Preparation of bioplastic from cassava starch/anthocyanin through melt mixing process for beef floss rancidity detection

Abstract

The freshness of food goods is a crucial attribute for consumers when selecting meals. The integration of anthocyanin into food packaging offers a distinct visual indicator to consumers regarding the product’s freshness. This work incorporated anthocyanin, an active compound present in purple sweet potatoes (PSP), into cassava starch-based food packaging using the industrial process of melt mixing. This study aims to investigate the impact of melt mixing on the incorporation of anthocyanin into starch-based bioplastics and their efficacy in detecting rancidity. Anthocyanin exhibited significant antioxidant activity (IC50 of 95.73 ppm), signifying superior oxidative markers. An exceptional outcome was achieved regarding the antioxidant efficacy of the package during storage, with a peroxide value remaining beneath the standard threshold (1.33 – 2.27 mEq O2/1,000 g of fat). A discernible color transition from red to green occurs during the packaging of beef floss, resulting from the oxidation process, signifying effective rancidity detection. These findings indicate that cassava starch-based bioplastics infused with anthocyanin exhibit significant potential as intelligent packaging materials for monitoring food rancidity.

Keywords

Smart packaging cassava starch anthocyanin melt-mixing food rancidity

Introduction

In today’s food industry, preserving the quality of food products is a significant challenge, particularly when dealing with rancidity. According to S. Chen et al., 2020, rancidity not only damages consumers but also results in significant food waste.¹ The primary obstacle lies in early rancidity detection, which makes preserving product quality extremely challenging. In order to enhance food product safety and quality, innovative packaging solutions must be developed. One potential early detection method for changes in product conditions, such as rancidity, is the implementation of smart packaging.² It is crucial to develop packaging materials that can adapt to these changes as a result.

Employing bioplastics as the primary material for smart packaging is a growing approach to not only enhance the functionality of the packaging but also ensure its environmental safety.³ Previous research has confirmed the feasibility of developing smart packaging by utilizing proteins, lipids, and polysaccharides as the foundation for biodegradable packaging.⁴ For instance, a study conducted in 2019 by Kaushalya et al. emphasized the importance of incorporating cassava starch into bioplastics as a means to improve their mechanical strength and biodegradability. Cassava starch not only meets the criteria for mechanical strength and biodegradability, but also presents novel opportunities for the environmentally conscious packaging industry to enhance the utilization of cassava plants.⁵

In the development of bioplastics, the incorporation of natural pigments from plant products and food waste is an important aspect. The use of natural pigments, such as anthocyanins,⁶ curcumin 7, betalains,⁸ chlorophyll, carotenoids,⁹ and tannins¹⁰ from plant and food materials, can be utilized in the development of biodegradable smart packaging materials. For example, research by Rahmadhia et al. in 2023, focused on packaging sheets that use naturally occurring pigment compounds that are pH-sensitive to enhance food safety. The color shift in the film indicator indicated that the flavonoid anthocyanin components from purple sweet potatoes (Ipomoea batatas L.) were sensitive to pH fluctuations.¹¹

Although pH indicators, color changes, and antibacterial compounds are commonly used to detect specific rancidity, the use of pH indicators, such as anthocyanins from sweet potato, is becoming relevant.¹² Anthocyanins were chosen because they are sensitive to chemical changes during the rancidity process, which is characterized by color changes as a visual response.¹³ As a result, anthocyanin extract from purple sweet potatoes was added to cassava starch-based films in this work as a pH indicator and was integrated into a cassava starch-based matrix.

The development of this product will proceed with the adaptation of the production process to an industrial scale, despite the fact that films containing anthocyanins have been the subject of substantial laboratory research. Finding an effective mass-marketable production method is one of the primary hurdles in this area. Melt mixing, which has been shown to be effective in the mass manufacture of a variety of polymeric materials, is one approach that shows promise in this regard. There isn’t much data on the application of this technique for producing starch films enhanced with anthocyanins. By investigating the possibility of the melt blending process in the manufacturing of starch/anthocyanin films as intelligent packaging for rancidity detection on an industrial scale, this study seeks to close the gap. Numerous assessments were carried out, such as the activity concentration test, thermal characteristics, morphological structure, functional group interaction, and antioxidant test. In this study, beef floss was employed as a test sample on a cassava starch-based film along with purple yam extract and anthocyanins, as an applicable example.

Material and method

Materials

Cassava starch purchased from PT. Surya Pati Kencana, Indonesia; purple sweet potato from Pangalengan, Bandung, Indonesia; glycerol; potatoes dextrose agar (PDA); salt agar (SA), sodium thiosulfate (Na₂S₂O₃); hydrochloric Acid (HCl); potassium iodate (KIO₃); potassium iodide (KI); ethanol 96%. The chemicals were purchased from Merck (Darmstadt Germany).

Extraction of purple sweet potatoes anthocyanins (PA)

To get rid of extra moisture, the fresh PSP was properly cleaned in distilled water and then dried in the shade. The PSP was then mashed in a commercial blender and sieved through an 80-mesh size after being dried in an oven at 40°C until a consistent weight was reached. Subsequently, the PSP powder was placed inside a zip lock bag and kept cold until the pigment was extracted.

500 g of crushed PSP were placed in a dark, light-resistant container. Then, 96% of acidified ethanol (1:4)—which included 0.1% v/v HCl—was added, and the mixture was allowed to settle at room temperature for 24 hours. An extract filtered with vacuum pump filter and remained solvent where to evaporate using a BUCHI R-300 Germany rotating vacuum evaporator. Prior to analysis, the concentrated filtrates were maintained in glass vials, sealed in a pouch made of laminated aluminum foil, and refrigerated. The recovery percentage of the concentrate was determined using equation (1)¹⁴

E x t r a c t i o n y i e l d (%) = \frac{w e i g h t o f c o n c e n t r a t e d (g)}{w e i g h t o f p o w d e r e d s a m p l e (g)} x 100

(1)

Preparation of film smart packaging

Using the melt-mixing method, cassava starch, glycerol, and PSP concentrate were incorporated to produce the smart packaging. Cassava starch and glycerol (3:1) were pre-mixed with (Phillips Blender HR2118) until smooth. Melt-mixing (Labo Plastomil 30R150 Toyoseiki) was used to incorporate various amounts of PSP concentrated (1.5: 3; and 4.5%, w/w) into the pre-mixed mixture for 3 minutes at 120°C. PSP concentration changes were represented by the synthetic film packaging, which was designated as FP-1.5, FP-3, and FP-4.5, respectively. And FP-0 is the sample without any PSP extract. Then, 5 g of the resulted sample were compression molded for 3 minutes at 135°C with a load of 50 kg/cm².

Characterization of purple sweet potato anthocyanin

pH stability test

2 mL of extract PSP were dissolved into various pH (2, 5, 8, 10, 14) using buffer solution, respectively. The color change when the extract is exposed with different pH was investigated. These color variations can be identified in a similar way as color changes that occur in smart packaging film.

Characterizations

Antioxidant activity test

The evaluation of the antioxidant activity of PSP concentrated was performed by DPPH (α, α, Diphenyl Picrylhydrazyl) free radical method. 0.1 M solution DPPH were prepared by adding 0.004 g of DPPH was into 100 mL of 98% methanol. A series (100, 200 and 500 ppm) of PSP concentrate and 0.1 M DPPH solution were prepared in test tube and vortex for about 10 s, respectively. The absorbance of each sample was measured using a UV-Vis Spectrophotometer at maximum wavelength of 700 nm (UV-Vis 2600 SHIMADZHU) to determine antioxidant activity, and ethanol was employed as the reference standard for comparative analysis. The DPPH radical was determined of percentage inhibition using equation (3)¹⁵ where ‘A_c’ denoted absorbance of the control (DPPH solution without sample) and ‘A_s’ absorbance of the sample with DPPH solution.

% i n h i b i t i o n = \frac{A_{c} - (A_{c} - A_{b})}{A_{c}} \times 100

(2)

Calculation of peroxide number

Determination of rancidity in smart packaging is determined by the iodometric titration method following standard procedures.¹⁶ 4 g of the film was put in an Erlenmeyer flask. 30 mL of acetic acid and chloroform (2:1) solution were added into Erlenmeyer. Subsequently 1 g KI was added, and the Erlenmeyer was immediately closed using aluminum foil. After giving the mixture a good shake, the slurry turned yellow, signifying the iodine molecules starting to form. Subsequently, the combination was titrated up to a pale-yellow using a standardized Na₂S₂O₃ solution (0.01 N). Three drops of the starch indicator solution were added in the interim. The titration was carried out until the endpoint’s dark blue-black color turned colorless. Sample smart packaging was used for the titration procedure following storage (7, 14, 21, 28, 35 days). Using equation (4), the peroxide number in the sample was determined. Where sample weight (Ws), normalcy (N), blanko titration volume (Vb), and sample titration volume (Vs).

P e r o x i d e n u m b e r = \frac{V_{s} - V_{b} \times N \times 1000}{W_{s}}

(3)

Fourier transform infrared

Determination of chemical interaction between cassava starch/ sweet potato anthocyanin (PA) in smart packaging film was carried out using attenuated total reflection mode (ATR)-FTIR spectrophotometer (Nicolet iS, 5 FTIR) The functional groups from FTIR were identified in the wavenumber range of 500 – 3500 cm⁻¹ with a resolution of 2 cm⁻¹/s

Differential scanning calorimetry

Differential scanning calorimetry was used to measure the thermal properties of packaging films (DSC 214 Polyma NETZCH). The film was heated under N₂ atmosphere from 40 to 200°C at heating and flow rates of 10°C/min and 40 mL/min, respectively.

Scanning electron microscopy

The morphology of packaging films were examined using a Scanning Electron Microscope (JEOL JSMIT300, Japan)., A sputter coating process using a 5 nm Au layer (∼10 nm) was applied to the film prior to testing. The films were submerged in liquid nitrogen and cut for cross-sectional imaging and the SEM images were taken under accelerating voltage of 20 kV.

Water contact angle

The hydrophilicity of the packaging film was evaluated through the sessile drop technique for measuring the water contact angle. The film (dimension of 1 × 1 cm²) was desiccated 24 hours prior to measurement. Next, a micro syringe was used to precisely dispense 5 μL of distilled water over the membrane’s surface. After that, side-view photos of the water droplets were taken. The testing was carried out five times. The ImageJ software was used to determine the angle between the water droplet and the membrane’s surface.

Water vapor transmission rate (WVTR)

The measurement of water vapor transmission rate (WVTR) was conducted according to the method reported by Xu et al., 2020a). A circular film disc (∅ 30 mm) was affixed onto the rim of a glass bottle (∅ 17.5 mm) containing 10 mL of distilled water using a Teflon tape. The bottle was heated at 40°C for 24 hours. The bottle was weighted. The WVTR was then determined using equation (4)

WVTR = \frac{(W_{1} - W_{2})}{A} \times T

(4)

Where W₁ and W₂ correspond to the initial weight and final weight of bottle after heating, respectively, A is the area of the glass bottle mouth, and T is time (24 hours).

Mechanical properties

Tensile test and elongation at break were measured using a universal strength tester (UCT-5T, Oriented Co. Ltd, Japan) according to the ASTM D882-12. The sample film was cut into dimension of 2 × 8 cm², with an initial grip distance of 4 cm and a crosshead speed of 1 mm/s. The measurement of each sample was conducted repeatedly for five times.¹⁷

Biodegradability test

The biodegradability assessment of packaging film was conducted according to ASTM G21.¹⁸ The salt agar and Aspergillus niger were used as the growth medium and the decomposing organism, respectively.¹⁹ The packaging film was cut into dimensions of 1.5 × 1.5 cm². Prior to the commencement of the experiment, all equipment was sterilized using an autoclave for 15 mins. Stock cultures were prepared by mixing 10 mL of sterile distilled water with A. niger using a wire loop. Subsequently, 1 mL of the stock culture was diluted with 9 mL of sterile distilled water to achieve a 10⁻¹ dilution. Further dilution was carried out by adding 1 mL of the 10⁻¹ culture to 9 mL of sterile distilled water to obtain a 10⁻² dilution. A volume of 100 µL from the 10⁻² dilution was then evenly spread onto sterile Petri dishes. Salt agar, totaling 20 mL, was added to each Petri dish. Once the salt agar solidified, the films were placed on its surface. Subsequently, 100 µL of the 10⁻² dilution culture was evenly distributed onto the surface of each film sample. The observation of biodegradability test was conducted over 14 days and replication of three times for each sample.

Shelf life of beef floss

The shelf-life test was carried out via observations for 35 days with temperature variations (15, 30, and 45°C) during storage period of beef floss. Observations were made by taking samples at different PA compositions for each observation and testing for color changes. The data obtained were plotted against time and three equations for different storage temperatures of the product, Y = bx+a, where x is the storage time (days), Y is the characteristic value of FP, a is the initial characteristic value of FP, and b is the rate of change in characteristics (slope). From each linear regression equation, the value of the rate of quality deterioration (K) is obtained. Then the value of ln K is plotted with 1/T (K⁻¹), so that the intercept and slope values of the linear regression equation are obtained ln K = ln K₀ -(E/R) (1/T). After the FP activation energy characteristics and the constant value of K₀ are obtained, the Arrhenius equation is calculated with equation (5).²⁰

k = k_{0} . e^{- E / RT}

(5)

Where K, K_0, E, T and R are denoted as degradation constant, constant (independent of temperature), activation energy, absolute temperature (K), and gas constant (1.986 cal/mol K), respectively.

Using the Arrhenius equation, the reaction rate (K) of the change in FP characteristics at a predetermined temperature (T) can be calculated. Determination of the shelf life of FP is done by selecting the highest correlation coefficient (R2) parameter. Furthermore, the K value obtained is entered into the reaction order equation (6).²⁰

At = Ao + Kt

(6)

where Ao and At are a value at the beginning and at the end of shelf life, respectively, ‘while t is the shelf-life. Hence, the shelf-life FP for each specified temperature will be obtained.

Results and discussions

Extraction yield

In this study, PA derived from PSP were successfully extracted using ethanol as much as 7.62 g /100 g. Because anthocyanins contain phenolic compounds and are polar, ethanol can interact with the hydroxyl groups on phenolic compounds to extract anthocyanins (PA) from PSP. This is why ethanol solvent is used in this work.²¹ Different extraction solvents can affect the solubility, extraction yield, and antioxidant activity of different bioactive components in tubers differently since these compounds have varied chemical properties.²² Chloroform extracts the highest yield of anthocyanins in comparison with methanol,²³ ethanol, and methanol.²⁴ Solvents containing methanol, water²⁵ and acetone²⁶ extracted less anthocyanins with the acetone extract being unstable and losing its color within 48 hours. Whereas water-based solvents (combined solvents)²⁷ and water were the least effective solvents in extracting anthocyanins. Although chloroform achieves the maximum yield, it is a more hazardous solvent than other alcohols, such as ethanol and methanol. Consequently, we employed a less hazardous solvent in our study.

Antioxidant propertiesof PSP extract

Evaluation of the antioxidant properties of PA through the DPPH radical scavenging assay provides a valuable insight into its antioxidant activity. In this assay, the DPPH was used to evaluate how effectively PA can neutralize the free radicals, which are reactive molecules that can cause oxidative damage to tissue cells. The higher PA extract concentration the higher inhibition percentage obtained (Figure 1). The IC₅₀ value (half-maximal inhibitory concentration) is a measure of the concentration of substance (in this case, PSP extract) required to inhibit 50% of DPPH radicals in the assay and was calculated from the graph of % inhibition versus sample concentration using a straight-line equation (y = mx + c), where y = dependent variable, x = independent variable, m = slope of the line and c = intercept.

Figure 1.

Antioxidant activity graph of purple sweet potato extract anthocyanins.

A stronger antioxidant effect is indicated by a lower IC₅₀ value, which means that less of the chemical is needed to neutralize half of the free radicals. At 95.73 ppm, the IC₅₀ value of PA suggests that it possesses potent antioxidant properties. In particular, PA interacts with free radicals and takes part in redox reactions. Its antioxidant activity allows it to destroy free radicals by either receiving or donating electrons. Thus, adding PA into packaging film enhances their antioxidant properties.

pH dependence of PA stability

A pH stability test was also carried out to demonstrate the presence of anthocyanins in the PSP extract. The PSP extract contains anthocyanins, as seen by the various color shifts (Figure 2), which include bright red at pH 5, purple tints at pH 8, and green at pH 10–14. The pH of the solution affects the color of anthocyanins. This is because anthocyanin molecules have an ionic structure.²⁸ Certain anthocyanins lose color and turn red in acidic environments. When the pH is neutral, anthocyanins are purple; when the pH rises, they become blue green. Red-colored anthocyanin pigments are primarily found as flavylium cations.²⁹ (Gamage & Choo, 2023) The flavylium cations make anthocyanins extremely soluble in water, thus making anthocyanins are more stable in lower pH solutions. Color stability is decreased when the concentration of water decreases because it speeds up the deprotonation rate of flavylium cations. These results inferred that pH of the solution affects the color of anthocyanins. Since anthocyanin molecules have an ionic structure, its stability is a pH dependence.

Figure 2.

Color change activity of purple sweet potato extract at various pH.

The structure of packaging films

Figure 3 shows a broad absorption band at 3292 cm⁻¹ which originates from O-H stretching of PA. In addition, other bands were also found at 1149 cm⁻¹ and 1627 cm⁻¹ which correspond to C-O and C = C bending vibrations. The O-H functional groups that appear in the anthocyanins spectra indicate the phenolic hydroxy groups and alcohols derived from the anthocyanins structure.³⁰ While starch is composed of a number of amylose and amylopectin formed by glucopyranose rings.³¹ The characteristic peak of starch is attributed to the C = C stretching of the glucopyranose ring. In addition, the absorption bands at wavenumbers 3000 cm⁻¹ and 1641 cm⁻¹ are common and prominent spectra for all samples derived from C-H stretching. The peak at 1000 cm⁻¹ in the addition of cassava starch (FP) indicates the O-H deformation. The addition of glycerol as plasticizer was observed at wavenumber 3310 cm⁻¹ which indicates O-H stretching, while the peaks at 2915 cm⁻¹ and 1439 cm⁻¹ indicate the vibration functional group of C-H bond which is a typical peak derived from glycerol.³²

Figure 3.

a) FTIR spectra for anthocyanin extract, FP with various anthocyanin composition and b) Interaction of starch with anthocyanins.

Moreover, the addition of PA caused a shift in the O-H functional group at wavenumbers 3301 to 3274 cm⁻¹ in all samples, as presented in the FTIR spectra for cassava starch, glycerol, and anthocyanin. This indicates the hydrogen bonding between functional moieties of anthocyanin and starch on the fabrication of packaging films (Figure 4).³³ This result is in a good agreement with previous study (Qin et al.,2019))

Figure 4.

DSC graphs of FP with different anthocyanin result.

Thermal analysis

Figure 4 demonstrates the DSC graphs of packaging films (FP) with addition of anthocyanin at different ratio where the endothermic points of FP decreased as an increase PA ratio (1.5 to 4.5%) from 92.1 to 89.1°C. Another result reported the endothermic point 106.5°C of cassava starch.³⁴ This result indicates the effect of PA on the thermal properties of the packaging film, which is likely due to the interaction between PA and cassava starch that forms complexes and disrupts the crystal structure and thermal properties of cassava starch resulting in the FP, associating the more susceptible structure toward temperature changes.

Table 1 illustrates the thermal properties of FP formulations obtained via DSC analysis, revealing notable concentration-dependent changes. As FP concentrations increased, the onset temperature (T_onset) decreased sharply, from 85°C in FP 1.5 to 55°C in FP 3.0 and 50°C in FP 4.5, indicating progressively lower thermal stability. The peak temperature (T_p) followed a similar trend, dropping from 92.1°C in FP 1.5 to 69.9°C in FP 3.0, before rising slightly to 89.1°C in FP 4.5. Variations in the endset temperature (T_endset) were also observed, with a decrease to 90°C in FP 3.0 but an increase to 130°C in FP 4.5, suggesting distinct ranges of thermal transitions.

Table 1.

Thermal properties of FP formulations (FP 1.5, FP 3.0, FP 4.5) from DSC analysis.

	FP 1.5	FP 3.0	FP 4.5
T onset (°C)	85.0	55.0	50.0
Tp (°C)	92.1	69.9	89.1
T end set (°C)	120.0	90.0	130.0
ΔH (J/g)	249.4	258.8	181.7

Enthalpy change (ΔH) peaked in FP 3.0 (258.8 J/g), surpassing FP 1.5 (249.4 J/g) and FP 4.5 (181.7 J/g), reflecting stronger molecular interactions in FP 3.0 and reduced stability in FP 4.5. These results underscore the significant influence of PA and FP concentration on thermal behavior. While PA disrupts the starch structure, leading to reduced thermal stability, FP 3.0 demonstrates superior thermal characteristics. However, FP 4.5’s lower stability restricts its use in applications requiring high thermal resistance.

Morphology of packaging films

Figure 5 displays surface and cross-sectional images of FP and FP films with varying PA loadings. Some intact starch granules were present, indicating that certain starch may not have fully gelatinized during the film-forming process. Consequently, to ensure the complete gelatinization of starch throughout the biopolymer synthesis process, the heating duration must be prolonged. There are no significant differences between the FP and the FP with varied anthocyanins on the film’s surface. However, the cross-section reveals that, as the concentration of PA increases, the starch granules become distinctly visible. The film surface displays granular structures and irregular protrusions following the incorporation of PA, suggesting an uneven or incomplete integration of the starch matrix, as depicted by the granular structures (emphasized by the yellow box). The smooth texture of cassava starch plastic film is anticipated due to starch granules functioning as stress concentrators inside the polymer matrix, hence affecting attributes like tensile strength and processability. The result shows a poor compatibility between starch and anthocyanin which can be a challenge in the future.

Figure 5.

Surface and cross-section morphology of FP and FP with various PA loadings.

Water contact angle (WCA)

Figure 6 shows the hydrophilicity of packaging film, where hydrophilicity and contact angle are inversely correlated; the more hydrophilicity is indicated by water contact angle (WCA) value < 90^o.³⁵ Overall, all the samples have hydrophilic nature where the FP has WCA of 67.1°. The addition of PA reduced the WCA value of FP up to 6.7 ° indicating an increase in hydrophilicity, that can affect the overall properties of the packaging film.³⁶ In addition, the formation of hydrogen bonds between PA and starch can enhance the film to absorb and retain water molecules, thus increasing its hydrophilicity. This is also evidenced by the SEM results, which indicate the presence of starch granules that can influence water absorption.

Figure 6.

(a) Water contact angle of FP and FP with various PA loadings (b) Water Drop in Water Contact Angle Measurement.

Water vapor transmission rate (WVTR)

WVTR is one of the important criteria for assessing the ability of packaging materials to maintain the quality of packaged food.³⁷ The ability of the packaging film to inhibit water vapor transmission between the surrounding environment and the wrapped food determines its effectiveness.³⁸ Figure 7 presents the WVTR values of various types of films.

Figure 7.

Water Vapor Transmission Rate of FP and FP with various PA loadings.

The WVTR value of the FP sample was 60.47 g/m² per day, indicating a lower water vapor transmission rate compared to the other anthocyanin-added films. This result is coherent with the previous WCA result which the incorporation of PA increases the hydrophilicity, thus increasing the WVP result.³⁹ The WVTR result is closely associated with the prior findings in SEM and water contact angles, indicating a significant level of hydrophilicity in the material.

Mechanical properties

Analysis of the mechanical properties of smart packaging is essential to assess the strength and durability of smart packaging under operational stress. This study evaluates the impact of PA addition on the mechanical properties of smart films. The addition of PA to smart packaging has an impact on mechanical properties (Figure 8). Smart packaging without PA addition (FP) showed the highest tensile strength and elongation break values of 4.81 MPa and 64.14%, which is due to its compact structure and lower porosity. The addition of PA into smart packaging shows hydrogen bonding between starch and PA which is confirmed by FTIR analysis causing an increase in the hydrophilic nature of smart packaging according to the water contact angle test. Increased hydrophilicity causes swelling which can weaken the structure, causing a decrease in tensile strength and elongation break values. Increasing the PA concentration in the film to 4.5% (FP-4.5), the tensile strength and elongation break values decreased slightly to 3.95 MPa and 60.62%, possibly due to excessive amount of ungelatinized starch. During melt mixing process, the semicrystalline part of the starch was ruptured and become amorphous. Incomplete gelatinization during processing will result in residual semicrystalline structure in the TPS, which will affect the material’s mechanical properties.⁴⁰

Figure 8.

Tensile strength of FP and FP with various PA concentrations.

Biodegradability test

Biodegradability assessments are crucial for evaluating the environmental implications of materials, especially those designed for transient uses such as packaging. These assessments measure a material’s capacity for natural decomposition via microbial activity, including that of fungus and bacteria.⁴¹ The justification for conducting such tests lies in ensuring that the materials used do not persist in the environment, contributing to long-term pollution. For smart packaging, biodegradability is a critical parameter because these materials are expected to degrade after fulfilling their purpose, thus aligning with sustainability goals.⁴²

In this study, the test was carried out using the fungus Aspergillus niger, a common microorganism known for its role in the natural decomposition of organic matter such as leaves, grass, and food waste. The extent of biodegradation was monitored by observing the growth of A. niger on the surface of packaging films with different polyphenol acid (PA) concentrations (FP-1.5, FP-3.0, FP-4.5), as shown in Figures 9 and 10.⁴³ Following a 7-day period, significant fungal growth was noted on the film samples. Samples devoid of PA demonstrated over 58% surface coverage by A. niger, but samples containing PA had even higher colonization, surpassing 60%. This signifies a pronounced vulnerability of the films to microbial deterioration. The results correspond with the water vapor transmission rate (WVTR) data, indicating elevated WVTR in samples containing PA. Given that fungal growth necessitates moisture, an elevated water vapor transmission rate (WVTR) enables increased water absorption, which fosters fungal proliferation and subsequently enhances biodegradability.⁴⁴

Figure 9.

The Aspergillus niger growth after 7 days in FP and FP with various PA loadings.

Figure 10.

Antioxidant Activity on FP and FP with various PA loadings after 35 days.

Antioxidant activity during storages

The antioxidant activity of smart packaging films was evaluated using the DPPH free radical capture method, a commonly used technique for measuring antioxidant capacity.⁴⁵ Figure 10 presents the antioxidant activity data of smart packaging films on days 0 to 7. As mentioned by Wang et al. (2023),⁴⁶ the greater the rate of DPPH radical annihilation, the greater the antioxidant capacity of the film.⁴⁷ From the results, it was observed that the antioxidant activity of all samples decreased during storage, which can be indicated by the increase in IC₅₀ values. When the PA addition reached 4.5%, the lowest IC₅₀ value of 1845.6 ppm was observed, indicating that the anthocyanins had diffused into the film matrix efficiently. Anthocyanins work by stopping the chain reaction of free radicals, providing active methylene groups that act as hydrogen donors to react with DPPH free radicals, thus allowing the composite film to exhibit high antioxidant activity.⁴⁷

Therefore, based on these results, it can be concluded that the sample with 4.5% concentration of purple sweet potato extract (FP-4.5) has the optimal antioxidant activity among the tested samples. Films with high antioxidant activity can help protect food products from oxidation that causes rancidity, and also act as an effective indicator of rancidity during storage. This is evident from the testing data of the antioxidant properties of purple sweet potato extract, which has the potential to protect products from oxidation that causes rancidity, as well as acting as an effective rancidity indicator.

Color change during storage

During the storage process, beef floss used two types of packaging, namely cassava starch bioplastic (FP) and smart packaging with the addition of purple sweet potato extract at various concentrations (FP-1.5, FP-3.0, FP-4.5). Figure 11 presents data on packaging color changes during 0-35 days of storage. Significant color changes occurred under specific storage conditions. While FP packaging maintained its color stability over a 0-35 Day period at various storage temperatures (as depicted in Figure 11), slight reddish color changes were observed in smart packaging FP-1.5 and FP-3.0. Moreover, FP-4.5 exhibited a reddish at 15°C and turned green at 30°C and 45°C.

Figure 11.

The color change of beef floss packaging with and without anthocyanin extract in span of 35 days with various storage temperatures.

The interaction between packaging environment and beef floss during storage plays a crucial role in this color variation, primarily driven by the auto-oxidation reaction of fatty acids in the beef.⁴⁸ Initiation involves the attachment of reactive oxygen, releasing hydrogen atoms from the methylene group (-CH₂) on unsaturated fatty acids.⁴⁹ Unsaturated fatty acids, especially those with double bonds, are more prone to oxidation, particularly influenced by triplet oxygen. The formation of peroxyl radicals leads to the generation of fatty hydroperoxides, indicative of propagation and auto-oxidation stages.

Temperature’s impact on packaging discoloration directly corresponds to storage environment conditions, with higher temperatures accelerating oxidation reactions in packaging components and subsequently enhancing color change rates. Extreme temperatures can compromise pigment or dye stability in packaging, resulting in more pronounced color variations. Meanwhile, the effect of PA concentration on packaging discoloration is attributed to the antioxidant properties of the pigment. Higher PA concentrations provide superior protection against oxidation and discoloration. Hence, packaging with elevated PA levels exhibits enhanced resistance to discoloration, especially under elevated storage temperatures.

Investigation of peroxide number

Figure 12(a)–(c) depict the alterations observed in the compound peroxide index throughout a storage period of 35 days. On the 35th day, the FP bioplastic packaging demonstrated the highest equivalent of 2.5670 mEq O2/1,000 g of fat, with FP-3.0 smart packaging following closely behind at 2.27 mEq O2/1,000 g of fat, FP-1.5 at 1.91 mEq O2/1,000 g of fat, and FP-4.5 at 1.33 mEq O2/1,000 g of fat. Although the peroxide levels of all packages exhibited an upward trend over time, they consistently remained below the established safe threshold specified in SNI 3741:2013, which is 10 mEq O2/1,000 g of fat.

Figure 12.

Graphs of (a)Peroxide number during storage 15°C, (b) Peroxide number during storage 30°C (c) Peroxide number during storage 45°C.

Peroxide measurement plays a critical role, with higher values indicating advanced oxidation. However, low peroxide numbers may not necessarily denote early oxidation, as the rate of peroxide formation could be slower compared to degradation into other compounds. The peroxide graphs of beef floss in FP-1.5, FP-3.0, and FP-4.5 demonstrated lower values compared to FP due to PA’s interaction in smart packaging with radical compounds, which inhibits oxidation during storage. Notably, peroxides also form during processing, particularly during deep frying, due to increased oil usage, which augments the number of double bonds and accelerates oxidation rates. The influence of storage temperature on peroxide levels is apparent, with higher temperatures correlating with increased peroxide concentrations. Additionally, oxidation rates are affected by factors like inhibitors or catalysts, reaction environment nature, and involved components. As color intensity changes during storage, it corresponds to increased peroxide concentration. This results from the formation of more peroxides, subsequently inducing color variations such as aging, darkening, or other changes as the product’s fat undergoes oxidation. Thus, the observed color changes during storage directly correlate with the degree of oxidation in the product.

Shelf life of Beef Floss

In this study, shelf-life analysis was carried out using the Arrhenius method, resulting in the data in Table 2 with sample variations FP, FP-1.5, FP-3.0, and FP-4.5 evaluated at storage temperatures of 15°C, 30°C, and 45°C.

Table 2.

Shelf-life of FP, FP-1.5, FP-3.0, and FP-4.5.

Sample variations	T (⁰C)	T (⁰K)	Q₀	Q_t (SNI 3741 2013)	K	Ea	Shelf life (days)
FP	15	288	0.1299 mEq O₂/1.000 g	Mak. 10 mEq O₂/1.000 g	0.0974	2807.61	101
	30	303			0.1242		79
	45	318			0.1548		64
FP-1.5	15	288	0.1299 mEq O₂/1.000	Mak. 10 mEq O₂/1.000 g	0.0886	1487.55	111
	30	303			0.1007		98
	45	318			0.1132		87
FP-3.0	15	288	0.1299 mEq O₂/1.000	Mak. 10 mEq O₂/1.000 g	0.0367	4005.96	269
	30	303			0.0520		190
	45	318			0.0711		139
FP-4.5	15	288	0.1299 mEq O₂/1.000	Mak. 10 mEq O₂/1.000 g	0.0371	3998.22	266
	30	303			0.0524		188
	45	318			0.0717		138

The analysis revealed a notable increase in the reaction rate coefficient (K) with rising storage temperatures. Likewise, the activation energy (Ea) signified the energy required to facilitate the oxidation reaction in beef floss packaging. Utilizing the Arrhenius formula for shelf-life calculation indicated that higher storage temperatures led to shorter shelf-life spans across all packaging variants. Particularly, packages incorporating PA (FP-1.5, FP-3.0, FP-4.5) exhibited prolonged shelf life compared to PA-absent FP packages at equivalent temperatures.

This underscores PA efficacy in enhancing beef floss packaging stability against oxidation under varying storage temperature conditions. Moreover, product shelf-life directly correlates with peroxide number analysis outcomes. Products with lower peroxide numbers tend to endure longer shelf lives as slower or minimal oxidation preserves their stability and quality over extended periods. Conversely, products with higher peroxide numbers tend to have abbreviated shelf lives due to rapid or heightened oxidation, resulting in diminished product quality within a shorter timeframe.⁵⁰

Consistent with color change observations during storage, where slower or minimal color changes in packaging correspond to longer shelf lives, while rapid or intense color changes (signifying high peroxide numbers) correspond to shorter shelf lives. Consequently, the conclusion can be drawn that PA supplementation fortifies beef floss packaging stability against oxidation, thereby extending the product’s shelf life.

Conclusions

The research effectively isolated anthocyanins (PA) from purple sweet potato extract (PSP) utilizing ethanol. The successful fabrication of bioplastic smart packaging has been realized by a melt mixing technique that integrates thermoplastic starch with anthocyanin extracted from purple sweet potatoes. Melt mixing was utilized due to its effectiveness in large production. The oxidative assessment of the anthocyanin extract is notably effective, demonstrated by its significant IC50 value of 95.73 ppm. The incorporation of anthocyanin led to a reduction in tensile strength from 4.81 MPa to 3.95 MPa, an enhancement in hydrophobicity, and a significant increase in water vapor transmission rate (WVTR) from 60.47 g/m² per day to 106.48 g/m² per day. The growth rate of A. niger covered more than 58% of the surface of film samples without PA, whereas film samples with PA exhibited even larger coverage, surpassing 60%, indicating that the film samples are more prone to degradation. The research examines alterations in beef floss packaging with and without anthocyanin extract during a 35-day period at varying storage conditions. The addition of purple sweet potato extracts at concentrations of 3.0% and 4.5% effectively induced a color change in the beef floss packaging, suggesting a highly promising option for biodegradable smart packaging for beef products. Even though, compatibility between anthocyanins and the polymer matrix remains a significant challenge.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the YKAAI and RUA were supported by BRIN through Research Assistant Scheme (Grant ID: 8/HK/II/2024).

ORCID iDs

Yeyen Nurhamiyah

Rossy Choerun Nissa

References

Chen

, et al. Development of pH indicator and antimicrobial cellulose nanofibre packaging film based on purple sweet potato anthocyanin and oregano essential oil. Int J Biol Macromol 2020; 149: 271–280.

Yates

Deeney

Rolker

, et al. A systematic scoping review of environmental, food security and health impacts of food system plastics. Nat Food 2021; 2(2): 80–87.

Jancikova

Jamróz

Kulawik

, et al. Furcellaran/gelatin hydrolysate/rosemary extract composite films as active and intelligent packaging materials. Int J Biol Macromol 2019; 131: 19–28.

Bhargava

Sharanagat

Mor

, et al. Active and intelligent biodegradable packaging films using food and food waste-derived bioactive compounds: a review. Trends Food Sci Technol 2020; 105: 385–401.

Kaushalya

RANC

Dhanushka

MKDT

Samarasekara

AMPB

, et al. Development of biodegradable packaging materials using polylactic acid (PLA) and locally extracted starch. In: 2019 Moratuwa Engineering Research Conference (MERCon). Piscataway: IEEE, 2019, pp. 610–615.

Zhang

Huang

T-S

Yan

, et al. Novel pH-sensitive films based on starch/polyvinyl alcohol and food anthocyanins as a visual indicator of shrimp deterioration. Int J Biol Macromol 2020; 145: 768–776.

Chen

Zhang

Bhandari

, et al. Novel pH-sensitive films containing curcumin and anthocyanins to monitor fish freshness. Food Hydrocoll 2020; 100: 105438.

Surendhiran

Roy

, et al. Smart packaging film prepared from subcritical water-modified oat starch and betalain of beetroot extract reinforced with cellulose nanofibrils. Sustain Chem Pharm 2023; 36: 101349.

de Oliveira Filho

Bertolo

MRV

Fernandes

, et al. Intelligent and active biodegradable biopolymeric films containing carotenoids. Food Chem 2024; 434: 137454.

10.

Mileti

Mammolenti

Baldino

, et al. Starch films loaded with tannin: the study of rheological and physical properties. Int J Biol Macromol 2024; 254: 127973.

11.

Safinta Nurindra Rahmadhia

SNR

Afist Azkiya Sidqi

AAS

Yanas Anggana Saputra

YAS

. Physical properties of tapioca starch-based film indicators with anthocyanin extract from purple sweet potato (Ipomea batatas L.) and response to pH changes. Sains Malays 2023; 52(6): 1685–1697.

12.

Jiang

Hou

Zeng

, et al. Preparation and characterization of indicator films from carboxymethyl-cellulose/starch and purple sweet potato (Ipomoea batatas (L.) lam) anthocyanins for monitoring fish freshness. Int J Biol Macromol 2020; 143: 359–372.

13.

Alizadeh-Sani

Mohammadian

Rhim

J-W

, et al. pH-sensitive (halochromic) smart packaging films based on natural food colorants for the monitoring of food quality and safety. Trends Food Sci Technol 2020; 105: 93–144.

14.

Gonfa

Teketle

Kiros

. Effect of extraction solvent on qualitative and quantitative analysis of major phyto-constituents and in-vitro antioxidant activity evaluation of Cadaba rotundifolia Forssk leaf extracts. Cogent Food Agric 2020; 6(1): 1853867.

15.

Islam

Kamal

Kabir

, et al. Phenolic compounds and antioxidants activity of banana peel extracts: testing and optimization of enzyme-assisted conditions. Measurement: Food 2023; 10: 100085.

16.

Rahanur Alam

Dey

Kobirul Islam

, et al. Determination of Iodine Content of Commercially Available Table Salts at the Retailer Level in Selected Areas of Bangladesh. Eur J Nutr Food Saf. Feb. 2020; 284–288. DOI: 10.9734/ejnfs/2019/v11i430177.

17.

Wei

Jia

, et al. Characteristics and bioactive functions of chitosan/gelatin-based film incorporated with ε-polylysine and astaxanthin extracts derived from by-products of shrimp (Litopenaeus vannamei). Food Hydrocoll 2020; 100: 105436.

18.

Masssijaya

Lubis

MAR

Nissa

, et al. Utilization of spent coffee grounds as a sustainable resource for the synthesis of bioplastic composites with polylactic acid, starch, and sucrose. J Compos Sci 2023; 7(12): 512.

19.

Zdanowicz

Rokosa

Pieczykolan

, et al. Study on physicochemical properties of biocomposite films with spent coffee grounds as a filler and their influence on physiological state of growing plants. Int J Mol Sci 2023; 24(9): 7864.

20.

Pulungan

Sukmana

Dewi

. Shelf life prediction of apple brownies using accelerated method. In: IOP Conference Series: Earth and Environmental Science. Bristol: Institute of Physics Publishing, 2018.

21.

Cai

Lan

, et al. Conventional, ultrasound-assisted, and accelerated-solvent extractions of anthocyanins from purple sweet potatoes. Food Chem 2016; 197: 266–272.

22.

Azabou

Sebii

Taheur

, et al. Phytochemical profile and antioxidant properties of tomato by-products as affected by extraction solvents and potential application in refined olive oils. Food Biosci 2020; 36(Aug): 100664. DOI: 10.1016/j.fbio.2020.100664.

23.

Solovchenko

Chivkunova

Merzlyak

, et al. “A Spectrophotometric Analysis of Pigments in Apples,” Russian Journal of Plant Physiology 2001; 48(5): 693–700. DOI: 10.1023/A:1016780624280.

24.

Karaaslan

Yaman

. Anthocyanin profile of strawberry fruit as affected by extraction conditions. Int J Food Prop 2018; 20: S2313–S2322.

25.

Neff

Chory

, “Genetic interactions between phytochrome A, phytochrome B, and cryptochrome 1 during arabidopsis development”. Plant Physiol 1998; 118(1): 27–35.

26.

Garcia-Viguera

Zafrilla

Tomás-Barberán

. The use of acetone as an extraction solvent for anthocyanins from strawberry fruit. Phytochem Anal 1998; 9(6): 274–277.

27.

Gauche

Malagoli

, et al. “Copigmentation of anthocyanins from grape extracts with organic acids Effect of pH on the copigmentation of anthocyanins from Cabernet Sauvignon grape extracts with organic acids”. Scientia Agricola 2010; 67(1): 90162010000100006.

28.

Turturică

Oancea

Râpeanu

, “Anthocyanins: naturally occuring fruit pigments with functional properties”. Annals of the University Dunarea de Jos of Galati 2015; 39(1): 9–24.

29.

Gamage

GCV

Choo

. Thermal and pH stability of natural anthocyanin colourant preparations from black goji berry. Food Chemistry Advances 2023; 2: 100236.

30.

Rostami

Rabiee

. Anthocyanins extract as a non-toxic and green fluorescent label for bioimaging of HER2-positive breast cancer cells. Environ Res 2023; 237: 116878.

31.

Weligama Thuppahige

Moghaddam

Welsh

, et al. Extraction and characterisation of starch from cassava (Manihot esculenta) agro-industrial wastes. LWT 2023; 182: 114787.

32.

Fernandes

Carlos Pinto

Cabral-Albuquerque

ECM

, et al. Free-radical polymerization of urea, acrylic acid, and glycerol in aqueous solutions. Polym Eng Sci 2015; 55(6): 1219–1229.

33.

Ferreira

DCM

Molina

Pelissari

. Biodegradable trays based on cassava starch blended with agroindustrial residues. Compos B Eng 2020; 183: 107682.

34.

Chen

Yao

Gao

, et al. Physicochemical properties comparative analysis of corn starch and cassava starch, and comparative analysis as adhesive. J Renew Mater 2021; 9(5): 979–992.

35.

Cheng

Cui

Yan

, et al. Effect of dual-modified cassava starches on intelligent packaging films containing red cabbage extracts. Food Hydrocoll 2022; 124: 107225.

36.

Qin

Yun

, et al. Impact of storage conditions on the structure and functionality of starch/polyvinyl alcohol films containing Lycium ruthenicum anthocyanins. Food Packag Shelf Life 2021; 29: 100693.

37.

Amaregouda

Kamanna

Gasti

. Fabrication of intelligent/active films based on chitosan/polyvinyl alcohol matrices containing Jacaranda cuspidifolia anthocyanin for real-time monitoring of fish freshness. Int J Biol Macromol 2022; 218: 799–815.

38.

Wei

Jia

39.

Kurek

Garofulić

Bakić

, et al. Development and evaluation of a novel antioxidant and pH indicator film based on chitosan and food waste sources of antioxidants. Food Hydrocoll 2018; 84: 238–246.

40.

Chaléat

Halley

Truss

. In: Halley

Avérous

LBT-SP

(eds). Chapter 7 - Mechanical Properties of Starch-Based Plastics. Amsterdam: Elsevier, 2014, pp. 187–209.

41.

da Silva Filipini

Romani

Guimarães Martins

. Biodegradable and active-intelligent films based on methylcellulose and jambolão (Syzygium cumini) skins extract for food packaging. Food Hydrocoll 2020; 109: 106139.

42.

Khalil

RKS

Sharaby

Abdelrahim

. Novel active edible food packaging films based entirely on citrus peel wastes. Food Hydrocoll 2023; 134: 107961.

43.

Rilda

Valeri

Drajat

, et al. Biosynthesis, characterization, and antibacterial activity of Ti-Doped Zno (Ti/ZnO) using mediated Aspergillus Niger. South African Journal of Chemical Engineering 2023; 45: 10–19.

44.

Heaton

LLM

López

Maini

, et al. Growth-induced mass flows in fungal networks. Proc Biol Sci 2010; 277(1698): 3265–3274.

45.

Gomes

Pires

Mateus

, et al. Pyranoflavylium-cellulose acetate films and the glycerol effect towards the development of pH-freshness smart label for food packaging. Food Hydrocoll 2022; 127: 107501.

46.

Wang

Y-C

Meng

F-B

, et al. Effect of honeysuckle leaf extract on the physicochemical properties of carboxymethyl konjac glucomannan/konjac glucomannan/gelatin composite edible film. Food Chem X 2023; 18: 100675.

47.

Roshandel-hesari

Mokaber-Esfahani

Taleghani

, et al. Investigation of physicochemical properties, antimicrobial and antioxidant activity of edible films based on chitosan/casein containing Origanum vulgare L. essential oil and its effect on quality maintenance of cherry tomato. Food Chem 2022; 396: 133650.

48.

Alizadeh-Sani

Tavassoli

Mohammadian

, et al. pH-responsive color indicator films based on methylcellulose/chitosan nanofiber and barberry anthocyanins for real-time monitoring of meat freshness. Int J Biol Macromol 2021; 166: 741–750.

49.

Jeyaraj

Lim

Choo

. Antioxidant, cytotoxic, and antibacterial activities of Clitoria ternatea flower extracts and anthocyanin-rich fraction. Sci Rep 2022; 12(1): 14890.

50.

Narayanan

Loganathan

Valapa

, et al. UV protective poly(lactic acid)/rosin films for sustainable packaging. Int J Biol Macromol 2017; 99: 37–45.