A system for developing a pH-based biosensor using anthocyanins from red cabbage and chitosan nanoparticles

Abstract

System for the development of a pH-based biosensor using anthocyanins from red cabbage (Brassica oleracea) and chitosan nanoparticles for monitoring food quality, comprising: ◯ Glassware for collecting and processing red cabbage samples, using a system for extracting anthocyanins by: • Thoroughly wash red cabbage with tap water and distilled water; • Chop the washed cabbage into fine pieces of about 1-2 cm; • Soaking 2 kg of shredded cabbage in 100 ml of acidified ethanol solution (ethanol:water:acetic acid, 70:29:1 v / v / v) for 24 hours; • Filter the extract using a vacuum filter and concentrate it under reduced pressure; • Store the extract at 4°C in a dark bottle; ◯ a device for the synthesis of chitosan nanoparticles via ionic gelation, comprising: • Prepare a 0.1% (w / v) chitosan solution in 1% acetic acid, adjusted to pH 4.6; • Add 0.1% tripolyphosphate (TPP) solution dropwise while stirring; • Collecting the nanoparticles by centrifugation and washing with deionized water; ◯ a system for introducing anthocyanins into chitosan nanoparticles by mixing the anthocyanin extract with the chitosan solution prior to TPP addition; ◯ a solution casting system for the production of a pH-sensitive biofilm by mixing anthocyanin-loaded nanoparticles with poly(vinyl alcohol); ◯ analytical instruments, including an Alpha E ATR-FTIR (Bruker, Germany) and an FEI QUANTA 450 scanning electron microscope, for characterizing the nanoparticles and biofilm; ◯ Design of a well diffusion test to assess antibacterial activity against Staphylococcus aureus using Mueller-Hinton agar plates.

Description

SCOPE OF THE INVENTION

[0001] The present disclosure relates to a system for the development of an active pH-based biosensor using anthocyanins from red cabbage (Brassica oleracea) and chitosan nanoparticles for real-time monitoring of food quality in smart food packaging applications. BACKGROUND OF THE INVENTION

[0002] Food spoilage is a major challenge in the food industry and requires innovative solutions for real-time quality monitoring. Biosensors based on natural pH-sensitive indicators, such as anthocyanins, offer a sustainable and effective approach to spoilage detection. Anthocyanins, naturally occurring pigments in red cabbage, exhibit pH-dependent color changes and are therefore ideally suited for biosensor applications. Furthermore, chitosan, a biodegradable and antimicrobial polysaccharide, can be used to create nanoparticles that enhance the stability and functionality of anthocyanins. These nanoparticles form a protective matrix that improves shelf life and the controlled release of anthocyanins in response to environmental changes.The integration of anthocyanins and chitosan nanoparticles into biofilms represents a promising approach for smart food packaging, combining pH sensitivity, antimicrobial properties and biodegradability.

[0003] Current methods for monitoring food quality often rely on synthetic indicators or complex electronic sensors, which may lack biocompatibility or cost-effectiveness. The use of natural extracts, such as anthocyanins from red cabbage, in combination with biocompatible chitosan nanoparticles offers an environmentally friendly and sustainable alternative.

[0004] The proposed system aims to address these challenges by developing a biosensor with improved stability, sensitivity, and antimicrobial properties for the real-time detection of spoilage in food packaging. SUMMARY OF THE INVENTION

[0005] The following is a simplified summary to provide a basic understanding of some aspects of the disclosed invention. This summary is neither a comprehensive overview nor is it intended to identify important / critical elements or to define the scope of the invention. Its sole purpose is to present some concepts in simplified form as a prelude to the more detailed description that follows.

[0006] This disclosure provides a system for developing a pH-based biosensor using red cabbage anthocyanins and chitosan nanoparticles. The system includes the extraction of anthocyanins from red cabbage, the synthesis of chitosan nanoparticles by ionic gelation, the incorporation of anthocyanins into these nanoparticles, and the formation of a biofilm for food packaging applications. The biosensor is characterized with respect to its pH sensitivity, antimicrobial activity, and structural properties, ensuring its suitability for real-time food quality monitoring. DETAILED DESCRIPTION

[0007] The embodiments described herein, along with their various features and advantageous details, are explained in more detail with reference to the non-limiting embodiments described below. Descriptions of known components and processing techniques are omitted to avoid obscuring the embodiments. The examples provided are intended to facilitate understanding of how the embodiments can be put into practice and to enable those skilled in the art to implement them. These examples are not to be understood as limiting the scope of the embodiments. Extraction of anthocyanins from red cabbage

[0008] Samples of red cabbage (Brassica oleracea) are sourced from a local farmers' market and thoroughly washed with tap and distilled water to remove impurities. The washed cabbage leaves are finely chopped into 1–2 cm pieces to maximize surface area. Approximately 2 kg of chopped cabbage is soaked in 100 ml of an acidified ethanol solution (ethanol:water:acetic acid, 70:29:1 v / v / v) for 24 hours at room temperature. The mixture is filtered through a vacuum filter, and the filtrate is concentrated under reduced pressure to obtain a deep purple anthocyanin extract. The extract is stored at 4 °C in a dark bottle to prevent degradation due to light and temperature. Production of chitosan nanoparticles

[0009] Chitosan nanoparticles are synthesized using the ionic gelation method. A 0.1% chitosan solution is prepared by dissolving chitosan in 1% acetic acid, adjusting the pH to 4.6. A 0.1% tripolyphosphate (TPP) solution is added dropwise to the chitosan solution while stirring continuously, facilitating the formation of nanoparticles through ionic interactions. The resulting nanoparticles are collected by centrifugation, washed with deionized water, and stored for further use. Incorporation of anthocyanins into chitosan nanoparticles

[0010] Anthocyanins are incorporated into chitosan nanoparticles by mixing the anthocyanin extract with the chitosan solution prior to the addition of TPP. The mixture is processed using the ionic gelation method described above, ensuring the encapsulation of the anthocyanins within the nanoparticle matrix. The resulting anthocyanin-loaded nanoparticles are then investigated for stability and controlled release properties. Biofilm production

[0011] The anthocyanin-loaded chitosan nanoparticles are incorporated into a biofilm using a solution casting process. To produce the biofilm, the nanoparticle suspension is mixed with a polyvinyl alcohol solution, then cast and dried to form a flexible, pH-responsive film suitable for food packaging. Characterization of the nanoparticles and the biofilm

[0012] The nanoparticles and the biofilm are characterized using Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The FTIR analysis, performed with an Alpha E ATR-FTIR (Bruker, Germany) in the range of 400–4000 cm⁻¹, 1 The molecular interactions between anthocyanins and chitosan, as well as key functional groups, were confirmed. SEM analysis, performed with an FEI QUANTA 450 scanning electron microscope at 4.00 kx magnification, examined the surface morphology of the freeze-dried biofilm after gold coating. These techniques confirmed the structural integrity and composition of the biosensor. Assessment of antibacterial activity

[0013] The antibacterial activity of anthocyanin-loaded chitosan nanoparticles against Staphylococcus aureus is evaluated using the well diffusion method. S. aureus is cultured in Mueller-Hinton broth to a turbidity of 0.5 McFarland standard. Sterile Mueller-Hinton agar plates are inoculated with the bacterial suspension, and 6 mm wells are filled with 50 µL of nanoparticle suspensions at concentrations of 10, 20, and 30 mg / ml. After a 24-hour incubation at 37 °C, the zones of inhibition are measured to determine antibacterial efficacy. pH sensitivity and performance of the biosensor

[0014] The pH sensitivity of the biosensor is evaluated by exposing the biofilm to solutions with pH values ​​between 2 and 9. The colorimetric reaction is observed, with the film exhibiting distinct color changes (red at pH 2, purple at pH 7, green at pH 9). The reaction time, reversibility, and reusability of the biosensor are tested to ensure its suitability for real-time food quality monitoring. Application in food packaging

[0015] The biosensor is applied to real food samples to evaluate its ability to detect spoilage. The biofilm is integrated into packaging materials and brought into contact with food samples under controlled conditions. Changes in pH due to microbial activity or chemical decomposition lead to visible color changes in the biosensor, indicating spoilage.

[0016] These and other aspects of the embodiments described herein will be better understood when considered in conjunction with the accompanying descriptions. Many changes and modifications can be made to the embodiments without altering their purpose, and the embodiments described herein include all such modifications.

Claims

[1] System for the development of a pH-based biosensor using anthocyanins from red cabbage (Brassica oleracea) and chitosan nanoparticles for monitoring food quality, comprising: ◯ Glassware for collecting and processing red cabbage samples, using a system for extracting anthocyanins by: • Thoroughly wash red cabbage with tap water and distilled water; • Chop the washed cabbage into fine pieces of about 1-2 cm; • Soaking 2 kg of shredded cabbage in 100 ml of acidified ethanol solution (ethanol:water:acetic acid, 70:29:1 v / v / v) for 24 hours; • Filter the extract using a vacuum filter and concentrate it under reduced pressure; • Store the extract at 4°C in a dark bottle; ◯ a device for the synthesis of chitosan nanoparticles via ionic gelation, comprising: • Prepare a 0.1% (w / v) chitosan solution in 1% acetic acid, adjusted to pH 4.6; • Add 0.1% tripolyphosphate (TPP) solution dropwise while stirring; • Collecting the nanoparticles by centrifugation and washing with deionized water; ◯ a system for introducing anthocyanins into chitosan nanoparticles by mixing the anthocyanin extract with the chitosan solution prior to TPP addition; ◯ a solution casting system for the production of a pH-sensitive biofilm by mixing anthocyanin-loaded nanoparticles with poly(vinyl alcohol); ◯ analytical instruments, including an Alpha E ATR-FTIR (Bruker, Germany) and an FEI QUANTA 450 scanning electron microscope, for characterizing the nanoparticles and biofilm; ◯ Design of a well diffusion test to assess antibacterial activity against Staphylococcus aureus using Mueller-Hinton agar plates. [2] System according to claim 1, wherein the glassware is washed with an acid / base solution and subsequently with Milli-Q water and dried in a hot air oven. [3] System according to claim 1, wherein the anthocyanin extract exhibits pH-dependent color changes, transitioning from red at pH 2 to violet at pH 7 and green at pH 9. [4] System according to claim 1, wherein FTIR spectroscopy confirms the incorporation of anthocyanins into chitosan nanoparticles by hydrogen bonding, esterification and amide bonding. [5] System according to claim 1, wherein the SEM analysis shows a uniform and well dispersed nanoparticle structure within the biofilm. [6] System according to claim 1, wherein the biosensor exhibits antibacterial activity with inhibition zones of 18 mm against Escherichia coli and 20 mm against Staphylococcus aureus in the well diffusion test. [7] System according to claim 1, wherein the biofilm has high tensile strength (34.2 MPa) and flexibility (15.4% elongation at break) for food packaging applications.