Calcined and Hydrated Shell Powder with Layered Porous Structures for Food Sterilization and Pesticide Residue Removal

Shell powder, a natural source product with excellent adsorption and antibacterial properties, has exhibited a broad application prospect in daily life. Herein, low-temperature calcined shell powder (LCSP) and high-temperature calcined and hydrated shell powder (HCSP) were prepared using shells as raw materials in different calcination processes. The surface structures and the chemical compositions were analyzed and the specific surface areas and pore size distributions were measured. The LCSP and HCSP reserved the unique layered porous structures of the shell. The main composition of LCSP is CaCO3, which also contains a small number of organisms. The main composition of HCSP is Ca (OH)2, which also contains CaO and a small amount of CaCO3. According to the better adsorption characteristic of the HCSP, the antibacterial activities and the removal abilities of pesticide residues of HCSP were systematically studied. The results indicated that HCSP exhibited excellent antibacterial activity (> 99.99%) and high efficiency in removing common pesticide residues (> 90%).


Introduction
For decades, detergents commonly used in household life consisted of chemical products containing large amounts of artificial surfactants, and 60% of these artificial surfactants entered the aquatic environment and caused great damage [1].With increasing attention to food safety and health, natural food cleaning technologies have begun to attract people's attention [2][3][4][5][6][7][8].Shell powder, reported excellent adsorption and antibacterial properties as well as its biological, eco-friendly nature, has exhibited a broad application prospect in daily life [2,3].
The main component of the shell is 95% calcium carbonate and about 5% organic matter by weight.After high-temperature calcination, the main component of shell powder is CaO [4].According to reports, the shells of oysters [5,6] and mussels [7] exhibit antibacterial activity after heat treatment.Recently, it has been proven that calcined shell powder particles have higher bactericidal, sporicidal, and viral effects [8,9].Hu et al. [10] have shown that calcined shell powder has a significant bactericidal and antibacterial effect on foodborne pathogens, and can be used as an antibacterial agent.Calcined shell powder has also been applied in food, successfully disinfecting fresh vegetables and bean sprouts [11,12], Frankfurt sausages [13], chicken [14], frozen meat [15,16], and fresh whole fish [17].Tsuruma et al. [18] successfully suppressed the formation of the CaCO3 scale by coating calcined shell powder with sorbitol, improving the hygroscopicity and treatment quality of calcined shell powder, while maintaining its antibacterial effect and preservation effect on fresh agricultural products.Jun et al. [3] hydrated scallop powder into a slurry after high-temperature calcination, exhibiting stronger bactericidal effects compared to NaOH at the same pH.In addition, calcined shell powder can be used as an efficient and eco-friendly pesticide residue removal.Ma et al. [2] developed an efficient mussel shell-based adsorbent, whose maximum adsorption capacity for bifenthrin and Cypermethrin can reach 1.05 and 1.79 mg/g, and the removal efficiency exceeds 40% and 70% respectively at the maximum initial concentration.
Therefore, expanding the use of shells in food sterilization and pesticide residue removal applications is particularly promising.Herein, we have proposed a method to obtain the calcined and hydrated shell powder reserving the unique layered porous structures of the shell.The surface structures and the chemical compositions were analyzed and the specific surface areas and pore size distributions were measured.The antibacterial activities and the removal abilities of pesticide residues of HCSP were systematically studied, which shows that HCSP has potential in food sterilization and pesticide residue removal.

Preparation of LCSP
First, scallop shells were scrubbed repeatedly with a brush to remove the impurities on their surface.Then, they were immersed in 5% NaOH aqueous for 1 h.After washing with deionized water continuously until the pH was neutral, they were dried in an oven and broke into pieces.Next, calcination of the pieces was carried out using a muffle furnace at 300 ℃ for 4 h.Once cooled completely, they were further crushed and transferred into a sealed container.Finally, deionized water (40 times the weight of the calcined shell powder) was added for complete hydration, thus, the low-temperature calcined shell powder was obtained.

Preparation of HCSP
The same treatments as above for the scallop shells were conducted to obtain pieces.Then, they were calcined by the muffle furnace at 1100 ℃ for 4 h.After cooling down, crushed and transferred into a sealed container, and added 40 times the weight of deionized water.Thus, complete hydration occurred to obtain the high-temperature calcined shell powder.

Scanning electron microscopy (SEM) Tests
LCSP and HCSP were placed on the sample stage and sputtered with platinum for 45 s using a sputter coater and vacuumed at a voltage of 15 kV.Then, their structures were observed by the scanning electron microscope (SU1000, Hitachi) at different magnifications.

Characterization of Brunner−emmet−teller measurements (BET)
The BET-specific surface area and DR pore size distribution of samples were determined using an automatic specific surface area analyzer.The conditions were as follows: a degassing temperature of 150 ℃, a degassing duration of 360 min, and a constant temperature bath of 77.3 K/1.8 (mm/H).

Fourier Transform Infrared Spectroscopy (FT-IR) Test
The samples were dried by infrared light, and then they were prepared by the tablet method.Then, they were measured by a Fourier Transform Infrared Spectrometer (Tensor27), the scanning range was 400 ~ 4000 cm with a resolution of 2 cm -1 .

X-ray Diffraction (XRD) Test
Using an X-ray diffractometer, the XRD measurement was carried out on the sample under the condition that the diffraction angle (2θ) was 10-90 °, λ=1.5418Å, Cu Kα target, and the scanning speed was 0.3 s -1 .

X-ray Photoelectron Spectroscopy (XPS) Test
An X-ray photoelectron spectrometer is used for detection and analysis.The vacuum degree of the analysis chamber was 8 × 10 -10 Pa.The ion gun type was Al K-Alpha, and Alka rays (hv = 1486.6eV) were used as the excitation beam.The working voltage was set at 12.5 kV, and the filament current was 16 mA.Five cycles of signal accumulation were performed.An ion gun was used to etch and thin the sample.The etching spot size was 650 µm, the step length was 1 eV, and the number of energy steps was 1361.

Antibacterial tests
Antibacterial activity evaluation was carried out according to the quantitative sterilization test operation procedure of China (2002 version).All the samples (including shell powder, calcium oxide, calcium hydroxide, calcium carbonate, baking soda, salt, and flour) were prepared into a 3.0 g/L suspension, respectively.4 mL suspension was added into a centrifuge tube and evenly mixed with 1mL bacterial suspension (2.5 × 10 8 cfu/mL).After incubating at 37 ℃ for 10 min, 100 μL solution and a series of 10fold dilutions were transferred onto the LB plate and incubated for 24 h to count the number of colonies.4 mL PBS was used as the control, and the antibacterial ratio was calculated according to the following equation: where CFUs and CFUc signified the number of colonies in the sample and control (PBS), respectively.4

Evaluation of pesticide residue removal
200 g of fruits and vegetables were put in a beaker, and 0.5 ppm of pesticide standard was evenly sprayed on the surface of the fruits and vegetables while shaking.Then, they were placed in a ventilated cabinet to allow the solvent to evaporate completely, and divided into two groups.One group was washed with deionized water for 5 min, while the other one was treated with a 0.1% shell powder aqueous suspension.After draining the water, pesticide residue tests were conducted by gas chromatography-mass spectrometry according to GB/T 232008-2016.All the samples should be pre-treated following GB/T 23200.113-2016.

Microstructures of LCSP and HCSP.
Figure 1 shows the scanning electron microscope (SEM) results of LCSP and HCSP magnified 1000 times, with the top right corner showing the SEM results magnified 10,000 times.Figure 1a revealed that the particle sizes of LCSP range from 2 to 10 μm, while Figure 1b showed that the particle sizes of HCSP range from 2 to 15 μm.This was because the LCSP was ground, and the HCSP was directly formed after hydration.As seen by the magnified SEM results, the structures of LCSP and HCSP reserved layered structures of shells, and more porous nanostructures were found in HCSP.This was the result of the decomposition of the organic components in the shell powder after high-temperature calcination.

LCSP and HCSP specific surface area analysis.
Figure 2 presents the N2 adsorption-desorption isotherms of LCSP and HCSP, corresponding to Type III and Type IV pore models [19], respectively.As shown in Figure 2a, the specific surface area of the LCSP was 4.60 m 2 /g, and the average pore diameter was 3.80 nm. Figure 2b revealed that the specific surface area of the HCSP was 13.40 m 2 /g, and the average pore diameter is 4.11 nm.As the decomposition of organic matter during high-temperature calcination, an increase in pore structures and pore diameter was found in HCSP.Hence, the HCSP shows more potential for adsorption because it exhibits a higher specific surface area and porosity than the LCSP.

FT-IR and XPS analysis of LCSP and HCSP
In Figure 3a, LCSP presented distinct absorption peaks at 875 cm -1 and 711 cm -1 , which were identified as the absorption peaks of CO3 2- [20].Compared to LCSP, a strong absorption peak near 3401 cm -1 identified as the -OH could be observed in the curve of HCSP, which was associated with the hydration process [21].These spectral features suggest the potential presence of calcium hydroxide within the HCSP compound.Based on the above analysis, the main components of LCSP include calcium carbonate and calcium oxide; the main component of HCSP is calcium hydroxide.
Furthermore, the elemental characteristic peaks of Ca, C, O, and N in LCSP and HCSP were identified in Figure 3b.Unlike LCSP, HCSP does not contain N elements.This was attributed to the hightemperature of the shell powder calcined process, which leads to a more thorough decomposition of organic matter [22].The content of C and O elements in HCSP was significantly reduced due to the high-temperature calcined process that converts most of the calcium carbonate into calcium oxide.Therefore, the main components of HCSP include calcium hydroxide, calcium oxide, and a small amount of calcium carbonate.In contrast, the main components of LCSP were calcium carbonate calcium oxide, and a small amount of organisms.Figure 3c shows the XRD results of LCSP and HCSP.A significant difference could be seen that the main component of the LCSP was CaCO3 (b), while one of the HCSP was fully transformed into Ca(OH)2 (a) [4,23,24] These results were consistent with the FT-IR and XPS results.

Antibacterial activity of the HCSP
According to the results of compositions and structures of LCSP and HCSP, the HCSP was chosen to systematically study the antibacterial activities and the removal abilities of pesticide residues.
Figure 4a presents the antibacterial activity of HCSP against E. coli and S. aureus with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 g/L).When the concentration of HCSP was under 1.0g/L, it exhibited poor antibacterial activity, which was 26.27% and 25.00% against E. coli and S. aureus, respectively.While the antibacterial ratio was up to 99.99% when the concentration was over 2.0 g/L.Furthermore, the excellent bactericidal performance of HCSP was also assessed by the comparison with daily chemical products.Figure 3b shows the different antibacterial activities of HCSP, calcium oxide, calcium hydroxide, calcium carbonate, baking soda, salt, and flour.It could be seen that baking soda, salt, and flour demonstrated no antibacterial activities against E. coli and S. aureus.LCSP presented an extremely poor antibacterial ratio of 6.00% and 3.66% against E. coli and S. aureus, respectively.HCSP exhibited high antibacterial ratios of 99.99%.This could be attributed to the alkaline effect caused by the hydration of calcium oxide in HCSP, which weakened the survival ability of microorganisms and destroyed the cell structure, thereby killing them [25,26].The detailed mechanism could be found in the previous works [27,28], when bacteria contacted the alkaline surface of HCSP, the transmembrane proton concentration gradient of bacteria was weakened, so its respiration action was disturbed.Meanwhile, the produced ⋅OH and ⋅O2 -in the environment would attack the cell membrane of the free bacteria, causing the lysis of them.

Removing agricultural residues performance of HCSP
Figure 5 exhibits the residual removal performance of HCSP against seven common pesticides (including propoxur, DDVP, cypermethrin, butachlor, DDT, fenpropathrin, and pyridoxine), pure water was used as control.It should be noted that DDVP and fenpropathrin were neutral pesticides, pyridoxine, cypermethrin, and DDT were acidic pesticides, and butachlor and propoxur were alkaline pesticides.The removal rates of pesticide residues in the pure water group were 48.75%, 26.70%, 29.26%, 5.83%, 25.77%, 5.57%, and 3.68%, respectively.In contrast, the ones in the HCSP group were 80.58%, 90.67%, 81.10%, 90.93%, 83.00%, 63.93%, and 88.83%, respectively, which were higher than the pure water group with significant increases of 31.83~85.15%.When compared to counterpart functional materials reported previously [4,29], there was still a slightly higher removal efficiency of HCSP than others, which was due to its high specific surface area and porosity after treating by high temperature calcination and hydration.This microstructure created favorable conditions for the adsorption of pesticide residues [2,25].More importantly, HCSP demonstrated a more stable removal ability than pure water, where the removal rates against acidic, alkaline, and neutral pesticides were all maintained at a relatively high level.

4.Conclusion
In summary, we have fabricated two kinds of calcined shell powder.One is calcined at low-temperature (LCSP), and the other is calcined at high-temperature (HCSP).SEM observations have indicated that unique layered porous structures of the shell were reserved both in LCSP and HCSP.The chemical compositions were analyzed by FT-IR, XPS, and XRD.It is noted that the main composition of LCSP is CaCO3, which also contains a small number of organisms.The main composition of HCSP is Ca (OH)2.BET results have shown the specific surface area of the LCSP is 4.60 m 2 /g (the average pore diameter: 3.80 nm), while the specific surface area of the HCSP is 13.40 m 2 /g (average pore diameter: 4.11 nm).According to the adsorption characteristics of two calcined shell powers, the antibacterial activities and the removal abilities of pesticide residues of HCSP were systematically studied.The results have indicated that HCSP exhibited excellent antibacterial activity (> 99.99%) and high efficiency in removing common pesticide residues (> 90%), which shows that the HCSP has potential in food sterilization and pesticide residue removal.

Figure 4 .
Figure 4.The antibacterial activities of the HCSP with different concentrations (a) and the different antibacterial activities among HCSP, LCSP, and daily necessities with a concentration of 2.0 g/L (b).

Figure 5 .
Figure 5.The removal abilities of HCSP to seven common pesticides.