antibacterial, physical and chemical tests, it has been seen that our eraser complies with
the limit values given in the studied standards and regulations, and in addition to these, it has an antibacterial effect value.
References
[1] Oz, Elif. Foto-“Click” Tepkimesiyle Hazırlanan Gümüş Nanopartikül İçeren Termoset Polimerlerin Endüstriyel Uygulamaları. Yüksek Lisans Tezi, Yalova Üniversitesi, 2016.
[2] US 10,314,312 B2, Vineet Sharma, Ashutosh Agarwal, ‘Synergistic antimicrobial composition of zinc pyrithione’, 2018-10-31, assigned to Jubilant Life Sciences Ltd
[3] US 2012/0276040 A1, Vincent T. Chuang, ‘Antimicrobial compositions and methods of making same’, 2012-11-01
[4] ISO 22196:2011 Measurement Of Antibacterial Activity On Plastics And Other Non-Porous Surfaces
[5] Michele Pittola *, Daiane Tomacheskia,b, Douglas Naue Simõesa,b, Vanda Ferreira Ribeiroa,b, Ruth Marlene Campomanes Santanab
(2017) ‘Antimicrobial Performance of Thermoplastic Elastomers Containing Zinc Pyrithione and Silver Nanoparticles.
[6] Product Safety Commission (AfPS) GS Specification Testing and assessment of Polycyclic Aromatic Hydrocarbons (PAHs) AfPS GS 2019:01 PAK
[7] CPSC-CH-C1001-09.4 Standard Operating Procedure for Determination of Phthalates January 17, 2018.
[8] BS EN 1122:2001 Plastics - Determination of Cadmium - Wet Decomposition Method
[9] Test Method: CPSC-CH-E1002-08.3 Standard Operating Procedure for Determining Total Lead (Pb) in Nonmetal Children’s Products.
[10] Çevre ve Şehircilik Bakanlığının 30105 Sayılı Kimyasalların Kaydı, Değerlendirilmesi, İzni ve Kısıtlanması Hakkında Yönetmelik
Elif Öz Çetin
R&D Specialist
MSc. Chemical Engineer
Adel Kalemcilik Ticaret ve Sanayi A.Ş.
Dr. Fatma Seda Güreli Feridun
Product Safety and Regulations Specialist
PhD Chemist
Adel Kalemcilik Ticaret ve Sanayi A.Ş.
