A novel PANI-pectin-based colorimetric assay for rapid total plate count estimation in raw milk

Introduction

The rapid detection and continuous monitoring of pathogenic microorganisms have become increasingly important due to the growing incidence of food-and water-borne disease outbreaks worldwide (Valenzuela-Amaro et al., 2023) [35] . Pathogenic and toxin-producing bacteria are responsible for significant public health concerns, causing illness and mortality across populations (Abebe et al., 2020) [1] . Their widespread occurrence in food, drinking water, and environmental matrices necessitates the development of reliable monitoring systems to ensure food safety and quality (Yao et al., 2022) [37] . Microbial quality assessment is particularly important in the dairy industry, where contamination can occur during production, processing, storage, and distribution (Fusco et al., 2020) [12] . Milk provides an ideal environment for the growth of various pathogenic and spoilage-causing microorganisms such as Coliforms, Bacillus spp., Pseudomonas spp., Listeria monocytogenes, Staphylococcus aureus, and Salmonella spp. (Haldar et al., 2022) [17] . Conventional microbiological methods, such as the Total Plate Count (TPC) method (IS 5402-1:2021), remain the regulatory standard for evaluating bacterial load. However, these methods are labour-intensive and typically require 48-72 h to produce results, limiting their suitability for modern dairy processing environments where rapid decision-making is essential. In India, the regulatory framework for milk and milk products is primarily governed by the Food Safety and Standards Authority of India (FSSAI), under the Food Safety and Standards Act (FSSA), 2006, which sets mandatory TPC standards for all categories of dairy products (Gandhi et al., 2020) [13] .

International Journal of Advanced Biochemistry Research 2026; SP-10(6): 1409-1416

To overcome these limitations, several advanced analytical techniques, including flow cytometry (D-Count), polymerase chain reaction (PCR), and the direct epifluorescent filter technique (DEFT), have been developed (Moldenhauer, 2008) [26] . Although these methods significantly reduce detection time, they require expensive instrumentation, specialized laboratory facilities, and trained personnel. Recent advances in molecular diagnostics, including recombinase polymerase amplification, CRISPRbased assays, and viability-PCR approaches, have further improved detection sensitivity and specificity (Shah et al., 2026) [31] . Nevertheless, these technologies remain largely restricted to centralized laboratories and are often impractical for routine on-site monitoring. Consequently, there is a growing demand for rapid, cost-effective, and user-friendly sensing platforms that can be deployed directly at dairy reception points and processing facilities (Mhapsekar et al., 2025) [25] . Colorimetric sensing has emerged as a promising alternative because of its simplicity, low cost, and ability to provide visual information without the need for sophisticated instrumentation (Kant et al., 2023) [21] . Such sensors can be incorporated into food packaging or used as standalone indicators to monitor microbial contamination and product freshness (Yousefi et al., 2019) [39] . Unlike solution-based colorimetric assays, which may suffer from interference due to sample turbidity, solid-state sensor formats offer greater practicality and ease of interpretation. Conducting polymers, particularly polyaniline (PANI), are attractive materials for colorimetric sensing applications because of their high sensitivity to pH changes and excellent environmental and thermal stability (Wang & Feng, 2022) [36] . PANI undergoes a reversible transition from its insulating emeraldine base (EB) form (blue) to its conductive emeraldine salt (ES) form (green) upon protonation. This unique property enables the direct transduction of bacterial metabolic activity into a visible color response (Thakur et al., 2015) [33] . Previous investigations using PANI-Pectin (PANI-Pec) nanoparticles have demonstrated their suitability for bacterial sensing applications. Anjali et al. (2022) [4] reported a PANI-Pec paper strip-based sensor for detecting Escherichia coli in milk and milk products, achieving a detection sensitivity of 0.52 ± 0.17 log CFU/mL within 10-21 h. More recently, Saha et al. (2025) [29] developed a PANI-Pec paper strip sensor for rapid TPC detection in milk and milk products, capable of detecting 4.0 ± 0.15 log CFU/mL of microorganisms within 4 ½ ± ¼ h at 30 ± 2 °C. These advances confirm the potential of PANI-Pec systems for dairy quality monitoring; however, further improvements in detection speed, format versatility, and assay simplicity are required. In the present study, a novel PANI-Pec-based colorimetric assay was developed using a 2 mL Eppendorf tube format distinct from paper strip configurations for rapid TPC estimation in raw milk. The sensing mechanism relies on the interaction between bacterial metabolic products and the pH-responsive PANI-Pec matrix embedded in agarose, resulting in a measurable color change correlated with microbial proliferation. By providing a rapid visual indication of bacterial activity within a compact, sterile, and self-contained tube format, the developed assay offers a practical, equipment-free, and cost-effective approach for real-time microbial quality assessment in dairy products.

Materials and Methods

Chemicals and reagents

Aniline (99%, Sigma-Aldrich), ammonium persulfate (Sigma-Aldrich), hydrochloric acid (37%, Sigma-Aldrich), ethanol (HPLC grade, Sigma-Aldrich), NaOH crystals (HiMedia), pectin (HiMedia), and Milli-Q water (Millipore water purification system) were used. Casein enzymatic hydrolysate, yeast extract, dextrose, and sodium chloride used in the study were obtained from HiMedia (India).

Synthesis of PANI-pec nanoparticles

PANI-Pec nanoparticles were synthesized using a methodology adapted from Anjali et al. (2022) [4] , and Thakur et al. (2015) [33] . Briefly, aniline was polymerized in the presence of pectin as stabilizer (aniline: pectin weight ratio of 1:2) using ammonium persulfate as the oxidizing initiator and hydrochloric acid as the doping agent. The reaction was carried out at room temperature under constant stirring for 4 h. The resulting PANI-Pec dispersion was precipitated in a 1:1 ethanol-water mixture, filtered, washed, and re-dispersed in Milli-Q water by ultrasonication. The final dispersion was adjusted to pH 7.0 using 1 N NaOH to convert the emeraldine salt (ES) form to the emeraldine base (EB) form, yielding a stable blue-colored nanoparticle dispersion.

Characterization of PANI-pec nanoparticles

Fourier-transform infrared (FTIR) spectra were recorded on a Shimadzu IR Affinity-1S (4000-400 cm⁻¹, 4 cm⁻¹ resolution). UV-Vis spectra were obtained using a TECAN Infinite M200PRO plate reader (300-1000 nm). Electrical conductivity was measured with a Eutech PC 2700 multiparameter bench meter. Particle size, polydispersity index (PDI), and zeta potential were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS90.

Fabrication of PANI-pec sensor

An optimized volume of PANI-Pec solution (3 mg/mL) was incorporated into 5 mL of 1× phosphate-buffered saline (PBS) containing 2% (w/v) agarose. The mixture was heated to approximately 70 °C with continuous stirring until a homogeneous dispersion was obtained. The optimized volume of the warm PANI-Pec-agarose composite was transferred into sterile 2 mL microcentrifuge (Eppendorf) tubes and allowed to solidify, forming a uniform gel layer. The resulting sensor matrices were sterilized by exposure to UV light for 20 min before further use.

Optimization of PANI-pec solution, growth medium, and sample volume 2.4.1. Optimization of PANI-pec volume

The volume of PANI-Pec nanoparticle suspension incorporated into the agarose matrix was optimized by evaluating volumes ranging from 100 to 400 µL. Each formulation was tested for its ability to produce a rapid and distinct color transition from blue to green in response to bacterial metabolic activity in the optimized growth medium. The volume exhibiting the fastest response time and the most pronounced visual color change was selected as the optimal concentration for subsequent experiments.

Optimization of growth media

Casein enzyme hydrolysate, yeast extract, dextrose, and sodium chloride were optimized by Response Surface Methodology (Design Expert v10.1). Fifteen medium combinations were evaluated against three criteria: highest optical density (600 nm), lowest pH after incubation at 37 °C, and the sensitivity and colour intensity of the resulting PANI-Pec colorimetric assay response.

Optimization of sample volume

Sample volumes ranging from 100-500 µL, spiked reconstitute milk powder to approximately 0.5 log CFU/mL, were evaluated to identify the volume yielding the most rapid colour change.

Sensitivity of the PANI-pec sensor

Skim milk was spiked with bacterial cell suspensions isolated from raw milk to achieve final counts ranging from 8 to 4 log CFU/mL. The optimized assay protocol was applied, and the time to visible colour change was recorded for each inoculum level.

Shelf-life study of sensor

Prepared sensors were stored in airtight containers at room temperature (25 ± 2 °C), 4 °C, and -20 °C. Sensors were evaluated for sensitivity and color intensity at 15-day intervals over a 6-month period using the optimized assay protocol.

Statistical analysis

Linear regression analysis was performed to establish the correlation between bacterial count (log CFU/mL) and detection time. Pearson correlation coefficient (R²) was calculated to assess linearity. All experiments were conducted in triplicate, and data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using appropriate software at a significance level of p<0.05. Conductivity of the PANI-Pec solution at 3 mg/mL was 6.51 ± 0.02 × 10⁻⁴ S/cm, consistent with values reported previously for PANI-Pec nanoparticles (Amarnath et al., 2014) [2] . Pectin served as both a stabilizer and biocompatible coating, facilitating the formation of uniformly sized particles with improved aqueous dispersibility (Fig. 1).

Principle of the colorimetric sensor

The sensing mechanism of the PANI-Pec Eppendorf tube sensor relies on the pH-responsive behavior of polyaniline. Under neutral or slightly alkaline conditions (pH ≥ 7), polyaniline exists predominantly in its emeraldine base (EB) form, exhibiting blue coloration. During bacterial growth, microorganisms metabolize carbon sources (particularly dextrose) in the optimized growth medium through glycolytic and fermentative pathways, releasing acidic byproducts including succinate, acetate, lactate, and formate. These metabolites create an acidic microenvironment within the sensor tube. The resulting protonation of the PANI-Pec matrix triggers the EB-to-ES transition, producing a distinctive and visually detectable color change from blue to Green (Fig. 2). The use of an agarose-embedded semi-solid matrix within sterile Eppendorf tubes offers a significant advantage over liquid-phase monitoring: it minimizes interference from bacterial cell turbidity while maintaining intimate diffusive contact between the sensor matrix and metabolites produced in the overlying liquid phase. This configuration enables reliable and reproducible color change detection across a wide range of bacterial concentrations.

Optimization of PANI-pec solution, growth medium, and sample volume

The optimized PANI-Pec nanoparticle volume was found to be 250 µL, which produced the most intense and visually distinguishable blue-to-green colour transition in response to bacterial metabolite-induced pH changes. This volume provided the highest assay sensitivity and was therefore selected for subsequent studies. RSM-based optimization of the growth medium identified an optimum composition of 2.75 gm/50 mL incorporating casein enzyme hydrolysate, yeast extract, dextrose, and sodium chloride. This medium supported enhanced production of acidic metabolites and ionic compounds during bacterial growth. One-way ANOVA confirmed significant differences among medium combinations (p<0.0001), with R² values of 0.9685 (OD at 600 nm) and 0.9452 (pH), indicating a strong correlation between predicted and experimental outcomes. A sample volume of 250 µL was found to be optimal for reliable sensor performance in the Eppendorf tube format, yielding the most rapid color change at 37 °C with a detection time of 2.0 ± 0.25 h at an initial bacterial concentration of 8.0 log CFU/mL (Fig. 3). This finding parallels the volume optimization reported for PANI-Pec paper strip sensors, where 500 µL was optimal for a larger paper strip format (Saha et al., 2025) [29] . When applied to raw milk samples, the PANI-Pec nanoparticle sensor produced a positive result (Fig. 4), evidenced by a clear blue-to-green color transition within 2.0 ± 0.25 h at 37 °C. This color shift was attributed to the acidic metabolites and ionic by-products released during active bacterial growth inherent in raw milk, which contains a naturally high and diverse microbial load. On the other hand, sterilized milk sample yielded a negative result (Fig. 4), with the sensor retaining its characteristic blue colour throughout the incubation period. The absence of a colour transition in sterilized samples reflected the significantly reduced bacterial population resulting from thermal processing. These findings demonstrate the ability of the PANI-Pec colorimetric assay to effectively discriminate between microbiologically unsafe raw milk and thermally treated pasteurized milk.

Sensitivity

The sensitivity of the PANI-Pec Eppendorf tube sensor was evaluated using raw milk spiked with bacterial cell suspensions at concentrations ranging from 8.0 to 4.0 log CFU/mL. A strong linear relationship (R² = 0.9584) was observed between bacterial count and detection time (Fig. 5), demonstrating the quantitative capability of the assay. At high inoculum (8.0 log CFU/mL), the sensor produced a visible color change within 2.0 ± 0.25 h, while at low inoculum (4.0 log CFU/mL), detection was achieved within 4.0 ± 0.25 h. This performance compares favorably with existing PANI-Pec paper strip sensors (detection at 4.0 ± 0.15 log CFU/mL within 4.5 ± 0.15 h) and with commercial systems such as the Soleris® NB-100 system (Neogen® Corporation). The absence of biological recognition elements antibodies, enzymes, or nucleic acids is a key advantage of this system, as it eliminates the risk of biocomponent degradation that typically limits the shelf life of conventional biosensors.

Discussion

The present study reports the development of a PANI-Pec (polyaniline-pectin) colorimetric assay for the rapid detection of total plate count (TPC) in raw milk, employing an Eppendorf tube-based semi-solid agarose matrix as a novel sensing platform. The fundamental sensing mechanism relies on proton doping of polyaniline (PANI) by organic acids generated through bacterial fermentative metabolism, causing a characteristic colour transition from green (emeraldine salt, oxidised conductive form) to yellow (emeraldine base, reduced insulating form) as the local pH decreases (Thakur et al., 2015; Saha et al., 2025) [29,33] . This acid-induced chromogenic response has been well established in PANI-based sensor literature (Janata and Josowicz, 2003; Bhadra et al., 2009) [7,19] . However, the current work introduces important architectural and medium-optimisation innovations that substantially enhance the practical utility and analytical performance of the PANI-Pec sensor relative to previously described paper strip formats (Thakur et al., 2015; Saha et al., 2025) [29,33] . A central innovation in this study is the transition from a PANI-Pec paper strip configuration to an Eppendorf tubebased semi-solid 2% agarose matrix. In liquid-phase biosensing systems, increasing microbial cell density inevitably leads to sample turbidity, which can confound optical measurements and impair visual colour discrimination (Lazcka et al., 2007) [23] . The semi-solid agarose gel physically confines the PANI-Pec sensor material at the base of the tube, creating a spatial separation between the turbid microbial suspension above and the sensing zone below, while permitting free diffusion of lowmolecular-weight metabolites primarily short-chain organic acids such as lactic acid, acetic acid, and formic acid to the sensor surface. This geometry-driven optical isolation is particularly important for raw milk samples, where the intrinsic opacity of the matrix compounds the turbidity problem noted in earlier PANI-Pec work (Thakur et al., 2015) [33] . Beyond turbidity management, the Eppendorf tube format offers a sealed, contamination-resistant, selfcontained reaction vessel that is readily scalable for highthroughput testing, aligning with the logistical demands of modern dairy quality assurance laboratories (Bhunia, 2014) [8] . The nutrient medium composition was systematically optimised to support broad-spectrum acid-producing microbial growth. The selected formulation casein enzyme hydrolysate (nitrogen source), yeast extract (vitamins, minerals, and co-factors), dextrose (fermentable carbon source), and sodium chloride (osmotic stabiliser) at a combined concentration of 2.75 g/50 mL was designed to support the metabolic requirements of the diverse milk microbiota, including fermentative Gram-positive bacteria (Lactobacillus, Streptococcus, Staphylococcus spp.) and Gram-negative bacteria (coliforms, Pseudomonas, Listeria spp.) (Boor et al., 2017; Quigley et al., 2013) [9,28] . Dextrose serves as the primary fermentable substrate whose catabolism via glycolysis and mixed acid fermentation yields organic acids that protonate the PANI backbone. This formulation mirrors the medium components and concentration ranges reported by Saha et al. (2025) [29] for paper strip-based TPC detection in milk, validating the cross-platform applicability of this medium design. The inclusion of yeast extract and casein enzyme hydrolysate ensures the availability of essential growth factors and assimilable nitrogen that might otherwise limit growth of fastidious organisms at early time points, thereby maximising the kinetic acid output that drives sensor actuation (Atlas, 2010) [5] . The detection performance of the PANI-Pec Eppendorf tube assay is notable across a range of initial bacterial concentrations. At an inoculum of 4.0 log CFU/mL representative of the lower regulatory threshold for raw milk microbial quality in many jurisdictions -a definitive colour change was recorded within 4.0 h. This compares favourably with the analogous PANI-Pec paper strip sensor, which required 4.5 ± 0.15 h to achieve a comparable endpoint at the same bacterial load (Saha et al., 2025) [29] , suggesting that the agarose matrix configuration confers a modest kinetic advantage, possibly attributable to the higher local concentration of metabolites at the gelliquid interface. At elevated bacterial loads of 8.0 log CFU/mL, the assay achieves detection within 2.0 h, approaching real-time monitoring capability. In contrast, conventional standard plate count (SPC) and total viable count (TVC) methods require 48-72 h of incubation before enumeration, a delay that is incompatible with perishable dairy product release decisions (Frank et al., 1992) [11] . Several other rapid methods, including ATP bioluminescence (Bautista et al., 1992) [6] , flow cytometry (Gunasekera et al., 2000) [16] , and impedance microbiology (Ur and Brown, 1975), reduce analysis time to 3-8 h but require expensive capital equipment and trained operators, limiting their deployment in resource-constrained or field settings. The PANI-Pec assay achieves comparable speed without instrumentation, representing a significant costperformance advantage. An important analytical feature of the assay is the linear relationship (R² = 0.9584) between detection time and initial bacterial count, which enables semi-quantitative estimation of microbial load from a simple visual time-to-positive readout. This inverse relationship shorter detection time at higher initial concentrations arises from the kinetics of acid accumulation: higher initial cell densities reach the threshold acid concentration required for PANI Protonation more rapidly (Dalgaard and Koutsoumanis, 2001) [10] . A similar linear detection time versus bacterial load relationship has been documented for impedimetric rapid methods (Gibson et al., 1988) [14] and for PANI-Pec paper strip sensors (Saha et al., 2025) [29] . The R² value obtained in this study (0.9584) is consistent with values reported for comparable colorimetric and impedimetric rapid methods, demonstrating adequate predictive capability for semiquantitative milk quality classification (Bhunia, 2014) [8] . Crucially, this semi-quantitative functionality is achieved without spectrophotometric or fluorometric instrumentation, relying solely on timed visual observation, which is a practical advantage in low-resource dairy testing environments. The broad-spectrum detection capability of the PANI-Pec assay is a key operational advantage in raw milk quality surveillance, where the microbiota is heterogeneous and dominated by diverse acid-producing species. The assay responds generically to all fermentative acid-producing bacteria encountered in raw milk, including coliforms, lactobacilli, streptococci, staphylococci, pseudomonads, and Listeria spp. (Quigley et al., 2013; Boor et al., 2017) [9,28] . This is consistent with the principle that TPC methodologies are inherently non-selective, measuring the collective metabolic activity of the viable microbial community rather than targeting individual pathogens (Jay et al., 2005) [20] . However, a recognised limitation of the present assay shared with PANI-Pec paper strip sensors (Saha et al., 2025) [29] and related colorimetric acid-response platforms is its inability to detect endospore-forming bacteria such as Bacillus cereus and Bacillus licheniformis within the standard assay window. These organisms characteristically exhibit extended lag phases and reduced acid production rates relative to vegetative fermentative bacteria (Andersson et al., 1995) [3] , and their spores are heat-resistant, rendering them a persistent concern in pasteurised dairy products. Future iterations of the assay could address this gap by extending the incubation window or by incorporating selective pre-enrichment steps for spore-formers (Griffiths, 2010) [15] . From a practical and economic perspective, the PANI-Pec composite material offers significant advantages over enzyme-based or antibody-based biosensors that are widely investigated for food safety applications. PANI and pectin are both commercially available, chemically stable compounds that can be synthesised and stored without refrigeration or specialised conditions (Stejskal and Gilbert, 2002; Thakur et al., 2015) [32,33] . Unlike Enzyme-Linked Immunosorbent Assays (ELISA) or lateral flow immunoassays, the PANI-Pec sensor does not require biological recognition elements that are subject to denaturation, cold-chain dependency, or batch-to-batch variability. The Eppendorf tube format is universally available in microbiology laboratories, requires no custom fabrication, and is compatible with standard biosafety practices. The overall assay cost is low, and the read-out is purely visual, requiring no reader device, power source, or data system. These attributes collectively position the PANI-Pec Eppendorf tube assay as a particularly suitable tool for quality control in small and medium-sized dairy operations, cooperative testing laboratories, and field survey settings in low-and middle-income countries (Lim et al., 2015) [24] . Contextualising the PANI-Pec assay within the broader landscape of colorimetric microbial detection technologies, several parallels and distinctions are noteworthy. Redox indicator dyes such as resazurin (Sarker et al., 2007) [30] and tetrazolium salts (Kuhn et al., 2015) [22] similarly transduce bacterial metabolic activity into a colour signal, but these are typically single-use, soluble dyes that offer no structural confinement and are subject to background interference from coloured food matrices. Carbon nanotube and graphene-based colorimetric sensors offer higher sensitivity but require sophisticated nanomaterial synthesis protocols and are not yet amenable to point-of-use implementation. The PANI-Pec system occupies an advantageous niche: analytically sufficient sensitivity and speed for regulatoryrelevant bacterial thresholds, combined with fabrication simplicity and a fully visual read-out. Future work should address the quantitative precision of the time-to-positive relationship through a larger multi-laboratory validation study, assess sensor performance across diverse raw milk compositions (fat content, somatic cell count, antibiotic residue presence), and explore miniaturised or multiplexed configurations that could enable simultaneous TPC and pathogen-specific detection (Neethirajan et al., 2018; Bhunia, 2014) [8,27] . Integration of the assay with a simple smartphone-based colour capture application could further standardise the visual end-point determination and facilitate remote data logging (Yetisen et al., 2013) [38] .

Conclusion

A novel PANI-Pectin nanoparticle-based colorimetric assay for rapid TPC estimation in raw milk operates on the principle of pH-driven proton doping of PANI-Pec nanoparticles embedded in an agarose gel matrix, producing a distinct and visually detectable color change from blue to green in response to acidic metabolites generated during bacterial growth. The PANI-Pec Eppendorf tube assay offers several significant advantages over conventional TPC methods and existing rapid technologies: it requires no specialized equipment, is low-cost, provides results within 2-4 h (compared to 48-72 h for plating methods), is compatible with diverse bacterial groups in raw milk, and delivers unambiguous visual results interpretable by the naked eye. From a regulatory and implementation standpoint, the PANI-Pec Eppendorf tube assay is particularly well-suited for deployment at Raw Milk Reception Docks (RMRDs) in India and other developing nations, where access to laboratory infrastructure is limited. The assay offers a compelling alternative to the Methylene Blue Reduction Test (MBRT), which is a conventional indirect rapid screening method but is semi-quantitative, operator-dependent, and unreliable for pasteurized or chilled milk. The PANI-Pec system, in contrast, provides consistent performance across both raw and pasteurized milk and eliminates subjectivity in result interpretation through its unambiguous blue-to-green color transition.

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