Edible Thai rice film incorporated with ginger extract by microwave extraction: 1 optimization of bioactive compounds and functional properties for antimicrobials 2

21 This study aimed to integrate microwave-assisted extraction (MAE) on the dry ginger extract 22 and to develop rice-based edible film incorporated with ginger extract. An efficient MAE was 23 developed to extract the dried ginger using a 3 2 full factorial design. The optimal condition was 24 a microwave power of 400W and an extraction time of 1 min. The extraction time was a 25 significantly effective factor than microwave power, whist power was not a significant factor 26 for yield, 6-gingerol, 6-shogaol, and paradol. A crude extract of dried-ginger has antimicrobial 27 activity against S. mutans DMST 18777 with minimum inhibitory concentration (MIC) and 28 minimum bactericidal concentration (MBC) of 0.49 mg/mL and 31.25 mg/mL, respectively. 29 The rice-based edible film incorporated with 32 mg/mL of ginger extract against S. mutans 30 DMST 18777 with a mean zone of inhibition of 12.69±0.07 mm. The functional property of 31 rice film was remarkably better than the original. Significant increases in TPC, antioxidant and 32 bioactive compounds were associated with increase ginger extract contents in rice film. The 33 main phenolic compounds including 6- gingerol 6-shogaol, paradol, and zingerone, and 34 essential oils including α-curcumene, α-zingiberene, γ-muurolene, α-farnesene, β-bisabolene, 35 and β-sesquiphellandrene were found in rice film strip fortify with crude ginger extract.


Introduction
Recently, natural additives from plant extracts incorporation in edible films or coating have gained increasing attention among researchers. Natural additives have been applied to improve texture, rheology, and functional properties such as antioxidant, antimicrobial, anti-browning, of the edible films 1 . Rice (Oryza sativa L.) is one of the main foods for almost half of the global population. Rice flour is widely used as food hydrocolloids due to its inexpensive, convenient, biodegradable, and easy processability 2 . Furthermore, rice flour can be formulated with other components to improve the physical, chemical, sensory, and nutritional properties of the product 3 . The use of rice flour to produce films or coating is generally transparent, odorless, colorless, and tasteless 2 .
Zingiber officinale Roscoe (ginger) as a member of the Zingiberaceae family, is commonly used as a spice in food and as a traditional medicine in Asian countries. Many bioactive compounds of ginger such as phenolic compounds (gingerols, shogerols, and paradols), terpenes (zingiberene, β-bisabolene, and α-curcumene), polysaccharides, lipids, and organic compounds have processed multiple biological activities nowadays, for example, antioxidant activity, anticancer, anti-diabetic, anti-inflammatory, antimicrobial activity, cardiovascular protective 4 . However, the use of a conventional method to extract the bioactive compounds required a long extraction time and it affects the quality of a final product by losing some of the volatile compounds which leads to low extraction efficiency 5 . The microwave-assisted technique has been widely employed to extract phenolic compounds from plants due to its small equipment size, simplicity, and rapidness. The efficiency of the MAE was found to be two times higher than that of the conventional method 6,7 .
In the previous study, 9-months of dried ginger at 60 °C for 308 min presented the highest TPC of 12.21 µmol tannic acid/g and 6-gingerol of 12.57 mg/g 8 . Furthermore, the nonvolatile compounds including gingerols, shogaols, paradols, and zingerone in ginger exhibit 5 used. The summary of the analysis of variance (ANOVA) representing results indicated that TPC, ABTS, and FRAP values were reliable and significant (p< 0.05) with R 2 values of 0.9758, 0.9267, and 0.09005, respectively (Table 2). Microwave power and extraction time were found to be non-significant (p> 0.05) on these responses, which means that the increase of microwave power and extraction time was decreased the TPC, ABTS, and FRAP values. However, the interaction of microwave power and extraction time was represented the positive signs on the observed value. Consistent with the ANOVA results, MAE presented a negative effect for TPC ( Fig. 1 B), ABTS ( Fig. 1 C), and FRAP ( Fig. 1 D) with a significant decrease in their values when a high microwave power (>500W) and long extraction time (>2 min) was applied.

Effect of microwave power and extraction time on bioactive compounds
The highest yield of 6-gingerol was obtained either with low microwave power and short extraction time (400, 1 min) and/or high microwave power and long extraction time (800 W, 5 min). The highest content of paradol could be produced when microwave power is in a range of 400-600 W. While, the highest yield of 6-shogaol and zingerone could be achieved when microwave power is 800 W and with the long extraction time of 5 min. 6-Gingerol and paradol were showed the highest value of 73.41±1.33 mg/g and 23.51±0.87 mg/g at low power (400 W, 1 min). Whilst, 6-shogaol, and zingerone represented the highest value at high power and longer extraction time (800 W, 5 min) with the values of 16.04±1.86 mg/g and 5.79±0.06 mg/g, respectively (Table 1). However, the use of MAE on extracted bioactive compounds from ginger could be generated the model for only 6-gingerol (p< 0.05, Table 2). High values for R 2 were achieved as 0.9267 and microwave power and extraction time were found to be nonsignificant (p> 0.05). Consistent with the ANOVA and contour plots of TPC, ABTS, and FRAP, MAE represented the negative effect for 6-gingerol with a significant decrease in their values when a high microwave power (>500W) and long extraction time (>2 min) was used ( Fig. 1 E).

Optimization and validation of MAE condition
The contour response surfaces were plotted to study the interactions between the factors on the significant responses to determine the optimum levels of each factor required to obtain maximum values of the yield, TPC, ABTS, FRAP, and 6-gingerol ( Figure 2). Based on the analysis and calculation, the validation experiment was conducted as microwave power of 400 W and 1 min of extraction time. The highest yield of crude ginger (7.66±0.66%) was closed to the predicted value of 7.61%. TPC and antioxidant activities including ABTS and FRAP values were 198.24±0.74 mg GAE/g, 106.42±3.13 mgTrolox/g, and 304.62±5.49 mgTrolox/g, respectively ( Table 3). The highest yield of 6-gingerol was obtained as 71.57±3.60 mg/g. The percentage of approximated error between predicted and experimental values was in the range of 0.07-4.38%, which not above 10% of the approximated error. This indicated that the results of the validation were acceptable and consistent with the predicted values.

Antimicrobial activity of rice-based edible film
The disc diffusion analysis results revealed that the crude extract of ginger (63-500 mg/mL) showed no significant difference (p<0.05) on antimicrobial activity against S. mutans DMST 18777 with an average inhibition zone of 9.50±0.71-11.00±1.41 mm ( Table 4). The MIC and MBC of the crude extract of ginger against S. mutans DMST 18777 were 0.49 mg/mL and 31.25 mg/mL, respectively. Thus, the highest concentration of crude ginger extract that was applied into the rice-based edible film was 32 mg/mL (3.2%, w/v). Therefore, inhibition zone diameters yield by rice-based edible film disks with various concentrations (0, 4, 8, 16, and 32 mg/mL or 0.4, 0.8, 1.6, and 3.2%, w/v) of crude ginger extract against S. mutans DMST 18777 are presented in Table 3. No inhibition zone against S. mutans DMST 18777 was observed for rice film without the incorporation of crude ginger extract, which indicating that rice film alone did not affect antimicrobial activity. Furthermore, the incorporation of crude ginger extracts less than 16 mg/mL in the rice film not enough to inhibit the growth of S. mutans DMST 18777.
The efficiency concentration was 3.2 % (w/v) with an inhibition zone of 12.69±0.07 mm.

Discussions
Ginger is common and widely used as a spice and herbal medicine for a long time. The bioactive compounds such as 6-gingerol and 6-shogaol were accounted for several bioactivities including antioxidant, anticancer, antimicrobial, and anti-inflammatory 10 . Ginger is known to harden the teeth because of indirect mineralization properties, thus, ginger was validated for oral care 11 . While rice is a staple food that is consumed by half of the global population.
Furthermore, there is no information about the effect of the combination of crude ginger extract on rice film properties. In this study, we optimized the MAE condition in order to increase the yield, antioxidant activity, and bioactive compounds of dried ginger, and then combined the crude extract into an oral rice film strip.
Generally, the increase of microwave power could be increased the extraction yield with a shorter extraction time 12 . The change of antioxidant activities might be due to the generation of free radicals such as H + , OH -, and electrons through microwave radiation 4 . The highest yield of 6-gingerol was obtained either with low microwave power and short extraction time (400, 1 min) and/or high microwave power and long extraction time (800 W, 5 min), which agreed on the results of Teng et al. 5 . Under high temperatures and/or high microwave power, 6-gingerol dehydrated water (H2O) from its structure and converted to 6-shogaol 4 . If the reduction of (CH2)2 occurred, 6-shogaol will be transformed into paradol. In another case, microwave power promoted retro-aldol reaction of 6-gingerol and proposed to generate zingerone constituents with an aldehyde to deliver the products (Figure 3). The competition of these reactions can be further demonstrated by the synthesis of the 6-shogaol, paradol, and zingerone constituent's yields. The content of 6-shogaol and zingerone was gradually increased under high microwave power (>600 W) and long extraction tine (>3 min). This indicated that 6-shogaol and zingerone were produced during high temperatures, high microwave power, and also by thermal degradation of gingerol 9 . 6-Shogaol and zingerone increased with increasing the microwave power and extraction time, which results in increased ABTS and FRAP values.
This phenomenon could be explained that 6-gingerol was dehydrated and generated H + and OHradical at high temperatures or high microwave power results in produced 6-shogaol, paradol, zingerone, and its derivatives 5,6 .
S. mutans is found in the oral cavity and formed a dental plaque to prevent the permission of antimicrobial agents 11 . The oral film strip is considered one of the most convenient routes for administration due to cost efficiency and ease of administration. In this study, we observed the inhibitory activity of crude ginger extract and found that the MIC and MBC of the crude ginger extract against S. mutans DMST 18777 were 0.49 mg/mL and 31.25 mg/mL, respectively, with the inhibition zone approximately 11.0 mm. These results agree with that reported by Mathai et al. 13 , where fresh ginger extract against S. mutans MTCC 497 with the inhibition zone of 11.72±0.62 mm. Furthermore, many studies also showed the inhibition zone of fresh ginger extract against S. mutans approximately 6-18 mm [14][15][16] . The incorporation of crude ginger extracts less than 16 mg/mL in the rice film not enough to inhibit the growth of S. mutans DMST 18777. This might be due to the immobilization of rice molecules within the film and a high number of bacteria (2.76×10 6 CFU/mL or 6.44 log CFU/mL) that exceed inhibition activity 17 . The antimicrobial activity of crude ginger against S. mutans was 5 logs CFU/mL 17 . The efficiency concentration was 32 mg/mL (3.2% w/v) with the inhibition zone of 12.69±0.07 mm. It can be concluded that the produced rice-based edible film with 32 mg/mL of ginger extract has the potential to be considered for anti-caries rice film. In contrast, the starch edible film with ginger essential oils (1-3% v/w) inhibited the growth of Escherichia coli with 1.00-9.73 mm of inhibition zone 18 . It seems that gram-negative bacteria (E. coli) were more resistant to lipophilic compounds as compared with gram-negative bacteria (S. mutans), which occupied a single peptidoglycan layer structure 19 .
The presence of 6-gingerol and 6-shogaol in the rice film strip could be imparted a pungent and aromatic taste to the film. 6-Gingerol, 6-shogaol, paradol, and zingerone can inhibit reactive oxygen species and maintain their antioxidant properties 9 . Thus, the antioxidant activity of rice film might be related to the presence of TPC and bioactive compounds. The essential oil of crude ginger was found to be 44 volatile compounds 8 . In this study, the main essential oil in the rice film strip was α-curcumene, α-zingiberene, γmuurolene, α-farnesene, β-bisabolene, and β-sesquiphellandrene, as identified by GC/MS.

Conclusions
MAE was successfully used to extract TPC, 6-gingerol, 6-shogaol, paradol, and zingerone from dried ginger, and increased antioxidant efficiency within shorten extraction time by 3 2 full factorial design. The optimal condition was microwave power of 400W and extraction time of 1 min and showed the responses which were close to the predicted responses. For antimicrobial activity, the crude extract of dried-ginger against S. mutans DMST 18777 with MIC and MBC values of 0.49 mg/ml and 31.25 mg/ml, respectively. Furthermore, it has been recently shown that antimicrobial activity of rice-based edible film incorporated with 3.2 % (w/v) ginger extract was preferable applied for anti-caries rice film with the bioactive compounds and essential oil that can exhibit the growth of bacteria. Rice film incorporated with 32 mg/mL ginger extract showed a significant antibacterial effect against S. mutans DMST 18777, which proved that ginger is being released from the film in the surrounding culture medium and that its antimicrobial activity has been preserved after the fortify in a polymer. The presence of phenolic compounds including 6-gingerol 6-shogaol, paradol, and zingerone, and essential oils including α-curcumene, α-zingiberene, γ-muurolene, α-farnesene, β-bisabolene, and βsesquiphellandrene in the rice-based edible film might be helpful for several therapeutic effects.
Thus, the development of rice-based edible film incorporated with dried ginger extract to the product may constitute an alternative way against the resistance to S. mutans.

Raw material
The fresh and 9 months matured rhizomes of ginger were obtained from the Hsu Chuan Foods

Microwave-assisted extraction (MAE) experimental design
The MAE was carried out on the laboratory microwave (Toshiba, Model ER-300C(S) Power Max 900, frequency 2.45 × 109 Hz, Japan). A 3 2 full factorial design was constructed to investigate the influence of two variables, including microwave power at 400, 600, and 800 W and the reaction time at 1, 3, and 5 min ( Table 1). The filtrate was collected and concentrated using a rotary evaporator under vacuum at 50 ± 4°C, finally, dry extract yield was calculated and expressed in percentage. The dried extract samples were kept at 4°C until further used.
The experimental data were analyzed using the response surface regression procedure to fit the following second-order polynomial model (Eq. 1).
Where Y is the predicted response variable, 0 is the constant coefficient, is the linear effect, is the squared effect, is interaction effects and and represent the independent variables respectively. Design-Expert version 6.0.10 (Stat-Ease Inc., Minneapolis, MN, USA) was applied to perform the experimental design and the data analysis.

Edible film preparation
The rice-based film was prepared by casting technique according to Miksusanti et al. 18  rice-based film with ginger extract was then cast in a petri dish (9 cm diameter) and dried at 50°C for 6 h. The films were peeled off from the casing plates and conditioned for 7 days at 25°C in a desiccator before all analysis.

Total phenolic content (TPC)
Total phenolic compounds were examined using the method described by Singleton & Rossi 23 . The ginger solution (200 µL) and 10% Folin-Ciocalteu reagent (1 mL) were mixed and 2% Na2CO3 was then added with water: methanol (4:6) diluting solvent to make a total volume of 10 mL. Absorbance was recorded at 740 nm after 30 min using a spectrophotometer (UV-Vis model 1601, Shimadzu, Japan).

Antioxidant activities DPPH radical-scavenging activity
Four mL of extract solution and 1 mL of DPPH solution were mixed (0.1 mM in methanol) by a vortex mixer and then stood at room temperature in dark storage for 30 min 24 . The absorbance was recorded at 517 nm. The percentage of scavenging effect was calculated using the following equation (1) shown below: where A0 was the absorbance of the control solution (DPPH without sample) and A1 was the absorbance of the ginger extract in DPPH solution.

ABTS method
A mixture between 7 mM ABTS and 2.45 mM potassium persulphate, the ABTS solution, was stood in a dark place for 14±2 h before use. Afterward, the ABTS solution was diluted with ethanol to measure the absorbance of 0.700 ± 0.02 at 734 nm 25 . Ginger extract (150 µL) was allowed to react with 4,850 µL of the ABTS solution for 6 min and then read by a spectrophotometer at 734 nm.

Ferric reducing ability power assay (FRAP assay)
The FRAP assay was determined by the modification method from Benzie & Strain 26 . The

Evaluation of zone of inhibition
The disc diffusion method was used to determine the zone of inhibition. The impregnated paper discs with 10 µL of ginger extracts and/or rice-based films (6 mm in diameter) were placed on the standard protocol described by the National Committee of Clinical Laboratory Standards (NCCLS) 27 . The plates were incubated at 37°C and the diameters of the inhibition zones were measured after 24 h. Filter paper discs containing DMSO without any test compounds served as a control and no inhibition was observed. Additionally, for comparative purposes, tetracycline (30 µg, 10 µL) was used as a reference standard. Each assay was performed in triplicate and repeated three times.

Determination of minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC)
The MIC and MBC of the ginger extract against S. mutans DMST 18777 were determined by the reference protocol of the NCCLS method 27  plates. All tests were carried out in triplicate.

Analysis of active compounds in ginger using a high-performance liquid chromatography method (HPLC)
After filtering through a 0.2 µm syringe filter, the final sample was used for injection. Standards of 6-gingerol, 6-shogaol, paradol, and zingerone were prepared. The method was performed on HPLC (HPLC, Agilent Technologies, Santa Clara, CA, USA) with a photodiode array detector. The HPLC system contained a C18 reverse-phase column (Water C18, 250x4.6 mm, 5 µm particle size). The gradient elution was acetonitrile and water at a flow rate of 1.0 mL/min and detection of 282 nm. The mobile phase contained water (A) and acetonitrile (B). The gradient elution program was set as follows: from 0 to 25 min, B was isocratic at 33%; from 25-35 min, B was changed from 33% to 55%; from 35 to 60 min, B followed changed linearly from 55% to 90%; from 60 to 65 min, B was a linear change from 90% to 33%; and from 65 to 70 min, B was isocratic at 33% 28 .

Headspace solid-phase microextraction (HS-SPME)
To evaluate the volatile compounds of the rice edible film incorporated with crude ginger extract, the compounds were extracted using carboxen/polydimethylsiloxane (CAR/PDMS) fiber. The sample headspace (5 g) was transferred into a 25 mL screw cap glass vial and extracted at 50 °C for 30 min. The bound volatiles was injected into GC-MS analysis 29 .
The measurement of the target analysts was performed using GC-MS (GC-17A, Shimadzu, Japan) coupled with mass spectrometry (QP 5050A, Shimadzu, Japan). Capillary BPX-5 (30 m × 0.25 mm × 1.00 µm; SGE, Melbourne, Australia) column was used for separation and run at 1.0 mL/min with helium as the carrier gas. The inlet temperature was 250 °C in split mode (1:50). The initially oven temperature was started from 80 °C for 1 min, heated to 220 °C at 5 °C/min and maintained for 10 min, and finally increased to 250 °C. The detector temperature was set at 300 °C.

Statistical analysis
All experiments were carried out according to the relevant guidelines and regulation. The data were shown as the mean and standard deviation for the triplicate analyses.