2.1.1. Materials and chemicals:
- Chitosan powder from Tin Cay joint stock company.
- Cinnamaldehyde Acros Organics, Belgium.
- Agar is produced by Vietxoco Vegetable and Fruit JSC Co., Ltd.
- Chloramphenicol antibiotic produced by Quang Binh pharmaceutical JSC.
- Nutrient Broth environment of Himedia, India.
- Glycerol, cinnamaldehyde, DPPH, 1 % acetic acid.
- 99.5 % ethanol provided by Cemaco Vietnam Co., Ltd.
- Gram – negative bacteria strain Escherichia coli ATCC 8739 was provided by Saigon University of Technology, Ho Chi Minh City, Vietnam.
- Aspergillus niger and Aspergillus fumigatus were isolated and grown for 2 weeks on potato dextrose agar by us and identified at the Center for Bioscience and Biotechnology of the University of Science.
2.1.2. Equipment:
- UV – Vis Spectrophotometer Hitachi UH – 5300 (Japan).
- 4 – digit analytical balance (Sartorius, Germany).
- Hand – held micrometerr (Germany).
- Laboratory inoculation chamber and incubator.
- Drying chamber.
- Testometric M350 – 10CT (England).
- Thermostatic shaker.
- Necessary tools such as becher, erlen, pipette, micropipette, volumetric flask, petri, ...
42 2.2. Researching methods:
2.2.1. Research diagram:
Figure 2.1. Research diagram.
43 2.2.2. Film preparation:
Figure 2.2. Diagram of CH - CNM film preparation.
CH solutions (2 %, w/v) were made by dispersing 10 g of CH in 500 mL of acetic acid solution (1 %, v/v) over a 20 – minute period with heat stirring 90oC. After the CH was entirely dissolved, the solution was filtered with a vacuum filter to confirm that it was
44
residue – free. As a plasticizer, 30 % (v/v) glycerol was added to the total solid weight in solution. CNM solutions of 1, 3, 5, 7, and 9 % (v/v) were made by dispersing in ethanol (99.5 %, v/v). CNM in various quantities was added to the polymer solutions. Finally, the respective CH – CNM solutions (20 mL) were cast over the petri dishes (diameter of 9 cm) for 48 hours in drying chamber at 50oC. The CH – CNM films were carefully peeled and stored in the saturated brine chamber 80 % RH for 48 hours for further experiments.
2.3. Content and analytical method:
2.3.1. Characterization of cinnamaldehyde - chitosan film:
2.3.1.1. Physical properties of film:
❖ Thickness
After being stored in a saturated NaCl medium for 24 hours, the thickness of the film samples was measured with a hand – held micrometer (Germany). Calculate the mean and standard deviation of 5 distinct membrane locations, which were taken along the length of each specimen. The hand – held micrometer has an accuracy of ± 0.01 mm.
❖ Tensile strength (TS) and elongation-at-break (E)
Mechanical properties of TS and E were measured with an Testometric Rochdale M350 – 10CT (England). The initial grip separation was set at 20 mm and the crosshead speed was set at 15 mm/min. TS was computed by dividing the highest load (N) by the initial cross – sectional area (m2) of the specimen and represented in MPa. E was computed as a percentage as the ratio of the elongation to the initial length of the specimen (20 mm) and multiplying by 100. For each type of film, the TS and E tests were repeated three times.
Tensile strength (TS) = 𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑙𝑜𝑎𝑑 (𝑁) 𝐶𝑟𝑜𝑠𝑠−𝑠𝑒𝑐𝑡𝑖𝑜𝑛 𝑎𝑟𝑒𝑎 (𝑚2) Elongation-at-break (E) = 𝐸𝑙𝑜𝑛𝑔𝑎𝑡𝑖𝑜𝑛
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑙𝑒𝑛𝑔𝑡ℎ × 100%
❖ UV-Vis absorption spectrum and optical transmittance
Using a UV – Vis (UH – 5300 Hitachi) spectrophotometer, the percentage of transmitted light was measured to determine the film's transparency. The film samples were prepared with size of 12 × 40 mm. The membranes perpendicular to the light beam in the cuvette slit of the spectrometer. Air was used as a control for measurements. Each membrane was examined from 200 to 1100 nm. The average percentage of light is calculated at each wavelength region: UV wavelengths between 200 and 380 nm, visible
45
light wavelengths from 380 to 700 nm, and infrared wavelengths between 700 and 1100 nm.
Equilibrium moisture content, water uptake, water solubility
Equilibrium moisture content, water uptake, water solubility were determined according to Nouri et al. [44]. However, there are a few minor tweaks that may be made to the experiment to make it more beneficial and acceptable for the existing laboratory conditions.
Figure 2.3. CH films with different CNM concentration (size 20 × 20 mm).
Cut the film to size 20 × 20 mm according to Figure 2.3 and weigh to get the original weight (W0). The weight loss of films was measured after drying until they reached a constant weight at 105°C (dry sample weight). After 24 hours, the films were taken out and weighed (W1). The following formula was used to compute the equilibrium moisture content (EMC):
Equilibrium moisture content (EMC) = 𝑊0−𝑊1
𝑊0 × 100%
The water uptake was calculated by immersing the films above in closed beakers containing 20 mL distilled water. All samples were maintained at room temperature for 24h. After that, the films were surface – dried with a clean paper towel and reweighed (W2). The film pieces were again placed in a drying chamber at 105°C for 24 h. The final weight is W3.
46
The water uptake (WU) was determined according to the following formula:
The water uptake (WU) = 𝑊2−𝑊3
𝑊3 × 100%
The water solubility (WS) was determined according to the following formula:
The water solubility (WS) = 𝑊1−𝑊3
𝑊1 × 100%
Each experiment should be repeated three times.
❖ Water vapor permeability (WVP)
Water vapor permeability (WVP) properties of the films were studied using the standard test method ASTM E 96 (ASTM E96-95 1995) according to Shekarabi et al. [45], with a few modifications. Glass vials, with an average diameter of 50 mm and a depth of 40 mm.
Food Science & Nutrition published by Wiley Periodicals, Inc. Novel Nano Edible Film A. S. Shekarabi et al. ingly used to determine WVP of films, instead of the standard cup.
The films CH – CNM were cut into discs with a diameter slightly larger than the vial's diameter and placed on top with varying concentrations. Anhydrous silica gel (0 % RH) was placed inside the cup, and a sodium chloride saturated solution (75 % RH, 30oC) was utilized in the desiccator to maintain a 75 % RH gradient across the film. [46] Wrapping film samples around the cup's mouth and securing them with a cap. A round hole with a diameter of 45 mm is drilled into the lid. Then, place a sample at room temperature. For 48 hours, weigh the glass vials every 1 hour. The rise in mass of the glass vials after 1 hour was used to determine water vapor permeability. The slope of the line would be determined by plotting a graph based on linear regression (r2 > 0.99) from data on increasing cup mass and time. The water vapor transmission rate (WVTR) is determined by dividing the slope K (g/h) by the moisture transmission area A (m2). WVP moisture permeability was determined according to the formula [47]:
WVP = 𝑊𝑉𝑇𝑅
𝑃×(𝑅𝐻1−𝑅𝐻2) × X (g.Pa-1.m-1.h-1) Where:
WVTR = 𝐾
𝐴 (A = 0.003181 m2)
P is the saturated steam pressure (Pa) at the test temperature. On the high – stream side of the film, the water vapor pressure was 3.2 kPa (water vapor pressure of saturated NaCl aqueous solution at 30oC) [48].
47
RH1 is the humidity outside the cup (75 % RH) and RH2 is the humidity inside the cup (0 %).
X is the thickness of the film (m).
All measurements performed in three replicates and the average reported.
2.3.1.2. Bioactivities assay:
❖ Antioxidant ability
Blois (1958) devised this method with the goal of determining antioxidant activity in a similar way by employing a stable free radical α,α – diphenyl – β – picrylhydrazyl (DPPH;
C18H12N5O6, M = 394.33). The antioxidants' ability to scavenge free radicals is tested in this experiment. By acquiring a hydrogen atom from antioxidants, the odd electron of the nitrogen atom in DPPH is reduced in hydrazine. The deep violet color, which has a 515 nm absorption in ethanol solution, is likewise caused by delocalisation. The reduced form of the DPPH solution is generated when it is coupled with a molecule that can donate a hydrogen atom, and the violet hue is lost [49].
The antioxidant capacity of the film was measured by radical scavenging activity method DPPH freedom by Yichao Ma et al (2017) with some changes [50].
Dissolve 2.5 mg of DPPH in 100 mL of 99.5 % ethanol to prepare 0.063 mM DPPH solution (stock solution) then stored until needed. Four types of solution are prepared as shown in table below:
Table 2.1. Formulation of four types solution.
Membrane (including CNM)
(g)
0.063 mM DPPH solution
(mL)
Ethanol 99.5 % (mL)
Membrane solution 0.4 7.0 0
Blank membrane
solution 0.4 0 7.0
Control solution 0 7.0 0
Blank control
solution 0 0 7.0
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Every 1 hour, measure the absorbance of the membrane solution cuvette and control solution cuvette. Each cuvette is measured with a separate blank. Measure the absorbance of the solutions at 515 nm for 24 h. The experiment was repeated 3 times.
The scavenging activity (SA) at time t is calculated by the formula:
SA (%) = (𝐴𝑜−𝐴𝑡)−(𝐴𝑜𝑐−𝐴𝑡𝑐)
𝐴𝑜 ×100%
Where: Ao: the absorbance of membrane solution at t0
At: the absorbance of membrane solution at t Aoc: the absorbance of control solution at t0
Atc: the absorbance of control solution at t
❖ Antimicrobial activity Bacteria strains
The antimicrobial activity of the samples against Gram – negative Escherichia coli (E.
coli) according to Wong et al. [51], with a few modifications. Escherichia coli strain ATCC 8739 was stored at a temperature below 4°C before being injected into Nutrient Broth (NB) and incubated for 24 hours in a 37°C thermostatic shaker. The initial density of the inoculum following incubation in the incubator was around 109 CFU/mL. Proceed to dilute the organism suspension with distilled water to reach an initial density of roughly 107 CFU/mL, dilute with a dilution factor of 10-5 and use this density to assess the film's antibacterial effectiveness.
Prepare a culture medium for E.Coli (2.2 % agar, 1.3 % NB). After sterilizing the medium and the petri dish, pour it into the petri dishes. Wait until the medium has solidified and reached room temperature before performing the spread plate method, adding 100 àL of bacterial suspension to the media and spreading the bacteria uniformly on the agar surface with the glass spreader. In the culture medium, place the prepared membranes (cut into 10 × 10 mm). After 24 hours, 48 hours, and 52 hours, check the antibacterial ability of the membrane. Record and take pictures of the results received.
– Application of CH – CNM films to packaging for pork slices:
Lean pork slices were provided from a meat shop nearby. A strip of film was placed on either side of the pork sample (3.00 g). For improved adherence, the edges of the films were pressed. Unwrapped pork pieces were employed as a control. For 6 days, all samples were kept at 4°C. Samples were obtained at various times for microbiological analyses.
49
Each experiment was carried out three times. This method is based on the research of Wang et al. (2017) [52] with some modifications.
– Microbiological Analysis:
On days 0, 3, 5, 7, and 9, the total viable count (TVC) was calculated. 3 g of material was taken for each treatment, combined with 27 mL of 0.9 % NaCl solution in a conical flask, and suitably oscillated. Plate count agar was used to plate appropriate serial dilutions, which were then incubated at 28°C for 48 hours. Log – transformed TVC measurements (CFU/g) were used.
Mold
We performed this study based on the method of Phan Thi Thanh Diem (2018)[53].
However, There are some slight variations in testing to achieve the best conditions for mold growth and laboratory conditions.
In this experiment, PDA medium (120 mL potato extract, 12 g glucose, 12 g agar, 0.0411 g Chloramphenicol 50 mg) were used to inoculate and isolate mould from fruits. Pour the medium into the petri dishes after sterilizing the medium and petris. Wait for the medium to solidify completely and cool to room temperature. Carrying out mold culture onto the medium and place the membranes (cut into squares measuring 1 × 1 cm) on the medium mold inoculation. Observe the growth of mold on the plate containing the membrane after 24 h, 48 h and 52 h. Record and take pictures of the results received.
The mold Aspergillus niger and Aspergillus fumigate were isolated by the method of Diem, Phan Thi Thanh et al. [54] with some modifications. We isolated Aspergillus niger from lemons (lemons were obtained from the local market and allowed to spoilage naturally) and Aspergillus famigatus from rice (rice was taken randomly and allowed to spoilage naturally). 1g of sample (parts of lemon or rice contain mold) was taken, then crushed, and added 99 mL of distilled water. Next stage, the suspension was shacked well.
The prepared suspension was aspirated 100 L of the aliquot into each petri dish containing a sterile PDA medium. The sterile glass spreader was used to spread the drop evenly over the surface of the agar. These petri dishes were wrapped and incubated in an incubator at 37oC for 3 – 4 days. After that, both types of mold were isolated by the streak plate method to obtain pure strains.
Mold strains used in this experiment had been isolated and grown for two weeks on PDA ourselves and identified at the Center for Bioscience and Biotechnology of the University of Science.
– Application of CH – CNM films to packaging for lemon fruit:
50
Lemon fruits (Citrus aurantifolia) were acquired from a local marke and selected for equal maturity stage, size, and lack of flaws or injuries. All lemons were washed with distilled water and dried in the open air. Make a wound with the tip of a needle on the rind.
The wound is soaked with a mold suspension (Aspergillus niger). Fruit from each treatment was soaked for 5 minutes in 0 % CH – CNM, 1 % CH – CNM, 3 % CH – CNM, 5 % CH – CNM, 7 % CH – CNM, and 9 % CH – CNM, with a control sample without membrane.
The fruits were kept in a saturated brine chamber with a RH of 80 % [42]. During 12 days of storage, we examined quality parameters and other analysis. All treatments were performed in triplicate.
– Measurement of weight loss:
The approach for determining lemon weight loss was based on earlier research [55].
Lemon's weight decrease was determined as follows:
Weight loss (%) = ((M0 – M1)/M0) × 100%
Where M1 is the final weight of lemon, and M0 is the initial weight of lemon.
2.4. Statistical processing methods:
All of the samples were taken in triplicate, and the data was analyzed using the one - way analysis of variance (ANOVA). To assess the significant differences between the data, statistical analysis was performed using SPSS software (SPSS Institute, Inc., Cary, NC, USA) with Duncan's Multiple RangeTest (p ≤ 0.05).
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CHAPTER 3: RESULTS
3.1. Appearance of cinnamaldehyde - chitosan film:
Figure 3.1. depicts the appearance of CH films. Pure CH films are stronger and more removable (from petri dish) than films with a high CNM component. The addition of CNM resulted in an increase in the yellowish tone of the CH films because of the existence of a conjugated double bond following the synthesis of the Schiff base (Figure 3.1) which is suggestive of the occurrence of the Schiff – base reaction [43, Fig. 3.2] [43]. The volatile aldehyde's covalent attachment to the polymer's backbone stabilizes the molecule, preventing losses during manufacturing and storage of the polymer film.
Figure 3.1. CH – CNM films.
1% CNM 0% CNM
5% CNM 3% CNM
7% CNM 9% CNM
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Figure 3.2. Schiff base is formed by the nucleophilic addition of the amino group of the CH backbone to the carbonyl group of CNM.
At current anticipated consumption levels, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) stated that cinnamaldehyde and other cinnamon compounds do not represent a food safety risk (JECFA 2001). Cinnamon poisoning is an uncommon occurrence. A seven – year old child was reported to develop severe gastrointestinal, central nervous system, and cardiovascular manifestations after intentionally ingesting 60 mL of cinnamon oil (Pilapil 1989). Therefore, the CNM concentration chosen in this paper is reasonable, because just studying CNM at low concentration will not result in a strong antibacterial impact.
3.2. Physical properties of films:
❖ UV – Vis absorption spectrum and optical transmittance
UV – Vis absorption spectrum and optical transmittance are an important property of the films, which helps to see the transparency of the film as well as the ability to resist UV rays – a barrier to protect the product.
53
Figure 3.3. UV – Vis absorption spectrum of chitosan films with various CNM concentration.
Table 3.1. Average percentage of absorpbance of CH films with various CNM concentration from 200 – 1000 nm.
CH samples with various CNM content
Transmittance
UV range (200-400 nm)
Visible range (400-700 nm)
Infrared range (700-1100 nm)
0% CNM 12.42 84.33 90.04
1% CNM 9.65 84.33 89.65
3% CNM 16.07 84.84 90.39
5% CNM 18.32 84.69 89.47
7% CNM 14.41 84.92 89.97
9% CNM 19.52 85.55 90.16
0 20 40 60 80 100
200 400 600 800 1000
%T
Wavelength (nm)
0% 1% 3% 5% 7%
54
In general, as can be seen in the table and the graph above there is not a remarkable difference in the transmittance between samples with divergent concentrations of CNM.
They fluctuated in the range of UV light and the nearby wavelength of visible light. Almost the range of visible light and infrared light remained unchanged when altering the CNM percent in the sample.
In the UV region, the light transmission is low which shows the ability to resist UV light. This is a good contribution in food preservation. Especially, in the wavelength from 250 to 300, the CH membranes illustrate the excellent barrier properties against UV (0 % absorption). The aromatic amino acids also absorb UV radiation. They both suggest that CH – based films have good UV barrier qualities [56]. Likewise, Asthana et al. also reported aldehyde derivatives that strongly absorb UV light at about 280 nm [57], which is consistent with our research results. The CH – based films with the addition of CNM increases the content of the aldehyde group, creating good conditions for the film to resist UV rays at wavelengths of about 270 – 280 nm. In the food system, UV light is the factor that causes lipid oxidation – the main reason of spoilage [58].
The data also illustrates that the implementation of CNM decreases the transparency of films. It is able to be realized by bare eyes. In the Table 3.1 the transmission of membranes grow gradually from 0 % CNM to 9 % CNM (from 84.33 % to 85.55 %). This can be explained by the fact that CNM is not well dispersed, forming emulsion – like droplets and Schiff's basic products absorb light leading to opaque.
❖ Equilibrium moisture content, water uptake and water solubility:
Crucial factors in packaging applications are the barrier properties of food packages to gases, vapors, and organic chemicals. Moisture and oxygen transition to the environment of the internal or external packaging produces a constant change in shelf life, food quality, and even spoiling [44]. Equilibrium moisture content, water uptake, water solubility significantly affect the function of active packaging. Depending on the purpose of using the membrane, non – absorbent or water – insoluble properties may be needed to enhance water resistance as well as preserve product. CH is a hydrophilic polymer with a high capacity for water retention [43]. As a result, the effect of grafting CNM into CH films on equilibrium moisture content, water uptake, water solubility of the films was investigated in this study. Table 3.2 and Figures 3.4, 3.5, 3.6 illustrate the equilibrium moisture content, water uptake, water solubility properties of the studied membrane samples.
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Figure 3.4. Equilibrium moisture content of CH films with various CNM content.
Figure 3.5. Water uptake of CH films with various CNM content.
e
d cd
bc
ab a
26 27 28 29 30 31 32 33
0 1 3 5 7 9
Equilibrium moisture content (%)
CNM concentration (%)
bc
a
c b
a
b
0 10 20 30 40 50 60 70
0 1 3 5 7 9
Water uptake (%)
CNM concentration (%)
56
Figure 3.6. Water solubility of CH films with various CNM content.
The solubility of films in water can provide information about their behavior in an aquatic environment and is a measure of their water resistance. This is also a key component in determining the biodegradability of films used as packaging materials. The water solubility percentages of CH films containing CNM at various concentrations are shown in Table 3.2. With different concentrations of CNM, the water solubility in the CH films dropped slightly. Because of their hydrophobic character, we predicted the films that included CNM to have a greater reduction in water solubility. However, the maximum fall was only around 4 %. Although the percentages of water solubility declined, the water solubility values remained very high. Therefore, the film’s solubility was not affected by the incorporation of CNM (p > 0.05). The addition of carvacrol (a hydrophobic chemical) into CH films was previously found to have no effect on the CH films' solubility [59].
Another study included montmorillonite nanoclay and rosemary essential oil into CH films, and it was discovered that increasing the essential oil did not always result in a linear drop in solubility [60]. Other researchers found similar outcomes when a hydrophobic substance, such as α – tocopherol or cinnamon essential oil, was added into CH films [61].
The moisture content and water uptake values for CH and CNM films are also shown in Table 3.2. The results of moisture content and water uptake values of five membrane samples with CNM added when compared with the corresponding membranes without CNM found that there was very little change. Due to flaws in experimental manipulation, the standard deviation of the values for moisture and water uptake is rather large. There was no statistical significance (p > 0.05) so the addition of CNM to the membrane did not affect the moisture content, water uptake values as well as water solubility of the membrane because of the CNM’s volatile compound and its hydrophobic nature [59].
b b ab ab
a
a
0 5 10 15 20 25
0 1 3 5 7 9
Water solubility(%)
CNM concentration (%)