Investigation of Galactomannan/deacetylated Chitosan Nanocomposite Films and their Antibacterial Properties

A little packaging is a wonderful thing, a lot of packaging is a nightmare, particularly when landlls around the world threaten to engulf our living space. The topic of edible packaging is still of interest to the food industry and other organization funding research to solve packaging dilemmas. In this research, galactomannan (GM) was used as raw material and deacetylated chitosan (DE-ChN) was used as strengthening modier to prepare GM based packaging lms. The chemical structure of the composite lm was analyzed with SEM and FTIR. The properties inuence for lms of different DE-ChN content were studied. The obtained GM/DE-ChN nanocomposite lms showed superior hydrophobicity and high tensile strength. The nanocomposite lms against Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae showed great antibacterial properties. Moreover, the GM and GM/DE-ChN nanocomposite lm showed no toxicity to RAW264.7 macrophage cells. The nal obtained GM/DE-ChN packaging lm provides a foundation for the potentials for futural plastic packaging alternatives.


Introduction
Consumers' growing awareness of healthy lifestyles has prompted people to explore new technologies that can extend the shelf life of food without using preservatives. Due to its ability to improve the quality of foods, edible lms and coatings are specially considered in food preservation. Today, traditional biobased polymers from biomass have been gradually applied to food packaging lms and coatings materials, and the commercialization of biopolymer lm and coating has become more profound (Galus and Kadzińska 2015). Natural substrates such as polysaccharides, proteins, and lipids can be used to make edible lms and coatings (Gutiérrez et al. 2015). The changes in the mechanical properties and barrier properties of the main components in the biopolymer matrix have aroused people's interest in composite structures, which enables people to explore the complementary advantages of each component and minimize its shortcomings (Lee et al. 2004).
Polysaccharides such as starch, cellulose, chitosan, pullulan and Tara gum are some of the most normally used materials for producing edible lms (Wu et al. 2012;Xiao et al. 2012). Galactomannan (GM) is a representative polysaccharide, GM derived from the Sesbania cannabina seed endosperm is a natural renewable polysaccharide, which can effectively reduce the use of non-renewable petroleum resources when preparing food packaging materials. At the same time, it can be used as a safe edible lm to reduce consumers' concerns about the safety of food packaging. However, the single GM lm has poor mechanical properties, and is brittle, which is not conducive to its application. It is often necessary to add hydroxyl groups and polyols to improve the tensile strength, exibility, etc. of the lm, avoiding the surface breakage of the lm after preparation, and making the lm surface smooth (Li et al. 2019).
Glycerin, polyvinyl alcohol and polysaccharides containing hydroxyl groups are commonly used plasticizers. These plasticizers are similar to polysaccharides because of their molecular structure.
Hydrogen bonding will occur between shared hydroxyl groups, but different plasticizer molecules differences in structure, size, C, O element content and spatial con guration lead to different lm-forming properties (Antoniou et al. 2014).
There have been a large number of literatures reported that chitosan has broad-spectrum antibacterial properties, and has a good inhibitory effect on a variety of bacteria, fungi, and even some viruses. Park et al. found that the fresh-keeping time of strawberries covered with chitosan lm was prolonged and the water loss was signi cantly reduced (Park et al. 2005). In addition, chitosan also has broad application prospects in the biomedical industry.
The antibacterial properties of DE-ChN are closely related to its degree of deacetylation, molecular weight, solvent and pH of the system. The surface of DE-ChN is rich in amino groups, which is the key to the antibacterial properties of DE-ChN. At present, a large number of scholars have studied the in uencing factors of chitosan's antibacterial properties (Sudarshan et al. 1992;Kong et al. 2010). Researchers have found that the source (Chien et al. 2016), deacetylation degree, molecular mass, and concentration of chitosan have a great in uence on the antibacterial effect Zheng and Zhu 2003). At the same time, the pH, water content, external factors such as solvents also have a signi cant impact on the antibacterial ability of chitosan.
In this paper, different amount of DE-ChN were added into GM to prepare GM/DE-ChN nanocomposite lms. The effects of different concentration of DE-ChN on the nanocomposite lm's mechanical properties, hydrophobicity, thermal stability and four common antibacterial properties were investigated.
The nal prepared GM based functional food packaging lm provides a foundation for the potentials for futural plastic packaging alternatives. The prepared functional food packaging lm based on GM can be used as a plastic packaging alternative.

Materials
Sesbania cannabina seed endosperm was purchased from Lianyungang city (Jiangsu Province, China). Chitin with a degree of deacetylation of 6.9% was puri ed from crab shells (Eriocheir sinensis) as described in the Liu et al work (Liu et al. 2016

Preparation of GM
In order to separate GM, the Sesbania cannabina seed endosperm was swelled in distilled water for 24 hours, and then mechanically crushed using a juicer. The resulting slurry was stirred at 50 o C for 24 h, and then centrifuged to separate the suspended solids. From the supernatant GM solution, GM was precipitated with 95% (v/v) ethanol (The volume ratio of GM and ethanol was 1:3) and washed with additional ethanol of 95% (v/v). The precipitated and washed GM was then freeze-dried to yield a powder after the ethanol was evaporated under a fume hood. Finally, the average molecular weight (Mw) of GM was calculated by gel permeation chromatography to be 430,000 Daltons (GPC, Agilent Technologies, Santa Clara, CA, USA), the extracted GM sample consisted of 91.43 ± 0.36 % galactomannan was determined by high performance liquid chromatography (HPLC, ICS 3000, Dionex, USA). The molar ratio of mannose to galactose was determined to be 2.10. These results indicated that the extracted GM was a biomacromolecular with higher purity and high molecular weight, and thus is an ideal matrix for making various materials.

Preparation of DE-ChN
Partially deacetylated chitin was prepared as described in a previous report (Fan et al. 2010). Puri ed chitin was suspended in a (30%wt) NaOH solution and heated at 90 o C for 4 h, partially deacetylated chitin with degree of deacetylation of about 25% was prepared successfully, after deacetylation, the sample was collected and washed with distilled water until the supernatant reached a neutral pH. Then the sample was freeze-dried.
To prepare DE-ChN, the sample after freeze-dried was dispersed in distilled water at a concentration of 0.4% (w/v), and the pH was adjusted to 3 using acetic acid under constant stirring; then, the suspension was homogenized at 10,000 rpm for 30 s by a homogenizer (T25, IKA, Germany) and the sonication procedure was conducted at 500 W for 5 min (an interval of 3 s) by an ultra-sonicator (VCX500, USA) and repeated for 5 times; nally the dispersion was centrifuged, and the supernatant was collected as the DE-ChN. For further use, DE-ChN dispersions with 0.5% (w/v) were prepared by dilution or concentration using rotary evaporation.

Preparation of GM/DE-ChN nanocomposite lms
To prepare GM/DE-ChN nanocomposite lms, the GM solution (10 g/L) was rst obtained at 50 o C with 3 h constant stirring. The GM/DE-ChN nanocomposite lms were prepared by ultrasonic dispersion and solution casting. A series of DE-ChN with mass fractions of 20%, 40% and 60% (according to the dry weight of GM) were gradually added to the GM solution under stirring. After ultrasonic defoaming treatment, the above mixture was poured into a polytetra uoroethylene mold diameter of 9 cm and dried in an oven at 40° C and the nanocomposites lms were obtained. All lms were stored at 25°C and 50% relative humidity (RH) for at least 48 h before measurement. The composite lms were named GM,

Characterization of GM/DE-ChN nanocomposite lms
The micro-morphology of nanocomposite lms was observed by scanning electron microscopy (SEM) using a Quanta 200 (FEI, American) microscope. The cross section of the lms was obtained by applying liquid nitrogen freeze-cracking composite lms. To prepare the sample for imaging, the lm was pasted onto a row of staples with the cross section facing upwards. The xed lm was then coated with a 20 nm gold layer before recording SEM images.
The light transmittance spectra of nanocomposite lms were detected in the range of 200-900 nm using a UV-vis spectrophotometer (Ultrospec 2100, Amersham Bioscience).

Antibacterial performance
To investigate the germicidal ability of the GM/DE-ChN nanocomposites lms, a shake ask method was used to test against B. subtilis, E. coli, S. aureus and S. pneumoniae. After cultivation in liquid lysogeny broth (LB) medium (containing 10 g/L peptone, 5 g/L yeast extract, and 10 g/L sodium chloride) for 12 h at 37°C, the microorganisms were diluted with the LB liquid culture medium to obtain a bacterial suspension with approximately 1.0×10 6 CFU/mL concentration. According to Xu et al work (Xu et al. 2019), 0.01g composites lm was immersed in 10 mL of bacterial suspension adjusted pH 5.5. The solution was then cultured in a shaker stirring at 150 rpm and at 37°C for 6 h. Then, a 100 µL of 7-fold serial dilutions was pipetted into agar plates. The mixture was incubated in incubator at 37°C for 12 h and the number of bacteria was obtained using the colony forming count method.

Statistical analysis
Statistical analysis was performed using a commercial SPSS program (SPSS 20.00, SPSS INC., Chicago, Illinois, USA). The data were expressed as mean ± standard deviation (SD) and p < 0.05 was taken as the minimal level of signi cance.

Results And Discussion
Morphology and UV absorption of GM/DE-ChN nanocomposite lms DE-ChN has been widely reported in the preparation of composite lm, which endows the composite lm excellent chemical and physical properties. Light transmittance is an auxiliary means to evaluate the compatibility of polymers. If the compatibility between DE-ChN and GM molecules in the GM/DE-ChN nanocomposite lm is poor, then light re ection or scattering will occur at the phase interface between them, the light transmittance of the lm is reduced. The lm prepared by mechanically mixing had a smooth surface and higher transparency due to its high level of uniformity (Gennadios et al. 1998). Digital images of the composite lm showed the transparency of the lm as can be seen from Fig. 1. The original GM lm was slight matt, and the light transmittance of the GM lm compounded with DE-ChN increased from 73% (original GM lm) to 86% (GM/60% DE-ChN lm). Both lms had no UV absorption. Figure.

FT-IR analysis of GM/DE-ChN nanocomposite lms
To investigate the effect of DE-ChN on the structure of the GM lms, the chemical bonds in the original GM lm, DE-ChN lm and the GM/DE-ChN nanocomposite lm were surveyed using ATR-FTIR. When a H atom was connected to O, F and other atoms with a small radius and strong electronegativity, the electron cloud moved and exposed the H atom nucleus, which was easily electrostatically attracted to other atoms with lone pairs of electrons, forming a strong dipolar effect, this was the hydrogen bond, which would affect the infrared spectrum. From Figure.  in methylcellulose-based lms signi cantly improved the tensile strength of lms with the reduction of viscoelasticity. The 50% chitosan containing starch-based lms was considered as the optimum because the lms had good strength (47 MPa). Moreover, the appearance of the lms was quite transparent. As the content of chitosan increased, the hydrogen bonding force between polysaccharide molecules increased, and the tensile strength of the corresponding lm also increased.

Antibacterial performance
The sterilization property against E. coli, B. subtilis, S. aureus and S. pneumoniae of composite lms was determined by the colony counting method. The bacterial suspension was inoculated in the solid medium and the numbers of total viable counts were visualized in Figure. Figure 10 showed the antibacterial effect of the lm on S. aureus, the GM/20% DE-ChN lm had an inhibitory rate of 13% against S. aureus, GM/40% DE-ChN and GM/60% lm had an inhibitory rate of 46% and 47% against S. aureus, and the inhibitory rate of DE-ChN lm against S. aureus was 57%, the antibacterial effect was particularly signi cant (P < 0.001). The antibacterial effect of the composite lm on S. pneumoniae was shown in Fig. 11. The bacteriostatic rate of GM/20% DE-ChN against S. pneumoniae was 29%, GM/40% DE-ChN, GM/60% DE-ChN and the inhibitory rates of DE-ChN against S. pneumoniae were signi cantly 88%, 92% and 94%, respectively (P < 0.001). This was because the amino group in chitosan was cat ionized at pH 5.5, then the interaction between chitosan and bacteria was activated (Raafat et al. 2008). The permeability of the bacterial cell membrane changes, causing internal osmotic imbalance, leading to the leakage of electrolytes such as K + and other low-molecular-weight protein components in the cell, and nally leading to the apoptosis of microorganisms (Liu et al. 2004). The different antibacterial activities of the lms against gram-positive bacteria and gram-negative bacteria might be due to the different cell wall structures of the two bacteria (Jing et al. 2007). The cell wall of gram-positive bacteria was composed of polypeptidoglycan, and chitosan could easily pass through the peptidoglycan network and directly act on the cell membrane. However, the cell wall of gramnegative bacteria contained not only an inner membrane composed of peptidoglycan and lipopolysaccharide, but also an outer membrane composed of lipoproteins and phospholipids. The outer membrane of gram-negative bacteria could act as an effective external barrier for macromolecules, which might prevent the macromolecule chitosan from reaching the plasma membrane. Therefore, chitosan showed a stronger antibacterial effect on gram-positive bacteria than gram-negative bacteria.

Conclusions
The GM/DE-ChN nanocomposite lms showed great antibacterial properties for the prohibition of E. coli, B. subtilis, S. aureus and S. pneumoniae. When the additional amount of DE-ChN was 60% into GM, the antibacterial rate of GM/DE-ChN nanocomposite lm against E. coli, B. subtilis, S. aureus and S. pneumoniae reached 33%, 99%, 47% and 92%. The obtained GM/DE-ChN nanocomposite lms had improved tensile strength (107 MPa), which was 343% higher than the original GM lm. The GM/DE-ChN nanocomposite lms had excellent hydrophobicity of 107 o , which was 147% higher than the original GM lm. Moreover, the GM/DE-ChN nanocomposite lm showed no toxicity to macrophage cells. The above conclusions indicated that the biocompatible DE-ChN had signi cant antibacterial properties against common bacteria. The physical and chemical properties of GM lms were improved. Therefore, combining DE-ChN with renewable and sustainable packaging materials to prepare packaging materials with antibacterial properties will provide a new method for food packaging without harmful additives.