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Plant-based natural saponins for Escherichia coli surface hygiene management
LWT - Food Science and Technology 122 (2020) 109018
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Plant-based natural saponins for Escherichia coli surface hygiene management
T
Rok Fink∗, Anja Potočnik, Martina Oder University of Ljubljana, Faculty of Health Sciences, Zdravstvena Pot 5, 1000, Ljubljana, Slovenia
A R T I C LE I N FO
A B S T R A C T
Keywords: Natural plant extracts Standard saponins E. coli Surface hygiene management Surface tension Emulsification index
Cleaning the surfaces enables reducing microbial populations and consequently the risk of food contamination. The aim of this research was to study the potential of natural extracts from soap nuts, quillaja bark, and horse chestnuts for the reduction of Escherichia coli on glass surfaces. Analogous to that, we tested the efficacy of standard saponins as the active components in the plants mentioned above. The results show that the numbers of bacteria cells are decreased by increasing concentrations of all cleaning product, except for quillaja standard saponin (p < 0.05). Furthermore, the findings indicate that natural plant extracts have greater efficacy than standard saponins do. In particular, quillaja bark extract shows the potential to reduce bacterial populations by up to 66%. Moreover, the research also demonstrates that natural plant extracts have excellent abilities to reduce water surface tension (22 mN/m for soap nut extract) and emulsification potential (74 for quillaja bark extract). Natural plant extracts are inexpensive, biodegradable, and residues are less toxic than conventional ones. The evidence of this study suggests that natural plant extracts are efficient against attached E. coli cells on the glass surface and are a good candidate for surface hygiene management.
1. Introduction Interactions between microorganisms and contact surfaces play an important role in food technology, catering as well as kitchen environments; in all these contexts, we attempt to keep the microbial population at the lowest level possible. Food contact materials can be contaminated with bacteria and are therefore great sources for transmission of foodborne pathogens (Fink et al., 2017). Glass is the most inert of common substances and has been used satisfactorily as food contact material for many years, the glass matrix being amongst the hygienic and highest quality materials available for the packaging of drinks and foodstuffs, e.g. bowls, jars, bottles and cutting boards (Bohinc et al., 2014). Cleaning is an operation through which soil on the surface is eliminated to prevent bacterial accumulation and growth (Giaouris & Simões, 2018). Thus, in order to control the microbial population on the surface, different approaches are used. The chemical method is the most predominant action; it works by separating the contaminants from the surface and by placing them in a solution or dispersion, so-called detergency. This is done by surface-active substances, also known as surfactants. Surfactants lower the surface tension, acts as detergents, wetting agent emulsifiers, foaming agents, and dispersants (Fink, 2015; González-Rivas et al., 2018). A specific group of surfactants are ∗
saponins, whose amphipathic nature enables them to act as surfactants with the potential to interact with bacterial cell membrane components, such as cholesterol and phospholipids, possibly making saponins useful for the development of cosmetics, drugs, and cleaning products (Sharma, Mulligan, & Mudhoo, 2014). The use of many cleaning products in food processing makes it possible to control microbial populations, but this approach has a negative impact on the global environment. Moreover, the problem of antimicrobial resistance and cross-resistance is not limited solely to the clinical environment; with the widespread use of cleaning products in food processing the problem has shifted into the industrial environment (Aiello & Larson, 2003). Research on industrial wastewater has shown that many chemicals are deposited in the environment (Wieck, Olsson, & Kümmerer, 2018). The growing global population and improving economies in many countries increase the global consumption of cleaning products and thereby the pressure on the environment; it is well recognized that there is an urgent need to reduce the impact per produced unit of cleaning product to sustain human needs without compromising natural resources (Elorriaga, Marino, Carriquiriborde, & Ronco, 2013). Great potential is found in natural cleaning products based on biopolymers, plant extracts, natural surfactants, and natural acids that have a broad spectrum of activity, high efficiency, and low bacterial resistance (Bassanetti et al., 2017; Bernal, Guzman, Illanes, &
Corresponding author. E-mail address: rok.fi[email protected] (R. Fink).
https://doi.org/10.1016/j.lwt.2020.109018 Received 12 September 2019; Received in revised form 20 December 2019; Accepted 2 January 2020 Available online 06 January 2020 0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
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Fig. 1. Cleaning products efficacy assessment flowchart.
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prepared in six different concentrations (10%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001%).
Wilson, 2018). Cui, Yuan, Li, and Lin (2017) demonstrated that edible film incorporated with chitosan and Artemisia annua oil inactivate E. coli O175:H7. Fink, Kulaš, and Oder (2018) tested standard sodium dodecyl sulphate and extracts from Mediterranean plants and reported higher efficacy for natural compounds in comparison to conventional ones. Cui, Ma, and Lin (2016) tested Co-loaded proteinase K/thyme oil liposomes for inactivation of E. coli O157:H7 biofilms and found 2.4 log reduction. In another study Cui, Bai, Marwan, Rashed & Lin (2018) showed antibacterial activity of clove oil/chitosan nanoparticles against E. coli O175:H7. Natural saponins can be found in soybeans, quillaja bark, asparagus, bean, peas, sugar beets, and other plants. For example, the plant active substance saponin escin can be found in horse chestnuts (Aesculus hippocastanum), quillaja saponin in quillaja bark (Quillaja saponaria) and sapogenin saponin in soap nuts (Sapindus saponaria) (Sharma et al., 2014). A considerable amount of studies has analysed the antimicrobial potentials of saponins (Fleck et al., 2019; Kose & Bayraktar, 2016; Montenegro, Salas, Pena, & Pizarro, 2009; Zhang, Li, & Lian, 2010). However, far too little attention has been paid to the reduction of bacterial cells on the surfaces, because any remains of dead cells (e.g., antimicrobial-resistant genes) on the surface can act as a source of cross-resistance (Verraes et al., 2013). The great advantage of natural surfactants is, therefore, their antibacterial activity, non-toxic residues and low production costs (Nitschke & Silva, 2018). Accordingly, the aim of this study was to examine the potential of natural plant extracts from quillaja bark, horse chestnuts, and soap nuts for application in surface hygiene management.
2.4. Efficacy of cleaning products ingredients for E. coli detaching At first step E. coli cells adhered on the glass surface (2.4.1) and after that cells were exposed to cleaning products (2.4.2) (Fig. 1). 2.4.1. Bacterial adhesion to glass surface A single colony of E coli was transferred from nutrient agar to the nutrient broth (Biolife, Italy) and incubated for 24 h at 37 °C. Next, overnight culture of the E. coli was diluted in a 1:300 ratio, with a fresh nutrient broth. Clean and sterile glass coupons (1 cm × 1 cm) (Isolab, Germany) were transferred to the sterile petri dish, and a prepared suspension of E. coli bacteria was added. The coupons were incubated for 1 h at 37 °C to achieve irreversible adhesion. After the incubation period, the medium was removed and washed with 3 mL of phosphate buffer saline (PBS). The attached bacterial cells on the coupons were stained with a 2% solution of crystal violet (Merck, Germany) for 5 min. The number of attached bacterial cells were counted using a light microscope Olympus CX40 and CCD CMOS camera. In this way, we determined the number of cells attached to the surface before the cleaning products were applied (Fig. 1). 2.4.2. Cleaning products exposure Coupons with attached E coli were exposed to the 3 mL of each cleaning product and each concentration for 10 min. After that, the cleaning product was removed, and the coupons were washed with the 3 mL PBS buffer. The rest of the attached bacterial cells were stained with a 2% crystal violet solution for 5 min and counted using an Olympus CX40 and CCD CMOS light microscopy camera (Fig. 1). The efficacy (%) of cleaning products was determined as the difference in the number of bacteria before and after exposure to cleaning products, calculated as follows:
2. Methods 2.1. Bacterial strain In this study, a standard strain of E. coli ATCC 35218, obtained from the Czech Collection of Microorganisms (Brno, Czech Republic) was used. E. coli is an important opportunistic pathogen and is often used in studies as a model organism and indicator of faecal contamination (Belluco, Barco, Roccato, & Ricci, 2016; Chien, Sheen, Sommers, & Sheen, 2017; Fink et al., 2018).
Efficacy of cleaning products no. of bacteria after exposure × 100 ⎞⎞ = ⎜⎛100 − ⎜⎛ ⎟⎟ (%) ⎝ no. of bacteria before exposure ⎠⎠ ⎝
2.2. Cleaning products
(1)
The experiments were obtained for each concentration of each cleaning products in three parallels and three repetitions.
The study analysed the efficacy of various natural extracts from plants, e.g., soap nuts (Greenwill, Greer, South Carolina, USA), horse chestnuts (Favn, Grosuplje, Slovenia) and quillaja bark (Sena, Santiago de Chile, Chile). Due to the chemical complexity of natural extracts, standard saponins were also tested. We tested sapogenin, quillaja saponin, and escin (Sigma-Aldrich, USA) to compare efficacy natural plant extracts analogous to standard active components. This approach allows testing the efficacy of natural extracts without any fractionations of natural extracts. As reported by Cos, Vlietinck, Berghe, and Maes (2006) any fractionation of natural extracts can lead to the reduction or loss of biological activity by compound break-down or loss of additive or synergistic effects between different extracts components.
2.5. Determination of cleaning product emulsification index (E-24 index) The cleaning product emulsification index was determined following a standard protocol (Lawrance et al., 2014). The E-24 index was determined by mixing 2 mL of each cleaning product at each concentration with 2 mL of paraffin liquid (Sigma-Aldrich, USA) in the different test tubes. The tubes with solutions were vigorously mixed in a vortex for 2 min. The resulting mixture was allowed to stand for 24 h to separate into an emulsified layer (hydrophobic phase) and the remaining aqueous layer (hydrophilic phase). The emulsification index was measured as the ratio of the height of the thickness of the emulsified layer to the total height of the solution, as shown in the equation:
2.3. Preparation of natural extracts
Emulsification Index (E − 24) =
Plant extracts were prepared using the method of (Pradhan & Bhattacharyya, 2017) and modified as follows. First horse chestnuts peel was removed, soap nuts were halved to remove seeds, and quillaja bark was cut in small pieces. In next step plants were crushed in a mortar and dried at room temperature (20 ± 2 °C) for 24 h. 10 g of each plant sample was added in volumetric flask and filled up with distilled water to the final 100 mL (1:10). In the next step, maceration was carried out for 24 h at 21 °C on a continuous magnetic stirrer at 200 rpm. The experiments were triplicated. The solution was then filtered through a cellulose acetate membrane filter with 84 g m−2 density (Sigma-Aldrich, USA). The extracts of all three plants were
Height of emulsified layer X 100 (/) Total height of the solution (2)
The E-24 of all selected cleaning products was measured for all six concentrations (10%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001%) and three repetitions. 2.6. Determination of the cleaning product surface tension To determine the surface tension of the selected cleaning products, a tensiometric method was used according to the (Basu, Basu, 3
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significance was found (Table 1). Surprisingly, comparing the efficacy of E. coli reduction between standard saponins and natural plant extracts show a substantial increase in efficacy at a 10% solution for both cleaning product groups. Therefore, we assume increasing the concentration > 10% would result in a higher bacterial reduction. We found that natural plant extracts are more effective against E. coli on glass surfaces in comparison to standard saponins. Results show statistically significant differences between cleaning products at all concentrations (p < 0.05). We assume that natural plant extracts contain mixtures of not only saponins, but also natural acids, phenols, tannins, flavonoids, and other compounds with antibacterial properties (Arabski, Węgierek-Ciuk, Czerwonka, Lankoff, & Kaca, 2012; Fink et al., 2018). An additional advantage of natural saponins in comparison to synthetic ones is the ability to pass through the cell wall more efficiently, causing disruption of the metabolism, and consequently cell detachment (Khan et al., 2018; Kralova & Sjöblom, 2009). For example, at 10% solution quillaja bark extract reduced the number of bacteria from 3.7 log no. bact./mm2 to 3.1 log no. bact./mm2, soap nut extract to 3.2 log no. bact./mm2, and horse chestnut extract to 3.3 log no. bact./mm2, respectively (p < 0.001). In contrast to that, standard sapogenin and escin reduced the number to 3.4 log no. bact./mm2 and quillaja saponin only to 3.5 log no. bact./mm2 indicating the natural plants are more efficient for E. coli reduction that standard saponins (Fig. 2, Table 1). In a comparable study, also Khan et al. (2018) found that bacterial reduction is concentration-dependent and that a 12% solution of saponins from green tea seeds is effective against E. coli cells. Furthermore, Kose and Bayraktar (2016) found that a 10% solution of soap nut extract has antimicrobial effects on E. coli. Among our tested natural plant extracts, the quillaja bark extract is the most effective (66%), followed by soap nut extract (62%), and horse chestnut extract (41%). The evidence from this study suggests that quillaja bark extract represent good candidate for cleaning product in food industry, but
Bandyopadhyay, & Chowdhury, 2015). The surface tension was measured using a Leconde Du Nouy Tensiometer, with which the tension required to lift the platinum ring out of the surface of the water was measured
Surface tension (σ ) =
Fmax (mN / m) (L x cos(θ))
(3)
where Fmax is the force exerted on the platinum ring, L is the characteristic length of the ring, and θ is the contact angle between the platinum ring and solution. The surface tension of all selected cleaning products was measured for all six concentrations (10%, 1%, 0.1%, 0.01%, 0.001% and 0.0001%) and three repetitions. 2.7. Statistical analysis Statistical analysis was run on R software version 3.1.3 (Bell Laboratories, New Jersey, U.S.). Normality was checked using the Shapiro-Wilk test (p > 0.05). One-way analysis of variance (ANOVA) and the Duncan statistical test were used to determine the significant differences at a significance level of p < 0.05. 3. Results and discussion The study assessed and compared the efficacy of saponin standards and plant derived saponins on reducing E. coli. The results show that before exposure to standard saponins and natural plant extracts the number of bacteria on the glass coupons was 3.7 ± 0.11 log no. bact./ mm2. The results for both standard saponins and natural extracts show that increasing the cleaning product concentrations results in a decreasing number of bacteria for all cleaning products (p < 0.05), except for standard quillaja saponin where no statistically significant
Table 1 Comparison of the number of bacteria, surface tension and emulsification index due to the type of cleaning product and concentration.
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Fig. 2. E. coli adhesion (log no. bact./mm2) on glass coupons after exposure to different concentrations of standard saponins and natural plant extracts.
Cartwright, and Bailey (2010) showed the antibacterial activity of a commercial extract rich in quillaja extract against S. Typhimurium, S. aureus, and E. coli. Moreover, the authors demonstrated that the pretreatment with quillaja bark extract increased the sensitivity of the bacterial cultures to peracetic acid, showing the potential of the hurdle approach. Another great advantage of quillaja extract is its low toxicity, as it is often used in the food industry as a humectant in baked goods, frozen dairy products, and puddings and as a foaming agent in soft drinks (Younes et al., 2019). Therefore, applying quillaja based cleaning products in the food industry would minimalize problems with cleaning residues on food contact materials assuring the safety of
further studies for implementation are needed. The study carried out by Berlowska, Dudkiewicz, Kregiel, Czyzowska, and Witonska (2015) on yeast cells showed that soap nut extract increased cell membrane permeability in different strains, lysis, and consequently cell detachment. Quillaja bark extract in our research shows some potential for reducing the number of cells on the glass surface. It is assumed that saponin interacts with lipopolysaccharide and thereby increases the permeability of the bacterial cell wall (Arabski et al., 2012). Similar to our study, Sewlikar and D'Souza (2017) tested quillaja bark extract on E. coli O157: H7 and found a reduction of cells from 7.5 CFU to 6.8 CFU, which is comparable to our results with E. coli reduction. Hassan, Byrd, 5
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Fig. 3. Emulsification index (E-24) of standard saponins and natural plant extracts vs concentration.
ones, which was also found in our study. The authors also report that foam from the soap nut is much more stable than an isolated one. Another study revealed a natural extract from soap nuts has a higher emulsification index than synthetic sodium dodecyl sulphate and Tween 80 do (Tmáková, Sekretár, & Schmidt, 2016). Moreover, a recent study demonstrated that natural saponins of C. enchinulata can even exceed an E-24 value of 80 (Andrade et al., 2018), which shows that natural extracts should be given more attention. A good surfactant can decrease the surface tension of water from 72 mN/m to 35 mN/m (Pradhan & Bhattacharyya, 2017). The results of surface tension show that by increasing the concentration for all tested
consumers. Emulsification ability is an essential property of good cleaning products. The amphiphilic nature helps surfactants to solubilise waterinsoluble substances, e.g., hydrocarbons. Results of the emulsification index demonstrate that increasing the concentration for all tested cleaning products increases E-24 (p < 0.001) (Table 1). Fig. 3 shows the highest value for a natural extract from quillaja bark up to 73, followed by standard quillaja saponin (70), horse chestnut extract (65), sapogenin (64), escin (60), and soap nut extract (58). The study by Pradhan and Bhattacharyya (2017) showed that natural extracts from soap nuts possess higher values of E-24 when compared to standard 6
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Fig. 4. Surface tension (mN/m) of standard saponins and natural plant extracts vs. concentration.
solution of standard saponins and natural plant extracts. The results show the quillaja bark extracts remove up to 66% of bacteria relative to the control, followed by sapogenin at 65%, escin at 51%, quillaja saponin at 45%, and horse chestnut extract at 41%, respectively (Fig. 5). In accordance with the present results, we have demonstrated that quaillaja bark extract can reduce the bacterial population on the surface, efficiently emulsify fats and decrease the water surface tension. The scope of this study was limited in terms of testing different mixture of natural plant extracts against E. coli, concentrations of active components in natural extracts, analysing efficacy on different microorganisms and variety of food contact materials.
cleaning products, surface tension decreases (p < 0.001) (Table 1). The most substantial decrease of surface tension can be observed at the highest concentration (10%) for soap nut extract (22 mN/m), quillaja bark extract, escin and sapogenin (23 mN/m) and the least for the horse chestnut extract and standard quillaja saponin (25 mN/m) (Fig. 4). This indicates that natural plant extracts have cleaning potential and that further research on this field is needed. Previous studies have shown a reduction of water surface tension by soap nuts only to 47 mN/m (Tmáková et al., 2016) and 35 mN/m (Pradhan & Bhattacharyya, 2017), which is less than the present study demonstrated. Images from the light microscope confirm the efficacy of 10% 7
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Fig. 5. Microscope image of E. coli cells before and after exposure to 10% solution of standard saponins and natural plant extracts.
4. Conclusions
emulsification index. All this indicates that quaillaja bark extract represents good candidate for future research. More research is needed to better understand effects of different concentrations and active components on variety of food pathogens, optimising plant extract procedure, mixture of different plant extracts and application of natural cleaning product in food industry.
Management of the bacterial population in the food industry has to be focused on searching for cleaning products with appropriate efficacy that leave low bacterial cells residues on the surface, but also take into the account environmental issues. This study has found that increasing the concentration of all tested extracts and standard compounds results in decreased number of bacteria, decreased water surface tension and also increased emulsification index. Moreover, our study demonstrated that among tested cleaning products, natural plant extract from quillaja bark is the most efficient by decreasing number of E. coli cells on glass surface up to 66%, followed by sapogenin (65%) and soap nut extract (62%). Furthermore, findings also show that quaillaja bark extract substantially decreases the water surface tension and increases the
Declaration of competing interest None. Acknowledgements The research was supported by the Slovenian Research Agency 8
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under Slovenia-United States of America bilateral project “Natural cleaning agents potential for biofilm control in food industry BI-US/1819-075”. The authors would like to express their gratitude to Terry Troy Jackson for grammar proof.
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References Aiello, A. E., & Larson, E. (2003). Antibacterial cleaning and hygiene products as an emerging risk factor for antibiotic resistance in the community. The Lancet Infectious Diseases, 3(8), 501–506. https://doi:10.1016/S1473-3099(03)00723-0. Andrade, R. F., Silva, T. A., Ribeaux, D. R., Rodriguez, D. M., Souza, A. F. S., Lima, M. A. B., et al. (2018). Promising biosurfactant produced by Cunninghamella echinulata UCP 1299 using renewable resources and its application in cotton fabric cleaning process. Advances in Materials Science and Engineeringe1624573. https://doi.org/10.1155/ 2018/1624573. Arabski, M., Węgierek-Ciuk, A., Czerwonka, G., Lankoff, A., & Kaca, W. (2012). Effects of saponins against clinical E. coli strains and eukaryotic cell line. BioMed Research International, 2012, e286216. https://doi:10.1155/2012/286216. Bassanetti, I., Carcelli, M., Buschini, A., Montalbano, S., Leonardi, G., Pelagatti, P., et al. (2017). Investigation of antibacterial activity of new classes of essential oils derivatives. Food Control, 73, 606–612. https://doi.org/10.1016/j.foodcont.2016.09.010. Basu, A., Basu, S., Bandyopadhyay, S., & Chowdhury, R. (2015). Optimization of evaporative extraction of natural emulsifier cum surfactant from Sapindus mukorossi—characterization and cost analysis. Industrial Crops and Products, 77, 920–931. https://doi:10.1016/j.indcrop.2015.10.006. Belluco, S., Barco, L., Roccato, A., & Ricci, A. (2016). Escherichia coli and enterobacteriaceae counts on poultry carcasses along the slaughterline: A systematic review and meta-analysis. Food Control, 60, 269–280. https://doi.org/10.1016/j. foodcont.2015.07.033. Berlowska, J., Dudkiewicz, M., Kregiel, D., Czyzowska, A., & Witonska, I. (2015). Cell lysis induced by membrane-damaging detergent saponins from Quillaja saponaria. Enzyme and Microbial Technology, 75, 44–48. http://doi:10.1016/j.enzmictec.2015. 04.007. Bernal, C., Guzman, F., Illanes, A., & Wilson, L. (2018). Selective and eco-friendly synthesis of lipoaminoacid-based surfactants for food, using immobilized lipase and protease biocatalysts. Food Chemistry, 239, 189–195. http://doi:10.1016/j.foodchem. 2017.06.105. Bohinc, K., Dražić, G., Fink, R., Oder, M., Jevšnik, M., Nipič, D., et al. (2014). Available surface dictates microbial adhesion capacity. International Journal of Adhesion and Adhesives, 50, 265–272. https://doi.org/10.1016/j.ijadhadh.2014.01.027. Chien, S. Y., Sheen, S., Sommers, C., & Sheen, L. Y. (2017). Modeling the inactivation of Escherichia coli O157:H7 and Uropathogenic E. coli in ground beef by high pressure processing and citral. Food Control, 73, 672–680. https://doi.org/10.1016/j.foodcont. 2016.09.017. Cos, P., Vlietinck, A. J., Berghe, D. V., & Maes, L. (2006). Anti-infective potential of natural products: How to develop a stronger in vitro ‘proof-of-concept’. Journal of Ethnopharmacology, 106(3), 290–302. https://doi.org/10.1016/j.jep.2006.04.003. Cui, H., Bai, M., Rashed, M. A. A., & Lin, L. (2018). The antibacterial activity of clove oil/ chitosan nanoparticles embedded gelatin nanofibers against Escherichia coli O157:H7 biofilms on cucumber. International Journal of Food Microbiology, 266, 69–78. http:// doi:10.1016/j.ijfoodmicro.2017.11.019. Cui, H., Ma, C., & Lin, L. (2016). Co-loaded proteinase K/thyme oil liposomes for inactivation of Escherichia coli O157:H7 biofilms on cucumber. Food & Function, 7, 4030–4040. https://doi.org/10.1039/c6fo01201ahttps://search.crossref.org/?q= Co-loaded+proteinase+K%2Fthyme+oil+liposomes+for+inactivation+of +Escherichia+coli+O157%3AH7+biofilms+on+cucumber. Cui, H., Yuan, L., Li, W., & Lin, L. (2017). Edible films incorporated with chitosan and Artemisia annua oil nanoliposomes for inactivation of E. coli O157:H7 on cherry tomatoes. International Journal of Food Science and Technology, 52, 687–698. https:// doi.org/10.1111/ijfs.13322https://search.crossref.org/?q=Edible+films +incorporated+with+chitosan+and+Artemisia+annua+oil+nanoliposomes +for+inactivation+of+E.+coli+O157%3AH7+on+cherry+tomatoes. Elorriaga, Y., Marino, D. J., Carriquiriborde, P., & Ronco, A. E. (2013). Human pharmaceuticals in wastewaters from urbanized areas of Argentina. Bulletin of Environmental Contamination and Toxicology, 90(4), 397–400. http://doi:0.1007/ s00128-012-0919-x. Fink, R. (2015). Higienically relevant biofilms (1st ed.). New York: Nova Science Publishers (Chapter 9). Fink, R., Kulaš, S., & Oder, M. (2018). Efficacy of sodium dodecyl sulphate and natural extracts against E. coli biofilm. International Journal of Environmental Health Research,
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Plant-based natural saponins for Escherichia coli surface hygiene management
T
Rok Fink∗, Anja Potočnik, Martina Oder University of Ljubljana, Faculty of Health Sciences, Zdravstvena Pot 5, 1000, Ljubljana, Slovenia
A R T I C LE I N FO
A B S T R A C T
Keywords: Natural plant extracts Standard saponins E. coli Surface hygiene management Surface tension Emulsification index
Cleaning the surfaces enables reducing microbial populations and consequently the risk of food contamination. The aim of this research was to study the potential of natural extracts from soap nuts, quillaja bark, and horse chestnuts for the reduction of Escherichia coli on glass surfaces. Analogous to that, we tested the efficacy of standard saponins as the active components in the plants mentioned above. The results show that the numbers of bacteria cells are decreased by increasing concentrations of all cleaning product, except for quillaja standard saponin (p < 0.05). Furthermore, the findings indicate that natural plant extracts have greater efficacy than standard saponins do. In particular, quillaja bark extract shows the potential to reduce bacterial populations by up to 66%. Moreover, the research also demonstrates that natural plant extracts have excellent abilities to reduce water surface tension (22 mN/m for soap nut extract) and emulsification potential (74 for quillaja bark extract). Natural plant extracts are inexpensive, biodegradable, and residues are less toxic than conventional ones. The evidence of this study suggests that natural plant extracts are efficient against attached E. coli cells on the glass surface and are a good candidate for surface hygiene management.
1. Introduction Interactions between microorganisms and contact surfaces play an important role in food technology, catering as well as kitchen environments; in all these contexts, we attempt to keep the microbial population at the lowest level possible. Food contact materials can be contaminated with bacteria and are therefore great sources for transmission of foodborne pathogens (Fink et al., 2017). Glass is the most inert of common substances and has been used satisfactorily as food contact material for many years, the glass matrix being amongst the hygienic and highest quality materials available for the packaging of drinks and foodstuffs, e.g. bowls, jars, bottles and cutting boards (Bohinc et al., 2014). Cleaning is an operation through which soil on the surface is eliminated to prevent bacterial accumulation and growth (Giaouris & Simões, 2018). Thus, in order to control the microbial population on the surface, different approaches are used. The chemical method is the most predominant action; it works by separating the contaminants from the surface and by placing them in a solution or dispersion, so-called detergency. This is done by surface-active substances, also known as surfactants. Surfactants lower the surface tension, acts as detergents, wetting agent emulsifiers, foaming agents, and dispersants (Fink, 2015; González-Rivas et al., 2018). A specific group of surfactants are ∗
saponins, whose amphipathic nature enables them to act as surfactants with the potential to interact with bacterial cell membrane components, such as cholesterol and phospholipids, possibly making saponins useful for the development of cosmetics, drugs, and cleaning products (Sharma, Mulligan, & Mudhoo, 2014). The use of many cleaning products in food processing makes it possible to control microbial populations, but this approach has a negative impact on the global environment. Moreover, the problem of antimicrobial resistance and cross-resistance is not limited solely to the clinical environment; with the widespread use of cleaning products in food processing the problem has shifted into the industrial environment (Aiello & Larson, 2003). Research on industrial wastewater has shown that many chemicals are deposited in the environment (Wieck, Olsson, & Kümmerer, 2018). The growing global population and improving economies in many countries increase the global consumption of cleaning products and thereby the pressure on the environment; it is well recognized that there is an urgent need to reduce the impact per produced unit of cleaning product to sustain human needs without compromising natural resources (Elorriaga, Marino, Carriquiriborde, & Ronco, 2013). Great potential is found in natural cleaning products based on biopolymers, plant extracts, natural surfactants, and natural acids that have a broad spectrum of activity, high efficiency, and low bacterial resistance (Bassanetti et al., 2017; Bernal, Guzman, Illanes, &
Corresponding author. E-mail address: rok.fi[email protected] (R. Fink).
https://doi.org/10.1016/j.lwt.2020.109018 Received 12 September 2019; Received in revised form 20 December 2019; Accepted 2 January 2020 Available online 06 January 2020 0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
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Fig. 1. Cleaning products efficacy assessment flowchart.
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prepared in six different concentrations (10%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001%).
Wilson, 2018). Cui, Yuan, Li, and Lin (2017) demonstrated that edible film incorporated with chitosan and Artemisia annua oil inactivate E. coli O175:H7. Fink, Kulaš, and Oder (2018) tested standard sodium dodecyl sulphate and extracts from Mediterranean plants and reported higher efficacy for natural compounds in comparison to conventional ones. Cui, Ma, and Lin (2016) tested Co-loaded proteinase K/thyme oil liposomes for inactivation of E. coli O157:H7 biofilms and found 2.4 log reduction. In another study Cui, Bai, Marwan, Rashed & Lin (2018) showed antibacterial activity of clove oil/chitosan nanoparticles against E. coli O175:H7. Natural saponins can be found in soybeans, quillaja bark, asparagus, bean, peas, sugar beets, and other plants. For example, the plant active substance saponin escin can be found in horse chestnuts (Aesculus hippocastanum), quillaja saponin in quillaja bark (Quillaja saponaria) and sapogenin saponin in soap nuts (Sapindus saponaria) (Sharma et al., 2014). A considerable amount of studies has analysed the antimicrobial potentials of saponins (Fleck et al., 2019; Kose & Bayraktar, 2016; Montenegro, Salas, Pena, & Pizarro, 2009; Zhang, Li, & Lian, 2010). However, far too little attention has been paid to the reduction of bacterial cells on the surfaces, because any remains of dead cells (e.g., antimicrobial-resistant genes) on the surface can act as a source of cross-resistance (Verraes et al., 2013). The great advantage of natural surfactants is, therefore, their antibacterial activity, non-toxic residues and low production costs (Nitschke & Silva, 2018). Accordingly, the aim of this study was to examine the potential of natural plant extracts from quillaja bark, horse chestnuts, and soap nuts for application in surface hygiene management.
2.4. Efficacy of cleaning products ingredients for E. coli detaching At first step E. coli cells adhered on the glass surface (2.4.1) and after that cells were exposed to cleaning products (2.4.2) (Fig. 1). 2.4.1. Bacterial adhesion to glass surface A single colony of E coli was transferred from nutrient agar to the nutrient broth (Biolife, Italy) and incubated for 24 h at 37 °C. Next, overnight culture of the E. coli was diluted in a 1:300 ratio, with a fresh nutrient broth. Clean and sterile glass coupons (1 cm × 1 cm) (Isolab, Germany) were transferred to the sterile petri dish, and a prepared suspension of E. coli bacteria was added. The coupons were incubated for 1 h at 37 °C to achieve irreversible adhesion. After the incubation period, the medium was removed and washed with 3 mL of phosphate buffer saline (PBS). The attached bacterial cells on the coupons were stained with a 2% solution of crystal violet (Merck, Germany) for 5 min. The number of attached bacterial cells were counted using a light microscope Olympus CX40 and CCD CMOS camera. In this way, we determined the number of cells attached to the surface before the cleaning products were applied (Fig. 1). 2.4.2. Cleaning products exposure Coupons with attached E coli were exposed to the 3 mL of each cleaning product and each concentration for 10 min. After that, the cleaning product was removed, and the coupons were washed with the 3 mL PBS buffer. The rest of the attached bacterial cells were stained with a 2% crystal violet solution for 5 min and counted using an Olympus CX40 and CCD CMOS light microscopy camera (Fig. 1). The efficacy (%) of cleaning products was determined as the difference in the number of bacteria before and after exposure to cleaning products, calculated as follows:
2. Methods 2.1. Bacterial strain In this study, a standard strain of E. coli ATCC 35218, obtained from the Czech Collection of Microorganisms (Brno, Czech Republic) was used. E. coli is an important opportunistic pathogen and is often used in studies as a model organism and indicator of faecal contamination (Belluco, Barco, Roccato, & Ricci, 2016; Chien, Sheen, Sommers, & Sheen, 2017; Fink et al., 2018).
Efficacy of cleaning products no. of bacteria after exposure × 100 ⎞⎞ = ⎜⎛100 − ⎜⎛ ⎟⎟ (%) ⎝ no. of bacteria before exposure ⎠⎠ ⎝
2.2. Cleaning products
(1)
The experiments were obtained for each concentration of each cleaning products in three parallels and three repetitions.
The study analysed the efficacy of various natural extracts from plants, e.g., soap nuts (Greenwill, Greer, South Carolina, USA), horse chestnuts (Favn, Grosuplje, Slovenia) and quillaja bark (Sena, Santiago de Chile, Chile). Due to the chemical complexity of natural extracts, standard saponins were also tested. We tested sapogenin, quillaja saponin, and escin (Sigma-Aldrich, USA) to compare efficacy natural plant extracts analogous to standard active components. This approach allows testing the efficacy of natural extracts without any fractionations of natural extracts. As reported by Cos, Vlietinck, Berghe, and Maes (2006) any fractionation of natural extracts can lead to the reduction or loss of biological activity by compound break-down or loss of additive or synergistic effects between different extracts components.
2.5. Determination of cleaning product emulsification index (E-24 index) The cleaning product emulsification index was determined following a standard protocol (Lawrance et al., 2014). The E-24 index was determined by mixing 2 mL of each cleaning product at each concentration with 2 mL of paraffin liquid (Sigma-Aldrich, USA) in the different test tubes. The tubes with solutions were vigorously mixed in a vortex for 2 min. The resulting mixture was allowed to stand for 24 h to separate into an emulsified layer (hydrophobic phase) and the remaining aqueous layer (hydrophilic phase). The emulsification index was measured as the ratio of the height of the thickness of the emulsified layer to the total height of the solution, as shown in the equation:
2.3. Preparation of natural extracts
Emulsification Index (E − 24) =
Plant extracts were prepared using the method of (Pradhan & Bhattacharyya, 2017) and modified as follows. First horse chestnuts peel was removed, soap nuts were halved to remove seeds, and quillaja bark was cut in small pieces. In next step plants were crushed in a mortar and dried at room temperature (20 ± 2 °C) for 24 h. 10 g of each plant sample was added in volumetric flask and filled up with distilled water to the final 100 mL (1:10). In the next step, maceration was carried out for 24 h at 21 °C on a continuous magnetic stirrer at 200 rpm. The experiments were triplicated. The solution was then filtered through a cellulose acetate membrane filter with 84 g m−2 density (Sigma-Aldrich, USA). The extracts of all three plants were
Height of emulsified layer X 100 (/) Total height of the solution (2)
The E-24 of all selected cleaning products was measured for all six concentrations (10%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001%) and three repetitions. 2.6. Determination of the cleaning product surface tension To determine the surface tension of the selected cleaning products, a tensiometric method was used according to the (Basu, Basu, 3
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significance was found (Table 1). Surprisingly, comparing the efficacy of E. coli reduction between standard saponins and natural plant extracts show a substantial increase in efficacy at a 10% solution for both cleaning product groups. Therefore, we assume increasing the concentration > 10% would result in a higher bacterial reduction. We found that natural plant extracts are more effective against E. coli on glass surfaces in comparison to standard saponins. Results show statistically significant differences between cleaning products at all concentrations (p < 0.05). We assume that natural plant extracts contain mixtures of not only saponins, but also natural acids, phenols, tannins, flavonoids, and other compounds with antibacterial properties (Arabski, Węgierek-Ciuk, Czerwonka, Lankoff, & Kaca, 2012; Fink et al., 2018). An additional advantage of natural saponins in comparison to synthetic ones is the ability to pass through the cell wall more efficiently, causing disruption of the metabolism, and consequently cell detachment (Khan et al., 2018; Kralova & Sjöblom, 2009). For example, at 10% solution quillaja bark extract reduced the number of bacteria from 3.7 log no. bact./mm2 to 3.1 log no. bact./mm2, soap nut extract to 3.2 log no. bact./mm2, and horse chestnut extract to 3.3 log no. bact./mm2, respectively (p < 0.001). In contrast to that, standard sapogenin and escin reduced the number to 3.4 log no. bact./mm2 and quillaja saponin only to 3.5 log no. bact./mm2 indicating the natural plants are more efficient for E. coli reduction that standard saponins (Fig. 2, Table 1). In a comparable study, also Khan et al. (2018) found that bacterial reduction is concentration-dependent and that a 12% solution of saponins from green tea seeds is effective against E. coli cells. Furthermore, Kose and Bayraktar (2016) found that a 10% solution of soap nut extract has antimicrobial effects on E. coli. Among our tested natural plant extracts, the quillaja bark extract is the most effective (66%), followed by soap nut extract (62%), and horse chestnut extract (41%). The evidence from this study suggests that quillaja bark extract represent good candidate for cleaning product in food industry, but
Bandyopadhyay, & Chowdhury, 2015). The surface tension was measured using a Leconde Du Nouy Tensiometer, with which the tension required to lift the platinum ring out of the surface of the water was measured
Surface tension (σ ) =
Fmax (mN / m) (L x cos(θ))
(3)
where Fmax is the force exerted on the platinum ring, L is the characteristic length of the ring, and θ is the contact angle between the platinum ring and solution. The surface tension of all selected cleaning products was measured for all six concentrations (10%, 1%, 0.1%, 0.01%, 0.001% and 0.0001%) and three repetitions. 2.7. Statistical analysis Statistical analysis was run on R software version 3.1.3 (Bell Laboratories, New Jersey, U.S.). Normality was checked using the Shapiro-Wilk test (p > 0.05). One-way analysis of variance (ANOVA) and the Duncan statistical test were used to determine the significant differences at a significance level of p < 0.05. 3. Results and discussion The study assessed and compared the efficacy of saponin standards and plant derived saponins on reducing E. coli. The results show that before exposure to standard saponins and natural plant extracts the number of bacteria on the glass coupons was 3.7 ± 0.11 log no. bact./ mm2. The results for both standard saponins and natural extracts show that increasing the cleaning product concentrations results in a decreasing number of bacteria for all cleaning products (p < 0.05), except for standard quillaja saponin where no statistically significant
Table 1 Comparison of the number of bacteria, surface tension and emulsification index due to the type of cleaning product and concentration.
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Fig. 2. E. coli adhesion (log no. bact./mm2) on glass coupons after exposure to different concentrations of standard saponins and natural plant extracts.
Cartwright, and Bailey (2010) showed the antibacterial activity of a commercial extract rich in quillaja extract against S. Typhimurium, S. aureus, and E. coli. Moreover, the authors demonstrated that the pretreatment with quillaja bark extract increased the sensitivity of the bacterial cultures to peracetic acid, showing the potential of the hurdle approach. Another great advantage of quillaja extract is its low toxicity, as it is often used in the food industry as a humectant in baked goods, frozen dairy products, and puddings and as a foaming agent in soft drinks (Younes et al., 2019). Therefore, applying quillaja based cleaning products in the food industry would minimalize problems with cleaning residues on food contact materials assuring the safety of
further studies for implementation are needed. The study carried out by Berlowska, Dudkiewicz, Kregiel, Czyzowska, and Witonska (2015) on yeast cells showed that soap nut extract increased cell membrane permeability in different strains, lysis, and consequently cell detachment. Quillaja bark extract in our research shows some potential for reducing the number of cells on the glass surface. It is assumed that saponin interacts with lipopolysaccharide and thereby increases the permeability of the bacterial cell wall (Arabski et al., 2012). Similar to our study, Sewlikar and D'Souza (2017) tested quillaja bark extract on E. coli O157: H7 and found a reduction of cells from 7.5 CFU to 6.8 CFU, which is comparable to our results with E. coli reduction. Hassan, Byrd, 5
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Fig. 3. Emulsification index (E-24) of standard saponins and natural plant extracts vs concentration.
ones, which was also found in our study. The authors also report that foam from the soap nut is much more stable than an isolated one. Another study revealed a natural extract from soap nuts has a higher emulsification index than synthetic sodium dodecyl sulphate and Tween 80 do (Tmáková, Sekretár, & Schmidt, 2016). Moreover, a recent study demonstrated that natural saponins of C. enchinulata can even exceed an E-24 value of 80 (Andrade et al., 2018), which shows that natural extracts should be given more attention. A good surfactant can decrease the surface tension of water from 72 mN/m to 35 mN/m (Pradhan & Bhattacharyya, 2017). The results of surface tension show that by increasing the concentration for all tested
consumers. Emulsification ability is an essential property of good cleaning products. The amphiphilic nature helps surfactants to solubilise waterinsoluble substances, e.g., hydrocarbons. Results of the emulsification index demonstrate that increasing the concentration for all tested cleaning products increases E-24 (p < 0.001) (Table 1). Fig. 3 shows the highest value for a natural extract from quillaja bark up to 73, followed by standard quillaja saponin (70), horse chestnut extract (65), sapogenin (64), escin (60), and soap nut extract (58). The study by Pradhan and Bhattacharyya (2017) showed that natural extracts from soap nuts possess higher values of E-24 when compared to standard 6
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Fig. 4. Surface tension (mN/m) of standard saponins and natural plant extracts vs. concentration.
solution of standard saponins and natural plant extracts. The results show the quillaja bark extracts remove up to 66% of bacteria relative to the control, followed by sapogenin at 65%, escin at 51%, quillaja saponin at 45%, and horse chestnut extract at 41%, respectively (Fig. 5). In accordance with the present results, we have demonstrated that quaillaja bark extract can reduce the bacterial population on the surface, efficiently emulsify fats and decrease the water surface tension. The scope of this study was limited in terms of testing different mixture of natural plant extracts against E. coli, concentrations of active components in natural extracts, analysing efficacy on different microorganisms and variety of food contact materials.
cleaning products, surface tension decreases (p < 0.001) (Table 1). The most substantial decrease of surface tension can be observed at the highest concentration (10%) for soap nut extract (22 mN/m), quillaja bark extract, escin and sapogenin (23 mN/m) and the least for the horse chestnut extract and standard quillaja saponin (25 mN/m) (Fig. 4). This indicates that natural plant extracts have cleaning potential and that further research on this field is needed. Previous studies have shown a reduction of water surface tension by soap nuts only to 47 mN/m (Tmáková et al., 2016) and 35 mN/m (Pradhan & Bhattacharyya, 2017), which is less than the present study demonstrated. Images from the light microscope confirm the efficacy of 10% 7
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Fig. 5. Microscope image of E. coli cells before and after exposure to 10% solution of standard saponins and natural plant extracts.
4. Conclusions
emulsification index. All this indicates that quaillaja bark extract represents good candidate for future research. More research is needed to better understand effects of different concentrations and active components on variety of food pathogens, optimising plant extract procedure, mixture of different plant extracts and application of natural cleaning product in food industry.
Management of the bacterial population in the food industry has to be focused on searching for cleaning products with appropriate efficacy that leave low bacterial cells residues on the surface, but also take into the account environmental issues. This study has found that increasing the concentration of all tested extracts and standard compounds results in decreased number of bacteria, decreased water surface tension and also increased emulsification index. Moreover, our study demonstrated that among tested cleaning products, natural plant extract from quillaja bark is the most efficient by decreasing number of E. coli cells on glass surface up to 66%, followed by sapogenin (65%) and soap nut extract (62%). Furthermore, findings also show that quaillaja bark extract substantially decreases the water surface tension and increases the
Declaration of competing interest None. Acknowledgements The research was supported by the Slovenian Research Agency 8
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under Slovenia-United States of America bilateral project “Natural cleaning agents potential for biofilm control in food industry BI-US/1819-075”. The authors would like to express their gratitude to Terry Troy Jackson for grammar proof.
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