Hand sanitizer is a practical and effective hand-washing product to inhibit the growth of bacteria such as Staphylococcus aureus and Escherichia coli. Hand sanitizer is a simple product because it is easy to use when soap and water are unavailable1,2. Meanwhile, hand sanitizer is selected for use because it effectively kills bacteria in a relatively fast time3. Hand sanitizer with an alcohol concentration of 60-70% inhibits the growth of bacteria on the hands for 1-12 minutes4. Alcohol inhibits bacterial growth by denaturing and coagulating bacterial cells5.

Excessive or continuous use of alcohol-based hand sanitizers can irritate the skin, causing a burning feeling on the skin6. One effort to reduce chemicals in a hand sanitizer product is to innovate alcohol-free hand sanitizer products using extracts from natural ingredients that have antibacterial properties, such as lotus seeds7, banana stems8, shell chitosan snails9, Binahong leaves10, Stevia leaves, and pineapple peel11. According to Ariningrum et al.12, hand sanitizer with Trembesi and Stevia leaf extract can reduce the number of bacteria on the hands by up to 88%.

In addition to extractives from natural ingredients, enzymes such as proteases, lipases, amylase, cellulases, peroxidases, and laccases have also been reported as having antibacterial properties13. Laccase is an enzyme with a wide range of substrates, so it is applied easily in various industrial fields such as the food, textile, pulp, cosmetic, and personal care industries14. Laccase is widespread in higher plants such as pear, radishes, cabbage, apples, potatoes, and asparagus15, insects such as Anopheles, Apis, Bombyx, Calliphora, Diploptera, Drosophila, and Lucilia16; bacteria such as Bacillus pumilus, Bacillus subtilis, Bacillus licheniformis, Thermus thermophilus, and Sinorhizobium meliloti; and fungi such as Trametes, Pleurotus, Lentinula, Pycnoporus, Phanerochaete, and Agaricus17. Laccase produced by bacteria and fungi is easier to obtain because secreted into the growth medium. Laccase produced by fungi has a higher potential redox value than bacteria. The potential redox value is related to the enzyme's ability to degrade high molecular-weight substrates18.

Laccase with 0.1-5.0 mg/L in activity produced by the fungi Myceliophthora thermophila and Polyporus pinisite can inhibit the growth of Gram-positive bacteria, Staphylococcus epidermidis, and Gram-negative bacteria, Pseudomonas aeruginosa up to 99%. The antibacterial properties of the laccase enzyme are applied potentially to detergent, disinfectant, food, beverages, or cosmetic products such as soap, shampoo, deodorant, mouthwash, and contact lens cleaning fluid19.

The laccase enzyme from Trametes hirsuta has never been studied before for its use in personal care products. This study aims to determine the effect of the laccase enzyme on the physicochemical properties and the antibacterial potential of the hand sanitizer gel against pathogenic bacteria. Laccase enzyme was used at various concentrations in the hand sanitizer gel formulation and was tested for its effectiveness in inhibiting the growth of S. aureus and E. coli. The stability test of the hand sanitizer gel was carried out at room temperature (27±3°C) for four weeks.

Materials

Oil palm empty fruit bunch (OPEFB) were taken from oil palm plantations in Cikasungka, West Java, Indonesia. Trametes hirsuta EDN 082 (NCBI GenBank accession number MT476912) was isolated from Taman Eden 100, North Sumatra, Indonesia. Potato dextrose agar, malt extract, glucose, peptone, sodium benzoate, and CuSO4 were purchased from Merck, Germany. Vanillin and 2,2-azino-bis-[3-ethyl benzothiazoline-6-sulphonic acid] (ABTS) were purchased from Sigma Aldrich. Petrifilm™ Staph Express Count Plate and E. coli Count Plate were purchased from 3M, US. Carbopol, triethanolamine (TEA), lemon oil, and propylene glycol was purchased from PT. Palapa Muda Perkasa, West Java, Indonesia.

Methods

Laccase production and extraction

Trametes hirsuta EDN 082 was cultured on Potato Dextrose Agar (PDA) and incubated at 27–30℃ for seven days. Laccase enzyme was produced according to Ningsih et al.20 using OPEFB as a substrate. After ten days of incubation time, the laccase on the OPEFB substrate was extracted by mixing with 0.1 M acetate buffer (pH 4.5) at a ratio of 1 : 3 (w/v) and homogenizing using an ACE AM-11 homogenizer (Nissei, Japan) at 10,000 rpm for 10 minutes under cold condition. The extraction process was conducted according to Anita et al21. The crude laccase was filtered using a nylon syringe filter of 0.22 µm and then was analyzed for its activity22.

Preparation of laccase hand sanitizer gel

Carbopol was weighed and dispersed into sterile distilled water. The mixture was then homogenized using a homogenizer at 150 rpm for 10 minutes. After 10 minutes, sodium benzoate, propylene glycol, laccase enzymes, lemon oil, vanillin, TEA, and remaining distilled water were added to the homogenizer. The mixture was then homogenized using a homogenizer at 150 rpm for 10 minutes. The preparation was then put into a container. Hand sanitizer gel was made in three formulations with different concentrations of the laccase enzyme as an antibacterial compound (Table I). The commercial hand sanitizer (F4) was used for comparison with 70% of ethanol as an active ingredient.

TableI. Formulation of laccase hand sanitizer gel.

Physicochemical properties of laccase hand sanitizer gel

The physicochemical properties of laccase hand sanitizer gel, including organoleptic, pH value, viscosity, and gel spreadability, were evaluated. The organoleptic test was color, shape, odor, and homogeneity. The pH value was measured using a pH meter (Metrohm, Switzerland). Spreading diameter gel was measured by applying the gel to a round glass, sandwiched between two round glasses, and subjected to a 150 g load for 1 minute. The viscosity was determined using a Viscometer (RheolabQC Anton Paar, Austria) equipped with a CC27 spindle with a constant shear rate of 26/s for 60 seconds23.

Antibacterial activity test of laccase hand sanitizer gel

The antibacterial activity test was carried out using the palm swab method. This method was carried out by taking two swabs, washing hands with running water (control), and using hand sanitizer gel after treatment24,25. First, hands were washed with water without using soap for 20 seconds. The palms were wiped using sterile wet cotton buds. After that, the cotton buds were dipped into a tube containing 10 mL of sterilized distilled water for dilution. A total of 1 mL of the dilution was inoculated into Petri films E. coli Count Plate and Staph Express Count Plate. The Petri films were then incubated at 37°C for 48 hours. Colony growth was calculated with a colony counter. The same method was used for hand sanitizer formulas F0, F1, F2, F3, and commercial (F4). The percentage of inhibition was calculated by Equation 1.

[1]

Physicochemical stability test of laccase hand sanitizer gel

Hand sanitizer preparations were stored at room temperature (27±3°C) for four weeks. The stability test, which included an organoleptic test, pH value, and spreadability, was carried out once a week, while the viscosity was carried out every two weeks.

The organoleptic test was carried out to evaluate the physical appearance by observing the hand sanitizer gel's color, odor, homogeneity, and shape (consistency). The color test showed that the more laccase enzyme added, the more yellow the color of the gel produced (Figure 1). The gel has a lemon-like smell. The odor was produced by the fragrance used. The addition of the laccase enzyme has not affected the odor of the hand sanitizer gel. All hand sanitizer gels formulation formed homogeneous, as indicated by the absence of coarse particles and no phase separation (between laccase and gel base) in the formulated hand sanitizer gel after the application on transparent glass23. However, the gel became more liquid when more laccase was added. Compared with commercial hand sanitizers, F0–F3 hand sanitizers look cloudy and not transparent. According to Asngad et al.8, adding glycerin can cause the hand sanitizers to become clear and transparent. Besides that, glycerin can be used as a moisturizer.

Figure1. Laccase hand sanitizer gel, without laccase (F0), laccase 4% (F1), laccase 7% (F2), laccase 10% (F3), and commercial hand sanitizer (F4).

The pH test aims to see the safety of the formulated hand sanitizer gel so that it does not irritate the skin when applied26. According to the Indonesian National Standard (SNI), the pH value of topical preparations is 4.5–827. The pH values of formulated hand sanitizer gels (F0–F3) were slightly higher than commercial hand sanitizers. However, the formulation F0–F3 had a pH in the skin pH range, and therefore it is safe to use. The variations of the laccase enzyme concentration in hand sanitizer had not significantly affected the pH value (Table II).

The viscosity test was carried out to determine the consistency and flowability of the gel formulation when applied to the skin. As shown in Table II, the more laccase enzyme added, the lower the formulated hand sanitizer gel viscosity. The viscosity values produced by all formulations are lower than commercial hand sanitizers. Asngad et al.8 reported that hand sanitizer gel that consists of banana stem extract, alcohol, triclosan, and glycerin produces viscosity values between 520–1.250 cPs. Factors that affect the low viscosity value of formulation gel include the formulas' pH, the extract's pH, the amount of Carbopol and TEA used8, and mixing or stirring during the formulation process28.

The spreadability test was carried out to determine the ability of the gel to spread on the skin surface. The greater the spreadability, the easier the gel to apply to the skin's surface. If the gel is too watery, then the gel will be challenging to stick to the skin. If the gel is too thick, then the gel is difficult to apply to the skin surface29. The higher the concentration of the laccase enzyme used, the greater the spreadability of the preparation (Table II). The lower the viscosity value, the higher the spreadability10,30.

TableII. The pH, viscosity, and spreadability values of hand sanitizer gel formulation.

The result of the antibacterial activity of laccase hand sanitizer gel can be seen in Figure 2. As shown in Figure 2, the higher the laccase concentration in the hand sanitizer formula, the greater the inhibition of bacterial growth against S. aureus and E. coli. The activity of the laccase enzyme stock used was 0.032 U/mL. The laccase enzyme activity detected in the hand sanitizer formulation with adding the laccase enzymes 4, 7, and 10% were 0.003, 0.005, and 0.013 U/mL, respectively. The higher the laccase enzyme concentration added, the higher the enzyme activity value detected in the preparation. The results revealed that the highest inhibition against S. aureus was shown by F2 (96.77%), while in F3, it decreased (95.63%). The effectiveness of F2 against S. aureus was the closest to the growth inhibition of the commercial product F4 (97.90%).

Different things happened to the growth of E. coli. The percentage of inhibition of E. coli for all concentrations from F1 to F3 was 100%, the same as that of commercial product F4. This result is different from antibacterial solid bath soap made from palm leaf, which is only able to inhibit the growth of S. aureus31. In most cases, antibacterial compounds are more resistant to Gram-negative than Gram-positive bacteria. This is because of the different composition and structure of the two bacteria's cell walls. Gram-positive bacteria have a simpler and thicker cell wall structure than Gram-negative bacteria, with a single layer that is 15-80 nm thick, low in lipid content (1–4%), and contains teichoic acid. At the same time, the Gram-negative bacteria have three-layered cell walls with a thin (10-15 nm) outer layer, a high lipid content (11–12%), and peptidoglycan located in the rigid inner layer with a small amount of about 10% dry weight and no teichoic acid32,33. The results of this study, which showed that E. coli was more sensitive than S. aureus, suggest that a hand sanitizer formula with laccase could more easily lyse the outer layer of Gram-negative bacteria's cell wall in the form of lipopolysaccharides. However, this hypothesis should be investigated further in future research.

These findings suggest that the laccase enzyme could be used as an active ingredient in hand sanitizers because it inhibits bacterial growth. Laccase has been shown to have the same antibacterial properties as hand sanitizers containing 60–95% alcohol24. Furthermore, laccase-containing hand sanitizers inhibit bacterial growth even more effectively than those containing 1.5–2% triclosan, which only suppresses bacterial growth by 62.94–64.5%34. Triclosan is an active substance that is generally added to antibacterial bath soaps35.

Figure2. The percentage of inhibition of hand sanitizer gel formulation against Gram-positive-bacteria, S. aureus, and Gram negative-bacteria, E. coli.

The stability test included an organoleptic test, pH value, viscosity, and spreadability. The organoleptic test revealed no color, odor, or shape differences (Table III). This demonstrates that the laccase enzyme active ingredient in the hand sanitizer gel is stable after four weeks of storage. During the storage, the pH of each formula did not change significantly (from 7.42 to 7.71). Despite this, the pH value met the SNI's requirements (4.5–8). Table III showed no significant differences in pH changes throughout the week. During storage, F0 and F1 spreadability ranged from 3.85 to 5.37 cm. Meanwhile, F2 and F3 spreadability ranged from 3.89 to 5.83 cm (Table III). The viscosity of the formula is inversely proportional to its spreadability36. The dispersion of each formula appears to fluctuate during storage. Small changes in spreadability indicate that the formula has good dispersion stability11.

The viscosity of each formula decreases as storage time increases until four weeks (Figure 3). Viscosity loss can be caused by factors such as light and humidity in the storage environment. Uncontrolled humidity in the storage room can cause the gel absorbs moisture from the air, thus lowering the gel's viscosity. Less impermeable packaging can cause the gel to absorb moisture (hygroscopic) from the outside, causing the volume of water in the gel formula to increase37.

TableIII. The physicochemical stability test of laccase hand sanitizer gel during storage.

W: white; WY: white yellow; L: lemon; G: gel; Lq: Liquid

Figure3. The viscosity of hand sanitizer gel formulation during storage time.

Laccase can be used as an active ingredient in hand sanitizer gel formulations. Variations in the concentration of laccase affect the characteristics of the hand sanitizer gel produced. Hand sanitizer gel formulation with laccase at concentrations of 4, 7, and 10% inhibited the growth of S. aureus to 94.11, 96.77, and 95.63%, respectively. All formulations are known to inhibit the growth of E. coli by 100%. Formula F2, with a laccase concentration of 7%, shows the best performance with the most significant inhibition on tested pathogenic bacteria.

The authors thank the Integrated Laboratory of Bioproducts (ILaB) at the National Research and National Agency (BRIN), Cibinong, Bogor, Indonesia, for the facilities and scientific and technical assistance. BRIN supported part of this research through DIPA 2021 and by Japan-ASEAN Science, Technology, and Innovation Platform (JASTIP) Network.

Sita Heris Anita: designed the study, analyzed data, supervised the experiment, wrote, reviewed, and edited the manuscript. Asishe: performed research, collected data, and wrote the initial manuscript. Vilya Syafriana: analyzed data, wrote, reviewed, and edited the manuscript. Amelia Febriani: analyzed data, wrote, reviewed, and edited the manuscript. Deni Zulfiana: analyzed data, supervised the experiment, reviewed, and edited the manuscript. Maulida Oktaviani: analyzed data and supervised the experiment. Oktan Dwi Nurhayat: analyzed data and supervised the experiment. Dede Heri Yuli Yanto: analyzed data, reviewed, and edited the manuscript.

None.

The authors declare there is no conflict of interest.

1. Muleba L, Van Wyk R, Pienaar J, Ratshikhopha E, Singh T. Assessment of Anti-Bacterial Effectiveness of Hand Sanitizers Commonly Used in South Africa. Int J Environ Res Public Health. 2022;19(15):9245. doi:10.3390/ijerph19159245

2. Golin AP, Choi D, Ghahary A. Hand sanitizers: A review of ingredients, mechanisms of action, modes of delivery, and efficacy against coronaviruses. Am J Infect Control. 2020;48(9):1062-7. doi:10.1016/j.ajic.2020.06.182

3. Atolani O, Baker MT, Adeyemi OS, Olanrewaju IR, Hamid AA, Ameen OM, et al. COVID-19: Critical discussion on the applications and implications of chemicals in sanitizers and disinfectants. EXCLI J. 2020;19:785-99. doi:10.17179/excli2020-1386

4. Manaye G, Muleta D, Henok A, Asres A, Mamo Y, Feyissa D, Ejeta F, et al. Evaluation of the Efficacy of Alcohol-Based Hand Sanitizers Sold in Southwest Ethiopia. Infect Drug Resist. 2021;14:547-54. doi:10.2147/idr.s288852

5. Huffer S, Clark ME, Ning JC, Blanch HW, Clark DS. Role of alcohols in growth, lipid composition, and membrane fluidity of yeasts, bacteria, and archaea. Appl Environ Microbiol. 2011;77(18):6400-8. doi:10.1128/aem.00694-11

6. Lachenmeier DW. Safety evaluation of topical applications of ethanol on the skin and inside the oral cavity. J Occup Med Toxicol. 2008;3:26. doi:10.1186/1745-6673-3-26

7. Cahyaningtyas FD, Ukrima ZA, Nora N, Amaria A. Pemanfatan Ekstrak Biji Teratai Sebagai Bahan Aktif Antibakteri Untuk Pembuatan Hand Sanitizer. Indones Chem Appl J. 2019;3(1):7. doi:10.26740/icaj.v3n1.p7-13

8. Asngad A, Bagas AR, Nopitasari N. Kualitas Gel Pembersih Tangan (Handsanitizer) dari Ekstrak Batang Pisang dengan Penambahan Alkohol, Triklosan dan Gliserin yang Berbeda Dosisnya. Bioeksperimen J Penelitian Biol. 2018;4(2):61–70. doi:10.23917/bioeksperimen.v4i2.6888

9. Umarudin U, Surahmaida S, Syukrianto S, Wulansari SA, Nurhaliza S. Aplikasi Hand Sanitizer Kitosan Cangkang Bekicot Sebagai Antibakteri dan Upaya Preventif Covid 19. Simbiosa. 2020;9(2):107–17. doi:10.33373/sim-bio.v9i2.2669

10. Rahmasari D, Hendradi E, Chasanah U. Formulation and evaluation of hand sanitizer gel containing infused binahong leaf (Anredera cordifolia) as antibacterial preparation. Farmasains J Farmasi Ilmu Kesehatan. 2020;5(1):23–30. doi:10.22219/farmasains.v5i1.13008

11. Maulana MR, Ariningrum ND, Nurjanah BAD, Harismah K. Uji Stabilitas Fisik Hand Sanitizer Antiseptik Berbasis Daun Stevia Dan Kulit Nanas (Ananas comosus (L.) Merr.). Prosiding SNPBS Seminar Nasional Pendidikan Biol Saintek. 2020;5:391–7.

12. Ariningrum ND, Anisa B, Nurjanah D, Maulana MR, Harismah K. Uji efektivitas gel hand sanitizer sebagai antiseptik tangan berbasis ekstrak daun trembesi (Albizia saman (Jacq.) Merr) dan stevia. Prosiding SNPBS Seminar Nasional Pendidikan Biol Saintek. 2020;5:506–13.

13. Raveendran S, Parameswaran B, Ummalyma SB, Abraham A, Mathew AK, Madhavan A, et al. Applications of Microbial Enzymes in Food Industry. Food Technol Biotechnol. 2018;56(1):16-30. doi:10.17113/ftb.56.01.18.5491

14. Zerva A, Simic S, Topakas S, Nikodinovic-Runic J. Applications of microbial laccases: Patent review of the past decade (2009-2019). Catalysts. 2019;9(12):1023. doi:10.3390/catal9121023

15. Dana M, Khaniki GB, Mokhtarieh AA, Davarpanah SJ. Biotechnological and industrial applications of laccase: A review. J Appl Biotechnol Reports. 2017;4(4):675–9.

16. Janusz G, Pawlik A, Świderska-Burek U, Polak J, Sulej J, Jarosz-Wilkołazka A, et al. Laccase properties, physiological functions, and evolution. Int J Mol Sci. 2020;21(3):966. doi:10.3390/ijms21030966

17. Bertrand B, Martínez-Morales F, Trejo-Hernández MR. Fungal laccases: Induction and production. Rev Mex Ing Quim. 2013;12(3):473–88.

18. Barber-Zucker S, Mateljak I, Goldsmith M, Kupervaser M, Aldalde M, Fleishman SJ. Designed High-Redox Potential Laccases Exhibit High Functional Diversity. ACS Catal. 2022;12(21):13164-73. doi:10.1021/acscatal.2c03006

19. Osma JF, Toca-Herrera JL, Rodríguez-Couto S. Uses of laccases in the food industry. Enzyme Res. 2010;2010:918761. doi:10.4061/2010/918761

20. Ningsih F, Yanto DHY, Mangunwardoyo W, Anita SH, Watanabe T. Optimization of laccase production from a newly isolated Trametes sp. EDN134. IOP Conf Ser Earth Environ Sci. 2020;572:012024. doi:10.1088/1755-1315/572/1/012024

21. Anita SH, Ardianti FC, Oktaviani M, Sari FP, Nurhayat OD, Ramadhan KP, et al. Immobilization of laccase from Trametes hirsuta EDN 082 in light expanded clay aggregate for decolorization of Remazol Brilliant Blue R dye. Bioresour Technol Rep. 2020;12:100602. doi:10.1016/j.biteb.2020.100602

22. Yanto DHY, Guntoro MA, Nurhayat OD, Anita SH, Oktaviani M, Ramadhan KP, et al. Biodegradation and biodetoxification of batik dye wastewater by laccase from Trametes hirsuta EDN 082 immobilised on light expanded clay aggregate. 3 Biotech. 2021;11(5):247. doi:10.1007/s13205-021-02806-8

23. Noviardi H, Himawan HC, Anggraeni R. Formulasi dan aktivitas antibakteri sediaan gel hand sanitizer dari ekstrak etanol biji mangga harum manis (Mangifera indica L.) terhadap Escherichia coli dan Staphylococcus aureus. J Farmamedika (Pharmamedica J). 2018;3(1):1–9. doi:10.47219/ath.v3i1.20

24. Radji M, Suryadi H. Uji Efektivitas Antimikroba Beberapa Merek Dagang Pembersih Tangan Antiseptik. Pharm Sci Res. 2007;4(1):1–6.doi:10.7454/psr.v4i1.3408

25. Wolfe MK, Lantagne DS. A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens. J Vis Exp. 2017;124:55604. doi:10.3791/55604

26. Fallica F, Leonardi C, Toscano V, Santonocito D, Leonardi P, Puglia C. Assessment of Alcohol-Based Hand Sanitizers for Long-Term Use, Formulated with Addition of Natural Ingredients in Comparison to WHO Formulation 1. Pharmaceutics. 2021;13(4):571. doi:10.3390/pharmaceutics13040571

27. Dolorosa MT, Nurjanah, Purwaningsih S, Anwar E. Utilization of Kappaphycus alvarezii and Sargassum plagyophyllum from Banten as cosmetic creams. IOP Conf Ser Earth Environ Sci. 2020;404:012008. doi:10.1088/1755-1315/404/1/012008

28. Binder L, Mazál J, Petz R, Klang V, Valenta C. The role of viscosity on skin penetration from cellulose ether-based hydrogels. Skin Res Technol. 2019;25(5):725-34. doi:10.1111/srt.12709

29. Kulawik-Pióro A, Miastkowska M. Polymeric Gels and Their Application in the Treatment of Psoriasis Vulgaris: A Review. Int J Mol Sci. 2021;22(10):5124. doi:10.3390/ijms22105124

30. Booq RY, Alshehri AA, Almughem FA, Zaidan NM, Aburayan WS, Bakr AA, et al. Formulation and evaluation of alcohol-free hand sanitizer gels to prevent the spread of infections during pandemics. Int J Environ Res Public Health. 2021;18(12):6252. doi:10.3390/ijerph18126252

31. Febriani A, Syafriana V, Afriyando H, Djuhariah YS. The utilization of oil palm leaves (Elaeis guineensis Jacq.) waste as an antibacterial solid bar soap. IOP Conf Ser Earth Environ Sci. 2020;572:012038. doi:10.1088/1755-1315/572/1/012038

32. Malanovic N, Lohner K. Antimicrobial Peptides Targeting Gram-Positive Bacteria. Pharmaceuticals. 2016;9(3):59. doi:10.3390/ph9030059

33. Syafriana V, Febriani A, Suyatno S, Nurfitri N, Hamida F. Antimicrobial Activity of Ethanolic Extract of Sempur (Dillenia suffruticosa (Griff.) Martelli) Leaves against Pathogenic Microorganisms. Borneo J Pharm. 2021;4(2):135–44. doi:10.33084/bjop.v4i2.1870

34. Wijaya JI. Formulasi Sediaan Gel Hand Sanitizer dengan Bahan Aktif Triklosan 1,5% dan 2%. Calyptra J Ilmiah Mahasiswa Univ Surabaya. 2013;2(1):1–14.

35. Weatherly LM, Gosse JA. Triclosan exposure, transformation, and human health effects. J Toxicol Environ Health B Crit Rev. 2017;20(8):447-69. doi:10.1080/10937404.2017.1399306

36. Kryscio DR, Sathe PM, Lionberger R, Yu L, Bell MA, Jay M, et al. Spreadability measurements to assess structural equivalence (Q3) of topical formulations--a technical note. AAPS PharmSciTech. 2008;9(1):84-6. doi:10.1208/s12249-007-9009-5

37. Basiak E, Lenart A, Debeaufort F. How Glycerol and Water Contents Affect the Structural and Functional Properties of Starch-Based Edible Films. Polymers. 2018;10(4):412. doi:10.3390/polym10040412