Abstract

Chitosan is an extremely valuable biopolymer and is usually obtained as a byproduct from the shells of crustaceans. In the current work, chitosan is obtained from an herbal source (Ganoderma lucidum spore powder (GLSP)) for the first time. To show this, both standard (thermochemical deacetylation, (TCD)) and emerging (ultrasound-assisted deacetylation (USAD)) methods of chitosan preparation were used. The obtained chitosan was characterized by elemental analysis, XRD (X-ray diffraction), FT-IR (Fourier transform infrared spectroscopy) and thermogravimetric measurements. The process resulted in chitosan possessing comparable values of DD, [η] and Mv¯ to the commercial product. Chitosan obtained via both processes (TCD and USAD) displayed excellent biocompatibility; although the USAD prepared biopolymer exhibited significantly improved fibroblast (L929 cell) viability and enhanced antibacterial zones for both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The findings of new herbal chitosan mark key developments of natural biomaterials; marking a potential shift from conventional sea-based organisms.

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Introduction

Chitin is a white linear polysaccharide and is insoluble in most common solvents¹. It is the main constituent of exoskeleton in several insects and shellfish (e.g. crabs and shrimps)². It’s well documented biodegradability and low toxicity have led to extensive use of the refined form in biomaterial fields³. Based on the molecular (chain) structure of N-acetyl-D-glucosamine linked by β- (1–4) glycosidic bonds, chitin is classified into three different polymeric forms; α, β and γ⁴. Since most abundantly used chitins are from seafood shells, which are more likely to contain allergenic contaminants and greater mineral content compared to fungi⁵; complex purification methods are often deployed to minimize consumer health risks^6,7. Furthermore, obtaining high quality chitin from discarded and spoiling seafood shells is also challenging with further impact on production time and health risk aspects.

Chitosan is a derivative of chitin; obtained after deacetylation. In this instance, the GlcNAc unit in chitin is converted to GlcN forming chitosan⁸ with the repeating units of both biopolymers. Like chitin, chitosan is a biopolymer which has generated extensive interest; surpassing research interest in cellulose⁴, largely due to its better aqueous solubility⁹. Chitosan possesses good biocompatibility, biodegradability and low toxicity¹⁰ and the materials absorptivity of heavy metal ions also makes it a valuable biopolymer¹¹. For these reasons it has been used in numerous fields including food¹², pharmaceutics and biomedical engineering¹³.

Due to increased demand for chitosan (emerging bio-applications and preference over existing materials) acetylation methods and processing of chitin have diversified¹⁴. Conventionally, thermochemical deacetylation (TCD) is used deploying high concentrations of aqueous sodium hydroxide¹⁵, elevated process temperatures (120 °C) and lengthy reaction times (~4 h)¹⁶. To overcome this, several methods converting chitin to chitosan (with greater product yield and quality) have been developed which include microwave assisted deacetylation¹⁷, ultrasound assisted deacetylation (USAD)¹⁶ and supercritical fluid extraction¹⁸. USAD has proven to be a crucial breakthrough, converting β-chitin into chitosan (with 83–94% deacetylation) at reduced synthesis temperatures (50–80 °C) and quicker reaction times (10–60 min), which also minimizes severe depolymerization risk¹⁹. Chitosan is used in numerous industrial applications and is conventionally sourced from sea organisms²⁰. In this regard, alternative sourcing of chitosan together with enhanced processing will provide timely developments.

Ganoderma lucidum, also known as the “Linghzi” Mushroom, belongs to a large group of fungi called polypore and has been used as a medicinal product for many millennia²¹. Furthermore, potential health benefits of Ganoderma lucidum spore powder (GLSP, Fig. 1) have recently been documented which include improved immuno-regulation, anti-inflammatory action, anti-cancer properties, radical scavenging ability and prevention of diabetes²². The powder constitutes a chitin bilayer structure with an overall particulate size range of ~6–12 μm. The GLSP component comprises polysaccharides, triterpenoids, nucleotides, sterols, steroids, ganoderic acids and proteins²², making this a potential nutritional source for various bioactive elements. To date several raw chitin products have been developed using mainly seafood byproduct sources (crab, shrimp and lobster shells) although with little to no emphasis on unlocking the nutritional resource following human consumption. Taking into account the growing demand (and applications) of the material, alongside its nutritional composition, it becomes imperative to explore opportunities to make such bioactive components accessible.

Figure 1

Results and Discussion

Deacetylation of Thermochemical Deacetylation (TCD) and Ultrasound-Assisted Deacetylation (USAD)

GLSP is an excellent source of chitin, indicating potential utilization and benefits in the biomedical and therapeutic arena. However, its conversion to chitosan from the raw herbal powder remains to be explored. In this study, conventional TCD and emerging USAD processes were utilized to obtain chitosan from fungal spores.

The chitosan morphological were changed following deacetylation using both methods (Fig. 2). Although many minute pores are observed on GLSP surface, the overall particulate structure is oval in shape and rigid (Fig. 2a), which suggests the polysaccharide extraction process does not impact chitin structure. However, morphological differences arose upon deacetylation and varied depending on the method deployed. The chitosan morphology post TCD and USAD (Fig. 2b,c), were lost the well-defined oval shape. The surface chitosan obtained using TCD (termed as C-T hereafter) is relatively smooth when compared to chitosan obtained using USAD (termed as C-U hereafter). C-T was obtained using solvent and heating based reaction under static conditions, whilst C-U involved vigorous ultrasound cavitation. In this regard, the USAD method broadens application scope of chitosan obtained from the herbal source since the irregular surface is beneficial; increasing the contact area with other materials.

Figure 2

Conclusion

The current work investigates the herbal based source for chitosan production. Both TCD and USAD successfully induced N-deacetylation reaction and converted chitin into chitosan and products were compared with a commercial source. The obtained chitosan were characterized by elemental analysis, XRD, FT-IR and thermogravimetric measurements. Chitosan derived using both processes displayed excellent biocompatibility and improved fibroblast cell viability and antibacterial activity. Compared to control and TCD groups, chitosan prepared via USAD displayed better viability and antibacterial zones. These interesting findings indicate an exciting breakthrough for herbal based chitosan for use in biomaterial science.

Materials and Methods

Materials

GLSP was provided by TianHe Agricultural Group (Zhe Jiang Long Quan, China). GLSP relics were used in this study following polysaccharide extraction as shown in previous work⁴². Hydrochloric acid, acetic acid, sodium chloride (troche) and sodium hydroxide (troche) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Modified eagle’s medium was obtained from (MEM, Gibco, USA) and fetal bovine serum (FBS) was obtained from Sijiqin (Sijiqin, Hangzhou, Zhejiang, China). Deionized water (DI) was produced using a Millipore Milli-Q Reference ultrapure water purifier (Millipore, Bedford, USA). Hydrogen peroxide 30% (H₂O₂) was obtained from Sinopharm Chemical Reagent Co., Ltd. (ShangHai, China). All chemicals were analytical grade without additional purification. For comparison, standard (commercial) chitosan (C_6nH_11nNO_4n) was purchased from Macklin (Macklin Biochemical Co., Ltd., Shanghai, China).

Preparation of GLSP sediment

GLSP relics obtained post polysaccharide extraction were dried in a vacuum oven (−0.095 MPa, D2F-6020AF, Tianjin GongXing Laboratory Instrument Co., Ltd., Tianjin, China) at 65 °C for 3 days prior to further treatment. Dried GLSP was then bleached with 30% H₂O₂ at 70 °C for 2 h according to a previous method⁴³. The pH of resulting suspension was adjusted to neutral using NaOH and universal indicator paper (pH 1–14, Shanghai SSS reagent Co., Ltd., Shanghai, China). Subsequently, the mixture was centrifuged at 7000 rpm for 10 min (Centrifuge 5810 R, Eppendorf, Germany), after which the GLSP sediment was transferred into a centrifuge tube and stored for further experimentation.

Thermochemical deacetylation (TCD) process efficiency

The GlcNAc unit in chitin is converted to GlcN forming chitosan with the repeating units of both biopolymers. In TCD process, NaOH solution (20 w/v%) was prepared by dissolving NaOH flakes in DI water. The alkali solution was then added to GLSP sediment (0.5 g) at a solid-liquid ratio of 1:15 (g: mL) into a conical flask. Individual GLSP suspensions were then heated at 90 °C (Thermostatic Water Bath, DK-8D, Jinghong Laboratory Instrument Co., Ltd., Shanghai, China) for pre-determined time periods (from 1 to 3 h). Subsequently, HCl was added in drop-wise fashion to adjust the pH, after which neutral suspensions were allowed to stand overnight (14 h). Suspensions were then centrifuged at 7000 rpm for 10 min and resulting sediments were collected and dried by in a vacuum oven (0.95 MPa, D2F-6020AF, GongXing Laboratory Instrument Co., Ltd., Tianjin, China) at 65 °C for 3 days, which was final product (chitosan).

Ultrasound-assisted deacetylation (USAD) process efficiency

NaOH solution (20 w/v %) was prepared by dissolving NaOH flakes in DI water. The alkali solution was then added to GLSP sediment (0.5 g) at a solid-liquid ratio of 1:20 (g: mL) into a centrifuge tube. Several identical suspensions were prepared and subjected to ultrasound irradiation for 5 to 75 min at the ambient temperature (25 °C) using the ultrasonic transducer (Branson Digital Sonifier 250, Danbury, Conn., USA). During experimentation, the temperature of deionized water was monitored every 5 min using a mercurial thermometer (MC, 30 cm, 0 °C–200 °C). The actual temperature of irradiated sample varied with irradiation time (Supplementary Fig. S1). For ultrasound irradiation process, the device was equipped with a ultrasonic probe (diameter = 1.3 cm, ν = 20 kHz). The distance from ultrasound probe to centrifuge tube was maintained at 2 cm, while the total volume of DI water (2 L) occupying the glass utensil (20 × 15 × 15 cm) was also kept constant. Following ultrasonic irradiation, HCl acid was added in dropwise fashion to suspensions until neutrality was obtained. Suspensions were then allowed to stand overnight (14 h). Resulting samples were subjected to centrifugation and sediments were collected and dried in a vacuum oven (−0.095 MPa, D2F-6020AF, Tianjin GongXing Laboratory Instrument Co., Ltd., Tianjin, China) at 65 °C for 3 days, after which the final product (chitosan) was obtained.

Morphology assessment

Field emission environmental scanning electron microscopy (SEM, Quanta FEG650, FEI, China) were used to study surface features of raw GLSP and resulting chitosan products. For SEM analysis, an accelerating voltage of 20 kV was used. Samples were fixed on metallic stubs using double-backed conductive tape. Prior to analysis, all samples were sputter-coated (108 Auto Cressington Sputter Coater, Ted Pella, INC.) with a thin layer of gold under vacuum for 60 s using a current intensity of 25 mA.

DD, [η] and ��¯ measurements

The DD (%) of chitosan was obtained using Eqs (1) and (2) as reported by Brugnerotto et al.⁴⁴:

A1320/A1420=0.3822+0.03133DA

(1)

DD(%)=1−DA

(2)

where DA is degree of acetylation, A₁₃₂₀ and A₁₄₂₀ absorbance values obtained at 1320 and 1420 cm⁻¹, respectively.

Chitosan was added to NaCl (40 mL, 0.2 M) and acetic acid (0.1 M) at a 1:1 solution volume ratio. The suspension was then subjected to ultrasound at the ambient temperature (25 °C) for 1 h. A viscometer (DV2TLVCJ0, Brookfield, USA) was used to determine [η] at 25 ± 0.5 °C. Each sample was measured in triplicate. [η] was calculated using Eqs (3–5)⁴⁵:

���=�−�0�0

(3)

�r=��0

(4)

[�]=1�2(���−ln��)

(5)

where,η₀ is solvent viscosity, η is solution viscosity, η_sp is a specific viscosity value and η_r is the ratio of η_r and η₀.

Mv¯ (viscosity average molecular weight) was calculated from [η] using the Mark-Houwink equation, shown as Eq. (6)⁴⁶, where κ, α are variable parameters dependent on solution and temperature⁴⁷.

[�]=�M¯��

(6)

here, the values κ = 1.81 × 10⁻³ L/g and α = 0.93 were used to calculate viscosity average molecular weight (Mv¯). Since Mv¯ is reduced upon chitin conversion to chitosan, polymer distribution and agglomeration are also affected. Therefore, a decrease in Mv¯ serves as a useful indicator for the chitosan conversion process.

Fourier transform infrared spectroscopy (FT-IR)

FT-IR spectroscopy (IR Affinity 1, Shimadzu, Japan) was used to confirm presence or absence of functional groups in materials. Samples were prepared using the KBr pellet pressing method⁴⁸, and a scanning range of 4000–400 cm⁻¹ was selected. In brief, 2 mg of C-C, C-T and C-U were mixed and grinded with ~200 mg KBr powder using a pestle and mortar. Mixtures were compressed into transparent pellets using powder compressing machine (FW-4A, TUOPU instrument Co., Ltd., Tianjin, China) under pressure (14 MPa) for 2 min. Spectra for each sample was acquired from 20 scans at a resolution of 4 cm⁻¹.

Thermogravimetric analysis

TGA was performed under atmospheric conditions using a TGA/DSC1 device (Mettler-Toledo, UK). A heating step rate of 10 °C/min was selected, and the temperature ranged from 31 to 600 °C. TGA was performed on C-C, C-T and C-U, with the initial weight of the three samples recorded as 2.94, 2.94 and 3.03 mg, respectively.

X-ray diffraction (XRD)

XRD analysis was performed at the ambient temperature (25 °C) on an X-ray diffractometer (Gemini A OHra, Oxford Varian, UK). Samples were measured using 1°of DivSlit and 10 mm of DivH.L.Slit, and examined at 40 kV/30 mA. Data was obtained in the 2θ range 3–60° at continuous scanning deploying a step of 0.02° at a step speed of 5°/min. The CrI was calculated using the following Eq. (7) ¹⁶:

CrI110=[(I110−Iam)/I110]×100

(7)

where, I₁₁₀ and I_am are the maximum diffraction and amorphous diffraction intensities at 2θ ≈ 20° and 13°, respectively.

Cell culture and viability assay

Cell viability was assessed using CCK-8 (cell counting Kit-8 reagent, Dojindo Laboratories, Kumamoto, Japan). L929 cells were cultured in modified eagle’s medium (MEM) (Gibco, Carlsbad, California US) supplemented with 10% fetal bovine serum (FBS, Gibco) in 6 cm diameter cell culture dishes, under standard conditions at 37 °C in a humid atmosphere (5% CO₂) for 48 h. A cell suspension (density = 1.4 × 10⁵cells/mL) was obtained. Selected materials were disinfected using UV irradiation for 2 h before addition to cell culture medium. 100 μL of cell suspension was pipetted into a 96-well plate, which was incubated at standard conditions for 24 h. GLSP, C-T, C-U or C-C (1, 0.1 and 0.01 mg/mL of each) were added to a 96-well plate and incubated at standard conditions for 24 h. 10 μL CCK-8 solution was then added and the absorbance value from 8 wells (post 4 h incubation, for each condition) was assessed using a microplate reader (spectra Max 190, NanoDrop, USA) at 450 nm. In addition, 1 mL of the original cell suspension was added to 2 mL MEM directly into a cell culture dish (Φ = 3 cm) and incubated at standard conditions for 24 h. The incubating medium was then removed and 3 mL of fresh medium was added along with 3 mg of GLSP, C-T, C-U or C-C and incubated for 24 h. Fluorescence microscopy (Olympus, BX61W1-FV1000, Tokyo, Japan) was used to observe the impact of biopolymer growth on cells. Alexar Fluor 546 phalloidin (Invitrogen, Carlsbad, California USA) and 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI, Invitrogen) staining reagents were used. Cell treatment was performed according to a previous method⁴⁹ prior to fluorescence microscopy. Stained cells were analyzed further using SEM to determine cell and biopolymer interaction. For SEM analysis, samples were coated with a layer of gold as described in 2.3.1, although an accelerating voltage of 15 kV was used.

Control groups consisted of cells incubated with MEM and CCK-8. Blank groups comprised mixtures of MEM and CCK-8 only. Cell viability in the control groups were defined as 100% and cell viability in treated groups was calculated using the following Eq. (8):

Cellviability(%ofuntreatedcells)=[(As−Ab)/(Ac−Ab)]×100%.

(8)

Here, As, Ac and Ab are the absorbance values for experimental, control and blank groups, respectively.

Antibacterial activity

Antibacterial activity of GLSP, C-T, C-U or C-C was based on a plate assay method. Inhibition zone assay was used to indicate antibacterial properties against two common pathogens; E. coli ((NW1014 (8099), Nanjing Maojie Microbiology Technology Co. Ltd., Jiangsu, China) and S. aureus (CMCC (B)26003, Shanghai Luwei Microbial SCI. & TECH. Co., Ltd., China). 300 mg of selected biopolymers (e.g. GLSP, C-T or C-U) were pressed into discs using a compressing machine (FW-4A, Tianjin TUOPU instrument Co., Ltd., Tianjin, China). 0.4 mL inoculums containing ~1.5 × 10⁶ CFU/mL of either E. coli or S. aureus were added to violet red bile agar (Qingdao Hope Bio-Technology Co., Ltd., Shandong, China) plates and baird-parker agar base (Qingdao Hope Bio-Technology Co., Ltd., Shandong, China) plates, respectively. Egg-yolk tellurite emulsion was then added to baird-parker agar base culture system (Qingdao Hope Bio-Technology Co., Ltd., Shandong, China). Compressed GLSP, C-T or C-U discs were placed on inoculated agar plates (10 cm diameter) after which they were incubated (SHP-080 Biochemical Incubator, Shanghai Jinghong Laboratory Instrument Co., Ltd., Shanghai, China) at 37 °C for 24 h. The diameter of inhibition zone was measured for all samples.

Fluorescence microplate reader analysis

0.4 mL inoculum containing ~1.5 × 10⁶ CFU/mL of either E. coli or S. aureus were incubated in nutrient broth (Qingdao Hope Bio-Technology Co., Ltd., Shandong, China) and 7.5% sodium chloride broth (Qingdao Hope Bio-Technology Co., Ltd., Shandong, China), respectively. E. coli and S. aureus were stained using 25 μg/mL propidium iodide (PI) (Solarbio Life Science, Beijing, China) for 20 min and 25 μg/mL fluorescein diacetate (FDA) (Solarbio Life Science, Beijing, China) for 20 min, respectively, at the ambient temperature (25 °C) prior to fluorescence analysis. Fluorescence analysis was carried out using Fluorescent microplate reader (FlexStation II, NanoDrop, USA). Control groups comprised bacteria incubated in broth only for 24 h at 37 °C.

Statistical analysis

All experiments were performed in triplicate and data is given as mean ± standard deviation (n = 3). Statistical analysis was performed using SPSS software (SPSS Statistics v18, IBM, UK). All statistical data was plotted using Origin software (OrginLab, USA).

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Acknowledgements

This research was financially supported by the National Nature Science Foundation of China (No. 81771960), the Fundamental Research Funds for the Central Universities (2017QNA5017) and Key Technologies R&D Program of Zhejiang Province (2015C02035).

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Authors and Affiliations

Key Laboratory for Biomedical Engineering of Education Ministry of China, Zhejiang University, Hangzhou, 310027, PR China

Li-Fang Zhu, Zhi-Cheng Yao & Ming-Wei Chang

Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou, 310027, PR China

Li-Fang Zhu, Zhi-Cheng Yao, Jing-Song Li & Ming-Wei Chang

Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester, LE1 9BH, UK

Zeeshan Ahmad

Contributions

Li-Fang Zhu performed the experimental work. Zhi-Cheng Yao, Zeeshan Ahmad and Jing-Song Li helped with experiments, and analysed the data. Ming-Wei Chang supervised and designed the research. All authors revised the manuscript and reviewed the manuscript.

Corresponding author

Correspondence to Ming-Wei Chang.

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